CHAPTER ONE

1.0 Introduction

The word “nano” is a Greek word which means dwarf. It means very small miniature size.

Nanotechnology is defined as the research and development of materials, device and system

exhibiting physical, chemical and biological characteristics that are differ from those found on

larger scale (Edison et al., 2012).

Nanotechnology deal with the study of structures having particles sizes of 1 to 100nm (Edison et

al., 2012). Nanotechnology is the treatment of individual atoms, molecules, or compound into

structures to produce materials and devices with special properties. Nanotechnology involves

reducing size of large structures to smallest structures (Salamat et al., 2008). Nanotechnology

produced materials of various types at nanoscale level).

The vision of nanotechnology was introduced in 1959 by late Nobel Physicist, Richard P

Feynman in dinner talk where he said “there is plenty room at the bottom” (Feynman, 1960).

Nanotechnology involve work from top down i.e., reducing size of large structure to smallest

structure e.g., top-down or bottom up which involves changing of individual atoms, molecules

and compound into nanostructures and more closely related to chemistry and biology (Mantovani

et al., 2011).

1.1 Nanoparticles

Nanoparticles are particles that have particle size of 100nm or less. Nanoparticles (NPs) are

wide class of materials that include particulate substances, which have one dimension less than

100nm at least (Laurent et al., 2010). Nanoparticles have been extensively exploited because of

its unique properties, depending on their size, shape, physical, chemical and biological properties

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which allow the most diverse applications (Ferreira et al., 2013). Fine particles have the range of

100-2500 nm while ultrafine has the size of 1-100 nm (Buzea et al., 2007).

NPs are not simple molecule itself and therefore composed of three layers i.e.

(a) the surface layer, which may be functionalized with a variety of small molecules, metal ions,

surfactants and polymers.

(b) the shell layers, which is chemically different material from the core in all aspect, and

(c) the core, which is essentially the central portion NP and are usually refers the NP itself (Shin

et al., 2016) (Figure 1).

1.1.1 Classification of nanoparticles

Nanoparticles are largely classified into various group based on their morphology, size and

chemical properties. Based on physical and chemical characteristics.

(a) Carbon based NPs: Fullerenes and carbon nanotube (CNTs) represent two major classes

of carbon based NPs. Fullerenes contain nanomaterial that are made of globular hollow

cage such as allotropic forms of carbon (Astefanei et al., 2015). CNTs are elongated,

tubular structure, 1-2 nm in diameter (Ibrahim 2013). Their structure is resembling to

graphite sheet rolling upon itself (Figure 2). The rolled sheet can be single, double, or

many walls and therefore they are named as single-walled (SWNTs), doubled-walled

(DWNTs), or multi-walled carbon nanotubes (MWNTs) respectively.

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Figure 1: Relative size of nanoparticles compared with familiar items

Source: Mozafari (2011)

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Figure 2: Rolling of graphite sheet into single-walled and multi-walled CNTs

Source: Khan et al. (2017)

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(b) Metals NPs: are made up of pure metals precursors, due to well-known localized surfaced

Plasmon resonance (LSPR) characteristics, these NPs possess unique optoelectrical

properties. NPs of the alkali and noble metals i.e., Cu, Ag and Au have a broad

absorption band in the visible zone of electromagnetic solar spectrum. The facet, size and

shape-controlled synthesis of metals NPs is important in present day cutting – edge

materials (Dreaden et al., 2012).

(c) Ceramics NPs: are inorganic nonmetallic solids, synthesized via heat and successive

cooling. They can be found in amorphous, polycrystalline, dense, porous or hollow forms

(Sigmund et al., 2006). Therefore, these NPs are getting great attention of researcher due

to their use in applications such as catalysis, photocatalysis, photodegradation of dyes,

and imaging applications (Thomas et al., 2014).

(d) Semiconductors NPs: semiconductors materials have properties between metals and non-

metals and therefore they found various applications due to this property (Ali et al., 2017;

Khan et al., 2017). Semiconductor NPs possess wide bandgaps and therefore showed

significant alteration in their properties and bandgap tuning. Therefore, they are very

important materials in photocatalysis, photo optics and electronic devices (Sun, 2000). As

an example, variety of semiconductor NPs are found exceptionally efficient in water

splitting applications due to their suitable band gap and bandedge positions (Hisatomi et

al., 2014).

(e) Polymeric NPs: these are normally organic based NPs and are termed as special polymer

nanoparticles (PNP). They are mostly nanospheres or nanocapsular shaped (Manshal et

al., 2017). The former are matrix particles whose overall mass is generally solid and the

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 other molecules are adsorbed at the outer boundary of the spherical surface. The PNPs

are readily functionalize and thus have bundles of application ( Abouelmagd et al., 2016).

(f) Lipid NPs: these contain lipid moieties and effectively using in many biomedical

applications. Generally, a lipid NPs is characteristically spherical with diameter ranging

from 10-1000 nm. Like polymeric NPs, lipid NPs possess a solid core made of lipid and a

matrix contains soluble lipophilic molecules. Surfactants or emulsifiers stabilized the

external core of these NPs (Rawat et al., 2011). Lipid nanotechnology (Mashaghi et al.,

2013) is a special field, which focus the designing and synthesis of lipid NPs for various

application such as drug carriers and delivery (Puri et al., 2009) and RNA release in

cancer therapy (Gujrati et al., 2014).

1.1.2 Characteristic of Nanotechnology

 It is very diverse

 It is based upon molecular self-assembly

 It is used in field of science

 It was used to create many new materials

 It has impact on environment and economics

 It is used to make high performance products (Khan et al. 2017).

1.1.3 Applications of Nanotechnology

 Nano capacitors-based filters

 Nano insulators

 Nano resistance

 Nano engineering materials

 Nano ZnO arrestors (Khan et. al. 2017)

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 CHAPTER TWO

2.0 Application of Nanotechnology in Biochemistry

Nanotechnology has a large area of application in field of biochemistry. Nanoparticles are being

deploy for several biomedical applications; carrier system, biomaterial, biosurfactants, drug

delivery, cellular targeting and imaging (Mozafari, 2011).

2.1 Carrier system

This controlled release system, sustained release carriers, colloidal drug delivery system,

bioactive carriers and particulate drug carriers (Mozafari, 2011).

2.1.1 Drug Delivery

In nanotechnology, nanoparticles are used for specific site drug delivery. In this technique

required drug dose is deliver in the optimum dosage range often resulting in increased

therapeutic efficiency of the drugs, weakened side effects and improved patient compliances

(Alexis et al., 2008). Micelles obtained from block co-polymer are used for drug encapsulation.

They transport small drug molecules to desired location. Similarly, nano electromechanical

systems are utilized for the active release of drugs. Iron nano particles or gold shells are finding

application in cancer treatment. The pharmacological and therapeutics properties of drugs can be

improved by proper designing of drug delivery systems, by the use of lipid and polymer-based

nanoparticles (Allen et al., 2004).

The strength of drug delivery systems is their ability to alter the pharmacokinetics and

biodistribution of drug. Nanoparticles are designed to avoid the body defense mechanism

(Bertrand et al., 2012). New complex drug delivery mechanisms are being developed, which can

get drugs through cell membranes into cell cytoplasm, thereby increasing efficiency. Drugs that

are placed in the body in the body can activate only on receiving a particular signal. A drug with

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poor solubility will only be replaced by a drug delivery system, having improved solubility due

to presence of both hydrophilic and hydrophobic environment (Balogh et al., 2012).

Tissue damage by drug can be prevented with drug delivery, by regulated drug release. With

drug delivery system larger clearance of drug from the body can be reduced by altering the

pharmacokinetics of the drug. Liposomes has been used as potential drug carrier instead of

conventional dosage forms because of their unique advantages which include ability to protect

drug from degradation, target to the site of action and reduce the noxiousness and other side

effects. Most of the semiconductor and metallic nanoparticles have immense potential for cancer

diagnosis and therapy on account of their surface Plasmon resonance (SPR)enhance light

scattering and absorption (Prashant et al., 2007).

(a) Liposome: This are microscopic spherical vesicles composed of a phospholipid bilayer

that are capable of encapsulating active agents (Figure 3). Liposomes are closed,

continuous bilayer structures made mainly of lipid and/or phospholipid molecules

(Mozafari et al., 2002). Liposomes have been extensively explored and most developed

nano carriers for novel and targeted drug delivery due to their small size, these are 50-

200nm in size. When dry, phospholipid is hydrated, closed vesicles are formed.

Liposomes are biocompatible, versatile, and have good entrapment efficiency. Its

application has long been used in circulatory and active and passive delivery of gene,

protein and peptide. The concept of utilizing liposomes as vehicle for drug delivery

system was introduced in 1970s and more recently use of liposomes has been extended to

immunological adjuvants and as delivery vehicles for vaccine especially to specific target

cells (Anthony et al., 2013). Both lipophilic and hydrophilic particles can be incorporated

into liposome and delivered to target sites within the host organism. Hydrophilic particles

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 including peptides, protein and nucleic acid can be entrapped within the inner aqueous

phase while lipophilic drugs such as adjuvants and lipopeptides can be incorporated onto

the outer phospholipid layer (Anthony et al., 2013).

(b) Dendrimers: Dendrimers is from two Greeks words, “Dendron” meaning tree and

“Metros” meaning parts. Structure of dendrimers has a well-defined size, shape and

define molecular weight (Anirudha et al., 2012). Dendrimers are hyper-branched,

globular, monodisperse and three-dimensional nanoscale synthetic polymers (Anirudha

et al., 2012). A dendrimer is a synthetic polymeric macromolecule (nm), consists of

several extremely pronged monomers that appear radically from the central core (Figure

4). Properties associated with these dendrimers such as their monodisperse size,

modifiable surface functionality, multivalency, water solubility and available internal

cavity make them attractive for drug delivery (Svenson and Tomalia, 2005). The easily

modifiable surface characteristic of dendrimers enables them to be simultaneously

conjugated with several molecules such as imaging contrast agents, targeting ligands or

therapeutics drugs, and yielding a dendrimer-based multifunctional drug delivery system

(Svenson and Tomalia, 2005).

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Figure 3: Structure of Liposome

Source: Farouk et al. (2018)

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Figure 4: Structure of dendrimer

Source: Janardana (2019)

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 Table 1: Types of nanocarriers for drug delivery

SYSTEM STRUCTURE CHARACTERISTICS EXAMPLES OF

COMPOUNDS

Polymeric Drugs are conjugated to (a) Water soluble, non-toxic, Albumin taxol
nanoparticles the side chain of a linear biogradable (abraxane)
(polymer drug polymer with a linker (b) Surface modification
conjugates) (deavable bond) (c) Specific targeting of cancer Poly (L-glutamate)
cells camptotheon

Polymeric Amphiphilic block (a) suitable carrier for water Polyethylene

micelles copolymers assemble and insoluble drugs glycol-pluronic-
form a micelle with a (b) ease of functional modification doxorubucin
hydrophobic core and (c) biocompatible, self-assembling,
hydrophilic shell biodegradable
(d) targeting potential

Dendrimers Radially hyperbranched (a) High structural and chemical Poly amidoamine-
synthetic and repeating homogeneity methotrexate
units (b) High ligand density and ease of
functionalization
(c) Controlled degradation

Carbon-cylinders (a) Water soluble and Carbon nanotube –

composed of benzene biocompatibility through methotrexate
Carbon
rings chemical modification
nanotubes Carbon nanotube –
(b) Multifunctionality
amphoteri O n B

Liposomes Self-assembling closed (a) Amphiphilic and biocompatible Pegylated

colloidal structures (b) Ease of modification liposomal DOX
composed of lipid bilayers (c) Targeting potential (doxil)

Non-pegylated
liposomal DOX
(myocet)

Source: Cho et al. (2008)

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2.1.1 Pharmacokinetics, targeting and sub-cellular transport of nanocarriers

(a) Circulation and Clearance

When administered in vivo, therapeutic agents are always recognized as foreign substances and

consequently cleared from the body ( Moghimi et al., 2001; Yoo et al., 2010). Clearance of

foreign compounds in the body occurs mainly by the reticuloendothelial system and other

elements of the immune system, as well as by renal filtration (Moghimi and Szebeni, 2003).

Resident macrophages in the alveoli remove substances administered into the lungs through the

respiratory tract, Kupffer cells in the liver sinusoids remove materials that enter the portal

circulation through the gastrointestinal epithelium, materials administered in the systemic

circulation are cleared mainly by spleen and liver, and the lymph nodes remove substances that

arrive to the tissue parenchyma by draining them through the lymphatics.

For most applications, including those designed for treatment of genetics condition, rapid

clearance is detrimental as it minimizes the chances of the delivered agent to reach its targets in

the body and accumulate there at amounts amenable to render significant efficacy (Moghimi and

Szebeni, 2003). One strategy to control this parameter consists of coupling to poly (ethylene

glycol) or PEG, known as PEGylation or stealth technology. PEG form a hydrophilic brush

around cargoes and/or their carriers, minimizing interactions with plasma opsonins, the

compliment, professional phagocytes and lymphocytes which provide specific immunity

(Moghimi and Szebeni, 2003), (Figure 5). As a consequence, certain physiochemical properties

of the cargo (such as hydrophobicity) are altered, allowing the platform to gain solubility and to

remain elusive from immune detection. This prolongs the circulation in the bloodstream for few

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hours to days, which favors lengthened medicinal effects and less frequent administrations

(Moghimi and Szebeni, 2003; Musacchio and Torchilin, 2011).

Figure 5: strategies of nanocarriers to minimize rapid clearance. (A) poly-ethylene glycol (PEG)
minimizes interaction with plasma proteins and binding to macrophages. (B) CD47 binding to
the surface of immune cells inhibits engulfment and phagocytosis.

Source: Janet and Muro (2011)

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Other strategies to minimize drug removal take advantage of the natural mechanism by which

red blood cells in the body avoid clearance by elements of the innate immune system. This is the

case for CD47 (Figure 5c), a transmembrane protein that act like a marker of the “self” by

binding to its cognate receptor expressed on leukocytes, inhibitory receptor signal regulatory

protein alpha (SIRPα). CD47 binding to SIRPα inhibits phagocytosis in part via regulation of the

cytoskeleton and inhibition of engulfing structures. Incorporation of CD47 on drugs carrier

surfaces reduces attachment to neutrophils and macrophages, therefore prolonging circulation

and inhibiting inflammation (Stachelek et al., 2011). In addition to controlling the solubility

level, half-life in circulation, and immune system recognition, nanocarries can also improve

control of the drug efficacy upon release in the case of therapeutics intervention where

administration is local. Localized implantation of bioactive agents embedded within porous

matrices/ or hydrogels capable of responding to microenvironment properties can provide

controlled release and effects (Tokarev and Minko, 2010).

(b) Targeting

Nanomedicines can improve the bioavailability and pharmacokinetics of diagnostics and

therapeutics agents, also protecting them from rapid degradation. In order to maximize their

efficacy, carriers can also be designed to help maximize bio adhesion to areas in the body where

their action is required, a strategy known as Targeting (Figure 6) (Janet and Muro, 2011). In

some cases, general enhanced delivery throughout the body is preferred, rather than specific

delivery to particular organs (Janet and Muro, 2011). This is the case for genetics condition that

affects multi-organs system due to ubiquitous distribution of the molecular markers or functions

15
affected, such as in many monogenic disorders with both peripheral and central nervous system

components. Since most therapeutics do not present intrinsic affinity to cells in the body,

coupling them to carriers with affinity properties provide advantages (Janet and Muro, 2011).

Hydrophilic and slightly positively-charged polymers provide affinity to the negatively charged

plasma of cell membranes (Panyam and Labhasetwar, 2003; El-sayed et al., 2005). Also, this

system can be coupled to affinity moieties that enhance bioadhesion. This is the case for

promiscuous affinity peptides such as Tat, antennapedia and other sequence that gain access to

the plasma membrane of cells due to their positively charged nature (Suk et al., 2006; Sawant

and Torchilin, 2010). (Figure 6Bi).

Specific localization of drugs cargoes and imaging agents is necessary for optimal effects, such

as the case of disease with more prominent symptoms in particular organs or cell types (Langer,

1998; Torchilin, 2006; Pardridge, 2010). Moreover, such specific targeting may also reduce

potential side effects of the therapeutics in non-intended targets (Janet and Muro, 2011). Also,

nanocarriers can gain preferential access to organs irrigated by discontinuous blood vessels

(Figure 6A) which do not pose a barrier from free diffusion of substances between circulation

and tissue, such as in the liver, an organ considered as a main therapeutics target for many

monogenic diseases that affect metabolic pathways (Janet and Muro, 2011). However, delivery

of therapeutics to most other sites in the body requires more complex and precise strategies of

active targeting. This can be achieved by coupling to affinity moieties that recognize specific

markers expressed by the cells which require intervention (Figure 6Bii), including natural

ligands of such markers, proteins and peptides, antibodies and their fragments, sugars and

aptamers (Muro and Muzykantov, 2005; Bareford and Swaan, 2007).

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Figure 6: Passive targeting (A) and Active targeting (B) of drugs carriers helps localized their
cargo within the body, non-specific interactions with the target cells (i), more specific
recognition of particular surface markers (ii)

Source: Janet and Muro (2011)

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(c) Sub-cellular transport

Targeting of selected cells and tissues is not sufficient to attain significant effects, the delivered

cargo must gain access to intracellular compartments where their molecular target is located

(Janet and Muro 2011). This is also the case for delivery of some chaperones, inhibitor or

activators, enzymes and other protein located in the cytosol or sub-cellular organelles such as

mitochondria, peroxixomes e.t.c. which can be redirected to these compartments by signals

peptides if delivered previously to the cytosol (Janet and Muro 2011).

Drug carriers can be targeted to cell surface receptors involved in endocytosis (Muro and

Muzykantov, 2005; Bareford and Swaan, 2007; Pardridge, 2010; Sahay et al., 2010). This term

refers to a group of processes by which cells engulf extracellular material with their plasma

membrane, followed by pinching off the resulting vesicles into the cytosol. Uptake by

endocytosis is regulated by numerous pathways (clathrin and caveolar-mediated mechanisms,

macropinocytosis, phagocytosis, and other non-classical mechanisms), and most commonly

result in transport of the internalized materials to endosomes and lysosomes (Bareford and

Swaan, 2007; Sahay et al., 2010).

This strategy is ideal in the case of delivery of therapeutic agents whose action is required at sub-

cellular compartments such as in the case of carrier assisted delivery of enzyme replacement for

lysosomal storage disorders (Figure 7) (Garnacho et al., 2008; Muro et al., 2008; Muro 2010;

Hsu et al., 2011). In those cases where delivery to the cytosol and access to other sub- cellular

compartment is required, carriers can be coupled to fusogenic peptides derived from bacteria

toxins (e.g., hemaglutinnin), which can induce poration of the endosomal membrane, such as in

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the case of pH sensitive (poly-acid) carriers and temperature responsive (poly-eletrolyte)

hydrogels (Yessine and Leroux, 2004; Stayton et al., 2005; Choi et al., 2006; Oishi et al., 2006).

Figure 7: sub –cellular delivery of nano-carriers. (A) Endocytosis of carriers can lead to transport
to endosomes and lysosomes, where acidic pH and hydrolases can degrade the carrier
components and release the therapeutic cargo. Alternatively, pH-sensitive carriers can destabilize
the endosome, releasing the cargo to the cytosol. (B) transport across cellular layers can take
place via either paracellular transport between adjacent cells or via transcytosis across the cell
body.

Source: Janet and Muro, (2011)

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2.1.2 Immune Response

The nano device bucky balls have used to alter the allergy/ immune response. They prevent mast

cells from releasing histamine into the blood and tissue, as these bind to free radicals better than

any anti-oxidant available, such as vitamin E (Abraham 2010). Liposomes are immunologically

advantageous due to their targeting and uptake by professional antigen representing cells, and

additionally antigens, adjuvants and antibodies can be attached to the outer surface of liposomes

to facilitate delivery into infected cells (Anthony et al., 2013).

Optimal combination of antigens, antibodies and adjuvant gives liposomes plasticity and allow

the opportunity for optimization of different drugs regimens. The detection of analytes in tissue

sections can be accomplished through antigen-antibody interaction using antibodies labeled with

fluorescent dyes, enzymes, radioactive compounds or colloidal Gold (Au) nanoparticles

(Khebtstov et al., 2010).

2.1.3 Anti-Malarial Efficacy Carrier

Nanocarriers with prolong blood circulation time i.e., stealth nanocarriers have been used for

delivering anti malaria drugs in order to increase the resident time in human body and to increase

probability of drug molecules to interact with infected red blood cells and parasites (Lawal et al.,

2019). Nano- drugs delivery systems provide protection to unstable drugs, cell adhesion

properties, and ability to conjugate specific ligands on their surface. Nano –drug carriers such as

mesoporous silica liposomes, polymeric nanoparticles, solid nanopartcles, dendrimers and

nanoemulsions are extensively studied for anti-malaria drug delivery (Weynom et al., 2019).

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(a) Mesoporous Silica as Nanocarriers for antimalaria drugs: The use of mesoporous silica

nanoparticles (MSNs) as a strategy in drug design application came as a better alternative

nanocarrier due to its inherent noble properties of large surface area, tunable pore size,

high thermal property, non-toxicity, biocompatibility which enables it cargo favorably

large drug proportions to the target cells in a controlled flux release (Panpan et al., 2011).

Beck et al., 1992, were the first to introduce MSN popularly referred to as MCM-41 IN

1992. But not until 2001, that Vallet-Regi et al. (2001), introduced MCM-41 for the first

time as a drug delivery system, and effort has been devoted to the design of versatile

MSNs for treating diverse pathologies.

Construction of nano-vector materials, capable of encapsulating anti-malarial drugs and

delivering them to plasmodium-infected red blood cell (Prbc) with high specificity

efficacy and at an affordable rate is of paramount importance. Urban et al. (2014),

reported the use of poly (amidoamines) (PAAs) as drug conjugate for the delivery of

chloroquine (CQ) and primaquine (PQ) to P.falciparium 3D7 in vitro with IC50 values of

14.6nM and 2.5µM respectively when the most effective PAA (having an intrinsic ant

plasmodial activity, IC50 of 13.7µM was used and to P.yoelli17XL in vivo with 96.5

reduction in parasitemia at the least dose when the most effective PAA was also used.

The construction of nano dendritic polymer as drug conjugate for CQ and PQ carriage to

plasmodium-infected red blood cell (pRBC) with a clear improvement in the vitro IC50

for CQ and PQ which were 4.0nM and 1.1µM respectively. In the research done by

Amolegbe et al., (2018), made use of mesoporous silica in the form of MCM-41

encapsulated quinine (MCM-41ƆQN)(1),3-phenylpropyl silane functionalized MCM-41

loaded QN(pMCM-41ƆQN)(2), MCM-41 encapsulated artesunate (MCM-41ƆATS) and

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 3-aminopropyl silane functionalized MCM-41 contained ATS(Amcm-41ƆATS) which

were synthesized, well characterized and were screened in vitro for their activity against

P.falciparium W2 strain, cytotoxicity against BGM cells and in vivo for their activity

against Plasmodium berghei. The result shows: Anti plasmodial activity and cytotoxicity

of the nanodrugs of MSN. The antiplasmodial and cytotoxicity for the nanodrugs: MCM-

41ƆQN(1) Pmcm-41ƆQN(2),MCM-41ƆATS(3) and Amcm-41ƆATS(4) show that with

the exception of MCM-41 encapsulated ATS, the other three encapsulated antimalarial

drugs were active against P.falciparium W2 strain and were not toxic against buffalo and

green monkey kidney(BGM) cells (Amolegbe et al., 2018).

(b) Liposomes as nanocarrier for antimalarial drugs: Liposomes have shown important effect

as nanocarrier for the prophylaxis and also for the treatment of malaria and as well as for

vaccine delivery for the prevention of malaria (Salvador et al., 2012). Encapsulation of

therapeutic agents within liposomes can favorably alter dose and distribution of drugs

within the body, which may significantly reduce unwanted toxic side effect, enhance

treatment efficacy and reduce the risk of drug resistance (Tiwari et al., 2009). Early

studies have identified that the liposomalization of the anti-malaria agent chloroquine

increases its maximal tolerable dose and its efficiency and activity against murine malaria

infections greater than just chloroquine alone (Farouk et al., 2018). The ability to increase

the doses of chloroquine per injection after liposome encapsulation allowed successful

treatment of infection with chloroquine resistant P .berghei which could be cured by a

seven day course with the maximum tolerable dose of free chloroquine (Moles et al.,

2015). Antibody coated liposomes loaded with anti malaria drugs such as primaquine and

chloroquine completely arrest human infecting parasite, P. falciparium growth in vitro

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 and clear infection (Biagini et al., 2005). This was attributed to the fact that liposome is

of dual therapeutic and prophylactic effect to target both infected and non-infected

erythrocytes (Biagini et al., 2005). Liposomes circumvent drug resistant malaria because

they are targeted for intracellular delivery which bypasses chloroquine transponder and

pass through cell membrane by alternative mechanisms such as membrane fusion or

entrapment of chloroquine in pH-sensitive liposomes (Moles et al., 2015). Ligands

conjugated to the surface of liposomes can be used to target and specifically bind

plasmodium infected cell (Urban et al., 2014), because the blood stage of plasmodium

infection is responsible for all symptoms and pathologies of malaria, plasmodium –

infected erythrocytes are the main anti-malaria therapeutic target. Erythrocytes was

targeted by liposomes using heparin and monoclonal antibodies to erythrocyte surface

protein s have been studied in vitro and have shown promise towards targeted drug

delivery. Liposomes coated with heparin and primaquine encapsulated was formulated

and demonstrated to have anti-malaria activity and specific binding affinity for

plasmodium-infected erythrocytes in vitro via heparin targeting of heparin-binding

protein in erythrocytes membrane (Longmuir et al., 2006). Antibody –mediated

erythrocytes targeting using liposomes are another promising strategy for targeted drug

release. Recently, drug carried by liposomes were shown to be specifically targeted in

vitro to P. falciparium infected erythrocytes relative to non-infected erythrocytes likely

by docking to infected cell surfaces to facilitate membrane fusion (Longmui et al., 2006).

This demonstrates the feasibility of constructing a carrier able to completely discriminate

infected from non-infected erythrocytes.

2.1.4 Anti-cancer nano carrier

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There are variety of nanoparticles currently investigated and are explored with some emphasis

for cancer therapeutics; some nanoparticles mainly gold and silver (Au & Ag) and some

magnetic oxides in particular (Fe3O4) receive much interest in cancer treatment (Bououdina et

al., 2013). Abraxane, is albumin bound paclitaxel, a nano particle used for the treatment of breast

and non-small cell lung cancer. Nanoparticles are used to deliver drug with enhanced

effectiveness for head and neck cancer, in mice model study, which was carried out from Rice

University and University of Texas MD Anderson Cancer Center. The reported treatment uses

were Cremophor EL which allows the hydrophobic paclitaxel to be delivered intravenously.

When the toxic cremophor is replaced with carbon nano particles, its side effects are lowered and

drug targeting was much improved and lower dose of toxic paclitaxel is needed (Hollmer, 2012).

Nanoparticle chain was used to deliver the drug doxorubicin to breast cancer cells in mice study

at Case Western Reserve University. The scientists prepared a 100nm long nano particle chain

by chemically linking three magnetic, iron oxide nano spheres, to one doxorubicin loaded

liposome. After penetration of the nano chains inside the tumor magnetic nanoparticles were

made to vibrate by generating, radiofrequency field which resulted in the rupture of the

liposomes, thereby dispersing the drug in its free form throughout the tumor. Tumor growth was

halted more effectively by nanotechnology than the standard treatment with doxorubicin and is

less harmful to healthy cells as very low doses of doxorubicin were used (Bauer et al., 2012;

Garde, 2012).

2.2 Biomaterial

These are substances and compound from biological origin used in therapeutics, diagnosis, tissue

repair and drug delivery.

2.2.1 Tissue engineering

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The use of nanotechnology in this area aims to apply the principles of cell transplantation and

engineering to construct biological substitutes which are in turn used in an attempt to restore and

maintain normal function of organs and tissue previously diseased or damage (Atala 2005;

Langer 1999). The focus of tissue engineering is to repair or reconstruct lost or damaged tissue

through the use of growth factors, cell therapy, injectable biopolymer, and biomaterials, which

serve as support for the development of the cells (Venugopal et al., 2012). Cell interact with the

environment through thousands of interactions on a nanometric scale, therefore the goal of tissue

engineering on a nanoscale is to create biomaterials that direct interactions between cells and

their micro-environment, by the creation of nanoscale molecular signal of biological interest.

Thereby, the cells receive, process, and respond to the information presented in the surrounding

environment, these actions being essential for the control of cell behavior (Wheeldon et al.,

2011).

Electrospinning is the most widely studied in the techniques used to construct biomaterials and it

has also been demonstrated to give the most promising results in term of tissue engineering

applications. It is highly versatile method of transforming solutions, mainly made from

polymers, in continuous filament with diameter ranging from a few micrometers to nanometers.

Through this method, the fibers can be obtained randomly or in an ordered way. Electrospinning

works by the electrostatic principle, where the solution is supplied to the system via a syringe

and is subjected to a difference in electrical voltage, yielding solid fiber at the end of the process

(Greiner et al., 2007).

Nanofibers scaffolds are very promising physical guidance substrates for regenerating nerves to

transverse larger nerve gaps in the peripheral nervous system. For this kind of regeneration, the

orientation of the fibers is one of the parameters most studied when considering only the

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morphological of the biomaterials. Subramanian et al. compared random and longitudinally

aligned scaffolds of poly (lactic-co-glycolic acid) (PLGA) for the cultivation of Schwann cells.

These results for morphological and cell proliferation rate than random fibers. The physic-

chemical parameters evaluated also showed better results for nerve regeneration than random

fibers.

2.2.2 Gene Therapy

Gene therapy is an introduced method for the treatment or prevention of genetic disorders by

correcting defectives genes responsible for disease development based on the delivery of

repaired genes or the replacement of incorrect ones (Ariga, 2006). The most common approach

for correcting faulty genes is insertion of a normal gene into a non- specific location within the

genome to replace a nonfunctional gene. An abnormal gene could also be swapped for a normal

gene through selective reverse mutation, which returns the gene to its normal function

(Hanakawa et al., 2005). Mammalian cell typically have a diameter of a few microns and their

organelles are within the nanometer range. The use of nanodevices has the advantage of entering

the cells more easily when compared to larger devices and they can, therefore, interact better

with cells or at least in a different way (Kompella et al., 2013).

The use of nanotechnology in gene therapy could be applied to replace the currently used viral

vectors with potentially less immunogenic nano size gene carriers. Delivery of repaired genes or

the replacement of incorrect genes are field in which nanoscale objects could be introduced

successfully (Sahoo et al., 2007). The use of nanotechnology, here exemplified as the use of

nanoparticles, has some advantages in gene delivery: the structure of the nanoparticles protects

the nucleic acids from degradation by nucleases and the environment; it also minimizes side

26
effect by directing the nucleic acid to the specific location of action; they facilitate cell entry of

nucleic acids and normally nanoparticles sustain gene delivery for longer periods when

compared to other vehicles (Kompella et al., 2013).

2.2.3 Renewable Surfactants

Renewable surfactants (also called biosurfactants) are surfactant obtained from fermentation and

surfactants synthesized from renewable raw materials (bio-based or natural surfactants).

Renewable surfactants are used for high added value applications in biochemistry (protein

crystallization, gene transfection) and in nanotechnology (Nano emulsions). Due to the self-

assembling nature, and their role in the moderation of interfacial tension, surfactant can be

applied to the formation of nano emulsion which is application of nanotechnology in

biochemistry. IUPAC defines nano emulsion as emulsion with a very small droplet size

(<300nm), which are thermodynamically unstable systems. Nevertheless, they considered as

kinetically stable system because the kinetics of destabilization are very slow (a month scale)

(Anton and Vandamme, 2011).

Nanoemulsion can be prepared using “high energy” methods (i.e., through ultrasound or high

pressure) or “low energy” emulsification method (Tadros et al., 2004). The nature and amount of

the surfactant and cosurfactant influence the size distribution and stability of the nanoemulsion

formulated. Generally, surfactants with high HLB values are used (Bouchemal et al., 2004).

Nano emulsion can be applied in drug delivery due to their specific properties: thermodynamics

stability, increased surface area, easy preparation, high bioavailability and optical transparency.

They can act as vehicle for the delivery of active pharmaceutical ingredients. Compared to

conventional drugs delivery system, nano emulsion offers a reduction of toxicity and irritant

potency, an improvement of consumer compliance, an enhancement of absorption, and a

27
prolongation of stability (Jaiswal et al., 2015). Nano emulsion are also useful for other

application such as cancer therapy. They can easily be targeted to the tumor area due to their

submicron size. They have the capability to increase biodistribution of therapeutic agents to

target organs and to improve the pharmacokinetics, which result in improved efficacy (Bangia

and Om, 2015). There has been great interest on sucrose esters for the formation of various nano

system. Sucrose esters can be used as emulsifier, and as stabilizing agent in nanoemulsion, in

nanosuspensions, or in solid lipid nanoparticles (Calderilla-Fajardo et al., 2006; Szuts and

Szabo-Revesz, 2012; Tagekami et al., 2008). Apart from sucrose esters, there are also report

about the use of other green surfactants, such as glycolipids (Ahmad et al., 2014) or fatty acid-

esters, (Hadzir et al., 2013) for the formulation of nano emulsions to be applied in drug delivery.

28
 CHAPTER THREE

3.1 Enzyme Immobilization

Enzymes are catalyst that catalyze many biochemical and chemical reaction. They are

universally present in plants and animals. Immobilization of enzyme was discovered in 1916

(Nelson et al., 1916). It was demonstrated so that activity of enzyme invertase does not get

hampered when it is adsorbed on solid matrix, such as charcoal or an aluminum hydroxide. This

aspect led to the development of currently available enzyme immobilization techniques (Brady

et al., 2009; Cantone et al., 2013).

Currently, commercial application of immobilized enzyme has been enhanced as they are highly

efficient (Cantone et al., 2013; Dicosimo et al., 2013). It is also resistance to various

environment changes such as pH or temperature has been increased immobilization of enzyme

on solid support (Cherry et al., 2003).

3.1.1 Methods of Immobilization

The selection of mode of immobilization is very important to prevent loss of enzyme activity by

not changing the chemical nature or reactive groups in the binding site of enzyme. The most

29
common procedure for enzyme immobilization is adsorption, covalent coupling, entrapment and

cross linking (Brady et al., 2009).

3.1.2 General Method for Immobilization

(a) Adsorption: Adsorption of enzymes unto insoluble support is a very old but simple method

which has wide application and high capability enzyme loading relative to other

immobilization methods. Enzymes can be immobilized by simply mixing the enzymes with

a suitable adsorbent, under appropriate condition of pH and ionic strength (Figure 8). After

washing off loosely bound and unbound enzyme, the immobilized enzyme is obtained in

directly unstable form. Adsorption process is based on Vander Waal forces, ionic and

hydrogen bonding as well as hydrophobic interactions which are very weak forces, but in

large number, impart sufficient binding strength (Spahn et al., 2008).

Adsorption by physical method leads to major change in protein microenvironment, and

typically involves multipoint protein adsorption between a single molecule and a number of

binding sites on the immobilization surface (Johnson et al., 1996). The main advantage of

this method is that enzyme is easily desorbed by factors like change in temperature, change

in substrate and ionic concentrations (Rao et al., 1998).

30
Figure 8: Adsorption method of enzyme immobilization

Source: Ahmad and Sardar (2015)

31
(b) Covalent binding: Covalent binding is covalent immobilization of enzyme involve the

formation of covalent bonds between the enzyme and support the matrix (Figure 9). The

functional groups present in the enzymes get linked to support matrix as these functional

group are not responsible for the catalytic activity. The binding reaction must be performed

under conditions that do not cause loss of enzymatic activity, and the active site of the

enzyme must be unaffected by the reagents used. Covalent association of enzymes to

support occurs owing to their side chain amino acid like arginine, aspartic acid, histidine and

degree of reactivity based on different functional groups like imidazole, indolyl, phenolic

hydroxyl etc. (D’Souza, 1999).

(c) Entrapment: Entrapment (Figure 10) This is done by restricting the movement of enzymes in

porous gel, yet keeping them as free molecules in solution. Entrapment of enzymes within

gels or fibers is a convenient method for use in processes involving low molecular weight

substrates and products. Enzymes have been entrapped in natural polymers like agar,

agarose and gelatin through thermo reverse polymerization, but in alginate and carrageenan

by ionotropic gelation (Datta et al., 2013).

32
Figure 9: Covalent binding of enzyme immobilization

Source: Ahmad and Sardar (2015)

33
Figure 10: Entrapment mode of enzyme immobilization

Source: Ahmad and Sardar (2015)

34
(d) Cross-linking: Cross-linking (figure 11), this method involves attachment of biocatalysts to

each other by bi- or multifunctional reagents or ligands (Datta et al., 2013). In this way, high

molecular weight typically insoluble aggregate is formed. Cross-linking is a relatively

simple process. It is not preferred method of immobilization as it does not use any support

matrix. So, they are gelatinous and not particularly firm. Since it involves a bond of the

covalent kind, biocatalyst immobilized in this way usually undergoes changes in

conformation with a resultant loss of activity.

3.1.3 Nanoparticles as Immobilization Matrix

Nanoparticles is an efficient material which provide support to enzyme immobilization, because

of their ideal characteristics for balancing the key factors that determine biocatalysts efficiency,

including specific surface area, mass transfer resistance, and effective enzyme loading (Ansari et

al., 2012; Feng et al., 2011; Gupta et al., 2011; Verma et al., 2013). Diffusion problem is

important when dealing with macromolecular substrate, and for such system nanoparticles are

suitable candidates (Hwang et al., 2013). Also, the enzyme bound nanoparticles show Brownian

movement, when scattered in aqueous solutions showing that the enzymatic activities are

comparatively better than that of unbound enzyme (Gupta et al., 2011). Furthermore, ymagnetic

nanoparticles possess additional advantage, i.e. can be separated easily using an external

magnetic field. Studies have shown that immobilization of enzymes to the nanoparticles can

reduce protein unfolding and improved stability and performance (Gupta et al., 2011).

35
Enzymatic immobilization on Au and Ag nanoparticles have been study using either whole cells

or isolated enzymes, which include lysozyme (Vertegel et al., 2004), glucose oxidase (Lan et al.,

2008) and alcohol dehydrogenase (Keighron et al., 2010). The immobilization of enzymes S

Carlsberg and Candida Antarctica lipase B (CALB) on fumed silica nanoparticles for application

in nonaqueous media was observed and catalytic activities were remarkably high (Cruz et al.,

2011). The immobilization of acetylcholinesterase on nickel nanoparticles and a highly sensitive

detection method for organophosphate pesticides was obtained (Ganesana et al., 2011).

All the activity of immobilized enzymes on micron-sized particles are inherited when

nanomaterials are used as solid supports. There are four main approaches to link enzyme to the

nanoparticles (Ahmad et al., 2015).

(a) Electrostatic adsorption: this is the most widely used linkage approach (Figure 12). This

is the simplest approach and is already used routinely as an electron dense marker in

histology (Geoghegan et al., 1977). The interaction between the nanoparticle and protein

may be modulated by the pH or charge screening by controlling the ionic strength of the

medium.

36
Figure 11: Cross linking mode of enzyme immobilization

Source: Ahmad and Sardar (2015)

Figure 12: Electrostatic adsorption of enzyme link to nanoparticle

Source: Ahmad and Sardar (2015)

37
(b) Covalent attachment to the surface modified nanoparticles: This is also general method

for nanoparticle –protein conjugation covalently linking a protein to the nanoparticle

ligand (Figure 13). This approach has been greatly advanced by extreme control over the

surface chemistry of the nanoparticles. For example, a variety of organic functional

groups can be introduced to the surface using mild conditions (Aubin-Tam et al., 2008).

The popular labeling chemistry utilizes the covalent binding of primary amines with

sulfo-NHS esters or R-COOH groups via reaction with EDC (Aubin-Tam et al., 2008).

Nanoparticles labeled with NHS esters can reacts to form covalent bonds with the

primary amine of lysine on a protein. Also, nanoparticles coated with maleimide groups

can react with the thiol of cysteine on a protein.

(c) Conjugation using specific affinity of protein: Nanoparticles-protein conjugation can also

be achieved by using specific labeling strategies (Figure 14). Example is streptavidin

coated nanoparticles can selectively bind biotin-labeled proteins and the antibody coated

nanoparticles selectively bind to the specific protein (Di Marco et al., 2010).

38
Figure 13: Covalent attachment to the nanoparticle ligand

Source: Ahmad and Sardar (2015)

39
Figure 14: Conjugation using specific affinity of protein

Source: Ahmad and Sardar (2015)

(d) Direct conjugation to the nanoparticles surface: A direct reaction of a chemical group on

the protein without the use of a linker is usually desired if the particle is used as biosensor

where electron transfer is used (Figure 15). For Au and Ag nanoparticles, this can be

achieved by the Au-thiol or Ag- thiol chemistry where an enzyme with a cysteine

covalently bonds to an Au or Ag nanoparticle. The conjugation requires incubation of the

protein and the nanoparticle together as the Au-S bond or Ag-S bond is strongly favored.

Similarly, for Sulphur containing nanoparticles such as ZnS/CdSe, cysteine can directly

form a disulfide bridge with the surface atom (Ahmad and Sardar, 2015).

40
Figure 15: Direct conjugation to the nanoparticle surface

Source: Ahmad and Sardar (2015)

41
3.2 Enzyme Therapy

The term enzyme therapy describes the administration of exogenous enzyme to replace the

defective endogenous counterparts (enzyme replacement therapy) and can also be applied

beyond the treatment of genetic diseases with impaired enzyme production, for example, in cases

where administration of additional enzymes that are not encountered endogenously in the human

body can help alleviate phenotypic symptoms. Examples of the first approach have been applied

to the treatment of lysosomal storage disorders (LSDs), mostly caused by genetic defects

affecting enzymes involved in lysosomal degradation of varied substrates (Burrow et al., 2007).

Second approach is illustrated in the administration of alginate lyase to degrade components of

infectious biofilms in cystic fibrosis (CF) (Lamppa et al., 2011) or delivery of uricase fur gout

treatment (Sherman et al., 2008). Preliminary attempt in this direction include the case of

delivery of PEG-modified dextranase, which achieved prolong activity by passing

immunorecognition in a mouse model of LSD mimicked by lysosomal accumulation of dextran

(Mumtaz and Bachhawat 1994). Similar strategies of PEGylation have been useful for delivery

of uricase for gout treatment in hyperuricemia (Sherman et al., 2008), non-mammalian

phenylalanine ammonia lyase for treatment of neuropathic phenylketonuria (PKU) (Ikeda et al.,

2005), or alginate lyase delivery targeted to CF infections (Lamppa et al., 2011)

Prolidase delivery by chitosan nanoparticles enhance absorptive endocytosis of the enzyme into

the cells, with subsequent pH-triggered release to the cytosol (Colonna et al., 2008), or enhanced

delivery of acid sphingomyelinase or α-galactosidase to lysosomal compartments by ICAM-1

42
targeted nanocarriers (Garnacho et al., 2008; Hsu et al., 2011; Muro et al., 2006). In the latter

example, ICAM-1 targeting provides means for switching the entry pathway of lysosomal

enzymes within the cells from clarithin-mediated endocytosis, typically induced by binding of

mannose or mannose-6-phosphate present on the naked lysosomal enzymes to the corresponding

cell targeting. This provides an advantage for those cases when the pathology itself tends to alter

the expression or function of cell receptors, such as the case of mannose-6-phosphate receptor in

some LSDs (Cardone et al., 2008; Dhami and Schucman, 2004). Finally, transport across cellular

barriers, greatly impaired for many enzymes therapies could be improved by formulating said

enzymes as their nanocarrier counterparts, enabling these treatments to reach the central nervous

system or to be converted into oral delivery modalities.

3.3 Protein Engineering

Protein engineering has been a powerful tool in producing a vast number of proteins/enzymes

used in industrial applications (Foo et al., 2012). This field of knowledge has expanded not only

in the industrial area, but also in medical field, due to its therapeutic potential in biosensors

development, cellular imaging and immunoassays (Foo et al., 2012; Tamerler et al., 2010).

Considering industrial applications, protein engineering has been used to improve the

performance of enzymes involved in lignocelluloses degradation, as well as the synthesis of

biofuels (Wen et al., 2009).

Nanoparticles play a core role by putting together different catalytic domains through a suitable

attachment strategy. Carneiro and Ward developed a paramagnetic nanocomposite coated with

chitosan and N-(5-amino-1-carboxy-pentyl) iminodiacetic acid which bind virtually any protein

or set of protein containing histidine tag (Carneiro et al., 2018). The binding of a 6-histidine tag

to the carboxy- or amino-terminal end of the polypeptide is the most simple and original example

43
of a successful protein engineering strategy to design a generalized purification scheme applied

virtually to any protein (Gaberc-Porekar et al., 2005).

Protein engineering strategies point out the rational design and construction of nanoscale protein

cages as a feasible and reachable strategy through conventional recombinant DNA technologies

and microbial protein over expression system. In therapeutics applications, naturally formed

nanostructures (such as viruses, flagella or protein oligomers) can be manipulated to acquire

specific characteristics of interest. For example, by the insertion of cell targeting agents, it is

possible to control biodistribution allowing specific delivery of associated drugs (Vazquez et al.,

2010).

3.4 Protein and peptide delivery

Protein and peptides are macromolecules and are called biopharmaceuticals. These have been

identified for treatment of various diseases and disorders as they exert multiple biological actions

in human body. Nanomaterials e.g., dendrimer are called nano biopharmaceutical and are used

for targeted or controlled delivery

Nanoparticles were found useful in delivering of myelin antigens, which induce immune

tolerance in a mouse model with relapsing multiple sclerosis. In this technique, biodegradable

polystyrene micro particles coated with the myelin sheath peptides will reset the mouse immune

system and thus prevent the reoccurrence of disease and reduce the symptoms as the protective

myelin sheath forms coating on the nerve fibers of central nervous system. This method of

treatment can potentially be used in treatment of various other autoimmune diseases (Laurance

2012; Miller et al., 2012)

3.5 Nanoencapsulation

44
Encapsulation is a process of enclosing the substances within an inert material which protects the

substances from the environment as well as control drug release. Two types of encapsulation

have been performed in several research: Nanoencapsulation and Microencapsulation.

Nanoencapsulation is defined as a technology to encapsulate substances in reduced scale and

refers to bioactive packing on at the nanoscale range (Lopez et al., 2006). The delivery of any

bioactive compound to various sites within the body is directly affected by the particle size

(Hughes, 2006). Thus, nanoencapsulation has the potential to enhance bioavailability, improve

controlled release, and enable precision targeting of the bioactive compounds in greater extent

than microencapsulation (Mozafari et al., 2006). Nanoparticles are colloidal-sized particles with

diameters ranging from 10 to 1000nm and are expressed both as nano capsules and nanospheres

(Konan et al., 2002). Nanocapsules are vesicular systems in which the bioactive compounds is

confined to a cavity surrounded by a unique polymer membrane, while nanospheres are matrix

systems where the bioactive compound is uniformly dispersed (Figure) (Couvreur et al., 1995).

(a) Core materials for Nanoencapsulation: Core materials such as lipophilic and

hydrophilic nutraceuticals compounds are used for nanoencapsulation. Hydrophilic

compounds are soluble in water but insoluble in lipids and organic solvents. Some

nanoencapsulated hydrophilic compounds are ascorbic acid, polyphenols e.t.c. (Lakkis et

al., 2007; Teeranachaideekul et al., 2007; Dube et al., 2010; Ferreira et al., 2007).

Nanoencapsulated lipophilic compounds includes lycopene, beta-carotene, lutein,

phytosterols and docosahexaenoic acid (Lakis et al., 2007; Heyang et al., 2009; Zimet et

al., 2009; Leong et al., 2011).

(b) Coating materials for Nanoencapsulation: Polymers are used in preparation of

nanoencapsulation, the polymers should be compatible with the body in the terms of

45
 adaptability (non-toxic) and (non-antigenicity) and should be biodegradable and

biocompatible (Ghosh et al., 2000). The most commonly used natural polymers in

preparation of polymeric nanoparticles are chitosan, gelatin, sodium alginate and albumin

(Nagavarma et al., 2012).

3.6 Application of Nanoencapsulation

The basic reason for nanoencapsulation is to protect the core material and to then release it when

it is needed (Suganya et al., 2017). It is applied to targeted drug delivery systems that release

drug only when the drug has arrived at the site in the body where it is required. Timed release

drug delivery where the nanoencapsulation material slowly allows the drugs to be released into

the body such as nasal delivery of insulin (Suganya et al., 2017). The coating material can be

customized to determine the rate of delivery. Nanoencapsulation can be applied to food additives

and food enhancements such as omega-3 fatty acid addition to bread that do not alter taste

(Suganya et al., 2017). Increasing shelf life and stability of products like vitamins (Suganya et

al., 2017).

46
 CHAPTER FOUR

4.0 Nanotechnology in food and nutritional biochemistry

Nanotechnology found a wide range in food sector (Acevedo et al., 2017; Faridi et al., 2016),

which includes food safety and quality, the targeted delivery of important compounds and

increased bioavailability, the development of new product, the control of technological

processes, packaging and sensory improvements, such as taste modification and texture (Huang

et al., 2010; McClement et al., 2009; Ranjan et al., 2014).

Innovative nanotechnology has revolutionized the food industry (Sanguansri and Augustine,

2006; Weiss et al., 2006; Chaudhry et al., 2008; Silverstre et al., 2011; Cushen et al., 2012;

Rossi et al., 2014; Thangave and Thiruvengadam, 2014). There is progressive improvement in

use of nanoparticles in food industry especially on food processing, packaging, storage and

development of innovative products. Nanoparticles aimed at enhancing bioavailability of nano-

sized nutraceuticals and health supplements, improving taste and flavor, consistency, stability

and texture of food product (Chaudhry et al., 2008; Chaudhry et al., 2010; Momin et al., 2013).

Due to antimicrobial activities of nanoparticles, it can be incorporated into the food packaging

materials to improve shelf life and keep it safer for human consumption. It is predicted that the

invasion of the food production market with nanoparticles will be significantly increased in the

47
near future (Heinlaan et al., 2008). Moreover, the use of encapsulated nanoparticles enables

development of nano-formulated agrochemicals such as pesticides, fertilizers, biocides,

veterinary medicines, additives, antimicrobials and detoxifying compounds. In human food

processing, nano capsules have been used as nona-sized ingredients, additives nutritional

supplements, and in functional food (Momin et al., 2013). Cushen et al. (2012) reported that

nanoencapsulation of food ingredients and additives have been carried out to provide protective

barriers, flavor and taste masking, controlled release, and better dispensability for water –

insoluble food ingredients and additives.

4.1 Nanotechnology in food processing

During food processing, nanoparticles have been applied to improve nutritional quality, flow

properties, flavor, color and stability or to increase shelf life. Indeed, nanotechnology might help

in development of healthier food with lower fat, sugar and salts to overcome many food-related

diseases. Recently, bulk amounts of SiO2 and TiO2 oxides have been permitted as food additives

(E551 and E171) (EFSA, 2000). Effective olive oil hydrolysis by the use of covalent

immobilization of porcine triacylglycerol lipase onto functionalized nanoscale SiO2 with reactive

aldehyde group for better reuse, adaptation and stability have been reported (Bai et al., 2006).

Several nano and micro- structured assemblies of nanoparticles have been designed for

encapsulation of food ingredients, additives, nutritional supplements as well as functional foods

(Augustin and Hemar, 2009).

4.1.1 Nano-sized additives and nutraceuticals

The potential of nanotechnology in functional food, design of nutritional supplements and

nutraceuticals containing nanosized ingredients and additives such as vitamins, antimicrobials,

antioxidants and preservatives are currently available for enhanced taste, absorption and

48
bioavailability (Momin et al., 2013). Some nutraceuticals incorporated in the carriers include

beta-carotene, lycopene and phytosterols are used in healthy foods to prevent the accumulation

of cholesterol (Mozafari et al., 2006).

4.1.2 Nanoencapsulation

Nanosized carriers’ systems or nanoparticles, in the form of micelles, liposomes or protein-based

carriers have been used as nano-food additives, nutritional supplements, mask the undesirable

taste, enhance bioavailability and allow for better dispersion of insoluble additives without need

for surfactants or emulsifiers (Morris et al., 2011; Cushen et al., 2012; Duran and Marcato,

2013). During nanoencapsulation, the food additives substances enclosed in nanocomposite

polymer e.g., Octenyl succinic anhydride-ε-polylysine ofr controlled release (Yu et al., 2009;

Sekhon, 2010).

Anti- cancer activity of curcumin was enhanced by encapsulation in hydrophobically modified

starch (Yu and Huang, 2010).

Also, the use of lipid based nanoencapsulation e.g., nanoliposomes, nano cochleates and

archaesomes as nano delivery system for nnutraceuticals, enzymes, food additives and

antimicrobials were reported (Mozafari et al., 2006; Mozafari et al., 2008). Nanoencapsulation

of probiotics to be targeted to specific region in gastro intestinal (GI) tract has been achieved

(Vidhyalakshmi et al., 2009).

Inorganic nano-sized additives and composites such as silver, iron, calcium, magnesium,

selenium, and silica have been used as preservative and additives to improve taste and flavors.

An example of nano-sized particles is the addition of food grade polypeptide, ε-polylysine as

antioxidant to protect oil from oxidation. These polylysine nanoparticles are much smaller than

phytoglycogenoctenyl succinate nanoparticles (Scheffler et al., 2010). Vargas et al, (2008)

49
described the design of a nanocoating to be used as carrier of functional ingredients during

nanoencapsulation process. Edible nanoparticle films based on chitosan obtained by

incorporating nanoparticles made from montmorillonites, nanosilver or silver zeolite (Rhim et

al., 2006).

(a) Encapsulation of polyphenol in food

There are numerous biologically active compounds that can be encapsulated, but the

encapsulation of polyphenol is highly important (Faridi et al., 2016). Polyphenol are widely

studied plant secondary metabolites due to their potential positive effects on human metabolic

processes (Alrgei et al., 2016; Stanisavljevic et al., 2016; Kostic, 2019). Phenolic compound has

significant antioxidant and antimicrobial characteristics, but they are very unstable and

susceptible to degradation processes, poorly soluble and in most cases, their bioavailability is

relatively low (Faridi et al., 2016). So, incorporating of plants phenolic compounds in food can

significantly affect their physiochemical properties, stability, solubility and bioavailabilty

(Acevado et al., 2017; Pesic et al., 2019).

Encapsulation can prevent the degradation of phenolic compounds due to its protective effects

against negative impact of light, oxygen and PH. Encapsulation can also prevent interaction

between polyphenol and other food components (Gomez-Estaca et al., 2012). Further,

polyphenol- loaded nanoparticles can be used for development of new types of functional food,

of which the consumption prevents various diseases (Madureira et al., 2015). Different

nanocarriers for phenolic components can be used as a protective barrier, and they can roughly

be divided into polysaccharide- and protein-based delivery systems (Aguirre et al., 2019).

Substances such as cyclodextrins, polymeric nanoparticles, nanomicelles (Faridi et al., 2016),

gelatin nanoparticles and films (Hu et al., 2017; Chen et al., 2010), food-protein nanoparticles

50
like casein, whey proteins, soybean proteins (Prakash et al., 2017; Tian et al., 2019), zein

nanoparticles (Wang et al., 2018), chitosan (Chen et al., 2010; Hu et al., 2017; Prakash et al.,

2017; Liang et al., 2017), lipid nanocarriers (Pimentel-Moral et al., 2018; Balanc et al., 2018), or

protein–polysaccharide complex nanoparticles (Chang et al., 2017) have proven to be suitable

carriers for the nanoencapsulation of polyphenolic compounds.

The choice of carrier material affect the physiochemical characteristics of the encapsulated

substances (Kalusevic et al., 2017). Food-protein nanoparticles and chitosan are the most

commonly used nanocarriers for the delivery of plant polyphenols because there is solid

evidence that they improve the intestinal absorption of phenolic compounds (Hu et al., 2017).

Recently, polysaccharide–protein nanocarriers have been reported as being promising for

polyphenols encapsulation [Jones and Mc-Clements, 2011; Veneranda et al., 2018). Active

components bind to the protein part of the nanocarrier via hydrogen bonding and hydrophobic

interactions, while polysaccharides contribute to the prevention of enzymatic protein degradation

in gastric conditions (Luo et al., 2015). Polysaccharide–bioactive peptide nanoparticles can also

be valuable nanocarriers for the encapsulation of small molecular polyphenols, providing better

bioavailability of these valuable components (Hu et al., 2012). Among polyphenols, the most

commonly encapsulated are catechins, quercetin, eugenol, epigallocatechin, epigallocatechin-

gallate (EGCG), curcumin, and polyphenols derived from teas or essential oils.

Polyphenols can interact with nanocarriers. For example, it has been shown that there is a certain

level of hydrogen bonding between catechin and unreacted amino groups of chitosan in their

encapsulates (Dudhani and Kosaraju, 2010). Catechin can also interact with proline-rich proteins

(Faridi and Jafari, 2016; Schwarz and Hofmann, 2010), or with the phenolic ring of EGCG with

prolines in β-casein (Faridi and Jafari, 2016; Jobstl et al., 2004). The mechanisms of

51
encapsulation enabling polyphenol–nanocarrier interactions include ionic gelation, coacervation,

liposome entrapment, inclusion complexation, co-crystallization, nanoencapsulation, freeze-

drying, yeast encapsulation, and emulsion (Zhang et al., 2016). These various mechanisms can

increase the bioavailability of polyphenols, prevent degradation in the gastrointestinal tract,

enhance the delivery of polyphenols directly to the targeted sites (Aguirre and Borneo, 2019), or

provide stability during storage or processing (Nedovic et al., 2011).

i. Use of Polyphenol-Loaded Nanoparticles in Food Processing: Polyphenol-rich

nanoparticles can be used in food processing for the improvement of physicochemical

characteristics, such as food stability, texture, and flavor profile, or functional properties

such as antioxidant or antimicrobial activity (Madureira et al., 2015). Nanoencapsulation

has become an important approach for preserving the properties of active components

during food processing and consumption (Sarkar et al., 2017). Further, nanotechnology-

based sensor systems have been developed as alternatives for conventional analytical

methods in safety and quality control (Zhang and Chen, 2019).

ii. Polyphenol-Loaded Nanoparticles for the Enhancement of Functional Properties of Food

 Anti-oxidants properties: The aim of polyphenolic-nutrient nanoencapsulation is to

protect and transport these active components and ensure their maximum bioavailability.

For example, the dominant polyphenols of tea catechin and its derivatives (−)-

epigallocatechin-3-gallate, (−)-epicatechin-3-gallate, (−)-epigallocatechin, and (−)-

epicatechin, have strong biological properties, such as antioxidant and anticancer

activities (Chen et al., 2010; Liang et al., 2016; Hu et al., 2008); on the other hand, if

they are not encapsulated, their bioavailability is low (Hu et al., 2008). Consequently,

there is a necessity for the development of effective delivery systems for improving of

52
 polyphenolic components’ bioavailability. In the study of Tang et al., (2013), chitosan/γ-

PGA (PGA-edible polypeptide (poly-γ-glutamic acid)) nanoparticles were prepared for

the oral delivery of tea catechins. Briefly, tea-catechin-loaded nanoparticles showed very

effective radical scavenging capacity and could serve as suitable carriers for the

transmucosal delivery of tea catechins. According to the same authors, nanoencapsulated

catechins could be used as food additives and dietary supplements. It has been found that

among tea polyphenols, EGCG has the largest scavenging capacity (Peng et al., 2006;

Puligundla et al., 2017), but in many studies, it has been indicated that EGCG has low

bioavailability because of its large-sized molecules and the number of hydrogen bonds

(Puligundla et al., 2017; Lante and Friso, 2013). Application of chitosan/β-lactoglobulin

nanocarriers provided sustained release of tea EGCG and thereby increased its absorption

in the human intestine (Liang et al., 2016). Nanocarriers such as dextran sulfate,

combined with N-dimethylhexadecyl carboxymethyl chitosan nanoliposome (DS-

DCMC-NL), are also a suitable carrier for EGCG, which possesses a high encapsulation

capacity and the potential for further application in food processing (Zou et al., 2015).

 Health-Promoting Properties: Besides antimicrobial properties, the potential health

benefits of polyphenols were in a much broader scope of numerous studies in recent

years. The beneficial effects of resveratrol, such as antioxidant, antitumor, anti-

inflammatory, and antiaging effects, can be improved by nanoencapsulation (Ahmadi et

al., 2019). However, among numerous investigations, only few in vivo studies were

performed to confirm the health-promoting properties of nanoencapsulated resveratrol,

indicating that further research is required (Santos et al., 2019). Curcumin also showed

antidiabetic properties when curcumin–metal (ZnO) complexes were loaded with

53
 chitosan (Chauhan et al., 2019). The addition of curcumin nanocapsules in food also

resulted in improved functional properties (better solubility and stability, and lower

degradation rate) while keeping its remarkable biological functions (Sivasami and

Hemalatha, 2018). The study of Di Costanzo and Angelico (2019), reviewed the

numerous possibilities of silymarin nanoencapsulation, a mixture of flavonolignan and

flavonoid polyphenolic compounds from the Silybum marianum seed, which has

significant biological potential (antioxidant, anti-inflammatory, anticancer, and antiviral

activities). The synergistic effect of polyphenols is being studied, and it is known that

certain polyphenol combinations have better health-promoting characteristics when

combined rather than with the sum of individual ones (Zhang et al., 2019). Nanogels

where tea polyphenols were encapsulated with lysozyme and carboxymethyl cellulose

showed antitumor effects and represent a promising carrier for functional food

ingredients (Liu et al., 2019).

4.2 Nanotechnology in food packaging

The crucial role of nanotechnology in food packaging process is considered as the largest

commercial application in food sector (Chaudhry et al., 2010). The use of nanoparticles might

help in production of new food packaging materials with improved mechanical barrier and

antimicrobial properties to increase shelf life (Chaudhry et al., 2008; Mihindukulasuriya and

Lim, 2014). Beside antimicrobial activities, nanoparticles can be as vehicle to deliver

antioxidants, enzymes, flavor, anti-browning agents and other material to extend shelf life, even

after opening (Cha and Chinnan, 2004; LaCoste et al., 2005; Weiss et al., 2006). Inorganic

nanomaterials of some metals and metal of oxides such as silver, iron, titanium dioxide, zinc

oxides, magnesium oxides as well as silicon dioxide and carbon nanoparticles have been used as

54
antimicrobial agents in food packaging and in some cases as food supplements (Sekhon et al.,

2010). TiO2 is widely used as a disinfecting agent as it generates highly reactive oxygen species

(ROS) that are toxic to pathogenic microorganisms (Mahmoud, 2015).

4.2.1 Use of Polyphenol-Loaded Nanoparticles in Food packaging

Nanoparticle incorporation into packaging materials improves their barriers (gas/water/aroma),

mechanical or oxygen-scavenging properties, thus enhancing food quality and prolonging the

shelf-life (Sarkar et al., 2017). In addition to improving the physical properties of packaging

materials, nanoparticles add other functions, such as antimicrobial and antioxidant properties, or

act as nanosensors/nanobiotracers, enabling better food security (Sarkar et al., 2017; Enescu et

al., 2019). There is a growing demand for the development of new environmentally friendly

films for food packaging based on biomaterials (Otoni et al., 2014).

Nanofilms compared to traditional food-packaging films should have another dimension: the

slow and controlled release of antimicrobial and other active components (Makwana et al.,

2014). This is especially important in the case of antioxidants that, as components of packaging

materials may provide prolonged shelf-life for food products. Nanoemulsions containing

polyphenols have been used for creating fruit-based edible films, which showed not only good

antimicrobial and physicomechanical properties (such as permeability to water vapor), but also

had an environmentally friendly dimension thanks to waste usage from the food industry.

Pectin/papaya puree/cinnamaldehyde nanoemulsion edible composite films could be

environmentally friendly antimicrobial packaging material for food applications (Otoni et al.,

2014).

It has also been demonstrated that the antibacterial properties of nanoencapsulated

cinnamaldehyde used for food coatings were better when compared with free cinnamaldehyde.

55
These films showed significant activity against E. coli and B. cereus (Makwana et al., 2014).

Besides coating, food shelf-life could be prolonged with protection against the oxidation of fatty

foods, using polyphenols such as EGCG in the form of nanosized particles (Liang et al., 2017).

A similar effect was obtained with green-tea polyphenol-loaded nanoparticles for the same

purpose (Wrona et al., 2017). The incorporation of sage extract (Salvia officinalisL.) topoly (ε-

caprolactone) films resulted in the formulation of potential food-packaging material with both

antioxidative and antimicrobial functions (Salevic et al., 2019).

Other food packaging nanoparticles with antimicrobial activity including; MgO (Stoimenov et

al., 2002). Cu2O (Yoon et al., 2007; Cioffi et al., 2005). Chitosan (Qi et al., 2004; Tan et al.,

2013).

CONCLUSION

Nanotechnology is very much predominant almost every facet of life. Nanotechnology is a new

useful technology that use to solve many activities. Its application in medicine is warranted and

becomes the new hop in diagnosis, treatment, and prevention. The applications of

nanotechnology can be seen in several fields of medicine and become the new things that

practitioner has to recognize and use.

The change in behaviour of material at nanoscale is dominated in the first place by quantum

mechanics and is additionally attributable to material confinement in small space, and the

increase in surface area per volume. At the nanoscale, biology, chemistry, physics, material

science, and engineering intersect toward the same principles and tools. Resulting the

development in nanoscience has very far-reaching impact. Nanoparticles have prospective

applications in the field of medical sciences including new diagnostic tools, imaging agents and

56
methods, targeted drug delivery, pharmaceuticals, bio implants and tissue engineering. Drugs

with more toxic possible like cancer chemotherapeutic drugs can be given with better safety

portrait with the profitability of nanotechnology.

Nanotechnology has been able to offer exciting approach in area of biochemistry for both living

systems, including characterizing distinctive phenotypes and enriching clinical relevant

biomarkers and perturbing living systems such as drug delivery modalities that can overcome

biological barriers. In nanotechnology, nanocarriers has been used for effective drug delivery

which shows more efficient and fast delivery of the drug to the targeted cells without the

complications accompanied by other routes of delivery thereby making them safer.

The use of Nanoparticles has emerged as a versatile tool for generating excellent supports for

enzyme stabilization due to their small size and large surface area. It has been observed that the

stability and activities of enzyme increase when immobilized on such materials.

57
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