Chapter 3

3.1 Introduction

Globally, cancer is ranked as the leading cause of death. Chemotherapy remains to be the primary cancer
treatment process in clinics, but the biggest challenge has been hindrance of chemotherapeutic agents by their
characteristically low aqueous solubility, toxicity, drug resistance, as well as the side effects. The
chemotherapeutic agents have reduced clinical efficacy which is attributed to non-specific bio-distribution after
entering the body [1, 2]. The low efficacy of Gemcitabine is particularly attributed to their poor selectivity and
to toxicity. Gem is a weak base (pKa 3.6) consists of deoxycytidine analog. It is a novel and potent anticancer
drug which used for multiple types of cancer treatments such as pancreatic cancer [57, 58, and 266]. As
discussed in chapter 2 Gem is a pro-drug that needs cellular uptake as well as intracellular phosphorylation.
Gem triphosphate is an active moiety which can inhibit the DNA synthesis within the cell [58].

Following a systematic administration of Gem, the drug undergoes rapid metabolization by cytidine deaminase
to form 2′-deoxy-2, 2′-difluorouridine - an inactive compound that is gotten rid of by the kidneys. Thereafter,
the excretion of the metabolite occurs via urine with a half-life of about 8 to 17 min. It is necessary to have
repeated administrations of the drug so as to achieve the required pharmacological effect [18]. As a result of
having a pharmacodynamic profile that is complicated, the drug’s anti-tumoral effects are largely controlled by
administration frequency, as opposed to dosage. One can only achieve a suitable therapeutic response following
a frequent administration, or prolonged infusion for specific cancer types [20]. Even though Gem comes with
improved therapeutic index compared to other anti-cancer medications, the drug is has some serious side effects
that have been observed in clinical trials, such as mild and transient neutropenia, anaemia, mild and transient
neutropenia, and thrombocytopenia [19]. Furthermore, there are reports that it reduces its clinical efficacy due to
multidrug resistance (MDR) [63]. More discussions on this will be in chapter 4.

In order to overcome these limitations, researchers have proposed a number of strategies to ensure efficient
delivery of gem and improve its efficacy, and also reduce therapeutic complications associated with the drug
[20, 21]. In the last few decades, there has been a considerable use of drug delivery systems that have high
potential to act as diagnostic and therapeutic agents for cancer imaging as well as treatment of the disease. These
drug delivery systems are less toxic and provide higher efficacy [3, 4]. With such novel drug delivery systems,
the therapeutic features of a drug are optimized, hence, protecting it against metabolic inactivation and
improving the drug’s plasma half-life, its therapeutic index, and in overall, improved efficacy [1, 2, and 3]. Gem
is capsulated in colloidal devices such as polymeric nanoparticles and liposomes, and then conjugated with
lipophilic compounds for the purpose of developing formulations that can enhance the drug’s biopharmaceutical
properties. Of the available nanomedicine platforms, liposome formulations are the ones that the US Food and
Drug Administration for cancer treatment has approved [24, 25].

Liposomes are self-assembled vesicles that are biologically compatible and degradable, and have a
supramolecular lipidic organization, characteristically similar to that in natural cell membranes as discussed in
chapter 1. The development of liposomal devices to aid in gem Gem delivery has contributed to the marketing
of additional formulations, such as liposomal doxorubicin [23]. When the liposomes are developed as part of
drug delivery system, limitations such as non-specificity, drug leakage, drawbacks in monitoring cellular events
etc. are encountered. Thus, there is increased urgency of designing and developing novel drug delivery systems
that provide multiple actions, like simultaneous stimuli-responsive drug release and tissue cell targeting [22].
Different stimuli like pH, temperature, enzymes, and photoirradiation have been successfully applied to act as
trigger agents for the release of encapsulated drugs. The pH-responsive system for treatment of cancer has
gained interest due to the fact that endosomes and extracellular tumours are more acidic (pH = 5.5 and pH=6.8
respectively) compared to the normal issues (with a pH = 7.4), which influence carriers to deliver the drug in a
manner that is dependent on the pH.

Liposomal system can be simply modified by changing the composition and increase the drug release according
to the environment conditions. In the case of the acidic environment of the endosomal system, liposomes can be
modified to control the release of their components suitable for acidic environment. These types of liposomes
are name as pH sensitive liposomes (PSL) [[1]. The PSL undertake fast destabilisation in low acidic medium
such as endosomes. Composition of these liposomes are usually coneshaped lipid dioleoylphosphatidyl-
ethanolamine (neutral) and cholesteryl hemisuccinate (CHEMS) (weakly acidic) [2]. Fusogenic and structural
behaviour of these liposomes are mainly due to the presence of dioleoylphosphatidyl-ethanolamine (DOPE).
There are other lipids which has ability to influence pH sensitivity such as Dioleoylphosphatidylcholine (DOPC)
[3] and N-succinyl-DOPE (5). These lipids have common characteristics such as negatively charge group which
can lead to destabilisation, neutralized with acidification, ability to fuse with endosomal membrane and release
of content [6].

PSL immensely enhance the intracellular delivery of Gem. Furthermore, it is advantageous in terms of
biocompatibility and biodegradability as it does not induce any side effects or accumulation. The use of PSL in
treating solid tumours has been demonstrated, particularly in the prevention of the drug molecule from
inactivation after intravenous administration which is associated with a reduction in drug accumulation in
healthy cells before it reaches the target site [23]. As a result, PSL reduces non-specificity toxicity and increases
drug concentration at the specific site of action [2]. Both in vitro and in vivo findings have shown that PSL can
increase the cytotoxicity of most anticancer drugs [130, 264, and 265]. Furthermore, some research studies have
shown that PSL may increase the cell transport and thereby increase the efficacy of the drug delivery of drugs
which require active transport such as gemcitabine [58].

In our research, we investigated a number of methods such as reverse phase evaporation vesicle (REV), thin
film hydration (TFH), and a remote loading method that utilize ammonium sulphate gradient, that can be used
for loading Gem into PSL [129, 130, 302]. Our research group concentrated on developing an alternative
approach to enhance the efficacy of Gem therapy through encapsulation of the drug into liposomes. Different in
vitro and in vivo studies showed promising, especially on cytotoxic activity [13-17]. For an adequate protection
of the drug, a method known as small volume incubation (SVI) was used to load Gem into PSL. SVI is a novel
method in which a small volume of a solution containing condensed drug is used for incorporating the drug into
liposome. From the above observations, we considered passive diffusion as the mechanism for loading the drug.
To add on this, the method was developed to raise the EE as well as the DL to 4.0% which still stands to be the
highest percentage of drug loading achieved to date. More so, the study findings show that it is imperative to
have a high drug loading for effective cytotoxicity in Gem-resistant pancreatic cell lines.

PSL has some limitations despite promising selective delivery of therapeutic agents to the required sites, such as
poor intracellular drug release that prevent adequate availability of the drug at target cells. Coating of PSL with
hyaluronic acid (HA) can effectively help the delivery of Gem to cancer cells and enhance the drug’s
bioavailability. This is attributed to the fact that histological studies from different tumours have shown that a
connective tissue matrix known as stroma (enriched with hyaluronan) surrounds virtually all human epithelial
tumours. The overexpression of CD44 - a principal HA cell surface receptor, makes the tissue a vector for
targeting procedure against cancer tumour.

HA was discovered by Meyer and Palmer in 1934 [13] and is typically made of natural acidic polysaccharide
macromolecules, which are non-toxic and biodegradable. Figure 18 shows a structure of HA as found in several
animal species, and all vertebrates [14, 15, and 16]. HA is a glycosaminoglycan polymer with a high molecular
weight (MW=106 Da), and is composed of N-acetyl-d-glucosamine units and also units of d-glucuronic acid, all
repeatedly linked together by alternating bonds of β-1,3 and β-1,4 glycosidic [23, 24]. The molecular weight
vary with chain length [15, 17].
 Figure 18: The structure of hyaluronic acid (HA)

HA acts as a targeting moiety, or a drug carrier, which is functional material or natural ligand for cancer cells in
which CD44 is overexpressed and can therefore, interact with CD44 receptors in most tumour cells [19]. HA
and CD44 receptors trigger intracellular signals that in turn influence cell differentiation, proliferation, and
migration. HA is important in extracellular matrix (ECM), and contributes to cell signalling, tissue structure and
tissue hydration, wound healing, and angiogenesis [15, 16 and 18]. Transmembrane CD44 is one receptor for
HA, which is a kind of glycoprotein that is found on many cell surfaces and is overexpressed in many tumour
cells [19]. The interactions between the HA ligand and CD44 receptors is important in the adhesion and
therapeutic treatment of pancreatic cancer. Sialylated carbohydrate ligands on PSL bind to selectin receptors
found on the surface of endothelial cells in order to stay attached to the microvasculature in other tissues [19,
28]. The mechanism of attachment by adhesion has recently been applied in biometric approaches to target PSL
through CD44 receptors under the condition of physiological flow [66, 97, and 98]. With such techniques, flow
cancer cells easily interact with apoptosis-inducing ligands, triggering programmed cell death by binding with
the receptors on cancer cells. The PSL undergoes such interactions based on the physical barrier called the
glycocalyx, which is found on the cell surface.

In this work, we have briefly discussed the structure of EC glycocalyx, since more details have been discussed
by other authors [71, 95, 101, and 129]. It is a very thin gel-like layer of macromolecules with a thickness of
about 150-500 mm, found on the vascular ECS (see figure 19A) [129]. Computational models and experimental
observations reveal that these spacing acts as “molecular sieve” for plasma proteins, creating a difference in the
plasma proteins concentration between the luminal surface of endothelium and a tissue [1, 45, 77, and 90].

Figure 19: The effects of glycocalyx on circulating tumour cells in a microvasculture; A: Under normal
physiological conditions, the endothelial cell glycocalyx is thicker than the length of most adhesion receptors.
Here, it hinders cell adhesion; B: Changes in inflammatory conditions, matrix metalloproteinase exposure, and
shear stress can result in remodeling and/or shedding of the glycocalyx, increasing the number of available
receptors for binding with adhesion ligands on CTCs.

Like the EC glycocalyx, apart from fibrous proteins like collagen, the tumour cell glycocalyx is made of
different proteoglycans and glycosaminoglycans (GAGs) [109]. However, there is frequent impairment of the
synthesis of hyaluronan in tumor cells during the transformation of malignant, which can lead to excessive
production of hyaluronan (see figure 19B) [39, 43, 48, and 65]. Hyaluronan in cancer cells incorporates into the
matrix of the surrounding cells by formation of aggregates of hyaluronan-binding molecules, and can control
cell growth, differentiation, adhesion, and motility. Hyaluronan synthase genes encode the major enzymes in
hyaluronan synthesis, and can regulate the formation of hyaluronan matrices and the molecular size of
hyaluronan [50]. It has been also indicated that the expression of hyaluronan CD44 receptors increase in tumour
cells (see figure 1B) [34, 57]. The CD44+ cells from neck and head squamous cell carcinoma may have
properties similar to those of cancer stem cells [96], including cell differentiation and renewal. Carcinomas of
epithelial origin have high expression of CD44 variant isoforms which relate to tumour growth and metastatic
potential of some types of cancer [38, 87, and 108]. Apart from these components, other cell and matrix
adhesion molecules such as selectin ligands and integrins are embedded in the glycocalyx.

After injection of PSL coated with HA into blood circulation in the body, the drug can be delivered to the target
tumour cells and prevent the clearance of the reticuloendothelial system, which may in turn increase circulation
time, thus, hindering leakage of the drug. With prolonged circulation of blood, PSL accumulates in the tumour
tissue through the effect of improved permeation and retention. The PSL would enrich the target cancer cells by
the interaction of HA and CD44 receptors. The pH sensitive carrier material would then deliver Gem
immediately the particle was placed in acidic conditions. The PSL retained Gem and prevented other foreign
molecules by shielding the drug until it reached the target cells, before disrupting the coating layer to release the
drug after cellular uptake. Hence, it could be observed that PSL coated with HA maximize drug efficacy and
minimize toxicity compared to uncoated PSL (see figure 20).

Figure 20: A diagrammatic illustration of sequential mechanisms of HA-PSL-GEM anti-cancer drug on

pancreatic tumor immediately after intravenous injection.

Active targeting of drug with PSL to tumour cells further increases the activity and specificity of the drug. In
preparing PSL targeted to tumour cells, it is important to consider the size when selecting the HA: high
molecular weight HA [26] and low molecular weight oligomers [27] (4800 and 12,000 Da). HA catabolite with
low-molecular-weight is known to have procancerous activity, while its counterpart with high-molecular-weight
has anticancer activity [26]. In this study we used low molecular weight HA to compare the ability of liposomes
decorated with the un-coating PSL to target tissue cells. Studies have demonstrated the role played by HA in
tumour metastasis, with variations in the expression of HA in different tumour milieu [27]. In the present study,
we describe the preparation, characterization, and evaluation of the biological properties of HA-coated
liposomes encapsulating a lipophilic GEM pro-drug on CD44 expressing human pancreatic adenocarcinoma cell
lines. For this purpose, we synthesized molecular conjugates between the phospholipid 1, 2-dipalmitoyl-sn-
glycero-3-phosphoethanolamine and HA, and incorporated them in the preparation of liposomal.

3.2 Aims and Objectives

3.3 Chemicals and Reagents

Both 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and Cholesterylhemisuccinate (CHEMS) were

acquired from Avanti Polar Lipids (Alabama, USA). N-(carbonyl-methoxypolyethylene-glycol-2000)-1, 2-
distearoyl-sn-glycero-3-phospho-thanolamine (DSPE-MPEG2000) was acquired from Lipoid (Steinhausen,
Switzerland). Cholesterol, gemcitabine (as HCl salt, purity > 98%) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyl-tetrazolium bromide (MTT) for cytotoxicity studies were obtained from Sigma (Auckland, New
Zealand). A water purification system (Millipore Corp., Bedford, MA, USA) was used to prepare milli-Q water.
The rest of materials for the study were of analytical grade.

3.4 Experimental Method

3.4.1 pH-sensitive Liposome Preparation

PSLs were prepared following the description in the literature [19, 26], with a molar ratio of DOPE: CHEMS:
cholesterol: DSPE-mPEG2000 at 6:2:2:0.3 or 6:2:2:0.5 [259]. Non-pH sensitive liposomes were also prepared
using the ratios of DPPC: cholesterol: DSPE-mPEG2000 as 6: 3: 0.28. To prepare thin film liposomes, the
mixture of lipids of DOPE: CHEMS: cholesterol: DSPE-mPEG2000 in the ratio of 6:2:2:0.3 was first weighed
to obtain 10 mg in total. The lipid mixture was then dissolved in 1.0 ml of chloroform with methanol using a v/v
ratio of 3:1. To obtain the thin film lipid, the organic solvent was removed using a rotary evaporator in vacuum
conditions. Flowing nitrogen through the thin film lipid at 45°C for about 1 hour ensured further removal of any
remaining traces of organic solvent. A phosphate buffer at a pH of 7.0, 50 mM and adjusted to isotonic using
NaCl was applied to hydrate the thin film before sonication for 2 minutes. After about 45 minutes of hydration
and stirring, extraction of liposomes was performed through a 200 nm pore size polycarbonate membrane with
the aid of a sterilized extrusion device obtained from Gastight, Hamilton, New Zealand, for size reduction.
Ultra-centrifugation was then performed at 186,000×g for 1 hr. at 4°C to obtain liposomal pellet.

3.4.2 Preparing HA-coating for pH-sensitive Liposome

The same method that was used to prepare blank liposome was employed in preparing hyaluronated liposomes.
The lipid films consisted of the molar ratios of PSL DOPE: CHEMS: cholesterol: DSPE-mPEG2000 as
6:2:2:0.3, and were hydrated using 50 Mm phosphate buffer at pH 7.0 for about 45 minutes. After freezing and
thawing for 7 cycles, the resulting suspension of liposome was extruded through a polycarbonate membrane of
200 nm pore size. The liposomal pellets were then obtained and coated with HA.

A mixture of 3 mg EDC, 3 mg HA, and 3 mg NHS was then dissolved in 500 µL of 0.1M sodium acetate buffer
at a pH of 4.5 before incubation at 37˚C for about 2 h in order to activate HA. After the incubation process,
500µL of HA solution was mixed with 500µL of 0.1M borate buffer at pH 9 to re-suspend 10 mg of liposome.
The liposome suspension was finally incubated at 37˚C for about 24 h, before shaking at 350 rpm.

3.4.3 Loading the Drug into PSL and HA-PSL

Loading of Gem onto the liposomes was completed using a modified Small Volume Incubation (SVI) method
that is previously reported [152]. In summary, 12.0 mg of hydrochloride salt was measured and dissolved in 3.0
ml of 10.0 mM PBS at pH 7.4 with NaCl, and the atmospheric pressure set to 320 mOsm to prepare accurate
concentrations of the drug loading solution. The prepared solution was made into equal divisions in tubes and
placed in the SC210A vacuum concentrator - tobtained from Thermo Scientific, USA, and left to dry. This
yielded Gem powder in the tubes that was then suspended again with 20.0 μl of Milli-Q water, producing 1.5
mg of Gem suspension. A small volume of 20.0 μl of Gem suspension was then added to pellets of liposomal,
and then vortex-mixed for up to 3 minutes and incubated for 4 h at 60°C for loading the drug. Hence, the name
‘small volume incubation (SVI)’ for this particular method.
It was proven that PBS stabilizes the pH of the solution containing the drug at 7.4. This is particularly important
in maintaining the stability of the PSL. Using a buffer with low concentration also provided the advantage of
lowering the characteristic cytotoxicity effects of blank liposomes, as indicated by the preliminary data which
showed that the cytotoxicity of blank liposomes decreased with decrease in the concentration of PBS.

Figure 21: Diagrammatic representation of the procedure to prepare PSL and HA-PSL with Gem loading

3.4.4 Characterisation of Particle Size, Zeta Potential and Morphology

We used a laser diffraction particle analyser (Nano-ZS Malvern Instruments Ltd, UK) to analyse particle size
distribution and zeta potential of HA coating and un-coating liposomes. The Milli-Q water was used as a
dilution media at 25°C. We measured at least 3 samples to get the average. For particle size distribution, the
polydispersity index was used as an indicator. Using negative staining transmission electron microscopy (FEI
Tecnai G2 Spirit Twin 120Kv), we examined the morphology of the liposomes. In brief, a 3.0 mm copper mesh
was placed on a drop of liposome suspension. After about 2 minutes of incubation, a filter paper was used to
remove the surplus and the sample of liposome stained with a solution of 2% uranyl acetate and further
incubated for 1 minute, then removed and dried.

3.4.5 Statistical Analysis

The data findings in this study are expressed as average ± SD. We used Analysis of Variance (ANOVA) with
Tukey’s multiple comparisons test by employing the Origin 8.0. The p value for significance was 0.05.

3.5 Results and Discussion

3.5.1 Characterisation of Particle Size, Zeta Potential and Morphology

The liposomes had a zeta potential of -15.0 ± 0.3 and -8.1 ± 0.4 mV for HA-PSL and PSL respectively. TEM
results indicate that both appeared to be homogenous in size (see figure 22) which was in agreement with the
data obtained from the zetersizer which indicated a particle size of 200 ± 5 nm (PDI, 0.07 ± 0.01) for HA-PSL,
and 145 ± 5 nm (PDI 0.05 ± 0.01) for PSL. Using a modified SVI method it was observed that the EE of Gem
increased to 30.0 ± 1% for HA-PSL, and 30.2 ± 0.5% (n = 3) for PSL. This are the highest values of EE ever
achieved. Similarly, Gem’s DL for HA-PSL and HA were the highest ever reported at 4.2 ± 0.1% (14.2 ± 0.2%
in molar ratio) for HA-PSL, and 4.5 ± 0.1%, w/w (16.1 ± 0.2% in molar ratio) the PSL.
 [Insert Figure]

Figure 22: Transmission electron micrographs (TEM) of PSL and HA-PSL suspensions.

For the preparation of liposome, it was critical to consider the use of PBS and the pH of the hydration medium
(pH = 7.4). At a pH < 6.5, a longer duration was needed for thin film hydration and high pressure for extrusion.
In addition, using a non-buffered saline solution as a hydration medium led to aggregation 2 h after extrusion.
This was attributed to the lower pH of the lipid mixture (pH 6.3) after the hydration process, which possibly
destabilized the vesicles.

3.6 Conclusions