Academic year 2013-2014

SYNTHESIS, PHYSICOCHEMICAL CHARACTERISATION AND

CYTOTOXICITY EVALUATION OF DIFFERENT PGA-PTX CONJUGATES

Pieterjan MERCKX

First Master of Drug Development

Promotor:

Prof. Dr. S. De Smedt

Co-promotor:

Dr. M. J. Vicent D’Ocon

Commissioners:

Prof. Dr. B. De Geest

Dr. K. Raemdonck

CENTRO DE INVESTIGACIÓN PRÍNCIPE FELIPE

ADVANCED THERAPIES RESEARCH PROGRAMME

Laboratory of Polymer Therapeutics

GHENT UNIVERSITY

FACULTY OF PHARMACEUTICAL SCIENCES

Department of Pharmaceutics

Laboratory of General Biochemistry

and Physical Pharmacy

Academic year 2013-2014

SYNTHESIS, PHYSICOCHEMICAL CHARACTERISATION AND

CYTOTOXICITY EVALUATION OF DIFFERENT PGA-PTX CONJUGATES

Pieterjan MERCKX

First Master of Drug Development

Promotor:

Prof. Dr. S. De Smedt

Co-promotor:

Dr. M. J. Vicent D’Ocon

Commissioners:

Prof. Dr. B. De Geest

Dr. K. Raemdonck

CENTRO DE INVESTIGACIÓN PRÍNCIPE FELIPE

ADVANCED THERAPIES RESEARCH PROGRAMME

Laboratory of Polymer Therapeutics

GHENT UNIVERSITY

FACULTY OF PHARMACEUTICAL SCIENCES

Department of Pharmaceutics

Laboratory of General Biochemistry

and Physical Pharmacy

COPYRIGHT

"The author and the promotor give the authorization to consult and to copy parts of this thesis

for personal use only. Any other use is limited by the laws of copyright, especially concerning

the obligation to refer to the source whenever results from this thesis are cited."

Promotor

Prof. Dr. Stefaan De Smedt

Author

Pieterjan Merckx

AUTEURSRECHT

“De auteur en de promotor geven de toelating deze masterproef voor consultatie beschikbaar

te stellen en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder de

beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting

uitdrukkelijk de bron te vermelden bij het aanhalen van de resultaten uit deze masterproef.”

Promotor

Prof. Dr. Stefaan De Smedt

Auteur

Pieterjan Merckx

SUMMARY

Today, paclitaxel serves as a widely used chemotherapeutic agent but clinical use is limited

by a series of holdbacks due to its physicochemical features. Conjugation of paclitaxel with the

biodegradable, watersoluble polymer polyglutamic acid (PGA-PTX) is nowadays investigated

as a promising new strategy to enhance pharmacokinetics and drug delivery of paclitaxel.

In this study, PGA-PTX conjugates of different PGA polymer lengths and different drug

loadings were synthesised. All synthesised polymer-drug conjugates were characterised by

physicochemical techniques to study the influence of length and drug loading on their

properties. Plasma stability, drug release kinetics and cytotoxicity were also evaluated.

PGA of three different lengths (25, 50 and 100 glutamic acid subunits/polymer) was

loaded with two different amounts of PTX, aiming a total drug loading (TDL) of 5 and 10 mol%.

Determination of TDL by 1H-NMR and UV-VIS spectrophotometry showed that for polymer-

drug conjugates of PGA25, a conjugation yield higher than 50% could not be reached. PGA25

polymer-drug conjugates were found to be more difficult to purify than polymer-drug

conjugates of PGA50 and PGA100.

GPC analysis of the PDI indicated a homogenous conjugation of PTX for polymer-drug

conjugates of different length and TDL. The CAC was determined out of DLS measurements in

PBS and showed that every polymer-drug conjugate could form aggregates in solution and

that the value for CAC for polymer-drug conjugates with different length and TDL was similar.

Temperature did not influence the size of aggregates of a polymer-drug conjugate in solution.

Total drug loading influenced the size of aggregates in solution.

Plasma stability studies showed all degradation in terms of hydrolysis took place within

the first 24 hours after dissolution in plasma for polymer-drug conjugates of different length

and TDL. Polymer-drug conjugates of PGA25 were less stable than polymer-drug conjugates of

PGA50 and PGA100. TDL did not influence stability in blood plasma. Drug release kinetics studies

in PBS showed that increase of temperature led to increase of drug release for all polymer-

drug conjugates. Polymer length and TDL were found to not influence drug release at constant

temperature. Study of cytotoxicity led to incoherent data so no conclusions were drawn.

It was generally concluded that polymer length of polymer-drug conjugates influenced

conjugation yield, purification and stability in blood plasma. Total drug loading influenced the

size of aggregates in PBS.

SAMENVATTING (SUMMARY IN DUTCH)

Hoewel paclitaxel in de huidige praktijk een veelgebruikt chemotherapeuticum is, wordt

het klinisch gebruik ervan bemoeilijkt door zijn fysicochemische eigenschappen. Conjugatie

van paclitaxel met het biologisch afbreekbare, wateroplosbare polymeer polyglutaminezuur

(PGA-PTX) wordt tegenwoordig onderzocht als een veelbelovende, nieuwe strategie om de

farmacokinetische eigenschappen en drug delivery van paclitaxel te verbeteren.

PGA-PTX conjugaten met verschillende polymeerlengtes en geneesmiddelenladingen

werden gesynthetiseerd en gekarakteriseerd met fysicochemische technieken, teneinde de

invloed van deze parameters op de eigenschappen van het conjugaat te bestuderen. Ook

werden plasmastabiliteit, drug release kinetiek en cytotoxiciteit geëvalueerd.

Polyglutaminezuur van drie verschillende lengtes (25, 50 en 100 glutaminezuur

subunits/polymeer) werd beladen met twee verschillende hoeveelheden PTX, strevend naar

een totale geneesmiddelenlading (TDL) van 5 en 10 mol%. De TDL werd bepaald met 1H-NMR

en UV-VIS spectrofotometrie en wees uit dat voor conjugaten van PGA25, een opbrengst van

meer dan 50% niet kon worden bereikt. Voor PGA25 conjugaten werd eveneens vastgesteld

dat opzuivering moeilijker verliep vergeleken met conjugaten van PGA50 and PGA100.

GPC-analyse van de PDI wees uit dat er een homogene conjugatie van PTX kon worden

bereikt voor alle conjugaten. CAC analyse via DLS toonde aan dat elk conjugaat aggregaten

vormt in oplossing en dat de CAC voor conjugaten met verschillende lengte en TDL gelijkaardig

was. Er was geen invloed van temperatuur op de grootte van aggregaten van een conjugaat.

De TDL beïnvloedde de grootte van aggregaten in oplossing.

Een studie van de plasmastabiliteit toonde aan dat voor conjugaten van verschillende

lengte en TDL, alle afbraak door hydrolyse plaatsvond binnen de eerste 24 uur. Conjugaten

van PGA25 werden minder stabiel bevonden dan conjugaten van PGA50 en PGA100. TDL had

geen invloed op stabiliteit in plasma. Drug release kinetiek in PBS wees uit dat

temperatuurstoename tot een toename in geneesmiddelenvrijstelling leidde voor alle

conjugaten. Polymeerlengte en TDL beïnvloedden geneesmiddelenvrijstelling bij constante

temperatuur niet. Evaluatie van de cytotoxiciteit leidde tot incoherente data.

Algemeen werd besloten dat voor polymeer-geneesmiddelconjugaten, polymeerlengte

invloed had op de conjugatieopbrengst, opzuivering en stabiliteit in plasma.

Geneesmiddelenlading had invloed op de grootte van aggregaten in oplossing.

ACKNOWLEDGEMENTS

I would like to thank Dr. María Jesus Vicent D'Ocon for giving me the opportunity

to partake in this project. It has been a fantastic four months in which I broadened

my knowledge on a wide variety of scientific fields.

I would like to give special thanks to my supervisor Dr. Julie Movellan for the

pleasant collaboration. I want to thank her for guiding me through the process by

helping, advising and correcting me during my experimental work. I also want to

thank her for all of her support while writing my report.

I want to thank everybody of I-36 for all of their support and advice, as well as for

integrating me in the Spanish culture. I also want to thank my fellow UGent

students Frauke and Géraldine for all of their support.

I would like to thank my Belgian and Valencian friends

for all of their support and interest.

Finally, I want to thank my parents, my brother Frederik and the rest of my family

for all of their support and care, as well as for their interest in my project.

TABLE OF CONTENTS

1. INTRODUCTION ..................................................................................................................1

GENERAL INTRODUCTION ............................................................................................1

POLYMER-DRUG CONJUGATES .....................................................................................2

1.2.1. Nanomedicines for improved drug delivery ...........................................................2

1.2.2. General concepts ..................................................................................................3

1.2.3. The Enhanced Permeation and Retention effect ...................................................4

1.2.4. Endocytosis ...........................................................................................................5

PACLITAXEL ..................................................................................................................7

1.3.1. General .................................................................................................................7

1.3.2. Solvent based formulation ....................................................................................9

1.3.3. Nanoparticle albumin bound-paclitaxel ...............................................................10

PACLITAXEL-POLYGLUTAMIC ACID CONJUGATES........................................................12

1.4.1. Polyglutamic acid ................................................................................................12

1.4.2. Paclitaxel-poliglumex ..........................................................................................12

2. OBJECTIVES ......................................................................................................................14

3. MATERIALS AND METHODS .............................................................................................15

3.1. CHEMICALS ................................................................................................................15

3.2. PGA-PTX SYNTHESIS FOR 5% AND 10% TOTAL DRUG LOADING ..................................16

3.3. PURIFICATTION ..........................................................................................................17

3.3.1. Work-up ..............................................................................................................17

3.3.2. Size Exclusion Chromatography ...........................................................................17

3.3.3. Freeze-drying for preservation ............................................................................19

3.4. CHARACTERISATION ...................................................................................................20

3.4.1. Total Drug Loading and impurities determination by 1H-NMR .............................20

3.4.2. Total Drug Loading determination by UV-VIS spectrophotometry .......................21

3.4.3. Molecular weight and polydispersity determination by GPC ...............................22

3.4.4. Determination of CAC and Hydrodynamic diameter by DLS .................................23

3.5. PLASMA STABILITY AND DRUG RELEASE KINETICS ......................................................24

3.5.1. Determination of drug release in plasma.............................................................24

3.5.2. Determination of drug release kinetics in PBS .....................................................25

3.6. CELL VIABILITY ASSAY .................................................................................................25

3.6.1. Culturing and subculturing ..................................................................................25

3.6.2. Seeding ...............................................................................................................27

3.6.3. Treatment ...........................................................................................................27

3.6.4. MTS/PMS cell viability assay................................................................................27

4. RESULTS AND DISCUSSION ................................................................................................28

4.1. PGA-PTX SYNTHESIS FOR 5% AND 10% TOTAL DRUG LOADING ..................................28

4.2. CHARACTERISATION ...................................................................................................28

4.2.1. Total Drug Loading and impurities determination by 1H-NMR .............................28

4.2.2. Total Drug Loading determination by UV-VIS spectrophotometry .......................30

4.2.3. Molecular weight and polydispersity determination by GPC ...............................32

4.2.4. Critical Aggregation Concentration by DLS ..........................................................34

4.2.5. Evaluation of aggregation and temperature influence by DLS .............................36

4.3. PLASMA STABILITY AND DRUG RELEASE KINETICS ......................................................37

4.3.1. Plasma stability assay ..........................................................................................37

4.3.2. Drug release kinetics ...........................................................................................39

4.4. CELL VIABILITY ASSAY .................................................................................................41

5. CONCLUSIONS ..................................................................................................................43

6. REFERENCES .....................................................................................................................45

LIST OF ABBREVIATIONS

CAC Critical Aggregation Concentration

DEHP di-(2-ethylhexyl) phtalate

DIC N,N'-diisopropylcarbodiimide

DIEA diisopropylethylamine

DLS Dynamic Light Scattering

DMAP 4’-dimethylaminopyridine

DMF N,N-dimethylformamide

DPBS Dulbecco's Phosphate Buffered Saline

EPR Enhanced Permeation and Retention

ESF European Science Foundation

FDA Food and Drug Administration

GPC Gel Permeation Chromatography

HOBT 1-hydroxybenzotriazol

HPLC High Pressure Liquid Chromatography

IC50 Half Maximal Inhibitory Concentration

MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carbomethoxyphenyl)-2-(4-

sulfophenyl)-2h tetrazolium

NMR Nuclear Magnetic Resonance

PBS Phosphate Buffered Saline

PDI Polydispersity Index

PGA polyglutamic acid

PMS phenazine methosulfate

PTX paclitaxel

PVC polyvinyl chloride

SEC Size Exclusion Chromatography

SPARC Secreted Protein Acid Rich in Cysteine

TDL Total Drug Loading

USP United States Pharmacopeia

UV-VIS Ultraviolet-Visible light

VEGF Vascular Endothelial Growth Factor

1

1. INTRODUCTION

GENERAL INTRODUCTION

According to the World Health Organisation, worldwide 8.2 million deaths are caused by

cancer every year, making it the world’s second biggest killer after cardiovascular disease.

Regarding 14 million cases of cancer in 2012 and an expected increase to 22 million within the

next 20 years, the development of effective strategies to prevent and cure cancer remains

essential. Despite the fact that scientific developments already went a long way, many

challenges still lay ahead. (1)

Cancer is caused by sequential alterations of oncogenes and tumor-suppressor genes,

which regulate growth, differentiation and survival of cells. Genetic alterations are the result

of hereditary DNA abnormalities or induced by external factors such as ultraviolet or ionic

radiation, microbiological infection or chemicals. (1) (2). This eventually leads to uncontrolled

cell proliferation, resulting in aggregates of malignant cells called tumors. As tumors increase

in size, they invade surrounding tissues and often, malignant cells are capable of affecting

other tissues through metastasis. In this way, normal physiological functions are impaired,

developing a life-threatening condition for the patient. Cancer can be cured by radiotherapy,

surgery and chemotherapy, in which the ultimate goal is prolonged duration of life together

with an increased quality of life. However, most radiotherapy and chemotherapies today

cause severe side effects and sometimes, surgery requisites sacrifice of healthy tissue. (3)

Ovarian cancer is the most lethal gynaecologic cancer in the United States and moreover,

it is the fifth most diagnosed cancer among American women. The most occurring subtype is

epithelial ovarian cancer, with more than 90 per cent of the cases. (4) Higher risk of developing

ovarian cancer is related to hormonal factors, genetic factors, medication, child bearing, family

history of breast or ovarian cancer and age. Vague symptoms and the fact that the disease is

often diagnosed when it is already at and advanced stage cause a high risk of bad prognosis.

Almost all stages of ovarian cancer can be treated with surgery. This involves either a total

hysterectomy, in which the uterus is completely removed, or removal of the ovaries, fallopian

tubes, omentum or abdominal fat tissue. In most cases, chemotherapy is used after surgery,

to eliminate any remaining cancerous tissue. The standard treatment is a combination of a

platinum compound, such as carboplatin or cisplatin, and a taxane, such as paclitaxel or

docetaxel. (5) (6)

2

POLYMER-DRUG CONJUGATES

1.2.1. Nanomedicines for improved drug delivery

Nanotechnology is the general term which describes all technological developments used

to create particles with dimensions within the nanoscale range, usually between 1 and

1000 nm. These particles comprise both chemical materials and devices. However, the exact

definition of the term ‘nanomaterial’ is globally still debated and many industries and sectors

tend to classify nanomaterials in different ways. (7) (8)

The European Science Foundation (ESF) Forward Look Nanomedicine, defines the term

‘nanomedicine’ in the following way: “Nanomedicine uses nano-sized tools for the diagnosis,

prevention and treatment of disease and to gain increased understanding of the complex

underlying patho-physiology of disease. The ultimate goal is improved quality-of-life.” In this

way, nanomedicine science can be considered as the application of nanotechnology for

healthcare purposes. Generally, nanomedicines can be roughly divided into three main

domains: nanopharmaceuticals, nanoimaging agents and nanotheranostics. However, the

classification remains artificial and some nanomaterials and –devices for healthcare purposes

are on the intersection of these domains. (7)

An important evolution of nanotechnology is the development of nanomedicines for

enhanced drug delivery. Drug delivery is the strategy to target pharmaceutically active

substances more specifically to the desired pathological site(s) in the human body.

Improved drug delivery is useful to overcome disadvantages of ‘classic’ formulations, in which

the active pharmaceutical ingredient is distributed more diffuse into the whole body. This

causes a bigger diffusion into healthy, non-pathological tissue. As a result, side effects are

caused, which is important for compounds with a narrow therapeutic index, such as

immunosuppressive, antitumor and antirheumatic drugs. It eventually makes doselimiting

necessary, which also decreases the therapeutic effect of the molecule. In addition, several

small molecules have a short half-life and a higher overall clearance. By formulating

therapeutically active small molecules as a nanomedicine, drug delivery can be improved

significantly. This is due to the fact that biological barrier crossing and penetration at a cellular

and subcellular scale, largely depend on the physicochemical properties of the nanoparticle

guiding the drug. Nanoparticles also permit the incorporation of ligands for specific biological

receptors, which contributes to further targeting of the system. (7) (9) (10) (11) (12)

3

1.2.2. General concepts

Polymer-drug conjugates are a class of nanomedicines in which one or more bioactive small

molecules are linked to a biodegradable polymer. Polymers are macromolecules consisting of

constitutional repeating units. The big advantage of conjugation with polymers is that the

‘fate’ of a small molecule drug in the human body can be influenced by optimizing the

properties of the polymer that guides it.

Figure 1.1.: The Ringsdorf model for polymer-drug conjugates. The polymer backbone carries a drug through

linkage with a cleavable spacer and can consist of a solubilising group and a targeting moiety. (12)

According to the Ringsdorf model (Figure 1.1.), one or more biologically active small

molecules can be conjugated to a biocompatible polymer backbone by a cleavable spacer. The

polymer types that can be used include polysaccharides, proteins and synthetic polymers. The

spacer is usually a ‘bioresponsive’ chemical bond, meaning that it undergoes dissociation

under certain biological conditions. These conditions are either chemical, such as a pH-shift,

or either the presence of a cleaving enzyme such as an esterase, lipase or protease. As a result,

the exposure to particular enzymes or a pH-shift after uptake in cells can eventually lead to a

more selective drug release. By keeping these conditions in consideration for the desired site

of action in the body, the most suitable spacer can be selected so cleavage is induced by these

conditions and drug release takes place in this particular site. (12) (13) (11)

In addition, other entities can be conjugated to enhance the pharmacokinetic and drug

delivery aspects of the polymer-drug conjugate. Solubilising agents can be linked in order to

improve the bioavailability and physicochemical properties of hydrophobic drugs. Moieties for

targeting such as antibodies or polysaccharides can be linked in order to target specific

biologic receptors or antigens, which is called active targeting. In contrast, polymers can be

targeted passively as well, meaning that it does not require a targeting moiety. The polymer

is then targeted based on its physical and chemical properties, such as its size. (12) (13) (11)

4

1.2.3. The Enhanced Permeation and Retention effect

The Enhanced Permeability and Retention (EPR) effect is an important starting point for

developing nanoparticle systems for drug delivery of antitumor drugs. It is considered as

becoming the “gold standard”, since nearly all newly designed macromolecular, lipidic and

micellar tumor targeting strategies are based on this effect.

The EPR effect (Figure 1.2.), as described by Matsumura and Maeda in the 1980s, is the

result of the structural and anatomical differences between tumoral and healthy vascularity.

When solid tumor cell aggregates exceed a total diameter of 1-2 mm, extensive angiogenesis

is driven by a variety of growth factors. This leads to a more efficient blood supply on which

tumor cells greatly depend for provision of oxygen and nutrients. (7) (14) The newly formed

blood vessels are described as ‘neovasculature’ and they significantly differ from non-

pathological mature vascular tissue, because of following abnormalities:

1) An increased vascular density as a result of extensive angiogenesis (hypervasculature)

2) Irregularity of vascular networks with big fenestrations and pores

3) Absence of a smooth muscle layer

4) Formation of pericytes

5) Extensive release of multiple mediators that induce extravasation: VEGF, NO, bradykinine,

prostaglandins, collagenase, peroxynitrine

6) A low amount of effective receptors for ATII and thus absence of ATII induced

vascoconstriction

7) A general lack of lymphatic vessels (14) (15)

Consequently, neovasculature can be considered as an irregular, incomplete structured

vascular tissue that is insufficiently sensitive to physiological responses. This is utilised, as the

previously described characteristics are in favour of drug delivery of macromolecular systems,

including polymer-drug conjugates. Characteristics 1-6 lead to enhanced permeability of the

tumoral vascular wall for polymers. Whereas the dimensions of small molecules permit them

to diffuse through the endothelial cell layer of both healthy and malignant vascular tissue,

macromolecules only diffuse through the more leaky and fenestrated tumoral vascular wall

(permeation). This results in a more selective delivery of antitumor drugs into their desired

site of action, sparing more non-tumoral tissue from exposure.

5

The lack of lymphatic vessels leads to an impaired overall lymphatic clearance, allowing

retention of polymers in the interstitial space and thus intratumoral accumulation (retention).

The intratumoral drug concentration is 10 to 100 fold bigger compared to an equivalent

conventional dose of the drug. (7) (14) (15) (16)

Figure 1.2.: The EPR-effect in tumoral neovasculature. Preferential accumulation in tumor tissue results

from higher permeability of the tumoral vascular wall in comparison with healthy vascularity. (17)

Still, drug delivery based on the EPR effect is limited due to the fact that tumor vasculature

and tissue greatly vary from tumor to tumor and from patient to patient. Even within the same

tumor, local variations in permeability and retention occur. (7)

1.2.4. Endocytosis

After permeation across endothelial cells, polymer-drug conjugates diffuse through the

interstitium to eventually reach the tumor cells. Whereas small molecules enter cells by

diffusion, for macromolecules, including polymer-drug conjugates, the molecular weight is too

high to pass the cell membrane by diffusion.

6

The mechanism of entering the cell is through endocytosis. The polymer-drug conjugates

are captured in invaginations of the cell membrane that get internalised by pinching off

inwards. In this way, intracellular endosomes are formed in which the polymer-drug

conjugates are entrapped.

The pH in endosomes is approximately 6.0 to 6.5. Endosomes fuse with lysosomes and the

polymer-drug conjugate gets exposed to lysosomal enzymes. Studies have shown that the

most important proteolytic enzyme degrading the polyglutamic acid backbone is cathepsin B,

a lysosomal protease. Other enzymes may contribute as well, though to a lower extent. Some

studies also have shown an upregulated level of cathepsin B in tumor cells, favouring

degradation of the polymer-drug conjugate. (18) (19) (16)

Compared to endosomes, the intralysosomal pH is approximately 5.0 to 5.5. It is proposed

that for drugs that are conjugated with an ester bond, the bond hydrolyses nonenzymatically

due to the pH-shift. Other studies suggest that lysosomal esterase catalyses the hydrolysis of

the ester bond. Nonenzymatic and/or enzymatic hydrolysis lead(s) to drug release and thus

free intracellular drug. After diffusion past the endolysosomal membrane, drugs can

eventually reach the nucleus to carry out their effect (Figure 1.3.). (19) (16)

Figure 1.3.: Cellular uptake of polymer-drug conjugates by endocytosis. A) internalisation of the polymer-drug

conjugate into endosomes; B) fusion with lysosomes containing lysosomal enzymes; C) degradation of polymer

backbone by lysosomal enzymes and drug release due to pH-shift and/or esterase; D) diffusion of the free drug

towards the nucleus of the cell (16)

A B

C

D

7

PACLITAXEL

1.3.1. General

Taxol (Figure 1.4.), originally extracted from the stem bark of the Taxus brevifolia, was

firstly identified in the early 1960s by the National Cancer institute as an antineoplastic drug

with a demonstrated inhibiting effect on proliferation in many tumor models. (20) (21) Since

its commercial development by Bristol-Myers Squibb, it has been renamed to paclitaxel.

Today, it serves as a widely used chemotherapeutic agent for several types of cancers,

including ovarian cancer, breast cancer, prostate cancer and non-small-cell lung cancer. (22)

Figure 1.4.: Chemical structure of paclitaxel.

Paclitaxel is classified as a tubulin binding agent and acts as a ‘microtubule stabiliser’,

meaning that it disturbs microtubule dynamics through stabilisation. Paclitaxel has been

shown to bind a hydrophobic pocket in microtubular beta-tubulin subunits. By binding these

subunits, it promotes polymerisation of microtubules during the cell cycle. As a result, the

process of disassembly into tubulin dimers, necessary to proceed the cycle, is inhibited. The

cycle arrests during the transition from the G0 into the G1 phase and from the G2 into the M

phase, eventually leading to cell death by apoptosis. (Figure 1.5.) (23) (24) (25)

As with other chemotherapeutics, systemic administration of paclitaxel affects all rapidly

dividing cells in the body, in which there is no differentiation between tumorous and non-

tumorous dividing cells. (26) Affected healthy cells include cells in the bone marrow, hair

follicles and the gastrointestinal tract, giving rise to a vast number of side effects. Following

side effects are experienced by more than 10 percent of patients treated with paclitaxel:

impaired immune system, anaemia, more frequent bleedings and bruises, arthralgia, myalgia,

allergic reactions, mouth sores and ulcers, fatigue, alopecia, numbness and diarrhea.

8

Clinically, it leads to health risks and a high level of discomfort for the treated patient,

illustrating the need for strategies that target tumor tissue more selectively. (27)

Figure 1.5.: Schematic representation of paclitaxel mechanism. Paclitaxel stabilises microtubules, eventually

leading to cell cycle arrest during G0/G1 transition and G2/M transition. (28)

The main issue regarding formulation of paclitaxel are its physicochemical features.

Paclitaxel is highly hydrophobic and thus poorly soluble, causing difficulties in formulation in

aqueous solvents which is required for intravenous administration. In addition, its

hydrophobicity leads to a series of unfavourable pharmacokinetic features. It shows to have

intense plasma protein binding, a big distribution volume, big tissue distribution and a short

distribution phase. Moreover, the plasma half-life of paclitaxel is relatively low compared to

other drugs. As a result, the relative exposure of a tumor to a biologically relevant paclitaxel

concentration is very limited compared to systemic exposure, leading to a low therapeutic

index and a high toxicity of the compound. (29) (22)

9

1.3.2. Solvent based formulation

A mixture of Cremophor EL® and dehydrated ethanol, was developed around 1980 in order

to solubilise paclitaxel in aqueous solutions for intravenous administration. Today, the solvent

based formulation of paclitaxel (sb-paclitaxel) is still used as standard formulation for

paclitaxel, despite having serious disadvantages. (30) (20)

Cremophor EL (polyoxythyl 35 castor oil USP, macrogolglycerol ricinoleate Ph.Eur.),

currently commercialised as Kolliphor ELP TM by BASF, is a nonionic surfactant used as an

emulsifying agent for formulation of hydrophobic molecules in aqueous solvents. (31) A

standard sb-paclitaxel intravenous formulation usually consists of 1 mg/mL paclitaxel

solubilised in a 1:1, V/V mixture of Cremophor EL and dehydrated ethanol. (29)

Despite having a good solubilising ability, Cremophor EL greatly influences pharmacokinetic

properties and drug delivery of paclitaxel. It is proposed that entrapment of paclitaxel in large

polar Cremophor EL micelles in the blood has a fencing effect on paclitaxel. Paclitaxel

entrapped in micelles is hindered from interaction with endothelial receptors. In addition,

micelles are not able to diffuse through biological membranes. The resulting impaired

transport from the vascular space into the interstitial space eventually leads to limited tumor

penetration and thus a decrease of tumoral exposure to the drug. Consequently, the

therapeutic effect of a dose of paclitaxel is reduced. Micellar entrapment also leads to

decreased metabolism and billairy excretion, causing a longer systemic exposure. As a result,

the systemic toxicity of paclitaxel is increased as well. In conclusion, the therapeutic index of

paclitaxel remains relatively low. (20) (32) (33) (34)

Several studies report hypersensitivity and peripheral neuropathies associated with

Cremophor EL and the amount used for solubilising paclitaxel is remarkably higher compared

to what is needed for other hydrophobic molecules. Therefore, the intravenous

administration of paclitaxel in this formulation clinically requires premedication with

corticosteroids and antihistamines, in order to decrease the severity of these side effects.

Nevertheless, among 41 to 44% of the patients, minor side effects such as flushing and rash

still occur and 1.5 to 3% even show potentially life threatening reactions (29) (35) (20).

Both ethanol and Cremophor EL interact with di-(2-ethylhexyl) phthalate (DHEP) which is

used as plasticiser in materials with polyvinylchloride (PVC). Because DEHP is hydrophobic, it

gets attracted by Cremophor EL which results in leaching of DEHP in the administered solution.

10

Some infusion bags and administration sets used for routine clinical administration consist

of PVC. Therefore, patients are exposed to substantial amounts of DEHP when treated with

sb-paclitaxel in this type of material. DEHP is hepatotoxic, carcinogen, teratogen and mutagen,

so chronic exposure to this plasticiser may cause considerable health risks. As a result,

alternative materials such as glass or polyolefin containers and nitro-glycerine tubings are

required for administration of sb-paclitaxel. (36) (37) (29)

1.3.3. Nanoparticle albumin bound-paclitaxel

The previously described shortcomings of the conventional formulation of paclitaxel

illustrated the importance of developing more efficient and biologically less active formulation

strategies. This led to the development of nanoparticle albumin bound paclitaxel

(nab-paclitaxel), commercialised as Abraxane ®. Nab-paclitaxel is a nanomedicine consisting

of paclitaxel bound to albumin. It is prepared by high pressure homogenisation of paclitaxel

and serum albumin, resulting in a colloidal suspension of conjugated nanoparticles with a

mean diameter of approximately 130 nm. The concentration of albumin in the formulation is

3 to 4%, making it similar to the concentration of serum albumin in human blood. The most

important feature is that Abraxane ® formulates paclitaxel without of use Cremophor EL,

making it hold some major advantages compared to the solvent based formulation. (34) (38)

(33)

Serum albumin acts as an endogenous carrier of hydrophobic compounds through the

blood such as vitamins and hormones, by binding them non-covalently. This allows transport

of hydrophobic components through the body and release at the cell surface. (32) Thus,

complexation of paclitaxel with albumin in a pharmaceutical formulation overcomes

previously described issues related to hydrophobicity of paclitaxel, without the use of

Cremophor EL. (20)

Taking these features in consideration, nab-paclitaxel holds significant advantages

compared to the rather classic solvent based formulation. Since the formulation is free of

Cremophor EL, there is no longer side effects due to pharmacological activity and leaching.

Premedication is no longer required for clinical paclitaxel administration. (34)

It is proposed that for nab-paclitaxel, enhanced penetration into tumor tissue is the result

of two possible mechanisms, the EPR effect and transcytosis through endothelial cells. They

both result in transport from the vascular space into the interstitial space. (33)

11

Transcytosis results from the interaction between albumin and albondin receptors

together with intracellular caveolin-1 of endothelial cells. These two proteins act as regulators

of the transcytosis mechanism, resulting in the transport of intravascular constituents into the

interstitial space. The albondin-receptor is found at the cell membrane of these vascular

endothelial cells. It is a glycoprotein with the size of 60 kDa (gp60) that is proposed to be a

docking site for albumin in blood vessels. Interaction between albumin and this receptor leads

to activation of the intracellular protein caveolin-1, which in turn stimulates invagination of

the membrane. During this process, both protein-bound and free compounds in the plasma

are trapped in so-called ‘caveolae’, which are the intracellular vesicles that result from this

invagination. The caveolae migrate towards the interstitial space, followed by release of the

entrapped compounds. (Figure 1.6.) In this way, nab-paclitaxel deals with the previously

described Cremophor EL fencing issues, in which paclitaxel is prevented from good tumor

penetration due to entrapment in large polar Cremophor EL micelles. (32) (34) (39) (40)

Figure 1.6.: Albumin receptor-mediated uptake of intravascular constituents and transcytosis across the

vascular endothelium. A) Albumin receptor (gp60) binds albumin, resulting in induction of caveolin-1; B)

caveolin-1 induces membrane internalisation, entrapping free and protein-bound plasma molecules; C)

formation of caveolae, leading to transcytosis and extravascular release of the caveolae content. (32)

Besides having affinity for the albondin-receptor, albumin also has been shown bind

secreted protein acid rich in cysteine (SPARC), also named osteonectine, which is a protein

that holds sequence homology with a glycoprotein 60. It is known that both caveolin-1 and

SPARC are overexpressed in several types of malignant cells, so paclitaxel in association with

albumin leads to a good tumoral accumulation in these cases. (34) (39)

12

PACLITAXEL-POLYGLUTAMIC ACID CONJUGATES

1.4.1. Polyglutamic acid

Polyglutamic acid (Figure 1.7.) (PGA) is a synthetic, non-toxic polypeptide. Structurally, it is

a homopolymer consisting of naturally occurring glutamic acid subunits, making the polymer

biodegradable. Its side chain carboxylic acid groups allow for conjugation of a vast amount of

compounds by esterification and moreover, free carboxylic acid groups make it negatively

charged at neutral pH. (7) (41)

Figure 1.7.: Chemical structure of the sodium salt form of polyglutamic acid

(n=number of monomer repeating units).

1.4.2. Paclitaxel-poliglumex

Paclitaxel poliglumex (CT-2013, OpaxioTM, previously called XytotaxTM) is currently

developed by Cell Therapeutics Inc. as a novel polymer-drug conjugate of PTX. In current FDA

clinical studies, OpaxioTM is in Phase 3 for first line treatment of ovarian cancer. (41)

Figure 1.8.: Chemical structure of paclitaxel-poliglumex. The ester bond between the γ-carboxylic acid side

chain of PGA and the 2’hydroxyl group of PTX, which is the tubulin binding site, makes the conjugate

pharmacologically inactive. The active site becomes available when PTX is released from the polymer.

2’

13

Paclitaxel poliglumex (Figure 1.8.) is a polymer-drug conjugate in which paclitaxel is

conjugated to α-poly (L) glutamic acid (PGA) as polymeric backbone (PGA-PTX). Conjugation is

obtained by esterification of the γ-carboxylic acid side chain of PGA to the 2’hydroxyl group of

paclitaxel. This results in PGA with approximately one paclitaxel bound per each 10.4 amino

acid subunits of the polymer, or 37% of the molecular weight (approximately 80 000 kDa).

Because of covalent linkage, paclitaxel poliglumex is classified as a new chemical entity.

However, the therapeutic effect is carried out by paclitaxel after intracellular dissociation from

PGA. (20) (42) (16)

By conjugating paclitaxel to PGA, solubility is enhanced without the use of toxic solvents

such as Cremophor EL. This also overcomes some of paclitaxel’s unfavourable

pharmacokinetic properties. Conjugation with PGA leads to a lower distribution volume and a

prolongation of the distribution phase compared to non-conjugated paclitaxel. Furthermore,

the elimination phase is prolonged as well. Another important feature is that paclitaxel which

is bonded to the polymer is pharmacologically inactive. In this way, systemic exposure to

paclitaxel is limited, since it does not circulate as an active drug in the bloodstream. After more

selective penetration into tumorous tissue due to the EPR effect, the drug is then released

once it is taken up in cells by endocytosis. In this way, any paclitaxel activity is ideally carried

out intracellularly in tumorous tissue. However, it should be taken in consideration that all

described pharmacokinetic enhancements only occur ideally when the PGA-PTX polymer is

well designed. (29) (16)

14

2. OBJECTIVES

Conjugation of paclitaxel with the biodegradable, watersoluble polymer polyglutamic acid

(PGA-PTX) is a promising new strategy to improve pharmacokinetics and drug delivery of

paclitaxel, without the use of cosolvents. As it is a new way to enhance PTX efficacy, it is of

interest to gain insight in its physicochemical and biological properties.

The principal objective of this project was the synthesis of PGA-PTX conjugates with

different PGA polymer lengths and different drug loadings, followed by characterisation by

different techniques to evaluate the influence of length and drug loading on their properties.

Secondary objectives were the study of polydispersity, solution conformation in PBS, stability

in plasma, drug release kinetics in PBS and cytotoxicity.

PGA of three different lengths (25, 50 and 100 glutamic acid subunits/polymer) was loaded

with two different amounts of PTX, aiming a total drug loading (TDL) of 5 and 10 mol%.

Synthesis was carried out until polymer-drug conjugates of each polymer length with a TDL

of < 5 mol% and > 5 mol% were obtained. Each synthesised polymer-drug conjugate was

purified by precipitation, liquid-liquid extraction and SEC.

Of each synthesised polymer-drug conjugate, the TDL was determined by 1H-NMR and

UV-VIS spectrophotometry. The conjugation yield in terms of TDL was evaluated. Impurities

were evaluated by 1H-NMR. The molecular weight and PDI were determined by GPC to study

the homogeneity of conjugation. The CAC and size of aggregates in PBS were studied by DLS.

The correlation between CAC and TDL and polymer length were studied. The correlation

between length and TDL and aggregate size was studied.

The correlation between TDL and length and stability in blood plasma was studied. The

release of PTX by ester bond hydrolysis over a period of 48 hours was measured by HPLC. Drug

release kinetics of different polymer-drug conjugates in PBS were studied. The release of PTX

by ester bond hydrolysis over a period of 15 days at 37 °C and 50 °C was measured by HPLC.

Correlation between temperature and drug release for each polymer-drug conjugate was

studied. Drug release of different polymer-drug conjugates at the same temperature were

compared. Correlation between drug release and TDL or length was studied. Cytotoxicity of

different polymer-drug conjugates was evaluated. A cell viability assay was performed.

15

3. MATERIALS AND METHODS

3.1. CHEMICALS

n-Butyl-polyglutamicacid (MW-range: 7600-8700 Da; average 50 subunits/polymer)

(PGA50), n-Butyl-polyglutamicacid (MW-range: 12600 – 13300 Da; average 100

subunits/polymer) (PGA100) and Npt-polyglutamicacid (MW-range: 3700-6100; average 25

subunits/polymer) (PGA25) were obtained from Polypeptide Therapeutic Solutions SL

(Valencia, Spain). Anhydrous N,N-Dimethylformamide (99,8% anhydrous) (anhydrous DMF)

was purchased from Scharlau (Sentmenat, Spain). N,N'-Diisopropylcarbodiimide (DIC) was

purchased from Iris Biotech GmbH (Marktredwitz, Germany). 1-Hydroxibenzotriazol

monohydrate (HOBt) was purchased from Iris Biotech GmbH (Marktredwitz, Germany). 4-

Dimethylaminopyridine (DMAP) was purchased from Sigma Aldrich (Saint Louis MO, United

States). Paclitaxel (PTX) was purchased from Xi'an Rongsheng Biotechnology (Shaanxi, China).

N,N-Diisopropylethylamine (DIEA) was purchased from Sigma-Aldrich (Saint Louis MO, United

States). N,N-Dimethylformamide (synthesis grade) was purchased from Scharlau (Sentmenat,

Spain). Ethyl acetate (analytical grade; ACS; Reag. Ph. Eur.) (ethylacetate) was purchased from

Scharlau (Sentmenat, Spain). Sodium bicarbonate (>99.5%) (sodium bicarbonate) was

purchased from Sigma-Aldrich (Saint Louis MO, United States). Dichloromethane was

purchased from VWR (Amsterdam, The Netherlands). Sephadex G-25 ® was purchased from

Sigma-Aldrich (Saint Louis MO, United States). Deionised Milli-Q ® ultrapure type 1 water

(Resistivity 18,2 MΩ.cm at 25 °C; TOC < 5 ppb) (deionised Milli-Q ® water) was purchased from

Merck Millipore (Billerica MA, United States). Liquid nitrogen (-196°C under atmospheric

pressure) was purchased from Air Liquide (Paris, France). Deuterium oxide (99.9 atom % D for

NMR analysis) was purchased from Sigma-Aldrich (Saint Louis MO, United States). Methanol

Emplura ® grade (methanol) was purchased from Merck Millipore (Billerica MA, United

States). Phosphate Buffered Saline with pH 7.4 was purchased from Sigma-Aldrich (Saint Louis

MO, United States). Diethyl ether (synthesis grade; stabilised with approx. 7 ppm BHT) (diethyl

ether) was purchased from Scharlau (Sentmenat, Spain). Acetonitrile (gradient 240 nm/far UV

HPLC grade) (acetonitrile) was purchased from Scharlau (Sentmenat, Spain). 4T1 ATCC® CRL-

2539™ cell line was purchased from ATCC (Manassas VA, United States). Dulbecco’s

Phosphate Buffered Saline (magnesium free; calcium free; sterile filtered) (DPBS) was

purchased from Sigma-Aldrich (Saint Louis MO, United States). 0.5% Trypsin/EDTA Gibco ®

(trypsin) was purchased from Life Technologies (Carlsbad CA, United States).

16

RPMI 1640 medium (containing L-Glutamine and 25 mM HEPES) completed with 10% fetal calf

serum (4T1 medium) was purchased from Life Technologies (Carlsbad CA, United States).

Trypan blue solution was purchased from Sigma-Aldrich (Saint Louis MO, United States). 3-

(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

(MTS) and phenazine methosulfate (PMS) were purchased from Promega (Madison WI, United

States).

3.2. PGA-PTX SYNTHESIS FOR 5% AND 10% TOTAL DRUG LOADING

Figure 3.1.: PGA-PTX synthesis.

Synthesis (Figure 3.1.) was carried out in a schlenk flask under continuous stirring and

nitrogen atmosphere. PGA (100 mg, 0.77 mmol monomer units) was dissolved in 5 mL

andhydrous DMF and added. DIC (1.1 equivalents compared to PTX) was added dropwise

using a micropipette. After 5 minutes, HOBt (1.1 equivalents compared to PTX) was added into

the reaction mixture. After 10 minutes PTX (5% or 10% mol percent compared to glutamyl

monomer subunits) was added, followed by direct addition of DMAP (catalytic amount,

previously dissolved in DMF at 1 mg/mL) using a micropipette. After total dissolution, DIEA

was added dropwise until pH 8 was reached.

After 24 hours under continuous stirring under nitrogen atmosphere, the presence of

unconjugated PTX was monitored by TLC using ethylacetate as mobile phase. A small amount

of reaction mixture was spotted and compared with a spot of PTX solution (Rf = 0.8 for free

PTX and Rf = 0 for polymer-drug conjugate). In case there was still free PTX in the reaction

mixture, the reaction was reactivated by adding 0.5 equivalents of DIC and HOBt. The pH was

adjusted to 8. The reaction was then stirred for another 24 hours under nitrogen atmosphere.

When the reaction was completed, DMF was concentrated and the crude product was dried

under high vaccuum.

DIC/HOBt DMAP

DIEA pH 8

17

3.3. PURIFICATTION

3.3.1. Work-up

After evaporation of DMF, the crude was redissolved in the least possible amount of DMF.

The crystals of urea formed during the reaction of DIC were filtered and the product was

precipitated by a dropwise addition into a 50 mL centrifuge tube of cold (4°C) diethylether for

precipitation. The mixture was centrifuged for 10 minutes at 4000 rpm and the supernatant

was then separated from the precipitate. The precipitate was redissolved in the least possible

amount of DMF and precipitated again until a white precipitate of polymer-drug conjugate

was obtained. The solid was dried under high vacuum to remove any trace of diethylether or

DMF.

After the first purification step, 2 mL of a 1M sodium bicarbonate solution was added to

the white precipitate. The mixture was vortexed and sonicated until complete dissolution. To

the dissolution of the salt form of the polymer (PGA-COONa, aqueous phase), 4 mL of

dichloromethane was added. The mixture was vortexed and centrifuged at 4000 rpm during

10 minutes to remove emulsification. The organic phase was separated from the aqueous

phase. The same extraction was repeated once.

3.3.2. Size Exclusion Chromatography

3.3.2.1. Size Exclusion Chromatography

Size Exclusion Chromatography (SEC) is a chromatographic method in which

macromolecules, including polymers, can be separated from small molecules or other

macromolecules of a different molecular weight. The stationary phase consist of a porous

matrix as spherical particles, which are usually physically very stable and chemically inert.

Separation results from the ability of small molecules to permeate into these pores, whereas

macromolecules do not have this ability due to their size. As a result, small molecules are

transported slower through the gel matrix, making them elute later and thus separately from

macromolecules. Given the fact that size is related to molecular weight of the polymers,

different polymers can be separated from each other based on their molecular weight. The

higher the molecular weight, the bigger the retention and thus the bigger the retention

volume. Separation is mostly described using volumes of retention (Figure 3.2.).

18

Figure 3.2.: Schematic representation of SEC with elution and molecular weight of polymers.

Elution is often described relatively by using the distribution coefficient K (43)

𝐾 =(𝑉𝑒 − 𝑉0)

(𝑉𝑡 − 𝑉0 )

In which:

Vt : Total column volume: the volume at which particles elute that are completely retained

V0 : Void volume: the volume at which particles elute that are not retained

Ve : Elution volume: the volume at which particles elute which are partially retained, the

volume of elution of a particular compound

3.3.2.2. Protocol

The salt form the polymer was loaded on a Sephadex G25 column which was previously

equilibrated with deionised Milli-Q ® water and SEC was performed. By dropwise elution of

the sample, the elute was collected in steps of 1,5 mL in 2 mL eppendorfs. The elution was

carried out until all polymer-drug conjugate was collected.

(3.1)

19

3.3.3. Freeze-drying for preservation

3.3.3.1. Freeze-drying

Freeze-drying or lyophilisation is a technique which is used to make a sample in solution

completely free of solvents and bound moisture. This results in the solid state of the sample,

avoiding the risk of solvent mediated or microbiological degradation. Also, the freeze-dried

product can be preserved without the need for freezing. It is a widely used technique for stable

and convenient preservation of samples over time. The solution can be reconstituted at any

time by dissolution of the solid sample in the original solvent.

The freeze-drying process consist of three stages, being the freezing stage, the primary

drying stage and secondary drying stage. First, the samples are frozen below the eutectic

temperature of the solution, normally using liquid nitrogen (freezing stage). The samples are

then added to a freeze-dry tube which is mounted to a lyophiliser. In order to start sublimation

of the solvent, heat is added and pressure is lowered in the environment of the sample. During

the process, temperature is carefully monitored so it is high enough for sublimation but below

the eutectic temperature so the rest of the sample remains in solid state.

The tube is connected to an ice collector, which is a chamber in which the pressure and

temperature is lower than in the freeze-dry tube. These conditions allow the gaseous phase

solvent molecules to migrate out of the freeze-dry tube as a result of the pressure difference

and then condense due to the decreased temperature (primary drying). As final stage in the

process, all bound moisture is eliminated from the sample by heating the centrifuge tube. The

temperature is usually above ambient temperature but with consideration of the stability of

the sample at high temperatures. The pressure is kept the same (secondary drying). (44)

3.3.3.2. Protocol

After purification, all eppendorfs were freeze-dried in liquid nitrogen and added to a

lyophilisation tube that was then mounted to a Benchtop K lyophiliser (VirTis SP Industries,

Warminster PA, United States). After a lyophilisation cycle of 24 hours (condenser

temperature: -80 °C; pressure: 150 µbar), all eppendorfs containing polymer were selected

and the polymer conjugate was collected for further characterisation

20

3.4. CHARACTERISATION

3.4.1. Total Drug Loading and impurities determination by 1H-NMR

3.4.1.1. Proton Nuclear Magnetic Resonance

Proton Nuclear Magnetic Resonance (1H-NMR) is a technique for identification and

quantification of organic molecules. The nucleus of an isotope has a characteristic spin (I),

which is 1/2 for 1H. When an external magnetic field is applied, the magnetic moment of the

spin can exist in two states, +1/2 and –1/2, in which +1/2 is the spin which is aligned with the

magnetic field and spin -1/2 is opposed. These two spin states have a different energy,

resulting in an energy difference between the two states. When the sample is irradiated with

radio frequency waves corresponding with the exact energy difference between the magnetic

moment of the two spin states, it results in excitation of the nucleus from the +1/2 into the -

1/2 state. After absorption of this energy, it is resonated as a wave.

Figure 3.3.: Schematic overview of a 1H-NMR apparatus.

In a 1H-NMR (Figure 3.3.), the radiofrequency wave is transmitted by a radio frequency

transmitter and two magnetic poles provide the magnetic fields. The field can be fluctuated

by the sweep coils which alter the strength of the field over a small interval. During this, the

radio frequency receiver detects the emission of absorbed energy as a radiofrequency wave

which is eventually amplified by the amplifier and digitalised by the control console and

recorder. Equivalently, the radiofrequency can be varied holding the magnetic field constant.

21

Every isotope has its specific isotope nucleus with its specific magnetic moment, but

depending on how and where a proton is bonded within an organic molecule, it will give a

resonance signal at a different magnetic field strength or radiofrequency, depending on which

one is varied. This is due to the fact that protons are surrounded by electrons when covalently

bounded. Electrons are charged, so they move whenever a magnetic field is applied, resulting

in a secondary magnetic field which shields the nucleus from the external field. For resonance

of the nuclei, the field must be increased compared to the necessary field when there would

be no electrons. In this way, every proton will give a resonance signal at a magnetic field

strength along the x-axis which is unique for the molecule in which it is covalently bounded,

making it useful for identification. The intensity of the resonance is shown at the y-axis and is

proportional to the molar concentration of the compound. (45)

3.4.1.2. Protocol

Of the lyophilised polymer-drug conjugate, 500 µL of a 5 mg/mL dissolution in

deuteriumoxide was prepared and was added to a 1H-NMR -tube. 1H-NMR (128 scans, 300

MHz) was performed using a 300 UltraShieldTM (Bruker, Billerica MA, United States).

3.4.2. Total Drug Loading determination by UV-VIS spectrophotometry

3.4.2.1. UV-VIS spectrophotometry

Ultraviolet-Visible (UV-VIS) spectrophotometry is a technique which is used to quantify the

molar concentration of an ultraviolet or visible light absorbing substance in solution. When

ultraviolet or visible light is beamed towards the solution, a part of its energy is absorbed and

used by the outer electrons of the substance to shift from ground to excited state. As a result,

the intensity of the light after passing the solution will be decreased. Given the fact that every

molecule has a unique electron composition, the wavelength of maximal absorbance is

molecule specific. For quantification, a monochromatic light beam with a specific wavelength

is illuminated towards the solution. Mostly, the wavelength of maximal absorbance is used.

The intensity of the light before (I0) and after (I) passing through the solution is measured. The

transmission (T) is defined as the ratio of both intensities:

𝑇 =𝐼

𝐼0

(3.2)

22

The absorbance (A) is defined as the negative logarithm of the transmission:

𝐴 = − 𝑙𝑜𝑔(𝑇) = − 𝑙𝑜𝑔 (𝐼

𝐼0)

The absorbance (A) is a function of the molar concentration of the absorbing substance (M)

as described by the Lambert-Beer law:

𝐴 = 𝜀𝜆𝑀𝑙

In which:

ελ: Molar extinction coefficient for light of the corresponding wavelength λ

d: Length of the cuvet (46)

3.4.2.2. Protocol

For calibration, paclitaxel was dissolved in methanol. For measurement of TDL, polymer-

drug conjugates were dissolved in deionised Milli-Q ® water. Absorbance was measured at

λ=240 nm with a quartz cuvette (10 mm light path) in a V-630 Jasco spectrophotometer (Jasco

Analytica, Madrid, Spain).

3.4.3. Molecular weight and polydispersity determination by GPC

3.4.3.1. Gel Permeation Chromatography

Gel Permeation Chromatography is a type of Size Exclusion Chromatography (cf. 3.3.2.)

which is automated and provided by a detector to detect elution of compounds to eventually

obtain a chromatogram. It has been shown that several parameters such as elution volume

are proportional to the molecular weight of the compound. By comparing elution to the

elution of a representative standard of a known molecular weight, an accurate estimation of

the molecular weight and polydispersity can be obtained. The polydispersity is a measure for

uniformity of molecular weight in a polymer sample. (43)

3.4.3.2. Protocol

Of each freeze-dried polymer-drug conjugate, an 8 mg/mL dissolution in PBS at pH 7.4 was

prepared and filtered. 100 µL of this dissolution was added to a GPC vial. GPC was performed

using a 2695 Separations module (Waters, Milford MA, United States).

(3.3)

(3.4)

23

For detection, a Triple Detector Viscotek Array 302 (Malvern Instruments ltd.,

Worcestershire, United Kingdom) was used. The used flow was 1 mL/min. The calibration was

carried out with a pullulan standard.

3.4.4. Determination of CAC and Hydrodynamic diameter by DLS

3.4.4.1. Dynamic Light Scattering

Dynamic Light Scattering (DLS) is a technique for measuring the size distribution of particles

that are suspended, including polymer-drug conjugates. The general principle is based on the

fact that spherical suspended particles scatter monochromatic light after illumination.

However, the intensity of the scattered light fluctuates because of the Brownian motion of

these particles. The Brownian motion is the random motion of suspended particles due to

collision with smaller particles, molecules or atoms in its direct surrounding. The bigger the

particle, the slower it will move through its surrounding.

Through analysis of the fluctuation of the intensity of the scattered light, the diffusion

coefficient (D) is calculated, which is a measure for the velocity of the Brownian motion of the

particle. Eventually, the hydrodynamic diameter (Rh) is then calculated using the Stokes-

Einstein equation:

𝐷 =𝑘𝐵𝑇

6𝜋𝜂𝑅ℎ

In which:

D: Diffusion coefficient

Rh: Hydrodynamic diameter

T: Absolute temperature

η: Viscosity of the medium

kB : Boltzmann constant

During measurement, a constant temperature is maintained in order to obtain a stable

signal for calculation of the diffusion coefficient. Any measurement comes with mentioning of

the exact temperature, because it also influences the viscosity of the medium. (47) (48)

(3.5)

24

3.4.4.2. Protocol

Every polymer drug-conjugate was dissolved at the studied concentration in PBS at pH 7.4.

Samples were filtered to avoid dust contamination and added to a disposable cuvette for DLS.

DLS was performed using the Zetasizer Nano ZS (Malvern Instruments, Worcestershire, United

Kingdom) with a 633 nm laser at measurement angle 173°. All measurements for CAC were

performed at 20 °C. Measurements for study of the influence of temperature on

hydrodynamic diameter were performed at the respective temperatures.

3.5. PLASMA STABILITY AND DRUG RELEASE KINETICS

3.5.1. Determination of drug release in plasma

3.5.1.1. High Pressure Liquid Chromatography

High Pressure Liquid Chromatography (HPLC) is a type of liquid chromatography in which

liquid samples are separated based on their affinity for a stationary phase. Samples are

injected into the device and are transported by a liquid (mobile phase) along a column packed

with porous particles (stationary phase). Reversed Phase HPLC is a type of HPLC in which the

stationary phase is hydrophobic and is mostly used for analysis of hydrophobic molecules. The

mobile phase consists of a polar solvent, usually water, and an organic modifier in order to

solubilise the hydrophobic molecules. Samples can be analysed by gradient elution, meaning

that the composition of the mobile phase changes over time. Also, isocratic elution is possible,

in which the composition of the mobile phase is constant.

Different molecules have a different affinity for a certain stationary phase due to different

chemical and/or physical interactions with the stationary phase. This eventually results in a

different time of elution and thus a separation of the mixture. Moreover, eluting molecules

can be identified and quantified by a detector. Eventually, the signal is outputted as a

chromatogram, allowing qualitative and quantitative evaluation of the sample. For analysis,

following parameters are used:

Rt: Retention time. This is a measure for at which point in time the molecule elutes. Being a

molecule specific parameter, it is used to identify the elution of a certain molecule (qualitative

analysis).

A: Peak area. The peak area of a peak at the given retention time is used as a measure for the

concentration of this molecule (quantitative analysis). (49)

25

3.5.1.2. Protocol

A 3 mg/mL solution of each polymer in pig blood plasma was prepared. Of each solution,

a 100 µL sample was taken and lyophilised directly after dissolution as a time = 0 sample. 3

light proofed eppendorfs with 300 µL solution were prepared and put in the Thermo-Shaker

TS-100 (Biosan, Riga, Latvia) under continuous mixing at 500 rpm at 37 °C. Samples were then

taken and directly freeze-dried at different points in time and samples were kept at -20°C for

preservation until their analysis.

After defreezing, 100 µL of acetonitrile was added to each sample in order to precipitate

proteins in the plasma. The sample was vortexed and centrifuged in the Centrifuge 5451R

(Eppendorf AG, Hamburg, Germany) at 14000 rpm during 15 minutes. 100 µL of the

supernatans was taken and added to a HPLC vial. The samples were analysed by HPLC. The

used pump was a 515 HPLC Pump (Waters, Milford MA, United States) controlled by a PC2

Pump Control Module (Waters, Milford MA, United States) and 717Plus Autosampler (Waters,

Milford MA, United States). A Photodiode Array Detector 2996 (Waters, Milford MA, United

States) was used for UV-detection at λ = 230 nm. The used stationary phase was a

LiCrospher 100 ® RP-18 column (Merck Millipore, Billerica MA, United States) (5 x 150 mm,

5 µm particle size). The samples were analysed by gradient elution with an acetonitrile/water

mixture as mobile phase (gradient: 35/65, V/V to 80/20, V/V; run time: 120 minutes; injection

volume: 20 µL, flow rate: 1 mL/min, temperature: 20°C)

3.5.2. Determination of drug release kinetics in PBS

A 3 mg/mL solution of each polymer in PBS at pH 7.4 was prepared and incubated at 37°C

and 50°C in light proofed eppendorfs of 100 µL each. At different points in time, a 100 µL

aliquot was taken and immediately freeze-dried to stop the degradation. Each point was

repeated three times. The samples were kept at -20 °C for preservation before HPLC analysis

(same method as plasma stability).

3.6. CELL VIABILITY ASSAY

3.6.1. Culturing and subculturing

The cell cultures were visually checked for confluence and microbiological and/or fungal

contamination using an inverted contrasting microscope DM IL (Leica, Wetzlar, Germany).

26

The medium was completely removed and the cells were washed twice with magnesium

and calcium free, sterile filtered Dulbecco’s Phosphate Buffered Saline (DPBS). After complete

removal of all DPBS, the cell surface was completely covered with 2 mL of trypsine. The culture

was incubated at 37°C at 5% CO2 atmosphere in a Hepaclass incubator (Brand GmbH,

Wertheim, Germany) for detachment of cells from the flask surface. Detachment was

microscopically evaluated by visually observing movement of the cells. Aggregation and shape

of the cells were evaluated visually. 8 mL of fresh 4T1 medium was added to obtain a 10 mL

2/8, V/V trypsine - 4T1 medium mixture in the flask. The mixture was resuspended until cells

were disaggregated. The cell suspension was transferred into a centrifuge tube and after

homogenisation, a sample was taken. The sample was diluted 2-fold by adding an equal

volume of trypan blue. After homogenisation, 10 µL was applied in the sink of a Bluebrand ®

counting chamber (Brand GmbH, Wertheim, Germany) and the total amount of cells of the 4

big cell rooms was counted. The cell concentration (cells/mL) was calculated with following

formula:

𝐶𝑒𝑙𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑐𝑒𝑙𝑙𝑠

𝑚𝐿) = 𝑚𝑒𝑎𝑛 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑝𝑒𝑟 𝑏𝑖𝑔 𝑐𝑒𝑙𝑙 𝑟𝑜𝑜𝑚 𝑥 2 𝑥 10 000

In which:

Mean amount of cells per big cell room: The amount of cells per big cell room was counted for

the 4 cell rooms in the counting grid. The mean was taken by dividing the total amount by

four.

2: The reciproque of the dilution factor after addition of trypan blue for counting

10 000: Factor for the volume of the sink

Out of the calculated concentration of cells in the flask, the mixture was diluted with 4T1

medium to obtain the desired cell concentration. The mixture was then added to a new flask

and incubated at 37°C at 5% CO2 atmosphere in a Hepaclass incubator (Brand GmbH,

Wertheim, Germany) until treatment. Subculturing was performed every two days with daily

follow up of growth and contamination. Subculturing as well as culturing, seeding, treatment

and MTS/PMS addition were performed in a certified Bio-II-A cabinet cell culture hood (Telstar

EN, Teressa, Spain). Every substance added to cells was previously brought to 37°C in a water

bath (VWR, Amsterdam, The Netherlands).

(3.6)

27

3.6.2. Seeding

A polystyrene, flat bottom 96 well plate (Corning Incorp., Corning NY, United States) was

used for seeding of cells. Every first and last well of each column was filled with 50 µL of

medium as a blank. All remaining wells in the column were filled with 50 µL of cell suspension

(seeding). Of columns that were not used, every well was filled with 50 µL of DPBS. The seeded

well plate was incubated at 37°C at 5% CO2 atmosphere in a Hepaclass incubator (Brand

GmbH, Wertheim, Germany) until treatment.

3.6.3. Treatment

24 hours after seeding, 50 µL of a solution of a cytotoxic drug was added in every well,

except for wells that were not used that contained DPBS. All wells of one column per well

plate were filled with 50 µL of medium as a control. Each time, one column per well plate was

filled with 50 µL of medium as a control. Cells were then incubated at 37°C at 5% CO2

atmosphere in a Hepaclass incubator (Brand GmbH, Wertheim, Germany) until MTS/PMS

assay.

3.6.4. MTS/PMS cell viability assay

3.6.4.1. MTS/PMS viability assay

When added to cells, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium (MTS) is reduced by reducing enzymes in cells into a formazan

product in the presence of phenazine methosulfate (PMS) as an electron coupling agent. The

concentration of formazan is measured colorimetrically and is a measure for the amount of

reducing enzymes and thus the amount of living cells in a well. The amount of living cells or

viability reflects the cytotoxicity of the evaluated drug. The viability is measured relatively to

blank wells containing only medium.

3.6.4.2. Protocol

The MTS/PMS cell viability assay was performed 48 hours after treatment. To each well,

10 µL of a 1/20, V/V PMS-MTS mixture was added. The cells were incubated at 37°C at 5% CO2

atmosphere in a Hepaclass incubator (Brand GmbH, Wertheim, Germany). 3 hours after

MTS/PMS addition, absorbance of each well was measured at λ=490 nm using a Victor 1420

microplate reader (Perkin Elmer/Wallac, Waltham MA, United States).

28

4. RESULTS AND DISCUSSION

4.1. PGA-PTX SYNTHESIS FOR 5% AND 10% TOTAL DRUG LOADING

Polyglutamic acid (PGA) of 3 different lengths (25, 50 and 100 subunits) was conjugated

with two different quantities of paclitaxel (PTX) corresponding to an aimed total drug loading

(aimed TDL) of 5 mol% and 10 mol% (cf. 3.2.). The TDL is the percentage of moles of conjugated

PTX to moles of glutamic acid subunits. The aimed total drug loading is the total drug loading

based on the quantity in moles of PTX to the quantity in moles of glutamic acid subunits as

put in the reaction, or the total drug loading in the ideal situation of 100% yield of conjugation.

Each polymer-drug conjugate batch resulting from one synthesis was referenced. Yield of the

reaction in terms of real total drug loading was evaluated by 1H-NMR and UV-VIS spectrometry

as a part of characterisation (cf. 4.2.). Synthesis of new batches was performed until for every

polymer length, a batch with TDL > 5% and < 5% was obtained (Table 4.1.).

Table 4.1.: Overview of synthesised polymer-drug conjugate batches.

Polymer reference

Polymer type Amount of

subunits Aimed TDL (mol%)

PM49 PGA100PTX10 100 10

PM70 PGA100PTX10 100 10

PM53 PGA100PTX5 100 5

PM63 PGA100PTX5 100 5

PM74 PGA50PTX10 50 10

PM85 PGA50PTX10 50 10

PM92 PGA50PTX5 50 5

PM65 PGA25PTX10 25 10

PM82 PGA25PTX10 25 10

4.2. CHARACTERISATION

4.2.1. Total Drug Loading and impurities determination by 1H-NMR

Of each synthesized polymer-drug conjugate, 1H-NMR of a 5 mg/mL dissolution in

deuterium oxide was performed (cf. 3.4.1.2.) after one purification. Peaks were identified for

protons of paclitaxel at δ = 7.25-8.25 ppm (m, arHPTX, 15) and protons of glutamic acid at

δ = 4.00-4.50 (m, CH-PGA, 100) and at δ = 1.75-2.5 ppm (m, CH2-PGA, 200). After normalising

the peak area at δ = 4.00-4.50 ppm to 100 for glutamic acid, the TDL was calculated as the

ratio of the peak area at δ = 7.25-8.25 ppm divided by 15 (15 aromatic protons for one PTX

molecule) to the peak area at δ = 4.00-4.50 ppm.

29

Impurities were identified at δ = 1.00-1.75 ppm and purification steps were repeated

whenever levels of impurity were too high (clear visible peaks at δ = 1.00-1.75 ppm)

(Figure 4.1.).

.

Figure 4.1.: 1H-NMR spectrum of PGA50PTX9 with peaks for PGA-PTX protons of interest assigned.

Polymer-drug conjugates of PGA25 were found to be more difficult to purify, with for every

polymer-drug conjugate of this polymer length three required repetitions of purification. For

polymer-drug conjugates of PGA50 and PGA100, repetitions of purification were not required.

A possible explanation is that PGA25 polymer-drug conjugates are smaller in size, which means

that the difference in SEC elution time with impurities is smaller so a higher fraction of

impurities is eluted along with the polymer-drug conjugate. In addition, smaller size makes it

less favourable to precipitate than PGA50 and PGA100.

arH PTX

-CH- PGA

-CH2- PGA

30

4.2.2. Total Drug Loading determination by UV-VIS spectrophotometry

4.2.2.1. Calibration

In order to quantify the concentration of loaded PTX in a polymer-drug conjugate sample,

a calibration curve for PTX was made. A dilution series of 1 to 100 µg/mL PTX in methanol was

prepared (cf. 3.4.2.2.) and of each solution, the absorbance was measured at λ = 240 nm. The

absorbance (A) was plotted as a function of concentration (C). After elimination of the

absorbance value for 60 µg/mL as an outlier, the calibration curve and equation as shown in

Figure 4.2. were obtained. Further quantification of PTX in polymer-drug conjugate samples

by UV-VIS spectrophotometry was performed with this calibration.

Figure 4.2.: Calibration curve and calibration equation for PTX in methanol.

4.2.2.2. Polymer-drug conjugate

Of each synthesised polymer-drug conjugate, a 100 µg/mL solution in deionised Milli-Q ®

water was prepared and absorbance was measured at λ = 240 nm (cf. 3.4.2.2.). The respective

concentration of PTX was calculated. The TDL was then calculated as the ratio of the molar

concentration of PTX to the molar concentration of glutamic acid subunits. Both values for

TDL as calculated with 1H-NMR and UV-VIS were compared to the aimed total drug loading as

shown in Figure 4.3..

A = 0,0198C + 0,0493R² = 0,9999

0

0,5

1

1,5

2

2,5

0 20 40 60 80 100

Ab

sorb

ance

Concentration PTX (µg/mL)

31

Figure 4.3.: Histogram illustrating values for TDL as obtained by 1H-NMR and UV-VIS

spectrophotometry compared to mean TDL of both values and aimed TDL.

Values for TDL as measured by 1H-NMR differ from values as measured by UV-VIS

spectrophotometry. The quantitative determination of the TDL by 1H-NMR is based on the

ratio between peak areas of PTX and PGA, equivalent with the concentration. As shown in

Figure 4.1., 1H-NMR spectra of PGA-PTX in deuterium oxide generally show peak broadening,

making integration and thus values for peak areas inaccurate. Peak broadening can be reduced

by performing 1H-NMR at a higher concentration. Future experiments can be performed with

more concentrated solutions to obtain a more accurate result. Accurate measurements by

UV-VIS spectrophotometry depend on accurate calibration of the spectrophotometer and

cuvette irregularities. For UV-VIS sample preparation, the accuracy of weighed polymer

depends on the weighed mass on the balance. When assumed that the error of the measured

mass on the theoretical mass is a constant value, the relative error to the weighed mass will

be higher for a smaller mass compared to a higher mass. For future experiments, more

accurate results can be obtained by weighing a larger mass to make the stock solutions for

calibration and solutions for measurement.

4,6

7,4

3,0

5,0 5,7

9,0

3,9

3,4 3,7

6,65,7

3,0

3,3

5,9 6,7

3,83,8

1,5

5,6

6,6

3,04,2

5,8

7,8

3,9

3,6 2,6

10 10

55

10 10

5

10 10

0,0

2,0

4,0

6,0

8,0

10,0

12,0

TDL

(mo

l%)

Polymer reference (polymer type)

TDL by NMR TDL by UV-VIS TDL mean Aimed TDL

32

Table 4.2.: Overview of conjugation yields for every synthesised polymer-drug conjugate batch.

For every synthesised polymer-drug conjugate, the conjugation yield was calculated as

shown in Table 4.2.. Results show that, for polymer-drug conjugates with PGA50 and PGA100,

for both aimed TDL’s (5 and 10 mol%), a conjugation yield of more than 50 % could be

obtained. Batches with TDL < 5 mol% and TDL > 5 mol% could be synthesised for polymer-drug

conjugates of PGA100 and PGA50. For polymer-drug conjugates of PGA25 with 10 mol% aimed

TDL, the maximum yield was 36%. Only a batch of TDL < 5 mol% could be synthesised. The

conjugation yield was found to be lower for PGA25 compared to PGA100 and PGA50.

4.2.3. Molecular weight and polydispersity determination

GPC was performed for the sodium salt form of the pure polymer before conjugation and

the resulting polymer-drug conjugate (Figure 4.4.). The evaluated polymers were

PGA100PTX4.2, PGA100PTX6.6, PGA50PTX7.8, PGA50PTX3.9 and PGA25PTX3.6. They were compared

with their polymer precursors at the protected stage analysed in DMF (PGA-COOBn) and with

the sodium salt form (PGA-COONa) analysed in PBS. The polydispersity index (PDI) for these

forms of the respective batches were provided by Polypeptide Therapeutic Solutions®.

For each polymer-drug conjugate evaluated, the PDI of an 8 mg/mL dissolution in 25mM

PBS at pH 7.4 was determined by GPC (cf. 3.4.3.2.). The PDI was compared to the PDI of the

sodium salt and the protected form of the pure polymer before conjugation with PTX. In this

way, it was evaluated to which extent the PDI was influenced by conjugation. Also, the

measured value for MW for every polymer-drug conjugate was compared to the theoretical

MW by calculation (Table 4.3.).

Polymer reference

Amount of subunits

Aimed TDL (mol%)

TDL (mol%) Conjugation

Yield (%)

PM49 100 10 5.60 56.0

PM70 100 10 6.55 65.5

PM53 100 5 3.00 60.0

PM63 100 5 4.15 83.0

PM74 50 10 5.80 58.0

PM85 50 10 7.82 78.2

PM92 50 5 3.87 77.4

PM65 25 10 3.60 36.0

PM82 25 10 2.59 25.9

33

Figure 4.4.: GPC chromatogram of PGA50PTX3,9 and PGA50-COONa.

Table 4.3.: Overview of MW and PDI values for different polymer-drug conjugates

and their synthesis precursors.

Polymer-drug

conjugate

Before conjugation

Polymer-drug conjugate (PBS) Protected form (DMF)

Salt form (PBS)

Mw PDI Mw PDI Mw PDI theoretical Mw

PGA100PTX4.2 22446 1.06 15086 1.21 67789 1.55 18706

PGA100PTX6.6 22446 1.06 15086 1.21 33248 1.23 20789

PGA50PTX7.8 12600 1.15 7369 1.65 13293 1.25 10895

PGA50PTX3.9 12600 1.15 7369 1.65 29093 1.42 9203

PGA25PTX3.6 6112 1.14 11135 1.26 4516

2014-03-25_12;13;59_PM92_01.vdt: Refractive Index

59.51

126.11

192.71

259.31

325.91

392.51

Re

fra

ctiv

e In

de

x (

mV

)

Retention Volume (mL)

0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00 36.00 40.00

2014-03-25_12;55;08_PGA_50_COONa_01.vdt: Refractive Index

53.21

143.53

233.84

324.16

414.47

504.79

Re

fra

ctiv

e In

de

x (

mV

)

Retention Volume (mL)

0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00 36.00 40.00

PGA50PTX3.9

PGA50-COONa

34

Results show moderate changes in PDI for every polymer-drug conjugate, meaning that

conjugation of PTX was homogenous. Increase in polydispersity is due to a heterogeneous

conjugation and conjugation yield for the different polymer molecules in the reaction mixture.

The further the synthesis proceeds, the more reactions are carried out, leading to an

increasing non-uniformity of molecules. PDI is an important quality parameter as it reflects

the heterogeneity of a polymer-drug conjugate batch. A higher PDI will lead to a broad

distribution in terms of molecular weight and thus in terms of TDL and polymer length. Batches

that are too heterogeneous would have a low repeatability in terms of dose and polymer

length whenever a certain mass of the batch would be weighed for formulation as a

pharmaceutical product. This would cause a high variability in effect and toxicity for

formulated units coming from the same batch, which is undesired when aiming for good

quality batches. In this way, it is important to monitor the PDI over different steps in the

synthesis. The reaction conditions for steps in the process that cause an unacceptable PDI

increase can then be identified and further optimised.

The measured values for MW greatly differ from the theoretically calculated MW. Calibration

of the used GPC device was performed with the polysaccharide pullulan, whereas a PGA-PTX

polymer-drug conjugate is a polyanion. Polyanions show different interaction with the solvent

and the column, impacting retention and thus signal. In this way, a possible error in the

determination of the Mw and PDI can be expected. Further optimisation of the method,

especially for the standard, is necessary to obtain more accurate results. A standard with a

more similar structure to PGA, such as a polypeptide, can be used for future calibrations.

4.2.4. Critical Aggregation Concentration by DLS

The Critical Aggregation Concentration (CAC) of different polymer-drug conjugates was

determined to study the conformation in solution. Of polymers PGA100PTX6.6, PGA50PTX7.8 and

PGA25PTX3.6, a dilution series of 0.01 mg/mL to 5 mg/mL in PBS was prepared. The mean count

rate of each dilution was determined by DLS (cf. 3.4.4.2). The value for mean count rate based

on intensity was plotted as a function of the polymer-drug conjugate concentration

(logarithmic). The CAC is defined as the concentration at which multimolecular aggregates

start to form, which is measured as the concentration at which the mean count rate of

particles starts to increase. The CAC was determined graphically as the x-value corresponding

to the intersection of both graphs before and after mean count rate increase (Figure 4.5.).

35

Figure 4.5.: CAC curves for PGA50PTX7.8, PGA100PTX6.6 and PGA25PTX3.6.

0

50

100

150

200

250

300

0,01 0,1 1 10

Mea

n c

ou

nt

rate

(n

m)

Polymer-drug conjugate concentration (mg/mL)

PGA50PTX7.8

0

50

100

150

200

250

300

350

400

0,01 0,1 1 10

Me

an c

ou

nt

rate

(n

m)

Polymer-drug conjugate concentration (mg/mL)

PGA100PTX6.6

0

100

200

300

400

500

600

0,01 0,1 1 10

Mea

n c

ou

nt

rate

(n

m)

Polymer-drug conjugate concentration (mg/mL)

PGA25PTX3.6

36

Table 4.4.: PDI and hydrodynamic diameter values for different polymer-drug conjugates before and after CAC.

Results show that for each polymer-drug conjugate, an increase in mean count rate could

be observed and a CAC could be determined. Conformationally, this shows that PGA-PTX

polymer-drug conjugates form aggregates in PBS at a concentration higher than the CAC. For

each polymer-drug conjugate type, the CAC lies between 0.4 and 0.5 mg/mL, which implies

that there is no significant difference in CAC between polymers of different length or TDL.

4.2.5. Evaluation of aggregation and temperature influence by DLS

For polymer-drug conjugates PGA25PTX3.6, PGA50PTX7.8 and PGA100PTX6.6, out of results of

CAC measurements, a dissolution of every polymer drug-conjugate with a concentration of

0.1 mg/mL (d < CAC) and 2 mg/mL (d > CAC) in PBS was prepared. For each dissolution, the

hydrodynamic diameter (d) was measured at 20°C by DLS (cf. 3.4.4.2). In this way, the size of

unimolecular particles (d < CAC) and multimolecular aggregates (d > CAC) in PBS could be

studied. For each dissolution, the polydispersity index (PDI) was also measured (Table 4.4.).

Results show that for each tested polymer-drug conjugate, the hydrodynamic diameter

was significantly lower at a concentration below the CAC than measured at concentrations

higher than the CAC. This confirms that polymer drug conjugates of all tested polymer-drug

conjugates form aggregates at a concentration higher than the CAC. Also, the higher the drug

loading, the higher the measured size, so it can be concluded that TDL influences the size of

polymer-drug conjugate aggregates in PBS.

For polymer PGA50PTX5.8, the particle size of a 2 mg/mL dissolution in PBS was measured

at 20 °C, 37°C and 50°C (Table 4.5.).

Table 4.5.: PDI and hydrodynamic diameter values for PGA50PTX5.8 at different temperatures.

Polymer-drug conjugate

20°C 37°C 50°C

d (nm) PDI d (nm) PDI d (nm) PDI

PGA50PTX5.8 121 0.274 127.2 0.265 129.1 0.255

Polymer-drug conjugate

d (nm) < CAC d (nm) > CAC

d (nm) PDI d (nm) PDI

PGA25PTX3.6 2.7 0.421 134.2 0.311

PGA50PTX7.8 2.3 0.336 306.2 0.278

PGA100PTX6.6 7.8 0.254 253.4 0.308

37

Results show no significant difference in hydrodynamic diameter at different temperatures,

meaning that for PGA50PTX5.8, temperature does not influence the size of aggregates in

dissolution.

4.3. PLASMA STABILITY AND DRUG RELEASE KINETICS

4.3.1. Plasma stability assay

4.3.1.1. Calibration

In order to quantify the free PTX concentration, a dilution series of PTX of 1 to 200 µg/mL

in a 1/1, V/V acetonitrile-water mixture was prepared and peak areas for each concentration

were measured by HPLC. The peak area was plotted as a function of the concentration of free

PTX, resulting in the calibration curve and equation as shown in Figure 4.6.. All measurements

of free PTX for plasma stability and drug release kinetics studies were based on this calibration.

Figure 4.6.: Calibration curve for free PTX in acetonitrile-water 1/1, V/V measured by HPLC.

4.3.1.2. Plasma stability

In order to evaluate the stability of different polymer-drug conjugates in blood, the

degradation in terms of ester bond hydrolysis of polymers with different lengths and different

drug loadings in blood was studied. Polymers PGA25PTX2.6, PGA100PTX6.6, PGA50PTX7.8 and

PGA50PTX3.9 were dissolved at 3 mg/mL in pig blood plasma and incubated at 37 °C. Samples

were taken at time 0 and after 6, 24 and 48 hours of continuous incubation at 37°C. Analysis

was performed by HPLC by calculating the concentration of free, non-conjugated paclitaxel

out of the peak area observed at Rt = 10.7 minutes (cf. 3.5.1.2.). A degradation profile of every

polymer-drug conjugate was made and the different profiles were compared (Figure 4.7.).

Area = 40428C + 43410R² = 0.9963

0

2000000

4000000

6000000

8000000

10000000

0 50 100 150 200

Pea

k A

rea

Concentration (µg/mL)

38

Figure 4.7.: Degradation profile of different polymer-drug conjugates over 48 hours.

Results show no additional PTX release between 24 and 48 hours of incubation at 37°C in

blood, so no additional degradation. All degradation takes places within 24 hours of

dissolution in plasma. A possible explanation is that in blood plasma, most enzymes and

proteins have a low stability, with most of the proteins and enzymes that are degraded and

thus inactive within the first 24 hours. In this way, there is no additional PTX release due to

hydrolysing enzymes after 24 hours of incubation. Additionally, it is proposed that in the three

dimensional conformation in solution, some conjugated PTX is trapped in a hydrophilic PGA

shell and some at the periphery of this shell. This makes that PGA-PTX ester bonds at the

periphery have a bigger contact surface with surrounding water molecules and enzymes than

the ones in the centre of the conformation. Ester bonds with a big contact surface are thus

more ‘accessible’ to water and enzymes and will be hydrolysed within the first 24 hours. Ester

bonds at the centre will remain stable for periods of time greater than 48 hours. In this way,

there is no observed difference in PTX release between 24 and 48 hours, with the accessible

bonds already hydrolysed and the protected bonds still stable after 48 hours.

0

2

4

6

8

10

12

14

16

18

20

0 6 24 48

% P

TX r

elea

sed

(m

/m)

Time (hours)

PGA100PTX6.6 PGA25PTX2.6 PGA50PTX7.8 PGA50PTX3.9

39

Results also show that for PGA25PTX2.6, a drug release of 12% to 14% could be observed,

whereas for other conjugates PGA100PTX6.6, PGA50PTX7.8 and PGA50PTX3.9, drug release was

below 8%. This could be explained by the fact that less subunits form a smaller hydrophilic

shell in the three dimensional conformation in solution. In this way, ester bonds have a larger

contact surface with surrounding molecules and are thus more ‘accessible’ to enzymes and

water molecules due to less protection by the PGA shell, leading to a higher degradation.

4.3.2. Drug release kinetics in PBS

A drug release kinetics study was performed to evaluate PTX release by ester bond

hydrolysis in PBS and to study if a different release occurs at 50 °C occurs in comparison with

37°C. The difference in hydrolysis rate was compared for the different temperatures.

Of polymers PGA100PTX5.6, PGA100PTX6.6, PGA50PTX5.8 and PGA50PTX3.9, a 3 mg/mL solution

of in PBS at pH 7.4 was made (cf. 3.5.2.). These solutions were incubated at 37°C and 50°C and

samples were taken after respectively 1, 2, 3, 4, 7, 10 and 15 days of continuous incubation at

the respective temperatures. The free, non-conjugated paclitaxel concentration was

determined by HPLC by calculating the concentration out of the peak area at Rt = 10.7 minutes.

A degradation profile was made for comparison. The percentage of released PTX to the total

loaded PTX over time illustrates the hydrolysis rate of the ester bond (Figure 4.8.).

Figure 4.8.: Drug release kinetics of different polymer-drug conjugates at 37°C and 50°C over 15 days.

50°C

37°C

0

5

10

15

20

25

0 5 10 15

%P

TX r

elea

sed

(m

/m)

Time (days)

PGA50PTX3.9

PGA50PTX5.8

PGA100PTX6.6

PGA100PTX5.6

40

(4.1)

Results show no difference between polymers of different TDL or a different polymer

length in terms of drug release at a constant temperature. Drug release was significantly

higher for all conjugate types at 50°C in comparison with drug release at 37°C. Increased

temperature led to increased drug release, whereas polymer length or drug loading did not

influence release kinetics for this experiment.

The observed higher hydrolysis rate at 50 °C compared to at 37 °C can be explained by the

dependence of reaction rate on temperature, in accordance to the Arrhenius’ equation:

𝑘 = 𝐴𝑒−𝐸𝐴𝑅𝑇

In which:

k: chemical reaction rate constant

A: frequency factor

EA: activation energy

R: gass constant

T: absolute temperature

Increased temperature leads to a higher chemical reaction rate constant and thus a higher

hydrolysis rate. (50) A higher drug release at increased temperature is of interest in studies of

the use of PGA-PTX conjugates in conditions of hyperthermia.

However, results of this experiment do not suffice in order to evaluate drug release in vitro,

as it only studies the influence of temperature on drug release. In PBS, the hydrolysis is lower

than it would be in the presence of endogenous occurring enzymes and in vivo conditions.

Cathepsin B has been shown to cleave peptide bonds between glutamyl subunits of PGA in

PGA-PTX conjugates. Identified metabolites are diglutamyl-PTX and monoglutamyl-PTX

fragments. (51) The proposed PGA-PTX conformation in aqueous solvents is a central

hydrophobic PTX core with a hydrophilic PGA shell. After cleavage by cathepsin B, the bound

can be considered to become more ‘accessible’ for hydrolysis, due to the fact that the

degradation products are loose fragments of PTX conjugated to one or two glutamyl subunits.

The conformation is lost and ester bonds have a bigger contact surface with surrounding water

molecules and enzymes. This results in a higher collision frequency and thus a higher

probability of reaction, compared to conditions in which cathepsin B is not present.

41

A second important enzyme which occurs in vivo is esterase, which catalyses hydrolysis of

ester bonds. As a result, the in vivo hydrolysis rate is probably higher, making further in vivo

studies or in vitro studies in the presence of cathepsin B and esterase necessary.

Hydrolysis of ester bonds is also influenced by the pH, so further tests at different pH are

necessary. It is important to study the difference in drug release between endosomes (pH 6.0-

6.5) and lysosomes (pH 5.0-5.5). These two cell components fuse after endocytosis of PGA-

PTX, thus the influence of this pH-shift should be studied in further experiments, as it is desired

that PTX is released intracellularly.

4.4. CELL VIABILITY ASSAY

For determination of cytotoxicity of different polymer-drug conjugates, a cell viability assay

was performed (cf. 3.6.). Plates were seeded with a 4T1 cell suspension corresponding to a

concentration of 1000 cells per well. For polymers PGA100PTX6.6, PGA50PTX7.8 and PGA50PTX3.9,

a dilution series in 4T1 medium was made. For every dilution of the series, the concentration

of the polymer-drug conjugate was in this way that the concentration of loaded paclitaxel was

respectively 0.1 nM, 0.5 nM, 1nM, 5 nM, 10 nM, 50 nM and 100 nM. Polymer-drug conjugates

of different length and TDL could then be compared mutually for delivery of an equal mass of

PTX. 24 hours after seeding, every column of one plate was treated with a different

concentration of the same polymer-drug conjugate. 48 hours after treatment, an MTS/PMS

assay was performed and absorbance of every well at λ=490 nm was measured.

Of every repetition of the same treatment in one column, the mean was taken as a value for

absorbance for one treatment. The standard deviation was also determined. The cell viability

of one column of the plate treated with a certain concentration of polymer-drug conjugate, is

the percentage of absorbance decrease in comparison with a blank column that was not

treated but seeded with an equal amount of cells. This represents the percentage of cells that

was killed by the treatment.

42

Figure 4.9.: Cell viability of a 4T1 cell line when treated with different polymer-drug conjugates.

As a parameter for overall cytotoxicity, it was the goal to determine the IC50. The IC50 is the

concentration of a certain drug to carry out 50% of the studied effect. As it was the goal to

determine the toxicity of the drug, the IC50 would be the concentration of polymer-drug

conjugate to kill 50% of all seeded cells per well, so the concentration at 50% cell viability.

Results show that for an equivalent PTX concentration of 0.1 nM, a viability value of more

than 100% was obtained for PGA50PTX7.8 and PGA50PTX3.9. This indicates data are incoherent

and no conclusions could be drawn from this experiment. However, other studies showed that

the IC50 of PGA-PTX conjugates could be determined with coherent data in similar conditions.

(22) Values for viability are calculated as a percentage of a blank that should normally contain

an equal amount of cells per well before treatment. A possible explanation is that during

seeding, not all wells were homogenously seeded, meaning that cell concentration could have

been lower in the blank wells than in the wells for treatment. When a percentage is calculated,

an unrealistic higher amount of cells after treatment with a low concentration is then possible

to be observed in other wells. As cells were seeded starting from a suspension in a centrifuge

tube, sedimentation of cells in the tube could lead to a heterogeneous distribution of cells in

the suspension while seeding. Future experiments can be performed in which the

homogeneity of the suspension is frequently checked and restored by shaking and tilting the

suspension regularly before seeding.

0

20

40

60

80

100

120

140

160

180

200

0,1 1 10 100

Cel

l via

bili

ty (

%)

Equivalent paclitaxel concentration (nM)

PGA100PTX6.6

PGA50PTX7.8

PGA50PTX3.9

43

5. CONCLUSIONS

PGA-PTX conjugates of three different lengths (25, 50, 100 subunits/polymer) and two

different quantities of PTX were synthesised. For PGA100 and PGA50 conjugates, a conjugation

yield of more than 50% could be obtained and batches with TDL < 5 mol% and > 5 mol% could

be synthesised. For PGA25 the maximum obtained yield was 36% for 10 mol% aimed TDL. Only

a batch with TDL < 5 mol% could be synthesized. Yield was lower for PGA25 polymer-drug

conjugates. Polymer-drug conjugates of PGA25 were more difficult to purify than PGA100 and

PGA50 conjugates. Values for TDL after measurement with UV-VIS and 1H-NMR differed.

The PDI and molecular weight for PGA100PTX4.2, PGA100PTX6.6, PGA50PTX7.8, PGA50PTX3.9 and

PGA25PTX3.6 showed moderate changes compared to the PDI of the sodium salt form and the

protected form of PGA before conjugation. The conjugation of PTX was homogenous for all

studied polymer-drug conjugates. Total drug loading and polymer length did not influence

homogeneity of conjugation. Measured molecular weight values greatly differed from the

theoretical molecular weight.

For PGA50PTX7.8, PGA100PTX6.6 and PGA25PTX3.6, a CAC could be observed and calculated for

every polymer-drug conjugate. Every polymer-drug conjugate formed aggregates in solution

in PBS. Polymer length and TDL did not influence CAC. All polymer-drug conjugates showed

increase in size at a concentration higher than the CAC. All studied polymer-drug conjugates

formed aggregates in solution whenever the CAC was exceeded. Temperature did not

influence the size of PGA50PTX5.8 aggregates in solution in PBS. TDL influenced the size of

polymer-drug conjugate aggregates in PBS.

For PGA25PTX2.6, PGA100PTX6.6, PGA50PTX7.8 and PGA50PTX3.9 there was no additional

paclitaxel release in plasma between 24 hours and 48 hours of incubation at 37°C. For every

studied conjugate, degradation over 48 hours took place within the first 24 hours after

dissolution in plasma. Polymer-drug conjugates of PGA25 were less stable in blood than

polymer-drug conjugates of PGA50 and PGA100. Polymer length influenced plasma stability. TDL

did not influence plasma stability.

44

For polymer-drug conjugates PGA100PTX5.6, PGA100PTX6.6, PGA50PTX5.8 and PGA50PTX3.9, drug

release in PBS over a period of 15 days was higher for all polymer-drug conjugates at 50 °C

compared to 37 °C. Temperature influenced drug release kinetics for all studied polymer-drug

conjugates in PBS. TDL and polymer length did not influence drug release kinetics in PBS at

constant temperature.

Generally, it can be concluded that polymer length of polymer-drug conjugates influenced

conjugation yield, purification and stability in blood plasma. TDL influenced the size of

polymer-drug conjugate aggregates in PBS. Polymer length and TDL did not influence

homogeneity of conjugation, CAC in PBS and drug release kinetics in PBS.

45

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