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GHENT UNIVERSITY CENTRO DE INVESTIGACIÓN PRÍNCIPE FELIPE FACULTY OF PHARMACEUTICAL SCIENCES ADVANCED THERAPIES RESEARCH PROGRAMME Department of Pharmaceutics Laboratory of Polymer Therapeutics Laboratory of General Biochemistry and Physical Pharmacy Academic year 2013-2014 SYNTHESIS, PHYSICOCHEMICAL CHARACTERISATION AND CYTOXICITY EVALUATION OF DIFFFERENT 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 GHENT UNIVERSITY CENTRO DE INVESTIGACIÓN PRÍNCIPE FELIPE FACULTY OF PHARMACEUTICAL SCIENCES ADVANCED THERAPIES RESEARCH PROGRAMME Department of Pharmaceutics Laboratory of Polymer Therapeutics Laboratory of General Biochemistry and Physical Pharmacy Academic year 2013-2014 SYNTHESIS, PHYSICOCHEMICAL CHARACTERISATION AND CYTOXICITY EVALUATION OF DIFFFERENT 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 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 Author Prof. Dr. Stefaan De Smedt 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 Auteur Prof. Dr. Stefaan De Smedt 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 polymerdrug conjugates of PGA25, a conjugation yield higher than 50% could not be reached. PGA 25 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 polymerdrug 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 PGA 25 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 Geneesmiddelenlading had invloed op de grootte van aggregaten in oplossing. in plasma. 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 DEHP DIC DIEA DLS DMAP DMF DPBS EPR ESF FDA GPC HOBT HPLC IC50 MTS NMR PBS PDI PGA PMS PTX PVC SEC SPARC TDL USP UV-VIS VEGF Critical Aggregation Concentration di-(2-ethylhexyl) phtalate N,N'-diisopropylcarbodiimide diisopropylethylamine Dynamic Light Scattering 4’-dimethylaminopyridine N,N-dimethylformamide Dulbecco's Phosphate Buffered Saline Enhanced Permeation and Retention European Science Foundation Food and Drug Administration Gel Permeation Chromatography 1-hydroxybenzotriazol High Pressure Liquid Chromatography Half Maximal Inhibitory Concentration 3-(4,5-dimethylthiazol-2-yl)-5-(3-carbomethoxyphenyl)-2-(4sulfophenyl)-2h tetrazolium Nuclear Magnetic Resonance Phosphate Buffered Saline Polydispersity Index polyglutamic acid phenazine methosulfate paclitaxel polyvinyl chloride Size Exclusion Chromatography Secreted Protein Acid Rich in Cysteine Total Drug Loading United States Pharmacopeia Ultraviolet-Visible light Vascular Endothelial Growth Factor 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) 1 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) 2 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) 3 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 nonpathological 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. 4 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. 5 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) B A C D 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) 6 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 G 0 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 nontumorous 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. 7 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) 8 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. 9 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) 10 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) 11 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) 2’ 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. 12 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) 13 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. 14 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). 4Dimethylaminopyridine (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® CRL2539™ 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). 15 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 DIC/HOBt DMAP DIEA pH 8 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. 16 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.). 17 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 ) (3.1) 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. 18 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 19 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. 20 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= 21 𝐼 𝐼0 (3.2) The absorbance (A) is defined as the negative logarithm of the transmission: 𝐼 𝐴 = − 𝑙𝑜𝑔(𝑇) = − 𝑙𝑜𝑔 ( ) 𝐼0 (3.3) The absorbance (A) is a function of the molar concentration of the absorbing substance (M) as described by the Lambert-Beer law: 𝐴 = 𝜀𝜆 𝑀𝑙 (3.4) 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, polymerdrug 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). 22 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 StokesEinstein equation: 𝐷= 𝑘𝐵 𝑇 6𝜋𝜂𝑅ℎ (3.5) 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) 23 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) 24 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). 25 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 ) = 𝑚𝑒𝑎𝑛 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑝𝑒𝑟 𝑏𝑖𝑔 𝑐𝑒𝑙𝑙 𝑟𝑜𝑜𝑚 𝑥 2 𝑥 10 000 𝑚𝐿 (3.6) 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). 26 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-(4sulfophenyl)-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). 27 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 PM70 PM53 PM63 PM74 PM85 PM92 PM65 PM82 PGA100PTX10 PGA100PTX10 PGA100PTX5 PGA100PTX5 PGA50PTX10 PGA50PTX10 PGA50PTX5 PGA25PTX10 PGA25PTX10 100 100 100 100 50 50 50 25 25 10 10 5 5 10 10 5 10 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. 28 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.). . -CH2- PGA -CH- PGA arH PTX 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. 29 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. 2,5 A = 0,0198C + 0,0493 R² = 0,9999 Absorbance 2 1,5 1 0,5 0 0 20 40 60 80 100 Concentration PTX (µg/mL) 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.. 30 12,0 10 TDL (mol%) 10,0 8,0 6,0 4,0 6,6 4,6 5,6 10 10 7,4 6,6 5,7 5 3,0 3,0 3,0 5,0 5 4,2 3,3 5,9 5,7 5,8 10 10 9,0 7,8 6,7 5 3,9 3,9 3,8 3,8 3,4 3,6 10 3,7 2,6 1,5 2,0 0,0 Polymer reference (polymer type) TDL by NMR TDL by UV-VIS TDL mean Aimed TDL 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. 31 Table 4.2.: Overview of conjugation yields for every synthesised polymer-drug conjugate batch. 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 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.). 32 392.51 PGA50PTX3.9 Refractive Index (mV) 325.91 259.31 192.71 126.11 59.51 0.00 4.00 8.00 12.00 16.00 20.00 Retention Volume (mL) 2014-03-25_12;13;59_PM92_01.vdt: 24.00 28.00 32.00 36.00 40.00 Refractive Index 504.79 PGA50-COONa Refractive Index (mV) 414.47 324.16 233.84 143.53 53.21 0.00 4.00 8.00 12.00 16.00 20.00 Retention Volume (mL) 2014-03-25_12;55;08_PGA_50_COONa_01.vdt: 24.00 28.00 32.00 36.00 40.00 Refractive Index 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. Before conjugation Polymerdrug conjugate PGA100PTX4.2 PGA100PTX6.6 PGA50PTX7.8 PGA50PTX3.9 PGA25PTX3.6 Protected form (DMF) Salt form (PBS) Polymer-drug conjugate (PBS) Mw PDI Mw PDI Mw PDI theoretical Mw 22446 22446 12600 12600 6112 1.06 1.06 1.15 1.15 1.14 15086 15086 7369 7369 1.21 1.21 1.65 1.65 67789 33248 13293 29093 11135 1.55 1.23 1.25 1.42 1.26 18706 33 20789 10895 9203 4516 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.). 34 Mean count rate (nm) 300 PGA50PTX7.8 250 200 150 100 50 0 0,01 0,1 1 10 Polymer-drug conjugate concentration (mg/mL) Mean count rate (nm) 400 PGA100PTX6.6 350 300 250 200 150 100 50 0 0,01 0,1 1 10 Polymer-drug conjugate concentration (mg/mL) Mean count rate (nm) 600 PGA25PTX3.6 500 400 300 200 100 0 0,01 0,1 1 Polymer-drug conjugate concentration (mg/mL) Figure 4.5.: CAC curves for PGA50PTX7.8, PGA100PTX6.6 and PGA25PTX3.6. 35 10 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.). Table 4.4.: PDI and hydrodynamic diameter values for different polymer-drug conjugates before and after CAC. d (nm) < CAC d (nm) > CAC Polymer-drug conjugate 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 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. 20°C 37°C 50°C Polymer-drug conjugate d (nm) PDI d (nm) PDI d (nm) PDI PGA50PTX5.8 121 0.274 127.2 0.265 129.1 0.255 36 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. 10000000 Area = 40428C + 43410 R² = 0.9963 Peak Area 8000000 6000000 4000000 2000000 0 0 50 100 Concentration (µg/mL) 150 200 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.). 37 20 18 % PTX released (m/m) 16 14 12 10 8 6 4 2 0 0 6 24 48 PGA50PTX7.8 PGA50PTX3.9 Time (hours) PGA100PTX6.6 PGA25PTX2.6 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. 38 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 R t = 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.). 25 PGA50PTX3.9 %PTX released (m/m) PGA50PTX5.8 20 50°C PGA100PTX6.6 PGA100PTX5.6 15 10 5 37°C 0 0 5 10 15 Time (days) Figure 4.8.: Drug release kinetics of different polymer-drug conjugates at 37°C and 50°C over 15 days. 39 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: 𝐸𝐴 𝑘 = 𝐴𝑒 −𝑅𝑇 (4.1) 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. 40 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 6.06.5) and lysosomes (pH 5.0-5.5). These two cell components fuse after endocytosis of PGAPTX, 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. 41 200 180 PGA100PTX6.6 Cell viability (%) 160 PGA50PTX7.8 140 PGA50PTX3.9 120 100 80 60 40 20 0 0,1 1 10 100 Equivalent paclitaxel concentration (nM) 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 IC 50 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. 42 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. 43 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. 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