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Formulation of hydrophobic anticancer drugs into intelligent polymer nanoparticles

This PhD dissertation focused on the design of polymer-based carrier systems to enhance both the
solubility and the delivery of taxane anti-cancer drugs. PART I of the thesis comprises a general introduction to scope the need for advanced chemotherapy. Chapter 1 reports on the current treatment modalities for cancer applied in clinic. In particular, anti-cancer drug treatment is described along with the issues of conventional chemotherapy, to illustrate the need for safer and more efficacious anti-cancer drug delivery. Next, the rationale and assets of nanomedicines (i.e. passive and active targeting, bioresponsive drug release) are explained, outlining a window of opportunity for the materials chemistry field to come up with alternative formulation strategies for improved anti-cancer drug delivery. Chapter 2 provides a concise background on taxane drugs and their first
approved commercial formulations. Both the discovery and early development of paclitaxel (PTX) and docetaxel (DTX) are sketched. Next, an overview is given on the sourcing and manufacturing processes of taxanes. The biological mechanism of action is covered. Finally, the physicochemical features of Taxol and Taxotere are elucidated, along with their clinical translation and toxicity issues.
PART II covers the experimental work on the design of two polymeric nanocarrier systems for physical drug entrapment. Though PTX was used within the scope of this dissertation, note that both systems could also be exploited for physical encapsulation of other hydrophobic drugs. Chapter 3 describes the formulation of PTXloaded poly(glycerol sebacate) (PGS) nanoparticles. To obtain the latter, a straightforward formulation procedure was developed which involves co-dissolution of PGS and PTX in ethanol (EtOH) and subsequent solvent displacement in water (H2O). The biocompatibility of all excipients (i.e. PGS, EtOH and H2O) is a crucial asset for biomedical applications. Dynamic light scattering (DLS) confirmed PTX/PGS particles within the 200 nm size range
with narrow dispersity and high colloidal stability in physiological aqueous medium. Additionally, the critical aggregation concentration (CAC) of the nanoparticles was very low (i.e. < 1 Pg/mL), allowing substantial dilution without particle disintegration. Flow cytometry (FACS) and confocal microscopy confirmed the capability of PGS nanoparticles of delivering hydrophobic compounds into cancer cells in vitro. Finally, PTX/PGS nanoparticles showed equal in vitro biological performance compared to 2 commercial, advanced PTX formulations (i.e. Abraxane and Genexol-PM), whilst empty PGS nanoparticles confirmed the expected high cytocompatibility. These promising in vitro data propose key incentives for further in vivo evaluation. So far however, only a modest PTX concentration
(i.e. 0.5 mg/mL) could be obtained in the developed formulation. Future optimization would be required to further increase the drug loading capacity of the system, allowing to formulate sufficient doses of PTX into a smaller formulation volume. The latter would render the administration (i.e. intravenous, intraperitoneal) of PTX/PGS nanoparticles more feasible in clinical setting.
Chapter 4 reports on the systematic design of amphiphilic, acid-degradable, block copolymer
nanoparticles, synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. To obtain a bioresponsive, acid-sensitive hydrophobic block, a hydrophobic, ketal-functionalized acrylate monomer (i.e. (2,2-dimethyl-1,3-dioxolane-4-yl)methyl acrylate (DMDMA)) was copolymerized with a hydrophilic monomer (i.e.hydroxyethyl acrylate (HEA)). The DMDMA:HEA molar ratio was varied to obtain 5 different block copolymers with increasing amount of DMDMA incorporated in the hydrophobic block. Next, the influence of the hydrophobic block design on the physicochemical properties was extensively investigated. A DMDMA content higher than 11 mol %
results in self-assembly into nanoparticles in aqueous medium. All nanoparticles showed substantial colloidal stability in phosphate buffered saline (PBS), with sizes and CAC ranging from 23 to 338 nm and from 241 to 10 U+03BCg/mL, respectively, proportional to the block copolymer DMDMA content. Under acidic conditions, the nanoparticles decomposed into soluble unimers of which the decomposition rate was inversely proportional to the block copolymer DMDMA content. FACS and confocal microscopy confirmed in vitro the utility of the designed nanoparticles as carrier vehicle for the delivery of hydrophobic compounds into cancer cells. The block copolymers showed no intrinsic cytotoxicity. When loaded with PTX however, a significant decrease in cell viability was
observed comparable to that of Abraxane and Genexol-PM. The main objective of this study was to deliver a proofof-concept, demonstrating that RAFT polymerization is a valuable technique for designing well-defined, smart drug delivery carrier systems. However, this system cannot yet be reckoned suitable for further in vivo evaluation. Modifications in polymer design have to be considered to obtain nanoparticles with faster degradation kinetics at biologically more relevant pH-values (i.e. 5 U+2013 6.5), to achieve higher drug loading and to ensure maintenance of particle integrity upon substantial dilution in complex biological matrices (e.g. blood). Promising strategies to accomplish the latter include cross-linking strategies and chemically attaching the drug to the nanocarrier vehicle.
Chemical conjugation of drugs to a polymeric carrier is a highly acknowledged technique for designing
advanced anti-cancer drug formulations. An important advantage of chemical conjugation over physical encapsulation is the possibility to more thoroughly restrict premature burst release and hence reduce side-effects. PART III encompasses the second experimental section of the thesis, describing the design of taxane-polymer conjugates based on a grafting-from-drug RAFT approach. Chapter 5 describes the first-generation of ester-based PTX-polymer prodrug conjugates developed by this approach. A RAFT chain transfer agent (CTA) was regioselectively conjugated to the C2' hydroxyl group of PTX. The latter is crucial for the biological activity of PTX. This drug-functionalized RAFT CTA was subsequently exploited for polymerization of the hydrophilic monomer N,Ndimethylacrylamide (DMA), yielding well-defined, amphiphilic PTX-polymer prodrug conjugates with high drug loading (i.e. 20 wt % PTX) and water-solubility (i.e. t 30 mg/mL conjugate). When dispersed in PBS, stable micellar particles were detected by DLS, 27 nm in size and with narrow dispersity. The relatively high CAC of the nanoparticles (i.e. 102 U+03BCg/mL) implicates that the conjugate will most likely switch from micellar to water-soluble state upon aqueous dilution. Additional modification of the obtained conjugate was demonstrated by U+03C9-end postfunctionalization with a fluorescent tracer molecule. In vitro experiments showed that this conjugate is readily taken up into endosomes where native PTX is efficiently cleaved off and subsequently reaches its subcellular target (i.e. microtubules), as confirmed by a similar cytotoxicity profile compared to Abraxane and Genexol-PM
which are based on mere physical encapsulation. These results encourage further evaluation in vivo, especially because the high drug loading allows for the administration sufficient PTX doses. Selecting the right mouse model will be crucial. Conventional subcutaneous primary tumor models might not illustrate the full potential of this system, as the water-soluble, low molecular weight conjugates will most likely not exert significant passive targeting, mediated by the enhanced permeability and retention (EPR) effect. A clinically more relevant model (i.e. orthotopic/metastatic mouse model) might be more suitable to investigate whether, compared to nanoparticles, these small soluble conjugates can more efficiently penetrate tumor tissue, featured by poor vascularization
and/or by significantly less fenestrated endothelia. For this purpose, it will be vital to collaborate with research groups, disposing of the required expertise. Chapter 6 reports on second-generation PTX-polymer conjugates based on a similar approach as described in Chapter 5. To obtain conjugates with acid-sensitive release properties, the gained expertise on acetal/ketal
synthesis in Chapter 4 was further exploited. PTX was linked to a RAFT CTA either through a cyclic or through a linear acetal moiety. In contrast to the regioselective esterification of PTX reported in Chapter 5, direct acetalization of PTX can occur at 2 sites (i.e. either at the C2U+2019 or at the C7 hydroxyl group), yielding 2 regio-isomers of PTX-functionalized RAFT CTA. The isomers based on the cyclic acetal could be separated by silica gel chromatography, whilst the isomers based on the linear acetal could be used as regio-isomeric mixture for subsequent RAFT polymerization. The properties of the resulting PTX-polymer conjugates were in accordance to
their first-generation counterparts in terms of polymer definition, drug loading and water-solubility. Both the firstand second-generation conjugates did not exert burst release in PBS, whether or not supplemented with 10 % fetal bovine serum (FBS). At pH 4 and 5, significant release of PTX was observed for the linear acetal-based PTX-polymer conjugate, whilst the cyclic acetal-based and first-generation conjugates showed limited and no release, respectively. Viability was decreased in vitro to the same extent as for the first-generation conjugates, however higher concentrations were required (i.e. roughly by factor 102 and 103 for the linear and cyclic acetal-based conjugates, respectively). These data clearly indicate that the linear acetal-based conjugates are more acidsensitive than their cyclic acetal-based counterparts, and that the release of PTX from the first-generation esterbased conjugates is predominantly occurring through an enzymatically catalyzed process.
As both the first- and second-generation PTX-polymer conjugates most likely form soluble unimers upon aqueous dilution, long circulation kinetics and EPR-mediated passive targeting are not to be expected in vivo. On the other hand, swift renal clearance of small hydrophilic molecules can be a great tool to minimize side-effects by the fraction of the dose that has not reached its target site. To still obtain efficient accumulation of short half-life PTX-conjugates in tumor tissue (less susceptible to EPR effect), active targeting strategies need to be considered.
Chapter 7 comprises the design of DTX-polymer prodrug conjugates, equipped with an active targeting ligand. Using the same approach as described in Chapter 5, a DTX-functionalized RAFT CTA was synthesized and used to obtain highly defined DTX-polymer prodrug conjugates. Additionally, S,S-2-(3-(5-amino-1-carboxypentyl)-ureido)-pentanedioic acid (ACUPA) was synthesized. The latter possesses high binding affinity towards prostate specific
membrane antigen (PSMA), a surface receptor often overexpressed by malignant prostate cells. ACUPA was further modified with a maleimide moiety and subsequently used for post-modification of the DTX-polymer prodrug conjugates. Successful synthesis of DTX-polymer-ACUPA prodrug conjugates was confirmed by size exclusion chromatography (SEC) and nuclear magnetic resonance (NMR) spectroscopy. Fluorescently labeled DTX-polymerACUPA analogues were prepared by incorporating a small fraction of fluorescent monomer during the polymerization step. DTX-polymer prodrug conjugates, post-modified with a non-targeting moiety were prepared as control. FACS showed that the DTX-polymer-ACUPA prodrug conjugates interact to a higher extent in vitro with
PSMA-positive prostate cancer cells compared to the control. Complementary in vitro experiments (i.e. confocal microscopy, competitive binding assays, MTT) should further clarify whether DTX-polymer-ACUPA, compared to the control, can efficiently deliver higher doses of DTX to PSMA-positive prostate cancer cells by receptor-mediated endocytosis and if this translates into higher and more specific cytotoxicity. In summary, this PhD thesis has explored various design, synthesis and formulation strategies for obtaining advanced, passively or active targeted polymeric taxane formulations. Though solvent displacement is a very simple technique that enables formulation of nanoparticles with narrow size dispersity, achieving sufficient drug loading proved to be challenging. Throughout the experimental part of this thesis, RAFT has proven its value as highly controllable and chemically versatile polymerization technique in designing well-defined, bioresponsive
drug delivery systems. The grafting-from-drug approach in particular has shown great promise. This
straightforward synthesis strategy results in polymeric taxane prodrug conjugates with high drug loading and limited burst release in vitro. The successful post-modification with an active targeting ligand further highlights the opportunities of the approach. The small size of these soluble conjugates could allow for efficient penetration into (metastatic) tumor tissue that is less susceptible to passive targeting by EPR. This can be investigated in vivo, though by careful selection of an appropriate and clinically relevant mouse model. Future in vitro and in vivo experiments will clarify whether this approach enables active targeted drug delivery. Overall, this thesis encompasses valuable insights for future rational design of advanced polymeric anti-cancer drug delivery systems based on physical encapsulation and chemical conjugation.
Date:1 Jan 2013  →  31 Dec 2016
Keywords:anti-cancer drugs, advanced chemotherapy
Disciplines:Pharmacology, Other pharmaceutical sciences, Biomarker discovery and evaluation, Toxicology and toxinology, Pharmacognosy and phytochemistry, Drug discovery and development, Pharmacotherapy, Pharmaceutics, Medicinal products