1. Lipid encapsulation of arsenic trioxide attenuates cervical cancer properties by
inhibiting expression of folate receptor-[alpha] and human papillomavirus-E6
oncogene to induce apoptosis within cells.
Haider A. Sheikh1a
, Zeshan Ahmed1b
, Anam Akhtar1c
, Scarlet X. Wang1d
, Xuesong
Wen1e
, Ivan M. Roitt1
*.
1
Centre for Investigative and Diagnostic Oncology, Department of Natural Sciences, School of Science and Technology,
Middlesex University, London, UK.
Haider A. Sheikh is the sole contributor and first author to the content of this thesis.
Correspondence; a
first author; haider.sheikh@hotmail.co.uk, Tel: +44 786 1958907. b
collaborator;
zeshan_ahmed@hotmail.co.uk, Tel: +44 796 0935796. c
collaborator; a.akhtar@mdx.ac.uk, Tel: +44 208 4114931.
d
supervisor; x.wang@mdx.ac.uk, Tel: +44 208 4112234. e
supervisor; x.wen@mdx.ac.uk, Tel: +44 208 4114931.
* Lead supervisor; i.roitt@mdx.ac.uk, Tel: +44 208 4115176.
The author confirms there were no conflicts of interest in this thesis.
A report submitted on, 13th
April, 2015, in Partial Fulfilment of the Requirements for the
Award B.Sc. (Hons) Biomedical Science

2. Page I 0
Contents
Abstract.........................................................................................................................................2
Introduction ..................................................................................................................................3
١ْ .Hallmarks of cancer: the next generation. ...................................................................3
٢ْ .Shortfalls in current cancer therapy.............................................................................4
٣ْ .The expression of folate-receptors in cervical carcinoma cells...................................5
۴ْ .Trends and developments in liposome drug delivery systems....................................7
۵ْ .Pharmacokinetics of pegylated liposomal drug delivery..............................................8
٦ْ .Potential of folate-targeted liposome conjugates. .......................................................9
Aims............................................................................................................................................ 10
Materials and methods ............................................................................................................. 11
١ْ .Cell lines and cultured conditions. ............................................................................ 11
٢ْ .Liposome preparation and arsenic trioxide loading.................................................. 11
٣ْ .Treating HPV-infected and HPV-negative cells and flow cytometry analysis. ......... 12
۴ْ .Double immunocytochemical staining and confocal fluorescent microscopy........... 13
Results ....................................................................................................................................... 14
١ْ .Apoptotic activity amongst cervical cancer cells treated with empty liposomes....... 14
٢ْ .HPV-E6 and FRα expression decreases in cells after liposome treatment.............. 15
٣ْ .Cationic liposomes encapsulated with AS2O3 upregulate cervical cell apoptosis fairly
more than free-AS2O3. .................................................................................................. 16
۴ْ .AS2O3 inhibits cell proliferation, FRα expression and HPV-E6 oncogene expression
in cervical carcinoma. ................................................................................................... 17
۵ْ .Increased induction of necrosis and apoptosis in HPV-positive cervical cells. ........ 18
Discussion ................................................................................................................................. 19
١ْ .FR-targeted empty liposomes induce apoptosis within cervical carcinoma. ............ 19
٢ْ . AS2O3 inhibits cell proliferation and HPV-E6 oncogene expression in cervical
carcinoma. .................................................................................................................... 21
٣ْ .The role of HPV is crucial for the development of invasive cervical carcinoma....... 22
۴ْ .Mechanistic limitations of the study and future liposomal advancements................ 23
۵ْ .Conclusion ................................................................................................................ 26
Bibliography .............................................................................................................................. 27
Appendices................................................................................................................................ 32
Appendix 1. Learning Log............................................................................................. 32
Appendix 2. Consideration of Ethical Issues and Research Governance.................... 39
Appendix 3. NSESC Application Form ......................................................................... 40
Appendix 4. Expression of folate receptor isoforms (FRα, FRβ & FRγ) in normal and
malignant cancerous tissue, adapted from (Antony, 1996). ......................................... 41
Appendix 5. A table from (Gabizon et al., 2004b) to show the properties of liposomes
and polymer therapy drug carriers. The table refers to value generated from non-
targeted liposomes and polymers................................................................................. 42
Appendix 6. Below are the solutions that were made to prepare the samples to
undergo ICP-OES (10ml each)..................................................................................... 43
Appendix 7. The table below has been adapted from (Munoz et al., 2006) to show the
HPV genome and the proteins encapsulated to multiply.............................................. 44
Appendix 8. Below are the six-well plate arrangements of the liposomal formulations,
and their microscopic images (x100
mag
) after 24hrs treatment..................................... 45
Appendix 9. LabRAT Risk Assessment........................................................................ 46

3. Page I 1
Acknowledgements
‫هذا‬َ‫يم‬ِ‫ق‬َ‫ت‬ْ‫س‬ُ‫م‬ْ‫ل‬‫ا‬َ‫ط‬‫ا‬َ‫ر‬ِّ‫الص‬۩ ‫ا‬۩ ۩
Foremost, I am grateful to my mother, father, two older sisters and my little brother for
their supportive and nurturing nature that has propelled me to what I am today. I am
indebted to them. Sincere thanks goes to Dr Xuesong Wen, for her continued
encouragement and calm nature to deal with my antics and mistakes throughout this
process. I am also grateful to Dr Scarlet X. Wang, Anam Akhtar and Professor Ivan M.
Roitt for assisting me to complete this thesis, at the Department of Centre for
Investigative and Diagnostic Oncology, Middlesex University School of Science and
Technology. I am extremely thankful and indebted to all my past teachers and lecturers
who have inspired and facilitated my appetite for knowledge, whilst sharing expertise,
sincere and valuable guidance to encourage me. I must take this opportunity to declare
love to Zeshan Ahmed, who has always been my wicked/ corrupted partner along with
the malingering Wardi and Nas. I also thank my many other friends who deserve a
mention but hopefully will understand my plight of limited space (see below) and the
encouragement, support and attention they have given me. Finally, a note to my future
self; keep bringing a smile to the faces of many and try not to laugh when reading back
on this. Keep working hard, your priorities in mind and always work towards a goal.
Seriously.
Author
Zeshan Ahmed
collaborator
Haider Ali Sheikh
first author
“Take advantage of today because tomorrow is not promised.”
- SONIA RICOTTI.
Honorary references; Supervisors
Dr Xuesong ‘Song’ Wen Dr Scarlet Xiaoyan Wang Professor Ivan M. Roitt

4. Page I 2
Abstract
OBJECTIVES: Human papillomavirus (HPV) is linked to proliferative cervical cancer
growth, and thus inhibiting expression of folate receptor-[alpha] (FRα) and high-risk
human papillomavirus-E6 oncogene (hrHPV-E6) have become prime therapeutic
targets. Arsenic trioxide (AS2O3) has shown treatment efficacy in remission of solid
cancers by inducing apoptosis and attenuating cancerous activity within HPV18-
positive HeLa cells in-vitro. Here, we explore the effect and action pathways of AS2O3-
induced apoptosis within HPV-infected (HeLa) and HPV-negative (C33A) cervical
cancer cells.
METHODS: Culture two cervical cancer cell lines (HeLa, C33A). Prepare two
liposomes; neutral and cationic, composed of different formulations of folic acid (FA)
and 5µM AS2O3, and treat for 24hrs. The sample cell viability and apoptosis were
observed by flow cytometry analysis, to determine the cellular effect of treatment.
Double-immunocytochemical cell staining, and confocal fluorescent microscopy, were
analysed to determine the HPV18-E6 and FRα expression within HeLa and C33A. The
confocal micrographs of HPV18-E6 expression elucidated links with tumour
suppressing p53 protein. A treatment sample of free-AS2O3 prepared to compare the
efficacy and effect of free-drug treatment on cervical cancer cells.
RESULTS: AS2O3 inhibited proliferation and induced apoptosis more in HPV-positive
HeLa than HPV-negative C33A, as upregulated p53 can diminish AP-1 and attenuated
E6-oncogene within HPV-infected cells. The presence of FRα expression in C33A cells
elucidated that AS2O3 acts in a FRα-dependent manner, as empty cationic liposomes
induce apoptosis by ERK signaling pathways stimulated transcription factors.
CONCLUSIONS: Liposome-loaded AS2O3 inhibited the expression of FRα and hrHPV-
E6 oncogene, which upregulated p53 that induced cell-cycle arrest and apoptosis
within cervical cancer cells, suggesting possible therapeutic clinical-AS2O3 application
that combine apoptosis and inhibition.

5. Page I 3
Introduction
١ْ .Hallmarks of cancer: the next generation.
Cancer is the leading cause of worldwide mortality and continues to be the spearhead
of medical enigmas (Baskar et al., 2012). The International agency for Research on
Cancer, IARC, forecast that cancer alone amounts to 7.6 million casualties per annum,
along with 12.7 million new global cases per year (Ferlay et al., 2010). Within the past
decade, a vast advance in understanding oncogenic pathways and mechanisms has
led to a cohesive push towards more effective cancer therapeutics, making cancer
become ever increasingly curable (Hanahan and Weinberg, 2011). It is hoped that
developing novel agents and delivery systems can transform therapy, from previously
delivering non-specific and near-toxic treatment doses, to safer ‘effective’ doses that
specifically target the tumorigenic tissue (Pietras and Ostman, 2010).
Prior to developing treatment plans, hallmarks of cancers should be understood to best
combat cancerous properties. (Hanahan and Weinberg, 2011) have identified biological
properties that cells attain before turning cancerous, giving rationale for the
complexities within cancer cells. Fig. 1 illustrates how genome instability is significant in
all the present-known hallmarks, and gives rise to cancer variation and subsequent
untreatable metastasis (Azam et al., 2010). Other cancer hallmarks include cellular
death resistance and passive immune destruction, which deregulate cellular energetics
and thus allow cancer cells to achieve replicative immortality and sustain their
proliferative signaling. The multiplication within cancer cells is further amplified as
growth suppressing cell processes are evaded (Ahmed and Bicknell, 2009).
Figure 1; Illustration by (Hanahan and Weinberg, 2011) showing the hallmarks of cancer cells along with their
therapeutic targets. Investigational drugs can be employed to negate or interfere with these cancerous acquired abilities.
Examples shown here are non-exhaustive and illustrative of only some current investigational drugs, as there are many
other candidate drugs with alternative targets and pathways that are also utilized to affect these hallmarks.

6. Page I 4
Furthermore, progressive angiogenesis coupled with active invasion and metastasis
creates tumorigenic growth that eventually compromises organ function (Nguyen et al.,
2009). Moreover, tumors have also shown to possess arsenal of ostensibly normal
cells that participate in establishing a ‘tumour microenvironment’, forming a proliferative
launchpad for cancerous tissue (Joyce and Pollard, 2009). Recognizing the treatment
methods being deployed for these hallmarks can provide widespread applicability to
develop novel means of treating cancer.
Present cancer therapeutics mostly comprise intrusive processes such as;
chemotherapy to shrink cancer mass, surgery to eliminate the tumour if possible, and
more chemotherapy to kill remaining cancer cells (Witsch et al., 2010). The efficacy of
chemotherapy depends on the multiplication rate of cancerous cells, as they proliferate
faster than healthy cells and thus become more susceptible (Pages et al., 2010). Albeit
recent chemotherapy improvements have increased patient survival, the perplex
relapse of aggressive chemotherapy-resistant cells remains (Vajdic and van Leeuwen,
2009). As treatment efficacy is measured by the ability to kill cancerous cells with
limited damage to healthy tissue, recent focus has shifted to developing carriers that
permit alternative dosing routes with new therapeutic targets, such as angiogenic blood
vessels (Shimoda et al., 2010). Although past decade has contributed vastly towards
understanding cellular circuitry and intercommunications, further elaboration upon this
knowledge can yield futuristic novel therapeutics that aid cancer research.
٢ْ .Shortfalls in current cancer therapy.
Current cancer therapy procedures mostly rely on chemotherapeutic drugs, called
cytostatics, which focus primarily onto stopping unregulated cancerous cell growth
(Kerbel, 2001a). Cytostatics control overt dissemination by indiscriminately disturbing
growth-stimulating pathways/processes of actively dividing cells (Kerbel, 2001a; Saijo
et al., 2000). Whilst these cells are mainly cancerous, healthy active cells can also get
targeted and acquire unwanted side-effects from genetic mutation (Korn et al., 2001).
When administered using systemic therapy via carrier drugs, cytostatics can direct cells
more adeptly, unlike invasive surgery or radiotherapy (Glockzin and Piso, 2012).
Unsurprisingly, sustained systemic therapy with different regiments can diminish the
benefits or response to treatment, as (Oh et al., 2014) report that less than 20% of
patients beneficially respond after the fourth chemotherapy cycle. One major
contributing factor that limits chemotherapeutic effectiveness is drug resistance,
illustrated in Fig. 2 below, which can become cross-resistant intrinsically or acquired
during treatment (Housman et al., 2014; Kerbel, 2001b). The complex mechanics of
drug resistance such as drug efflux and drug inactivation, via ATP binding cassettes
(ABC) and metabolites, can reduce the drug amount available to bind to its aim and
therefore off-balance the effective dosage (Zahreddine and Borden, 2013; Michael and
Doherty, 2005). The change within the drug target-site can reduce the maximal drug

7. Page I 5
efficiency as fewer conjugates occur, i.e. polymorphisms in Thymidylate synthase (TS)
can reduce efficiency of drugs using the fluorodeoxyuridine monophosphate (FdUMP)
inhibitor (Takiuchi et al., 2007). DNA damage repair or cell death inhibition can reverse
the cytotoxic effects of the drug and immortalize the cancerous cell, causing epigenetic
effects (Stavrovskaya, 2000). Cancerous epithelial cells can also adhere to stromal
cells and travel to other body regions by mesenchymal transition (EMT), forming solid
tumorigenic tissue by metastasis (Shang et al., 2013; Chaffer and Weinberg, 2011).
Figure 2; Illustration by (Zwicke et al., 2012) that shows the drug resistant mechanisms, present inside cancerous cells,
that contribute to deplete chemotherapeutic efficacy. The cytotoxic effects of the therapeutic drug, cellular death, can
only result when external influences, such as the drug resistant mechanisms, can be negated successfully.
As drug resistance can be multi-factorial, novel ways have to be developed to better
regulate the cellular response to chemotherapy (Wilson et al., 2006; Suh et al., 2013;
Huang et al., 2012). Drug resistance is a major problem as the emergence of cross-
resistant cancer types, and subsequent new resistant strains, can undo decades of
research (Byler et al., 2014). One such strategy to negate drug resistance is to guide
drug delivery into cancer cells expressing folate receptor, thereby specifically targeting
the tumour and predicting the response to chemotherapy (Garcia-Bennett et al., 2011).
٣ْ .The expression of folate-receptors in cervical carcinoma cells.
Tumour growth and initiation is fuelled by the cellular component called folate/folic acid
(FA), a vital vitamin B required by both healthy and cancerous cells (Kelemen, 2006).
Folate receptors (FR) are glycoproteins that are membrane-bound by the glycosyl-
phosphatidylinositol anchors, with affinity to endocytose extrinsic FA into the cells
(Henderson, 1990). Isoforms of FR, such as folate receptor-alpha (FR-α), are
expressed on epithelial cellular membranes and becomes elevated in some
carcinomas, such as; ovarian, cervical or epithelial malignancies (Antony, 1996; Parker
et al., 2005). Other isoforms, such as folate receptor-beta (FR-β) is a myeloid indicator
of variation and elevates in nonepithelial carcinomas, whereas folate receptor-gamma
(FR-γ) and its truncated FR-γ‫׳‬ form, are extrinsically released in response to depleted
glycosyl-phosphatidylinositol signaling and found in hematopoietic tissues (Shen et al.,
1995; Elnakat and Ratnam, 2004; Ross et al., 1994). Cancerous cells tend to be overly
reliant on FA metabolism to replicate and distribute, thus making FA pathways a viable
therapeutic strategy (Yang, T. et al., 2014; Malhi et al., 2012).

8. Page I 6
As illustrated in Fig. 3, FA is endocytosed into the cell by FR due to the affinity of the
FR binding protein. FA is essential to the genetic synthesis of new cells, particularly in
actively replicating cells as it transports a carbon molecule to initiate methylation
reactions (Choi and Mason, 2000; Stern et al., 2000). As cancerous cells have high
affinity to FA for dissemination, the FA-FR interaction can be focused for imaging or
treatment, particularly as FA remains stable across a large range of temperatures and
pH (Antony, 1996). As shown in Fig. 3, FA is also an effective ligand that can recruit
several foreign bodies yet sill conjugate with the FR, providing an efficient way to
internalize therapeutic molecules.
Figure 3; Illustration by (Garcia-Bennett et al., 2011) to show endocytosis of folate-targeted liposome (FTL) ligands by
FR. 1) FTL binds onto FR at the cell membrane due to the affinity of the FA. 2) Internalization of the FTL inside the cell
by endocytosis. 3) Drug and imaging agent disbandment from the FTL due to pH decrease inside the vesicular
endosome. 4) The therapeutic drug and the imaging agent are released into the cytoplasm. 5) FR recycles and returns
back onto the surface of the cell to endocytose more FA into the cell. 6) Therapeutic drug inside the interstitial fluid,
classed as ‘free drug’, can otherwise be endocytosed into the cell by the efflux pump down the concentration gradient.
Appendix.1 shows the specific FR isoforms that can be expressed within different
malignant tissues, making FRα an exploitation gateway that delivers cytotoxic agents
into cancerous cells with least non-target cytotoxic effects. (Leamon and Low, 1991;
Stevens et al., 2004) state that FRα-based drug deliverance is dependent on
specifically aiming at substrates conjugated with FA towards FRα. This was shown in
the study by (Ke et al., 2004), as dose uptake reduced cellular protein synthesis in a
time-sequenced manner, proving that folate-targeted liposomes (FTL) can be
successfully delivered into the cytoplasm whilst being functionally active. Another study,
by (Low and Antony, 2004), states that FRα drug delivery is significantly advantageous
as it avoids being destroyed by lysosomes but is instead consensually internalized into
the cellular cytoplasm, away from the lysosomes, to be released into the endocytotic
cellular compartments. FTL therapies that utilize FRα-positive cancerous cells such as;
drug entrapped liposomes, antisense oligonucleotides or immunotherapeutic agents,
are being advanced to make inroads into cancer treatment (Leamon and Low, 2001).
One particular therapy; FA-conjugated liposomes can encapsulate cytotoxic agents
inside liposomes (bound onto FA) and deliver cargo loads of therapeutic drug inside
the cellular cytoplasm (Gabizon et al., 2004a). The therapy enhances optimal
liposome-tumour cell targeting due to prolonged liposomal circulation time, saturating
; Folic Acid (folate)
; Imaging agent
; Therapeutic drug
; Folate receptor
; Folate-targeted
liposome (FTL)

9. Page I 7
the tumorigenic tissue mass (Gabizon et al., 2004a; Zhao and Lee, 2004). The
accumulation of FA-conjugated liposomes, inside angiogenic vessels, causes
extravasation into the tumorigenic interstitial fluid (Fig. 5) where FTL bind onto the FRα
and are then internalized into the cancer cells (Zhao and Lee, 2004).
۴ْ .Trends and developments in liposome drug delivery systems.
Current molecular cancer therapy directs cytotoxic agents towards the tumorigenic
tissue mass, but can also have detrimental effects on patient quality of life as losses in
therapeutic dose depletes the efficacy of therapy (Kim and Yoo, 2014). Subsequent
solutions, to reduce these peripheral toxicities and negate compromising drug efficacy,
have identified that liposomal encapsulation can facilitate delivering anti-tumour agents
through specific cell receptors (Mamasheva et al., 2011). FTL now encompasses the
novel field of nanomedicine, producing ‘nano-scale’ mechanisms (Duncan, 2006), to
exploit the ‘enhanced permeability and retention’ (EPR) effect and guide drug delivery
(Maeda et al., 2000). The EPR effect follows two main principles; 1) unsystematic
layout of the tumour angiogenic vasculature enhances hyper-permeability as the
porous endothelial lining leaks circulating nanoparticles, 2) the clogged lymphatic
drainage is compromised which subsequently permits the accumulation of
nanomedicinal molecules (Maeda et al., 2013; Maeda, 2012). Consequently, the
molecules bind onto their specific cell receptors and thus initiate the cascade of actively
targeting the cancerous cells (Bazak et al., 2014) Fig. 5 presents a diagrammatic
representation of the EPR effect inside tumorigenic tissue.
Figure 4; Schematic of pegylated liposome modified from (Gabizon et al., 2010) to show liposome compartments with
enclosed therapeutic drug. The polyethylene-glycol (PEG) coating reduces opsonisation and thus increases circulation
time to enable accumulation by enhanced permeability and retention (EPR). Drug.1; Present inside the liposomal polar
cavity in which the water soluble, hydrophilic, drugs can be encapsulated. Drug.2; Present inside the liposomal
hydrophobic cavity, in which the lipid-soluble drugs can be encapsulated within the hydrophobic phospholipid bilayers.
Liposomes are a spherical assembly of phospholipid bilayers, enclosing an interior
compartment that is isolated from the external water content (Hirai et al., 2013). The
liposomes provide a great delivery mechanism as different therapeutic drugs can be
Lipid Membrane
(Phospholipid +
Cholesterol) + Drug.1
Polyethylene-Glycol
coating
Drug.2
85-100 nm

10. Page I 8
encapsulated within the compartments; the water soluble drugs within the inner
compartment and the hydrophobic drugs within the lipid bilayer (Gao et al., 2013). The
Fig. 4 by (Gabizon et al., 2010), shows the structural layout of a liposome with its
compartments. Liposomes are being combined with several different ligands to
selectively deliver drugs, thereby guiding the liposomes towards the specific receptor-
expressing cell. A liposomal property table for non-targeted liposomes is presented in
appendix 2 for further insight into liposome therapeutics.
۵ْ .Pharmacokinetics of pegylated liposomal drug delivery.
During the early 1970’s, therapeutic liposomes were prone to releasing their content
within the circulatory phase, only for it to be dispensed by the macrophages residing in
the reticulo-endothelial system (RES) (Gregoria.G and Ryman, 1971; Cai et al., 2012;
Dayani and Malmstadt, 2011). Extensive research and studies into optimizing
liposomal drug delivery, in the early 1990’s, led to the discovery of attaching a
hydrophilic polymer polyethylene-glycol (PEG) conjugate onto liposomes to greatly
reduce immunogenicity and enhance liposomal circulation time (Woodle and Lasic,
1992; Mei et al., 2014). As seen in Fig. 4, PEG is conjugated with a lipid anchor
amongst the liposomal phospholipid bilayer, called pegylation (Gabizon et al., 2010).
Pegylation has been found to increase the steric stability of liposomal vesicles and
prevent opsonization by macrophages, causing delayed RES removal, hence
pegylated liposomes are now termed as ‘stealth’ liposomes (Yang, G. et al., 2014).
Figure 5; Diagrammatic representation by (Gabizon et al., 2010) showing pegylated folate-targeted liposomes (FTL)
targeting FR expressing cells through the enhanced permeability and retention (EPR) effect. 1) Extravasation of the
pegylated liposomes from the fenestrations within the tumour angiogenic vessel and into the interstitial fluid. 2) FTL
begin to accumulate within the interstitial fluid. 3) Damaged liposomes release disclosed content as ‘free drug’ due to
environment change or macrophage attack, which is endocytosed by the efflux pump, embedded inside the cellular
membrane, down the concentration gradient. 4) Pegylated FTL bind onto FR by FA present on their membranes. 5)
Intact FTL are internalized by FR inside the endosome. 6) Release of drug content within FTL to provide cytotoxic effect
to the cell, prompting subsequent cell death.
Coupled with prolonged circulation time and drug retention, PEG-coated FTL can
capitalize on the aforementioned EPR effect (Fig. 5) to amass within tumors (Mei et al.,
2014; Chen et al., 2011). The vast fenestrations within the micro-vessels of the
angiogenic vasculature allow extravasation of macromolecules, which accumulate due

11. Page I 9
to lack of adequate lymphatic drainage and get internalized by FR cells (Rajora et al.,
2014; Nehoff et al., 2014). However, the bioavailability of the drug within the interstitial
fluid can also be in the form of ‘free drug’, which leaks out of damaged pegylated
liposomes due to the physico-chemical environment of the interstitial fluid or liposome-
engulfing macrophages (Maeda et al., 2001).
٦ْ .Potential of folate-targeted liposome conjugates.
Historically, FA targeting through pegylated FTL has mostly been directed towards
enhancing tumour selectivity (Kim and Yoo, 2014). Whilst drug accumulation within the
interstitial fluid is not significantly enhanced by FA targeting, especially within ascetic
tumors, it is still significantly higher than non-FA targeted liposomes comparatively
(Bazak et al., 2014; Maeda, 2012). Furthermore, FTL are significantly more capable of
altering intra-cellular drug delivery as more drug content reaches the objective cells
(Duncan, 2006). The uncomplicated procedure of encapsulating a therapeutic drug
within the cavities of the liposome is by far the most capable way to exploit
pharmacological advantages of FTL compared to other non-targeted liposomes (Bazak
et al., 2014; Duncan, 2006; Maeda, 2012).
Further critical review of FTL shows that the liposomal size is far greater than the
glomerular kidney filtration threshold and thus prevents FTL being filtered out of
circulation. This is crucial as the luminal kidney tubular cells also express FRα and thus
FTL elimination and subsequent exposure to the tubular cells can dispose the
encapsulated drug, causing serious toxicity damage to kidneys (Maeda, 2012). On the
contrary, FTL are also bulky structures that are incumbent when infiltrating solid tumour
masses, therefore their ability to reach cells further away from the angiogenic
vasculature is limited (Gabizon et al., 2010; Dayani and Malmstadt, 2011). Conclusively,
rational clinical approach dictates that benefits of FTL therapy convincingly trumps the
relative disadvantages and ligand-mediated targeting can provide significance to
clinical applications that target cancer, albeit requiring adjustments within.
As illustrated in Fig. 4, both the inner and outer liposomal compartments can be
incorporated with chemotherapeutic drugs of opposing polarities; polar drugs inhabit
the water-soluble inner cavity, non-polarized drugs inhabit the hydrophobic
phospholipid bilayers. In order for the encapsulated liposomes to deliver cytotoxic
drugs into objective cancerous tissue, they can be charged to exploit the differences
between blood vessels in healthy tissue and angiogenic blood vessels (Krasnici et al.,
2003). Healthy blood vessels consist of a thin single layer of epithelial cells, packed
tight to avoid extravasation out of the vessel. However, angiogenic blood vessels (Fig.
5) tend to have fenestrations which permit extravasation, as the small-sized nature of
drug-encapsulated liposomes can penetrate through these pores and aim towards
cancerous tissue by the EPR effect (Perche and Torchilin, 2013). Angiogenic
vasculature is also densely lined with negatively-charged particles, which attract

12. Page I 10
positively-charged cationic liposomes (CLs) in circulation and make them very
responsive to the drug within (Krasnici et al., 2003; Campbell et al., 2002). Therefore,
encapsulating a proficient drug within a positively-charged CLs can simultaneously
target specific tumorigenic tissue, whilst also permit the therapeutic drug to accumulate
in quantities that can be effective (Perche and Torchilin, 2013; Krasnici et al., 2003).
Arsenic trioxide (AS2O3) has been shown to be a substantially efficient cytotoxic drug to
encapsulate within liposomes and inflict cancerous cells (Ahn et al., 2010). As a
singular agent, lipid-encapsulated AS2O3 can affect numerous intracellular transduction
pathways such as protein-kinase signaling and cellular metabolism, which alter the
functionality of enzymes and associate molecules that influence gene expression
(Winter et al., 2011; Kallinteri et al., 2004). Then, cellular pathways such as apoptosis,
anti-angiogenesis and dissemination inhibition can also be induced (Zhao et al., 2008).
The cytotoxic nature of AS2O3, along with its ability to affect many cellular physiological
pathways, has transpired widespread AS2O3-targeting for malignancies derived from
variety of tissue types (Winter et al., 2011). Whilst cancerous cells are susceptible to
AS2O3, further mechanistic research should determine the specific biological pathways
of AS2O3 to fulfil their potential synergic application with other chemotherapeutic agents,
initiating improved cancer therapeutics (Zhang et al., 2012).
Aims
Our aims are to investigate the efficacy of folate-targeted liposomes as delivery
vehicles, to encapsulate cytotoxic drugs such as AS2O3, and specifically target tumors.
The screening of FRα-expression on HeLa and C33A cervical cancer cell lines, can
guide the therapeutic liposomal-drug conjugates towards tumor vasculature to dispose
the drug. HeLa and C33A cell lines will be targeted using neutral and cationic
liposomes, with differing formulations, to determine the effect of treatment on cell
viability and apoptosis within cervical cancer cells. The presence of necrotic cells, late-
apoptotic cells, and early-apoptotic cells will be measured by flow cytometry analysis.
The expression of HPV18-E6 oncogene within HeLa cells can determine the
relationship between cellular apoptosis and E6-oncogene downregulation. Confocal
microscopy will be utilized to visualize the expression of both HPV-E6 and FRα surface
receptors. The effect of free-ATO treatment on cell viability and mortality will also be
explored to determine if passive targeting increases in efficiency.

13. Page I 11
Materials and methods
١ْ .Cell lines and cultured conditions.
Two cervical cancer cell lines were purchased from American Type Culture Collection,
ATCC®
, (UK); HeLa (human cervix epithelial adenocarcinoma, FRa+
, HPV-18(+)
) and
C33A (human cervix epithelial carcinoma, HPV(-)
) were routinely cultured in RPMI-1640
medium from Sigma-Aldrich, Sigma (UK), containing 10% fetal calf serum (FCS) with
100 U/ml penicillin and 100 µg/ml streptomycin; incubated at 37ºC in humidified
conditions of 5% CO2 and 95% air. Cells were cultured in 75cm2
flasks or on sterile
coverslips inside six-well plates, and grown until confluency of 75%-90% was reached.
The cells were checked daily at about 15hrs and medium was changed accordingly.
٢ْ .Liposome preparation and arsenic trioxide loading.
The reagents used to prepare liposome samples were purchased from ThermoFisher
Scientific (UK) and Sigma (UK). The materials used were Soya Phosphatidylcholine
(PC), Cholesterol (Chol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
[biotinyl(polyethylene glycol)-2000]2772m/w
(DSPE-PEG2000), 1,2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000]2772m/w
- folic acid441m/w
(DSPE-PEG2000-FA)3213m/w
and Di-methyl-dioctadecyl-ammonium bromide630m/w
(DB).
1}(LIPO1-CT), neutral control liposome composition: PC:Chol:DSPE-PEG2000=
54.5:45.0:0.5 mol% of 13.8mg of (PC), 5.7mg of (Chol) and 0.5mg of (DSPE-PEG2000)
were dissolved in 0.5ml dichloromethane:methanol (2:1 ratio) at room temperature
(RT). 2}(LIPO2-CT), positive control liposome composition; PC:Chol:DSPE-PEG2000:
DB= 50.5:45.0:0.5:4.0 mol% of 13.3mg (PC), 5.5mg (Chol), 0.45mg (DSPE-PEG2000),
0.81mg (DB) were dissolved in 0.5ml dichloromethane:methanol (2:1 ratio) at RT.
3}(LIPO2-FA), positive folate-conjugated liposome composition; PC:Chol:DSPE-
PEG2000-FA:DB= 50.5:45.0:0.5:4.0 mol% of 13.3mg (PC), 5.5mg (Chol), 0.45mg
(DSPE-PEG2000-FA), 0.81mg (DB) were dissolved in 0.5ml dichloromethane:methanol
(2:1 ratio) at RT. The DSPE-PEG2000-FA was synthesized and provided by Dr Scarlet.
The dichloromethane and methanol were evaporated using nitrogen gas until the lipids
become solid. The dried lipid was then hydrated in 0.5ml 730mM Ni(OAc)2 aqueous
solution and rolling for 30mins. The step was repeated for another 30mins with another
0.5ml 730mM Ni(OAc)2 aqueous solution added in, The suspensions were subjected to
10 freeze-and-thaw cycles; 1ml of liposome suspension in a glass vial frozen in liquid
nitrogen for 3mins and then thawed in 37ºC water for 3mins. The liposome suspension
was then extruded by passing it through a 0.1µm Whatman Anotop filter once using a
1ml syringe. Subsequently, 1ml liposome was dialyzed in 14KDa dialysis tube against
0.01M NaH2PO4/Na2HPO4, at pH 7, overnight at RT to remove non-encapsulated
Ni(OAc)2. The suspension was diluted to 1.5ml after dialysis.

14. Page I 12
The cytotoxic AS2O3
197.8m/w
(ATO) was purchased from Sigma (UK). 4}(ATO), 20mM
free-ATO solution was prepared by dissolving ATO in 0.01M NaH2PO4/Na2HPO4, at pH
7, overnight in 37ºC water bath. 5}(LIPO2-ATO), positive ATO-encapsulated liposome
prepared by; before dialysis add 0.5ml of 20mM ATO to 0.75ml LIPO2-(Ni) with gentle
mixing on a roller for 150mins at RT; dialysis of 1ml LIPO2+ATO in 14KDa dialysis tube
against 0.01M NaH2PO4/Na2HPO4 at pH 7 for 5.0hrs at RT to eliminate excess ATO.
6}(LIPO2-FA-ATO), positive folate-conjugated and ATO-encapsulated liposome
prepared by; before dialysis add 0.5ml of 20mM ATO to 0.75ml LIPO2-FA-(Ni) with
gentle mixing on a roller for 150mins at RT; dialysis of 1ml LIPO2-FA-ATO in
tubing14kDa
against 0.01M NaH2PO4/Na2HPO4 at pH 7 for 5.0hrs at RT to eliminate
excess ATO. The concentrations of phosphate (P), nickle (Ni) and arsenic (AS) were
found by ICP-OES, Inductively Coupled Plasma-Optical Emission Spectroscopy.
Appendix 3 shows the solutions made to prepare ICP-OES samples.
٣ْ .Treating HPV-infected and HPV-negative cells and flow cytometry analysis.
The two cervical cancer cell lines were seeded in 5x104
/ml on coverslips in six-well
plates and grown for 24hrs before treatment inside 5ml cell culture flasks. The wells
were arranged as follows; 1} Control; 4.0ml solution made up of cells inside RPMI-1640
media. 2} LIPO1; 10.0µl of neutral control liposome inside 3990.0µl solution of cells
inside RPMI-1640 media, with dilution factor of 1:400. 3} LIPO2; 10.0µl of positive
control liposome inside 3990.0µl solution of cells inside RPMI-1640 media, with dilution
factor of 1:400. 4} LIPO2-FA; 10.0µl of positive folate-conjugated liposome inside
3990.0µl solution of cells inside RPMI-1640 media, with dilution factor of 1:400. 5}
LIPO2-ATO; 5.0µl of positive ATO-encapsulated liposome inside 3995.0µl solution of
cells inside RPMI-1640 media, with dilution factor of 1:760. 6} LIPO2-FA-ATO; 9.0µl of
positive ATO-encapsulated liposome inside 3991.0µl solution of cells inside RPMI-1640
media, with dilution factor of 1:440. 7} ATO; To achieve a dilution factor of 1:7880,
serial dilution was utilised for AS2O3: Tube A; 10.0µl in 3990.0µl solution of cells inside
RPMI-1640 media with dilution factor of 1:400, Tube B; 100.0µl in 3900.0µl solution of
cells inside RPMI-1640 media with dilution factor of 1:20. The arrangement of
liposomal formulations in six-well plates is shown in appendix 5, and their respective
microscopic images (x100mag
) after 24hrs treatment.
After 24hrs treatment, cell growth was inhibited using trypsin and then harvested into
15ml falcon tubes along with 2ml of PBS to wash trypsin from cells. The cells were then
centrifuged at 1000rpm for 3mins at RT, after which the supernatant was removed and
washed with PBS twice. Afterwards, binding buffer was added with 5µl of Annexin-
V36.0kDa
and 5µl of Propidium Iodide668.4m/w
(PI). The tubes were kept in the dark for
15mins before Flow Cytometry analysis, with BD CellQuestTM
Pro programme, which
also calculated the statistical analysis used throughout this thesis.

15. Page I 13
۴ْ .Double immunocytochemical staining and confocal fluorescent microscopy.
The two cell lines were seeded at 5x104
/ml on cover slips in a six-well plate and grown
for 24hrs, reaching a confluency of 75-90%, and treated with same plate arrangement
as before. After 24hrs of treatment, cells were then fixed by 4% Paraformaldehyde
(Sigma, Dorset, UK) for 8mins and later washed using Phosphate buffer solution239.2m/w
(PBS) three times, for 1min each. The cells were then placed in a detergent, 0.1%
Triton (Sigma, UK), in PBS to punch tiny holes within the cell membrane, and then
washed using PBS three times, for 1min each. The cells were then incubated with 50%
horse serum (HS), Sigma (UK), for 8mins to prevent unspecific binding and excess HS
was subsequently removed.
The 1st
antibody (monoclonal Anti-Human Papillomavirus 16 (E6), anti-HPV18 E6) with
dilution factor of 1:100, purchased from Santa Cruz Biotechnology (Heidelberg,
Germany), was applied to saturate the whole coverslip for 1hr. The cells were washed
using PBS three times, for 1min each and the 2nd
antibody, Biotinylated Goat Anti-
Rabbit IgG, purchased from Avidin/Biotin Complex (ABC) Universal kit (Vector Lab,
UK), was added for 30mins and subsequently cells were washed in three PBS cycles
for 1min each.
Next, a 3rd
antibody, HRP-Avidin66.0kDa
(ABC universal kit), was added and cells were
left to incubate for 20mins and then washed in three cycles of PBS for 1min each. Then
Cyanine 5 (Cy-5), purchased from TSA™ Plus Cyanine 5 system (PerkinElmer, USA),
with a dilution factor of 1:100 in a solution of 600µl for 5mins, with absorbance of
650nm fluorescence signal. The cells were washed in PBS for three cycles at 1min
each and then covered, to block unspecific binding sites, with goat serum (Sigma) for
8mins. Next, the second 1st
antibody (Rabbit anti-Human poly), purchased from Pierce
(UK), was added and left at RT for 1hr. Then, the wells were washed with three cycles
of PBS solution, at 1min each, and then 2nd
antibody (Goat anti-Rabbit IgG), with
absorbance of 520nm fluorescence signal, Alexa Fluor® 488 dye was loaded with
Fluorescein Isothiocyanate389.4m/w
(FITC) with Tyramide Signal Amplification (TSA™) for
90mins.
The cells were again washed in three 1min PBS cycles and covered in 4',6-diamidino-
2-phenylindole277.3m/w
(DAPI) containing mounting media, with absorbance of 300nm
fluorescence signal, for 5mins, and kept in a cool dark place. A confocal microscope
(Leica Microsystems, Wetzlar, Germany) was used to capture images of double
fluorescent staining; HPV-E6 (red), FRα (green), and counter-staining by 6-diamidino-
2-phenylindole (blue nuclei). The images were taken after 2 days after staining, both
HeLa and C33A were double stained by HPV/FRa.

16. Page I 14
Results
١ْ .Apoptotic activity amongst cervical cancer cells treated with empty liposomes.
Flow cytometry analysis was utilized to quantitatively determine the induction of
apoptosis within 10000 cells after treatment. Cells were stained with Annexin V-FITC
(AnV-F), x-axis, for fluorescent detection of apoptotic cells that labeled the
phosphatidylserine sites on the membrane surface. The propidium iodide (PI), y-axis,
attached to the cellular DNA in necrotic/ late apoptotic cells where the membrane was
compromised. The field was dissected into four quads; upper right (UR), upper left (UL),
lower right (LR) and lower left (LL), at quad location 400,400. This combination allows
differentiation of cells; early apoptotic cells:LR (AnV-F(+)
, PI(-)
), late apoptotic cells:UL
(AnV-F(-)
, PI(+)
) necrotic cells:UR (AnV-F(+)
, PI(+)
), and viable cells:LL (AnV-F(-)
, PI(-)
).
Figure 6; Apoptosis assay using flow cytometry after staining with Annexin V-FITC (AnV-F) and propidium iodide (PI).
HeLa and C33A cells were treated with neutral liposome control (LIPO1-CT) and positive liposome control (LIPO2-CT).
The scatter plots are arranged as following; A) HeLa control. B) C33A control. C) HeLa LIPO1-CT. D) C33A LIPO1-CT.
E) HeLa LIPO2-CT. F) C33A LIPO2-CT. The percentage (%) was calculated from 10000 events.
Fig. 6 shows the scatter plots obtained by flow cytometry after HeLa and C33A cells
were treated with LIPO1-CT and LIPO2-CT for 24hrs. Fig. 6A shows 96% of viable
HeLa with 2.3% in early apoptosis and 1.3% in late apoptosis stage, whilst the necrotic
HeLa C33A
Control(CT)
A) B)
LIPO1Control(LIPO1-CT)
C) D)
LIPO2Control(LIPO2-CT)
E) F)
96.0 2.28
0
1.34 0.38
99.9 0.03
.50
0.05 0.00
93.2 5.77
0.47 0.56
0
99.3
.5
0.60
.50
0.09 0.02
93.1 5.18
0.86 0.86
92.2
6.5
6.09
.50
1.49
.5
0.16
.50
%
10000 events
%
10000 events
%
10000 events
%
10000 events
%
10000 events
%
10000 events
Annexin V
PI
Annexin V
PI
Annexin V
PI
Annexin V
PI
Annexin V
PI
Annexin V
PI

17. Page I 15
HeLa cells amount to 0.38%. The Fig. 6B displays 99.9% viable C33A cells, with 0.05%
cells in late apoptosis and 0.03% in early apoptosis stage, whilst no necrotic cells were
present in the quadrant. When treated with the neutral control liposome, LIPO1-CT, the
number of viable HeLa cells decreased to 93.2%, with 0.47% late apoptosis, 5.77%
early apoptosis and 0.56% necrotic cells, shown in Fig. 6C. This was also witnessed
with C33A cells, Fig. 6D, as viable cells decreased to 99.3%, with 0.09% late apoptotic,
0.6% early apoptosis and 0.02 necrotic cells. When cells were treated with a CLs,
LIPO2-CT, the number of viable cells decreased further to 93.1% in HeLa and 92.2% in
C33A, whereas cells in early apoptosis and late apoptosis increased sharply; 5.18% &
0.86% in HeLa (Fig. 6E) and 6.09% & 1.49% in C33A (Fig. 6F).
٢ْ .HPV-E6 and FRα expression decreases in cells after liposome treatment.
Confocal micrographs were used to determine the uptake of LIPO1-CT and LIPO2-CT
by the cells to use immunofluorescence to tag HPV-E6 (red), FRα (green) and DAPI
counter-stained nuclei (blue), and show a merge of all pictures at lower-right in the
micrographs, at x4000mag
, from HeLa and C33A cells after being treated for 24hrs.
Figure 7; Confocal microscopic examination of HeLa and C33A cells after 24hrs treatment with LIPO1-CT and LIPO2-
CT. The micrographs are arranged as following; A) HeLa control. B) C33A control. C) HeLa LIPO1-CT. D) C33A LIPO1-
CT. E) HeLa LIPO2-CT. F) C33A LIPO2-CT. The colours represent; HPV-E6 (red) and FRα (green), DAPI (blue) was
used to counter stain and reveal nuclei/DNA location. The magnification used to image the samples was x4000mag
.
HeLa C33A
Control(CT)
A) B)
LIPO1Control(LIPO1-CT)
C) D)
LIPO2Control(LIPO2-CT)
E) F)

18. Page I 16
٣ْ .Cationic liposomes encapsulated with AS2O3 upregulate cervical cell
apoptosis fairly more than free-AS2O3.
Flow cytometry analysis was carried out on HeLa and C33A cells after they were
treated using the CLs conjugated with FA and ATO treatments. The scatterplots were
dissected into four quads; upper right (UR), upper left (UL), lower right (LR) and lower
left (LL), at quad location 400,400.
HeLa C33A
LIPO2FA
A) B)
LIPO2ATO
C) D)
LIPO2FA-ATO
E) F)
ATO
G) H)
Figure 8; Apoptosis assay using flow cytometry after staining with Annexin V-FITC (AnV-F) and propidium iodide (PI).
HeLa and C33A cells were treated with positive liposome conjugated with FA and ATO. The scatter plots are arranged
as following; A) HeLa LIPO2-FA. B) C33A LIPO2-FA. C) HeLa LIPO2-ATO. D) C33A LIPO2-ATO. E) HeLa LIPO2-FA-
ATO. F) C33A LIPO2-FGA-ATO. G) HeLa ATO. H) C33A ATO. The percentage (%) was calculated from 10000 events.
95.3 4.04
0.40 0.24
97.5
.5
2.43
.50
0.06
.5
0.00
.50
94.2
.5
4.68
.50
0.53 0.60
98.5
1.5
1.43
.50
0.06 0.00
.50
93.6 5.28
.50
0.41 0.68
99.6
4.5
0.33
.50
0.03 0.00
.50
96.5
.5
2.42
.50
0.66 0.41
.50
99.6
3.5
0.06
.50
0.31 0.00
0.50
%
10000 events
%
10000 events
%
10000 events
%
10000 events
%
10000 events
%
10000 events
%
10000 events
%
10000 events
Annexin V
PI
Annexin V
PI
Annexin V
PI
Annexin V
PI
Annexin V
PI
Annexin V
PI
Annexin V
PI
Annexin V
PI

19. Page I 17
The cells were stained with Annexin V-FITC (AnV-F), x-axis, to fluorescently detect
membrane phosphatidylserine sites of apoptotic cells and propidium iodide (PI), y-axis.
Fig. 8 shows the scatter plots that were obtained to determine, quantitatively, the
induction of apoptosis within 10000 cells after treatment; LIPO2-FA, LIPO2-ATO,
LIPO2-FA-ATO and ATO. This combination allowed the differentiation of cells; early
apoptotic cells:LR (AnV-F(+)
, PI(-)
), late apoptotic cells:UL (AnV-F(-)
, PI(+)
) necrotic
cells:UR (AnV-F(+)
, PI(+)
), and viable cells:LL (AnV-F(-)
, PI(-)
).
۴ْ .AS2O3 inhibits cell proliferation, FRα expression and HPV-E6 oncogene
expression in cervical carcinoma.
Figure 9; Confocal micrographs of HeLa and C33A cells after 24hrs treatment with LIPO2-FA, LIPO2-ATO, LIPO-FA-
ATO and ATO. The micrographs are arranged as following; A) HeLa LIPO2-FA. B) C33A LIPO2-FA. C) HeLa LIPO2-
ATO. D) C33A LIPO2-ATO. E) HeLa LIPO2-FA-ATO. F) C33A LIPO2-FGA-ATO. G) HeLa ATO. H) C33A ATO. The
colours represent; HPV-E6 (red) and FRα (green), DAPI (blue) was used to counter stain and reveal nuclei/DNA
location and a merge of all variables was shown at LR. The magnification used to image the samples was x4000mag
.
HeLa C33A
LIPO2FA
A) B)
LIPO2ATO
C) D)
LIPO2FA-ATO
E) F)
ATO
G) H)

20. Page I 18
Confocal micrographs, Fig. 9 (x4000mag
), were used to determine the cellular
expression of HPV18 (in HeLa) and FRα (in HeLa and C33A) when treatment was
alternated between FA and ATO. The HeLa (FRa+
, HPV-18(+)
) and C33A (HPV(-)
) cells
were treated for 24hrs with CLs, conjugated with; LIPO2-FA (Fig. 9A & Fig. 9B), LIPO2-
ATO (Fig. 9C & Fig. 9D) and LIPO2-FA-ATO (Fig. 9E & Fig. 9F). The cells were also
treated with free-ATO (Fig. 9G & Fig. 9H) which showed a significantly reduced HPV-
E6 and FRα expression compared to LIPO2-CT (Fig. 7E & Fig. 7F). Furthermore, the
confocal micrographs dictated that the presence of ATO in treatment, albeit with FA,
showed greater reduction in HPV18 and FRα expression compared to treatment
without ATO.
۵ْ .Increased induction of necrosis and apoptosis in HPV-positive cervical cells.
The results from the flow cytometry analysis were used to distinguish the effect of
treatment on both HeLa (FRa+
, HPV-18(+)
) and C33A (HPV(-)
) cells after 24hr treatment.
Fig. 10 shows early apoptosis (stripe white) data seems to be significant within HeLa
samples, ranging from 2.3% (HeLa-CT) to 5.8% (HeLa LIPO1-CT), whereas the early
apoptosis trend in C33A cells pales in comparison, ranging between 0.03% (C33A-CT)
to 6.09% (C33A LIPO2-CT). The late apoptosis (grey) activity in HeLa cells tends to be
at around 0.6%, whilst peaking to 1.3% (HeLa-CT), but significantly lower in C33A cells
comparatively, average of 0.07% whilst peaking at 1.5% (HeLa LIPO2-CT). Finally,
necrotic activity (black) within HeLa cells seems to be substantial, as every sample has
necrosis, whereas necrotic activity within C33A cells is near enough non-existent, with
the only substantial value being 0.2% (C33A LIPO2-FA) whilst all other samples having
either negligible necrosis data or none at all.
Figure 10; A bar graph to show the percentage of cell death due to; necrosis (black), late apoptosis (grey) or early
apoptosis (stripe white), in HeLa and C33A cells after being administered with 24hrs treatment. The data was generated
from a singular experiment using flow cytometry analysis of 10000 events. The bar graphs shown are of samples from
both HeLa and C33A cells; CT, LIPO1-CT, LIPO2-CT, LIPO2-FA, LIPO2-ATO, LIPO2-FA-ATO and ATO. The standard
error bars were also included to show 5% value error from the data. * - negligible data (<0.02), ** - data is zero (0.0).
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
percentageofcells(%)
HeLa C33A
Early apoptosis
Late apoptosis
Necrosis
*
* ** * ** ** *** **

21. Page I 19
Discussion
This study has shown that the expression of FRα and HPV-E6, on HeLa and C33A cell
lines, can be utilised to guide therapeutic liposome-encapsulated delivery of cytotoxic
AS2O3. The subsequent endocytotic internalization and release of liposomal-AS2O3
content has shown to incite apoptotic and necrotic effects within cancerous cells, which
can result in cellular death (Zhang et al., 2012). Therefore, successful identification of
surface characteristics of cancer cells can provide gateway approaches that deliver
cytotoxic agents, whilst also increasing the efficacy and specificity of anti-cancer
treatments (Yamashita et al., 2014; Yang, S. H. et al., 2014). The study by (Zheng et
al., 1999) has already utilized AS2O3 to instigate apoptosis within cervical carcinoma,
infected with HPV-18, by de-regulating E6 oncogene. This in-vitro study was set out to
determine the sensitivity of HPV-infected (HeLa) and non-HPV infected (C33A) cells to
5µM AS2O3 therapy, loaded with different liposomal formulations.
١ْ .FR-targeted empty liposomes induce apoptosis within cervical carcinoma.
Apoptosis is a regulated, yet prudent, process of cellular death that occurs as a normal
response to development, cell turnover or aberrant cell death (Hao et al., 2014).
However, incorrectly regulated apoptosis can have serious implication within various
disease states and cancers (Xu et al., 2014). Whilst apoptosis is a planned procedure
of autonomous cellular dismantling, it is much distinguishable from necrosis, an
unregulated and passive cell death, by its morphologic and biochemical features that
include disintegration of the nuclear chromatin, cytoplasmic contraction, and loss of
lipid membrane asymmetry (Pan et al., 2000). Within healthy viable cells,
phosphatidylserine (PS) is primarily found within the cytosolic inner-side of the plasma
membrane, which aids cell signalling pathways (Kim et al., 2015; Dong et al., 2015).
Upon apoptotic initiation, PS dismisses its asymmetric distribution and translocates
towards the outer extracellular membrane, exposing itself to the extrinsic surroundings
(Appelt et al., 2005). AnV is a calcium-dependent phospholipid-binding human cellular
protein, which serves as an anticoagulant with a heightened affinity for PS. The
labelling of AnV with FITC (AnV-F) can recognise apoptotic cells by attaching onto the
exposed PS, providing a quick and suitable assay for apoptosis (Yoshida et al., 2012).
The utilisation of the red-fluorescent PI, a nucleic binding dye which is permeant only
towards nucleic acid in dead cells, along with AnV-F can distinguish within cellular
populations in flow cytometry (Appelt et al., 2005).
The scatter graphs shown in Fig. 6 show the cell mortality in 10000 events of HeLa and
C33A cells, with samples treated with neutral LIPO1-CT and cationic LIPO2-CT. The
cells within C33A-CT sample were predominantly viable (99.9%), which was as
expected because cancer cells untreated with cytotoxic drug or liposomes should not
incur apoptosis (Kenis et al., 2006). Similarly, cell viability in C33A LIPO1-CT was

22. Page I 20
calculated at 99.3%, as a barren neutral-charged liposome should also not induce
apoptosis. However, empty cationic C33A LIPO2-CT did induce early apoptosis
(6.09%), suggesting that CLs might carry a degree of toxicity when applied to cultured
cancer cells (Yoshida et al., 2012). This trend was also observed with HeLa LIPO2-CT
sample, as early apoptosis was measured at 5.18%. This could have occurred because
when minimal CLs dosage was employed within cultured cells by (Kenis et al., 2006),
the transfection rate was minimally greater than raw gene delivery, thus indicating the
need to administer a higher dosage of CLs to become cytotoxic. Albeit early apoptosis
within HeLa-CT (2.28%) and HeLa LIPO1-CT (5.77%) was also substantial, it can be
deduced that HeLa was more susceptible to liposomal treatment than C33A and that
CLs treatment had indeed increased early apoptosis within both HeLa and C33A.
Figure 11; Illustration by (Shen et al., 2001) that shows the process by which phosphorylated proto-oncogene tyrosine-
protein kinase (Src) can induce extracellular signal-regulated kinase (ERK) signalling pathways. The latter stimulating of
various transcription factors, such as c-Jun and c-Fos, thus becomes vital to cervical carcinoma cell proliferation.
The increased apoptotic activity in HeLa cells, with empty LIPO1-CT and LIPO2-CT,
can be explained by the link between cervical cancer and extracellular signal-regulated
kinase (ERK) signalling pathways (Tai et al., 2014). (Chaudhury et al., 2012) report that
empty FR-targeted liposomes show a substantial increase in ERK phosphorylation.
(Shen et al., 2001) have also argued that endocytotic uptake of FA by FRα can
phosphorylate the proto-oncogene tyrosine-protein kinase (Src), which further activates
ERK signalling pathway. Subsequent nucleic infiltration of phosphorylated ERK1 &
ERK2 further stimulates many transcription factors such as c-Fos and c-Jun, which are
highly expressed specifically in cervical cancers and vital for proliferation and apoptosis
(Fig. 11). This trend is also seen in Fig. 8A & Fig. 8B as HeLa and C33A cells treated
with LIPO2-FA, without any cytotoxic AS2O3, had early apoptosis of 4.04% and 2.43%
respectively, proving that silencing FRα can instigate apoptosis amongst cervical
cancer cells. Therefore the activation of ERK signalling pathways, by down-regulating
FRα, can stem the development of cervical cancer and inhibit HeLa biological clock
which induces apoptosis (Chaudhury et al., 2012; Shen et al., 2001).
Extracellular
Matrix
Nucleus

23. Page I 21
٢ْ . AS2O3 inhibits cell proliferation and HPV-E6 oncogene expression in cervical
carcinoma.
The use of AS2O3 as a viable therapeutic strategy has shown substantial efficacy in
inducing complete cancer cell remission, with minimal myelosuppression (Zheng et al.,
1999). Investigations that elucidate upon these cellular response mechanisms have
shown that AS2O3 degrades aberrant intracellular signal transduction pathways that
modify cellular function, which can result in apoptosis and subsequent inhibition of
angiogenic growth (Lin et al., 2000). A study by (Wang et al., 2014) has shown that the
widespread effect on cellular and physiological pathways has made a variety of
malignancies, derived from several tissue types, susceptible to AS2O3.
The flow cytometry analysis of HeLa and C33A cells in scatter graphs (Fig. 8) showed
that CLs encapsulated with AS2O3 induced substantial early apoptosis within HPV+
HeLa cells, whilst having a negligible effect on HPV(-)
C33A cells. Early apoptosis within
cells treated with HeLa LIPO2-ATO showed 4.68% and 5.28% when treated with HeLa
LIPO2-FA-ATO, whilst the early apoptosis in HeLa free-ATO was 2.42%. A similar
early apoptosis trend, albeit considerably negligible, was seen in C33A LIPO2-ATO
(1.43%) and C33A LIPO2-FA-ATO (0.33%), whilst early apoptosis within C33A ATO
was non-existent (0.06%). The trend suggests that albeit free-ATO did induce early
apoptosis within C33A and HeLa cells, it was considerably lower than early apoptosis
induced by LIPO2-FA and LIPO2-FA-ATO. Therefore, it can be deduced that HPV18
expressing cervical cancer cells have intensified response to AS2O3 treatment
compared to HPV-negative cell lines, whilst treatment with free-ATO is not effective.
The heightened response of cervical cancer cells to AS2O3 treatment has been
reported by numerous studies, such as (Hu et al., 2013; Wang et al., 2014; Wu et al.,
2004). AS2O3 can suppress the proliferation of cancerous cells by cell cycle arrest,
within the G1 or G2/M phase, stimulating cyclin-dependent kinase inhibitors and
cellular apoptosis (Hu et al., 2013). The HPV-infected HeLa cell line was more
responsive to AS2O3 treatment due to the anticipated consequences of inhibiting HPV-
E6 oncogene expression (Wu et al., 2004). After HPV viral incorporation into the
nucleus, the HPV-E6 oncogene of high risk HPV’s attaches onto p53 and degrades it.
The subsequent loss of functional or active p53 tumour suppressor gene instigates the
immortalisation of the cervical cell, over-riding the regulating apoptotic mechanisms
(Wang et al., 2014; Wu et al., 2004). Therefore, the inhibition of HPV-E6 will
accumulate the presence of p53, causing subsequent G2/M cell cycle arrest and
apoptosis. Similarly within HPV-negative C33A cell line, p53 is still targeted but instead
is mutated and non-functional, therefore requiring a higher dosage to initiate
cytotoxicity and thus the early apoptosis is lower than HeLa cells. This can also be
indicative that CLs delivery to HPV-negative C33A is not as efficient compared to HPV+
HeLa cells (Wang et al., 2014).

24. Page I 22
٣ْ .The role of HPV is crucial for the development of invasive cervical carcinoma.
The discovery of human papillomavirus (HPV) infection has been established to be the
main cause of cervical cancer development, optimising the role of HPV treatment and
prevention in carcinomas (Diao et al., 2015). High risk genital HPVs represent a cohort
of non-enveloped dsDNA viruses that replicate within the cell nuclei, situated at the
basal layer of stratified squamous epithelium (Wen et al., 2012). The pathogenesis of
HPV infection is directed by six major non-structural proteins; E1, E2, E4, E5, E6 and
E7, which direct the initial expression of the HPV genome, and two main structural
proteins; L1 and L2 (Wang et al., 2013). The function of all eight of these proteins is
shown in appendix 4. The high-risk HPV-E6 and HPV-E7 oncogenes are essential for
the initiation and conservation of cervical cancer, and their repression is sought to be a
promising treatment of HPV-positive tumours (Wen et al., 2012). The subjugation of E6
and E7 activates tumour suppressor targets, p53 and retinoblastoma protein (pRb),
initiating the gradual deterioration of cervical cancer cells (Wang et al., 2014). Methods
targeting the upstream regulatory region (URR), which regulates E6/E7 expression,
can target p53 for proteasomal degradation by its connection with the E6-associated
protein (E6-AP), an ubiquitin ligase (Scheffner et al., 1993).
Immunocytochemistry was utilised to evaluate the presence of specific proteins, within
cultured or suspended cells, by using a specific antibody to bind to the proteins and
thus allowing visual under the confocal microscope (Diao et al., 2015).
Immunocytochemical analysis was done using DAPI-containing media, which gave
blue fluorescence to counter-stain and locate the nuclei. Both HeLa and C33A cell lines
were also double stained using anti-HPV18 E6 (red fluorescence) and anti-FRα (green
fluorescence) when forming conjugates. The confocal micrographs, shown in Fig. 7,
were obtained from both HeLa and C33A cells after 24hrs treatment with LIPO1-CT
and LIPO2-CT. The results were as expected, as HeLa cells are known to express both
HPV+
and FRα+
, displayed all three fluorescence types. Hence, this confirmed the
anticipated expression of HPV-E6, FRα and nuclei within the samples when treated
with HeLa-CT (Fig. 7A), HeLa LIPO1-CT (Fig. 7C) and HeLa LIPO2-CT (Fig. 7E).
Similar to expectation, HPV-negative C33A cells showed blue nuclei fluorescence and
an absence of red HPV fluorescence, but also expressed green FRα expression. This
was interesting as the relationship between FRα and C33A is unclear, as only some
studies have reported the presence of FRα in C33A (Wen et al., 2012). Therefore the
expression of FRα in C33A-CT (Fig. 7B), C33A LIPO1-CT (Fig. 7D) and C33A LIPO2-
CT (Fig. 7F) has confirmed that albeit mild, in comparison to HeLa cells, C33A does
express FRα. This trend was also seen in Fig. 9A & Fig. 9B as HeLa and C33A cells
expressed same fluorescence with LIPO2-FA, without any cytotoxic AS2O3, proving
that FRα was expressed within C33A cervical cancer cells and that it is HPV(-)
.

25. Page I 23
The confocal micrographs of HeLa and C33A cells in Fig. 9 have showed that CLs
encapsulated with AS2O3 inhibited the expression of HPV-E6 within HPV+
HeLa cells,
whilst having also reducing the expression of FRα in HPV(-)
C33A cells. The cells
treated with HeLa LIPO2-ATO showed mild HPV-E6 expression and none when
treated with HeLa LIPO2-FA-ATO, whilst the HPV18 expression in HeLa free-ATO was
also mild. This was mirrored by the flow cytometry results in Fig. 8 as the reduction of
HPV18 expression induced apoptosis within the respective HeLa samples. Similarly,
the FRα expression was also mild in both HeLa LIPO2-ATO (Fig. 9C) and HeLa LIPO2-
FA-ATO (Fig. 9E), whilst absent with free-ATO (Fig. 9G). The ATO upregulation of p53
within HPV-positive cancer cells has been documented within many studies (Wang et
al., 2014). This was correlated in our results as AS2O3 decreased the proliferation and
viability of HeLa cells, whilst p53 accumulation inhibited HPV-E6 expression. AS2O3
specifically targeted the AP-1 site, attached to the HPV18 URR promotor (appx.4),
which inactivated AP-1 and allowed the build-up of p53 (Lin et al., 2000). The anti-
metastatic effect of AS2O3 is hence down to disarming AP-1, an MMP-1 and MMP-3
activation transcription factor, by the apoptotic mechanism of p53 (Wang et al., 2014).
In C33A LIPO2-ATO (Fig. 9D) and C33A LIPO2-FA-ATO (Fig. 9F), the expression of
FRα was evident, however the FRα fluorescence was absent in free-ATO (Fig. 9H).
This indicates that treatment with AS2O3 reduced the expression of HPV-E6 and FRα
within HeLa cells whilst C33A cells lost FRα expression after treatment with free-ATO.
DAPI-blue fluorescence was present in all of the confocal micrographs to give evidence
that cells were present in all micrographs. This signifies the ability of AS2O3 to
significantly target HPV-E6 oncogene expression, incurring apoptotic effects, and that
free-ATO can better down regulate expression compared to liposomes encapsulated
with FA-ATO. It is also worth mentioning that the absence of FRα expression in HeLa
and C33A cells, with free-ATO, indicates that AS2O3 induces apoptosis in FRα-
dependent manner, whilst also targeting HPV-E6 within HPV-infected cells. This
suggests that FRα expression within cervical cancer can be exploited to target
carcinoma cells with cytotoxic agents.
۴ْ .Mechanistic limitations of the study and future liposomal advancements.
Improvement in flow cytometry analysis and statistical analysis
Flow cytometry is specialised analysis tool that can obtain information regarding
various cellular processes, surface marker expression, signalling proteins, or the cell
cycle phase (Jahan-Tigh et al., 2012). Its ability to recognise the complex interplay of
cellular biological processes amongst the heterogeneous cell population makes it vital
for detecting treatment response (Herzenberg et al., 2006). However, there are various
limitations that accompany the use of flow cytometry. Foremost, the cells must be
suspended within a single-cell heterogeneous sample, as flow cytometry relies on
passing these cells via a fluid stream, and restricts the analysis to only suspension

26. Page I 24
solutions (Roederer, 2001). Also, the resulting data from flow cytometry analysis is
given to a wholesome aggregate level, thus individual cell behaviour cannot be
observed. The detection of synchronized multiple markers is required to validate the
results that increase specificity, whilst the lack of set-up standardization in both assay
and instrument limits the analysis of flow data (Jahan-Tigh et al., 2012). Finally, trained
operatives are required to gate and make sense of the hefty data amount produced by
flow however mechanical multi-dimension visual and gating, along with post-analysis
data accumulation models are being developed. Studies by (Puga Yung et al., 2012; Li
et al., 2014) have shown that our results could be made more specific if flow cytometry
was combined with transcription profiling or adding more ‘detectors’ with mass
spectroscopy, which along with real-time polymerase chain reaction (rt-PCT) could
quantify mRNA levels and detect the gene transcripts of HPV-18 or apoptosis.
The study protocol to treat cells once (24hrs) and not in triplicates made the
subsequent flow cytometry analysis inept to generate statistical data, as calculating
data mean was not possible (Jahan-Tigh et al., 2012; Roederer, 2001). Therefore, any
subsequent attempts to utilise p-value or other statistical tests were redundant (Jemal
et al., 2006). Similar to the study by (Ling et al., 2008), triplicate experimentation
produces p-values which could have determined if the difference within cell viability
was significant. Other statistical tests such as power curve 2-sample T-tests can
generate data means which can allow further statistical analysis, such as 2-sample t-
test or Mann-Whitney, which can be utilised to elucidate significance within cell
populations and thus accept or reject null hypothesis regarding data (Herzenberg et al.,
2006). Finally, the length of treatment could also have been extended (>48hrs) to allow
comparison within dosage and time. Studies by (Rajmani et al., 2015; Sun et al., 2014)
have found links between AS2O3 treatment and HeLa survival and proliferation, in a
time and dosage dependent manner, to obtain the optimal circulation time and dose
amount (Jahan-Tigh et al., 2012; Jemal et al., 2006).
Limitations of florescent confocal microscopy and alternatives
The confocal light microscope was used to visualise the location of fluorescence within
small contiguous sample of fluorescent-stained molecules (Nwaneshiudu et al., 2012).
The fundamental limits of fluorescent confocal microscopy are the non-permanent
nature of the fluorescent dye, which can fade overtime due to photobleaching. The
antibody-labelled samples also require chemical fixation and thus treatment with
detergents, such as Triton, can infiltrate cell membranes and introduce artefacts
(Roederer, 2001). Another limitation of confocal microscopy is the overlapping dye
colours that can overwhelm each other, thus making visual examination inaccurate.
This was especially true for the red from cyanine-5 which diminished the visibility of
green Alexa Fluor®
hence studies by (Guitera et al., 2009) have shown that using less
Cy-5 concentration can negate this problem. On contrary, study by (Liang et al., 2006)
has found that fluorescence imaging using two-photon excitation microscopy is a

27. Page I 25
superior alternative to confocal microscopy, due to its ability to efficiently detect light
within finer wavelengths and reduced phototoxicity/ photobleaching.
Improving the intravascular release of liposomal drug into tumours
Current therapeutic liposomal treatments target bio-distribution to decrease free-drug
cytotoxicity and accumulate within tumours, by passive extravasation from
hyperpermeable angiogenic vessels (EPR effect, Fig. 5). Whilst these methods have
upregulated drug delivery, evidence suggest that liposomal conjugates are too large to
extravasate into tumour vasculature, as vessels permeable to 100nm liposomes can be
susceptible to variances of inter/intra-tumour properties and have limited outreach of 1-
2 cell layers from the vessel (Manzoor et al., 2012; Yuan et al., 1994). Also, the slow
release of encapsulated drug can desensitise the effect of treatment as tumour cells
are not exposed to optimal cytotoxic dose that can result in cell mortality.
Study by (Manzoor et al., 2012) has suggested that use of thermally sensitive
liposomes (TSLs) can rapidly release encapsulated drug when exposed to 40-42°C, as
clinical applications showed 30times higher doxorubicin free-drug release. The
enhanced drug delivery thus contributed to 5times greater anti-tumour efficacy and
treatment, compared to traditional liposomal strategies (Yarmolenko et al., 2010). The
preheating of tumours and subsequent hyperthermic TSL administration increased
drug deliverance and optimised intravascular liposomal drug release, overcoming the
limited heterogeneous vascular penetrability attributed towards EPR effect (Lindner et
al., 2004). Instead TSLs timed the release of their sequestered drug until faced with a
specific and localized tumour trigger, upregulating tumour site drug uptake as free-drug
penetrated deeper from angiogenic vessels (Manzoor et al., 2012; Kong et al., 2001).
Further pegylation of liposomes by PEG-coating can increase the circulatory time of
TSLs, by decreased opsonisation (Fig. 4). Therefore, future liposomal delivery can
utilise cationic pegylated-TSLs, encapsulated with cytotoxic agent, guided by surface
ligands specific to the tumour tissue (Al-Ahmady et al., 2014). Drug augmentation
within tumour specific tissue can also be achieved by conjugating gold nanoparticles
(AuNP) onto liposomes, prepared by loading within the bilayer of
dipalmitoylphosphatidylcholine (DPPC) liposomes, or by exploiting the molecular
charge of the liposomes (Gasselhuber et al., 2012). The interaction of drug and lipid
membrane bilayer can dictate liposomal construction as electrostatic interaction,
positive or negative, can augment hydrophilic AS2O3 delivery (Zhang et al., 2013).
Novel nanoparticle formulation of AS2O3 can reduce cytotoxicity.
The success of AS2O3 within hematologic cancers has yet to be duplicated within solid
tumours, as inept pharmacokinetics fail to stabilise liposomes (Ahn et al., 2010). The
liposomal-AS2O3 is aqueous at physiologic pH which permeants lipid bilayers and
subsequently leaks within healthy tissue (Baj et al., 2002). Current strategies to

28. Page I 26
stabilise pegylated 100nm liposomal-AS2O3 (arsenic-nanobins [NB(Ni,As)]) has led to
nickel acetate (Ni2+
) inclusion within the hydrophilic core, causing stabilised
precipitation of (Ni,As) (Yu et al., 2007). The development of nanoparticle AS2O3
constructs by (Ahn et al., 2010), also strive to amass within tumour vasculature for
efficient treatment. The NB core is densely packed with arsenic (>270 mmol/L), which
augments drug activity, as the release of arsenic is instigated by the low pH found in
tumour cells. Albeit AS2O3 treatments have long been limited by renal pathway filtration
and dose-limiting toxicity, this novel liposomal preparation method modifies the surface
with disialoganglioside-2 (GD2)-specific antibody, which prepared alongside CD19-
specific antibodies can optimize treatment (Ahn et al., 2010; Yu et al., 2007).
۵ْ .Conclusion
This thesis has shown that lipid-encapsulated AS2O3 can inhibit proliferation of cervical
cancer cells, whilst also inducing apoptosis. The response of HPV-positive and HPV-
negative cervical cells has revealed the differential therapeutic pathways adopted by
AS2O3, as upregulated tumour-suppressor p53 deregulates AP-1, to attenuate E6-
oncogene within HPV-infected HeLa. The confocal micrographs confirmed FRα
expression in C33A cells, elucidating that AS2O3 induces apoptosis in FRα-dependent
manner. The promising method of folate-tethered AS2O3 is possible by stable loading
using transmembrane gradients of (Ni2+
and Co2+
) ions which show heightened cervical
anti-cancer efficacy against FRα-overexpressing carcinomas, which are fairly
insensitive to free- AS2O3 drug treatments. The results showed that empty cationic
liposomes induced apoptotic effects within both cervical cancer cells, whilst HeLa was
more susceptible due to ERK signaling pathways stimulating transcriptional factors.
The response of HPV-infected HeLa cells to AS2O3 was intensified compared to HPV-
negative C33A cells, whilst free-ATO treatment was not as efficient within both cell
lines. Possible improvements, such as flow cytometry with mass-spec transcription
profiling, along with 2-sample t-test or Mann-Whitney statistical analysis can clarify
significance within treatments and cell viability. Our results have given a rationale for
liposomal-drug delivery that exploits folate-mediated endocytosis to target cervical
carcinoma cells, however further in-vitro studies into molecular AS2O3 mechanisms on
other HPV-infected carcinomas are warranted to aptly target different tumour specific
receptors.

29. Page I 27
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Appendices
Appendix 1. Learning Log
Meeting 1 Date: 28th
January 2015

35. Page I 33
Meeting 2. Date: 30th
January 2015

36. Page I 34
Meeting 3. Date: 22nd
January 2015