Thermoresponsive and biocompatible polymers attend nowadays great attention in research areas like DNA nanotechnology, continuous monitoring of analytes in biosensing assays and gene therapy as non viral vectors. Therefore, it is crucial to design and adjust the molecular architecture of such polymers to the corresponding requirements of the targeted application, e.g., by the specific structure of the monomers, by the choice of comonomer composition, and by the introduction of functional groups within the backbone or at the chain ends. The main focus of this thesis was directed towards the synthetic development of thermoresponsive polymer systems and study of their applicability as polymer-DNA-conjugates, as biosensor matrix, and as non-viral transfection vectors.
The design of polymer-DNA conjugates to achieve complex structure formation by hierarchical self-assembly with respect to responsive and reconfigurable matter combines the self-assembly pathways of block copolymers and DNA. Such conjugates allow the design of scaffolds with arbitrary and programmable shape, which can be applied e.g., as templates for the fabrication of nanoobjects with specific geometries. Thus, thermoresponsive polymer-DNA conjugates and their resulting superstructures are intensely studied in various research disciplines to understand their self-assembly mechanism. In the context of the collaboration with Dr. Emmanuel Stiakakis at the Forschungszentrum Jülich, synthetic routes based on cationic ring-opening polymerization (CROP) of 2-alkyl-2-oxazolines were developed in this thesis to obtain well-defined and thermoresponsive poly(2-oxazoline)s as co- and terpolymers carrying azide end groups. These functional end groups were introduced via 3-azidopropyl tosylate as initiator and sodium azide as termination agent. Furthermore, thermoresponsive NiPAm-based polymers were synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization, in which an azide-functionalized chain transfer agent was employed. A two-step post-functionalization strategy was developed, which includes the transformation of the respective azide group into an amino group by a Staudinger reduction and subsequent coupling of dibenzylcyclooctyne (DBCO)-groups via the N-hydroxysuccinimide (NHS) active ester. It was found that this efficient two-step post-functionalization strategy could be exploited for both, the poly(2 oxazoline) and the poly(NIPAm) derivatives carrying azide moieties. Efficient coupling of the azide- and DBCO-functionalized polymers with the respective azide-modified DNA derivative was demonstrated by gel electrophoresis experiments by the Stiakakis group in Jülich. These experiments showed also that the poly(2 oxazoline)s react more efficiently with the DNA than the poly(NIPAm) derivatives.
The application of hetero-telechelic poly(2 oxazoline)s as flexible linkers in surface plasmon resonance (SPR) spectroscopy-based sensors are considered for the design of different biosensing assay architectures that allow continuous monitoring of small analytes. This research topic was investigated in collaboration with Dr. Jakub Dostalek at AIT in Vienna. For this purpose, well-defined, thermoresponsive and hetero-telechelic poly(2-oxazoline) copolymers were prepared by CROP, which carried an azide end group on one side for coupling to a fluorescent dye. The other chain end was modified with either an amine- or a (protected) thiol-end group for coupling to the sensor surface. Successful coupling of the thermoresponsive copolymers to the sensor surface could be demonstrated after deshielding the amine- or thiol-end groups, followed by conjugation of the fluorescent dye Alexa Fluor 647 to the azide groups. In order to mimic affinity interactions, temperature-modulated surface plasmon-enhanced fluorescence spectroscopy (SPFS) experiments were conducted with these polymer layers in the labs of the Dostalek group. A promising sensor design was identified, in which the fluorescence intensity could be reversibly switched upon modulation of the temperature. However, in all performed SPFS measurements photobleaching was observed as undesired side effect.
In another research focus, a novel polymer architecture based on poly(2-oxazoline)s bearing protected thiol functionalities was investigated, which can be selectively liberated by irradiation with UV light. Whereas free thiol groups often suffer from oxidation or other spontaneous reactions that degrade polymer performance, this strategy with masked thiol groups offers the possibility of photodeprotection on demand with spatio-temporal control while maintaining the polymer integrity. In order to gain access to thiol-containing poly(2-oxazoline)s as gel precursors, a novel oxazoline monomer 2-{2-[(2-nitrobenzyl)thio]ethyl}-4,5-dihydrooxazole (NbMEtOxa) carrying a 2 nitrobenzyl-shielded thiol group was synthesized and copolymerized with 2-ethyl-2-oxazoline (EtOxa) in varying ratios. Moreover, the concept of tandem network formation was exemplarily demonstrated by using the photoinitiator 2 hydroxy-2-methylpropiophenone (HMPP) and pentaerythritol tetraacrylate (PETA), a commercially available, tetrafunctional vinyl crosslinker. The crosslinking experiments indicate that a minimal ratio of ~10% NbMEtOxa repeat units in the polymer backbone is necessary for network formation and in-situ gelation in N,N dimethylformamide.
Novel non-viral polymer-based vectors in gene therapy require several attributes like biocompatibility, the ability for strong DNA complexation, and an appropriate molar mass range. Further, the balance between hydrophobic moieties and charge influences their performance. The ability for complexation and the hydrophobicity of such carriers can be adjusted e.g., by varying the spacer lengths of the side chains carrying a positive charge or the number of amino groups at the side chain ends. The size of such a carrier molecule can be tailored with controlled polymerization techniques. Therefore, a series of copolymers composed of the biocompatible (2-hydroxyethyl) acrylamide (HEAm) and one of four different masked amino-functionalized acrylamides with different side chain lengths were prepared by RAFT polymerization with a targeted ratio of 30% side chains containing amino group. After removal of the amino protecting groups, the copolymers were scrutinized in in vitro experiments with different cell lines and compared to polyethyleneimine (PEI) as commonly adopted reference. The cell experiments were performed in collaboration with Dr. Tony Le Gall in the laboratories at the Université de Bretagne Occidentale in Brest. These studies demonstrated that the specifically synthesized polymers were tolerated by all tested cell types even at high polymer/DNA mass ratios. DNA complexation assays corroborated the ability to bind DNA for all tested polymers, in which the polymers carrying amino groups that are connected through a C12-spacer to the polymer backbone showed the best results. Further, in cell transfection experiments, the copolymers with the amino groups bound to the backbone by a C6-spacer yielded the best gene transfection efficiency. These results suggested that complexation of DNA outside of the cells and its release once inside target cells was best achieved with the latter compound under the experimental conditions explored. However, the transfection efficiency was lower compared to the gold standard polyethyleneimine (PEI).