Physicochemical and biochemical characterizations of Chitosan-g-poly(N-[3-(dimethylamino)propyl] methacrylamide) polymers synthesized with RAFT and EDC/NHS coupling reactions

Abstract

Since ancient times, diseases and their treatment methods have been and still are the subject of much research. Through interdisciplinary research, numerous diagnostic and therapeutic approaches have been established over time. Factors such as low bioavailability, inadequate dosage, toxic effects, and short half-life in conventional treatment methods have necessitated the design of novel application systems. Innovative treatment methods such as smart drug delivery systems and gene therapy have emerged with modern medicine. Polymers, which are frequently used in industrial fields, draw attention with the advantages they offer in biological applications such as drug/gene delivery systems. Polymeric systems designed for biomedical applications can exhibit high mechanical strength in systemic circulation and high transfection efficiency. On the other hand, they are sensitive to the target and contain functional groups suitable for modification, which is highly advantageous for biological applications. Polymers can create biocompatible, biodegradable, and highly bioavailable carrier systems. In addition, their availability for modification due to their functional groups allows them to be customized physicochemically and biochemically. Natural polymers are generally preferred when synthesizing polymeric carriers that enable the creation of target-sensitive systems. Although they provide high biocompatibility, biodegradability, and bioavailability in biological applications, natural polymers can cause problems such as poor mechanical strength and solubility when used alone. Such disadvantages of natural polymers limit their application areas, especially in biological studies. Natural polymers can be modified with synthetic polymers to overcome these limitations and improve their physicochemical/biochemical properties. In addition, polymeric systems intended for use in biological applications should not have hemolytic activity. In addition to the high blood compatibility of polymeric systems intended for use in intracellular studies, they must also be able to interact with and enter the cell, escape the endosomal cycle, and release the biological macromolecule they carry. Chitosan (Cs), a polysaccharide with low cytotoxicity, high biocompatibility, and biodegradability, is frequently used in biological applications. In addition, its structure is open to modifications due to the functional groups it contains, such as -NH2/-OH. Although chitosan is widely used in biological applications due to its advantages, it also has disadvantages, such as dissolving only in weak acidic solutions, poor mechanical strength, and low transfection efficiency. Modifying chitosan, which will be used for biological applications, is very popular to overcome these limitations. The graft copolymerization method, which enables modification via amine groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/ N-hydroxysuccinimide (NHS) coupling agents, is frequently used among the various chemical methods used to modify chitosan. Synthetic monomers derived from acrylate and acrylamide are generally preferred in the modification of chitosan. Such monomers increase the solubility of chitosan, improve cell interactions, and increase transfection efficiency, thus expanding its application areas. Besides, chitosan provides advantages in cell-targeted studies due to its cationic structure, shows a specific buffer capacity thanks to the protonable -NH2 groups it contains in its structure, and can contribute to endosomal escape. This thesis aimed to improve the physicochemical and biochemical properties of chitosan by using a synthetic polymer, poly(N-[3-(dimethylamino)propyl]methacrylamide) (PDMAPMAAm). Its monomer, DMAPMAAm, is widely used in biological applications due to its greater biocompatibility and lower cytotoxicity compared to its derivatives. It is essential to determine the branch lengths and molecular weights of both the main chains and the graft copolymers of the structures targeted for use in biological applications. Thus, 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPADB) was used as a raft agent to polymerize the DMAPMAAm monomer by reversible addition-fragmentation chain transfer (RAFT), and PDMAPMAAm macroCTA structures with carboxyl ends were obtained. A series of experiments was carried out to find the optimal conditions for synthesizing PDMAPMAAm macroCTAs. Factors such as the type of solvent used, initiator, raft agent, and pH were examined, and the most suitable conditions were selected according to the type of monomer and raft agent used. In addition, necessary arrangements have been made to solve the problem of whether the CPADB is hydrolyzed or not, to work most efficiently, and to manage controlled polymerization. The synthesized PDMAPMAAm macroCTA structures were characterized by UV-VIS spectrophotometry and 1H-NMR spectroscopy. Due to the selected raft agent, the maximum absorbance peak of the benzene ring at the end of the macroCTAs was used to determine the molecular weight and degree of polymerization. Targeted short-chain (20, 30, 40, 50 units) PDMAPMAAm macroCTAs were synthesized, and the results were found to be reproducible. The carboxyl ends of macroCTAs synthesized in desired chain lengths were activated with EDC/NHS coupling agents, and graft copolymerization was carried out by forming amide bonds with the amine groups of chitosan. Chitosan-graft-poly(N-[3-(dimethylamino)propyl]methacrylamide) graft copolymers (CsgD) were synthesized using chitosans of three different molecular weights degraded in the presence of KPS initiator and characterized by 1H-NMR spectrophotometry and GPC. Depending on the type of main chains and chitosan used, graft copolymers with different branch numbers and molecular weights were obtained. The suitability of the graft copolymers for intracellular studies was investigated. Thus, hemolytic activity and buffer capacity tests were performed. It was observed that the graft copolymers and the parent polymers, except the lowest molecular weight chitosan and the graft copolymers synthesized from it, had no hemolytic activity below specific concentrations. When the buffer capacities were examined, it was seen that the graft copolymers had a certain buffer capacity in the endosomal space, and their use in biological applications was evaluated. In addition, complexes with TPP molecules (CsgD/TPP) were formed to examine their interaction with biomolecules in their intracellular use, and solution/nanogel/aggregate boundaries were determined visually. Then, size, PDI, and zeta potentials were examined with DLS. All characterizations and tests of the graft copolymers, which are aimed at being a preliminary study in terms of carrier polymers to be designed for biological applications, have been completed. The results are thought to provide a wide range of sources in terms of synthesis conditions and intracellular studies for future studies.