Smart polymer platforms for in vitro drug screening assays based on drug-loaded nanoparticles

Abstract

The present PhD thesis represents a contribution to the IndiTreat project which started on January 1st2011 supported by the Danish Council for Strategic Research. The main goal of the project is toimprove chemotherapeutic treatment of patients suffering from colorectal cancer (CRC). In this thesiswe investigate the use of polymers for drug screening assays. The aim of this study is the developmentof a polymer platform that enables to overcome some of the limitations that characterize the existingscreening methods, by requiring very small amounts of tissues and permitting a fast and low-costscreening of individual drugs as well as combined drugs. Human colorectal adenocarcinoma cell line(HT-29) has been selected as cell culture model because easy to handle and phenotypically stable. Theresponsiveness of HT-29 cells to the individual and combined drug regimens normally selected forcolorectal cancer therapy is finally evaluated using our technologies. Two main platforms areproposed: one is based on the use of poly(ethylene glycol) diacrylate (PEGDA) hydrogels for controlleddrug release, and the second one consists on the use of poly(3,4-(1-azidomethylene)-dioxythiophene)(PEDOT-N3) micro-electrodes for co-localization of drug-loaded nanoparticles (liposomes) and cancercells. PEGDA hydrogels are widely used in different fields including tissue engineering and in vivo drug delivery. A home-made setup for the fabrication of PEGDA hydrogels through visible-light photopolymerization is described in detail. The method is utilized to create gels in which various clinically relevant drugs are embedded within the polymer network. The release profile of these molecules is determined and the anti-proliferative effect of passively released drugs is evaluated on HT-29 cultures. Individual drugs and combinations of chemotherapeutics are embedded within PEGDA gels as free molecules or loaded into nanoparticles (liposomes). The release of molecule from liposomes is temporally controlled through heat treatment and stable nanoparticles embedded within polymer networks are achieved. With the developed technology, various hydrogel architectures and sizes are produced allowing the confinement of targeted molecules within defined volumes of gel. From our described findings, the PEGDA-based hydrogel platform can ideally be applied for the screening of different compounds and to explore in vitro, various pathological cell lines. In this thesis is presented also an alternative methodology for drug screening purposes, consisting on the fabrication of a micro- and nano-platform for drug-loaded liposomes immobilization and cell capture. PEDOT-N3 micro-electrodes are fabricated through printed dissolution that, contrarily to the traditional lithography processes, preserves the biochemical properties of the exposed substrates. Cyclic olefin copolymers (COC) supports are used for PEDOT-N3 deposition and in situ polymerization. Various chemical modifications to create a coating onto the support surface are also explored. A variety of highly hydrophilic poly(ethylene glycol) (PEG)-based molecules are photo-chemically grafted onto COC supports to form protein repellent surfaces. Post-fabrication covalent modifications of polymer micro-electrodes are investigated and the use of “click-chemistry reaction” permits to modify free available azide groups presented at the surface of micro-electrodes. By covalently attaching appropriate bi-functional molecules onto a thin PEDOT-N3 film, chemical reactive liposomes (~100nm in diameter) are immobilized onto electrodes.  In addition to these findings, an extensive optimization of electrodes fabrication, liposomes stability and chemical engineering of the support areas between the electrodes for cell capture purposes, is necessary. We find that it would be highly relevant for future studies to explore the mechanisms involved on co-localization of liposomes, that are triggered for drugs release, and specific cell lines.