Collected Thesis

General Introduction
Introduction
Breast cancer is the most common cancer type in women worldwide

(Sharma et al., 2010). It is the leading cause of cancer death in women in less
developed countries and the second leading cause of cancer death in american
women, exceeded only by lung cancer (Jemal et al., 2011).
Anatomically, the breast is made of glands called lobules and thin tubes
called ducts. Ducts carry the milk from the lobules to the nipple. Breast tissue also
contains fat and connective tissue, lymph nodes, and blood vessels (figure I)
(Chaurasia and Pal, 2014).
Figure I: Anatomy of a normal breast (Chaurasia and Pal, 2014).
Breast cancer is classified into two types: non invasive (in situ) and invasive
carcinoma. The first type is further sub-classified into ductal or lobular carcinoma
(Malhotra et al., 2010). In situ ductal carcinoma is the most common type of breast
cancer in which abnormal cells are found in the lining of the ducts but are not spread
outside (Sharma et al., 2010). Breast cancer can also begin in the cells of the lobules
and in other tissues in the breast. Breast cancer that spreads to surrounding tissues is
called invasive breast cancer. It is manifested as infiltrating ductal, invasive lobular,
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ductal/lobular, mucinous (colloid), tubular, medullary and papillary carcinomas

(Malhotra et al., 2010).
A variety of causes contribute to the occurrence of breast cancer e.g.

inherited mutation in the breast cancer gene type 1 or 2 (BRCA1 or BRCA2)
(Chaurasia and Pal, 2014), high breast density on a mammogram, radiation
exposure and frequent X-rays in youth (Chaurasia and Pal, 2014). Furthermore
factors affecting hormonal status have been associated with increased risk of breast
cancer as late menopause (Dumalaon-Canaria et al., 2014), postmenopausal
hormone use of estrogen or estrogen plus progesterone, never having children, a late
first pregnancy (after the age of 35), or not breast-feeding (Sauter and Daly, 2010).
Early breast cancer is usually asymptomatic. As the cancer grows, symptoms

start to appear and are manifested as: swelling or mass in the breast and the
underarm, nipple discharge (clear or bloody), pain in the nipple, persistent
tenderness of the breast, and unusual breast pain. In advanced stage, several
symptoms appears such as bone pain, shortness of breath, drop in appetite,
unintentional weight loss, headache, neurological pain and weakness due to
metastasis to other organs as bone, lung and liver (Sharma et al., 2010).
Breast cancer progresses from non metastatic hormone dependent cells to

metastatic hormone independent cells developing mixed populations of hormone
dependent and independent cells with variable proliferation rates (Kumar et al.,
2006; Clarke et al., 1989). In case of organ-confined disease, mastectomy is the
preferred treatment (Kumar et al., 2006). Patients are also treated with radiation
and/or chemotherapy, in addition to hormone ablation depending on breast cancer
type and stage (Stebbing et al., 2011). In estrogen (ER) positive breast cancer
patients, estrogen ablation is achieved by tamoxifen treatment, aromatase inhibitors
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or ovariectomy (Fabian, 2007). Invasion and metastasis are the main reasons for the
high mortality rates and poor clinical outcomes associated with breast cancer.
Therefore, control of invasion and metastasis is an important target to prolong
patient survival (Liu et al., 2013).
Most of the chemotherapeutic drugs used in breast cancer therapy interfere

with cancer cells proliferation: cyclophosphamide destroys genetic material
controlling tumor cell growth, methotrexate, 5-fluorouracil (5-FU), antimicrotubule
reagents as paclitaxel, docetaxel, vincristine and vinblastine prevent cell division
and doxorubicin which is a cytotoxic antibiotic (Kumar et al., 2006). Cytotoxic
drugs used in breast cancer therapy are most commonly given by intravenous (IV)
route. Currently used chemotherapeutic drugs are not selective for cancer cells. To
reach therapeutically effective concentration at the tumor site, high systemic dosages
are required. This usually leads to severe side effects and peripheral tissue damage
as bone marrow suppression cardiotoxicity, nephrotoxicity, hepatotoxicity, and
hematotoxicity, beside other immediate side effects as nausea, alopecia, and fatigue
(Kumar et al., 2006; Yoon et al., 2016). Because of the high toxicity and poor
specificity of currently used drugs, increasing the chemotherapeutic dosages is not
possible. Moreover, it has also been found that patients in relapse suffered from
acquired drug resistance which decreased the efficacy of chemotherapy. Multidrug
resistance is attributed to different mechanisms such as P-glycoprotein (Pgp)
transmembrane efflux pump that actively excretes cytotoxic drugs and reduces its
accumulation through an ATPase mechanism, alteration of enzymatic activities such
as topoisomerase or glutathione S-reductase activity and gene controlling apoptosis
(Krishna and Mayer, 2000). Lack of selectivity, severity of side effects during
administration of chemotherapeutic drugs and occurrence of multidrug resistance
present the major problems for anticancer drugs (Kumar et al., 2006).
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To solve the above problems, several strategies are now being adopted: the
first relies on increasing delivery systems specificity to tumor tissues via the use of
smart engineered systems. The second strategy involves the use of natural
products with wide safety margin on normal cells and high cytotoxic activity on
cancer cells. Improved efficiency of these low toxiciy products is the task of
formulators and is also performed nowadays through engineered multifunctional
smart nanocarriers.
Recent advancement in nanotechnology has shown that nanoparticles (NPs)

are promising drug delivery systems possessing unique physicochemical,
mechanical, optical and biological properties (Yu et al., 2016; Khan et al., 2017).
NPs have different applications in biological systems in general and as drug delivery
systems in particular since most cells are 10,000–20,000nm in diameter and so NPs
can enter cells, even nuclear compartments, and interact with DNA and cellular
proteins. Their important and unique features, such as surface to mass ratio, quantum
properties and ability to adsorb and carry other compounds are the reason to be
attractive in medicinal applications (De Jong and Borm, 2008). They can be
fabricated at different sizes and surface modifications, which determine their
properties in biological systems. The prefix “nano” is commonly used for particles
that are up to several hundred nanometers in size (Wilczewska et al., 2012).
However, particles less than 100nm have longer circulation times, large effective
surface areas, low sedimentation rates, and they show enhanced diffusion potential
and are easily internalized in tumor cells through the membrane pores.
Agglomeration of NPs has to be avoided to prevent thrombosis. Furthermore small
particles have access to capillaries and are more resistant to the macrophage uptake
of the reticuloendothelial system (RES) (Cref et al., 1994).
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NPs applicable for cancer treatment need to counter the adverse effects of the
current chemotherapeutic therapy. NPs have the potential of improving drug
solubility and bioavailability, enhancing drug release and delivering the
pharmacologically required concentration of the drug and so less dose is required
(Bhatia, 2016). NPs also increase the stability of drug and formulation and provide
better formulation opportunities for drugs and increase patient compliance, increase
drug concentration at the target site, overcome multidrug resistance, reduce side
effects to vital organs by reducing systemic exposure, avoid immune response and
hematopoietic toxicity, destroy malignant cells specifically, sparing healthy cells,
kill primary tumors inaccessible to surgery and dormant cells and detect cancers at
an early stage (Leuschner and Kumar, 2005; Bhatia, 2016).
NPs can be classified into polymeric NPs, lipid NPs as solid lipid NPs,
nanostructured lipid carriers, lipid drug conjugates and vesicular systems (Müller et
al., 2002; Wissing et al., 2004; Kovacevic et al., 2011) and inorganic NPs as iron
oxide magnetic NPs (Vatta et al., 2006; Dobson, 2006), gold NPs, silver NPs, silica
NPs (Echeverría et al., 2010; Di Pasqua et al., 2009; Kwon et al., 2013) and carbon
nanotubes (CNTs) (Beg et al., 2011).
To enhance their in vivo application, several criteria should be considered in

designing NPs: biocompatibility of particles and coatings, particle size (PS), surface
properties and functionality, drug incorporation and release, formulation stability
and shelf life, immunogenicity, biodistribution, targeting and degradation properties
and the possible adverse effects of residual material after the drug delivery. In this
respect biodegradable, biocompatible and non toxic NPs with a limited life span as
long as therapeutically needed would be optimal (Rahoui et al., 2017; De Jong and
Borm, 2008). Factors affecting the toxicity of NPs include hydrodynamic size,
shape, amount, surface chemistry, the route of administration, reaction of the
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immune system and residence time in the blood stream. So toxicological studies of
each new DDS formulation are needed (Ai et al., 2011). NPs injected into biological
systems are rapidly coated with plasma proteins such as immunoglobulins and
fibronectin and form aggregates. This process is called opsonization. Opsonized
particles are recognized by the (RES) or mononuclear phagocytic system (MPS), of
the resident macrophages of the liver, spleen, lymph nodes, nervous system, and
bones. These macrophages internalize the opsonized NPs through phagocytosis
within 0.5–5min, thus removing the active NPs from the circulation and prevent their
access to the tumor tissue (Kumar et al., 2006).
As nanocarriers suffer from non-specific uptake and potential degradation in

macrophages, targeting is important for maximizing drug efficacy and minimizing
side effects. Targeting of NPs is achieved by passive targeting, active targeting, or a
combination of both strategies to achieve tumor-specific particle accumulation.
Figure II shows a schematic diagram representing the accumulation of nanocarriers
in tumor sites by passive or active targeting.
In passive targeting, size-selective accumulation of drug loaded NPs at

interstitial space of tumor is obtained due to the defective, leaky structure of tumor
vessels and the impaired lymphatic system. This is called an enhanced permeability
and retention (EPR) effect (Masood, 2016). To take full advantage of the EPR effect,
it is important to incorporate several properties into the design of nanocarriers to
escape body defense and clearance systems resulting in longer circulation time (≥6h)
in the blood stream with subsequent extravasation and drug accumulation in target
tumor tissue (Bazak et al., 2014). The threshold size for extravasation in tumors was
found to be ~400nm in diameter and it has been reported that nanocarriers ˂200nm
in diameter are preferred. Since, NPs smaller than 10nm are significantly filtered by
the kidneys, therefore, the PS of NPs between 10 and 200nm is considered optimal
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for tumor targeting (Yu et al., 2010). Surface charge of nanocarriers is another
important parameter. As both highly positive (˃+30) and highly negative (˂-30)
charged nanocarriers are susceptible to rapid clearance by RES with the great
cytotoxicity of cationic NPs, it is important to design nanocarriers with either a
neutral (±10) or a slight negative zeta potential (Clogston and Patri, 2011). Coating
the NPs with hydrophilic biodegradable matrices such as PEG renders them invisible
and no longer recognized by the MPS (known as stealth effect) and increases their
circulation half-life (Jokerst et al., 2011).
Figure II: Schematic diagram representing the accumulation of nanocarriers

in tumor sites by passive and active targeting (Yu et al., 2010).
In active targeting, specific biological processes such as ligand-receptor

recognition were used to deliver drug to the target cells. A ligand is attached to the
drug delivery system and acts as a homing device that takes the delivery system to
the target. Various types of targeting ligands have been employed to actively target
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NPs including: antibodies, antibody fragments, aptamers, peptides, whole proteins

(e.g. transferrin) and different receptor ligands (e.g. folic acid) (Kamaly et al., 2012).
As ligand and its receptor are complimentary to each other, the receptor-ligand
interaction enables surface binding and cellular internalization of NPs by receptor-
mediated endocytosis (Bazak et al., 2015). Furthermore, active targeting inhibits
multidrug resistance (MDR) via inhibiting or bypassing Pgp-mediated drug efflux
(Yu et al., 2010; Bazak et al., 2015).
Trastuzumab (TZB) also called herceptin (HER) is a humanized IgG1

monoclonal antibody that directly targets the epidermal growth factor receptor
(HER2 or ErbB2) (Dean, 2012). Schematic structure of trastuzumab is shown in
figure III.
Figure III: Schematic structure of trastuzumab (Ho et al., 2010).
Patients with HER2 overexpressed breast cancer are likely to have aggressive
tumor. This type of cancer is characterized by high proliferation rate and
angiogenesis, inhibition of apoptosis, rapid development of metastasis, decreased
expression of ER and PR and so a poor prognosis (Hortobagyi, 2005). HER2 is
overexpressed in breast cancer on the primary tumor as well as on metastatic sites
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and is minimally expressed by normal tissues. TZB conjugated NPs showed a

specific targeting to HER2 overexpressing cells with cellular uptake by receptor-
mediated endocytosis. It was also found that the internalization ability of HER2
allows an efficient uptake of the antibody alone as well as conjugated to drugs or
drug carrier systems (Steinhauser et al., 2006). TZB itself is considered the first-
line option for the treatment of early and advanced HER2 positive breast cancer
(Maximiano et al., 2016). TZB having high binding affinity to the EGFR, inhibited
binding of the natural ligand (EGF) to the receptor, thus inhibited receptor
phosphorylation down regulating the signaling pathways (Hortobagyi, 2005). This
led to increased cell cycle arrest and apoptosis and suppression of cell proliferation
and metastasis (Maximiano et al., 2016). A previous study showed that TZB, when
directed to HER2 receptor, suppressed growth of human breast and ovarian cancer
cells overexpressing p185HER2 and when combined with cisplatin (DNA damaging
drug) therapy, TZB blocked DNA repair and increased sensitivity to cisplatin in
drug-resistant ovarian carcinoma cells producing a synergistic decrease in cell
growth (Pietras et al., 1994). Another study on the effect of interaction between TZB
and different cytotoxic drugs on SK-BR-3 breast carcinoma cells demonstrated a
synergistic interaction between mAb HER2 and alkylating agents (thiotepa),
platinum analogs (cisplatin) and topoisomerase II inhibitors (etoposide) as well as
an additive cytotoxic effect with anthracyclines (doxorubicin), taxane (paclitaxel),
methotrexate and vinca alkaloid (vinblastine). On the other hand, an antagonist
effect was obtained with 5-FU (Pegram et al., 1999). The synergistic effect of the
combination of TZB with radiotherapy was also reported. TZB modulated the repair
of radiation-induced DNA damage and enhanced radiosensitivity of human breast
cancer cells overexpressing HER2 (Pietras et al., 1999). TZB was also used in
several studies for targeting different types of NPs to breast cancer cells
overexpressing HER2. Magnetic NPs modified via PEGylation followed by
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immobilization of TZB showed remarkable enhanced hyperthermia effect in cultured

SK-BR-3 cells in vitro as well as in vivo tumor bearing mice model (Almaki et al.,
2017). The surface decoration of paclitaxel-loaded PLGA/montmorillonite
(PLGA/MMT) with TZB was reported to provide synergistic therapeutic effects, to
achieve targeted chemotherapy for HER2-positive breast cancer and to reduce
paclitaxel side effects (Sun and Feng, 2008). TZB grafted polyamidoamine
(PAMAM) dendrimers improve site specific delivery of docetaxel to HER2
positive breast cancer cells and significantly improve its pharmacokinetic profile
(Kulhari et al., 2016). HER immobilized PLGA NPs encapsulating salinomycin
revealed 16.2% increase in cell uptake within 60min by MCF-7 cells (Aydın, 2013).
To overcome toxicity from chemotherapeutic agents, attention has been given

to the use of drugs with a wide safety margin. Recently, special focus had been given
to the formulation of novel drug delivery systems loaded with natural
extracts/compounds possessing anticancer activity with low or no side effects. Large
variety of active phytochemicals such as carotenoids, flavonoids, ligands,
polyphenolics, terpenoids, sulfides and plant sterols has been identified in different
types of herbs as ginko, goldenseal, ginseng, garlic, echinacea, aloe vera, saw
palmetto and magnolia along with others (Shareef et al., 2016, McKeown and
Hurta, 2015). These herbs defend the body from malignancy by different
mechanism of actions: enhancing body detoxification, preventing cancer cells
proliferation by modifying the activity of precise hormones and enzymes, preventing
lethal side effects and complications of chemotherapy and radiotherapy and
improving function of the body’s immune cells (Richard et al., 2015).
Magnolol (Mag), with the chemical structure shown in figure IV, is a

polyphenolic compound isolated from the root and stem bark of Magnolia
officinalis. A plant that belongs to the family Magnoliaceae distributed over China,
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Japan, and South Korea. M. officinalis had been used for over 1,000 years as a folk
remedy in Asia to treat asthma, digestive problems and emotional distress (Patočka
et al., 2006).
Previous preclinical studies had shown that Mag possesses anti-oxidative,

anti-inflammatory, anti-tumorigenic, anti-diabetic, anti-microbial, anti-depressant
and anti-neurodegenerative properties. Mag can also provide analgesic as well as
cardiovascular and liver protective effects (Chen et al., 2011a).
IUPAC name:4-Allyl-2-(5-allyl-2-hydroxy-phenyl)phenolical
Emperical formula: C18H18O2
Figure IV: Magnolol chemical structure, name & formula (Chen et al.,
2011a).
Recent studies have shown that Mag exhibits anti-cancer properties by

countering proliferation, inducing differentiation and apoptosis, and inhibiting
angiogenesis, metastasis and multidrug resistance (Liu et al., 2013). At the same
time, Mag has shown no side effect and to be non-toxic even at the highest doses in
several studies (McKeown and Hurta, 2015). Previous studies showed the
antiproliferative effect of Mag on various cancer cell lines such as human colon and
liver cancer cells by inhibiting DNA synthesis and activating apoptosis (Lin et al.,
2002), gallbladder cancer cells through the p53 pathway (Li et al., 2015) and human
lung carcinoma (Yang et al., 2003). Other studies reported that Mag pretreatments
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prevent UVB-induced skin cancer development by enhancing apoptosis (Seo et al.,

2011).
It was also found that Mag potently inhibited proliferation and induced
apoptosis in MCF-7 human breast cancer cells (Liu et al., 2013). Mag induced
apoptosis in MCF-7 cells via the intrinsic pathway with release of apoptosis-
inducing factor (AIF) from mitochondria and G2/M phase arrest pathway (Zhou et
al., 2013). It was also found that overexpression of the human epidermal growth
factor (HER2) oncogene contributes to tumor cell invasion, metastasis and
angiogenesis and correlates with poor prognosis. Previous studies showed that Mag
inhibited cell growth and HER2-mediated tumor metastasis in human HER2-
overexpressing cancer cells by the ability of Mag to down-regulate HER2 and its
downstream pathway (Chuang et al., 2011). Furthermore, it also suppressed the
expression of downstream target genes, vascular endothelial growth factor (VEGF),
matrix metalloproteinase 2 (MMP2) and cyclin D1 (Chuang et al., 2011).
Triple-negative breast cancers (TNBC) (15% to 20% of breast cancers) are

characterized by the absence of estrogen (ER), progesterone receptor (PR), and
HER2 expression and result in high mortality due to their rapid invasive potential
and acquired treatment resistance. Systemic treatment options for TNBC are limited
to cytotoxic chemotherapy (Arnedos et al., 2012). Strategies for preventing or
suppressing cancer invasion and metastasis can improve the survival of triple-
negative breast cancer patients. Liu et al., 2013 showed that Mag could inhibit
phorbol 12-myristate 13-acetate (PMA) induced breast cancer cell invasion by
inhibiting the nuclear factor NF-κB signaling pathway and the suppression of MMP-
9 at the mRNA and protein levels as shown in figure V.
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Figure V: Diagram of magnolol signal pathway of inhibiting breast cancer cell

invasion and migration (Liu et al., 2013).
These findings suggest that Mag is a novel promising anti-cancer and anti-
invasion compound. Moreover, Mag might be considered a new potential therapeutic
agent for patients with breast cancer, particularly highly invasive breast cancer.
In spite of its potent anticancer activity against cancer cells with wide safety
on normal cells, Mag suffers from poor aqueous solubility and very low oral
bioavailability (Tsai et al., 2015). It has been shown in previous studies that the
bioavailability of Mag following oral administration lies in the range (4-9%) due to
extensive first pass metabolism (Tsai et al., 1992; Lin et al., 2002). Mag had also
been reported to be a strong quencher which binds to human serum albumin
(HSA)/bovine serum albumin (BSA) with high affinity. This binding is
predominantly driven by hydrophobic and electrostatic interaction between Mag and
HSA/BSA (Liu et al., 2003).
Therefore, novel drug delivery systems are needed to deliver Mag to its site
of action (breast cancer cells). In this thesis, polymeric (PLGA) and metallic (gold)
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NPs will be used to load and enhance the delivery of Mag for the treatment of breast
cancer cells. Improving the NPs selectivity will be achieved by combining both
passive and active targeting strategies. Tailoring the size and charge of the NPs will
help to provide the passive targeting, while engineering the surface by attaching the
ligand (HER) will allow enhancing the selectivity to breast cancer cells via active
targeting.
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Scope of work
Scope of work
Breast cancer became a life-threatening disease for women worldwide. The

available treatment strategies are non specific, usually accompanied with severe side
effects leading to poor prognosis. So, in this thesis, we tried to find a novel strategy
that relies on the use of less toxic product with wide safety margin on normal cells
and high cytotoxic activity on cancer cells like several phytochemicals drugs; along
with an engineered targeted nanocarrier system to increase specificity to breast
cancer cells and improve efficiency. Through the work in this thesis, we will study
two types of nanocarrier based systems: polymeric based nanocarriers and inorganic
nanocarriers especially gold nanoparticles. To fulfill our goal, the work in this thesis
will focus on:
▪ Preparing magnolol (Mag) loaded NPs with PS less than 200nm suitable to
achieve passive targeting (EPR effect).
▪ Coating NPs with hydrophilic polymer to provide stealth effect.
▪ Providing selectivity by the attachment of targeting moiety to the prepared
Mag NPs.
▪ Challenging the system on MCF-7 breast cancer cells.
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Chapter 1 Introduction
Introduction
Polymeric nanoparticles (PNPs) designed for cancer treatment via IV route

are usually prepared from biocompatible and biodegradable polymers. The drug is
dissolved, entrapped, encapsulated or attached to a NP matrix. Depending on the
method of NPs preparation, nanospheres or nanocapsules can be obtained.
Nanocapsules are systems in which the drug is confined to a cavity surrounded by a
unique polymeric membrane, while nanospheres are matrix systems in which the
drug is physically and uniformly dispersed, see figure VI (Soppimath et al., 2001;
Nagavarma et al., 2012). Polymeric NPs effectively carry drugs, proteins, and DNA
to target cells and organs. Their nanometer-size promotes effective permeation
through cell membranes and stability in the blood stream. PNPs have many
advantages as enhancing the stability of pharmaceutical agents, ease and cheap
manufacturing, the ability to deliver a higher drug concentration to the desired
location and the ability to modify drug release.
Figure VI: Difference between nanospheres and nanocapsules (Nagavarma et

al., 2012).
Several methods are used for the preparation of PNPs. These techniques are
classified according to whether the particle formation involves a polymerization
reaction or the NPs are formed directly from a macromolecule or preformed polymer
or ionic gelation method (Rao and Geckeler, 2011). Emulsification and solvent
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evaporation/extraction, nanoprecipitation (solvent-displacement), supercritical anti-

solvent method and salting-out are among the most common techniques, which are
widely used for fabrication of PNPs (Masood, 2016). Figure VII shows various
techniques used for the preparation of PNPs.
Solvent
evaporation Emulsion
Nanoprecipitation Mini emulsion
Salting out Microemulsion
Dialysis Interfacial
SCF C/LR
SCF: supercritical fluid technology, C/LR: controlled/living radical.

Figure VII: Schematic representation of various techniques used for the
preparation of polymeric NPs (Rao and Geckeler, 2011).
Nanoprecipitation, also called solvent displacement method, involves the
precipitation of a preformed polymer from an organic solution and the diffusion of
the organic solvent in the aqueous medium in the presence or absence of a surfactant
(Fessi et al., 1989). The method is based on drop-wise addition of organic phase
(polymer dissolved in volatile water-miscible solvent) into an aqueous phase (with
or without stabilizer/surfactant). It can be applied to various polymeric materials
(Masood, 2016). NPs formation is instantaneous and the entire procedure is carried
out in only one step. The NPs synthesized are finally collected by ultracentrifugation
or high speed centrifugation, washed with Millipore water two to three times,
lyophilized in the presence of cryoprotective agents (sugars such as glucose and
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trehalose) to assess the redispersibility of the colloidal system and to prevent the
aggregation of NPs during the freeze‑drying process (Krishna et al., 2006). Only
lipophilic drugs can be encapsulated into PNPs via nanoprecipitation (Reis et al.,
2006). High drug entrapment efficiency, narrow size distribution, no need of
homogenization, and easy scale-up are the main advantages of this method (Reis et
al., 2006). Figure VIII shows a schematic representation of the nanoprecipitation
technique (Nagavarma et al., 2012).
Figure VIII: Schematic representation of nanoprecipitation technique

(Nagavarma et al., 2012).
An ideal polymeric carrier for NPs should ideally be easy to synthesize and
characterize, inexpensive, biocompatible, biodegradable, non-immunogenic and
non‑toxic (Krishna et al., 2006). The polymers used are classified into: natural
hydrophilic polymers such as proteins (gelatin, albumin, lecithin and legumin) and
polysaccharides (alginate, dextran, chitosan, agarose and pullulan) and synthetic
hydrophobic polymers such as poly(lactic acid) (PLA), poly(cyanoacrylates)
(PACA), poly(acrylic acid), poly(anhydrides), poly(amides), poly (ortho esters),
poly(ethylene glycol), poly(isobutylcynoacrylate) (PIBCA), poly(ethylene oxide)
(PEO) and poly(caprolactone) (PCL) and Poly-(lactic-co-glycolic acid) (PLGA)
(Muhamad et al., 2014). Natural polymers have certain limitations such as poor
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batch to batch reproductivity, in addition to the susceptibility to degradation and

potential antigenicity (Bhatia, 2016).
Drug loading in the nanoparticulate system can be done by two methods: the
first relies on incorporating the drug at the time of NPs production (incorporation
method). On the other hand, the drug is adsorbed after the formation of NPs by
incubating the carrier with the concentrated drug solution (incubation method) (Yih
and Al‑Fandi, 2006).
PLGA a copolymer of lactic acid and glycolic acid is considered one of the
most successfully used biodegradable polymers (Danhier et al., 2012). PLGA is
FDA-approved synthetic, biodegradable, biocompatible and non-toxic polymer
(Muhamad et al., 2014). Depending on the ratio of lactide to glycolide used for the
polymerization, different forms of PLGA can be obtained as: PLGA 75:25, PLGA
50:50, PLGA 25:75 etc. PLGA biodegrades by hydrolysis of its ester linkages, figure
IX (Makadia and Siegel, 2011). Mechanical strength and biodegradation rate of the
polymer are varied according to the degree of crystallinity of the PLGA, which
depend on the molar ratio of polylactic acid (PLA) and polyglycolic acid (PGA) in
the copolymer chain. Higher content of PGA leads to reduction in degree of
crystallinity and increase in degradation rate with an exception of the ratio 50:50 of
PLA/PGA exhibiting the fastest degradation (Makadia and Siegel, 2011).
(x is the number of lactic acid units and y is number of glycolic acid units)
Figure IX: Hydrolysis of poly lactic-co-glycolic acid (Makadia and Siegel,

2011).
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The use of biodegradable materials for nanoparticle preparation allows for

sustained drug release within the target site over a period of days or even weeks.
Biodegradable nanoparticles formulated from PLGA and PLA have been developed
to provide sustained drug delivery at the tissue site. In the majority of cases, drug
release follows more than one type of mechanism: release from the surface of
particles, diffusion through the swollen rubbery matrix and NPs erosion (Figure X)
(Jawahar and Meyyanathan, 2012).
Figure X: Drug release mechanisms from polymeric NPs (Jawahar and

Meyyanathan, 2012).
Biodistribution and cellular uptake of PNPs depend on size, shape, material
and surface characteristics, as well as the cell type (Kuhn et al., 2014). Figure XI
illustrates the effect of PS and surface charge on NPs biocompatibility and
biodistribution. NPs with optimum size (10-100nm) avoid renal elimination and
MPS and preferentially access tumors through their leaky vasculature and
accumulate in the interstitial compartment of the tumors due to the lack of lymphatic
clearance in the tumor tissue (known as the EPR effect) (Sadat et al., 2016). Cationic
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NPs bind more efficiently than anionic charged or neutral molecules to the
negatively charged plasma membrane of target cells. However cationic NPs are often
more cytotoxic than anionic and neutral ones, as the positive charged NPs surface
can cause more pronounced disruption of plasma-membrane integrity, stronger
mitochondrial and lysosomal damage, increased number of autophagosomes in
addition to the formation of complexes with the negatively charged nucleic acids
raising genotoxicity concerns (Gamucci et al., 2014). On the other hand, neutral
particles show lower interaction with the cell membrane than charged NPs, due to
the lower number of electrostatic interactions between NP surface and charged cell
membranes (Gamucci et al., 2014).
Figure XI: Effect of PS and surface charge on NPs biocompatibility and

biodistribution (Sadat et al., 2016).
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It has been demonstrated in previous work that most NPs, including PNPs, are
taken up by endocytosis (Cartiera et al., 2009). However passive penetration of
small NPs through the lipid bilayer may occur as an alternative process (Wang et
al., 2012). Figure XII briefly shows the classification of endocytic trafficking and
different mechanisms of endocytosis. Endocytotic uptake refers to two different
cellular uptake mechanisms: pinocytosis, which is responsible for the uptake of
fluids and molecules within small vesicles and phagocytosis responsible for
engulfing large particles (Kuhn et al., 2014). The pinocytosis route is further
subdivided as clathrin-mediated endocytosis (receptor mediated endocytosis),
caveolae-mediated endocytosis, clathrin/caveolae-independent endocytosis, and
macropinocytosis (Yameen et al., 2014). Endocytosis strongly depends on particle
size, surface characteristics and cell type. Figure XIII shows the effect of PS on the
internalization pathway of NPs (Sadat et al., 2016). Positive charged NPs are highly
uptaken by endocytosis or direct penetration due to the electrostatic interaction
between cationic NPs and anionic phospholipids, protein and glycan on the cell
surface (Sadat et al., 2016).
Polymeric PLGA NPs are internalized in cells partly through fluid phase
pinocytosis and also through clathrin-mediated endocytosis. The opsonization of
NPs by binding to plasma protein leads to attachment of NPs to macrophages and
subsequently their internalization by phagocytosis (Mahakalkar and Hatwar,
2014).
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Figure XII: Nanoparticles internalization pathways in mammalian cells (Kou et

al., 2013).
Figure XIII: Effect of particle size on NPs cell uptake (Hirota and Terada, 2012).
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Two strategies were adopted in this thesis to enhance cell uptake and better
internalization. The first is surface modification by providing a hydrophilic layer at
the surface rendering NPs invisible to RES and escaping phagocytosis. The second
is the targeting of tumor cells to increase selective cellular binding and
internalization through receptor-mediated endocytosis (Danhier et al., 2012).
The surface modification of NPs with nontoxic and blood compatible

material is essential in order to avoid recognition by macrophages and to prolong
blood circulation time. Protein opsonization not only enhances particle recognition
by the host immune cells but also increases the hydrodynamic diameter which
greatly affect NPs accumulation and tissue distribution (Gamucci et al., 2014).
Pegylation strategy had been found an ideal option to stealth the polymeric NPs and
to show resistance against opsonization and phagocytosis for longer residence time
in blood compared to the NPs prepared without PEG and reduces the risk of side
effects developed by the unmodified polymers (Shah et al., 2010). Polyvinyl alcohol
(PVA), polyethylene glycol (PEG), monomethoxy poly-(ethylene glycol) (mPEG),
polysorbate and d- α- tocopheryl polyethylene glycol 1000 succinate (TPGS) are
examples of hydrophilic, non-toxic, blood compatible polymers that are used for the
surface modification of PNPs (Masood, 2016) .
D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a water-

soluble derivative of natural vitamin E, which is formed by esterification of vitamin
E succinate with polyethylene glycol (PEG) 1000 (figure XIV). It has an average
molecular weight of 1513, an amphiphilic structure of lipophilic alkyl tail and
hydrophilic polar head with a hydrophilic/lipophilic balance value of 13.2 and a
relatively low critical micelle concentration (CMC) of 0.02% w/w (Zhang et al.,
2012). As one of the novel nonionic surfactants and FDA approved pharmaceutically
safe adjuvant, TPGS has been applied in drug delivery systems (DDS) as an
24
absorption enhancer, emulsifier, solubilizer, additive, permeation enhancer and

stabilizer (Guo et al., 2013; Mustafa et al., 2017; Yang et al., 2018). TPGS has
served for overcoming MDR by inhibiting Pgp to increase the effectiveness of
anticancer drugs. TPGS itself acts as an anticancer agent to induce apoptosis and
develops a synergistic effect with other anticancer drugs (Guo et al., 2013).
IUPAC name: 1-O-(2-hydroxyethyl)4-O-[2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-3,4-

dihydrochromen-6-yl] butanedioate
Emperical formula: C35H58O6
Figure XIV: TPGS chemical structure, name & formula (Mu and Feng,
2003a).
It is worthy to mention that the exploitation of the combination of PLGA and

TPGS in preparing hybrid NPs with possible stealth properties had been recently
proposed in literature (Vijayakumar et al., 2016). Replacing conventional stabilizers
with TPGS provided better emulsification with higher drug entrapment efficiency
(Ma et al., 2010; McCall and Sirianni, 2013; Gaonkar et al., 2017). In this thesis,
TPGS is used as stabilizer in the preparation of PLGA NPs by nanoprecipitation
method to provide hydrophilic NPs surface to prevent opsonization.
25
The surface modification by addition of targeting moieties not only influences

the biodistribution of NPs increasing cellular specificity and reducing toxicity and
side effects but also has a great potential in enhancing internalization by target cells.
The modification of the NPs surface with antibodies, peptides, aptamers, or other
motifs that specifically recognize a cell-surface receptor, leading to internalization
of NPs via receptor mediated endocytosis is an ideal strategy for NPs targeting
(Voigt et al., 2014). In this work, the mAb TZB was used as a homing molecule for
NPs to MCF-7 breast cancer cells.
The type of antibody used for targeting nanovehicles to specific cell types was
chosen based on surface antigens they presented (Steinhauser et al., 2006). As
targeting agents, antibodies have high selectivity and binding affinity due to the
presence of two epitope binding sites in a single molecule (Yu et al., 2010).
Two fundamentally different approaches have been developed to link ligands

such as antibodies, proteins, or enzymes to NPs surface: passive adsorption and
covalent conjugation. The passive adsorption, the commonly used method, relies
primarily on hydrophobic attractions and electrostatic interactions between the
antibody and the NPs surface to produce non-covalent NP protein conjugates (Yu et
al., 2010). Although the simplicity of the method, it suffers from non specificity,
lack of stability and activity due to conformational change or shielding of the
antigen-binding site (Aubin-Tam, 2013). The second approach uses covalent linkage
between the protein and NPs surface. Thioether, disulfide, and amide are the
frequently used covalent bonds. The conjugation can be performed either before or
after nanocarrier formation (Yu et al., 2010). In comparison to adsorption, chemical
conjugation provides a stable linkage between targeting ligands and nanocarriers.
Both site specificity and protein activity remain a challenge in NP conjugation. The
covalent coupling using chemical linkers that react with specific chemical groups on
26
the molecule to be conjugated provides a more specific and controllable method that
preserves protein tertiary structure and so its activity (Yih and Al‑Fandi, 2006).
In this chapter Mag encapsulated PLGA NPs (Mag-PLGA NPS) were first
prepared by nanoprecipitation method using PVA or TPGS as stabilizers, followed
by NPs targeting using mAb TZB as targeting moiety by both adsorption and
covalent conjugation methods.
27
Chapter 1 Experimental
Experimental
1. Materials:
• 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC):

Thermo Fisher Scientific, UK.
• 50/50 Poly(DL-lactide-co-glycolide) (PLGA): Grade 5002A, viscosity 0.2dl/g,
acid terminated, kindly provided by Corbion Purac Biomaterials, The
Netherlands.
• Acetonitrile: Merck, Germany.
• Bicinchonininc acid (BCA) protein assay reagent kit: Pierce, Rockford, USA
• D-α-tocopherol vitamin E polyethylene glycol 1000 succinate (TPGS):
Kindly provided by Isochem S.A.S, France.
• Dimethyl sulfoxide (DMSO): For proton magnetic resonance use, Merck, USA.
• Herceptin® (Trastuzumab (TZB)): F. Hoffmann-La Roche Ltd, Switzerland.
• Magnolol (Mag): purity >99%, Xian Lyphar Biotech Co., Ltd, China.
• Nanoseps: Centrifuge tube fitted with an ultra-filter: MWCO 100 KDa, Pall Life
Sciences, Port Washington, NY.
• N-hydroxysuccinimide (NHS): 98% (research grade), Sigma Aldrich, USA.
• Polyvinyl alcohol (PVA): Mw 30000, 98% hydrolyzed, Sigma Aldrich, Poole,
UK.
• Sodium dodecyl sulphate (SDS): BDH Laboratories Supplies, UK.
• Trehalose dihydrate: Fisher Scientific, Fairlwan, New Jersey.
• Vivaspin® 6: 6mL, MWCO 10 KDa, Sartorius Stedim Lab Ltd, UK.
• Bovine Serum Albumin (BSA), Cellulose dialysis tubing (MWCO 14 KDa,
15.9mm diameter), Syringe filters (0.45µm, Polytetrafluoroethylene (PTFE)):
Thermo Fisher Scientific, UK.
28
• Acetone, disodium hydrogen phosphate, glacial acetic acid, methanol

potassium chloride, potassium dihydrogen phosphate, sodium acetate,
sodium chloride and sodium hydroxide: El Nasr co., Egypt.
• All other chemicals and reagents used were of analytical grade.
2. Equipment:
• Balance, digital: Sartorius AY123, USA.
• Cooling centrifuge: Centurion K241, Germany.
• Fourier transform- infrared (FT-IR): Thermonicolet Nexus spectrometer,

Thermo Electron Corporation, USA.
• Freeze dryer: Christ alpha 1-2 LD plus, Christ, Germany.
• pH meter (Digital): JENWAY 350, UK.
• High performance liquid chromatography (HPLC) with UV detector: Jasco,

2080 plus, Japan. Column: Platinum C18, 100A, particle size 5µm, length
150mm.
• Spectrophotometer (double beam): Model UV-1601PC, Shimadzu, Kyoto,

Japan.
• Stirrer: Yellow line Ika magnetic stirrer with heater, MAG HSG HS7,
Wilmington, USA.
• Transmission electron microscope (TEM): JEM 1400 plus, electron

microscope, JEOL, Japan.
• Zetasizer Nano ZS: Malvern Instruments Ltd, UK.
29
3. Methodology:
3.1. Preparation and optimization of blank PLGA NPs
Blank PLGA NPs were prepared by the nanoprecipitation method previously

described in literature (Wang et al., 2016a). Briefly, 25mg PLGA were accurately
weighed and dissolved in 2mL organic solvent forming a 1.25% w/v PLGA solution.
This organic phase was added dropwise to 4mL water containing a SAA as stabilizer
maintained under stirring at 250rpm on a magnetic stirrer. The organic solvent was
then removed by stirring at 100rpm for 4h at room temperature. The NPs were
purified via two cycles of centrifugation at 30,000rpm for 30min at 4°C and washing
with deionized water. The NPs were then dispersed in 2mL water containing 2% w/v
trehalose. The NPs dispersions were first frozen at -20˚C and then placed in the
freeze dryer for 48h to yield the lyophilized NPs powders. The temperature of the
freeze dryer was set at -80°C and the vaccum was below 0.07mBar.
During preparation of PLGA NPs, various formulation parameters were

optimized in order to achieve the smallest uniform PS. Consequently, blank PLGA
formulae, F1to F10 were obtained as displayed in table 1. The optimization was
conducted by varying the following parameters. For the purpose, the effect of each
factor was evaluated while keeping all the other factors constant.
3.1.1. Organic solvent type:
Two water miscible organic solvents namely, acetone and acetonitrile were
tried.
3.1.2. SAA concentration and type:
Blank PLGA NPs prepared by nanoprecipitation method using acetone as

organic solvent and with constant polymer amount and organic to aqueous phase
30
ratio were tried with two different stabilizers, PVA and TPGS, in different
concentrations. Aqueous PVA and TPGS solutions were used in the respective
following concentrations 0.05, 0.1, 0.5 and 1% w/v and 0.03, 0.06, 0.12, 0.18, 0.24%
w/v (Esmaeili et al., 2007).
NPs showing optimum properties viz PS and PDI were selected for drug
loading.
Table 1: Composition of blank PLGA NPs prepared by nanoprecipitation for

the optimization of formulation parameters.
Formula Organic
SAA type SAA concentration (% w/v)
code solvent
F1 Acetonitrile 0.1
F2 0.1
F3 PVA 0.05
F4 0.5
F5 1
F6 Acetone 0.03
F7 0.06
F8 TPGS 0.12
F9 0.18
F10 0.24
All NPs formulae were prepared using a 1.25% w/v PLGA in an organic solvent and an organic to aqueous volume
ratio of 1:2.
3.2. Preparation and optimization of Mag-PLGA NPs
Plain formulae showing optimum PS and PDI (F4 and F9) were selected for
the incorporation of Mag. The drug loaded PLGA NPs were prepared using the same
method previously described in section 3.1. Accurately weighed Mag and PLGA
were dissolved in 0.5mL and 1.5mL acetone, respectively. The organic phase was
prepared by mixing Mag solution to PLGA solution and the procedure was
completed as before. Following two cycles of centrifugation at 30,000rpm for 30min
at 4°C and washing, lyophilization of NPs colloidal dispersion was performed using
trehalose as cryoprotectant in a concentration of 2% w/v (Holzer et al., 2009). NPs
31
dispersions were first frozen at -20˚C and then placed in the freeze dryer for 48h to
yield the lyophilized NPs powders as previously described.
In order to achieve the highest drug loading, while maintaining optimum size
and uniformity, formulation parameters were varied as shown in table 2. The
variables were as follows:
3.2.1. SAA type:
Mag-PLGA NPs were prepared with either PVA or TPGS in their previously
selected optimum concentrations.
3.2.2. SAA solvent phase:
The best SAA was chosen in its optimum concentration. Trials were made to
change the solvent phase of the SAA and to dissolve it in the organic phase. Polymer,
drug and SAA amounts and ratio of organic to aqueous phase were kept constant.
3.2.3. Organic to aqueous phase ratio:
Different organic to aqueous phase ratios were tried: 1:2 and 1:1 v/v. Mag-
PLGA NPs were prepared using optimum SAA type in its optimum concentration
and solvent location, while keeping all the other formula parameters unchangeable.
3.2.4. Mag theoretical loading amount:
The optimum formula (F14) was prepared using different amount of Mag: 1,
5 and 10mg corresponding to 4, 20 and 40% w/w of the polymer used as shown in
the procedure described above. Other parameters were kept constant.
PS, PDI and ζ were determined on the fresh NPs dispersions while EE was
performed on freeze dried NPs.
32
Table 2: Composition of Mag-PLGA NPs prepared by nanoprecipitation for

the optimization of formulation parameters.
SAA SAA
Formula Mag
SAA type concentration placement o:aq(a)
code (% w/w)(b)
(% w/v)
F11 PVA 0.5 Aqueous
1:2
F12 20
F13
F14 TPGS 0.18 20
Organic
F15 1:1 4
F16 40
(a)
The organic phase was composed of 25mg PLGA dissolved in 2mL acetone (1.25% w/v). Organic to aqueous phase
volume ratio; (b) calcutaled based on polymer weight.
3.3. Preparation of immuno-PLGA NPs
The main goal of the present study was to synthesize and characterize PLGA
NPs modified with the highly immunogenic anti-HER2 protein (herceptin or TZB).
A primary optimization was first done using the model protein, BSA due to the low
cost and the availability of the same functional groups. The surface of the particles
was thus modified to elicit non-specific (BSA) and specific (TZB) interactions with
cells (Barua et al., 2013). TZB and BSA were immobilized on the particle surface
by both adsorption and covalent binding, and the protein amounts bound to the
particles surfaces were quantified.
Freeze dried blank PLGA NPs were used for optimizing the modification
conditions for NPs surface modification with BSA via either direct adsorption or
chemical conjugation methods. Blank PLGA NPs were prepared by the
nanoprecipitation method previously described in section 3.2. Briefly, accurately
weighed 7.5mg TPGS and 25mg PLGA were dissolved in 2mL acetone. This organic
phase was added dropwise to 2mL water maintained under stirring at 250rpm on a
33
magnetic stirrer and the procedure was completed as before. Table 3 shows a
summary of the conditions tested for both methods which are detailed in the
following sections.
3.3.1. Adsorption method:
Freeze dried blank PLGA NPs, were dispersed in 5mL sodium acetate buffer,
pH 4.5, at a concentration of 5mg/mL and were mixed with 500μL BSA solution in
sodium acetate buffer, pH 4.5 (10mg/mL) at room temperature for a specific period
of time. The NPs were separated from free BSA by centrifugation using nanoseps
MWCO 100KDa at 6,000rpm for 1h at 4ºC. The obtained pellet was washed twice
with deionized water, then re-dispersed in 2mL of PBS, pH 7.4. The control was run
in the same way as the sample but acetate buffer pH 4.5 was used instead of BSA
solution to be used as blank in the protein content assay (Kocbek et al., 2007; Barua
et al., 2013). The two optimized parameters were:
3.3.1.1. Incubation time:
Three different times for incubation were tested for BSA adsorption: 2, 4 and
24h at pH 4.5 and at room temperature, while maintaining the ratio of BSA to NPs
at 0.1:1 w/w. The amount of adsorbed protein was detected after each time interval
using BCA protein assay described later under section 3.4.7.
3.3.1.2. BSA:PLGA weight ratio:
Different BSA to PLGA NPs weight ratios namely 0.1:1, 0.2:1 and 0.4:1 w/w
were tried at the selected optimum incubation time. The effects on PS, ζ and amount
of BSA loaded were evaluated.
34
Table 3: Preparation conditions of BSA modified blank PLGA NPs by different

coupling methods.
Preparation condition Adsorption Conjugation
Blank PLGA NPs 5mg/mL in acetate buffer 5mg/mL in acetate buffer
concentration (pH 4.5) (pH 4.5)
10mg/mL in acetate buffer 10mg/mL in acetate buffer
BSA concentration
(pH 4.5) (pH 4.5)
0.1:1
BSA:PLGA weight ratio 0.2:1 0.1:1
0.4:1
Incubation time 2, 4, 24h 4h
3.3.2. Covalent binding method:
Blank PLGA NPs prepared were dispersed in 5mL acetate buffer, pH 4.5,
(5mg/mL) containing 0.4% w/v of each of EDC and NHS. The colloidal dispersion
was mixed at room temperature at 150rpm for 30min followed by dialysis against
100mL deionized water using dialysis membrane (MWCO 14KDa) at room
temperature overnight with replacement of water every 30min for the first 3h to
discard excess NHS and EDC (Manoochehri et al., 2013). Then 250µL BSA
solution (10mg/mL) in acetate buffer, pH 4.5 was added to the activated NPs with
stirring at room temperature at 50rpm for 4h. BSA bound NPs were separated from
free BSA by centrifugation at 6,000rpm using nanoseps MWCO 100KDa for 1h at
4ºC. NPs were then washed with deionized water, recentrifuged then redispersed in
2mL of PBS, pH 7.4. The control was run the same way as the sample but acetate
buffer, pH 4.5 was used instead of BSA solution.
35
3.3.3. Preparation of immuno- Mag-PLGA NPs:
3.3.3.1. Preparation of BSA modified Mag-PLGA NPs (Mag PLGA-

BSA NPs):
Selected Mag-PLGA NPs formula (F14) previously prepared in section 3.2

were decorated with BSA using optimum BSA/NPs ratio as described in modified
blank NPs using both non covalent (adsorption) and covalent binding.
3.3.3.2. Preparation of TZB modified Mag-PLGA NPs (Mag PLGA-

TZB NPs) by covalent conjugation:
Mag-PLGA NPs previously prepared in section 3.2 were decorated with TZB
using optimum protein to NPs ratio (0.1:1 w/w) as described in BSA modified blank
NPs. Briefly, freeze dried optimized Mag-PLGA NPs (F14) were dispersed in 5mL
Millipore water to obtain final concentration of 5mg/mL. For activation of PLGA
carboxyl end groups, the pH of water was adjusted to 5 using 0.1N HCL and the
colloidal dispersion was mixed with 20mg of each of EDC and NHS at room
temperature at 150rpm for 30min. Excess NHS and EDC were separated by
dialysis against 100mL deionized water using dialysis membrane (MWCO
14KDa) at room temperature overnight with replacement of water every 30min for
the first 3h (Manoochehri et al., 2013). NPs were concentrated using vivaspin
MWCO 10KDa for 30min at 4ºC, resuspended in 5mL PBS, pH 7.4 and then 250µL
of TZB (10mg/mL) solution in PBS pH 7.4 was added to the activated pellet with
stirring at room temperature at 50rpm for 4h. TZB bound NPs were separated from
free TZB by centrifugation at 6,000rpm for 1h at 4ºC using nanoseps 100KDa. NPs
were washed with deionized water, then re-dispersed in 2mL of PBS, pH 7.4. The
control was run in the same way as the sample but PBS, pH 7.4 was used instead of
TZB solution.
36
3.4. NPs Characterization
All the prepared blank, Mag-PLGA NPs, surface modified blank and surface
modified Mag-PLGA NPs were characterized as described here in.
3.4.1. PS analysis:
The PS analysis was performed by dynamic light scattering (DLS) using

Malvern Zetasizer 4. The DLS yielded the hydrodynamic average diameter of the
main population and the polydispersity index (PDI) which is the measure for the
width of the PS distribution. All the fresh samples were diluted with distilled water
until the particles count reached 200-300 kilo count per seconds (KCPs) before
analysis. Results were performed in triplicates of different batches and the results of
PS were expressed in terms of intensity distribution.
3.4.2. Ζeta potential (ζ) determination:
The surface charge of selected NPs was determined by the measurement of

the ζ of the freshly prepared NPs dispersion using zetasizer 4. ζ was calculated
according to Helmholtz Smoluchowsk from NPs electrophoretic mobility
(Duzgunes et al., 2012). Before determination, all samples were diluted with
distilled water to the same concentration used in PS analysis. Results were
performed in triplicates of different batches.
3.4.3. Morphological examination by transmission electron microscope

(TEM):
Mag-PLGA NPs were observed by TEM. A drop of NPs dispersion was

applied on carbon-coated copper grid to allow its absorption in the carbon film and
37
after 2-3min, the excess was drawn off with filter paper. NPs were examined at a
power of 120kV.
3.4.4. Determination of Mag entrapment efficiency (EE) and loading

capacity (LC):
Amount of Mag in PLGA NPs was determined using a RP-HPLC method

coupled with UV-Vis detector as described in the following sections.
3.4.4.1. U.V scanning of Mag in 1% w/v aqueous sodium hydroxide

(NaOH) solution containing 0.5% w/v SDS:
A stock solution of 1mg/mL of Mag was prepared in methanol. Further
dilutions in 1% w/v aqueous sodium hydroxide (NaOH) solution containing 0.5%
w/v SDS was prepared and scanned spectrophotometrically in the UV range from
200 to 400nm using UV-visible spectrophotometer. The wavelength of maximum
absorbance (λmax) was determined using the corresponding solvent as blank.
3.4.4.2. Chromatographic conditions:

Chromatographic separations were carried out by a reversed-phase HPLC
chromatographic method using a platinum C18 column at 25ºC. Samples were eluted
with an isocratic system composed of acetonitrile: 0.1% w/v phosphoric acid (65:35)
at flow rate of 1mL/min with UV detection at the predetermined λ max (Tsai and
Chen, 1992).
3.4.4.3. Construction of Mag calibration curve in 1% w/v aqueous

NaOH solution containing 0.5% w/v SDS using high-
performance liquid chromatography (HPLC):
Serial dilutions of Mag solutions yielding concentrations of 2.5, 5, 10, 20, 25
and 50µg/mL were prepared in aqueous solution containing 1 and 0.5% w/v of
38
NaOH and SDS, respectively. HPLC with photodiode array UV detection was used
for the determination of Mag.
An accurately weighed (10mg) of freeze dried Mag loaded NPs was

reconstituted in 4mL of 1%w/v NaOH aqueous solution containing 0.5% w/v SDS.
The NPs dispersion was then sonicated and left for 24h at 37˚C on a magnetic stirrer
to allow for NPs degradation and the resulting solution was filtered through a
0.45µm syringe filter. The concentration of Mag was determined using a reversed
phase HPLC coupled with UV detector after appropriate dilution. The mean value
for three replicate determinations and their mean SD were reported.
EE%= (Amount of Mag in NPs/ initial amount of Mag) X 100 Eq (1)
LC%= (Amount of Mag in NPs/ NPs weight) X 100 Eq (2)
3.4.4.4. Validation of the analytical method:

The parameters used in assay validation include selectivity, linearity, range,
recovery, accuracy, precision and limit of quantitation (ICH Q2 (R1), 2005).
• Selectivity:
Selectivity is the ability of an analytical method to differentiate and quantify the
analyte in the presence of other components in the sample.
• Linearity and range:

Linearity was determined by calculating the regression line using a mathematical
treatment of the peak area vs Mag concentration in aqueous solution containing 1
and 0.5% w/v of NaOH and SDS, respectively.
The range of the method is the interval between the upper and lower levels of an
analyte concentration that have been determined with acceptable precision, accuracy
39
and linearity. It was determined on a linear response curve and was expressed in the
same units as the test results.
• Recovery:
The recovery of Mag was assessed by adding known amounts of Mag to aqueous
solution containing 1 and 0.5% w/v of NaOH and SDS, respectively to give
concentrations of 2.5, 7.5, 15 and 40µg/mL. Recovery percentage was calculated by
comparing the obtained concentration with the theoretical concentrations.
• Accuracy and precision:

Assay performance was evaluated according to intra- and inter-day accuracy and
precision, determined from replicate analysis of control specimens at the following
concentration ranges of 2.5-50μg/mL on a single day to assess within-day assay
(intra-day). Inter-day reproducibility was determined by comparing the data
obtained from samples analysis during three different days. Accuracy is a measure
of the closeness of test results obtained by a method to the theoretical value.
Accuracy was determined by using the following equation:
𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑐𝑜𝑛𝑐−𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑐𝑜𝑛𝑐

𝐴𝑐𝑐𝑢𝑟𝑎𝑐𝑦 = ( 𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑐𝑜𝑛𝑐
) ∗ 100 Eq (3)
Precision is a measure of the reproducibility of the whole analytical method
(including sampling, sample preparation and analysis) under normal operating
circumstances. Precision was determined by repeating the method of assay 6 to 10
times and was then expressed as coefficient of variation (CV%).
• Limit of Quantitation (LOQ):
This is the lowest concentration of analyte in a sample that can be determined
with acceptable precision and accuracy. It was quoted as the concentration yielding
a signal-to-noise ratio of 10:1.
40
3.4.5. In vitro drug release:
3.4.5.1. U.V scanning of Mag in PBS (pH 7.4) containing 0.5% w/v
SDS:
A stock solution of 1mg/mL of Mag was prepared in methanol. Further
dilutions in PBS pH 7.4 containing 0.5% w/v SDS was prepared and scanned
spectrophotometrically in the UV range from 200 to 400nm using UV-visible
spectrophotometer. The wavelength of maximum absorbance (λmax) was determined
using the corresponding solvent as blank.
3.4.5.2. Construction of Mag calibration curve in PBS (pH 7.4)

containing 0.5% w/v SDS using UV-Vis spectrophotometer:
Serial dilutions of Mag solutions yielding concentrations of 5, 10, 15, 20, 25
and 30µg/mL were prepared in PBS pH 7.4 containing 0.5% w/v SDS. UV-Vis
spectrophotometer detection at the predetermined λmax was used for the
determination of Mag.
3.4.5.3. Determination of percent cumulative in vitro drug release:

The dialysis bag diffusion technique was used to study the in vitro drug release
of Mag from PLGA NPs (Jawahar et al., 2009). The release study was carried out
using dialysis membrane of MWCO 14 kDa. An accurately weighed amount of Mag-
PLGA NPs equivalent to 2mg Mag, dispersed in 2mL PBS pH 7.4, was placed inside
the dialysis sac, which was then tied at both ends. The bag was immersed in a beaker
containing 50mL of PBS pH 7.4 containing 0.5% w/v SDS to ensure sink conditions
(Güç et al., 2010; Derakhshandeh and Soleymani, 2014). The beaker was then
placed in a shaker water bath at 80 stokes/min and incubated at 37˚C (Grumezescu
et al., 2016). One mL of dissolution medium was withdrawn at predetermined time
intervals and replaced with fresh medium. Blank NPs were treated similarly to be
used as blank in UV detection. Samples were analyzed using UV-Vis
41
spectrophotometer at the predetermined λmax and using the corresponding calibration

curve. Drug release data was calculated by converting drug concentration in solution
to percentage cumulative drug release.
3.4.6. Fourier transform-infrared (FT-IR) analysis:
FT-IR spectra were obtained by Thermonicolet Nexus spectrometer. Freeze

dried samples prepared with no cryoprotectant were pressed into potassium bromide
(KBr) pellets and recorded at frequencies from 4000 to 200cm-1 with resolution of
4cm-1.
3.4.7. Determination of protein on the surface of modified NPs using

protein assay:
The loading amount of protein (BSA and TZB) was detected via an indirect
quantification method using micro BCA protein assay kit using UV-Vis
spectroscopy at 562nm as per the manufacturer’s instructions (Yap et al., 2014). The
loading amount was determined by measuring the concentration of free protein in
supernatant which was obtained following the ultracentrifugation of NPs dispersion
in nanoseps 100 KDa at 6,000rpm for 1h at 4ºC.The amount of protein on the surface
of NPs was evaluated as the difference between the initial and the residual amount
of protein in the supernatant (Mattu et al., 2013).
3.4.7.1. Preparation of Micro BCA working reagent (WR):
WR was prepared by mixing 25 parts of Micro BCA reagent MA and 24 parts

of reagent MB with 1 part of reagent MC (25:24:1, reagent MA:MB:MC).
42
3.4.7.2. Construction of BSA calibration curve in acetate buffer (pH

4.5):
The content of one standard ampoule of BSA (2mg/mL) was diluted into
several clean vials, using acetate buffer pH 4.5 to prepare a set of diluted BSA
standards of 0.5 to 200µg/mL. A volume of 1mL of the WR was added to 1mL of
each standard and mixed well. The tubes were then covered and incubated at 60°C
in a water bath for 1h. Blank concentrations were prepared similarly using 1mL
PBS pH 7.4 instead of BSA standard solution. Subsequently, all tubes were cooled
to room temperature (RT) by direct immersion in a cold water bath and the
absorbance of all the samples were measured within 10min using UV-visible
spectrophotometer set to 562nm. The average absorbance reading of the blank
standard replicates was subtracted from the 562nm reading of all other individual
standards. The standard curve was constructed by plotting the average blank-
corrected 562nm reading for each BSA standard vs its concentration in µg/mL. The
standard curve was then used to determine the protein concentration of each
unknown sample (Smith et al., 1985).
3.4.7.3 Determination of BSA bound to the surface of blank NPs:
One mL of supernatant containing free unbound BSA was added to 1mL of

WR and procedure was repeated as previously discussed under calibration curve
preparation.
% BSA on NPs surface= (Initial protein amount – amount in supernatant) X 100. Eq (4)
Initial protein amount
Same procedure was applied for TZB determination but PBS pH 7.4 was used
instead of acetate buffer, pH 4.5
43
3.4.8. Proton nuclear magnetic resonance (1H-NMR):
The protein conjugation to PLGA NPs was confirmed via 1H-NMR. The
sample was dissolved in 0.2mL dimethyl sulfoxide (DMSO) and vortexed for 48h
to enhance dispersion in the solvent. The scan was done using 3mL NMR tube,
frequency 400MHZ, pulse width 12W and scan number 16.
3.5. Statistical Analysis
All formulations were prepared and reported in triplicates. Results are

expressed as mean ± SD (standard deviation). The statistical signiﬁcance of
difference between groups were evaluated by one-way ANOVA and Tukey's post
hoc test with a significance level of p<0.05.
44
Chapter 1 Results and discussion
4. Results and Discussion
4.1. Blank PLGA NPs
Blank PLGA NPs were prepared by the nanoprecipitation method due to its
suitability to produce NPs with PS smaller than 200nm (Zhang et al., 2013). This
size was reported to be efficient for cancer cells uptake relying on passive targeting
(Kumar, 2012).
4.1.1. Effect of organic solvent type:
Acetone and acetonitrile, two of the most commonly used organic solvents for
fabrication of PLGA NPs by the nanoprecipitation method, were tried in this work
(Sah and Sah, 2015). To evaluate the effect of the type of solvent on PS and PDI of
the NPs using the stabilizer PVA, formulae F1 and F2 were prepared using
acetonitrile and acetone as organic solvent, respectively. The ratio of organic to
aqueous phase was maintained at 1:2 and the concentration of PVA at 0.1% w/v. As
shown in table 4, a significantly lower PS of 158.3±1.18nm with a more homogenous
distribution (p˂0.05), PDI 0.184±0.004, was obtained using acetone (F2). In case of
F1 prepared with acetonitrile, PS and PDI were 196.9±1.25nm and 0.251±0.006,
respectively. Therefore, acetone was selected for further work.
4.1.2 Effect of SAA concentration and type:
Using acetone as solvent for PLGA and keeping polymer concentration and
ratio of organic to aqueous phase constant, formulae F2 to F5 were prepared using
PVA in different concentrations. Based on literature survey, PVA concentrations of
0.05, 0.1, 0.50, and 1% w/v were selected (Guhagarkar et al., 2009; Mehrotra and
Pandit, 2012; Sharma et al., 2016). Similarly, formulae F6 to F10 were prepared
45
using TPGS as SAA in the following concentrations: 0.03, 0.06, 0.12, 0.18, 0.24%
w/v (Esmaeili et al., 2007).
From table 4, it is obvious that increasing SAA concentration from 0.1 to 0.5%
w/v resulted in more particles stabilization manifested by a significantly (p˂0.05)
smaller PS with better distribution (111.7±6.75 vs 158.3±1.18nm and 0.12±0.016
vs 0.184±0.004, respectively). Further increase in PVA concentration to 1% w/v
(F5) didn’t significantly affect the PS and PDI. It is noteworthy to mention that at a
concentration of 0.05% w/v (F3) aggregates formation was evidenced indicating
insufficient amount of SAA for NPs stabilization (Nafee et al., 2007). The obtained
results imply that an optimum concentration of SAA is required to obtain NPs with
an optimum size and stealth effect. Previously, it was reported that optimum
concentrations of SAA reduced NPs size with a better stabilizing effect (Allémann
et al., 1992; Mainardes and Evangelista, 2005). Conversely, other authors reported
increased PS and wider PDI with increased stabilizer concentrations, which was
explained in terms of an increased viscosity (Benita et al., 1984; Maaz et al., 2011).
However, literature recommended the use of lowest optimum PVA concentration
(0.5% w/v) to optimally stabilize the NPs due to its toxicity (Mu and Feng, 2003b).
In general, TPGS produced NPs with smaller PS than those obtained with
PVA. Using TPGS at low concentration, 0.03% w/v, resulted in the production of
NPs with average size of 110.6±3.99nm, F6 (p˂0.05). Increasing TPGS
concentration up to 0.18% w/v led to insignificant effect on PS as seen by comparing
F6 to each of F7, F8 and F9 (p˃0.05). Further increase in concentration to 0.24%
w/v showed a significant increase in PS reaching 125nm±5.52 in case of F10
(p˂0.05). All formulae showed uniform PS distribution as evidenced from the small
PDI.
46
Table 4: Characterization of blank PLGA NPs prepared using different

formulation parameters.
SAA
Formula Organic SAA PS
concentration PDI
code solvent type (nm)
(% w/v)
F1 Acetonitrile 0.1 196.9±1.25 0.251±0.006
F2 0.1 158.3±1.18 0.184±0.004
F3 PVA 0.05 ND ND
F4 0.5 111.7±6.75 0.12±0.016
F5 1 119.3±2.62 0.12±0.013
F6 Acetone 0.03 110.6±3.99 0.11±0.003
F7 0.06 109.3±3.59 0.11±0.005
F8 TPGS 0.12 114.9±2.34 0.12±0.011
F9 0.18 118.8±3.68 0.10±0.011
F10 0.24 125.8±5.52 0.11±0.021
A PLGA concentration of 1.25% w/v was used in the organic phase and a ratio of 1:2 of organic to aqueous phase
volume was maintained. Results are mean of three determinations ±SD. ND: not determined due to visible aggregation.
Previous researchers in various works used TPGS as an emulsifier in low

concentrations ranging from 0.01 to 0.3% w/v due to its 67 fold higher
emulsification effect compared to PVA during the preparation (Turk et al., 2014;
Sharma et al., 2016; Lee and Ooi, 2016). Our results showed that beyond a TPGS
concentration of 0.18% w/v, the PS significantly increased (p<0.05). This might be
due to viscosity enhancement of aqueous phase which resulted in a decreased net
shear stress and subsequently increased PS (Rachmawati et al., 2016). In contrast to
our results, others reported that increasing concentration of TPGS from 0.01 to 0.3%
w/v resulted in smaller particles (McCall and Sirianni, 2013).
The previous results revealed the superior emulsification properties of TPGS

in comparison to PVA as shown by the smaller size of NPs fabricated using the
former SAA. This was also reported when the solvent/evaporation technique was
used (Turk et al., 2014; Lee and Ooi, 2016; Sharma et al., 2016). Furthermore, and
47
as previously mentioned, TPGS aids in inhibiting Pgp mediated multi-drug

resistance (Esmaeili et al., 2007). Hence, larger amount of TPGS would be
beneficial and therefore, F9, with small PS at the higher TPGS concentration was
selected for subsequent work.
Based on this study, formulae F4 and F9 prepared with PVA and TPGS,
respectively, were selected for drug loading study. Their respective PS were
111.7±6.75 and 118.8±3.68nm.
4.2. Mag-PLGA NPs
Mag-PLGA NPs were prepared by the optimized nanoprecipitation method

using different formulation variables.
4.2.1. HPLC assay of Mag:
HPLC method was chosen for the accuracy of determination of content of

Mag following degradation of PLGA using an alkaline hydrolysis method (Park et
al., 1998).
4.2.1.1. Validation of the HPLC method:
• Selectivity:
Figure 1 shows a representative chromatogram of Mag at a concentration of
10μg/mL. Using acetonitrile: phosphoric acid 0.1% w/v (65:35) as mobile phase
at flow rate of 1mL/min, Mag was well separated with a mean retention time of
3.8min. None of the other formulation components showed a peak at this
retention time under the applied chromatographic conditions.
48
Figure 1: HPLC chromatogram of Mag at 292nm.
• Linearity and range:

Standard plots obtained for Mag in 1% w/v aqueous NaOH solution
containing 0.5% w/v SDS were linear in the range of 2.5-50μg/mL (figure 2).
Linear regression analysis of the standard calibration plots yielded the equation:
y=15811x+12433 where y and x are peak area and Mag concentration,
respectively and the coefficient of determination R2 value was found to be 0.999.
900000
800000
700000
600000
Peak area
y = 15811x + 12433
500000 R² = 0.9997
400000
300000
200000
100000
0
0 10 20 30 40 50 60
Conc (µg/mL)
Figure 2: Calibration curve of Mag in 1% w/v aqueous NaOH solution

containing 0.5% w/v SDS determined by HPLC.
49
• Recovery:
The recovery for Mag is shown in table 5, the average recovery percentage
of Mag was found to be 103.86%.
• Accuracy and Precision:
Data concerning intra- and inter- day accuracy and precision were determined
and are listed in tables 5 and 6. They show that intra-day accuracy ranged from
4.74 to 8.95% while the respective values of precision expressed as %CV range
was 0.46 to 8.95%.
Inter-day accuracy values were in the range of 0.15 to 6.51%. The calculated
%CV values were found to be in the range 2.25 to 9.42% denoting good
precision.
Table 5: Recovery and intra-day accuracy and precision of Mag in 1% w/v

aqueous NaOH solution containing 0.5% w/v SDS.
Theoretical Recovery
Mean measured concentration* CV
concentration Accuracy %
(μg/mL±SD) %
(μg/mL)
2.5 2.35±0.87 6.00 3.71 94.00
7.5 7.85±0.03 4.74 0.46 104.74
15 16.16±0.48 7.78 2.97 107.78
40 40.15±0.52 3.92 1.29 100.37
* Mean of six measurements.
Table 6: Inter-day accuracy and precision of Mag in 1% w/v aqueous NaOH

solution containing 0.5% w/v SDS.
Theoretical concentration Mean measured concentration*
Accuracy CV%
(μg/mL) (μg/mL±SD)
2.5 2.32±0.15 7.20 6.29
7.5 7.69±0.24 2.53 3.12
15 16.44±0.07 9.99 0.44
40 40.59±0.73 1.49 1.80
* Mean of six measurements
50
• Limit of Quantitation:
The limit of Quantitation (LOQ) for Mag was 2.5μg/mL. Such a limit was
adequate for drug EE determination.
4.2.2. Effect of formulation variables on Mag-PLGA NPs properties:
The effects of the following four variables: SAA type in its optimum
concentration, SAA solvent phase, ratio of organic to aqueous phase and the drug
loading amount were evaluated on PS, surface charge of the NPs and %EE. Mag-
PLGA NPs were prepared using different formulation variables and table 7 shows
the characterization results of the different Mag-PLGA NPs formulae.
Table 7: Characteristics of Mag-PLGA NPs prepared using optimized

surfactants concentrations.
SAA type/ Mag
Formula SAA o:aq PS EE LC
concentration (a) (% w/w) PDI
code placement (nm) (% w/w) (% w/w)
(b)
(% w/v)
PVA /
F11 131.65±0.35 0.133±0.03 85.09±2.50 11.35±0.33
0.5% Aqueous
1:2 20
F12 115.13±2.21 0.101±0.008 85.18±0.89 11.36±0.12
F13 119.47±2.75 0.104±0.046 80.38±4.35 10.72±0.58
TPGS /
F14 20 118.45±4.79 0.104±0.012 85.69±1.32 11.43±0.18
0.18% Organic
F15 1:1 4 113.03±4.97 0.089±0.009 95.88±8.79 2.95±0.27
F16 40 185.70±4.58 0.093±0.097 45.08±4.99 10.61±1.17
A PLGA concentration of 1.25% w/v was used in the organic phase. Results are mean of three determinations ±SD.
(a)
organic to aqueous phase volume ratio, (b) theoretical Mag percentage based on polymer weight. ND: not determined.
4.2.2.1. SAA type used in its optimized concentration:
Mag incorporation in NPs F4 and F9, produced F11 and F12 Mag loaded NPs,
respectively, prepared with PVA and TPGS in their optimum concentrations (table
7). Drug loading was found to significantly increase (p˂0.05) the PS of the NPs only
51
in the case of PVA (F4 vs F11). F12 had significantly smaller PS with better size
distribution than F11 (p˂0.05). Non significantly different EE were obtained in both
SAA (p˃0.05). This was in agreement with previous works who found that the use
of TPGS resulted in lower PS compared to PVA (Saadati and Dadashzadeh, 2014).
However, Mu and colleagues reported that PVA stabilized NPs were relatively
smaller than the TPGS stabilized ones, while the NPs prepared with TPGS as SAA
demonstrated higher drug EE compared to those prepared with PVA (Mu et al.,
2004).
It could be concluded that TPGS was a more effective SAA than PVA since
its optimum concentration was lower than that of PVA (0.18 vs 0.5% w/v).
4.2.2.2. SAA solvent phase:
As TPGS is an amphiphilic polymer sharing solubility in both aqueous and

organic medium, trials were made to study the effect of changing its solvent phase.
Comparing F12 with F13 containing TPGS in aqueous or organic phase
respectively, proved non-significant changes in PS, PDI and %EE (p˃0.05). While
a significant decrease in ζ from -25.36±1.23 to -17.5±1.31mV was obtained when
TPGS was dissolved in organic phase (p˂0.05). The decrease in ζ might be due to
the coating of TPGS onto the PLGA NPs surface. Accordingly, decision was made
to keep TPGS in the organic phase in closer contact with the polymer to enable a
possible stealth effect on Mag NPs.
4.2.2.3. Organic to aqueous phase volume ratio:
Insignificant differences in PS, PDI and %EE were evident upon changing the
ratio of organic to aqueous volume from 1:2 in F13 to 1:1 in F14, using TPGS as
stabilizer as shown in table 7 (p˃0.05). On the contrary, others showed that
52
increasing the organic phase volume ratio led to increase in PS (Sharma et al., 2016).
The use of the solvent acetone with a lower viscosity compared to water (0.3 and
1cP, respectively at 25ºC) with the low polymer concentration (1.25% w/v) probably
accounted for the lack of significant effect upon changing phase volume ratio
(Kuriyan et al., 2012). Furthermore, no significant effect was seen with the EE due
to the hydrophobic nature of Mag under study. Accordingly, organic to aqueous
phase ratio 1:1 will be adopted in the following studies.
4.2.2.4. Theoretical drug loading:
The results of loading different Mag amounts can be seen in table 7.

Decreasing the theoretical drug loading from 5mg (20% w/w of polymer used) to
1 mg (4% w/w) in F14 and F15, respectively, caused no significant effect on PS
(p>0.5). However, the PS increased significantly to 185±4.58 nm when the amount
of drug increased to 10mg (F16) (p<0.5). These results were in agreement with a
previously published study where a 50% increase in PS was observed when the drug
concentration was increased from 0.2 to 1.0% w/w (Krishnamachari et al., 2007).
Increase in amount of drug may have resulted in a more viscous dispersed phase
resulting in larger particles (Mao et al., 2008).
In accordance with previously published studies, EE exhibited an insignificant

(p>0.5) downward trend (from 95.88±8.79 to 85.69±1.32 % w/w) with increasing
amount of drug in the formulation as could be evidenced in F14 and F15 prepared
at theoretical drug loadings of 5 and 1mg, respectively (Sharma et al., 2016). Further
increase in loading amount to 40% w/w was accompanied with significant decrease
in %EE to 45.08±4.99 % w/w (F16) (P˂0.5). Generally, the drug content in the NPs
is affected by the drug–polymer interactions and the drug miscibility in the polymer.
Higher drug miscibility leads to higher drug incorporation (Panyam et al., 2004).The
53
decrease in EE seen in this study, might be due to the fixed amount of polymer
available to encapsulate the drug.
Formula F14 containing Mag (5mg) and showing small PS, high %EE with
the highest %LC was selected for further characterization and preparation of
immuno-NPs.
4.2.3. Characterization of the selected formula:

4.2.3.1. Ζeta potential (ζ):
ζ of the selected Mag-PLGA NPs, F14, was found to be -14.75±0.64 mV. The
negative value of ζ was probably due to the presence of terminal carboxyl groups in
the polymer. Charged particles with a repulsive interaction produced more stable
particles with more uniform size distribution (Feng et al., 2016). Furthermore, the
negative ζ is an important feature that could increase the chance for Ab attachment.
4.2.3.2. Morphological examination using transmission electron

microscope (TEM):
The TEM micrographs, figure 3, reveal that the prepared Mag-PLGA NPs
were spherical, uniform in shape with size ~100nm. The NPs size observed by TEM
correlated well with the hydrodynamic diameter measured by DLS (figure 3).
Figure 3: TEM micrographs of Mag-PLGA NPs, F14, at different

magnifications.
54
4.2.3.3. In vitro drug release:
4.2.3.3.1. UV assay of Mag:
UV scanning of Mag in PBS pH 7.4 containing 0.5% w/v SDS, displayed in

figure 4, revealed that the drug showed absorption peak at 292nm. This was in
accordance with previously published data. The calibration curve of Mag in PBS,
pH 7.4, containing 0.5% w/v SDS determined using UV spectroscopy at λmax 292nm
is illustrated in figure 5. A linear relation between UV absorbances and
concentrations in the range of 5-30µg/mL with coefficient of determination (R2) of
0.999 was obtained.
0.9
0.8
0.7
0.6
Absorbance
0.5
0.4
0.3
0.2
0.1
0
200 250 300 350 400
Wavelength (nm)
Figure 4: Mag UV spectrum in PBS (pH 7.4) containing 0.5% w/v SDS.
55
1
0.9 y = 0.0283x + 0.0185
0.8 R² = 0.9994
0.7
Absorbance
0.6
0.5
0.4
0.3
0.2
0.1
0
0 5 10 15 20 25 30 35
Conc (µg/mL)
Figure 5: Calibration curve of Mag in PBS (pH 7.4) containing 0.5% w/v SDS
at 292 nm determined spectrophotometrically.
4.2.3.3.2. In vitro Mag release:
To achieve sink conditions in the dissolution medium PBS pH 7.4 containing

0.5% w/v SDS was used due to the drug hydrophobic nature (Wang et al, 2016b).
As could be seen from table 8, Mag powder was 100% w/w dissolved during
48h. The release profile for Mag–PLGA NPs (F14) in PBS pH 7.4 containing 0.5%
w/v SDS presented in figure 6 shows only a 10% w/w burst release during the first
four hours. The release rate then gradually decreased yielding a 40% w/w cumulative
release during the first day. Slower release was then noted where only less than 80%
w/w was released in 40 days.
The initial burst could be due to the diffusion of Mag distributed near or at the
surface of the NPs. The relative fast Mag release (~40% w/w) from the PLGA/TPGS
NPs during the first day, might be attributed to the amphiphilic nature of the TPGS,
56
causing rapid absorption of water molecules into the polymeric matrix, hence
promoting the drug diffusion through polymeric matrix of the NPs (Mu et al., 2004;
Ma et al., 2010; Chen et al., 2011b). This was an another advantage of PLGA/TPGS
NPs over the traditional PLGA NPs, which were found to slowly release the drug
below therapeutic needs in some cases (Su et al., 2017). The PLGA NPs prolonged
release could be explained based on the nature and slow erosion of the polymer
PLGA (Ma et al., 2010). In contrast to our observation, Mu and colleagues reported
that TPGS tended to form a compact domain inside the pores of PLGA matrix
causing a dense structure which can slow the erosion of the matrix sustaining the
drug release (Mu et al., 2004).
Table 8: Cumulative percent of Mag released from Mag powder and Mag-
PLGA NPs.
Cumulative percent Mag released (% w/w)
Time (h) from
Mag powder Mag-PLGA NPs

1 9.50±2.42 3.87±5.47
2 12.62±1.61 6.52±1.94
3 12.62±1.88 9.39±1.60
4 18.33±2.27 10.74±2.03
24 35.33±0.97 39.64±2.46
48 97.23±2.16 54.70±1.60
72 98.00±1.32 58.00±1.58
168 ND 57.52±2.31
504 ND 63.79±1.73
720 ND 77.81±1.92
960 ND 77.41±2.45
ND: not determined. Results are mean of three determinations ±SD.
57
120
Mag-PLGA NPs
Mag powder
100
Cumulative Mag released (% w/w)
80
60
120
Cumulative Mag released (% )
100
40 80
60
40
20 20
0
0 20 40 60 80
Time (h)
0
0 5 10 15 20 25 30 35 40 45
Time (days)
Figure 6: Release profiles of Mag in PBS (pH 7.4) containing 0.5% w/v SDS.
4.2.3.4. FT-IR analysis:
To characterize the matrix formed, The FT-IR spectra of PLGA, TPGS, Mag,
PLGA NPs and Mag-PLGA NPs were obtained and are shown in figure 7. From
PLGA spectrum, the C-O-C stretching peak is evident at 1089cm-1, C-H stretching
in methyl group at 1456cm-1, C=O at 1758cm-1, CH, CH2 and CH3 stretching
58
vibrations between 2850 and 3000cm-1, and OH stretching ~3500cm-1 (Wang, 2013).
In The FT-IR spectrum of TPGS, the carbonyl band appears at 1738cm−1. For the
PLGA blank NPs containing TPGS, the carbonyl band of PLGA apparently was
merged with that of TPGS and a new shifted peak with a lower intensity appeared at
1759cm−1. Overlapping of the CH stretching band of PLGA at 2952cm−1 and that of
TPGS at 2868cm−1 was also noticed. Furthermore, the change in position of the
absorption band of the PLGA terminal hydroxyl groups at 3500–
3650cm−1 evidenced its interaction with TPGS (Ma et al., 2010). This confirmed the
formation of a new matrix containing both PLGA-TPGS. Similar changes were
reported when Ma and colleagues chemically synthesized a matrix blend of PLGA
and TPGS (Ma et al., 2010). The introduction of TPGS as a component of the matrix
material with PLGA was previously attempted to achieve high emulsifying effects
and desired PS, size distribution, surface morphology and in vitro release kinetics
(Mu and Feng, 2003a).
The spectrum of Mag shows an intense absorption band at 3157cm-1 attributed

to the hydroxyl stretching vibration. The characteristic absorption band appearing at
1637cm-1 represents the allyl C=C stretching vibration and the absorption peaks at
1496cm-1 could be attributed to the C=C aromatic stretching. Other sharp bands
appear at 2973cm-1 (C-H aliphatic stretching vibrations), 1229cm-1 (C-O stretching
vibrations and 820cm-1 (Aryl-H bending vibrations) (Stefanache et al., 2017).The
spectrum of Mag-PLGA NPs shows disappearance of Mag fingerprint, indicating
successful encapsulation into the PLGA/TPGS matrix as has been similarly deduced
in other studies (Shukla et al., 2012). A reduction in OH stretching peak ~3500cm-
1
is observed in Mag-PLGA NPs spectrum compared to PLGA NPs spectrum
indicating a possible interaction between Mag and PLGA NPs.
59
Mag-PLGA/TPGS NPs
1750
2921
Mag powder
% Transmittance
2973
820
3157 Blank PLGA-TPGS NPs 1229

N 1637
3490
2996
2950
1759
TPGS
2868
2923
1738
3452
PLGA
powder
3644
3518
2952
2998
1456 1089
1758
4000 3500 3000 2500 2000 1500 1000 500 0

Figure 7: FT-IR spectra of PLGA, TPGS, blank PLGA-TPGS NPs, Mag
powder and Mag-PLGA NPs.
60
4.3. Immuno-NPs
Surface modification of the NPs with TZB was attempted by both physical
adsorption and covalent binding methods. BSA was used as a model protein in the
optimization process as previously explained.
4.3.1. BSA modified blank PLGA NPs (PLGA-BSA NPs) by adsorption

method:
It has always been known that the extent of protein adsorption to PLGA NPs
is highly dependent on the pH of its aqueous solution (Meissner et al, 2015). When
the pH equals the protein isoelectric point (Ip), Ab molecules with a zero net charge
could approach each other more closely and form a more compact conformation
resulting in more effective adsorption. Whereas away from this pH, higher net
charge on the protein molecules could result in repulsion within and between
peptides which could affect its adsorption onto NPs surface. Thus, maximal
adsorption was obtained around the Ip (Meissner et al, 2015), and hence in our study
a pH close to the protein Ip, 4.5 for BSA was selected for adsorption study.
In order to assess BSA (model protein) loading on PLGA NPs,

Bicinchonininc acid (BCA) assay kit was employed and the calibration curve of BSA
in PBS pH 7.4 was first prepared as described in section 3.4.7 and is graphically
illustrated in figure 8. Linear relation between absorbance and concentration of BSA
obeying Beer Lambert’s law with coefficient of determination (R2) of 0.996 was
obtained.
61
1.2
0.8
Absorbance
0.6 y = 0.022x + 0.129

R² = 0.996
0.4
0.2
0
0 10 20 30 40 50
Conc (µg/mL)
Figure 8: Calibration curve of BSA in PBS (pH 7.4) using BCA protein assay
kit.
4.3.1.1. Incubation time:
The optimal incubation time for surface modification was determined on blank
PLGA NPs previously prepared in section 3.3, by detecting the amount of BSA
present on the surface of blank PLGA NPs following incubation for three different
time periods (2, 4 and 24h). As could be seen from figure 9, using a weight ratio of
0.1:1 BSA to NPs, the amount of adsorbed BSA on the NPs surface, increased
significantly (p˂0.05) from 55.30±0.74 to 61.17±1.15% w/w with increasing
incubation time from to 2 to 4h. Further time increase led to significant decrease
(p˂0.05) in protein adsorption to 41.11±2.38% w/w due to BSA desorption that
occurred after long incubation period. Hence, an incubation time of 4h was selected
to prepare BSA-decorated NPs loaded with Mag.
62
70
61.17%
B S A a d s o r b e d ( % w /w )
55.3%
60
50
41.11%
40
30
4
2
2
T im e ( h )
Figure 9: Effect of incubation time on BSA percentage adsorbed on blank

PLGA NPs prepared at 0.1:1 BSA to NPs weight ratio.
4.3.1.2. BSA to PLGA weight ratio:
The effect of amount of BSA initial loading on adsorption efficiency was

checked by varying the BSA to NPs weight ratio (0.1:1, 0.2:1 and 0.4:1). From figure
10, we can depict that increasing the ratio from 0.1:1 to 0.2:1 by increasing the
amount of BSA caused a significant (p˂0.05) decrease on % BSA adsorbed from
61.17±1.15 to 40.85±1.85% w/w, respectively. However, further increase in the
BSA:NPs weight ratio to 0.4:1 caused an insignificant effect on the % w/w of BSA
adsorbed. A ratio of 0.1:1 of BSA:NPs was used subsequently.
70
61.17%
B S A lo a d in g (% w /w )
60
50
42.65%
40.85%
40
30
:1
:1
:1
.1
.2
.4
0
B S A /P L G A N P s w e ig h t r a t io
Figure 10: Effect of BSA to PLGA NPs weight ratios on BSA adsorbed on
blank PLGA NPs.
63
4.3.2. PLGA-BSA NPs by the covalent binding method:
BSA coupling by covalent conjugation using carbodiimide chemistry with

EDC and NHS is shown in figure XV. EDC is a convenient carbodiimide used to
form a variety of chemical conjugates, provided the presence of a primary amine and
a carboxylic group on the reacting molecules. EDC reacts with the carboxylic acid
group to form a highly reactive O-acylisourea intermediate which will attack a
primary amine (nucleophile) to form an amide bond. Being water soluble, EDC is
directly added to the reaction mixture without prior organic solvent dissolution
(Kocbek et al., 2007). NHS is often included in EDC coupling protocol to improve
efficiency and create dry stable (amine-reactive) intermediates. EDC couples NHS
to carboxyls forming an NHS ester considerably more stable than O-acylisourea
intermediates, allowing efficient conjugation to primary amines at physiological pH
as illustrated in figure XV. Excess reagent and byproduct formed of the cross-linking
reaction are water-soluble and can be easily removed (Kocbek et al., 2007).
The percent of BSA bound to the surface of NPs by adsorption and covalent
binding were 61.17±2.71 and 73.4±1.98% w/w, respectively. This was in agreement
with previous literature where a 9% w/w increase in the protein content was found
in covalently bound samples compared to unconjugated ones (Kocbek et al., 2007).
The significant increase (p˂0.05) in the amount of conjugated proteins was probably
due to the fact that the conjugation is done in an also physical adsorption favorable
buffer. This resulted in competing immobilization with chemical reaction and
electrostatic interaction (Qu et al., 2014).
64
EDC
PLGA NPs Stable intermediate

Unstable intermediate
(acid ended)
Ab conjugated PLGA NPs
Figure XV: Reaction scheme for the preparation of protein modified PLGA
NPs using carbodiimide coupling method. NPsconjugate
4.3.3. BSA modified Mag-PLGA NPs (Mag PLGA-BSA NPs):
Mag loaded NPs bound to BSA prepared by both adsorption and conjugation
methods using optimum Ab/NPs ratio (0.1:1) were characterized and the results are
displayed in table 9.
Worth to mention that the %EE of Mag and %BSA bound could not be
determined in BSA bound Mag-PLGA NPs as an interaction occurred between Mag
and BSA leading to decrease in their absorbance and difficulty of determination of
their real amount in the sample (Liu et al., 2003).
Following BSA adsorption, Mag-PLGA NPs exhibited aggregation with the

formation of micro rather than NPs, and non consistent results are shown in table 9
for the PS. The formation of large aggregates with protein-adsorbed NPs had been
previously reported by other workers (Jahan and Haddadi, 2015). The decrease in
ζ values around neutrality accounted for the noted aggregation.
65
Table 9: Characteristics of Mag-PLGA NPs prepared with and without protein

surface modification.
Protein
Formula Ab:NPs Binding ζ
PS (nm) PDI bound
code Ab ratio method (mV)
(% w/w)
F14 - - 118.5±4.79 0.104±0.01 -14.75±0.64 NA
F17 BSA Adsorption 1490.0±1199.96 0.377±0.38 0.24±0.53 ND

0.1:1
F18 110.4±1.13 0.096±0.02 -4.35±0.75 ND
Covalent
F19 TZB 145.1±1.77 0.108±0.02 -6.53±1.70 60.97±5.15
Results are expressed as mean of three determinations ±SD. Mag was used at a theoretical Mag loading of 5mg. NA:
not applicable, ND: not determined.
Conversely, insignificant change was noticed in the size and PDI of the
chemically conjugated BSA to the NPs (table 9, F14 and F18). Furthermore
negatively charged particles were obtained in case of chemical conjugation, with a
significant decrease in the negative ζ compared to unconjugated NPs (-4.35±0.75 vs
-14.75±0.64mV). This could be ascribed to the depletion of PLGA carboxyl groups
at the surface by their interaction with the amine termini of the protein (Jahan and
Haddadi, 2015). Similarly, previous studies reported that the mean ζ of uncoated
drug loaded PLGA NPs was approximately -14.75mV in water and this value
decreased to -4.35mV upon coating with BSA (Barua et al., 2013). Particles with ζ
more positive than +30mV and more negative than -30mV are normally considered
stable for colloidal dispersion (Feng et al., 2016). This suggests that our fabricated
particles should not be stored in a liquid suspension form and rather they should be
lyophilized (Mukherjee et al., 2008).
Results of the conjugation method proved to be more efficient in the protein

binding to NPs based on the PS and protein content.
66
4.3.4. TZB modified Mag-PLGA NPs (Mag PLGA-TZB NPs):
Similarly to BSA conjugation to NPs, TZB targeted Mag-PLGA NPs were

prepared using optimized conditions in section 3.3.3.2 and then characterized for PS,
PDI, surface charge measured by ζ, surface morphology by TEM, FT-IR, 1H-NMR
and %conjugation efficiency (CE).
In order to assess TZB loading on Mag-PLGA NPs, the calibration curve of

TZB in PBS pH 7.4 was first prepared as described in section 3.4.7 and is graphically
illustrated in figure 11. Linear relation between absorbance and concentration of
TZB obeying Beer Lambert’s law with coefficient of determination (R 2) of 0.974
was obtained.
0.45
0.4
0.35
0.3
0.25
Absorbance
0.2 y = 0.0131x - 0.0302

0.15
R² = 0.9743
0.1
0.05
0
0 5 10 15 20 25 30 35
Conc (µg/mL)
Figure 11: Calibration curve of TZB in PBS (pH 7.4) using BCA protein assay
kit.
TZB conjugated NPs, F19, showed a significant increase in PS by comparing
F19 to non- targeted Mag-PLGA NPs of F14 as seen in figures 12 (a and b) and table
67
9. Similarly, an increase in size for TZB conjugated NPs by approximately 30nm

had been previously reported (Zhou et al., 2015).
Similar to Mag PLGA-BSA, Mag PLGA-TZB NPs show a significant

decrease in the extent of the negative ζ from -14.75 to -6.52mV for unconjugated
and TZB conjugated Mag-PLGA NPs, respectively (table 9).
(a)
(b)
Figure 12: PS distribution plot of (a) Mag-PLGA NPs (F14), (b) Mag PLGA-
TZB NPs (F19).
68
Immuno NPs prepared by TZB showed similar %CE as obtained by BSA

surface modified blank NPs ~60% w/w. Due to the interaction between Mag and
BSA, we could not compare with the loaded NPs.
4.4. Characterization of immuno- Mag-PLGA NPs
4.4.1. FT-IR analysis:
FT-IR analysis was performed to confirm antibody attachment on PLGA NPs.

In the present study, the FT-IR spectra of BSA, TZB, blank PLGA NPs, BSA
modified blank NPs, BSA and TZB modified Mag-PLGA NPs are illustrated in
figure 13.
As previously explained, PLGA acid bond absorption peak can be observed

at 1759cm-1. The protein, BSA/TZB, shows the main peaks contributed by the
functional groups of molecules such as carboxylic acid O-H stretching (2500–
3000cm−1), overlapped amine and amide N-H stretching (3300–3500cm−1 and 3500–
3700cm−1), amide I C = O stretching (1600–1690cm−1) and amide II (1540cm− 1 )
which is attributed to the N-H bending and C-N stretching vibrations of the peptide
backbone (Mukherjee et al., 2008; Derman et al., 2015).
In figure 13 (a, b and c), the change in the shape of PLGA carboxylic acid OH
stretching peak (~3490cm-1) was accompanied with a shift to higher wavelength in
case of conjugated NPs compared to unconjugated NPs; evidencing the presence of
more carboxyl groups in addition to carboxylic groups of protein.
69
3653.4
3633.8
3513.7
1614.5
1756.7
Transmittance (%)
1625.3
3490.8
1759.3
PLGA-BSA NPs
3308.4
1535.2
Blank PLGA NPs

1656.0
BSA powder
4000.00 3500.00 3000.00 2500.00 2000.00 1500.00 1000.00 500.00 0.00
Wavenumbers cm-1
Figure 13(a): FT-IR spectra of BSA, blank PLGA NPs and PLGA-BSA NPs.
70
3410.5
1638.4
1751.6
Transmittance (%)
1638.6
1750.8
3308.4
1656.0 Mag PLGA-BSA NPs

1535.2
Mag-PLGA NPs
BSA powder
4000 3500 3000 2500 2000 1500 1000 500 0
Wavenumbers cm-1
Figure 13(b): FT-IR spectra of BSA, Mag-PLGA NPs and Mag PLGA-BSA
NPs.
71
Mag PLGA-TZB NPs
TZB
Mag-PLGA NPs
1639.52
3415.99
1760.07
1664.68
1538.61
3398.9
3293.11
1637.59
3488.32
1759.11
1637.59
4000 3500 3000 2500 2000 1500 1000 500 0
Wavenumbers (cm-1)
Figure 13(c): FT-IR spectra of TZB, Mag-PLGA NPs and Mag PLGA-TZB
NPs.
72
4.4.2. 1H-NMR:
Figure 14(a) shows typical 1H-NMR spectrum of TPGS. The peak at 3.65ppm
was assigned to the –CH2 protons of TPGS (Chen et al., 2011). Figure 14(b),
distinctly shows PLGA proton peaks at 5.10, 4.82 and 1.655ppm, corresponding
to the –CH, –CH2 and–CH3 protons of PLGA segments (Choi et al., 2016; Chen et
al., 2011) along with TPGS proton peaks at 3.65ppm confirming the formation of a
blend matrix of both components.
1
H-NMR spectra of BSA and TZB, figure 14(c and e), reveal the presence of
a peak at 3.5ppm corresponding to the protons from the amine (-NH2) group. TZB
exhibits characteristic δ values at 7.3, 7.2, 7.2ppm corresponding to TZB aromatic
group (Vivek et al., 2014).
BSA peaks following conjugation in figure 14(d) were not easily detected,
because the relative BSA signal was masked by the signal from the polymer chain.
This has previously been observed in NMR analysis of high molecular weight
polymer chains with end-group conjugation (Townsend et al., 2007).
1
H-NMR, seen in figure 14(f), reveals peaks corresponding to the benzene
moiety of TZB [δ = 7.311, 7.277, 7.242ppm] in Mag PLGA-TZB NPs and their
absence in unmodified NPs confirming the conjugation of Ab to the surface of NPs
(Vivek et al., 2014).
73
Figure 14(a): 1H-NMR spectrum of TPGS.
Figure 14(b): 1H-NMR spectrum of Mag-PLGA NPs.
74
Figure 14(c): 1H-NMR spectrum of BSA.
Figure 14(d): 1H-NMR spectrum of Mag PLGA-BSA NPs.
75
Figure 14(e): 1H-NMR spectrum of TZB.
Figure 14(f): 1H-NMR spectrum of Mag PLGA-TZB NPs.
76
4.4.3. TEM:
TEM micrographs of Mag PLGA-TZB NPs (figure 15) correlated well with
the hydrodynamic diameter measured by DLS. The increase in immune NPs size
compared to non targeted NPs in figure 3 confirmed the conjugation of TZB to the
surface of NPs. We can also observe a denser and thicker “corona” surrounding the
surface of Ab coated NPs, which was absent in the uncoated ones in figure 3. This
denoted the presence of the Ab on the surface of the NPs (Moura et al., 2014;
Thamake et al., 2010).
Figure 15: TEM micrographs of Mag PLGA-TZB NPs at different

magnifications.
To recapitulate, Mag-PLGA NPs were successfully prepared by

nanoprecipitation method. The use of TPGS as SAA in the organic phase
successfully produced NPs with adequate size for cell internalization and a new
matrix blend of PLGA and TPGS was obtained. The formation of the new matrix
was confirmed via FT-IR studies. The surface of selected NPs was then modified
with, TZB, a monoclonal antibody known to be selective to HER2 over-expressing
cells, like breast cancer cells. Ab loading by physical adsorption were excluded due
to the formation of large aggregated NPs, while NPs with adequate size for cell
77
uptake were obtained while conjugating the Ab amino groups with the PLGA
carboxyl groups. Surface modification was successfully achieved, as demonstrated
by changes in size and zeta potential of the particles. The amount of TZB on the
surface, measured by (BCA) protein assay, confirmed the presence of the antibody
on NPs surface.
78
Chapter 1 Conclusions
Conclusions
From the previous findings, the following could be concluded:
1. Acetone produced NPs with more uniform smaller PS compared to

acetonitrile when used as organic solvent for PLGA during the preparation of
NPs by the nanoprecipitation method.
2. The increase in PVA concentrations in the aqueous phase caused a

concentration dependent decrease in NPs size and a 0.5% w/v was found to
be optimum for the production of uniform, NPs with a size less than 150nm.
3. The use of TPGS in concentrations of 0.03 to 0.18% w/v in the aqueous phase
resulted in the production of NPs smaller than 120nm. Lower concentrations
of TPGS were used to produce small NPs compared to PVA.
4. Theoretical drug concentrations of 1 to 4% w/w were optimum to produce

NPs with acceptable PS and max EE.
5. The change in organic to aqueous phase volume ratio from 1:2 to 1:1 and the
TPGS solvent phase from aqueous to organic phase was shown optimum for
the fabrication of NPs.
6. The selected formula exhibited ζ of -14.75± 0.64mV indicating the formation

of stable NPs.
7. According to the TEM micrographs, Mag-PLGA NPs were spherical uniform

in shape with size ~100nm.
79
8. FT-IR and 1H-NMR studies confirmed the formation of a new blend matrix
composed of PLGA and TPGS. The new matrix was able to molecularly
disperse and encapsulate the hydrophobic drug Mag.
9. The release profiles for Mag from the new matrix showed a small burst
release of 10% w/w during the first four hours, 40% w/w Mag release during
the first day and then a slower release was then noted where less than 80%
w/w were released in 40 days.
10. TZB modified NPs showed a significant decrease in the negative ζ compared
to unmodified NPs confirming Ab attachment.
11. Immuno NPs prepared by TZB showed %CE of ~60% w/w.
80
Introduction
Gold nanomaterials have received great interest for their use in cancer
theranostic applications over the past two decades (Arvizo et al., 2010). Gold
nanoparticles (GNPs) represent a versatile, potent, selective, and highly multi-
functional anti-cancer technology. They are characterized by particular and unique
physical, chemical and photonic properties (Han et al., 2007).
As shown in figure XVI, GNPs have different size, shape, and structure (Lee
et al., 2014). Gold nanospheres are characterized by their simplicity and ease of
fabrication which contribute to their extensive applications. Also known as gold
colloids, gold nanospheres are solid balls of gold that range in diameter from only
2nm to more than 100nm and with absorption wavelength ranging from 500 to
600nm. The maximum absorption wavelength of a gold nanosphere shifts to longer
wavelength with increase in diameter (Li and Gu, 2010; Lee et al., 2014). Gold
nanoshells are spherical structures comprising a silica core and thin layer of gold,
50–150nm in size. The diameter of gold nanoshell is determined by the diameter of
silica core and the shell thickness is controlled by the amount of gold deposited on
the surface of the core. Their optical properties can be tuned by modifying the core
diameter and shell thickness (Li and Gu, 2010; Lee et al., 2014). Gold
nanorods(GNRs) have two dimensions: length and diameter. By manipulating the
aspect ratio, changes in peak absorbance wavelength are obtained. The absorption
wavelength of GNRs has two peaks depending on the orientation of the particle to
an incident beam of light (Lee et al., 2014). Nanocages are hollow structures with
thin, porous, and robust gold wall. Gold nanocages with controllable wall thickness
and absorption wavelengths ranging from visible to near infra-red (NIR) regions
could be obtained by varying the amount of gold chloride. The thicker the gold wall,
81
the longer the maximum absorption wavelength (Li and Gu, 2010). Hollow gold
nanospheres are spherical GNPs with an interior hollow. They are synthesized by
the cobalt NP mediated reduction of chloroauric acid. They represent second
generation gold nanostructures with unique structural features: small size (outer
diameter 30–50nm), spherical shape, hollow interior, and a strong and tunable
absorption band at a NIR region, optimal optical properties, and efficient
photothermal profiles (You et al., 2012).
Figure XVI: Different shapes of GNPs (Lee et al., 2014).
In the presence of an electromagnetic field at a certain wavelength, GNPs with

size much smaller than the wavelength of incident light can induce a resonance of
the free electrons across the particle, known as surface plasmon resonance (SPR)
(figure XVII). As a result of SPR, the particle absorbs and scatters the
electromagnetic radiation intensely. The SPR of GNPs depends strongly on the size,
shape, and surface composition of the particle, and the dielectric properties of the
surrounding medium (Huang and El-Sayed, 2010). Due to the SPR oscillation, the
light absorption and scattering are orders of magnitude stronger than the strongest
absorbing organic dye molecules and the emission is more enhanced compared to
the most strongly fluorescent molecules. According to El-Sayed and co-workers the
ratio of the scattering to absorption increases dramatically for particles with larger
size. This fact can guide the choice of GNPs for biomedical applications. For
imaging, larger NPs are preferred because of higher scattering efficiency, whereas
for photothermal therapy (PTT), smaller nanoparticles are preferred as light is
82
mainly absorbed by the particles and thus efficiently converted to heat for cell and
tissue destruction (Huang and El-Sayed, 2010). GNPs absorb light in both the
visible and the NIR regions. By changing the size/shape/surface of GNPs, the
wavelength of their plasmon absorption can be tuned to coincide in the NIR window
(~650–900nm), where penetration of 10cm in depth through breast tissue, even at
low laser power densities, can be achieved (Dreaden et al., 2012). In those spectral
regions, the attenuation of photons by tissues and physiological fluids is minimal
and the increase in the local temperature using laser PTT is sufficient to induce rapid
tumor cell death (necrosis) with minimal damage to surrounding tissues (Dreaden
et al., 2012). Also, the attractive optical and electronic properties of GNPs
themselves or of the GNPs co-labeled with imaging contrast agents can be used as
radiation therapy contrast, photo-imaging contrast and spectrochemical diagnostic
contrast in diverse areas such as in vitro assays, in vitro and in non-invasive in vivo
imaging (Cai et al., 2008).
Figure XVII: SPR of GNPs (Huang and El-Sayed, 2010).
Various methods have been developed for the synthesis of GNPs including
chemical, physical and biological methods (Shah et al., 2014). Turkevich method is
a commonly used method for synthesis of spherical GNPs in the size range of 10-
20nm. The principle of this method involves reduction of gold ions (Au 3+) to gold
atoms (Au0 ) in the presence of reducing agents like citrate, amino acids, ascorbic
83
acid or UV light (Shah et al., 2014). The bond strength between the gold surface
and citrate anions in Turkevich method is similar to a hydrogen bond in strength and,
so it is easily displaced by more strongly bound thiols or amines (Ulman A., 1996).
Brust method is used to produce spherical GNPs (1-3nm) in organic liquids
immiscible with water. It is a two phase process in which gold salt is transferred
from aqueous solution to an organic solvent as toluene using tetraoctylammonium
bromide (TOAB) as phase transfer agent followed by gold reduction using sodium
borohydride (NaBH4) in the presence of dodecanethiol (Brust et al., 1994). Seeding
growth method is applied to obtain GNPs in other shapes such as rods, cubes, tubes.
Gold seed particles are first produced by reducing gold chloride solution using a
strong reducing agent like sodium borohydride. Then the formed gold seed particles
are added as catalyst to a solution of gold chloride containing ascorbic acid (weak
reducing agent) and cetyltrimethyl ammonium bromide (CTAB) (structure directing
agent to accelerate the anisotropic growth of GNPs) (Hedkvist, 2013).Green
synthesis of GNPs is using plants extracts as Garcinia mangostana fruit peels, leaf
extract of Nepenthes khasiana and microorganisms as the bacterium Deinococcus
radiodurans for the reduction of gold (Bhau et al., 2015; Li et al., 2016a; Xin Lee
et al., 2016 ).This method has the advantage of being clean, eco-friendly and non-
toxic (Singh et al., 2016). Miscellaneous methods using ultrasonic waves, laser
ablation, solvothermal method, electrochemical reduction and photothermal
reduction are also applied for the preparation of GNPs (Shah et al., 2014). From all
the above methods of GNPs preparations, the Turkevich reduction method is
considered the most representative and popularly used procedure to synthesize
GNPs, because of its simplicity, reproducibility and the loose shell of citrates on the
NP surfaces which is easily replaced by other desired ligands with valuable function
(Khaled et al., 2016). Although the nanosurface of GNPs prepared by Turkevich
method showed overall negative charge, no decrease in uptake was found
84
(Turkevich et al., 1951). The negative citric acid groups desorb from the NP surface
by nonspecific adsorption of serum proteins and was replaced by high concentrations
of positive primary and secondary amines on the NP surface which allow for uptake
to take place (Dreaden et al., 2012).
As nanocarriers, GNPs suffer from non-specific uptake and potential

degradation in macrophages. Surface covering of GNPs with biocompatible
polymers as PEG elicits low immunogenic responses and longer circulatory half-
lives (Owens and Peppas, 2006).Targeting is important for maximizing drug
efficacy and minimizing side effects. Targeting of GNPs is achieved by passive
targeting, active targeting, or a combination of both strategies to achieve tumor-
specific particle accumulation. Size-selective accumulation at tumor sites due to the
enhanced permeability and retention effect is a particular advantage of anti-cancer
GNPs (Maeda et al., 2000). GNPs of 5nm in size are rapidly cleared by the kidneys
and those of 100nm are easily removed by immune system. Size in between showed
best accumulation and penetration in tumor. The control of surface charge and the
presence of stealthing agent is essential to achieve the desired behavior. Also the
shape is very important. Spherical GNPs are the best choice when used as carrier
due to the simple synthesis and ease of functionalization (Farré, 2013). Active
targeting is achieved by conjugating GNPs with various tumor-targeting agents, such
as antibodies, peptides, and small molecules such as folic acid. Once accumulated
in tumor tissue, GNPs enter the cell, by an energy-dependent process, mainly by
endocytosis. This approach has been employed to increase the targeting specificity
of chemotherapy conjugated in an effort to avoid off-target toxicity (Nicol et al.,
2015).
GNPs functionalization with cell-penetrating peptides and specific targeting

antibodies represent one area of particular interest. Different conjugation strategies
85
are used for attaching antibodies and other molecules to GNPs surface. Physical
interaction between antibodies and GNPs depends on the ionic attraction between
the negatively charged gold and the positively charged antibody, the hydrophobic
attraction between the antibody and the gold surface as well as the dative binding
between the gold conducting electrons and amino acid sulfur atoms of the antibody.
Chemical interactions between antibodies and NP surface are achieved in different
ways such as the chemisorption via thiol derivatives, the use of bifunctional linkers
or the use of adapter molecules like streptavidin and biotin.
GNPs followed three main pathways for the cellular uptake which includes
receptor mediated endocytosis, phagocytosis and fluid phase endocytosis (Khan et
al., 2014). Cellular uptake is dependent on charge, surface chemistry, size and shape
(Lee et al., 2014). Clathrin-dependent endocytosis is believed to be the dominant
uptake pathway for spherical particles, while those with larger dimensions are
predominantly internalized by macropinocytosis and phagocytosis. The tumor
uptake of GNPs in vivo is significantly lessened by the opsonization of the NPs with
plasma proteins and their subsequent phagocytosis by reticuloendothelial system
(RES) components such as monocytes and macrophages. Thus, most injected GNPs
are sequestered in the liver and spleen. Coating the NPs with polyethylene glycol
(PEG) prevents the uptake of NPs by the RES by acting like a stealth, thus
prolonging their circulation time and increasing their concentration in tumor tissue
(Mady et al., 2015). Studies showed that 50nm citrate-capped spherical GNPs
exhibited optimal uptake (Murugan et al., 2015).
The toxicity of GNPs depends on the size, shape, synthesis method and
surface charge. But as GNPs are considered to be non-toxic agents, thus overall
cytotoxicity of GNPs was found to be in acceptable level (Khan et al., 2014).
86
GNPs received great attention especially in the field of biological tagging,

chemical and biological sensing, photothermal therapy, biomedical imaging, DNA
labelling, microscopy and photoacoutisc imaging, surface-enhanced Raman
spectroscopy (SERS), tracking and drug delivery, catalysis and cancer therapy
(Khan et al., 2014).
Primary goal of developing new drug delivery system for anticancer drugs is
to minimize the various side effects caused by their cargo when loaded in
conventional dosage forms and to improve the drug selectivity and efficiency (Khan
et al., 2014). The inherent properties of GNPs including controlled preparation of
various sized particles (1–150nm), chemical stability, inherent biocompatibility
and low cytotoxicity make them a very promising vehicle for drug delivery (Li et
al., 2016b). GNPs have strong binding attraction for thiols, proteins, carboxylic acid,
aptamers and disulfides. This diverse functional possibility allows for a variety of
approaches for drug delivery design especially in the field of cancer therapy (Paciotti
et al., 2016). GNPs can be used to deliver drugs and imaging agents that exhibit low
solubility, poor pharmacokinetics, susceptible to enzymatic degradation, as well as
those that exhibit poor intracellular penetration (e.g., siRNA) (Giljohann, 2009). It
was reported that GNPs can deliver small drug molecules, peptides, proteins, and
nucleic acids such as DNA or RNA to their targets (Khan et al., 2014). Drugs are
loaded on by noncovalent interaction or through covalent conjugation. Hydrophobic
drugs can be loaded onto GNPs through non-covalent interactions without structural
modification for drug release. Covalent conjugation to the GNPs was done through
cleavable linkages to deliver prodrugs to the cell and then the drug was released by
external or internal stimuli (Duncan et al., 2010). GNPs have the advantage over
conventional liposomes and PLGA NPs by the ability of surface functionalization
87
with active ligands at densities of (1.0 × 106µm) which is 100- and 1000- fold higher,
respectively (Dreaden et al., 2012).
Most of the biosensors depend on localized surface plasmon resonance

(LSPR) of GNPs. GNPs based sensors can detect various metal ions depending on
the color change due to the aggregation of GNPs (Aldewachi et al., 2018).
Such types of sensors have been widely used for the detection of copper,
mercury, lead and arsenic in water (Pacławski et al., 2012). NPs combined with
biomolecules such as functionalization of GNPs with oligonucleotides and
mercaptopropionic acid have been used for the biosensing applications as the
biosensing of proteins and polynucleotides (Rastogi et al., 2012).
The atomic number of gold is much higher than that of the currently used
transverse cut (CT) contrast material iodine which permit CT imaging at lower
patient doses and with better sensitivity and good specificity. GNPs show extended
blood circulation time and significant CT contrast enhancement by accumulation of
NPs within the tumor and in areas surrounding it by the EPR effect (Shilo et al.,
2012). Raman spectroscopy is the most promising imaging technique for GNPs
based contrast agents (Cai et al., 2008).
Nowadays several researches studied the use of GNPs in Photo-induced cell

killing. This technique provides a safe and effective way to selectively eradicate
target cells such as cancerous cells while avoiding systemic toxicity and side effects
on healthy tissues because only the lesion under light irradiation is treated compared
to traditional chemotherapy and radiotherapy. Moreover, this type of therapy is more
beneficial to patients in which location or size of lesions limits the acceptability of
conventional therapy (Hong et al., 2016). Photo-induced cell killing could be
88
achieved either by photodynamic therapy (PDT) or photothermal therapy (PTT).

PDT and PTT will be discussed in detail later in chapter 3.
Several previous studies had shown GNPs promise in the advancement

of cancer therapy. Craig et al., 2012 showed that cisplatin could be successfully
conjugated to GNPs using PEG-based linkers and that cellular uptake and
cytotoxicity were significantly improved compared to free drug. GNPs conjugated
with different chemotherapeutic drugs as cisplatin, doxorubicin, capecitabine
reduced the chemoresistance of hepatocellular carcinoma-derived cancer cells and
increased susceptibility of these drugs for cell penetration (Tomuleasa et al., 2012;
Du et al., 2018). 5-fluorouracil (5-FU) conjugated to GNPs using two thiol ligands
(thioglycolic acid and glutathione) had a 2-fold higher anticancer effect compared
with free 5-FU (Safwat et al., 2016). Manivasagan et al., 2016 demonstrated the
multifunctional doxorubicin-loaded fucoidan capped GNPs for drug delivery and
photoacoustic imaging as it induced both early and late apoptosis in a concentration-
dependent manner compared with untreated control cells and was found to be a good
contrast agent for in vitro breast cancer imaging. Quercetin reduced GNPs were
successfully nanoencapsulated with PLGA to increase bioavailability and showed
enhanced apoptosis and inhibition of cancer cell proliferation on several cancer cell
lines in vitro (Bishayee et al., 2015). PLGA nanocomposites were developed by
incorporating a photosensitizer, verteporfin and GNPs into the polymeric matrix via
solvent evaporation single-emulsion method. The efficiency of PDT on epithelioid
carcinoma PANC-1 cell line under 405nm laser irradiation, was higher compared
with PLGA containing photosensitizer only (Deng et al., 2016).
The aim of the present work was to prepare Mag-GNPs suitable for IV
administration. The developed multifunctional nano-carriers that work
simultaneously for combined photothermal therapy and drug delivery vehicle to
89
target breast tumor cells was set as the ultimate goal. Characterizing the proposed
system and testing its release and stability is also undertaken in this chapter.
90
Experimental
1. Materials:
• Gold chloride: Sigma Aldrich., USA.
• Fetal bovine serum (FBS): GIBCOTM, UK.
• Nanoseps: Centrifuge tube fitted with an ultra-filter: MWCO 100KDa, Pall Life
Sciences, Port Washington, NY.
• [50/50 Poly(DL-lactide-co-glycolide) (PLGA): Grade 5002A, viscosity 0.2dl/g,
acid terminated, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC), acetonitrile, acetone, bicinchonininc acid (BCA)
protein assay reagent kit, bovine Serum albumin (BSA), cellulose dialysis
tubing (MWCO 14KDa, 15.9mm diameter), D-α-tocopherol vitamin E
polyethylene glycol 1000 succinate (TPGS), disodium hydrogen phosphate,
glacial acetic acid, Herceptin® (trastuzumab), Magnolol (Mag): purity >99%,
methanol, N-hydroxysuccinimide (NHS): 98% (research grade), potassium
chloride, potassium dihydrogen phosphate, sodium acetate, sodium chloride,
sodium hydroxide, sodium dodecyl sulphate (SDS) and syringe filters (0.45µm,
polytetrafluoroethylene (PTFE)) ] are previously described in chapter 1.
• All other chemicals and reagents used were of analytical grade.
2. Equipment:
• Transmission electron microscope equipped with energy dispersion X-ray

analyzer (EDX-TEM): JEM 1400 plus, JEOL, Japan.
• Homogenizer: Heidolf, Germany.
91
• [Balance, cooling centrifuge, fourier transform- infra red (FT-IR), freeze

dryer, pH meter (Digital), high performance liquid chromatography
(HPLC) with UV detector, spectrophotometer (double beam), stirrer,
zetasizer Nano ZS] are previously described in chapter one.
3. Methodology:
3.1. Synthesis of citrate capped-gold nanoparticles (GNPs)
All glasswares were first washed with aqua regia and then cleaned
thoroughly with deionized water before use (Jayalakshmi et al., 2014).
Colloidal GNPs were synthesized using gold chloride (HAuCl4) as precursor

and sodium citrate as reducing agent according to the Turkevich method as
illustrated in the following reaction (Turkevich, 1951).
3 (H2CCOOH)2 C(OH)COO− + 2AuCl− 3 (H2CCOOH)2 C=O + 2Au + 8Cl− + 3CO2 + 3H+
Briefly, 100mL of 1mM HAuCl4 were boiled in a conical flask under

continuous stirring followed by addition of 10mL of 1% w/v sodium citrate solution.
The dispersion was stirred and heated under reflux until a red colour was obtained.
The colloidal dispersion was allowed to cool at room temperature and was then
subjected to particle size (PS), zeta potential (ζ) and spectral analysis (Mohamed et
al., 2012).
3.2. Preparation and optimization of magnolol loaded gold

nanoparticles (Mag-GNPs)
Mag loading was accomplished by direct adsorption of Mag onto the surface
of citrate GNPs as previously described elsewhere (Curry et al., 2015). Briefly, Mag
92
solutions were prepared in ethanol according to the set concentration. A volume of

ethanolic solution containing the required amount of Mag was then added to 1mL of
the previously prepared GNPs colloidal solution. This solution was kept under
stirring at room temperature for a specified time in a closed stoppered vial and then
the lid was opened to allow for ethanol evaporation (Mohamed et al., 2012). Table
10 shows the composition of the prepared formulae where the following variables
were investigated:
3.2.1. Ethanol volume:
The theoretical Mag loading amount was fixed at 500µg and was dissolved in
different ethanol volumes: 250, 500 and 1000µL corresponding to drug
concentrations of 2, 1 and 0.5mg/mL, respectively. The set ethanol volume
containing 500µg of Mag from each of the prepared solutions was added to 1mL of
the previously prepared GNPs colloidal solution and the solution was kept under
stirring for 2h. The procedure was then completed as described in the previous
section.
3.2.2. Theoretical Mag loading amount:
Different drug amount namely: 0.25, 0.5 and 1mg were tried while the
optimum drug and gold concentration were kept constant. Stirring was allowed for
2h followed by ethanol evaporation at room temperature.
3.2.3. Loading time:
A 12h loading time was also tried instead of 2h while keeping previously
optimized parameters constants.
93
The optimum conditions were chosen depending on percent association

efficiency (%AE) and the best formula (F6) was further characterized for PS, PDI,
surface morphology, FT-IR and in vitro drug release.
Table 10: Mag-GNPs formulae.
Formula Mag amount Volume of Mag concentration Loading time

code (µg) ethanol (µL) (mg/mL) (h)
F1 250 2
F2 500 500 1
F3 1000 0.5 2
F4 250 250
F5 1000 1000 1
F6 500 500 12
All formulae were prepared in 1mL of 1mM GNPs colloidal solution.
3.3. Preparation of immuno- Mag-GNPs
As previously explained in chapter 1, a pilot study was conducted on BSA

(model protein) to prepare bovine serum albumin (BSA) modified Mag-GNPs.
3.3.1. Preparation and optimization of BSA modified Mag-GNPs:
Loading of BSA on the surface of GNPs was attempted by direct adsorption

as previously described elsewhere with slight modification (Barua et al., 2013).
Briefly, a solution of 10mg/mL BSA (Ip = 4.7) was prepared in sodium acetate buffer
(pH 4.5), from which different volumes of BSA solutions corresponding to 250, 500
and 1000µg were added to one mL of the previously prepared Mag-GNPs as shown
in table 11. Mixing was applied for 4h at room temperature. The unbound BSA was
separated from BSA-coated GNPs by two cycles of centrifugation at 30,000rpm for
15min at 4ºC with washing with deionized water before the second cycle.
94
Table 11: BSA modified Mag-GNPs formulae.

Formula code BSA amount (µg)
F6 -
F7 250
F8 500
F9 1000
All formulae were prepared from 1mL GNPs (1mM) and 500µg Mag theoretical loading.
3.3.2 Preparation and optimization of PLGA encapsulated Mag and

GNPs (Mag-GNPs/PLGA NPs) formula:
The nanoprecipitation method was used for the preparation of Mag-

GNPs/PLGA NPs. Trials were made to disperse GNPs either in the aqueous or
organic phase. Besides, the amount and concentrations of the GNPs were varied as
shown in table 12. It is to be highlighted that TPGS was always added with the
polymer in the organic phase.
3.3.2.1. Preparation by dispersing the GNPs in the aqueous phase:
Accurately weighed amounts of Mag (5mg), PLGA (25mg) and TPGS

(7.5mg) were dissolved in 2mL of acetone as organic phase at room temperature.
The aqueous phase was composed of 250µL of colloidal GNPs solution mixed with
1750µL water. Then, the organic phase was added dropwise to the aqueous phase
under magnetic stirring at 400rpm. Following addition of the organic phase, stirring
continued at 250rpm at room temperature until the organic solvent had evaporated
completely. The NPs were purified by two cycles of centrifugation at 30,000rpm for
30min at 4°C and washing with deionized water.
95
3.3.2.2. Preparation by dispersing the GNPs in the organic phase:
The method adopted was based on a previous reported study by Bishayee and
co-workers with slight modification (Bishayee et al., 2015). Different volumes of
the previously prepared GNPs dispersion (1mM), were concentrated to 10-fold using
ultrafiltration with nanoseps of MWCO 10KDa, by centrifugation at 4000rpm at
room temperature for 10min. Water (2mL) only constituted the aqueous phase.
Different volumes of GNPs dispersion (10mM) was mixed with the organic phase
composed of PLGA and TPGS dissolved in acetone and was dropped on the aqueous
phase at a volume ratio of 1:1. Then, the procedure was completed as previously
reported in chapter 1, section 3.2. NPs showing optimum colloidal properties were
selected for TZB decoration.
Table 12 shows the different Mag-GNPs/PLGA NPs formulae prepared with

different methods and formulation parameters.
Table 12: Mag-GNPs/PLGA NPs formulae prepared with different methods

and formulation parameters.
Formula GNPs volume GNPs concentrationa

GNPs dispersion phase
(µL) (mM)
P1 250 1 Aqueous phase

P2 25 10
Organic phase
P3 50 10
All formulae were prepared at a theoretical Mag loading of 5mg with PLGA 1.25% w/v of the solvent used acetone
and TPGS 30% w/w of the polymer and the ratio of organic to aqueous phase was maintained at 1:1. a represents the
initial concentration of added GNPs based on its preparation method.
96
3.3.3. Preparation of TZB modified PLGA encapsulated Mag and GNPs

(Mag-GNPs/PLGA-TZB NPs) formula:
Mag-GNPs/PLGA NPs (P2) previously prepared in section 3.3.2 were

decorated with the monoclonal antibody trastuzumab (mAb TZB) using covalent
binding with TZB at a 1:10 ratio of TZB:Mag-PLGA NPs. The same procedure used
in chapter 1, section 3.3.3, was adopted and the spectral analysis, PS, PDI, ζ, surface
morphology, FT-IR, Mag %EE and mAb %CE.
3.4. Characterization of the prepared NPs
3.4.1. Spectral analysis of GNPs:
The analysis was determined by scanning the freshly prepared citrate-GNPs,

Mag-GNPs and Mag-GNPs/PLGA NPs using the UV/Vis spectrophotometer in the
range of 400 to 800nm.
3.4.2. PS analysis:
The PS analysis was performed by DLS using Malvern zetasizer 4 as

previously described in chapter 1. All the samples were diluted with distilled water
until the particles count reached 200-300 kilo count per seconds (KCPs) before
analysis. Results were performed in triplicates of different batches.
3.4.3. Transmission electron microscope (TEM) examination:
TEM images were recorded on TEM equipped with energy dispersion X-ray
analyzer (EDX). The samples for TEM and EDX analysis were prepared by placing
a drop of the synthesized colloids onto a carbon-coated Cu grid followed by slow
evaporation of solvent at ambient condition. The average PS and standard deviation
(SD) were calculated by counting 200 particles from more than five TEM images of
97
different areas of the Cu grid. All images for TEM were obtained at 120Hz. Energy-
dispersive X-ray (EDX) analysis was done to confirm the presence of gold in the
particles.
3.4.4. Surface charge determination:
The NPs surface charge was determined by the measurement of ζ of the

freshly prepared NPs dispersion using zetasizer 4 as previously explained. Analysis
was performed on triplicates of different batches.
3.4.5. Determination of drug loading:
3.4.5.1. U.V scanning of Mag in a 50:50 v/v methanol:water mixture:
Spectrophotometric scanning of various concentrations of Mag prepared in

methanol-water (50:50 v/v) in the UV range from 200 to 400nm using UV-visible
spectrophotometer was performed. The wavelength of maximum absorbance (λ max)
was determined using the corresponding solvent as blank.
3.4.5.2. Construction of calibration curve of Mag in methanol-water

(50:50 v/v):
Stock solution of 1mg/mL Mag was prepared by dissolving 50mg Mag in 25

mL methanol in a 50mL volumetric flask and the volume was made up to mark with
distilled water. Serial dilutions of Mag solutions yielding concentrations of: 5, 10,
15, 20, 25 and 30µg/mL were prepared in methanol-water (50:50 v/v). UV-Vis
spectrophotometer detection at the predetermined λmax was used for the
determination of Mag and a calibration curve is constructed by plotting the
concentration of Mag vs the absorbance.
98
3.4.5.3. Mag association efficiency (AE):
Drug AE for Mag-GNPs was calculated indirectly by estimating Mag content

in the supernatant. Briefly 2mL of Mag-GNPs dispersion were centrifuged at
30,000rpm in a cooling centrifuge for 15min at 4ºC. The drug concentration in
supernatant was determined by UV/Vis spectrophotometer at the predetermined
λmax. The %AE of Mag on GNPs was estimated using the following formula:
AE% = [(Wintial Mag - Wfree Mag)/ Wintial Mag] x100 (Eq. 5)
Where "Winitial Mag" is the total amount of the drug used and "Wfree " is the amount of
free drug detected in supernatant after centrifugation of the aqueous dispersion.
While %EE of Mag in Mag-GNPs/PLGA NPs was determined directly using

HPLC-UV method after degradation of NPs as previously described in chapter1,
section 3.4.4.
3.4.6. FT-IR analysis:
FT-IR spectra of selected formulations were obtained by Thermonicolet

Nexus spectrometer. Samples were pressed into potassium bromide (KBr) pellets
and recorded at frequencies from 4000 to 400cm-1 with resolution of 4cm-1.
3.4.7. In vitro Mag release:
The release of Mag from selected formulations was carried out using dialysis
membrane of MWCO 14kDa as previously described in chapter 1, section 3.4.5.
99
3.4.8. Determination of protein on the surface of modified NPs using

protein assay:
The same procedure used in chapter 1, section 3.4.7 was applied for TZB
determination.
3.4.9. Stability study of the NPs in serum:
To evaluate the colloidal NPs stability of the selected formula in simulated

biological environment following IV administration, 0.5mL NPs colloidal
dispersion were suspended in 1mL 50% v/v FBS aqueous solutions, followed by
incubation at 37°C. The PS was determined at different time intervals (0, 1, 2 and
24h) using DLS (Pooja et al., 2014).
3.5. Statistical analysis
All formulations were prepared and reported in triplicates. Results are

expressed as mean ± SD. The statistical signiﬁcance of difference between groups
were evaluated by one-way ANOVA and Tukey's post hoc test with a significance
level of p<0.05.
100
4. Results and Discussion
4.1. Citrate capped GNPs synthesis
In this work, GNPs were synthesized using the Turkevich method. The
reduction of gold using citrate ions is considered as one of the most representative
and popularly used procedures to synthesize GNPs (Khaled et al., 2016). In this
simple, reproducible reduction method, the loose shell of citrates on the NPs surfaces
can be easily replaced by other desired ligands with valuable function (Khaled et al.,
2016).
At the beginning of the synthesis procedure, gold salt solution was yellow.
Following sodium citrate addition, a blueish color appeared, indicating the formation
of gold nuclei. A few minutes later, the solution turned ruby red denoting the
successful formation of the colloidal GNPs (Mohamed et al., 2012).
4.1.1. Spectral analysis of citrate capped GNPs:
As previously mentioned in the introduction of this chapter, GNPs exhibit a

unique surface plasmonic resonance (SPR) in the visible region. Figure 16 shows
that the maximum SPR peak is positioned at 519nm. Based on previous literature,
this detected SPR position and color indicated small size distribution of about 20nm
in diameter as well as good NPs stability (Burda et al., 2005). In fact, different sizes
of GNPs exhibit different colors; ruby red color of dispersion indicates GNPs size is
less than 50nm while formation of blue or black gold dispersion is generally
associated with larger size NPs formation (Hu et al., 2006). As seen in figure 17(b),
a colloidal gold solution with a characteristic red color is obtained denoting small
GNPs. Following preparation, synthesized GNPs were characterized by spectral
UV-Vis scan, PS and ζ determination.
101
1.8
1.6
519nm
1.4
1.2
1
Absorbance
0.8
0.6
0.4
0.2
0
400 500 600 700 800
Wavelength (nm)
Figure 16: UV-Vis spectrum of GNPs prepared by Turkevich method.
(a) (b)
)
Figure 17: Photograph of gold dispersion before (a) and after (b) NPs
formation.
4.1.2. PS analysis and morphology of GNPs:
PS distribution of GNPs was determined by DLS. The average hydrodynamic

diameter of citrate GNPs was found to be 23.4±0.71nm with a PDI of 0.440±0.03.
Previous works reported different GNPs with different PDI values: 0.406, 0.301,
0.223 (Verma et al., 2014; Srinath and Rai, 2015; Das et al., 2017). As the PDI
value was less than 0.7, the prepared GNPs didn’t have a broad size distribution and
102
were suitable for DLS technique (Das et al., 2017). Size and PDI of GNPs depend
on initial gold salt concentration and the molar ratios of citrate to gold salt (Zabetakis
et al., 2012). Figure 18 shows a PS distribution chart obtained from the zeta sizer
showing a single peak.
Figure 18: PS distribution plot obtained from the zeta sizer for plain GNPs.
The size and shape of the colloidal GNPs have been examined by TEM. As
seen in figure 19, the NPs are mostly spherical and the average PS is ~20nm.
Figure 19: TEM micrograph of plain citrate capped GNPs.

103
4.1.3. Surface charge determination:
The magnitude of the ζ is predictive of the colloidal stability. ζ of citrate

capped GNPs was found to be -57.20±2.24mV indicating NPs stability (Mukherjee
et al., 2008). Using similar gold and sodium citrate concentrations, others reported
comparable ζ (Arjmandi, 2012; Lim, 2014). The negative value resulted probably
due to the presence of three deprotonated anionic carboxyl groups of citrate ions
(Kiroula et al., 2016).
4.2. Mag-GNPs
4.2.1. Drug association efficiency (AE):
4.2.1.1. Calibration curve of Mag in methanol-water (50:50 v/v):
In accordance with previous literature, UV scanning of Mag in 50:50 v/v

methanol/water revealed that the drug showed ʎmax at 292nm (Stefanache et al.,
2017). The calibration curve of Mag in 50:50 v/v methanol/water is graphically
illustrated in figure 20. Linear relationship between absorbance and concentrations
in the range of 5-30µg/mL with coefficient of determination (R2) of 0.999 is
obtained.
1
0.8
Absorbance
y = 0.0274x + 0.0198
0.6 R² = 0.9993
0.4
0.2
0
0 10 20 30 40
Conc (µg/mL)
Figure 20: Calibration curve of Mag in 50:50 (v/v) methanol/water at 292nm.

104
4.2.1.2 Effect of ethanol volume:
The effect of the various formulation variables on the amount of Mag

adsorbed on GNPs surface had been investigated and the results are shown in table
13. Maintaining a constant theoretical drug loading amount, 500µg, while increasing
ethanol volume from 250 to 500µL corresponding to a drug concentration decrease
from 2 to 1mg/mL leads to a significant increase (p˂0.05) in drug AE from
9.66±1.18 to 20.75±3.65% w/w, as seen in F1 and F2, respectively. It is noteworthy
to mention that a white precipitate was observed upon using the higher drug
concentration (2mg/mL) (F1). This might be due to Mag crystallization which
occurred due to low alcohol content and was the cause of the observed low AE.
Increasing ethanol volume to 1000µL, accompanied with decrease in drug
concentration, resulted in a non-significant effect on AE (p˃0.05) depicting a value
of 16.42±2.83% w/w in F3.
Hence, a drug concentration of 1mg/mL was chosen for subsequent work.
Table 13: AE% of Mag in Mag-GNPs prepared using different formulation

variables.
Drug Mag AE
Formula Mag amount Ethanol Loading
concentration (% w/w)
code (µg) volume(µL) time (h)
(mg/mL)
F1 250 2 9.66±1.18
F2 500 500 1 20.75±3.65
F3 1000 0.5 2 16.42±2.83
F4 250 250 4.16±0.47
F5 1000 1000 1 20.00±1.41
F6 500 500 12 42.04±0.33
105
4.2.1.3. Effect of Mag loading amount:
From table 13, it is obvious that maintaining the drug concentration at

1mg/mL and decreasing the initial drug loading to 250µg was accompanied with a
decrease in %AE from 20.75±3.65 to 4.16±0.47% w/w, as could be seen in F2 and
F4, respectively. On the other hand, the increase in drug loading amount to 1000µg
did not show any significant effect (p˃0.05) on drug AE% as seen in F5 in
comparison to F2.
Mag is a hydrophobic drug with very poor aqueous solubility, less than
80µg/mL at 37ºC (Qiu et al., 2016) needing ethanol as a cosolvent. Addition of Mag
ethanolic solution on the GNPs aqueous dispersion resulted in drug deposition on
GNPs surface with the possibility of drug precipitation if the solubility limits were
exceeded. Beside being important for drug solubility, ethanol was found to affect
drug adsorption process. Structuring of water had been found to be responsible for
the nonspecific adsorption of drugs onto GNPs surface (VanDer et al., 2005).
Increasing ethanol concentration decreased water structuring around the drug and
probably decreased the driving force for the nonspecific adsorption. It could
therefore be deduced that at a specific drug concentration, in that case, 1mg/mL, an
optimum ethanol volume is required to dissolve a certain amount of the drug without
affecting water structuring and drug deposition on the surface of GNPs.
A maximum AE% of about 20% w/w was only achieved following the 2h of
incubation of Mag 500µg in 500µL ethanol.
4.2.1.4. Effect of loading time:
It is obvious from table 13 that the loading time had a significant effect on
Mag loading on GNPs (p˂0.05). Maximum loading amount of 210.2±0.33µg
106
corresponding to %AE of 42.04±0.33% w/w was obtained in F6 prepared using

500µL of 1mg/mL Mag in ethanol for 12h loading.
Studying the adsorption of drugs on the surface of GNPs, variable results were
reported in literature. Curry and co-workers found that the adsorption of doxorubicin
was fast and time independent (Curry et al., 2015). Conversely, Mane and
colleagues found that hydrophobic drugs, might require time to produce interfacial
tension with hydrophilic GNPs resulting into better adsorption after 12h (Mane et
al., 2016). This time dependence might be explained by the time taken by Mag to
replace the citrate ions on the surface of GNPs.
4.2.2. Spectral analysis of Mag-GNPs:
To have an insight onto the changes occurring during drug loading, spectral
analysis was performed on formulae F2, F4, F5 & F6 to investigate the change in
SPR as result of drug loading at various times and conditions. Upon loading Mag
onto citrate capped GNPs, a gradual color change from red to purple was seen in F2,
F4 & F5. Drug loading was manifested by a concentration dependent decrease in
intensity of the SPR band of spherical GNPs at 519nm and a slight shift in its λ max.
Shifts to 529, 533 and 536nm corresponding to 250, 500 and 1000µg Mag,
respectively are seen in figure 21. The observed shifts could be explained by the
change in the dielectric constant of the solution resulting from particles
agglomeration. The increase in the amount of the drug led to further agglomeration
(assembly) of the gold nanocrystals. When the interparticle distance in the assembly
decreases to less than about the average particle diameter, the electric dipole–dipole
interaction and coupling between the plasmons of neighboring particles in the
assembly causes bathochromic shift of the absorption band, called plasmon coupling
band (Mohamed et al., 2012). The bathochromic shift normally observed in UV-
107
visible spectra is an indication of an increase in the size of the particle or

agglomeration of NPs or both (Madhusudhan et al., 2014). Increasing the amount
of Mag had thus a significant effect on the degree of the assembly and the
nanocrystals agglomeration kinetics. Similarly, a shift of about 8nm in wavelength
was seen upon loading 5-FU on GNPs (Mohamed et al., 2012). In F6, where higher
amount of Mag was loaded (210µg) and expected larger PS, a band broadening is
obvious due to the dominate contributions from higher order electron oscillations
(Huang et al., 2010).
Plain GNPs
1.8
250ug Mag (2h), F4
1.6 500ug Mag (2h), F2

1000ug Mag (2h), F5
1.4 500ug Mag (12h), F6
1.2
1
Absorbance
0.8
0.6
0.4
0.2
0
400 500 600 700 800
Wavelength (nm)
Figure 21: UV-vis absorption spectra of plain and Mag loaded GNPs.
108
4.2.3. PS and Surface charge:

DLS was used to characterize the hydrodynamic size of selected GNPs
formula before and after Mag loading. The average hydrodynamic diameter of
citrate-GNPs significantly increased from 23.4±0.71 to 115.9±0.99nm after addition
of 500µg Mag (1mg/mL) on one mL GNPs colloidal dispersion for 12h as shown in
F6. An example of PS chart is shown in figure 22. Similar to the previously reported
data, DLS thus confirmed the GNPs aggregation into large nanoclusters of Mag-
GNPs (Li et al., 2014). When Mag was adsorbed onto the surface of the GNPs, the
negatively charged citrate anions, originally adsorbed on GNPs surfaces and
providing electrostatic stabilization, are replaced by Mag. This will result in GNPs
aggregation due to gold’s high density and strong van der Waals forces between
particles in solution. Further confirmation of this explanation was noticed by the ζ
value obtained with F6 (-35.0±1.29mV).
Figure 22: PS distribution plot of Mag-GNPs, F6, determined by DLS.
109
4.3. Immuno- Mag-GNPs

4.3.1. BSA modified Mag-GNPs:
BSA, a model protein in this work, was loaded on Mag-GNPs by direct

adsorption using three different BSA amounts: 250, 500 and 1000µg from BSA
stock solution (10mg/mL) in acetate buffer pH 4.5. The NPs were incubated with
the protein for 4h and the PS and ζ were monitored. As previously mentioned, due
to the reported interaction between Mag and BSA, the amount of free BSA could not
be determined and the results of NPs characterization are presented in table 14.
Table 14: Effect of BSA adsorption on the physical characteristics of Mag-

GNPs prepared with different BSA amounts.
Formula BSA (µg) PS (nm) PDI ζ (mV)
GNPs - 23.4±0.71 0.440±0.03 -57.20±2.24
F6 - 115.9±0.99 0.329±0.13 -35.00±1.29
F7 250 127.8±1.71 0.359±0.17 -27.31±1.56
F8 500 109.2±2.07 0.491±0.10 -21.20±2.14
F9 1000 99.8±1.96 0.321±0.09 -10.20±1.07
Formula F6 to F9 were prepared at a theoretical Mag loading of 5mg/mL of 1mM GNPs.
4.3.1.1. PS analysis:
It could be observed from table 14 that PS of BSA decorated Mag-GNPs in

F7 is significantly greater (127.8±0.71nm) in comparison to unmodified NPs, F6
with PS 115.9±0.99nm (p˂0.05). BSA adsorbed on Mag-GNPs led to increase in
hydrodynamic size. BSA was expected to interact with the particles surfaces by
many mechanisms: van der Waals forces, hydrogen bridges, hydrophobic interaction
or electrostatic attraction. At a pH 4.5, slightly lower than the protein Ip, BSA was
positively charged and so both partners became oppositely charged. Following
110
adsorption, the protein can be irreversibly immobilized by those forces or a

combination of them (Gole et al., 2001).
Further increase in amount of BSA added leads to significant decrease in PS

as shown in F8 and F9, table14. The interaction between weakly bound Mag on
GNPs leading to desorption of the former from GNPs surface could probably
account for such decrease in PS. This was in agreement with other investigators who
previously reported that 18% w/w of doxorubicin was desorbed in the presence of
0.7mM BSA over 50min (Curry et al., 2015). Such interaction with common blood-
borne constituents is of significant impact for in vivo drug delivery research.
Physiologically-relevant molecules like glutathione (GSH) and BSA were found to
rapidly desorb some drugs from the NPs surface at physiological concentrations
(Curry et al., 2015). An important point to consider for in vivo drug delivery and
controlled release studies.
4.3.1.2. Surface charge of modified GNPs:
The mean ζ of unmodified Mag-GNPs was −35.00±1.29mV. Addition of BSA

to NPs dispersion caused a concentration dependent decrease in the ζ to -27.31±1.56,
-21.20±2.14 and -10.20±1.07mV for F7, F8 and F9 modified with 250, 500 and
1000µg BSA, respectively (Table 14). The decrease indicated the effect of BSA on
GNPs surface and in accordance with previous works, the amount of BSA adsorbed
increased with increasing its initial added amount (Barua et al., 2013, Shubhra et
al., 2014).
Despite the simplicity of the Mag/GNPs interaction for drug loading, the
nanoconjugate remains vulnerable to the physiological conditions. Stabilization
methods such as polymer coating should be considered. Hence to prevent desorption
111
of Mag from surface of GNPs, attempts were done to protect Mag by encapsulation
in the biodegradable matrix PLGA.
4.3.2. PLGA encapsulated Mag and GNPs formula (Mag-GNPs/PLGA

NPs):
The interest in using the polymer-coated GNPs for the delivery of

chemotherapeutic has been increased in the recent years. Various biocompatible
polymers were used for coating GNPs for various purposes such as to improve the
stability of the NPs, increase the payloads, impart biocompatibility, prolong
systemic circulation and promote cellular uptake to utilize the GNPs as drug/nucleic
acid delivery system for cancer therapy (Muddineti et al., 2015). As previously
discussed in section 4.3.1, Mag-GNPs suffered from instability in biological fluid
due to easy desorption by BSA and GSH in serum and low payloads. Hence, coating
of Mag-GNPs by PLGA would provide a good stabilization method in biological
fluids and increase loading. Scarce work had been reported on the preparation of
PLGA encapsulated GNPs (GNPs/PLGA NPs) (Basu et al., 2012; Dixon et al.,
2014; Khandelia et al., 2014; Wang et al., 2014; Chauhan and Srivastava, 2015;
Wang et al., 2016c). Most of these works described the preparation of GNPs/PLGA
NPs using emulsion solvent evaporation method. Hence, preliminary trials had been
done to encapsulate the GNPs in PLGA using the emulsion solvent evaporation
technique by adding GNPs to the external aqueous phase in an o/w emulsion or as
internal phase in a w/o/w emulsion. However, a stable emulsion system was not
formed and instantaneous precipitation of black precipitate of GNPs was always
evident.
Mag-GNPs/PLGA NPs were prepared by nanoprecipitation method under the

optimized conditions previously reported in chapter one. First, the aqueous colloidal
112
GNPs solution was mixed with the aqueous phase. However, a black ppt indicating
the GNPs precipitation was seen.
Secondly, we attempted to mix colloidal GNPs with the organic phase

containing PLGA to provide better opportunity for GNPs encapsulation.
Concentration of GNPs aqueous colloidal solution using ultrafiltration through
nanoseps, allowed the use of a smaller aqueous volume containing the same amount
of GNPs, thus preventing PLGA and Mag precipitation. Increasing the volume of
GNPs (10Mm) from 25 to 50µL was accompanied with the appearance of black ppt
again, indicating the presence of excess free GNPs.
Table 15 shows the outcome of this study.
Table 15: Physical characteristics of Mag-GNPs/PLGA NPs.

GNPs GNPs
Formula GNPs PS ζ Mag EE
concentration placement PDI
(µL) (nm) (mV) (% w/w)
(mM)
Aqueous
P1 250 1 120.2±1.09 0.396±0.04 -23.28±1.25 88.66±0.85
Phase
P2 25 127.4±0.21 0.078±0.06 -23.80±2.83 87.15±2.42
Organic
10
P3 50 phase 129.3±1.92 0.122±0.04 -24.66±0.85 88.74±0.72
The organic phase was composed of 25mg PLGA and 5mg Mag dissolved in 2mL acetone. TPGS 30% w/w of polymer
was used as SAA. Organic to aqueous phase volume ratio is 1:1. All results are mean of three measurements±SD.
4.3.2.1. PS analysis:
From table 15, it is obvious that PS of formula P1 containing GNPs in the

aqueous phase was significantly (p˂0.05) smaller than the other formulae where
GNPs was added to the organic phase. Although the small PS (120.2±1.09nm) of
P1, it showed the largest PDI (0.396±0.041) indicating the instability of the system
manifested by the precipitation when GNPs were dispersed in the aqueous phase.
When GNPs were added to the organic phase, better PDI results were obtained as
113
observed in P2 & P3. Formula P2 containing 25µL GNPs (10mM) showed the
lowest PDI (0.078±0.006) as illustrated in figure 23.
Figure 23: PS distribution plot of Mag-GNPs/PLGA NPs containing 250µL

0.001M GNPs.
4.3.2.2. Surface charge of Mag-GNPs/PLGA NPs:
Formulae P1-P3 exhibited a ζ ranging from -24.66±0.85 to -23.28±1.25mV

(table 15). It is obvious that ζ of all the prepared Mag-GNPs/PLGA NPs is negative
and did not significantly differ among each other (p˃0.05). The negative value of ζ
was probably due to the presence of terminal carboxyl groups in the polymer (Feng
et al., 2016). It is worthy to mention that Mag-GNPs/PLGA NPs exhibited a lower
negative ζ than Mag-PLGA NPs prepared in chapter 1 and this might be due to the
adsorption of some GNPs on the surface of PLGA NPs.
4.3.2.3. Mag entrapment efficiency (EE):
All the prepared formulae showed high EE ~ 88% w/w similar to that obtained
with Mag-PLGA NPs without gold. It is obvious from table 15 that gold amount had
no significant effect on the %EE.
114
Formula P3 containing higher amount of GNPs showed black precipitate of

excess non coated GNPs. So they were excluded from further study and formula P2
was selected for further characterization.
4.3.2.4. Characterization of the selected NPs formula (P2):
4.3.2.4.1. Spectral analysis:
Plain GNPs show the SPR band ~520nm in the visible region. The SPR band
is affected by the PS. Increased PS due to the aggregation of GNPs after Mag loading
in Mag-GNPs red shifts the SPR wavelength to 600nm (figure 24). Scanning of P2
(Mag-GNPs/PLGA NPs) revealed a shift of ~80nm in the ʎmax accompanied with
broadening in the SPR starting from 500 to 670nm compared to blank GNPs and
Mag-GNPs as shown in figure 24. A similar shift in the absorption peak and change
in the scattering spectrum of a GNPs/PLGA NPs aqueous solution was reported in
previous work. This change is due to the clustering of GNPs into spherical polymeric
nanoconstructs (Iodice et al., 2016). Literature reported that the band broadening
was obvious in particles larger than 100nm due to the dominate contributions from
higher order electron oscillations (Huang et al., 2010).
1.8
Plain GNPs
1.6
Mag-GNPs
1.4
Mag-GNPs/PLGA NPs
1.2
Absorbance
1
0.8
0.6
0.4
0.2
0
400 500 600 700 800
Wavelength (cm-1)
Figure 24: UV-Vis spectra of various formulations of GNPs.
115
4.3.2.4.2 TEM-EDX:
EDX is an x-ray technique used to identify the elemental composition

of materials and estimate their proportion at different positions. All elements from
atomic number 4 to 92 could be detected (Joshi et al., 2008). EDX systems are
attached to TEM instruments where the imaging capability of the microscope
identifies the specimen of interest. The data generated by EDX analysis consists of
spectra showing peaks corresponding to the elements making up the true
composition of the sample being analyzed.
TEM-EDX was conducted to confirm encapsulation of GNPs inside PLGA.

TEM examination of NPs revealed nanoscale particles (~100 nm) encapsulating
GNPs as shown in figure 25. The tabulated EDX analysis results provide a semi-
quantitative view of the elemental composition in the inspection field in units of both
weight percent and atomic percent. Table 16 and figure 26 reveal that O and Au are
the main elements present within the inspection field and so confirm the presence
and encapsulation of gold in the prepared sample. The %C was omitted as the
copper grid used in EDX analysis was carbon coated.
a. b.
Figure 25: TEM micrographs of Mag-GNPs/PLGA NPs at different

magnifications.
116
Table 16: Percent of O and Au elements in EDX of Mag-GNPs/PLGA NPs.
O: oxygen, Au: gold, K: K level of the X-ray emission lines for oxygen, M: M level of the
X-ray emission lines for gold.
Figure 26: EDX of Mag-GNPs/PLGA NPs.
4.3.2.4.3. FT-IR analysis:
The FT-IR spectrum of plain GNPs, figure 27, depicts characteristic bands of
citrate at 3527.83 and 1635.62cm-1. The first was assigned to the stretching vibration
of OH group and the second was indicative of C=O stretching of carboxylate ions of
citrate (Kiroula, 2016).
The spectrum of Mag-GNPs/PLGA NPs shows, in addition to the previously

explained features, in chapter 1, a broadened peak at about 3400-3500 cm-1
117
corresponding probably to the citrate OH group. The C=O peak at 1635cm-1 was
probably also merged with the Mag-PLGA peak at 1638cm-1 forming one broad peak
with lower intensity compared to the plain GNPs, (figure 28). This confirms the
presence of gold in Mag-GNPs/PLGA NPs.
Figure 27: FT-IR spectrum of Plain GNPs colloidal dispersion.
120
Mag-GNPs/PLGA NPs
Mag-GNPs/PLGA Mag-PLGA NPs
100
1638.8
1750.8
2921.7
80
Transmittance (%)
60
1639.5
40
1759.1
3488.32
2998
2950
20
0
4000 3500 3000 2500 2000 1500 1000 500 0
Wavenumbers (cm-1)
Figure 28: FT-IR spectra of Mag-PLGA NPs and Mag-GNPs/PLGA NPs.
118
4.3.2.4.4. In vitro drug release:
The in-vitro release of Mag from PLGA NPs based formulations was studied
and the results were compared to the dissolution of Mag powder. The obtained
profiles are displayed in table 17 and illustrated in figure 29. Mag-GNPs showed
much faster Mag release compared to Mag powder. The release pattern of
conjugated system showed an initial burst release for the first hour, and then a
sustained release up to 24h. The loosely bound drug might release fast at the initial
stage followed by its sustained release. The adsorption of Mag on GNPs allowed
greater than a two-fold increase in the rate of drug release over the free drug during
24h. Literature showed that the attachment to GNPs became a general strategy for
solubilizing and enhancing a wide variety of therapeutic agents in aqueous media
(Zhang et al., 2011; Dreaden et al., 2012).
In contrast to pure Mag which showed 97% w/w release within 48h, the PLGA
NPs successfully sustained the drug release suggesting the potential of the PLGA
NPs as a sustained drug delivery system.
Table 17: Cumulative percent of Mag released from Mag powder, Mag-GNPs,
Mag-PLGA NPs and Mag-GNPs/PLGA NPs.
Time Cumulative drug released (% w/w)
(h) Mag powder Mag-GNPs (F6) Mag-PLGA NPs (F14) Mag-GNPs/PLGA NPs (P2)
1 9.50±2.42 32.98±2.12 3.87±2.47 0±0.47
2 12.62±1.61 37.00±1.94 6.52±1.94 1.76±0.37
4 15.25±1.88 47.31±2.45 9.39±1.60 5.19±1.49
6 18.33±2.27 52.73±1.88 10.74±2.03 5.95±1.86
24 35.33±0.97 80.32±0.13 39.64±2.46 47.00±1.93
48 97.23±2.16 98.56±2.15 54.70±1.60 50.24±2.41
All results are expressed as mean of 3 determinations±SD.
119
100
90
80
Cumulative Mag released (% w/w)
70
60
50
40
30
Mag powder
20
Mag-PLGA NPs
Mag-GNPs
10
Mag-GNPs/PLGA NPs
0
0 10 20 30 40 50
Time (h)
Figure 29: Release profiles of Mag from various formulations in PBS (pH 7.4)
containing 0.5% (w/v) SDS at 37ºC.
4.3.3. TZB modified PLGA encapsulated Mag and GNPs

formula (Mag-GNPs/PLGA-TZB NPs):
TZB was used for active targeting of Mag-GNPs/PLGA NPs to breast cancer
cells. Mag-GNPs/PLGA NPs were conjugated to TZB by covalent method as
previously described in chapter 1 section 3.3.3 using the selected Ab/NPs ratio. NPs
prepared were characterized for PS, PDI, surface charge measured by ζ, FT-IR,
TEM, %EE of Mag and % conjugation efficiency (CE) of TZB. Characterization
results are illustrated in table 18.
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Table 18: Characterization of Mag-GNPs/PLGA NPs before and after covalent

modification with TZB.
Formula TZB:NPs PS Ζ % Mag EE % TZB CE
PDI
code* ratio (nm) (mV) (w/w) (w/w)
P2 - 127.4±0.21 0.078±0.066 -23.80±2.83 86.85±2.53 -

P4 0.1:1 136.1±1.27 0.109±0.009 -8.15±0.95 81.4±1.82 66.8±3.73
Results are expressed as mean of three determinations±SD, Mag was used at a theoretical Mag loading of 5mg. *P2
denotes the unmodified particles while P4 denotes TZB modified particles.
4.3.3.1. PS and PDI:
DLS was used to determine the average hydrodynamic diameters of particles.

The size of Mag-GNPs/PLGA NPs (P2) and Mag-GNPs/PLGA-TZB NPs (P4) were
found to be 127.4±0.21 and 136.1±1.27nm, respectively (Figure 30). The increase
in NPs size in immuno- NPs when compared to unconjugated NPs confirmed the
conjugation of TZB to the surface of NPs (Vivek et al., 2014).
Figure 30: PS distribution plot of Mag-GNPs/PLGA-TZB NPs obtained by

zeta sizer.
121
4.3.3.2. Surface charge of Mag-GNPs/PLGA-TZB NPs:
For mAb-targeted NPs (P4), ζ was found to be −8.15±0.95mV as shown in

table 20. This result shows that the absolute value of ζ of the NPs is lower than that
previously reported for non modified Mag-GNPs/PLGA NPs (P2)
(−23.80±2.83mV). The decrease in absolute value of ζ may be due to screening of
the negative charge because of the antibody conjugated to the NPs surface. These
results were in agreement with previously reported results in chapter 1 section 4.3.
Although the small ζ is an indication of poor physical stability but neutral and
slightly negatively charged NPs were proved in literature to escape opsonization and
to have longer circulation lifetimes and less accumulation in the aforementioned
organs of the MPS (Blanco et al., 2015). Also studies have shown that charged NPs
are more cytotoxic than neutral charged NPs (Misra et al., 2014). To overcome their
poor stability, the prepared NPs should be lyophilized and reconstituted immediately
before administration (Jahan and Haddadi, 2015).
4.3.3.3. Drug entrapment efficiency:
In unmodified NPs (P2), the %EE of Mag was 86.85±2.53% w/w as seen in
table 18. In TZB modified NPs (P4), the EE significantly decreased (p˂0.05) to
81.4±1.82 % w/w. This decrease may be due to loss or release of drug during the
conjugation process. These results were in accordance with previously published
studies (Kulhari et al., 2016).
4.3.3.4. Conjugation efficiency of TZB to the surface of Mag-

GNPs/PLGA NPs:
The amount of TZB on NPs surface was evaluated with the micro BCA
protein assay kit. From table 18, It is obvious that 66.8±3.73% w/w of the mAb
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molecules were attached to PLGA encapsulated GNPs. This result was in accordance
with the Mag PLGA-TZB NPs prepared in chapter 1. The presence of gold has no
effect again on surface modification. This confirmed that GNPs were encapsulated
inside PLGA NPs.
4.3.3.5. FT-IR analysis:
FT-IR was done to confirm conjugation of TZB to the surface of PLGA NPs
in formula P4. In the present study, the FT-IR spectra of unmodified Mag-
GNPs/PLGA NPs and immuno TZB conjugated NPs are illustrated in figure 31.
The FT-IR spectrum of free TZB sample showed the characteristic peaks at
3398.93, 3293.1, 1644.68 and 1538.61cm−1 corresponding to carboxylic acid O-H
stretching, overlapped amine and amide N-H stretching, amide I and amide II peaks
of the peptide backbone (Mukherjee et al., 2008; Derman et al., 2015).
Mag-GNPs/PLGA NPs showed the characteristic bands of PLGA and GNPs

at 3488.32, 1759 and 1637.59cm-1 as explained previously in section 4.3.2.4.
The change in the FT-IR spectrum of Mag-GNPs/PLGA-TZB NPs was found

to be similar to Mag PLGA-TZB NPs previously reported in chapter 1 section 4.4.
A 65cm-1 shift in the PLGA carboxylic acid OH stretching peak from 3488 to
3423cm-1 is obvious in the conjugated NPs compared to unconjugated NPs,
respectively (figure 31), evidencing the interaction between TZB and PLGA.
Furthermore, the finger print region in TZB modified NPs shows clearly the
presence of TZB on the NPs surface.
123
TZB
TZB
250
Mag-GNPs/PLGA
PLGA encapsulatedNPs
Mag-GNPs
Mag-GNPs/PLGA-TZB NPs Mag-GNPs
Immuno-PLGA encapsulated
200
2999.36
1497.75
1639.52
2951.14
150
3423.71
1760.07
100
1637.59
50
3488.32
1759.11
1496.79
2998.40
2950.17
-50
Transmittance (%)
-100
2907.66
2943.88
1538.61
-150
3293.11
3398.93
1644.68
-200
4000 3500 3000 2500 2000 1500 1000 500 0
Wavenumbers (cm-1)
Figure 31: FT-IR spectra of free TZB, Mag-GNPs/PLGA NPs and Mag-
GNPs/PLGA-TZB NPs.
124
4.3.3.6. TEM:
TEM micrographs of TZB modified NPs (P4) showed that the NPs were
nearly spherical and homogeneous in size (Figure 32). The presence of GNPs as
electron rich darker spheres can also be seen inside the PLGA matrix. Also the mAb-
conjugated NPs were larger in size (140nm) as shown in figure 32 compared to
unmodified NPs (∼105nm) (figure 25). Conjugation may be a reason for the larger
size of the targeted nanoparticles (Koopaei et al., 2011).
Figure 32: TEM micrographs of Mag-GNPs/PLGA-TZB NPs.
4.3.3.7. Stability study of the NPs in serum:
The stability in serum was evaluated for 24h for the time of persistence in the
blood stream. The aggregation and the dimensional growth produced by deposition
of serum proteins on the particles surface were assessed by the change in NPs
dimension monitored by DLS (Ruozi et al., 2015).
Mag-GNPs/PLGA-TZB NPs did not show any significant change in PS as

shown in table 19 excluding aggregation and confirming NPs stability.
125
Table 19: Stability study results of Mag-GNPs/PLGA-TZB NPs.

Incubation time (h) PS (nm)
0 136.1±1.27
1 141.4±5.83
2 137.1±2.62
24 144.0±6.81
To sum up, in this chapter, multifunctional polymeric NPs, composed of

chemotherapeutic agents (therapeutic mAb and anticancer drug) were fabricated for
systemic chemotherapy, and GNPs for localized imaging and photothermal
treatment. Spectral analysis and TEM-EDX confirmed the presence of GNPs in the
PLGA NPs. The incorporation of GNPs into the PLGA NPs increased Mag payload
and provided sustained release of the drug compared to the uncoated Mag-GNPs.
Moreover, the mAb TZB was successfully conjugated by covalent binding to the
surface of PLGA NPs enhancing efficacy through active targeting of cancer tissues
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Conclusions
From the previous findings, the following could be concluded:
1. Citrate GNPs were successfully prepared using Turkevich method. The particles
were mostly spherical with average PS ~20nm and ζ was -57.20±2.24mV
indicating good NPs stability.
2. Mag-GNPs prepared by adsorption method using 500µL of 1mg/mL Mag in

ethanol for 12h showed maximum %AE of 42.04±0.33% w/w.
3. Mag-GNPs exhibited a drug dissolution rate greater than a two-fold that of the
free drug.
4. Mag-GNPs/PLGA NPs formula with organic phase composed of 25µL of

concentrated GNPs solution (10mM), 25mg PLGA, 7.5mg TPGS and 5mg Mag
dissolved in 2ml acetone was considered the best formula based on PS and PDI
(formula P2).
5. The encapsulation of gold inside PLGA was confirmed by TEM-EDX and FT-
IR analysis.
6. Similar to Mag-PLGA NPs, Mag-GNPs/PLGA NPs showed sustained drug

release.
7. TZB conjugation was confirmed by FT-IR, increase in PS, decrease in ζ and

%CE of TZB. An increase of ∼8nm in the hydrodynamic diameter of Mag-
GNPs/PLGA NPs was obtained after addition of TZB. The negative value of ζ
of the modified NPs (−8.15±0.95mV) was lower than that previously reported
for unmodified Mag-GNPs/PLGA NPs (−23.80±2.83mV).
127
8. The %TZB conjugated to Mag-GNPs/PLGA NPs measured by BCA protein

assay was found to be 66.8±3.73% w/w, which is correlated to the results of
PLGA NPs without gold prepared in chapter 1. Gold did not affect the extent
of conjugation.
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Introduction
New developed drug delivery systems (DDS) for cancer therapy should be
monitored to ensure biological safety and activity prior to patient administration. For
these testing purposes, cell cultures are considered an exceptionally powerful, rapid
and economic tool, that avoids ethical and legal issues associated with animal
handling in comparison to in vivo models (Costa et al., 2013).
Many in vitro cell-based assays have been developed to rapidly determine the
cytotoxic activity and potency of new anticancer formulations in human cancer cell
lines (Florento et al., 2012). The lactate dehydrogenase leakage assay (LDH),
protein assay, neutral red and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide assay (MTT) are the most common employed cytotoxicity assays (Fotakis
and Timbrell, 2006). The LDH leakage assay is based on the measurement of LDH
activity. The decrease in intracellular LDH is an indication of irreversible cell death
due to cell membrane damage. This test provides reliable, rapid and simple
evaluation of cell viability (Fotakis and Timbrell, 2006). The neutral red assay
depends on the uptake of a neutral red dye only by viable cells. So a decrease in the
number of viable cells will be accompanied with a change in the amount of dye
incorporated in the cells (Fotakis and Timbrell, 2006). The protein assay is an
indirect measurement of cell viability, that measures the protein content of viable
cells left after washing the plates (Fotakis and Timbrell, 2006).The MTT assay is
considered the gold standard of cytotoxicity assays due to its high sensitivity and
high-through output (96-well plates) and hence has been widely adopted in a large
number of studies ( Niles et al., 2008; Van Tonder et al., 2015 ). MTT is a yellow
water soluble tetrazolium salt, which is converted to an insoluble purple formazan
129
by cleavage of the tetrazolium ring by succinate dehydrogenase within the

mitochondria as shown in figure XVIII (Zhang, 2014).
Figure XVIII: Reduction of MTT in the presence of NADPH.
The MTT assay depends on the ability of living cells to reduce a water-soluble
yellow dye, MTT, to a purple colored water-insoluble formazan product by
mitochondrial enzyme succinate dehydrogenase. The purple colored formazan
product possesses an absorbance maximum near 570nm. The formazan product is
impermeable to the cell membranes and accumulates in healthy cells. When cells
die, they lose the ability to convert MTT into formazan and so the increase or
decrease in the number of viable cells is linearly related to mitochondrial activity
(Van Meerloo et al., 2011; Riss et al., 2016). The MTT assay is suitable for the
measurement of drug 50% inhibitory concentration (IC50) which is determined by
plotting the concentration response curve between log of drug concentration and
percentage cell viability. The IC50 is the concentration of drug at 50% position on Y
axis (Denizot and Lang, 1986). The amount of formazan produced is dependent on
several parameters such as the concentration of MTT, the incubation period, the
number of viable cells and their metabolic activity.
130
The cytotoxicity of cells incubated with Mag were determined in previous

studies by both LDH and the colorimetric MTT assay. Many studies showed that
Mag induces G1 phase arrest in cultured colon and induces cell apoptosis in liver
cancer cells (Li et al., 2007). Kim and co-workers showed that Mag induced
apoptosis in endothelial cells by the generation of reactive oxygen species (ROS) by
mitochondria and the activation of cleaved caspase-3 (Kim et al., 2013). Both MTT
and LDH assay confirmed that low Mag concentrations (≤25 μM) did not inhibit the
growth of the endothelial cells, but concentrations of Mag >50 μM did. Yang and
colleagues, using tryptan blue assay, found that Mag inhibited proliferation of
human lung squamous carcinoma CH27 cells at low concentrations (10–40 μM), and
induced apoptosis at high concentrations (80–100 μM) (Yang et al., 2003). Although
most cytotoxicity studies for NPs are conducted in vitro, they are limited in their
ability to mimic the complexity of the in vivo environment (Adjei et al., 2014).
The enhancement in cytotoxicity by the use of drugs loaded in NPs delivery

system instead of free chemotherapeutic drugs has been shown to be promising in
cancer chemotherapy due to higher cell internalization compared to micron size
particles (Suh et al., 1998). NPs-cell interaction and internalization depend on
physical characteristics of NPs: shape, size, surface charge and the presence of cell-
penetrating targeting ligands on the NPs surface, as well as cell-membrane
properties: membrane fluidity, receptor type, receptor density, and recycling rate
of receptors (Nel et al., 2009).
Passive targeting facilitates NPs accumulation and their efficient localization

preferentially in the tumor tissues as a result of EPR phenomenon but cannot further
promote their uptake by cancer cells. NPs uptake is achieved by actively targeting
NPs to receptors overexpressed on target cancer cells as previously discussed.
131
NPs can enter cells via phagocytotic or non-phagocytotic pathways (clathrin-

mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, other
endocytotic pathways). Phagocytosis is generally undesirable pathway as the drug
taken up in this way is usually eliminated from the body. To avoid this internalization
mechanism, the particles should be small with hydrophilic surface. However, the
most successful approach is to attach ligands to the NPs surface, directing the uptake
through pinocytosis mechanisms (Plajnsek et al., 2012). Functionalization of drug
loaded NPs can be achieved by small molecule ligands as folic acid and thiamine,
peptides as aspartic acid, glycine and arginine, sugars as galactose, aptamers,
proteins as transferrin and antibodies such as anti-HER2. As human epidermal
growth factor receptor (HER2) is overexpressed over 20-30% of invasive breast
cancer, so NPs functionalization with TZB is considered one of the most promising
strategies for targeting breast cancer cells and was widely applied in research studies.
Previous studies proved the efficiency of HER2 positive breast cancer cell targeting
and uptake by active targeting using TZB. TZB conjugated antisense
oligonucleotides NPs showed a specific targeting to HER2-overexpressing cells
with cellular uptake by receptor-mediated endocytosis into HER2-positive BT-474
cells (Spänkuch et al., 2008). Others reported the efficient targeting and
internalization of TZB functionalized docetaxel PLA-TPGS/TPGS-COOH by SK-
BR3 overexpressing HER2 breast cancer cells (Sun and Feng, 2009). Herceptin
targeting was found to enhance uptake of camptothecin/mucic acid containing
polymeric NPs by BT-474 breast cancer cell lines as well as to increase tumor
efficacy (Han and Davis, 2013). Aydın, 2013 showed that the HER functionalized
salinomycin encapsulated PLGA NPs were successfully uptaken by MCF7 cells.
The cell uptake of functionalized nanocarriers is very important in order to

address their selectivity and biological activity to cancer cells. It is therefore
132
desirable to study the localization of NPs within cells and organs. Several techniques
were used to monitor the cellular uptake of NPs as confocal laser scanning
microscopy (CLSM) and flow cytometry (Ducat et al., 2011; Adjei et al., 2014).
CLSM allows the acquisition of images at high resolution from selected depths using
the process of optical sectioning (Mach et al., 2010; Paddock, 2000). CLSM was
widely used to study the cellular uptake and the intracellular location of NPs. CLSM
coupled with Z-stacking became a powerful tool for tracking intracellular
localization of NPs. Z-stacking is a digital image processing method which combines
several images captured at several focal distances (Wallrabe and Barroso, 2005).
The quality of the images of CLSM shows higher resolution, improved signal to
noise image, reduced background fluorescence and information from multiple
depths in the specimen is not superimposed, and is restricted to a well-defined plane
compared to conventional optical or fluorescence microscopy (Ducat et al., 2011;
Zhang and Monteiro-Riviere, 2013). NPs should be labeled with fluorescent dyes
to follow their internalization. Fluorescent dye can be labeled on the NPs surface or
encapsulated inside NPs (Gao et al., 2009). Dyes are also used to label intracellular
structures to properly localize NPs as the 4',6-diamidino-2-phenylindole (DAPI)
which is used to stain the nuclei (Tsai et al., 2008). CLSM is used to localize rather
than to compare and quantify NPs that can be achieved with flow cytometry. Wang
et al., 2011 showed that Fluorescein isothiocyanate (FITC) labeled Mag loaded
chitosan NPs were internalized by vascular smooth muscles within 2h of incubation.
In Wartlick et al., 2004, CLSM demonstrated an effective internalization of the NPs
by HER2-overexpressing cells via receptor-mediated endocytosis. Another study
using CLSM showed that uncoated nanospheres exhibited higher uptake in BT-474
cells compared to nanorods and nanodisks after 2h incubation, implying that
nanosphere is a favorable shape for cellular entry and that uptake increased after
coating with TZB (Barua et al., 2013).
133
Flow cytometry is a technique for counting and sorting heterogeneous

populations of cells suspended in a stream of fluid but it does not provide the
localization of NPs within the cell (Mach et al., 2010).
Photo-induced cell killing including photodynamic therapy (PDT) and

photothermal therapy (PTT) is a non-invasive therapeutic strategy. PDT involves
photosensitizer drugs and external light. The activation of photosensitizer drugs with
specific wavelengths of light leads to energy transfer to oxygen molecules or other
substrates in the surrounding areas. The generated cytotoxic reactive oxygen species
(ROS) can trigger apoptotic and necrotic cell death (Hong et al., 2016). The majority
of photosensitizer drugs are hydrophobic with poor solubility in water. When
aggregated under physiological conditions, they lower the quantum yields of ROS
production. Also their accumulation selectivily at target tissues remains insufficient
for successful clinical use (Hong et al., 2016). GNPs enhance the solubility of
photosensitizer drugs in water and thus increase their cellular uptake. When loaded
onto GNPs, they can achieve passive targeting to tumor by EPR effect. Also surface
modification of GNPs with targeting moieties, such as antibodies, peptides, and
aptamers increases cell-specificity, improves bioavailability of photosensitizer drugs
and reduces their undesirable side effects to surrounding health tissues (Hong et al.,
2016; Calavia et al., 2018). Gold by itself is an ideal photosensitizer due to its high
extinction coefficient which is orders of magnitude higher than that of strongly
absorbing organic dyes (Coomassie blue) (Huang and El-Sayed, 2010).
Photothermal therapy (PTT) is a promising strategy for ablation of the

localized malignant tissues that cannot be removed by surgical resection (Huang
and El-Sayed, 2011). It is a noninvasive therapy that aims to replace the problems
of thermal therapy. Different energy sources were employed in thermal therapy as
microwaves, ultrasound and laser light. All of them have a common limitation that
134
the heating is non-specific and damage is also affecting surrounding normal tissue.
PTT employs the heat generated from the absorbed optical energy by light-
absorbing agents accumulated in the tumor to ablate cancer cells and so achieve
more controlled and selective heating of the target area, thereby confining thermal
damage to the tumor (Iodice et al., 2016). The absorbed light is converted into heat
through a series of photo-physical processes. Then the heat is dissipated from the
particles into the surrounding environment by photon-photon relaxation to heat up
the area surrounding the NPs. When the NPs are internalized into cancer cells, the
heat can destroy them depending on the amount of heat generated by the hot NPs
(Huang and El-Sayed, 2011). For photothermal agents to be effective, they need to
have an enhanced light absorption and efficient light-to heat conversions (Thakor
and Gambhir, 2013). Traditional used photothermal agents as natural chromophores
and external dyes (indocyanine green) suffer from low absorption, and rapid
photobleaching, thus they are not widely used (Huang and El-Sayed, 2011; Thakor
and Gambhir, 2013).
With the development of nanotechnology, metal NPs in particular GNPs was

found to be an ideal agent for enhancing laser-based ablation therapies mostly
because of their tunable optical properties and surface plasmon resonance with their
multiple targeting strategies and accumulation in tumor cells (Iodice et al., 2016;
Riley and Day, 2017). GNPs had been specifically delivered to cancer cells by both
passive and active targeting due to ease of functionalization of the NPs surface with
tumor targeting moieties. In addition, GNPs showed superior light absorption
efficiency over conventional dye molecules. Upon light irradiation with frequency
overlapping the nanoparticle SPR absorption band, strong surface fields are induced
due to the coherent excitation of the electrons in the nanogold followed by electron
relaxation producing strong localized hyperthermia capable of destroying
135
surrounding targeted cancer cells. The photothermal therapy (PTT) induced by

plasmonic GNPs is called plasmonic photothermal therapy (PPTT) (Huang and El-
Sayed, 2011; Yao et al., 2016; AL-Jawad et al., 2018). The localized surface
plasmon resonance (LSPR) peaks of GNPs strongly depend on PS, shape, surface,
and aggregation state. Depending on the LSPR wavelength, GNPs can absorb and
scatter light either in the visible region using spherical GNPs or in the near infrared
region (NIR) as gold nanorods, nanoshells and nanocages (Li and Gu, 2010). By
changing the structure and shape, the LSPR frequency of GNPs can be tuned to NIR
region. For in vivo applications, NIR lasers are favorable over visible light in PTT
as they penetrate deeper into fluids and tissues due to the optical window in the NIR,
where hemoglobin, melanin and water NIR absorption is reduced. Visible light was
applied only to superficial epithelial cells (Huang and El-Sayed, 2011). It was
recently found that small spherical GNPs, which do not absorb light in the NIR,
become aggregated within tumors into nanoclusters, resulting in increased local
absorption and red-shifting and produce hyperthermia when exposed to NIR
(Zharov et al., 2005). To promote NPs aggregation in tumors, GNPs should be
specifically targeted using antibodies and rapidly internalized by receptor mediated
endocytosis into tumor cell endosomes and lysosomes characterized by the acidic
medium and the presence of enzymes. The protonation of the carboxyl groups at the
low pH of endosomes/lysosomes and the lysosomal degradation of the targeting
antibody induce particle aggregation. Tumors thus act as catalysts for GNPs
aggregation. This is called “bio-nano amplification” strategy (Hainfeld et al.,
2014).
Laser light and light emitting diodes (LED) were commonly used in PTT.
Although laser is monochromatic coherent and provide a narrow beam of high
intensity light photons with minimal power loss and great precision, laser is available
136
for certain wavelengths only: Nd: YAG 532nm and Ti: Sapphire laser 800nm. Also,
laser heating with high power can result in thermal cell vaporization and production
of shock waves in cells so local temperature control is requested. On the other hand,
LED are monochromatic non-coherent light source which is easily portable,
economically feasible and suitable for in vivo applications for deep tumors via
coupling with optical fibers. As the coherence of light was found to be non-important
in low-power laser so LED was found to be a successful alternate for laser in PTT
(Gananathan, 2016).
PTT induced cell death via apoptosis or necrosis depending on both the light
dosage, type, irradiation time and the subcellular location of the GNPs. The laser
energy required for cell ablation is ten times lower when the NPs are internalized
inside the cytoplasm than those located on cytoplasm membrane (Huang and El-
Sayed, 2011).
The efficiency of PTT was widely studied in previous researches. El Sayed

and his team showed that the energy needed for irradiation of two oral squamous
carcinoma cell lines (HSC 313 and HOC 3 Clone 8) incubated with anti-epithelial
growth factor receptor (EGFR) antibody conjugated GNPs using continuous visible
argon ion laser at 514nm was found to be less than half the laser energy required to
kill the benign cells (HaCaT) under same conditions (El-Sayed et al., 2006).
Gananathan, 2016 hypothesized that PPTT leads to apoptosis using green LED of
30mW with 30nm GNPs. Huang, in 2006, demonstrated the efficiency of in vitro
PTT when selectively delivered gold nanospheres to oral squamous carcinoma cells
overexpressing EGFR were irradiated near their plasmon resonance absorption
(530nm) (Huang, 2006). Others evaluated tumor killing efficacy of metallic GNPs
(15nm) in comparison to 5-FU on MCF-7 cells after irradiation with green LED
(Gomaa et al., 2015). The encapsulation of small GNPs into larger spherical
137
nanoconstructs made out of a PLGA core stabilized by a superficial lipid-PEG

monolayer enhanced photothermal ablation and could favor tumor accumulation
(Iodice et al., 2016). The photothermal enhancement of chemotherapeutic
doxorubicin in breast cancer by visible irradiation of GNPs was recently reported by
Mendes et al., 2017. Although the combination of therapeutic approaches
(photothermal ablation and chemotherapy) against cancer has become a crucial
step to improve treatment efficacy, very few studies were found.
The aim of this chapter was to investigate the in vitro cytotoxic effect of
TZB modified PLGA encapsulated Mag and GNPs (Mag-GNPs/PLGA-TZB NPs)
on MCF-7 breast cancer cell lines along with the synergistic effect of the use of
PTT. Also, the cell uptake and targeting efficiency of the NPs will be also studied
to confirm the successful NPs cell internalization.
138
Experimental
1. Materials:
• 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, (MTT):
Sigma, Poole, Dorset, UK.
• Citifluor PVP plus antifade: Citifluor Ltd, UK.
• Paraformaldehyde 16% aqueous solution: Alfa Caesar, UK.
• Phosphate buffered saline tablets (PBS): Oxoid, Basingstoke, UK.
• Penicillin G sodium (sterile) and Streptomycin sulfate (sterile): Sigma, Poole,

Dorset, UK.
• Diamidino-2-phenylindole dihydrochloride (DAPI) for nuclear staining,

dimethyl Sulfoxide (DMSO), Nile red and trypsin-EDTA: Sigma-Aldrich,
UK.
• Roswell Park Memorial Institute medium (RPMI-1640), phenol red free

RPMI-1640 and fetal bovine serum (FBS): GIBCOTM, UK.
• Other materials used in this work are previously described in chapter 1 and 2.
2. Equipment:
• Balance, digital: Sartorius AY123, USA.
• Centrifuge: Beckman, UK.
• Confocal light scanning microscope equipped with an argon/krypton laser:

Zeiss LSM 510 Meta, Germany.
• Haemocytometer 0.1 mm deep chamber: Pierce, USA.
139
• Humid CO2 incubator (37°C, 95% RH, 5% CO2): Heraeus, Germany.

• Inverted microscope: Olympia, Germany.
• Monochromatic Light emitting diode sources (LED) with 530nm and
650nm peak wavelength, 200mW power: National institute of laser, Egypt
• Microplate Reader: Wallac Victor 1420 multilabel counter, UK.
• Multipoint pipette with 8 channel aspiration manifold 20-200μL: Eppendorf,
UK.
• Multi-well tissue culture plates: 24 well plates: Costar, USA.
• Sterile T- flasks, 25 and 75cm2 and sterile transfer pipettes 5, 10mL: Greiner
Bio-One GmbH, Germany.
• Cell Culture Clusters (96 well) and round cover slides: Corning, USA.
3. Methodology:
3.1. Cytotoxicity evaluation by MTT assay

Cytotoxicity of each of the selected formulae (F14, F19, P2 and P4) along
with plain PLGA NPs and the free drug as well as TZB were evaluated on the breast
cancer cells (MCF-7) using MTT assay.
Breast cancer cells (MCF-7) were maintained in Roswell Park Memorial

Institute medium (RPMI-1640) supplemented with 10% fetal bovine serum (FBS)
and streptomycin/penicillin antibiotics (100µg/mL) in humidified air atmosphere
(5% CO2, 95% RH, 37˚C). Cultivated cells were visualized using an inverted
microscope to detect any cross contamination or visible microbial contamination.
The cells were sub-cultured every 2 days and split at ratio of 1:6 to 1:3. Cells between
15 through to 18 passages were used for all the experiments.
140
In short, the MTT assay experiment was conducted as follows: the cells at
75% confluency, were removed from the flask by trypsinization (trypsin-EDTA
solution) and were counted under the microscope using a haemocytometer and
properly diluted with complete growth medium. The cells were seeded in 96-well
plates at a density of 5x103 cells/well in 200µL of the growth medium. Cells were
permitted to adhere for 24h till confluency in a 5% CO2 incubator. Stock solution of
Mag in dimethyl sulfoxide (DMSO), was diluted with serum free RPMI-1640 to
achieve concentrations ranging from 100 to 0.39µg/mL. Control cells treated with
the same concentration of DMSO in serum free RPMI was also done. Starting with
a drug concentration of 100µg/mL, an amount of particles containing the equivalent
amount of drug or an equivalent weight from blank particles were also prepared in
serum free RPMI. Serial dilutions of NPs were prepared by diluting the stock with
serum free culture medium. The culture medium from each well was aspirated,
replaced by 200µL of the prepared serial dilutions of NPs using multichannel pipette
followed by incubation for 24h at 37˚C. The culture supernatant was removed, and
the cells were washed with sterile PBS, then 200µL of fresh culture medium were
dispensed in each well and left for 24h incubation to stabilize the cells. The medium
was then aspirated and replaced with fresh medium. Then, 10µL of MTT solution
(5mg/mL in PBS, pH 7.4) were added on the cells in each well, incubated at 37˚C
for 4h. At the end of incubation, the MTT solution was removed and then 200µL of
DMSO were added to each well. The cells were incubated with DMSO for 15min at
37˚C to solubilize the formed formazan crystals. The absorbance was detected at
570nm using a microplate reader. Figure 33 shows the experimental set up and the
equipment used.
A control of untreated cells was made in the absence of test compound.

Untreated cells were used as negative control. The results were determined by three
141
independent experiments performed in triplicate (n=9). The percentage of the

viability was calculated according to the following equation.
% cell viability = [A(test)/A(negative control)] x 100 Eq (5)
Where A(test) is the absorbance obtained for each of the concentrations of the
test substance, A(negative control) is the absorbance obtained for untreated cells.
The later reading was assumed to correspond to 100% cell viability.
A one-way ANOVA analysis followed by Dunnett's post test was performed

using the Graph Pad Instat Anova software. Significant differences were considered
at p≤0.05.
IC50 was defined by the concentration that caused a 50% absorbance decrease
of drug-treated cells compared with untreated cells (Qiu et al., 2016). The IC50 was
calculated according to the equation for Boltzman sigmoidal concentration–response
curve using the nonlinear regression fitting models (Graph Pad, Prism version 5).
(a) (b) (c)
Figure 33: (a) Sterile T- flaks in a carbon dioxide incubator, (b) sterile 96 well
plates seeded with cells and (c) plate reader.
142
3.2. Photothermal (PT) effect
The aim of this study was to assess the potential of GNPs as a suitable agent
for plasmonic photothermal therapy (PPTT). MCF-7 cells treated with either plain
GNPs, Mag-GNPs/PLGA NPs or Mag-GNPs/PLGA-TZB NPs were exposed to a
light energy with a specific wavelength prior to MTT assay. This was performed by
application of light to dark and light control experiments for different exposure
periods and different light-emitting diode (LED) sources on MCF-7 cells.
3.2.1. Photothermal effect of plain GNPs:
MCF-7 breast cancer cells were seeded in 96-well plates containing 200µL of
phenol red free RPMI at a density of 5x103 cells /well. Cells were permitted to adhere
for 24h till confluence in a 5% CO2 incubator. Serial dilutions from 500µM to 3.9µM
of plain GNPs were prepared by diluting the aqueous stock solution with the culture
medium. The culture medium from each well was aspirated and replaced by 200µL
of medium containing GNPs using multichannel pipette. After 24h of incubation,
they were washed twice with PBS to remove non-internalized NPs, then 200µL fresh
phenol red free medium was added. Cells were then subjected to monochromatic
light from a LED while incubated in maintenance medium. Two types of light were
studied: green light source of 530nm wavelength and red light source of 650nm at
200mW power for 20 and 45min. A 24h incubation time was given to stabilize the
cells. The medium was then aspirated and replaced with fresh medium. MTT assay
was used to determine cell viability as described before.
A control of untreated cells was made in the absence of test compound and
was used as negative control representing 100% cell viability. The results were
determined by three independent experiments performed in triplicate (n=9) and the
IC50 was calculated.
143
3.2.2. Photothermal effect of Mag-GNPs/PLGA NPs and Mag-

GNPs/PLGA-TZB NPs:
Photothermal ablation therapy of both non-targeted and targeted drug loaded

GNPs were investigated in the same way described above using the selected light
source (650nm). Starting with an amount of NPs equivalent to a Mag concentration
of 100µg/mL, serial dilutions were prepared by diluting the stock with culture
medium with no FBS. Application of each therapeutic modality was followed by
evaluation of tumor cells viability using MTT assay.
3.3. Cell uptake and internalization studies
3.3.1. Preparation of Nile red labeled PLGA encapsulated GNPs (Nile

red-GNPs/PLGA NPs):
Nile red-GNPs/PLGA NPs were prepared by the modified nanoprecipitation
method, previously described in chapter 2. Briefly, accurately weighed, 7.5mg
TPGS and 25mg PLGA were dissolved in 1965μL acetone. A 1mg/mL stock
solution of Nile red in acetone was prepared. From the stock solution, 10μL of Nile
red were added to the PLGA/TPGS solution. Then 25μL of GNPs (10mM) was
mixed with the organic solution. This organic phase was added dropwise to 2mL
water maintained under stirring at 250rpm on a magnetic stirrer. The organic solvent
was then removed by stirring at 100rpm for 4h at room temperature. The NPs were
purified by two cycles of centrifugation at 30,000rpm for 30min at 4°C and washing
with deionized water. To ensure complete removal of free Nile red, the dynamic
dialysis technique was used against an aqueous ethanolic solution containing 20%
ethanol. Frequent changing of the ethanolic solution was performed until no red
fluorescence was visually detected in the dialysate (Banerjee et al., 2015).
144
3.3.2. Preparation of Nile red labeled TZB surface modified PLGA

encapsulated GNPs (Nile red-GNPs/PLGA-TZB NPs):
To prepare Nile red-GNPs/PLGA-TZB NPs, the same previous procedure was
adopted. Then, the surface of the NPs was decorated with TZB by applying the same
procedure used in chapter 1, section 3.3.3.
3.3.3. In vitro cellular uptake:
Breast cancer cells MCF-7 were used to study the NPs cell uptake. The cells
were maintained in complete medium consisting of RPMI-1640 supplemented with
10% FBS and streptomycin/penicillin antibiotics (100µg/mL) in humidified air
atmosphere (5% CO2, 95% RH, 37˚C).
For the uptake experiments, the cultured cells were washed, collected by
centrifugation and counted using a haemocytometer. The cells were seeded in 24
wells plate (5x104 cells/200 µL/well) on 35mm glass cover slips, incubated for 24 h
and then washed with RPMI-1640 to remove non-adherent cells. The cells were then
incubated with Nile red loaded NPs for 15, 60 and 120min to allow for cell uptake.
After incubation, the medium was aspirated, and the cells were washed 3 times with
PBS, fixed with 4% paraformaldehyde solution for 15min at room temperature. The
cells were then washed three times with PBS.
For nuclear staining, the cells were just covered with 300nM of DAPI
prepared in PBS. Following 15min of incubation at room temperature in the dark,
the cells were washed three times with PBS with 5min incubation for each wash.
For microscopical examination, the cover slips were mounted with aqueous
polyvinyl alcohol citifluor reagent mixed with AF100 antifade reagent (1:10). Slides
were examined under the confocal microscope (Zeiss LSM 510 Meta). Red channel
145
for Nile red: excitation: 561nm, emission collected: 570–620nm; blue channel for
DAPI: excitation: 405nm, emission collected: 425–475nm (Cartiera et al., 2009; Li
et al., 2017). Z-series of optical sections were acquired at spacing steps of 0.6µm
from the surface through the vertical axis of the specimen by a computer-controlled
motor drive.
3.4. Evaluation of targeting efficiency
For confirming targeting efficiency, MCF-7 cells were pretreated with excess
free TZB (100µg/mL) before incubation with targeted NPs in MTT assay previously
described in section 3.1.
3.5. Statistical analysis
Results are expressed as mean ± SD. One way ANOVA was used to test the
differences between treatments. In cases where the differences were significant,
post hoc pairwise comparisons were done using Dunnett and/or Student-Neuman-
Keuls for multiple comparison tests. A p-value ≤ 0.05 was taken as significant. The
IC50 was calculated according to the equation for Boltzman sigmoidal
concentration–response curve using the nonlinear regression fitting models (Graph
Pad, Prism version 5).
146
4. Results and discussion
4.1. Cytotoxicity by MTT assay

The cytotoxicity was assessed via determination of the cell viability using a
metabolic activity assay, MTT, in microtiter plates. The in vitro cytotoxicity of the
following formulations was tested on MCF-7 cells: Mag-PLGA NPs, Mag-
GNPs/PLGA NPs and TZB modified NPs. Furthermore, MTT assay was also
performed for free Mag, TZB (mAb) and plain PLGA NPs formulation. The IC50 of
Mag in its solution form and when encapsulated in PLGA NPs, PLGA/GNPs and
PLGA targeted NPs following 24h incubation with MCF-7 breast cancer cells was
also determined.
Table 20 shows that MCF-7 cell treated with free Mag for 24h shows
significant concentration dependent decrease in cell viability. The increase in Mag
concentration from 0.39 to 100µg/mL was accompanied with gradual decrease in
cell viability to 10.56±0.09% as illustrated in figure 34. The half maximal inhibitory
concentration (IC50) value calculated according to the equation for Boltzman
sigmoidal concentration–response curve, figure 35, using the nonlinear regression
fitting models was found to be 2.92µg/mL following 24h treatment. Variable
reported IC50 values after 24h incubation with Mag were 36.4 and 58.27µM which
were equivalent to 9.71 and 15.5µg/mL (Zhou et al., 2013; Liu et al., 2013).
Mag had been found to inhibit the proliferation of MCF-7 human breast cancer
cells by arresting the cell cycle at the G2/M phase and by induction of apoptosis
(Zhou et al., 2013). G2/M phase arrest was found to be associated with up-regulation
of the tumor suppressor protein p53 and p21 (p53 target gene) and down-regulation
of cyclin B1-cyclin-dependent kinase 1 (B1/CDK1) complex. Induction of apoptosis
147
was associated with increased ROS generation, disruption of matrix

metalloproteinase (MMP), release of cytochrome c and apoptosis inducing factor
(AIF), modulation of B-cell lymphoma 2 (Bcl-2) family proteins and cleavage of
poly (ADP-ribose) polymerase (PARP) in MCF-7 cells (Zhou et al., 2013).
Therefore, Mag might be a potential lead compound in the therapy of breast cancer.
Table 20: Cell viability after incubation of MCF-7 with free Mag and TZB in
solutions.
Concentration Cell viability (%)
(µg/mL) Mag TZB
200 ND 74.75±6.43
100 10.56±0.09 82.30±5.37
50 10.59±0.28 102.90±18.36
25 12.81±3.14 107.50±11.69
12.5 21.53±0.13 93.37±1.85
6.25 35.58±0.16 97.20±5.52
3.12 63.12±9.49 109.97±4.95
1.56 80.30±0.71 102.90±12.45
0.78 96.57±2.96 114.00±9.64
0.39 101.48±2.86 105.00±5.57
Results are expressed as mean± SD, n=3 experiments, three replicates per experiment at each test concentration.
On the other side, TZB started to show decrease in cell viability only at the
high concentrations 100 and 200µg/mL as shown in table 20. The IC50 value
following incubation with TZB was found to exceed 200μg/mL. Consistent with our
results, Yamaguchi and colleagues reported that 24h incubation with TZB alone (0
to 1000nM) had no significant effect on the cell cycle of MCF-7 cells (Yamaguchi
et al., 2005). The effect of TZB had been reported to be mainly in preventing cell
proliferation rather than inducing cell death and an IC50 of >200 µg/mL for TZB was
previously found (Rodríguez et al., 2015; Vesci et al., 2015).
148
Table 21 and figure 34 show that no decrease in cell viability following

incubation with blank PLGA NPs at all tested concentrations confirming that the
matrix used was safe and biocompatible. As an FDA approved polymer, PLGA was
reported to be a highly biocompatible and biodegradable (Makadia and Siegel,
2011). The backbone of the PLGA is made up of lactic acid and glycolic acid, which
are the key components involved in cell metabolism. The polymer degrades into
individual units which are metabolized by the cells (Jaidev et al., 2015).
In spite of the presence of TPGS in the matrix, no cytotoxicity was noted at

the tested concentrations. The concentration range of used TPGS was not enough to
induce any toxicity on MCF-7cells. An IC50 of >6.5mg/mL was previously found for
TPGS cytotoxicity on MCF-7 cells (Si-shen et al., 2014). Conversely, others showed
that TPGS can induce G1/S cell cycle arrest and apoptosis in breast cancer cell lines
(MCF-7 and MDA-MB-231) but not in "normal" (non-tumorigenic) immortalized
cells (MCF-10A and MCF-12F) and reported an IC50 of 40µg/mL for TPGS on
MCF-7 cells (Neophytou et al., 2014). TPGS has been shown to greatly enhance the
performance of NPs, resulting in much higher cellular uptake of the drug as well as
more desirable in vivo pharmacokinetics. Importantly, emerging evidence in the
literature suggests that TPGS not only may be useful in cancer chemotherapy as a
carrier drug but also may act synergistically or enhance the effect of anticancer drugs
(Neophytou and Costantino, 2015).
Similar to free Mag as solution, both Mag-PLGA NPs and Mag-GNPs/PLGA

NPs exhibited concentration dependent cytotoxic behavior. Detailed analysis of
cytotoxic effect (figures 34 and 35) indicated that Mag loaded NPs elicited
significantly more cell death than free Mag at equivalent dose and same incubation
time. The 24h dose-dependent (0-100µg/mL) study using MCF-7 cells shows a
gradual decrease in viability from 100% to 9.41±0.8 and 8.73±1.19% for Mag-
149
PLGA NPs and Mag-GNPs/PLGA NPs, respectively (table 21 and figure 34). The
viability of cells exposed to Mag-PLGA NPs and Mag-GNPs/PLGA NPs were
insignificantly different among each other (p˃0.05) and significantly (p˂0.05) lower
than pure Mag solution.
Table 21: Cell viability after incubation of MCF-7 with various plain and Mag
loaded PLGA NPs formulae.
Cell viability (%)
Concentration
(µg/mL) Plain PLGA Mag-PLGA Mag-GNPs/PLGA Mag-GNPs/PLGA-TZB
NPs NPs NPs NPs
100 105.40±4.76 9.41±0.80 8.73±1.19 7.13±1.13
50 104.21±11.80 10.08±0.38 8.86±1.32 8.43±0.33
25 99.69±3.79 10.97±0.75 12.98±2.05 10.28±0.52
12.5 101.71±7.78 16.99±1.22 13.03±1.30 12.99±2.16
6.25 95.64±5.43 21.16±0.45 18.90±2.34 17.81±0.27
3.12 102.13±8.73 53.09±5.24 54.47±2.25 47.87±14.67
1.56 100.31±2.14 64.18±1.67 62.28±4.58 70.74±3.54
0.78 99.90±8.28 72.85±2.04 79.87±1.94 103.19±5.30
0.39 99.79±7.07 98.64±5.04 106.38±9.21 99.58±7.14
Table 22 summarizes the calculated IC50 values of NPs formulations

compared to free Mag solution (figure 35). As depicted in table 22, the calculated
IC50 value of free Mag solution, 2.9μg/mL, was higher than Mag-PLGA NPs and
Mag-GNPs/PLGA NPs with respective IC50 of 1.8 and 2.2μg/mL. Cellular
internalization of the NPs may be responsible for the increased cytotoxic effect.
Literature previously reported that intracellular localization and interaction of NPs
probably led to the increased cytotoxic behavior (Gaonkar et al., 2017). Anticancer
potential of Mag can thus be enhanced by nanoencapsulation. The presence of TPGS
could also have contributed to the observed enhanced cytotoxicity. Vitamin E-
150
combined therapy had become a therapeutic strategy for common

chemotherapeutics, not only to improve the efficacy of anticancer drugs but also to
provide higher safety levels. The superior efficacy is obtained by increasing
chemotherapeutic drugs’ bioavailability or by acting in a synergistic manner. TPGS
has a multifunctional nature, it acts as a solubilizer, absorption and permeation
enhancer, and also as a potent Pgp inhibitor (Neophytou and Constantinou,
2015). Also The ability of TPGS to induce apoptosis in triple negative breast cancer
cells (TNBC) via the PI3K/AKT pathway had been previously shown (Neophytou
and Constantinou, 2015).
Apparently, the presence of GNPs did not affect the cytotoxicity as evidenced
by the non-significantly different cell viabilities obtained with Mag-PLGA NPs and
Mag-GNPs/PLGA NPs at all drug concentrations tested. Hence, the effect of GNPs
will be reevaluated in conjunction with photothermal (PT) effect as will be shown
later.
Similarly, conjugating TZB to the NPs did not considerably change the cell
viability as depicted by the non-significantly different cell viability (%) in case of
Mag-GNPs/PLGA NPs and targeted Mag-GNPs/PLGA NPs (100μg/mL).
Furthermore, the IC50 values for TZB conjugated NPs and Mag-GNPs/PLGA NPs
were found to be 1.75 and 2.22μg/mL, respectively (table 22).
Table 22: IC50 of various Mag loaded formulations.

Formula IC50 (µg/mL)
Mag solution 2.92
Mag-PLGA NPs 1.81
Mag-GNPs/PLGA NPs 2.22
Mag-GNPs/PLGA-TZB NPs 1.75
151
Mag
F r e esolution
M ag Mag-PLGA
M NPs
a g -P L G A NPs
TZB
F r e solution
e TZB Mag-GNPs/PLGA
P L G A /M a g - G NPs
NPs
150
Blank NPs*
b la n k NPs Mag-GNPs/PLGA-TZB
T Z B m o d ifie d P LNPs
G A /M a g - G N P s
100
V ia b ilit y ( % )
50
0
9
.5
0
5
0
.3
.7
.5
.1
.2
0
2
5
2
1
0
1
Conc (µg/mL)
*Blank NPs were prepared at the same solid content concentration of their equivalent medicated counterpart with no drug content.
Figure 34: MCF-7 breast cancer cell viability measured by MTT cytotoxicity assay after exposure to
increasing concentrations of Mag solution, TZB solution, blank NPs, Mag-PLGA NPs, Mag-GNPs/PLGA
NPs and Mag-GNPs/PLGA-TZB NPs.
152
120
100
80
Cell viability (%)
60
Mag solution
Mag-PLGA NPs
40 Mag-GNPs/PLGA NPs
Mag-GNPs/PLGA-TZB NPs
20
0
-0.5 0 0.5 1 1.5 2 2.5
Log [ Mag concentration (µg/mL)]
Figure 35: Cytotoxicity results of Mag solution, Mag-PLGA NPs, Mag-

GNPs/PLGA NPs and Mag-GNPs/PLGA-TZB NPs against MCF-7 cancer cell
lines.
153
4.2. Photothermal (PT) effect
This study was undertaken to study the effect of hyperthermia on the

cytotoxicity of GNPs containing formulae. LED, a monochromatic non coherent
light source was used due to its ease of portability, economical feasibility, and
efficient light source to be used in PTT to overcome the limitations of laser
(Gananathan, 2016).
4.2.1. Photothermal effect of plain GNPs:
The effect of hyperthermia of different GNPs concentrations was followed.

The optimum light exposure time was first determined in a preliminary study by
application of light at 530nm for 20 and 45min to dark and light experiments to
MCF-7 cells treated with GNPs. No decrease in cell viability was noted following
irradiation for 20min. It was only at 45min that the decrease in cell viability was
significant and data are not shown. Hence, exposure time for 45min was selected.
This result was in accordance with previous works (Gomaa et al., 2015; Iodice et
al., 2016; Gananathan, 2016).
When untreated MCF-7 were exposed to monochromatic LED at both 530

(green light) and 650nm (red light) wavelengths for 45min, no decrease in cell
viability was observed indicating the safety of the used light source at the tested
power, 200mW, on MCF-7 cells.
In dark experiment, MTT results, following 24h incubation of plain GNPs,

proved that GNPs between 50 and 250µM did not cause any apparent toxicity on
MCF-7 cancer cells. GNPs had no intrinsic cytotoxic or anti-proliferative effects on
cells (Gannon et al. 2008; Corr et al., 2012). In contrast to our results, Selim and
Hendi, 2012 showed that GNPs produced dose dependent cytotoxicity to MCF-7
154
cells in the concentration range of 25-200µg/mL suggesting that GNPs may induce
apoptosis in MCF-7 cells via different pathways (Selim and Hendi, 2012). Similarly,
some researchers reported an IC50 of 110μg/mL for GNPs (Vijayakumar and
Ganesan, 2012). While with others, GNPs cytotoxicity was seen even at
concentrations of 2μg/mL (Priya and Iyer, 2015). The difference in the output of
the different studies might be explained based on the difference in the size of GNPs
tested. It has been previously postulated that particles of 1–2nm were highly toxic
while larger 15nm gold colloids were nontoxic, irrespective of the cell type tested
(Pan et al., 2007).
From Table 23, The MTT result in dark shows 105.75±8.84% of viability after
24h incubation with 3.9µM GNPs, which decrease successively to 79.25±0.49%
with increasing GNPs concentration to 250µM. While in photo-irradiation at 530nm,
the cell viability was decreasing from 100.24±9.62 to 69.42±6.4% with increasing
drug concentration from 3.9 to 250µM. it could be depicted that green LED
irradiation at 530nm in presence of GNPs did not show any significant different
effect from dark test (p˂0.05). The calculated IC50 in dark could not be determined
accurately by the used software.
Conversely, following irradiation of MCF-7 cell with red LED in presence of

plain GNPs, the cells showed a significant decrease (p˃0.05) in %viability compared
to green irradiation and dark experiments. The cell viability was 91.55±0.77% at
3.9μM concentration and decreased to 48.37±8.39% for 250μM concentration. The
calculated IC50 of plain GNPs irradiated with red LED (305.3μM) was lower than
that obtained with plain GNPs irradiated with green LED (385.9μM) under same
conditions (table 24).
155
As previously shown in chapter two, the prepared GNPs with an average size
of 20nm exhibited maximal spectral absorption at ~520nm and so green LED at
530nm was thought to be suitable for irradiating GNPs (Gananathan, 2016). But
the clustering of GNPs in MCF-7 cells provided a significant shift of plasmonic
resonance to NIR range that may make 650nm application more effective with
spherical GNPs (Shao et al., 2013). Furthermore, literature showed that
photothermal cancer therapy can also be performed with GNPs at short NIR.
Previous works suggested that GNPs had a tendency for self-assembly in
clusters in vitro as well as in vivo. This aggregation was accompanied by a red-
shift of their plasmonic resonances with simultaneous enhancement of NIR
absorption (Shao et al., 2013; Mendoza-Nava et al., 2013). This plasmonic
phenomenon allows more effective application of PT therapy. The NIR window
is ideally suited for in vivo applications because of the high penetration due to the
minimal light absorption by hemoglobin and water (Mendoza-Nava et al., 2013).
Table 23: Cell viability of MCF-7 incubated with plain GNPs in dark and after
LED irradiation at 530 and 650nm.
Gold Cell viability (%)
concentration
(µM) Dark PTT at 530 nm PTT at 650 nm
500 50.45±6.29 58.56±1.02 39.34±5.88
250 79.25±0.49 69.42±6.40 48.37±8.39
125 90.65±1.06 82.30±15.26 73.90±0.58
62.5 93.60±5.23 93.96±9.36 70.47±5.26
31.25 98.10±0.99 93.81±11.80 76.92±5.67
15.6 103.05±9.83 89.57±8.65 77.38±2.88
7.8 99.90±7.21 104.75±6.31 89.42±2.47
3.9 105.75±8.84 100.24±9.62 91.55±0.77
156
Table 24: Calculated IC50 of MCF-7 incubated with plain GNPs in dark and
after LED irradiation at 530 and 650nm.
Condition IC50 (µM)
Dark ND
LED irradiation at 530nm 385.9
LED irradiation at 650nm 305.4
ND: not determined
4.2.2. Photothermal effect of Mag-GNPs/PLGA NPs and Mag-

GNPs/PLGA-TZB NPs:
To examine the effect of application of hyperthermia on Mag-GNPs/PLGA

NPs and Mag-GNPs/PLGA-TZB NPs , MCF-7 breast cancer cells were incubated
with different NPs concentrations equivalent to Mag concentrations from 0.39 to
100µg/mL and exposed to red LED of 650nm irradiation for 45min.
Mag-GNPs/PLGA NPs showed decrease in %viability from 98.06.58±1.64

to 5.40±1.59% with increasing NPs concentration equivalent to Mag concentration
from 0.39 to 100µg/mL after irradiation (table 25 and figure 36). The calculated IC50
of Mag-GNPs/PLGA NPs was 1.34µg/mL which is significantly lower than that
previously obtained with Mag-GNPs/PLGA NPs with no LED exposure (2.22
µg/mL). Similarly, Mag-GNPs/PLGA-TZB NPs showed a decrease in IC50 from
1.75 (no PT) to 1.1 µg/mL (with PT) (table 26). To obtain more effective PTT, higher
GNPs concentration is required. As it is impractical to increase the amount of
encapsulated GNPs, so it is advised to use higher Mag-GNPs/PLGA NPs
concentration, with adjusted Mag loaded amount to reach the targeted IC 50.
157
Table 25: Cell viability of MCF-7 incubated with Mag-GNPs/PLGA NPs and
Mag-GNPs/PLGA-TZB NPs following LED irradiation at 650nm for
45minutes.
Mag Cell viability (%)
concentration
(µg/mL) Mag-GNPs/PLGA NPs Mag-GNPs/PLGA-TZB NPs
100 5.40±1.59 4.06±0.67
50 5.50±0.32 5.47±0.98
25 5.04±0.5 6.38±0.44
12.5 6.60±0.77 10.69±4.75
6.25 6.00±3.79 12.42±5.24
3.12 47.72±1.33 43.74±1.56
1.56 60.46±6.98 53.09±0.96
0.78 89.95±0.15 95.84±10.20
0.39 98.06±1.64 102.05±3.94
Table 26: Calculated IC50 of Mag loaded NPs following exposure to light of
650nm for 45 minutes.
Formula IC50 (µg/mL)
Mag-GNPs/PLGA NPs 1.348
Mag-GNPs/PLGA-TZB NPs 1.100
158
120
110 Mag-GNPs/PLGA-TZB
TZB NPs
modified PLGA/Mag-GNPs
withoutPTT
without PTT
100 Mag-GNPs/PLGA-TZB
TZB NPs with
modified PLGA/Mag-GNPs
PTTPTT
with
Mag-GNPs/PLGAwithout
PLGA/Mag-GNPs NPs without
PTT
90
PTT
Mag-GNPs/PLGAwith
PLGA/Mag-GNPs NPsPTT
with PTT
80
Cell viability (%)
70
60
50
40
30
20
10
0
-0.5 0 0.5 1 1.5 2 2.5
-10
Figure 36: Cytotoxicity results of Mag-GNPs/PLGA NPs and Mag-

GNPs/PLGA-TZB NPs against MCF-7 cancer cell lines with and without
photothermal treatment using LED at 650nm for 45min.
159
4.3. Cell uptake and internalization of PLGA encapsulated

GNPs (GNPs/PLGA NPs) in MCF-7 cell line
GNPs/PLGA internalization by cancer cells was investigated using the human

breast cancer cell line MCF-7. To determine the uptake of the NPs in MCF-7 cancer
cells, the hydrophobic fluorescent dye, Nile red was encapsulated with the same
procedure followed for the preparation of Mag-GNPs/PLGA NPs. The fluorophore
exhibits emission at wavelength ˃590nm.
Fluorescent images of MCF-7 cells loaded with fluorescent Nile red labeled
PLGA encapsulated GNPs (Nile red-GNPs/PLGA NPs) are shown in figure 37. The
nuclei are shown in blue due to the fluorescent dye DAPI and Nile red-GNPs/PLGA
NPs are shown in red fluorescence.
The confocal microscopic images with respect to time showed the uptake of
GNPs/PLGA NPs inside the cell and their localization in the cytoplasm (figures 37-
41). A time dependent increase in the fluorescence intensity until 2h post incubation
of the cells with NPs was observed. After 2h incubation, NPs have been observed to
be localized near the nucleus, the Nile red labeled NPs were well distributed around
the blue stained nucleus indicating NPs localization near the nucleus (Davda and
Labhasetwar, 2002).
Confocal microscopy images show that TZB targeted NPs undergo much
faster uptake compared to the non-targeted analog. The TZB-targeted formulation
bound to the MCF-7 cell membrane very rapidly (within 15min of incubation). An
active interaction between the mAb present on the nanovehicle surface with the high
HER2 expression on the MCF-7 cell surface might be expected. The intensity of red
fluorescence increased with incubation time, suggesting increased uptake of the NPs
160
by lysosomes followed by possible release of Nile red into the cytoplasmic

compartment. These results indicate that the TZB targeted NPs can undergo
receptor-mediated rapid internalization in HER2 overexpressing MCF-7 cells. In
literature, the internalization of NPs by cells was reported to be facilitated by energy
dependent endocytosis mechanism (Jaidev et al., 2015).
Due to the absence of the Ab on the surface of NPs in the non targeted
formulation, NPs cannot undergo receptor mediated internalization, and the cellular
uptake is through slower processes like lipid rafts, membrane fusion, pores, as well
as through caveolae. Furthermore, a better cellular uptake of PLGA nanospheres
with smaller dimensions is foreseek. In the present study, the average size of
GNPs/PLGA NPs (˂200nm) may have enabled it to enter the cell through the more
fluid cell membrane, a known characteristic of cancer cells and it is likely that one
of those listed mechanisms might have contributed to the uptake of PLGA
nanospheres (Master et al., 2013; Jaidev et al., 2015). Hence for this group, the red
fluorescence is seen within the cells only around 60min.
The negatively charged cell membrane has a tendency to interact with positive
charged or neutral NPs. For non targeted and targeted GNPs/PLGA NPs , the zeta
potential is -23.80.1±2.83 and -8.15±0.95mV, respectively, indicating a slight
negative surface charge. The electronic repulsion between PLGA NPs and cells was
expected to be very weak and so NPs were capable of gaining intracellular access in
various cell lines investigated (Dhankar et al., 2011).
In order to confirm that the NPs were taken up into the MCF-7 cells and not
only adsorbed onto the surface of the cell membrane, their uptake was demonstrated
by acquiring 0.1µm-thick image slices of the cell monolayer that were stacked and
analyzed, together with cross-sectional slices perpendicular to the plane of the cell
161
monolayer midpoint (z-axis) (Nkabinde et al., 2014). The z-slices (figures 42-45)
show that both non targeted and TZB modified Nile red-GNPs/PLGA NPs were
present in different planes throughout the thickness of the monolayer. Serial z-
sections of the cells, each ~0.1µm in thickness, demonstrated high fluorescence
activity in sections between 3.71 and 8.36 µm from the surface of the cells indicating
that NPs were transported from the apical surface of the cell membrane towards the
basolateral membrane. Cross-sectional slices confirmed that NPs were indeed inside
the cytoplasm and not simply adsorbed to the outer surface (Davda and
Labhasetwar, 2002).
Thus, the confocal images showed thus a difference in the speed of uptake
between TZB modified and unmodified NPs in spite of the small difference in cell
cytotoxicity.
162
Figure 37: Confocal images of MCF-7 cells treated with Nile red-GNPs/PLGA
NPs for 15min: (a) blue filter, (b) red filter, (c) no filter and (d) merged filters.
The nuclei were stained with DAPI and the NPs were labeled with Nile red.
163
NPs for 60min: (a) blue filter, (b) red filter, (c) no filter and (d) merged filters.
The nuclei were stained with DAPI and the NPs were labeled with Nile red.
164
NPs for 120min. The nuclei were stained with DAPI and the NPs were labeled
with Nile red.
165
Figure 40: Confocal images of MCF-7 cells treated with Nile red-
GNPs/PLGA-TZB NPs for 15min: (a) blue filter, (b) red filter, (c) no filter and
(d) merged filters. The nuclei were stained with DAPI and the NPs were
labeled with Nile red.
166
Figure 41: Confocal images of MCF-7 cells treated with Nile red-
GNPs/PLGA-TZB NPs for 60min: (a) blue filter, (b) red filter, (c) no filter and
(d) merged filters. The nuclei were stained with DAPI and the NPs were
labeled with Nile red.
167
Figure 42: Z-staked confocal images of MCF-7 cells treated with Nile red-
GNPS/PLGA NPs for 60min. The nuclei were stained with DAPI and the NPs
were labeled with Nile red.
168
GNPs/ PLGA NPs for 120min. The nuclei were stained with DAPI and the
NPs were labeled with Nile red.
169
GNPs/PLGA-TZB NPs after for 60 min. The nuclei were stained with DAPI
and the NPs were labeled with Nile red.
170
GNPs/PLGA-TZB NPs for 120min. The nuclei were stained with DAPI and
the NPs were labeled with Nile red.
171
4.4. Targeting efficiency

The goal of this study was to show the efficacy of the active targeting of Mag-
GNPs/PLGA NPs with TZB conjugation. It could be inferred from the previous
studies that the presence of TZB enhanced the rate of particles uptake by the used
cancer cells as seen from confocal cells. This was manifested in MTT assay by a
small decrease in IC50 from 2.22.to 1.75µg/mL for non targeted and targeted NPs,
respectively.
Literature reported that MCF-7 cells overexpress PR in addition to ER as well

as moderately express HER2 (Lee et al., 2015; Subik et al., 2010). MCF-7 breast
cancer cell line was also reported to be heterogeneous due to the presence of different
phenotypes within a tumour population that differ in their relative expression of
receptors such as PR, ER, EGFR and HER2 (Baguley and Leung, 2011).
TZB, is a humanized recombinant monoclonal antibody that selectively binds

to the extracellular domain of the human epidermal growth factor receptor 2, HER2,
a transmembrane protein overexpressed in 25-30% of breast cancers.
Results in table 27 show that the cytotoxicity of the targeted NPs without free
Ab was greatly enhanced relative to those obtained with excess free Ab. The IC 50
increased by 1.3 fold after pretreatment with excess free TZB as shown in table 28.
In accordance with our results, Zheng et al., 2010 previously reported the decrease
in the cellular uptake of the transferrin NPs by the addition of excess transferrin.
From figure 46, it is obvious that a Mag concentration dependent decrease in

cell viability was obtained before treatment with free TZB. While after treatment
with free mAb, the cell viability decreased with increasing drug concentration up to
12.5µg/mL, further increase in concentration had no effect on cell viability. This
might be due to that the targeting was affected by the blockage of receptors by the
172
excess TZB in the media. This showed evidence that targeting of NPs depended on
HER2 expression on MCF-7 cells and that the efficient cellular uptake of targeted
NPs was due to TZB conjugation. Previous literature reported that TZB was
effective for targeting HER2 overexpressing MCF-7 breast cancer (Diessner et al.,
2014; Zhou et al., 2015).
Recently, TPGS-based NPs conjugated with herceptin were used for the
targeting of anticancer drugs such as docetaxel to cancer cells overexpressing the
HER2 receptor. A study showed that the NPs synthesized using TPGS with chain
length PEG 1000 resulted in the best therapeutic effects (Neophytou and
Constantinou, 2015).
Table 27: Cell viability of MCF-7 incubated with Mag-GNPs/PLGA-TZB NPs

before and after cells treatment with free TZB.
Mag Cell viability (%)
Concentration
(µg/mL) Before treatment with free TZB After treatment with free TZB
100 7.13±1.13 36.06±6.78

50 8.43±0.33 29.54±6.46
25 10.28±0.52 27.61±9.02
12.5 12.99±2.16 26.81±1.81
6.25 17.81±0.27 56.89±1.11
3.12 47.87±14.67 55.56±5.32
1.56 70.74±3.54 64.74±3.89
0.78 103.19±5.30 73.85±4.01
0.39 99.58±7.14 85.70±17.4
173
Table 28: Calculated IC50 of Mag-GNPs/PLGA-TZB NPs before and after cells
treatment with free TZB.
Cell condition IC50 (µg/mL)
Cells without treatment 1.755
Cells pretreated with free TZB 2.303
120
before treatment with free TZB
after treatment with free TZB
100
80
Cell viability (%)
60
40
20
0
-1 -0.5 0 0.5 1 1.5 2 2.5
Figure 46: Cytotoxicity results of Mag-GNPs/PLGA-TZB NPs before and

after MCF-7 cells treatment with free TZB.
174
To summarize up:
The Mag-GNPs/PLGA NPs afforded much higher anti-tumor efficiency

when compared to Mag alone. Besides, NPs also showed enhanced PTT after
illumination with NIR compared to plain GNPs. Both targeted and non targeted NPs
show efficient cell uptake and internalization as seen in confocal images. Finally,
blocking HER2 receptors with excess free TZB, evidenced the success of active
targeting with TZB and confirmed the internalization with receptor mediated
endocytosis. The Mag-GNPs/PLGA-TZB NPs showed notable combined targeted
drug delivery system and photothermal potential for breast cancer therapy.
175
Conclusions
• Cytotoxicity results on MCF-7 cell line confirmed the safety of blank PLGA
NPs and free TZB.
• Mag loaded NPs showed significant decrease in cell viability when compared
to free Mag with recorded IC50 1.81 and 2.92µg/mL, respectively.
• Dark cytotoxicity study did not show any significant difference in cell
viability between Mag-PLGA NPs and Mag-GNPs/PLGA NPs. Also
targeting with TZB has no effect on cytotoxicity.
• Irradiation of MCF-7 cell with green LED at 530nm in presence of blank
GNPs did not show any significant different effect from dark test (p>0.05).
While significant decrease (p<0.05) was obtained after red LED irradiation at
650nm.
• Mag-GNPs/PLGA NPs significantly (p<0.05) reduced MCF-7 cell viability
compared to plain GNPs after irradiation with red LED at 650nm with
calculated IC50 of 1.38 and 305.4µg/mL, respectively.
• The confocal microscopic images with respect to time confirmed the uptake
of both targeted and non targeted Nile red-GNPs/PLGA NPs inside the cell
and their localization into the cytoplasm with targeted NPs showing relatively
faster uptake.
• The efficacy of the active targeting of Mag-GNPs/PLGA NPs with TZB had
been proved by the decrease in cytotoxicity after blocking HER2 receptors
with free TZB before targeting.
176
General conclusion
General conclusion
The developed TZB modified GNPs/PLGA NPs system was able to boost
the cytotoxic activity of the natural anticancer drug, Mag. Higher cytotoxicity was
achieved following PT application using NIR light. The specificity to breast cells
was evidenced by the faster uptake of the targeted NPs compared to the
unmodified one with a compromised cytotoxicity following HER-2 receptors
saturation.
Mag-GNPs/PLGA-TZB NPs could be considered a safe breast cancer

multifunctional therapy showing notable combined targeted anticancer drug
delivery system with a photothermal potential.
177
Future perspectives
Future perspectives
▪ Testing in vivo antitumor activity of Mag-GNPs/PLGA-TZB NPs especially to

prove the suitability of the developed system for active targeting.
▪ Testing this developed engineered system in bioimaging and theranostics.
178
Summary
Summary
Breast cancer is the most common cancer in women worldwide and the
leading cause of cancer death in women. Invasion and metastasis are the main
reasons for the high mortality rates and poor clinical outcomes associated with breast
cancer. Treatment strategies depend on the type and stage of breast cancer. In case
of organ-confined disease, mastectomy is the preferred treatment. Patients are also
treated with radiation and/or chemotherapy, in addition to hormone ablation. Lack
of selectivity, high toxicity, severity of side effects and occurrence of multidrug
resistance present the major problems for anticancer drugs.
Our aim was to provide better breast cancer therapy that solve the
conventional chemotherapy problems. Two strategies are adopted in this thesis: the
first involved the use of natural products with wide safety margin and high cytotoxic
activity on cancer cells. The second strategy relied on increasing delivery systems
specificity to tumor tissues via the use of smart engineered systems which can target
cancer cells passively and/or actively. HER2 receptors are overexpressed in breast
cancer on the primary tumor as well as on metastatic sites and are minimally
expressed by normal tissues. Trastuzumab (TZB), also called herceptin was used in
this work as a targeting ligand to provide specific targeting to HER2 overexpressing
cells. Attaching this ligand to the surface of the NPs was attempted to promote NPs
cellular fast uptake and internalization via receptor-mediated endocytosis.
In this thesis, Magnolol (Mag), the natural anticancer polyphenolic compound

isolated from the root and stem bark of the chinese plant Magnolia officinalis was
used in this work. Mag exhibits anti-cancer properties by apoptosis, and inhibiting
proliferation, angiogenesis, metastasis, and multidrug resistance. However, Mag
suffers from high lipophilicity, low solubility and high affinity for interaction with
179
Summary
plasma proteins which limit its delivery options. We hypothesized that TZB surface
modified polymeric (PLGA) containing metallic (gold) NPs and loaded with Mag
can provide a multifunctional system that will guarantee the specific and enhanced
delivery of Mag to breast cancer cells. This, in combination with the specific
plasmonic photothermal effect of targeted GNPs, will help to boost the anticancer
activity of the drug. Accordingly, the work in this thesis was divided into three
chapters:
• Chapter 1: Preparation and optimization of magnolol loaded targeted PLGA

nanoparticles.
• Chapter 2: Preparation and optimization of magnolol and gold nanoparticles
encapsulated in targeted PLGA nanoparticles.
• Chapter 3: Biological evaluation and photothermal effect of magnolol and gold
nanoparticles encapsulated in targeted PLGA nanoparticles.
Chapter 1: Preparation and optimization of magnolol loaded targeted PLGA

nanoparticles.
The work in this chapter aimed at designing breast cancer cells targeted PLGA
NPs incorporating the anti-cancer drug Mag. To achieve our goal, the work in this
chapter encompassed the following:
1. Preparation and optimization of blank PLGA-NPs using nanoprecipitation

method. The effects of some formulation parameters on the properties of the
obtained NPs were studied. The effect of each factor was evaluated, while keeping
all other factors constants during NPs preparation. The following factors were
investigated:
1.1. Organic solvent type.

180
Summary
1.2. Surfactant (SAA) concentration and type.

2. Preparation and optimization of Mag loaded PLGA NPs (Mag-PLGA NPs). The
effects of the following factors were investigated:
2.1. SAA type (PVA or TPGS).
2.2. SAA solvent phase: aqueous or organic phase.
2.3. Organic to aqueous phase volume ratio.
2.4. Theoretical Mag loading amount.
3. Formulation of antibody modified NPs. BSA was first used to optimize the
process and then the mAb, TZB, was used to achieve the required targeting. The
following methods were employed to load the protein on the surface of pre-
fabricated PLGA NPs:
3.1. Non covalent (adsorption) binding.
3.2. Covalent binding.
All the prepared blank, Mag-PLGA NPs, surface modified blank and surface
modified Mag-PLGA NPs were characterized viz PS, ζ, morphological examination
by TEM, %EE, in vitro drug release, FT-IR analysis, 1H-NMR and determination of
protein on the surface of modified NPs using protein assay.
From the results obtained, it was found that:
➢ Acetone produced smaller NPs with more uniform smaller PS compared to

acetonitrile when used as organic solvent for PLGA during the preparation of
NPs by the nanoprecipitation method.
➢ The increase in PVA concentrations in the aqueous phase caused a concentration
dependent decrease in NPs size and a 0.5% w/v was found to be optimum for the
production of uniform NPs with a size less than 150nm.
➢ Compared to PVA, TPGS resulted in the production of small NPs at lower
concentrations and 0.18% w/v concentration was found optimum.
181
Summary
➢ The use of TPGS in concentrations of 0.03 to 0.18% w/v in the aqueous phase
resulted in the production of NPs smaller than 120nm. Lower concentrations of
TPGS were used to produce small NPs compared to PVA.
➢ The increase in theoretical drug amount from 1 to 4% w/w did not affect neither
the PS nor the drug EE. Further increase in drug amount to 20% w/w of the
polymer used caused a significant increase in size with a decrease in drug EE.
➢ The change in organic to aqueous phase volume ratio from 1:2 to 1:1 or the
change of TPGS solvent phase did not affect the NPs characteristics.
➢ ζ of Mag-PLGA NPs containing TPGS was found to be -14.75± 0.64mV
indicating the formation of stable NPs.
➢ According to the TEM micrographs, Mag-PLGA NPs were spherical uniform in
shape with size ~100nm.
➢ FT-IR and 1H-NMR studies confirmed the formation of a new blend matrix
composed of PLGA and TPGS. The new matrix was able to molecularly disperse
and encapsulate the hydrophobic drug Mag.
➢ The release profile for Mag from the new matrix showed a small burst release of
10% w/w during the first four hours, a 40% w/w Mag release during the first day
and then a slower release was then noted where less than 80 % w/w were released
in 40 days.
➢ An incubation time of 4h with a ratio of 0.1:1 of BSA:NPs provided higher % of
BSA adsorption.
➢ Unstable aggregating particles with very low ζ were obtained upon trying to load
BSA and TZB by adsorption on the surface of PLGA NPs.
➢ BSA and TZB coupling was carried through covalent conjugation using
carbodiimide chemistry with EDC and NHS. TZB and BSA attachment to Mag-
PLGA NPs was confirmed by FT-IR, 1H-NMR, PS, ζ and % CE.
182
Summary
➢ BSA and TZB modified NPs showed a significant decrease in the negative ζ
compared to unconjugated NPs confirming Ab attachment.
➢ Immuno- NPs prepared by TZB showed similar percent of Ab bound as obtained
by BSA ~60% w/w.
Chapter 2: Preparation and optimization of magnolol and gold nanoparticles

encapsulated in targeted PLGA nanoparticles.
The aim of this chapter was to prepare targeted Mag-GNPs suitable for IV
administration. The proposed delivery system was designed specifically for
combining imaging targeted therapeutic system in a single procedure. Hence,
developing multifunctional nano-carriers that work simultaneously for combined
photoacoustic imaging, photothermal therapy and drug delivery vehicle to target
breast tumor cells was set as the ultimate goal. To achieve our goal, the work in this
chapter encompassed the following:
1. Synthesis of citrate-capped gold NPs (citrate GNPs) by the reduction of

chloroauric acid (HAuCl4) with sodium citrate using Turkevich method.
2. Preparation and optimization of Mag-GNPs. The effects of some formulation
parameters on the properties of the obtained NPs were studied. Each factor
was evaluated while keeping all other factors constant during NPs preparation.
These are:
2.1. Effect of ethanol volume.
2.2. Effect of theoretical Mag loading amount.
2.3. Effect of drug loading time.
3. Preparation and optimization of BSA modified Mag-GNPs. BSA was used as a
model protein to optimize the protein adsorption on GNPs surface.
183
Summary
4. Preparation and optimization of PLGA encapsulated Mag and GNPs (Mag-

GNPs/PLGA NPs). In this study, both GNPs and the hydrophobic drug Mag were
encapsulated in PLGA NPs. The effects of formulation parameters on the
properties of the obtained NPs were studied:
4.1. The presence of GNPs in aqueous or organic phase.
4.2. The amount of GNPs.
5. Preparation of immuno-PLGA encapsulated Mag and GNPs. Loading of the Ab,
TZB, was done by conjugation method.
The prepared citrate-GNPs, Mag-GNPs and Mag-GNPs/PLGA NPs and
immuno- NPs were character ized viz spectral analysis, PS, ζ, morphological
examination by TEM, %EE, in vitro drug release, FT-IR analysis, protein assay.
➢ Citrate GNPs prepared were ruby red in colour and exhibited a unique SPR at
519nm. The particles were mostly spherical with average PS ~20nm and ζ was -
57.20±2.24mV indicating good NPs stability.
➢ Mag-GNPs (F6) prepared by adsorption method using 500µL of 1mg/mL Mag in
ethanol for 12h showed maximum %EE of 42.04±0.33 % w/w.
➢ Drug loading on GNPs was confirmed by the change in the colour of the colloidal
solution from red to purple and the decrease in intensity of the SPR band of
spherical GNPs at 519nm with the slight shift in its λmax. PS increased to
115.91±0.99nm confirming GNPs aggregation into large nanoclusters of Mag-
GNPs while ζ significantly decreased to −35.0mV.
➢ Mag-GNPs exhibited greater than a two-fold increase in release over the free
drug.
184
Summary
➢ BSA decoration of Mag-GNPs led to desorption of Mag from the surface of NPs
which is confirmed by the decrease in PS and the concentration dependent
decrease in the ζ.
➢ Mag-GNPs/PLGA NPs were prepared to protect from Mag desorption and
increase the drug payloads.
➢ Formula P2 with organic phase composed of 25µL of concentrated GNPs
solution (10mM), 25mg PLGA,7.5mg TPGS and 5mg Mag dissolved in 2ml
acetone was considered the best formula based on PS and PDI.
➢ The encapsulation of gold inside PLGA was confirmed by TEM-EDX and FT-
IR analysis.
➢ Similar to Mag-PLGA NPs, Mag-GNPs/PLGA NPs showed delayed burst effect
at 24hr to release 58% w/w followed by sustained release.
➢ Mag-GNPs/PLGA NPs were conjugated to TZB by covalent methods using
optimum Ab/NPs ratio (1/10).
➢ TZB conjugation was confirmed by FT-IR, PS, ζ and % Ab bound: An increase
of ∼8 nm in the hydrodynamic diameter of Mag-GNPs/PLGA NPs was obtained
after addition of TZB. The negative value of ζ of the modified NPs
(−8.15±0.95mV) was lower than that previously reported for unmodified Mag-
GNPs/PLGA NPs (−23.80±2.83mV).
➢ The change in the FT-IR spectrum in Mag-GNPs/PLGA-TZB NPs was found to
be similar to Mag PLGA-TZB NPs previously reported in chapter 1.
➢ The %EE of Mag was 86.85±2.53 % w/w in unconjugated NPs, while in TZB
conjugated NPs, the %EE significantly decreased to 81.4±1.82% w/w due to loss
or release of drug during the conjugation process.
➢ The %TZB conjugated to Mag-GNPs/PLGA NPs measured by BCA protein
assay was found to be 66.8±3.73% w/w, which is correlated to the results of
PLGA NPs without gold.
185
Summary
Chapter 3: Biological evaluation and photothermal effect of magnolol and gold

nanoparticles encapsulated in targeted PLGA nanoparticles.
1. Cytotoxicity evaluation by MTT assay

Cytotoxicity of each of the selected formulae in chapter 1 and 2 along with
their plain ones and the pure drug in solution was evaluated on the breast cancer cells
(MCF-7) using MTT assay.
2. Photothermal therapy (PTT)

This study was applied to study the potential of GNPs as a suitable agent for
plasmonic photothermal therapy (PPTT). The MTT assay was performed on MCF-
7 cells treated with either plain GNPs, Mag-GNPs/PLGA NPs and Mag-
GNPs/PLGA-TZB NPs exposed to a LED with a specific wavelength.
3. Cell uptake and internalization studies

Nile red labeled plain and targeted GNPs/PLGA NPs were prepared by the
nanoprecipitation method, as in chapter 1. The MCF-7 breast cancer cells were then
incubated with Nile red labeled NPs for 15, 60 and 120min to allow for cell uptake.
Confocal microscope was used to assess NPs uptake and internalization.
4. Evaluation of targeting efficiency

For confirming targeting efficiency, MCF-7 cells were pretreated with excess
free TZB (100µg/mL) before incubation with targeted NPs in MTT assay.
➢ Cytotoxicity results confirmed the safety of blank PLGA NPs and free TZB.
➢ Mag loaded NPs showed significant decrease in cell viability when compared
with free Mag with recorded IC50 of 1.81 and 2.92µg/mL, respectively.
186
Summary
➢ Dark cytotoxicity study didn’t show any significant difference in cell viability
between Mag-PLGA NPs and Mag-GNPs/PLGA NPs. Also targeting with TZB
had no effect on cytotoxicity.
➢ Irradiation of MCF-7 cell with green LED at 530nm in presence with spherical
blank GNPs did not show any significant different effect from dark test (p>0.05).
While significant decrease (p<0.05) was obtained after red LED irradiation at
650nm due to the clustering of GNPs in MCF-7 cells providing a significant
shift of plasmonic resonance to NIR range that made NIR more effective with
spherical NPs.
➢ Mag-GNPs/PLGA NPs significantly (p<0.05) reduced MCF-7 cell viability
compared with plain GNPs after irradiation with red LED at 650nm with reported
IC50 of 1.38 and 305.4µg/mL, respectively.
➢ The confocal microscopic images with respect to time confirmed the uptake of
both targeted and non targeted Nile red-GNPs/PLGA NPs inside the cell and their
localization into the cytoplasm with targeted NPs showing relatively faster
uptake.
➢ The efficacy of the active targeting of Mag-GNPs/PLGA NPs with TZB had been
proved by the decrease in cytotoxicity after blocking HER2 receptors with free
TZB before targeting.
187
References
References
1. ADJEI, I. M., SHARMA, B. & LABHASETWAR, V. 2014. Nanoparticles:

cellular uptake and cytotoxicity. Adv Exp Med Biol, 811, 73-91.
2. AI, J., BIAZAR, E., JAFARPOUR, M., MONTAZERI, M., MAJDI, A.,
AMINIFARD, S., ZAFARI, M., AKBARI, H. R. & RAD, H. G. 2011.
Nanotoxicology and nanoparticle safety in biomedical designs. Int J Nanomed, 6,
1117-1127.
3. AL-JAWAD, S. M., TAHA, A. A., AL-HALBOSIY, M. M. & AL-BARRAM, L.

F. 2018. Synthesis and characterization of small-sized gold nanoparticles coated
by bovine serum albumin (BSA) for cancer photothermal therapy. Photodiagnosis
Photodyn Ther, 21, 201-210.
4. ALDEWACHI, H., CHALATI, T., WOODROOFE, M. N., BRICKLEBANK, N.,

SHARRACK, B. & GARDINER, P. 2018. Gold nanoparticle-based colorimetric
biosensors. Nanoscale, 10(1), 18-33.
5. ALLÉMANN, E., GURNY, R. & DOELKER, E. 1992. Preparation of aqueous

polymeric nanodispersions by a reversible salting-out process: influence of
process parameters on particle size. Int J Pharm, 87(1-3), 247-253.
6. ALMAKI, J. H., NASIRI, R., IDRIS, A., NASIRI, M., MAJID, F. A. A. &
LOSIC, D. 2017. Trastuzumab-decorated nanoparticles for in vitro and in vivo
tumor-targeting hyperthermia of HER2+ breast cancer. J Mater Chem B, 5(35),
7369-7383.
7. ARJMANDI, N., VAN ROY, W., LAGAE, L. & BORGHS, G. 2012. Measuring
the electric charge and zeta potential of nanometer-sized objects using pyramidal-
shaped nanopores. Anal Chem, 84(20), 8490-8496.
188
References
8. ARNEDOS, M., BIHAN, C., DELALOGE, S. & ANDRE, F. 2012. Triple-

negative breast cancer: are we making headway at least? Ther Adv Med Oncol,
4(4), 195-210.
9. ARVIZO, R., BHATTACHARYA, R. & MUKHERJEE, P. 2010. Gold

nanoparticles: opportunities and challenges in nanomedicine. Expert Opin Drug
Del, 7, 753-763.
10. AUBIN-TAM, M-E. 2013. Conjugation of nanoparticles to proteins. Methods

Mol Biol, 1025, 19-27.
11. AYDıN, R. 2013. Herceptin‑decorated salinomycin‑loaded nanoparticles for

breast tumor targeting. J Biomed Mater Res A, 101(5), 1405-1415.
12. BAGULEY, B. C. & LEUNG, E. 2011. Heterogeneity of phenotype in breast

cancer cell lines. 2011. Breast cancer-carcinogenesis, cell growth and signalling
pathways. In Tech, 11, 245-256.
13. BANERJEE, R., PARIDA, S., MAITI, C., MANDAL, M. & DHARA, D. 2015.
pH-degradable and thermoresponsive water-soluble core cross-linked polymeric
nanoparticles as potential drug delivery vehicle for doxorubicin. Rsc Adv, 5,
83565-83575.
14. BARUA, S., YOO, J.-W., KOLHAR, P., WAKANKAR, A., GOKARN, Y. R.
& MITRAGOTRI, S. 2013. Particle shape enhances specificity of antibody-
displaying nanoparticles. P Natl Acad Sci, 110(9), 3270-3275.
15. BASU, S., MUKHERJEE, B., CHOWDHURY, S. R., PAUL, P.,

CHOUDHURY, R., KUMAR, A., MONDAL, L., HOSSAIN, C. M. & MAJI, R.
2012. Colloidal gold-loaded, biodegradable, polymer-based stavudine
189
References
nanoparticle uptake by macrophages: an in vitro study. Int J Nanomed, 7, 6049-

6061.
16. BAZAK, R., HOURI, M., EL ACHY, S., HUSSEIN, W. & REFAAT, T. 2014.
Passive targeting of nanoparticles to cancer: a comprehensive review of the
literature. Mol Clin Oncol, 2(6), 904-908.
17. BAZAK, R., HOURI, M., EL ACHY, S., KAMEL, S. & REFAAT, T. 2015.
Cancer active targeting by nanoparticles: a comprehensive review of literature. J
Cancer Res Clin, 141, 769-784.
18. BEG, S., RIZWAN, M., SHEIKH, A. M., HASNAIN, M. S., ANWER, K. &
KOHLI, K. 2011. Advancement in carbon nanotubes: basics, biomedical
applications and toxicity. J Pharm Pharmacol, 63(2), 141-163.
19. BENITA, S., BENOIT, J., PUISIEUX, F. & THIES, C. 1984. Characterization
of drug‑loaded poly (d, l‑lactide) microspheres. J Pharm Sci, 73(12), 1721-1724.
20. BHATIA, S. 2016. Nanoparticles types, classification, characterization,

fabrication methods and drug delivery applications. Natural Polymer Drug
Delivery Systems, 2, 33-93
21. BHAU, B., GHOSH, S., PURI, S., BORAH, B., SARMAH, D. & KHAN, R.
2015. Green synthesis of gold nanoparticles from the leaf extract of Nepenthes
khasiana and antimicrobial assay. Adv Mater Lett, 6, 55-58.
22. BISHAYEE, K., KHUDA-BUKHSH, A. R. & HUH, S.-O. 2015. PLGA-loaded

gold-nanoparticles precipitated with quercetin downregulate HDAC-Akt
activities controlling proliferation and activate p53-ROS crosstalk to induce
apoptosis in hepatocarcinoma cells. Mol Cells, 38(6), 518-527.
190
References
23. BLANCO, E., SHEN, H. & FERRARI, M. 2015. Principles of nanoparticle

design for overcoming biological barriers to drug delivery. Nat Biotechnol, 33(9),
941-951.
24. BREWER, S. H., GLOMM, W. R., JOHNSON, M. C., KNAG, M. K. &

FRANZEN, S. 2005. Probing BSA binding to citrate-coated gold nanoparticles
and surfaces. Langmuir, 21(20), 9303-9307.
25. BRUST, M., WALKER, M., BETHELL, D., SCHIFFRIN, D. J. & WHYMAN,
R. 1994. Synthesis of thiol-derivatized gold nanoparticles in a two-phase liquid–
liquid system. J Chem Soc Chem Commun, 7(7), 801-802.
26. BURDA, C., CHEN, X., NARAYANAN, R. & EL-SAYED, M. A. 2005.

Chemistry and properties of nanocrystals of different shapes. Chem Rev, 105(4),
1025-1102.
27. CAI, W., GAO, T., HONG, H. & SUN, J. 2008. Applications of gold
nanoparticles in cancer nanotechnology. Nanotechnol Sci Appl, 1, 17-32.
28. CARTIERA, M. S., JOHNSON, K. M., RAJENDRAN, V., CAPLAN, M. J. &

SALTZMAN, W. M. 2009. The uptake and intracellular fate of PLGA
nanoparticles in epithelial cells. Biomaterials, 30(14), 2790-2798.
29. CHAUHAN, D. S. & SRIVASTAVA, R. 2015. Synthesis and characterization

of gold encapsulated and tamoxifen loaded PLGA nanoparticles for breast cancer
theranostics. 9th IEEE International Conference on Nano/Molecular Medicine &
Engineering (Nanomed), 143-146.
30. CHAURASIA, V. & PAL, S. 2014. Data mining techniques: To predict and
resolve breast cancer survivability. Int J Comp Sci Mobile Computing, 3(1), 10-
22.
191
References
31. CHEN, H., ZHENG, Y., TIAN, G., TIAN, Y., ZENG, X., LIU, G., LIU, K., LI,
L., LI, Z. & MEI, L. 2011b. Oral delivery of DMAB-modified docetaxel-loaded
PLGA-TPGS nanoparticles for cancer chemotherapy. Nanoscale Res Lett, 6(1),
4-14.
32. CHEN, Y.-H., HUANG, P.-H., LIN, F.-Y., CHEN, W.-C., CHEN, Y.-L., YIN,
W.-H., MAN, K.-M. & LIU, P.-L. 2011a. Magnolol: a multifunctional compound
isolated from the chinese medicinal plant Magnolia officinalis. Eur J Integr Med,
3(4), 317-324.
33. CHOI, S. Y., PARK, S. J., KIM, W. J., YANG, J. E., LEE, H., SHIN, J. & LEE,
S. Y. 2016. One-step fermentative production of poly (lactate-co-glycolate) from
carbohydrates in Escherichia coli. Nat Biotechnol, 34(4), 435-440.
34. CHUANG, T.-C., HSU, S.-C., CHENG, Y.-T., SHAO, W.-S., WU, K., FANG,
G.-S., OU, C.-C. & WANG, V. 2011. Magnolol down-regulates HER2 gene
expression, leading to inhibition of HER2-mediated metastatic potential in
ovarian cancer cells. Cancer Lett, 311(1), 11-19.
35. CLARKE, R., BRÜNNER, N., KATZENELLENBOGEN, B. S., THOMPSON,

E. W., NORMAN, M. J., KOPPI, C., PAIK, S., LIPPMAN, M. E. & DICKSON,
R. B. 1989. Progression of human breast cancer cells from hormone-dependent to
hormone-independent growth both in vitro and in vivo. P Natl Acad Sci, 86(10),
3649-3653.
36. CLOGSTON, J. D. & PATRI, A. K. 2011. Zeta potential measurement in:

Characterization of nanoparticles intended for drug delivery, McNeil, Scott E.,
697, 63-70.
192
References
37. CORR, S. J., RAOOF, M., MACKEYEV, Y., PHOUNSAVATH, S., CHENEY,
M. A., CISNEROS, B. T., SHUR, M., GOZIN, M., MCNALLY, P. J. &
WILSON, L. J. 2012. Citrate-capped gold nanoparticle electrophoretic heat
production in response to a time-varying radio-frequency electric field. J Phys
Chem C, 116, 24380-24389.
38. COSTA, E. C., GASPAR, V. M., MARQUES, J. G., COUTINHO, P. &

CORREIA, I. J. 2013. Evaluation of nanoparticle uptake in co-culture cancer
models. Plos One, 8(7), 70072-70085.
39. CRAIG, G. E., BROWN, S. D., LAMPROU, D. A., GRAHAM, D. &

WHEATE, N. J. 2012. Cisplatin-tethered gold nanoparticles that exhibit enhanced
reproducibility, drug loading, and stability: a step closer to pharmaceutical
approval? Inorg Chem, 51(6), 3490-3497.
40. CREF, R., MINAMITAKE, Y., PERACCHIA, M., TRUBETSKOY, V.,

TORCHILIN, V. & LANGER, R. 1994. Biodegradable long circulating polymer
nanospheres. Science, 263(5153), 1600-1603.
41. CUN, D., FOGED, C., YANG, M., FRØKJÆR, S. & NIELSEN, H. M. 2010.
Preparation and characterization of poly (DL-lactide-co-glycolide) nanoparticles
for siRNA delivery. Int J Pharm, 390(1), 70-75.
42. CURRY, D., CAMERON, A., MACDONALD, B., NGANOU, C.,

SCHELLER, H., MARSH, J., BEALE, S., LU, M., SHAN, Z. &
KALIAPERUMAL, R. 2015. Adsorption of doxorubicin on citrate-capped gold
nanoparticles: insights into engineering potent chemotherapeutic delivery
systems. Nanoscale, 7, 19611-19619.
193
References
43. DANHIER, F., ANSORENA, E., SILVA, J. M., COCO, R., LE BRETON, A.
& PRÉAT, V. 2012. PLGA-based nanoparticles: an overview of biomedical
applications. J Control Release, 161(2), 505-522.
44. DAS, T., KOLLI, V., KARMAKAR, S. & SARKAR, N. 2017.

Functionalisation of polyvinylpyrrolidone on gold nanoparticles enhances its anti-
amyloidogenic propensity towards hen egg white lysozyme. Biomedicine, 5(2),
19-33.
45. DAVDA, J. & LABHASETWAR, V. 2002. Characterization of nanoparticle

uptake by endothelial cells. Int J Pharm, 233, 51-59.
46. DE JONG, W. H. & BORM, P. J. 2008. Drug delivery and nanoparticles:

applications and hazards. Int J Nanomed, 3(2), 133-149.
47. DEAN, L. 2015. Trastuzumab (herceptin) therapy and ERBB2 (HER2)

genotype. Medical Genetics Summaries.
48. DENG, W., KAUTZKA, Z., CHEN, W. & GOLDYS, E. M. 2016. PLGA
nanocomposites loaded with verteporfin and gold nanoparticles for enhanced
photodynamic therapy of cancer cells. Rsc Adv, 6, 112393-112402.
49. DENIZOT, F. & LANG, R. 1986. Rapid colorimetric assay for cell growth and
survival: modifications to the tetrazolium dye procedure giving improved
sensitivity and reliability. J Immunol Methods, 89, 271-277.
50. DERAKHSHANDEH, K. & SOLEYMANI, M. 2014. Formulation and in vitro

evaluation of nifedipine-controlled release tablet: Influence of combination of
hydrophylic and hydrophobic matrix forms. Asian J Pharm, 4, 185-193.
194
References
51. DERMAN, S., MUSTAFAEVA, Z. A., ABAMOR, E. S., BAGIROVA, M. &

ALLAHVERDIYEV, A. 2015. Preparation, characterization and immunological
evaluation: canine parvovirus synthetic peptide loaded PLGA nanoparticles. J
Biomed Sci, 22, 89-101.
52. DHANKAR, R., RATHEE, P., JAIN, A. K., ARORA, S., KUMAR, M. S.,
RATH, G., SAXENA, A. K., SHARMA, P., CHASHOO, G. & GOYAL, A. K.
2011. HER2 targeted immunonanoparticles for breast cancer chemotherapy. J
Appl Pharm Sci, 3, 132-139.
53. DI PASQUA, A. J., WALLNER, S., KERWOOD, D. J. & DABROWIAK, J. C.

2009. Adsorption of the PtII anticancer drug carboplatin by mesoporous silica.
Chem Biodivers, 6(9), 1343-1349.
54. DIESSNER, J., BRUTTEL, V., STEIN, R., HORN, E., HÄUSLER, S., DIETL,
J., HÖNIG, A. & WISCHHUSEN, J. 2014. Targeting of preexisting and induced
breast cancer stem cells with trastuzumab and trastuzumab emtansine (T-DM1).
Cell Death Dis, 5, 1149-1162.
55. DIXON, D., MEENAN, B. & MANSON, J. 2014. PEG Functionalized Gold
Nanoparticle loaded PLGA Films for Drug Delivery. J Nano R, 27, 83-94.
56. DOBSON, J. 2006. Magnetic nanoparticles for drug delivery. Drug Develop
Res, 67, 55-60.
57. DREADEN, E. C., AUSTIN, L. A., MACKEY, M. A. & EL-SAYED, M. A.

2012. Size matters: gold nanoparticles in targeted cancer drug delivery. Ther
Deliv, 3(4), 457-478.
195
References
58. DU, Y., XIA, L., JO, A., DAVIS, R. M., BISSEL, P., EHRICH, M. &
KINGSTON, D. G. I. 2018. Synthesis and evaluation of doxorubicin-loaded gold
nanoparticles for tumor-targeted drug delivery. Bioconj Chem, ahead of print.
59. DUCAT, E., EVRARD, B., PEULEN, O. & PIEL, G. 2011. Cellular uptake of
liposomes monitored by confocal microscopy and flow cytometry. J Drug Deliv
Sci Tec, 21(6), 469-477.
60. DUMALAON-CANARIA, J. A., HUTCHINSON, A. D., PRICHARD, I. &

WILSON, C. 2014. What causes breast cancer? A systematic review of causal
attributions among breast cancer survivors and how these compare to expert-
endorsed risk factors. Cancer Cause Control, 25(7), 771-785.
61. DUNCAN, B., KIM, C. & ROTELLO, V. M. 2010. Gold nanoparticle platforms
as drug and biomacromolecule delivery systems. J Control Release, 148(1), 122-
127.
62. DUZGUNES, N. 2012. Nanomedicine: cancer, diabetes, and cardiovascular,

central nervous system, pulmonary and inflammatory diseases. Academic Press,
508.
63. ECHEVERRÍA, J. C., ESTELLA, J., BARBERÍA, V., MUSGO, J. &

GARRIDO, J. J. 2010. Synthesis and characterization of ultramicroporous silica
xerogels. J Non-Cryst Solids, 356(6-8), 378-382.
64. EL-SAYED, I. H., HUANG, X. & EL-SAYED, M. A. 2006. Selective laser

photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody
conjugated gold nanoparticles. Cancer Lett, 239(1), 129-135.
196
References
65. ESMAEILI, F., ATYABI, F. & DINARVAND, R. 2007. Preparation of PLGA

nanoparticles using TPGS in the spontaneous emulsification solvent diffusion
method. J Exp Nanosci, 2(3), 183-192.
66. FABIAN, C. 2007. The what, why and how of aromatase inhibitors: hormonal
agents for treatment and prevention of breast cancer. Int J Clin Pract, 61(12),
2051-2063.
67. FARRÉ, J. C. 2013. Gold nanoparticles as drug delivery agents. Detoxifying the
chemotherapeutic drug cisplatin. Ph D thesis, Universitat Autònoma de
Barcelona.
68. FENG, B., ASHRAF, M. A. & PENG, L. 2016. Characterization of particle

shape, zeta potential, loading efficiency and outdoor stability for chitosan-
ricinoleic acid loaded with rotenone. Open Life Sci, 11(1), 380-386.
69. FESSI, H., PUISIEUX, F., DEVISSAGUET, J. P., AMMOURY, N. &

BENITA, S. 1989. Nanocapsule formation by interfacial polymer deposition
following solvent displacement. Int J Pharmaceut, 55(1), R1-R4.
70. FLORENTO, L., MATIAS, R., TUAÑO, E., SANTIAGO, K., DELA CRUZ, F.
& TUAZON, A. 2012. Comparison of cytotoxic activity of anticancer drugs
against various human tumor cell lines using in vitro cell-based approach. Int J
Biomed Sci, 8(1), 76-80.
71. FOTAKIS, G. & TIMBRELL, J. A. 2006. In vitro cytotoxicity assays:

comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines
following exposure to cadmium chloride. Toxicology Lett, 160(2), 171-177.
197
References
72. GAMUCCI, O., BERTERO, A., GAGLIARDI, M. & BARDI, G. 2014.

Biomedical nanoparticles: overview of their surface immune-compatibility.
Coatings, 4(1), 139-159.
73. GANANATHAN, P. 2016. Plasmonic phototherapy of gold nanoparticles with

light emitting diode. Int J Biome Res, 7(7), 511-519.
74. GAO, J., ZHONG, W., HE, J., LI, H., ZHANG, H., ZHOU, G., LI, B., LU, Y.,
ZOU, H. & KOU, G. 2009. Tumor-targeted PE38KDEL delivery via PEGylated
anti-HER2 immunoliposomes. Int J Pharmaceut, 374(1-2), 145-152.
75. GAONKAR, R. H., GANGULY, S., DEWANJEE, S., SINHA, S., GUPTA, A.,
GANGULY, S., CHATTOPADHYAY, D. & DEBNATH, M. C. 2017. Garcinol
loaded vitamin E TPGS emulsified PLGA nanoparticles: preparation,
physicochemical characterization, in vitro and in vivo studies. Sci Rep, 7(1), 530-
544.
76. GILJOHANN, D. A., SEFEROS, D. S., PRIGODICH, A. E., PATEL, P. C. &

MIRKIN, C. A. 2009. Gene regulation with polyvalent siRNA− nanoparticle
conjugates. J Am Chem Soc, 131, 2072-2073.
77. GOLE, A., DASH, C., RAMAKRISHNAN, V., SAINKAR, S., MANDALE,
A., RAO, M. & SASTRY, M. 2001. Pepsin− gold colloid conjugates: preparation,
characterization, and enzymatic activity. Langmuir, 17, 1674-1679.
78. GOMAA, I. E., GABER, S. A. A., BHATT, S., LIEHR, T., GLEI, M., EL-
TAYEB, T. A. & ABDEL-KADER, M. H. 2015. In vitro cytotoxicity and
genotoxicity studies of gold nanoparticles-mediated photo-thermal therapy vs 5-
fluorouracil. J Nanopart Res, 17, 102-113.
198
References
79. GRUMEZESCU, A. 2016. Nutraceuticals in: Nanotechnology in the food

industry, volume 4.
80. GÜÇ, E., GÜNDÜZ, G. & GÜNDÜZ, U. 2010. Fatty acid based hyperbranched
polymeric nanoparticles for hydrophobic drug delivery. Drug Dev Ind Pharm,
36(10), 1139-1148.
81. GUHAGARKAR, S. A., MALSHE, V. C. & DEVARAJAN, P. V. 2009.

Nanoparticles of polyethylene sebacate: a new biodegradable polymer. AAPS
Pharm Sci Tech, 10(3), 935-942.
82. GUO, Y., LUO, J., TAN, S., OTIENO, B. O. & ZHANG, Z. 2013. The
applications of Vitamin E TPGS in drug delivery. Eur J Pharm Sci, 49(2), 175-
186.
83. HAINFELD, J. F., O'CONNOR, M. J., LIN, P., QIAN, L., SLATKIN, D. N. &
SMILOWITZ, H. M. 2014. Infrared-transparent gold nanoparticles converted by
tumors to infrared absorbers cure tumors in mice by photothermal therapy. Plos
one, 9(2), 88414-88428.
84. HAN, G., GHOSH, P. & ROTELLO, V. M. 2007. Multi-functional gold

nanoparticles for drug delivery. Adv Exp Med Biol, 620, 48-56.
85. HAN, H. & DAVIS, M. E. 2013. Single-antibody, targeted nanoparticle delivery

of camptothecin. Mol Pharm, 10(7), 2558-2567.
86. HEDKVIST, O. 2013. Synthesis and characterization of gold nanoparticles. J

Organomet Chem, 693, 1043-1048.
199
References
87. HIROTA, K. & TERADA, H. 2012. Endocytosis of particle formulations by

macrophages and its application to clinical treatment in Molecular Regulation
Of Endocytosis, edited by Brian Ceresa-Open access book, chapter 16, 413-428.
88. HO, S. V., MCLAUGHLIN, J. M., CUE, B. W. & DUNN, P. J. 2010.

Environmental considerations in biologics manufacturing. Green Chem, 12,
755-766.
89. HOLZER, M., VOGEL, V., MÄNTELE, W., SCHWARTZ, D., HAASE, W. &
LANGER, K. 2009. Physico-chemical characterisation of PLGA nanoparticles
after freeze-drying and storage. Eur J Pharm Biopharm, 72(2), 428-437.
90. HONG, E. J., CHOI, D. G. & SHIM, M. S. 2016. Targeted and effective
photodynamic therapy for cancer using functionalized nanomaterials. Acta Pharm
Sin B, 6(4), 297-307.
91. HORTOBAGYI, G. N. 2005. Trastuzumab in the treatment of breast cancer. N

Engl J Med, 353, 1734-1736.
92. HU, M., CHEN, J., LI, Z.-Y., AU, L., HARTLAND, G. V., LI, X., MARQUEZ,
M. & XIA, Y. 2006. Gold nanostructures: engineering their plasmonic properties
for biomedical applications. Chem Soc Rev, 35(11), 1084-1094.
93. HUANG, X. & EL-SAYED, M. A. 2010. Gold nanoparticles: optical properties

and implementations in cancer diagnosis and photothermal therapy. J Adv Res,
1(1), 13-28.
94. HUANG, X & EL-SAYED, M. A. 2006. Gold nanoparticles used in cancer cell
diagnostics, selective photothermal therapy and catalysis of NADH oxidation
reaction. Ph D thesis, Georgia Institute of Technology.
200
References
95. HUANG, X. & EL-SAYED, M. A. 2011. Plasmonic photo-thermal therapy

(PPTT). Alexandria J Med, 47(1), 1-9.
96. ICH H. T. guideline, 2005. Validation of analytical procedures: text and

methodology Q2 (R1). International Conference on Harmonization, Geneva,
Switzerland, 2005, 1-13.
97. IODICE, C., CERVADORO, A., PALANGE, A., KEY, J., ARYAL, S.,
RAMIREZ, M. R., MATTU, C., CIARDELLI, G., O’NEILL, B. E. & DECUZZI,
P. 2016. Enhancing photothermal cancer therapy by clustering gold nanoparticles
into spherical polymeric nanoconstructs. Opt Laser Eng, 76, 74-81.
98. JAHAN, S. T. & HADDADI, A. 2015. Investigation and optimization of

formulation parameters on preparation of targeted anti-CD205 tailored PLGA
nanoparticles. Int J Nanomed, 10, 7371.
99. JAIDEV, L., KRISHNAN, U. M. & SETHURAMAN, S. 2015. Gemcitabine

loaded biodegradable PLGA nanospheres for in vitro pancreatic cancer therapy.
Mater Sci Eng C Mater Biol Appl, 47, 40-47.
100. JAWAHAR, N. & MEYYANATHAN, S. 2012. Polymeric nanoparticles for

drug delivery and targeting: a comprehensive review. Int J Health Allied Sci, 1(4),
217-223.
101. JAWAHAR, N., EAGAPPANATH, T., VENKATESH, N., JUBIE, S. &

SAMANTA, M. 2009. Preparation and characterisation of PLGA-nanoparticles
containing an anti-hypertensive agent. Int J Pharm Tech Res, 1, 390-393.
102. JAYALAKSHMI, K., IBRAHIM, M. & RAO, K. V. 2014. Effect of pH on the

Size of Gold Nanoparticles. Int J of Electron Elec Eng, 7(2), 159-164.
201
References
103. JEMAL, A., BRAY, F., CENTER, M. M., FERLAY, J., WARD, E. &
FORMAN, D. 2011. Global cancer statistics. CA Cancer J Clin, 61(2), 69-90.
104. JOKERST, J. V., LOBOVKINA, T., ZARE, R. N. & GAMBHIR, S. S. 2011.

Nanoparticle PEGylation for imaging and therapy. Nanomedicine, 6(4), 715-728.
105. JOSHI, M., BHATTACHARYYA, A. & ALI, S. W. 2008. Characterization

techniques for nanotechnology applications in textiles. Indian J Fiber Text Res,
33(3), 304-317
106. KAMALY, N., XIAO, Z., VALENCIA, P. M., RADOVIC-MORENO, A. F. &

FAROKHZAD, O. C. 2012. Targeted polymeric therapeutic nanoparticles:
design, development and clinical translation. Chem Soc Rev, 41(7), 2971-3010.
107. KHALED, A., MOHAMED A., WAEL A., GAMAL Z, MONTASSER S. &
Mohamed S. 2016. Synthesis and characterization of gold nanoparticles loaded
with 5-fluorouracil. Int J Pharm Pharm Res, 6, 412-422.
108. KHAN, A., RASHID, R., MURTAZA, G. & ZAHRA, A. 2014. Gold
nanoparticles: synthesis and applications in drug delivery. Trop J Pharm Res, 13,
1169-1177.
109. KHAN, I., SAEED, K. & KHAN, I. 2017. Nanoparticles: properties,

applications and toxicities. Arab J Chem, in press.
110. KHANDELIA, R., JAISWAL, A., GHOSH, S. S. & CHATTOPADHYAY, A.

2014. Polymer coated gold nanoparticle–protein agglomerates as nanocarriers for
hydrophobic drug delivery. J Mater Chem B, 2(38), 6472-6477.
111. KIM, G. D., OH, J., PARK, H.-J., BAE, K. & LEE, S. K. 2013. Magnolol
inhibits angiogenesis by regulating ROS-mediated apoptosis and the
202
References
PI3K/AKT/mTOR signaling pathway in mES/EB-derived endothelial-like cells.

Int J Oncol, 43(2), 600-610.
112. KIROULA, N., NEGI, J., SINGH, K., RAWAT, R. & SINGH, B. 2016.
Preparation and characterization of ganciclovir-loaded glutathione modified gold
nanoparticles. Indian J Pharm Sci, 78, 313-319.
113. KOCBEK, P., OBERMAJER, N., CEGNAR, M., KOS, J. & KRISTL, J. 2007.
Targeting cancer cells using PLGA nanoparticles surface modified with
monoclonal antibody. J Control Release, 120(1-2), 18-26.
114. KOOPAEI, M. N., DINARVAND, R., AMINI, M., RABBANI, H., EMAMI,
S., OSTAD, S. N. & ATYABI, F. 2011. Docetaxel immunonanocarriers as
targeted delivery systems for HER 2-positive tumor cells: preparation,
characterization, and cytotoxicity studies. Int J Nanomed, 6, 1903-1912.
115. KOU, L., SUN, J., ZHAI, Y. & HE, Z. 2013. The endocytosis and intracellular
fate of nanomedicines: Implication for rational design. Asian J Pharm Res, 8(1),
1-10.
116. KOVACEVIC, A., SAVIC, S., VULETA, G., MÜLLER, R. & KECK, C. M.
2011. Polyhydroxy surfactants for the formulation of lipid nanoparticles (SLN and
NLC): effects on size, physical stability and particle matrix structure. INT J
Pharmaceut, 406(1-2), 163-172.
117. KRISHNA, R. & MAYER, L. D. 2000. Multidrug resistance (MDR) in cancer:

mechanisms, reversal using modulators of MDR and the role of MDR modulators
in influencing the pharmacokinetics of anticancer drugs. Eur J Pharm Sci, 11(4),
265-283.
203
References
118. KRISHNA, R., SHIVAKUMAR, H., GOWDA, D. & BANERJEE, S. 2006.

Nanoparticles-a novel colloidal drug delivery system. Indian J Pharm Edu, 40, 15-
21.
119. KRISHNAMACHARI, Y., MADAN, P. & LIN, S. 2007. Development of pH-

and time-dependent oral microparticles to optimize budesonide delivery to ileum
and colon. Int J Pharmaceut, 338(1-2), 238-247.
120. KUHN, D. A., VANHECKE, D., MICHEN, B., BLANK, F., GEHR, P.,
PETRI-FINK, A. & ROTHEN-RUTISHAUSER, B. 2014. Different endocytotic
uptake mechanisms for nanoparticles in epithelial cells and macrophages.
Beilstein J Nanotech, 5, 1625-1636.
121. KULHARI, H., POOJA, D., SHRIVASTAVA, S., KUNCHA, M., NAIDU, V.,
BANSAL, V., SISTLA, R. & ADAMS, D. J. 2016. Trastuzumab-grafted
PAMAM dendrimers for the selective delivery of anticancer drugs to HER2-
positive breast cancer. Sci Rep, 6, 23179-23192.
122. KUMAR KHANNA, V. 2012. Targeted delivery of nanomedicines. ISRN

Pharmacol, 2012, 571394-571401.
123. KUMAR, C. S., HORMES, J. & LEUSCHNER, C. 2006. Nanofabrication

towards biomedical applications: Techniques, tools, applications, and impact,
Angew Chem, 117(29):4504-4505.
124. KURIYAN, J., KONFORTI, B. & WEMMER, D. 2015. The molecules of life:
Physical and chemical principles. Yale J Biol Med, 88(1): 101–102.
125. KWON, S., SINGH, R. K., PEREZ, R. A., NEEL, E. A. A., KIM, H.-W. &
CHRZANOWSKI, W. 2013. Silica-based mesoporous nanoparticles for
controlled drug delivery. J Tissue Eng, 4: 2041731413503357, 1-18.
204
References
126. LEE, A. V., OESTERREICH, S. & DAVIDSON, N. E. 2015. MCF-7 cells—

Changing the course of breast cancer research and care for 45 years. J Natl Cancer
Inst, 107(7), 1-4.
127. LEE, C. Y. & OOI, I. H. 2016. Preparation of temozolomide-loaded

nanoparticles for glioblastoma multiforme targeting—ideal vs reality.
Pharmaceuticals, 9(54), 1-11.
128. LEE, J., CHATTERJEE, D. K., LEE, M. H. & KRISHNAN, S. 2014. Gold
nanoparticles in breast cancer treatment: promise and potential pitfalls. Cancer
Lett, 347(1), 46-53.
129. LEUSCHNER, C. & KUMAR, C. S. 2005. Nanoparticles for cancer drug

delivery, in: Nanofabrication towards biomedical applications: techniques, tools,
applications, and impact. Edited by Kumar, C, Hormes, J, Leuschner, C. John
Wiley & Sons. Chapter 11, 289-326.
130. LI, H.-B., YI, X., GAO, J.-M., YING, X.-X., GUAN, H.-Q. & LI, J.-C. 2007.
Magnolol-lnduced H460 cells death via autophagy but not apoptosis. Arch Pharm
Res, 30(12), 1566-1574.
131. LI, J., LI, Q., MA, X., TIAN, B., LI, T., YU, J., DAI, S., WENG, Y. & HUA,
Y. 2016a. Biosynthesis of gold nanoparticles by the extreme bacterium
Deinococcus radiodurans and an evaluation of their antibacterial properties. Int J
Nanomed, 11, 5931-5934.
132. LI, J.-L. & GU, M. 2010. Gold-nanoparticle-enhanced cancer photothermal

therapy. IEEE J Sel Top Quant, 16, 989-996.
133. LI, M., ZHANG, F., WANG, X. A., WU, X., ZHANG, B., ZHANG, N., WU,
W., WANG, Z., WENG, H. & LIU, S. 2015. Magnolol inhibits growth of
205
References
gallbladder cancer cells through the p53 pathway. Cancer Sci, 106(10), 1341-
1350.
134. LI, N., CHEN, Y., ZHANG, Y.-M., YANG, Y., SU, Y., CHEN, J.-T. & LIU,
Y. 2014. Polysaccharide-gold nanocluster supramolecular conjugates as a
versatile platform for the targeted delivery of anticancer drugs. Sci Rep, 4, 4164-
4171.
135. LI, S., ZOU, R., TU, Y., WU, J. & LANDRY, M. P. 2017. Cholesterol-directed
nanoparticle assemblies based on single amino acid peptide mutations activate
cellular uptake and decrease tumor volume. Chem Sci, 8(11), 7552-7559.
136. LI, W., ZHAO, X., DU, B., LI, X., LIU, S., YANG, X.-Y., DING, H., YANG,
W., PAN, F. & WU, X. 2016b. Gold nanoparticle–mediated targeted delivery of
recombinant human endostatin normalizes tumour vasculature and improves
cancer therapy. Sci Rep, 6, 30619-30630.
137. LIM, J., LEE, N.-E., LEE, E. & YOON, S. 2014. Surface modification of
citrate-capped gold nanoparticles using CTAB micelles. Bull Korean Chem Soc,
35, 2567-2569.
138. LIN, S. Y., LIU, J. D., CHANG, H. C., YEH, S. D., LIN, C. H. & LEE, W. S.
2002. Magnolol suppresses proliferation of cultured human colon and liver cancer
cells by inhibiting DNA synthesis and activating apoptosis. J Cell Biochem, 84(3),
532-544.
139. LIU, J., TIAN, J.-N., ZHANG, J., HU, Z. & CHEN, X. 2003. Interaction of
magnolol with bovine serum albumin: a fluorescence-quenching study. Anal
Bioanal Chem, 376(6), 864-867.
206
References
140. LIU, Y., CAO, W., ZHANG, B., LIU, Y.-Q., WANG, Z.-Y., WU, Y.-P., YU,
X.-J., ZHANG, X.-D., MING, P.-H. & ZHOU, G.-B. 2013. The natural compound
magnolol inhibits invasion and exhibits potential in human breast cancer therapy.
Sci Rep, 3, 3098-3107.
141. MA, Y., ZHENG, Y., LIU, K., TIAN, G., TIAN, Y., XU, L., YAN, F.,
HUANG, L. & MEI, L. 2010. Nanoparticles of poly (lactide-co-glycolide)-da-
tocopheryl polyethylene glycol 1000 succinate random copolymer for cancer
treatment. Nanoscale Res Lett, 5(7), 1161-1169.
142. MAAZ, A., ABDELWAHED, W., TEKKO, I. & TREFI, S. 2011. Influence of
nanoprecipitation method parameters on nanoparticles loaded with gatifloxacin
for ocular drug delivery. Int J Acad Sci Res, 3(1), 1-12
143. MACH, W. J., THIMMESCH, A. R., ORR, J. A., SLUSSER, J. G. & PIERCE,
J. D. 2010. Flow cytometry and laser scanning cytometry, a comparison of
techniques. J Clin Monitor, 24(4), 251-259.
144. MADHUSUDHAN, A., REDDY, G. B., VENKATESHAM, M.,

VEERABHADRAM, G., KUMAR, D. A., NATARAJAN, S., YANG, M.-Y.,
HU, A. & SINGH, S. S. 2014. Efficient pH dependent drug delivery to target
cancer cells by gold nanoparticles capped with carboxymethyl chitosan. Int J Mol
Sci, 15(5), 8216-8234.
145. MADY, M. M., AL-SHAIKH, F. H., AL-FARHAN, F. F., ALY, A. A., AL-
MOHANNA, M. A. & GHANNAM, M. M. 2015. Enhanced Ehrlich tumor
inhibition using DOX-NP™ and gold nanoparticles loaded liposomes. Pak J
Pharm Sci, 28, 2321-2325.
207
References
146. MAEDA, H., WU, J., SAWA, T., MATSUMURA, Y. & HORI, K. 2000.
Tumor vascular permeability and the EPR effect in macromolecular therapeutics:
a review. J Control Release, 65(1-2), 271-284.
147. MAHAKALKAR, A. & HATWAR, B. 2014. Biophysicochemical

characteristics & applications of nanoparticles: mini review. Am J Drug Deliv
Ther, 1(1), 35-41.
148. MAINARDES, R. M. & EVANGELISTA, R. C. 2005. PLGA nanoparticles

containing praziquantel: effect of formulation variables on size distribution. Int J
Pharm, 290(1-2), 137-144.
149. MAKADIA, H. K. & SIEGEL, S. J. 2011. Poly lactic-co-glycolic acid (PLGA)

as biodegradable controlled drug delivery carrier. Polymers, 3(3), 1377-1397.
150. MALHOTRA, G. K., ZHAO, X., BAND, H. & BAND, V. 2010. Histological,
molecular and functional subtypes of breast cancers. Cancer Biol Ther, 109(10),
955-960.
151. MANE, S., PONRATHNAM, S. & CHAVAN, N. 2016. Interfacial tension

approach toward drug loading with two-dimensional crosslinked polymer
embedded gold: Adsorption kinetics evaluation. Int J Polym Mater Polym
Biomaterf, 65(4), 168–175.
152. MANIVASAGAN, P., BHARATHIRAJA, S., BUI, N. Q., JANG, B., OH, Y.-
O., LIM, I. G. & OH, J. 2016. Doxorubicin-loaded fucoidan capped gold
nanoparticles for drug delivery and photoacoustic imaging. Int J Biol Macromol,
91, 578-588.
153. MANOOCHEHRI, S., DARVISHI, B., KAMALINIA, G., AMINI, M.,

FALLAH, M., OSTAD, S. N., ATYABI, F. & DINARVAND, R. 2013. Surface
208
References
modification of PLGA nanoparticles via human serum albumin conjugation for

controlled delivery of docetaxel. Daru, 21(1), 58-68.
154. MAO, S., SHI, Y., LI, L., XU, J., SCHAPER, A. & KISSEL, T. 2008. Effects
of process and formulation parameters on characteristics and internal morphology
of poly (d, l-lactide-co-glycolide) microspheres formed by the solvent evaporation
method. Eur J Pharm Biopharm, 68(2), 214-223.
155. MASOOD, F. 2016. Polymeric nanoparticles for targeted drug delivery system
for cancer therapy. Mat Sci Eng C, 60, 569-578.
156. MASTER, A., MALAMAS, A., SOLANKI, R., CLAUSEN, D. M.,

EISEMAN, J. L. & SEN GUPTA, A. 2013. A cell-targeted photodynamic
nanomedicine strategy for head and neck cancers. Mol Pharm, 10(5), 1988-1997.
157. MATTU, C., PABARI, R., BOFFITO, M., SARTORI, S., CIARDELLI, G. &
RAMTOOLA, Z. 2013. Comparative evaluation of novel biodegradable
nanoparticles for the drug targeting to breast cancer cells. Eur J Pharm Biopharm,
85(3 Pt A), 463-472.
158. MAXIMIANO, S., MAGALHÃES, P., GUERREIRO, M. P. & MORGADO,

M. 2016. Trastuzumab in the treatment of breast cancer. Biodrugs, 30(2), 75-86.
159. MCCALL, R. L. & SIRIANNI, R. W. 2013. PLGA nanoparticles formed by

single-or double-emulsion with vitamin E-TPGS. J Vis Exp, 82, 51015-51097.
160. MCKEOWN, B. T. & HURTA, R. A. 2015. Magnolol affects cellular

proliferation, polyamine biosynthesis and catabolism-linked protein expression
and associated cellular signaling pathways in human prostate cancer cells in vitro.
Functional Foods in Health and Disease, 5(1), 17-33.
209
References
161. MEHROTRA, A. & PANDIT, J. 2012. Critical process parameters evaluation

of modified nanoprecipitation method on lomustine nanoparticles and cytostatic
activity study on L132 human cancer cell line. J Nanomed Nanotechnol, 3(8), 1-
8.
162. MEISSNER, J., PRAUSE, A., BHARTI, B. & FINDENEGG, G. H. 2015.

Characterization of protein adsorption onto silica nanoparticles: influence of pH
and ionic strength. Colloid Polym Sci, 293(11), 3381-3391.
163. MENDES, R., PEDROSA, P., LIMA, J. C., FERNANDES, A. R. &

BAPTISTA, P. V. 2017. Photothermal enhancement of chemotherapy in breast
cancer by visible irradiation of gold nanoparticles. Sci Rep, 7, 10872-10880.
164. MENDOZA-NAVA, H., FERRO-FLORES, G., OCAMPO-GARCÍA, B.,

SERMENT-GUERRERO, J., SANTOS-CUEVAS, C., JIMÉNEZ-MANCILLA,
N., LUNA-GUTIÉRREZ, M. & CAMACHO-LÓPEZ, M. A. 2013. Laser heating
of gold nanospheres functionalized with octreotide: in vitro effect on HeLa cell
viability. Photo Med Laser Surg, 31(1), 17-22.
165. MISRA, R., UPADHYAY, M. & MOHANTY, S. 2014. Design considerations

for chemotherapeutic drug nanocarriers. Pharm Anal Acta, 5(1), 279, 1-10.
166. MOHAMED, M., ADBEL-GHANI, N., EL-BORADY, O. & EL-SAYED, M.

2012. 5-Fluorouracil induces plasmonic coupling in gold nanospheres: new
generation of chemotherapeutic agents. J Nanomed Nanotechno, 3(7), 1-7.
167. MOURA, C. C., SEGUNDO, M. A., DAS NEVES, J., REIS, S. &
SARMENTO, B. 2014. Co-association of methotrexate and SPIONs into anti-
CD64 antibody-conjugated PLGA nanoparticles for theranostic application. Int J
Nanomed, 9, 4911-4922.
210
References
168. MU, L. & FENG, S. 2003a. PLGA/TPGS nanoparticles for controlled release
of paclitaxel: effects of the emulsifier and drug loading ratio. Pharm Res, 20(11),
1864-1872.
169. MU, L. & FENG, S. 2003b. A novel controlled release formulation for the
anticancer drug paclitaxel (Taxol®): PLGA nanoparticles containing vitamin E
TPGS. J Control Release, 86(1), 33-48.
170. MU, L., CHAN-PARK, M. B.-E., YUE, C. Y. & FENG, S. 2004.

Pharmaceutical properties of nanoparticulate formulation composed of TPGS and
PLGA for controlled delivery of anticancer drug. Innovation in Manufacturing
Systems and Technology, 1-7.
171. MUDDINETI, O. S., GHOSH, B. & BISWAS, S. 2015. Current trends in using
polymer coated gold nanoparticles for cancer therapy. Int J Pharm, 484(1-2), 252-
267.
172. MUHAMAD, I. I., SELVAKUMARAN, S. & LAZIM, N. A. M. 2014.

Designing polymeric nanoparticles for targeted drug delivery system. Nanomed,
11, 287-313.
173. MUKHERJEE, B., SANTRA, K., PATTNAIK, G. & GHOSH, S. 2008.

Preparation, characterization and in-vitro evaluation of sustained release protein-
loaded nanoparticles based on biodegradable polymers. Int J Nanomed, 3(4), 487-
496.
174. MÜLLER, R., RADTKE, M. & WISSING, S. 2002. Nanostructured lipid

matrices for improved microencapsulation of drugs. Int J Pharm, 242(1), 121-128.
175. MURUGAN, K., CHOONARA, Y. E., KUMAR, P., BIJUKUMAR, D., DU

TOIT, L. C. & PILLAY, V. 2015. Parameters and characteristics governing
211
References
cellular internalization and trans-barrier trafficking of nanostructures. Int J

Nanomed, 10(1), 2191-2206.
176. MUSTAFA, S., DEVI, V. K. & PAI, R. S. 2017. Kanamycin sulphate loaded
PLGA-Vitamin-E-TPGS long circulating nanoparticles using combined coating
of PEG and water-soluble chitosan. J drug deliv, 2017, 1-10.
177. NAFEE, N., TAETZ, S., SCHNEIDER, M., SCHAEFER, U. F. & LEHR, C.-
M. 2007. Chitosan-coated PLGA nanoparticles for DNA/RNA delivery: effect of
the formulation parameters on complexation and transfection of antisense
oligonucleotides. Nanomed Nanotechnol, 3(3), 173-183.
178. NAGAVARMA, B., YADAV, H. K., AYAZ, A., VASUDHA, L. &

SHIVAKUMAR, H. 2012. Different techniques for preparation of polymeric
nanoparticles—a review. Asian J Pharm Clin Res, 5(3), 16-23.
179. NEL, A. E., MÄDLER, L., VELEGOL, D., XIA, T., HOEK, E. M.,
SOMASUNDARAN, P., KLAESSIG, F., CASTRANOVA, V. & THOMPSON,
M. 2009. Understanding biophysicochemical interactions at the nano–bio
interface. Nat Mater, 8(7), 543-557.
180. NEOPHYTOU, C. M. & CONSTANTINOU, A. I. 2015. Drug delivery

innovations for enhancing the anticancer potential of Vitamin E isoforms and their
derivatives. Biomed Res Int, 2015, 1-16.
181. NEOPHYTOU, C. M., CONSTANTINOU, C., PAPAGEORGIS, P. &

CONSTANTINOU, A. I. 2014. D-alpha-tocopheryl polyethylene glycol succinate
(TPGS) induces cell cycle arrest and apoptosis selectively in Survivin-
overexpressing breast cancer cells. Biochem Pharmacol, 89, 1, 31-42.
212
References
182. NICOL, J. R., DIXON, D. & COULTER, J. A. 2015. Gold nanoparticle surface
functionalization: A necessary requirement in the development of novel
nanotherapeutics. Nanomed, 10(8), 1315-1326.
183. NILES, A. L., MORAVEC, R. A. & RISS, T. L. 2008. Update on in vitro

cytotoxicity assays for drug development. Expert Opin Drug Dis, 3(6), 655-669.
184. NKABINDE, L., SHOBA-ZIKHALI, L., SEMETE-MAKOKOTLELA, B.,

GROBLER, A. & HAMMAN, J. 2014. Poly (D, L-lactide-co-glycolide)
nanoparticles: Uptake by epithelial cells and cytotoxicity. Express Polym Lett,
8(3), 197-206.
185. OWENS, D. E. & PEPPAS, N. A. 2006. Opsonization, biodistribution, and

pharmacokinetics of polymeric nanoparticles. Int J Pharm, 307(1), 93-102.
186. PACIOTTI, G. F., ZHAO, J., CAO, S., BRODIE, P. J., TAMARKIN, L.,
HUHTA, M., MYER, L. D., FRIEDMAN, J. & KINGSTON, D. G. 2016.
Synthesis and evaluation of paclitaxel-loaded gold nanoparticles for tumor-
targeted drug delivery. Bioconj Chem, 27(11), 2646-2657.
187. PACŁAWSKI, K., STRESZEWSKI, B., JAWORSKI, W., LUTY-BŁOCHO,

M. & FITZNER, K. 2012. Gold nanoparticles formation via gold (III) chloride
complex ions reduction with glucose in the batch and in the flow microreactor
systems. Colloid Surface A, 413, 208-215.
188. PADDOCK, S. W. 2000. Principles and practices of laser scanning confocal

microscopy. Mol Biotechnol, 16(2), 127-149.
189. PAN, Y., NEUSS, S., LEIFERT, A., FISCHLER, M., WEN, F., SIMON, U.,
SCHMID, G., BRANDAU, W. & JAHNEN‑DECHENT, W. 2007. Size‑
dependent cytotoxicity of gold nanoparticles. Small, 3(11), 1941-1949.
213
References
190. PANYAM, J., WILLIAMS, D., DASH, A., LESLIE‑PELECKY, D. &

LABHASETWAR, V. 2004. Solid‑state solubility influences encapsulation and
release of hydrophobic drugs from PLGA/PLA nanoparticles. J Pharm Sci, 93(7),
1804-1814.
191. PATOČKA, J., JAKL, J. & STRUNECKÁ, A. 2006. Expectations of

biologically active compounds of the genus Magnolia in biomedicine. J Appl
Biomed, 4(4), 171-178.
192. PEGRAM, M., HSU, S., LEWIS, G., PIETRAS, R., BERYT, M.,
SLIWKOWSKI, M., COOMBS, D., BALY, D., KABBINAVAR, F. &
SLAMON, D. 1999. Inhibitory effects of combinations of HER2/neu antibody
and chemotherapeutic agents used for treatment of human breast cancers.
Oncogene, 18(13), 2241-2251.
193. PIETRAS, R., FENDLY, B., CHAZIN, V., PEGRAM, M., HOWELL, S. &
SLAMON, D. 1994. Antibody to HER2/neu receptor blocks DNA repair after
cisplatin in human breast and ovarian cancer cells. Oncogene, 9(7), 1829-1838.
194. PIETRAS, R., POEN, J. C., GALLARDO, D., WONGVIPAT, P. N., LEE, H.
J. & SLAMON, D. J. 1999. Monoclonal antibody to HER2/neureceptor modulates
repair of radiation-induced DNA damage and enhances radiosensitivity of human
breast cancer cells overexpressing this oncogene. Cancer Res, 59(6), 1347-1355.
195. PLAJNSEK, K. T., KOCBEK, P., KREFT, M. E. & KRISTL, J. 2012.

Mechanisms of cellular uptake of nanoparticles and their effect on drug delivery.
ZDR Vestn, 81(3), 225-235.
214
References
196. POOJA, D., PANYARAM, S., KULHARI, H., RACHAMALLA, S. S. &

SISTLA, R. 2014. Xanthan gum stabilized gold nanoparticles: characterization,
biocompatibility, stability and cytotoxicity. Carbohydr Polym, 110, 1-9.
197. PRIYA, M. K. & IYER, P. R. 2015. Anticancer studies of the synthesized gold
nanoparticles against MCF 7 breast cancer cell lines. Appl Nanosci, 5(4), 443-
448.
198. QIU, N., SHEN, B., LI, X., ZHANG, X., SANG, Z., YANG, T., AN, L., LIU,
J., CHEN, L. & WANG, L. 2016. Inclusion complex of magnolol with
hydroxypropyl-β-cyclodextrin: characterization, solubility, stability and cell
viability. J Incl Phenom Macro, 85(3-4), 289-301.
199. QU, Z., CHEN, K., GU, H. & XU, H. 2014. Covalent immobilization of
proteins on 3D poly (acrylic acid) brushes: mechanism study and a more effective
and controllable process. Bioconjugate Chem, 25(2), 370-378.
200. RACHMAWATI, H., YANDA, Y. L., RAHMA, A. & MASE, N. 2016.

Curcumin-Loaded PLA Nanoparticles: Formulation and Physical Evaluation. Sci
Pharm, 84(1), 191-202.
201. RAHOUI, N., JIANG, B., TALOUB, N. & HUANG, Y. D. 2017. Spatio-
temporal control strategy of drug delivery systems based nano structures. J
Control Release, 255, 176-201.
202. RAO, J. P. & GECKELER, K. E. 2011. Polymer nanoparticles: preparation

techniques and size-control parameters. Prog Polym Sci, 36(7), 887-913.
203. RASTOGI, L., KORA, A. J. & ARUNACHALAM, J. 2012. Highly stable,

protein capped gold nanoparticles as effective drug delivery vehicles for amino-
glycosidic antibiotics. Mat Sci Eng C, 32(6), 1571-1577.
215
References
204. REIS, C. P., NEUFELD, R. J., RIBEIRO, A. J. & VEIGA, F. 2006.

Nanoencapsulation I. Methods for preparation of drug-loaded polymeric
nanoparticles. Nanomed-Nanotechnol, 2, 8-21.
205. RICHARD, T. S., NWABO KAMDJE, A. & MUKHTAR, F. 2015. Medicinal

plants in breast cancer therapy. J Dis Med Plants, 1(1),19-23.
206. RILEY, R. S. & DAY, E. S. 2017. Gold nanoparticle‑mediated photothermal

therapy: applications and opportunities for multimodal cancer treatment. Wiley
Interdiscip Rev Nanomed Nanobiotechnol, 9(4), 1449-1465.
207. RISS, T. L., MORAVEC, R. A., NILES, A. L., DUELLMAN, S., BENINK, H.
A., WORZELLA, T. J. & MINOR, L. 2016. Cell Viability Assays, in: Assay
Guidance Manual (ebook), Sittampolam et al.
208. RODRÍGUEZ, C. E., REIDEL, S. I., DE KIER JOFFÉ, E. D. B., JASNIS, M.

A. & FISZMAN, G. L. 2015. Autophagy protects from trastuzumab-induced
cytotoxicity in HER2 overexpressing breast tumor spheroids. Plos One, 10(9),
0137920-0137936.
209. RUOZI, B., BELLETTI, D., SHARMA, H. S., SHARMA, A., MURESANU,
D. F., MÖSSLER, H., FORNI, F., VANDELLI, M. A. & TOSI, G. 2015. PLGA
nanoparticles loaded cerebrolysin: studies on their preparation and investigation
of the effect of storage and serum stability with reference to traumatic brain injury.
Mol Neurobiol, 52(2), 899-912.
210. SAADATI, R. & DADASHZADEH, S. 2014. Marked effects of combined

TPGS and PVA emulsifiers in the fabrication of etoposide-loaded PLGA-PEG
nanoparticles: in vitro and in vivo evaluation. Int J Pharm, 464(1-2), 135-144.
216
References
211. SADAT, S. M., JAHAN, S. T. & HADDADI, A. 2016. Effects of size and
surface charge of polymeric nanoparticles on in vitro and in vivo applications. J
Biomater Nanobiotechnol, 7, 91-108.
212. SAFWAT, M. A., SOLIMAN, G. M., SAYED, D. & ATTIA, M. A. 2016. Gold
nanoparticles enhance 5-fluorouracil anticancer efficacy against colorectal cancer
cells. Int J Pharm, 513(1-2), 648-658.
213. SAH, E. & SAH, H. 2015. Recent trends in preparation of poly (lactide-co-
glycolide) nanoparticles by mixing polymeric organic solution with antisolvent. J
Nanomater, 16(1), 1-22.
214. SAUTER, E. R. & DALY, M. B. 2010. Breast cancer risk reduction and early
detection, Springer.
215. SELIM, M. E. & HENDI, A. A. 2012. Gold nanoparticles induce apoptosis in

MCF-7 human breast cancer cells. Asian Pac J Cancer Prev, 13(4), 1617-1620.
216. SEO, J.-U., KIM, M.-H., KIM, H.-M. & JEONG, H.-J. 2011. Anticancer
potential of magnolol for lung cancer treatment. Arch Pharm Res, 34(4), 625-633.
217. SHAH, M., BADWAIK, V., KHERDE, Y., WAGHWANI, H. K., MODI, T.,
AGUILAR, Z. P., RODGERS, H., HAMILTON, W., MARUTHARAJ, T. &
WEBB, C. 2014. Gold nanoparticles: various methods of synthesis and
antibacterial applications. Front Biosci, 19, 1320-1344.
218. SHAH, M., NASEER, M. I., CHOI, M. H., KIM, M. O. & YOON, S. C. 2010.
Amphiphilic PHA–mPEG copolymeric nanocontainers for drug delivery:
preparation, characterization and in vitro evaluation. Int J Pharm, 400(1-2),165-
175.
217
References
219. SHAO, J., GRIFFIN, R. J., GALANZHA, E. I., KIM, J.-W., KOONCE, N.,
WEBBER, J., MUSTAFA, T., BIRIS, A. S., NEDOSEKIN, D. A. & ZHAROV,
V. P. 2013. Photothermal nanodrugs: potential of TNF-gold nanospheres for
cancer theranostics. Sci Rep 3, 1293-1302.
220. SHAREEF, M., ASHRAF, M. A. & SARFRAZ, M. 2016. Natural cures for
breast cancer treatment. Saudi Pharm J, 24(3), 233-240.
221. SHARMA, G. N., DAVE, R., SANADYA, J., SHARMA, P. & SHARMA, K.
2010. Various types and management of breast cancer: An overview. J Adv Pharm
Technol Res 1(2), 109-126.
222. SHARMA, N., MADAN, P. & LIN, S. 2016. Effect of process and formulation
variables on the preparation of parenteral paclitaxel-loaded biodegradable
polymeric nanoparticles: A co-surfactant study. Asian J Pharm Sci, 11(3), 404-
416.
223. SHILO, M., REUVENI, T., MOTIEI, M. & POPOVTZER, R. 2012.

Nanoparticles as computed tomography contrast agents: current status and future
perspectives. Nanomedicine, 7(2), 257-269.
224. SHUBHRA, Q. T., TÓTH, J., GYENIS, J. & FECZKÓ, T. 2014. Surface
modification of HSA containing magnetic PLGA nanoparticles by poloxamer to
decrease plasma protein adsorption. Colloid Surface B, 122, 529-536.
225. SHUKLA, A. K., PATRA, S. & DUBEY, V. K. 2012. Nanospheres

encapsulating anti-leishmanial drugs for their specific macrophage targeting,
reduced toxicity, and deliberate intracellular release. Vector-Borne Zoonotic Dis,
12(11), 953-960.
218
References
226. SINGH, P., KIM, Y.-J., ZHANG, D. & YANG, D.-C. 2016. Biological
synthesis of nanoparticles from plants and microorganisms. Trends Biotechnol,
34(7), 588-599.
227. SI-SHEN, F., CHONG, S. & ROMPAS, J. 2014. Chemotherapeutic

Engineering: collected papers of Si-Shen Feng—A Tribute to Shu Chien on His
82nd Birthday, CRC Press.
228. SMITH, P. K., KROHN, R. I., HERMANSON, G., MALLIA, A., GARTNER,
F., PROVENZANO, M., FUJIMOTO, E., GOEKE, N., OLSON, B. & KLENK,
D. 1985. Measurement of protein using bicinchoninic acid. Anal Biochem 150(1),
76-85.
229. SOPPIMATH, K. S., AMINABHAVI, T. M., KULKARNI, A. R. &

RUDZINSKI, W. E. 2001. Biodegradable polymeric nanoparticles as drug
delivery devices. J Control Release, 70(1-2), 1-20.
230. SPÄNKUCH, B., STEINHAUSER, I., WARTLICK, H., KURUNCI-

CSACSKO, E., STREBHARDT, K. I. & LANGER, K. 2008. Downregulation of
Plk1 expression by receptor-mediated uptake of antisense oligonucleotide-loaded
nanoparticles. Neoplasia, 10, 223-234.
231. SRINATH, B. & RAI, V. R. 2015. Biosynthesis of highly monodispersed,

spherical gold nanoparticles of size 4–10nm from spent cultures of Klebsiella
pneumoniae. Biotech, 5(5), 671-676.
232. STEBBING, J., DELANEY, G. & THOMPSON, A. 2011. Breast cancer (non-
metastatic). BMJ Clin Evid, 2011, 102-158.
233. STEFANACHE, A., IGNAT, M., PEPTU, C. A., DIACONU, A., STOLERIU,
I. & OCHIUZ, L. 2017. Development of a prolonged-release drug delivery system
219
References
with magnolol loaded in amino-functionalized mesoporous silica. Appl Sci, 7(3),

237-250.
234. STEINHAUSER, I., SPÄNKUCH, B., STREBHARDT, K. & LANGER, K.

2006. Trastuzumab-modified nanoparticles: optimisation of preparation and
uptake in cancer cells. Biomaterials, 27(28), 4975-4983.
235. SU, Y., HU, J., HUANG, Z., HUANG, Y., PENG, B., XIE, N. & LIU, H. 2017.
Paclitaxel-loaded star-shaped copolymer nanoparticles for enhanced malignant
melanoma chemotherapy against multidrug resistance. Drug Des Devel Ther, 11,
659-668.
236. SUBIK, K., LEE, J.-F., BAXTER, L., STRZEPEK, T., COSTELLO, D.,
CROWLEY, P., XING, L., HUNG, M.-C., BONFIGLIO, T. & HICKS, D. G.
2010. The expression patterns of ER, PR, HER2, CK5/6, EGFR, Ki-67 and AR
by immunohistochemical analysis in breast cancer cell lines. Breast Cancer
(AUCKL), 4, 35-41.
237. SUH, H., JEONG, B., LIU, F. & KIM, S. W. 1998. Cellular uptake study of
biodegradable nanoparticles in vascular smooth muscle cells. Pharm Res, 15(9),
1495-1498.
238. SUN, B. & FENG, S. 2008. Trastuzumab decorated nanoparticles for targeted
chemotherapy of breast cancer. Advances in Science and Technology, 2008.
Trans Tech Publ, 57, 160-165.
239. SUN, B. & FENG, S. 2009. Trastuzumab-functionalized nanoparticles of

biodegradable copolymers for targeted delivery of docetaxel. Nanomedicine, 4(4),
431-445.
220
References
240. THAKOR, A. S. & GAMBHIR, S. S. 2013. Nanooncology: the future of cancer

diagnosis and therapy. CA Cancer J Clin, 63(6), 395-418.
241. THAMAKE, S., RAUT, S., 2, RANJAN, A., GRYCZYNSKI, Z. &

VISHWANATHA, J. 2010. Surface functionalization of PLGA nanoparticles by
non-covalent insertion of a homo-bifunctional spacer for active targeting in cancer
therapy. Nanotechnology, 22(3), 035101-0351011.
242. TOMULEASA, C., SORITAU, O., ORZA, A., DUDEA, M., PETRUSHEV,
B., MOSTEANU, O., SUSMAN, S., FLOREA, A., PALL, E. & ALDEA, M.
2012. Gold nanoparticles conjugated with cisplatin/doxorubicin/capecitabine
lower the chemoresistance of hepatocellular carcinoma-derived cancer cells. J
Gastrointestin Liver Dis, 21(2), 187-196.
243. TOWNSEND, S. A., EVRONY, G. D., GU, F. X., SCHULZ, M. P., BROWN,
R. H. & LANGER, R. 2007. Tetanus toxin C fragment-conjugated nanoparticles
for targeted drug delivery to neurons. Biomaterials, 28(4), 5176-5184.
244. TSAI, S.-W., CHEN, Y.-Y. & LIAW, J.-W. 2008. Compound cellular imaging
of laser scanning confocal microscopy by using gold nanoparticles and dyes.
Sensors, 8(4), 2306-2316.
245. TSAI, T., KAO, C.-Y., CHOU, C.-L., LIU, L.-C. & CHOU, T.-C. 2016.
Protective effect of magnolol-loaded polyketal microparticles on
lipopolysaccharide-induced acute lung injury in rats. J Microencapsul, 33(5),
401-411.
246. TSAI, T.-H. & CHEN, C.-F. 1992. Identification and determination of honokiol
and magnolol from Magnolia officinalis by high-performance liquid
221
References
chromatography with phtodiode-array UV detection. J Chromatogr A, 598(1),

143-146.
247. TURK, C. T. S., OZ, U. C., SERIM, T. M. & HASCICEK, C. 2014.

Formulation and optimization of nonionic surfactants emulsified nimesulide-
loaded PLGA-based nanoparticles by design of experiments. AAPS Pharm Sci
Tech, 159(1), 161-176.
248. TURKEVICH, J., STEVENSON, P. C. & HILLIER, J. 1951. A study of the

nucleation and growth processes in the synthesis of colloidal gold. Discuss
Faraday Soc, 11, 55-75.
249. ULMAN, A. 1996. Formation and structure of self-assembled monolayers.

Chem Rev, 96 (4), 1533-1554.
250. VAN MEERLOO, J., KASPERS, G. J. & CLOOS, J. 2011. Cell sensitivity
assays: the MTT assay. Methods Mol Biol, 731, 237-245.
251. VAN TONDER, A., JOUBERT, A. M. & CROMARTY, A. D. 2015.

Limitations of the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium
bromide (MTT) assay when compared to three commonly used cell enumeration
assays. BMC Res Notes, 8, 47-57.
252. VANDER KAMP, K. A., QIANG, D., ABURUB, A. & WURSTER, D. E.

2005. Modified Langmuir-like model for modeling the adsorption from aqueous
solutions by activated carbons. Langmuir, 21, 217-224.
253. VATTA, L. L., SANDERSON, R. D. & KOCH, K. R. 2006. Magnetic

nanoparticles: properties and potential applications. Pure Appl Chem, 78(9),
1793-1801.
222
References
254. VERMA, H. N., PRAVEEN, S. & CHAVAN, R. 2014. Gold nanoparticle:

synthesis and characterization. Vet World, 7(2), 72-77.
255. VESCI, L., CAROLLO, V., ROSCILLI, G., AURISICCHIO, L., FERRARA,
F. F., SPAGNOLI, L. & DE SANTIS, R. 2015. Trastuzumab and docetaxel in a
preclinical organotypic breast cancer model using tissue slices from mammary fat
pad: Translational relevance. Oncol Rep, 34(3), 1146-1152.
256. VIJAYAKUMAR, M. R., KOSURU, R., SINGH, S. K., PRASAD, C. B.,

NARAYAN, G., MUTHU, M. S. & SINGH, S. 2016. Resveratrol loaded PLGA:
D-α-tocopheryl polyethylene glycol 1000 succinate blend nanoparticles for brain
cancer therapy. RSC Adv, 6(78), 74254-74268.
257. VIJAYAKUMAR, S. & GANESAN, S. 2012. In vitro cytotoxicity assay on

gold nanoparticles with different stabilizing agents. J Nanomater, 2012, 14, 1-9.
258. VIVEK, R., THANGAM, R., NIPUNBABU, V., REJEETH, C.,

SIVASUBRAMANIAN, S., GUNASEKARAN, P., MUTHUCHELIAN, K. &
KANNAN, S. 2014. Multifunctional HER2-antibody conjugated polymeric
nanocarrier-based drug delivery system for multi-drug-resistant breast cancer
therapy. ACS Appl Mater Interfaces, 6(9), 6469-6480.
259. VOIGT, J., CHRISTENSEN, J. & SHASTRI, V. P. 2014. Differential uptake

of nanoparticles by endothelial cells through polyelectrolytes with affinity for
caveolae. Proc Natl Acad Sci USA, 111(8), 2942–2947.
260. WALLRABE, H. & BARROSO, M. 2003. Confocal FRET microscopy-5:

study of clustered distribution of receptor-ligand complexes in endocytic
membranes. Biophys J, 85(1), 559-571.
223
References
261. WANG, J., KANG, Y.-X., PAN, W., LEI, W., FENG, B. & WANG, X.-J.
2016a. Enhancement of anti-inflammatory activity of curcumin using
phosphatidylserine-containing nanoparticles in cultured macrophages. Int J Mol
Sci, 17(5), 969.
262. WANG, J., ZHANG, G., LI, Q., JIANG, H., LIU, C., AMATORE, C. &
WANG, X. 2013. In vivo self-bio-imaging of tumors through in situ
biosynthesized fluorescent gold nanoclusters. Sci Rep, 3, 1157.
263. WANG, T., BAI, J., JIANG, X. & NIENHAUS, G. U. 2012. Cellular uptake of
nanoparticles by membrane penetration: a study combining confocal microscopy
with FT-IR spectroelectrochemistry. ACS Nano, 6(2), 1251-1259.
264. WANG, Y. J., STROHM, E. M., SUN, Y., NIU, C., ZHENG, Y., WANG, Z.
& KOLIOS, M. C. 2014. PLGA/PFC particles loaded with gold nanoparticles as
dual contrast agents for photoacoustic and ultrasound imaging. Proc Spie, 8943,
1-7.
265. WANG, Y., LI, P., TRUONG-DINH TRAN, T., ZHANG, J. & KONG, L.
2016b. Manufacturing techniques and surface engineering of polymer based
nanoparticles for targeted drug delivery to cancer. Nanomaterials Basel, 6(2), 26-
44.
266. WANG, Y., STROHM, E. M., SUN, Y., WANG, Z., ZHENG, Y., WANG, Z.
& KOLIOS, M. C. 2016c. Biodegradable polymeric nanoparticles containing gold
nanoparticles and Paclitaxel for cancer imaging and drug delivery using
photoacoustic methods. Biomed OPT Express, 7(10), 4125-4138.
267. WANG, Y.-J., CHIEN, Y.-C., WU, C.-H. & LIU, D.-M. 2011. Magnolol-
loaded core–shell hydrogel nanoparticles: drug release, intracellular uptake, and
224
References
controlled cytotoxicity for the inhibition of migration of vascular smooth muscle

cells. Mol Pharm, 8(6), 2339-2349.
268. WARTLICK, H., MICHAELIS, K., BALTHASAR, S., STREBHARDT, K.,

KREUTER, J. & LANGER, K. 2004. Highly specific HER2-mediated cellular
uptake of antibody-modified nanoparticles in tumour cells. J Drug Target, 12(7),
461-471.
269. WILCZEWSKA, A. Z., NIEMIROWICZ, K., MARKIEWICZ, K. H. & CAR,

H. 2012. Nanoparticles as drug delivery systems. Pharmacol Rep, 64(5), 1020-
1037.
270. WISSING, S., KAYSER, O. & MÜLLER, R. 2004. Solid lipid nanoparticles
for parenteral drug delivery. Adv Drug Deliv Rev, 56(9), 1257-72.
271. XIN LEE, K., SHAMELI, K., MIYAKE, M., KUWANO, N., BT AHMAD
KHAIRUDIN, N. B., BT MOHAMAD, S. E. & YEW, Y. P. 2016. Green
synthesis of gold nanoparticles using aqueous extract of garcinia mangostana fruit
peels. J Nanomet, 2016, 1-7.
272. YAMAGUCHI, Y., HIRONAKA, K., OKAWAKI, M., OKITA, R.,

MATSUURA, K., OHSHITA, A. & TOGE, T. 2005. HER2-specific cytotoxic
activity of lymphokine-activated killer cells in the presence of trastuzumab.
Anticancer Res, 25(2A), 827-32.
273. YAMEEN, B., CHOI, W. I., VILOS, C., SWAMI, A., SHI, J. &
FAROKHZAD, O. C. 2014. Insight into nanoparticle cellular uptake and
intracellular targeting. J Control Release, 190, 485-99.
225
References
274. YANG, S. E., HSIEH, M. T., TSAI, T. H. & HSU, S. L. 2003. Effector
mechanism of magnolol‑induced apoptosis in human lung squamous carcinoma
CH27 cells. BR J Pharmacol, 138(1), 193-201.
275. YAO, C., ZHANG, L., WANG, J., HE, Y., XIN, J., WANG, S., XU, H. &
ZHANG, Z. 2016. Gold nanoparticle mediated phototherapy for cancer. J
NANOMET, 2016, 1-29.
276. YAP, W. T., SONG, W. K., CHAUHAN, N., SCALISE, P. N., AGARWAL,
R. & SHEA, L. D. 2014. Quantification of particle-conjugated or-encapsulated
peptides on interfering reagent backgrounds. Biotechniques, 57(1), 39-44.
277. YIH, T. & AL‑FANDI, M. 2006. Engineered nanoparticles as precise drug

delivery systems. J Cell Biochem, 1184-1190.
278. YOON, H. J., KIM, K. H., KIM, J. Y., PARK, H. J., CHO, J. Y., HONG, Y. J.,
PARK, H. W., KIM, J. H., AHN, Y. & JEONG, M. H. 2016. Chemotherapy-
induced left ventricular dysfunction in patients with breast cancer. J Breast
Cancer, 19(4), 402-409.
279. YOU, J., ZHANG, R., ZHANG, G., ZHONG, M., LIU, Y., VAN PELT, C. S.,
LIANG, D., WEI, W., SOOD, A. K. & LI, C. 2012. Photothermal-chemotherapy
with doxorubicin-loaded hollow gold nanospheres: A platform for near-infrared
light-trigged drug release. J Control Release, 158, 319-328.
280. YU, B., TAI, H. C., XUE, W., LEE, L. J. & LEE, R. J. 2010. Receptor-targeted
nanocarriers for therapeutic delivery to cancer. Mol Membr Biol, 27(7), 286-298.
281. YU, X., TRASE, I., REN, M., DUVAL, K., GUO, X. & CHEN, Z. 2016. Design
of nanoparticle-based carriers for targeted drug delivery. J Nanomet, 2016,
1087250, 1-15.
226
References
282. ZABETAKIS, K., GHANN, W. E., KUMAR, S. & DANIEL, M.-C. 2012.
Effect of high gold salt concentrations on the size and polydispersity of gold
nanoparticles prepared by an extended Turkevich–Frens method. Gold Bull,
45(4), 203-211.
283. ZHANG, L. W. & MONTEIRO-RIVIERE, N. A. 2013. Use of confocal

microscopy for nanoparticle drug delivery through skin. J Biomed Opt, 18(6),
061214-061214.
284. ZHANG, X. C. 2014. Inorganic Biomaterials: Structure, Properties and

Applications, Smithers Rapra.
285. ZHANG, X.-Q., XU, X., LAM, R., GILJOHANN, D., HO, D. & MIRKIN, C.
A. 2011. A strategy for increasing drug solubility and efficacy through covalent
attachment to polyvalent DNA-nanoparticle conjugates. ACS Nano, 5(9), 6962-
6970.
286. ZHANG, Y., CHAN, H. F. & LEONG, K. W. 2013. Advanced materials and
processing for drug delivery: the past and the future. Adv Drug Deliv Rev, 65(1),
104-20.
287. ZHANG, Z., TAN, S. & FENG, S.-S. 2012. Vitamin E TPGS as a molecular
biomaterial for drug delivery. Biomaterials, 33(11), 4889-4906.
288. ZHAROV, V. P., GALITOVSKAYA, E. N., JOHNSON, C. & KELLY, T.

2005. Synergistic enhancement of selective nanophotothermolysis with gold
nanoclusters: potential for cancer therapy. Laser Surg, 37, 219-226.
289. ZHENG, Y., YU, B., WEECHARANGSAN, W., PIAO, L., DARBY, M.,
MAO, Y., KOYNOVA, R., YANG, X., LI, H. & XU, S. 2010. Transferrin-
227
References
conjugated lipid-coated PLGA nanoparticles for targeted delivery of aromatase

inhibitor 7α-APTADD to breast cancer cells. Int J Pharm, 390, 234-241.
290. ZHOU, Y., BI, Y., YANG, C., YANG, J., JIANG, Y., MENG, F., YU, B.,
KHAN, M., MA, T. & YANG, H. 2013. Magnolol induces apoptosis in MCF-7
human breast cancer cells through G2/M phase arrest and caspase-independent
pathway. Pharmazie, 68, 755-762.
291. ZHOU, Z., BADKAS, A., STEVENSON, M., LEE, J.-Y. & LEUNG, Y.-K.
2015. Herceptin conjugated PLGA-PHis-PEG pH sensitive nanoparticles for
targeted and controlled drug delivery. Int J Pharm, 487(1-2), 81-90.
228
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General Introduction

Breast cancer is the most common cancer type in women worldwide

Figure I: Anatomy of a normal breast (Chaurasia and Pal, 2014).

ductal/lobular, mucinous (colloid), tubular, medullary and papillary carcinomas

A variety of causes contribute to the occurrence of breast cancer e.g.

Early breast cancer is usually asymptomatic. As the cancer grows, symptoms

Breast cancer progresses from non metastatic hormone dependent cells to

Most of the chemotherapeutic drugs used in breast cancer therapy interfere

Recent advancement in nanotechnology has shown that nanoparticles (NPs)

To enhance their in vivo application, several criteria should be considered in

As nanocarriers suffer from non-specific uptake and potential degradation in

In passive targeting, size-selective accumulation of drug loaded NPs at

Figure II: Schematic diagram representing the accumulation of nanocarriers

In active targeting, specific biological processes such as ligand-receptor

NPs including: antibodies, antibody fragments, aptamers, peptides, whole proteins

Trastuzumab (TZB) also called herceptin (HER) is a humanized IgG1

Figure III: Schematic structure of trastuzumab (Ho et al., 2010).

and is minimally expressed by normal tissues. TZB conjugated NPs showed a

immobilization of TZB showed remarkable enhanced hyperthermia effect in cultured

To overcome toxicity from chemotherapeutic agents, attention has been given

Magnolol (Mag), with the chemical structure shown in figure IV, is a

Previous preclinical studies had shown that Mag possesses anti-oxidative,

Emperical formula: C18H18O2

Recent studies have shown that Mag exhibits anti-cancer properties by

prevent UVB-induced skin cancer development by enhancing apoptosis (Seo et al.,

Triple-negative breast cancers (TNBC) (15% to 20% of breast cancers) are

Figure V: Diagram of magnolol signal pathway of inhibiting breast cancer cell

Breast cancer became a life-threatening disease for women worldwide. The

Polymeric nanoparticles (PNPs) designed for cancer treatment via IV route

Figure VI: Difference between nanospheres and nanocapsules (Nagavarma et

evaporation/extraction, nanoprecipitation (solvent-displacement), supercritical anti-

SCF: supercritical fluid technology, C/LR: controlled/living radical.

Figure VIII: Schematic representation of nanoprecipitation technique

batch to batch reproductivity, in addition to the susceptibility to degradation and

Figure IX: Hydrolysis of poly lactic-co-glycolic acid (Makadia and Siegel,

The use of biodegradable materials for nanoparticle preparation allows for

Figure X: Drug release mechanisms from polymeric NPs (Jawahar and

Figure XI: Effect of PS and surface charge on NPs biocompatibility and

Figure XII: Nanoparticles internalization pathways in mammalian cells (Kou et

The surface modification of NPs with nontoxic and blood compatible

D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a water-

absorption enhancer, emulsifier, solubilizer, additive, permeation enhancer and

IUPAC name: 1-O-(2-hydroxyethyl)4-O-[2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-3,4-

Emperical formula: C35H58O6

It is worthy to mention that the exploitation of the combination of PLGA and

The surface modification by addition of targeting moieties not only influences

Two fundamentally different approaches have been developed to link ligands

• 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC):

• Acetone, disodium hydrogen phosphate, glacial acetic acid, methanol

• Cooling centrifuge: Centurion K241, Germany.

• Fourier transform- infrared (FT-IR): Thermonicolet Nexus spectrometer,

• Freeze dryer: Christ alpha 1-2 LD plus, Christ, Germany.

• pH meter (Digital): JENWAY 350, UK.

• High performance liquid chromatography (HPLC) with UV detector: Jasco,

• Spectrophotometer (double beam): Model UV-1601PC, Shimadzu, Kyoto,

• Transmission electron microscope (TEM): JEM 1400 plus, electron

• Zetasizer Nano ZS: Malvern Instruments Ltd, UK.

Blank PLGA NPs were prepared by the nanoprecipitation method previously

During preparation of PLGA NPs, various formulation parameters were

3.1.1. Organic solvent type:

3.1.2. SAA concentration and type:

Blank PLGA NPs prepared by nanoprecipitation method using acetone as

Table 1: Composition of blank PLGA NPs prepared by nanoprecipitation for

3.2. Preparation and optimization of Mag-PLGA NPs