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

Page I 0

Contents

Abstract .........................................................................................................................................2

Introduction ..................................................................................................................................3

١ .Hallmarks of cancer: the next generation. ...................................................................3

٢ .Shortfalls in current cancer therapy. ............................................................................4

٣ .The expression of folate-receptors in cervical carcinoma cells. ..................................5

۴ .Trends and developments in liposome drug delivery systems. ...................................7

۵ .Pharmacokinetics of pegylated liposomal drug delivery. .............................................8

٦ .Potential of folate-targeted liposome conjugates. .......................................................9

Aims ............................................................................................................................................ 10

Materials and methods ............................................................................................................. 11

١ .Cell lines and cultured conditions. ............................................................................ 11

٢ .Liposome preparation and arsenic trioxide loading. ................................................. 11

٣ .Treating HPV-infected and HPV-negative cells and flow cytometry analysis. ......... 12

۴ .Double immunocytochemical staining and confocal fluorescent microscopy. .......... 13

Results ....................................................................................................................................... 14

١ .Apoptotic activity amongst cervical cancer cells treated with empty liposomes. ...... 14

٢ .HPV-E6 and FRα expression decreases in cells after liposome treatment.............. 15

٣ .Cationic liposomes encapsulated with AS2O3 upregulate cervical cell apoptosis fairly more than free-AS2O3. .................................................................................................. 16

۴ .AS2O3 inhibits cell proliferation, FRα expression and HPV-E6 oncogene expression in cervical carcinoma. ................................................................................................... 17

۵ .Increased induction of necrosis and apoptosis in HPV-positive cervical cells. ........ 18

Discussion ................................................................................................................................. 19

١ .FR-targeted empty liposomes induce apoptosis within cervical carcinoma. ............ 19

٢ . AS2O3 inhibits cell proliferation and HPV-E6 oncogene expression in cervical carcinoma. .................................................................................................................... 21

٣ .The role of HPV is crucial for the development of invasive cervical carcinoma. ...... 22

۴ .Mechanistic limitations of the study and future liposomal advancements. ............... 23

۵ .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

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Acknowledgements

۩الصراط المستقيم هذا 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

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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.

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Introduction

١ .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.

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.

٢ .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

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).

٣ .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).

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)

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).

۴ .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

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.

۵ .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

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).

٦ .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

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.

Page I 11

Materials and methods

١ .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.

٢ .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.

Page I 12

The cytotoxic AS2O3197.8m/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.

٣ .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.

Page I 13

۴ .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.

Page I 14

Results

١ .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

C

on

tro

l (C

T)

A)

B)

LIP

O1 C

on

tro

l (L

IPO

1-C

T)

C)

D)

L

IPO

2 C

on

tro

l (L

IPO

2-C

T)

E)

F)

96.0 2.280

1.34 0.38

99.9 0.03.50

0.05 0.00

93.2 5.77

0.47 0.560

99.3.5

0.60.50

0.09 0.02

93.1 5.18

0.86 0.86

92.26.5

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

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).

٢ .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 x4000

mag.

HeLa C33A

C

on

tro

l (C

T)

A)

B)

LIP

O1 C

on

tro

l (L

IPO

1-C

T) C) D)

L

IPO

2 C

on

tro

l (L

IPO

2-C

T)

E) F)

Page I 16

٣ .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

L

IPO

2 F

A

A)

B)

L

IPO

2 A

TO

C)

D)

L

IPO

2 F

A-A

TO

E)

F)

A

TO

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

98.51.5

1.43.50

0.06 0.00.50

93.6 5.28.50

0.41 0.68

99.64.5

0.33.50

0.03 0.00.50

96.5.5

2.42.50

0.66 0.41.50

99.63.5

0.06.50

0.31 0.000.50

%

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

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(-)).

۴ .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 x4000

mag.

HeLa C33A

L

IPO

2 F

A

A)

B)

L

IPO

2 A

TO

C) D)

LIP

O2 F

A-A

TO

E) F)

A

TO

G) H)

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.

۵ .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

pe

rcen

tag

e o

f cell

s (

%)

HeLa C33A

Early apoptosis

Late apoptosis

Necrosis

*

* ** * ** ** ** * **

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.

١ .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

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

Page I 21

٢ . 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).

Page I 22

٣ .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(-).

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.

۴ .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

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

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

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).

۵ .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.

Page I 27

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Page I 32

Appendices

Appendix 1. Learning Log Meeting 1 Date: 28th January 2015

Page I 33

Meeting 2. Date: 30th January 2015

Page I 34

Meeting 3. Date: 22nd January 2015

Page I 35

Meeting 4 Date: 11th February 2015

Page I 36

Meeting 5. Date: 17th February 2015

Page I 37

Meeting 6. Date: 4th March 2015

Page I 38

Meeting 7. Date: 20th March 2015

Page I 39

Appendix 2. Consideration of Ethical Issues and Research Governance

Consideration of Ethical Issues and Research Governance

All students must review the ethical implications of their proposed work whether they are collecting primary data or analysing published work. You are required to discuss research governance and ethics with your supervisor, complete any required risk assessments and apply for ethical approval from the Natural Science Ethics Sub-Committee (NSESC). Once you have completed your ethics approval and risk assessment documents, you send it to your supervisor who will submit on your behalf.

You must complete Part I and Part II of this form. PART I I have discussed research governance and ethical implications of my study with my supervisor and: Ethical Clearance is NOT required for my project. Signed:

Student

Supervisor

I have applied for research ethics clearance from the NSESC. Ethical Approval has

been granted and the confirmation is appended in the learning log. Signed:

Student

Supervisor

PART II I have considered the need for completion of a risk assessment and: I do not require a risk assessment. Signed:

Student

Supervisor

I have completed and submitted a risk assessment which is appended in the learning log. Signed:

Student

Supervisor

Page I 40

Appendix 3. NSESC Application Form

Page I 41

Appendix 4. Expression of folate receptor isoforms (FRα, FRβ & FRγ) in normal and malignant cancerous tissue, adapted from (Antony, 1996).

System

FRα FRβ FRγ/ FRγ׳

Normal tissues

Genitourinary Placenta Kidney Bladder Testes

Placenta

Central Nervous

Choroid plexus Cerebrospinal fluid

Hematopoetic Spleen Thymus Neutrophils

Hematopoetic

Gastrointestinal Submandibular salivary glands Colorectum

Respiratory Lung

Endocrine Human milk

Malignant tissues

High expression

Nasopharyngeal epidermoid carcinoma Non-mucinous ovarian carcinoma Cervical carcinoma Uterine carcinoma Metastatic endometrial carcinoma Primary renal cell carcinoma Metastatic pancreatic carcinoma

Leukemia Lymphoma

Leukemia

Low or negligible expression

Mucinous ovarian carcinoma Primary endometrial carcinoma Metastatic renal cell carcinoma Lung carcinoma and adenocarcinoma Primary breast carcinoma5 Primary bladder carcinoma Primary pancreatic carcinoma Colorectal carcinoma Primary prostate carcinoma Primary brain carcinoma Primary liver carcinoma Primary head and neck carcinoma

Page I 42

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.

Liposome therapeutics Polymer therapeutics

Size (nm) 50-1000 10-100 a

Circulation half-life Up to 3-4 days 1-24 hours

Penetration into tumors Low

Bulky structures that have

difficulty penetrating the

heterogeneous solid

tumors

High

‘Elastic’ structure that

facilitate penetrating

heterogeneous solid

tumors

Drug loading High Low

Immunogenicity Attract opsonins (non-

pegylated)

Non-immunogenic

Drug-carrier association Drug physically

encapsulated in liposomes

Drug covalently chemically

linked to the polymer

Clearence Not filtered out by the

kidneys

Renal excretion

Drug release Drug leakage at

formulation- dependent

rate; drug might leak in

circulation

Controlled drug release

from the polymeric

backbone via hydrolysis of

pH-sensitive linkers or

enzymatic cleavage

Mechanisms of cellular

uptake

Endocytosis – liposome

breakdown in lysosome

required for cytoplasmic

drug release

Endocytosis-drug released

from the polymer in the

endosome or lysosome

FDA approval (no

targeted liposomal

drugs or polymer-drug

conjugates have been

approved yet)

Examples: DOXIL,

Ambisone®, DepoCyt®,

DaunoXome

Only polymer-protein

conjugates have been FDA

approved (Examples:

SMANCS, Oncaspar,

Neulasta®, PEGasys®,

PEGItron®, Cimzia®)

a Excluding pegylated proteins which may have half-lives of about 2-weeks.

Page I 43

Appendix 6. Below are the solutions that were made to prepare the samples to undergo ICP-OES (10ml each).

300115 Samples:

1. ATO in 0.02M NaH2PO4/Na2HPO4, in pH 7 at 20mM.

50µl of sample + 1.5ml HNO3 + 0.5ml H2O (1:41 dilution) → 20µl + 10ml H2O Dilution factor of 1:510, in a solution of 10ml. 2. Nickel acetate in dH2O, at 730mM.

50µl of sample + 1.5ml HNO3 + 0.5ml H2O (1:41 dilution) → 2.5µl + 10ml H2O

Dilution factor of 1:4001, in a solution of 10ml.

3. HeLa/C33A LIPO1-CT (Ni) after dialysis.

50ul of sample + 1.5ml HNO3 + 0.5ml H2O (1:41 dilution) → 100µl + 10ml H2O Dilution factor of 1:101, in a solution of 10ml. 4. HeLa/C33A LIPO2-CT (Ni) after dialysis.

50µl of sample + 1.5ml HNO3 + 0.5ml H2O (1:41 dilution) → 100µl + 10ml H2O Dilution factor of 1:101, in a solution of 10ml. 5. HeLa/C33A LIPO2-ATO.

25µl Sample + 0.75ml HNO3 + 9.25ml H2O Dilution factor of 1:410, in a solution of 10ml.

6. HeLa/C33A LIPO2-FA-ATO.

25µl Sample + 0.75ml HNO3 + 9.25ml H2O Dilution factor of 1:410, in a solution of 10ml.

Page I 44

Appendix 7. The table below has been adapted from (Munoz et al., 2006) to show the HPV genome and the proteins encapsulated to multiply.

Figure 12; Schematic presentation adapted from (Munoz et al., 2006) of the HPV genome, which infiltrates the host

DNA with their circular plasmid to initiate the process of high risk/low risk HPV infection. The non-coding region of the

plasmid is referred to as the upstream regulatory region (URR). The open reading frames (ORFs) encode the early (E),

and late (L) viral proteins. The genome of HPV is estimated to be about 7900bp.

Protein Role in the virus lifecycle

E1 Genome replication: ATP-dependent DNA helicase

E2 Genome replication, transcription, segregation, encapsidation. Regulation of cellular gene expression; cell cycle and apoptosis regulation

E4 Remodels cytokeratin network; cell cycle arrest; virion assembly

E5 Control of cell growth and differentiation; immune modulation

E6 Inhibits apoptosis and differentiation; regulates cell shape, polarity, mobility and signalling

E7 Cell cycle control; controls centrosome duplication

L1 Major capsid protein

L2 Minor capsid protein; recruits L1; virus assembly

Page I 45

Appendix 8. Below are the six-well plate arrangements of the liposomal formulations, and their microscopic images (x100mag) after 24hrs treatment.

Plates 1A & 1B; HeLa

HeLa CT

HeLa LIPO1

HeLa LIPO2

HeLa LIPO2-FA

HeLa LIPO2-ATO

HeLa LIPO2-FA-ATO

HeLa ATO

- -

- - -

Plates 2A & 2B; C33A

C33A CT

C33A LIPO1

C33A LIPO2

C33A LIPO2-FA

C33A LIPO2-ATO

C33A LIPO2-FA-ATO

C33A ATO

- -

- - -

Page I 46

Appendix 9. LabRAT Risk Assessment

Included in hard copy.