Von funktionalen polymerbasierten Transfektionsagenzien und enzymatischen Antikörpermodifizierungen zur bioorthogonalen Konjugation
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Abstract
Within the research area of nanoparticle-based tumor immune therapy, the de-velopment of polymer-based transfection vectors for efficient delivery of nucleic acids offers great potential in anti-tumor vaccination. It seems very promising to activate the body’s own immune system with pDNA or mRNA vaccines to combat malignant cells of solid tumors or even metastasis. Therefore, a main topic in this thesis was the further development of poly-(L-lysine)-block-poly(N-(2-hydroxypropyl)methacrylate) block copolymers as transfection vectors for dendritic immune cells. The combination of ring-opening polymerization of N-E-(Boc)-L-lysine N-carboxyanhydride and RAFT polymerization (reversible addition-fragmentation chain transfer polymerization) of pentafluorophenyl methacrylate enabled the syn-thesis of p(Lys)-b-p(PFPMA) reactive ester precursor block copolymers. After several post-polymerization modification steps, multifunctional p(Lys)-b-p(HPMA) block copolymers were obtained. The complexation of negatively charged nucleic acids, such as pDNA or mRNA, was achieved by electrostatic interactions with the cationic poly(L-lysine) block. The most important advantage of using poly(HPMA) as suitable shielding block for the charged polyplex is the straightforward synthesis via the reactive ester approach. Besides the conversion with 2-hydroxypropylamine the introduction of many different functionalities like fluorescent dyes by sequential aminolysis of the poly(PFPMA) block shows great benefit. In order to enhance transfec-tion efficiencies, the incorporation of an acid-labile hydrazone moiety within the p(Lys)-b-p(HPMA) block copolymer was evaluated. The hydrolytic cleavage of the hydrazone in the acidic endosomal compartments should facilitate the polyplex’ endosomal escape and consequently contribute to improved transfection efficien-cies. Furthermore, the poly(L-lysine) block of p(Lys)-b-p(HPMA) block copolymers was statistically modified with thiol-reactive 2-(pyridyldithio)-propionate moieties for reversible core cross-linking. In general, a disulfide cross-linking is known to improve the extracellular stability of the polyplex and in this work it was used for the additional incorporation of endosomolytic LAH4-L1 peptide as dithiol cross-linker.
With regard to a cell type-specific uptake of p(Lys)-b-p(HPMA)-based nanovac-cines by dendritic immune cells, the attachment of DEC205 antibodies as targeting ligands onto the polyplex’ surface was investigated. For this purpose, strain-promoted alkyne-azide-cycloaddition was used as bioorthogonal conjugation strategy. The synthesis of two azide-functionalized block copolymers was per-formed by post-polymerization modification. The resulting p(Lys)-b-p(HPMA)-N3(stat) and p(Lys)-b-p(HPMA)-N3(end) differ in number and position of azide moieties and were both employed for polyplex formation. DBCO-functionalization of aDEC205 was conducted by a two-step enzymatic antibody modification approach, using bacterial transglutaminase (BTG). This enzymatic strategy enables the site-specific modification at two distinct glutamine side chains within the IgG antibody and was applied for different BTG-substrates in bioorthogonal antibody conjugations.