The Synthesis And Properties Of Poly(PEGMA)-biomolecule Conjugates Via RAFT Polymerization

reversible addition/fragmentation chain transfer polymerization (RAFT) is one method of the living radical polymerisation (LRP). Polymerization is suitable for use with many dfferent monomers and does not require highly rigorous removal of oxygen and other impurities. RAFT polymerization has successfully synthesized a wide range of polymers with controlled molecular weight and low polydispersities (between1.05and1.4for many monomers). In addition, RAFT polymerization can also be used for a variety of radical polymerization mode:solution, emulsion and suspension polymerization. Dithioester derivatives SC-(Z) in the RAFT polymerization, usually joined to the S-R as a chain transfer agent. Sleep intermediates can self-cleavage, the release of the new active radical R·from the corresponding sulfur atoms, combined with the monomer to form a growth chain, addition, or fracture rate than the chain growth rate is much faster, dithioester derivatives rapid transfer between active radicals and dormant radicals, so that the molecular weight distribution to reduce, so that aggregation reflects the controlled/"living" characteristics.The RAFT process allows the synthesis of polymers with specific macromolecular architectures such as block, gradient, statistical, comb, brush, star, hyperbranched, and network copolymers. These properties make RAFT useful in many types of polymer synthesis. In recent years, the application of RAFT polymerization method design and synthesis of new polymer materials, such as gene/drug delivery materials, biological materials, biological materials, biological molecules-polymer complexes, organic inorganic hybrid materials, nanoparticles, and so on. made great progressProteins have been used as therapeutics and received rapid development recently with the discovery of novel pharmaceutical proteins and better understanding of their mechanism in vivo. However, there are several limitations for the direct use of protein-based medicines. For example, proteins in vivo are vulnerable to renal excretion or digestion by the proteolytic system, negating their therapeutic effect. Linking water-soluble polymers on the protein surface can protect proteins from recognition by the immune system and hence increase the protein circulation time. Poly(ethylene glycol)(PEG), a neutral, nontoxic, biocompatible and nonimmunogenic polymer, has been covalently linked on protein surfaces (PEGylation) since initial research in the1970s, and the PEGylated proteins have been clinically proven to be less toxic and have clearly prolonged plasma circulation time, which are both critical factors in reducing the frequency of administration. Consequently, immunological side effects are reduced. At the same time, attachment of PEG by covalent bonds to pharmaceutical proteins also improves the proteins' solubility, stability and minimizes the proteolytic degradation, i.e. enhancing the efficiency and safety of the proteins therapy. Thus, the PEGylation technology has been developed rapidly and has made commercial success in the pharmaceutical market. However, despite the obvious benefits of PEGylation, namely improved bioavailability and reduced immunogenicity in vivo, there are still some inherent limitations of conventional PEGylation technology, such as, the onerous synthesis and purification procedure used to modify the chain end of PEG, the side-effect of the PEG degradation in vivo and the dramatical drop of biological activity of protein-PEG conjugates. Thus, new PEGylation methods have been invented to enhance the bioactivity of the PEGylated proteins. One of these is the site-specific PEGylation approach, resulting in the retention of higher bioactivities than simpler unselective PEGylation methods. However, site-specific PEGylation requires the availability of amino acid residues, such as free cysteine groups, on the protein surface at certain locations. As this is extremely rare in native proteins, genetic engineering has been invoked to introduce specific amino acids into the protein sequence for attachment purposes, which is a costly and inconvenient approach. Thus simple, high effective alternative methods are promising for practical pharmaceutical application. It has been proven that the polymer structure affects the conjugation reaction and subsequent protein bioactivity significantly. Previous studies compared bioconjugates made from different structural PEGs (linear or branched with similar molecular weights), and demonstrated that the branched PEG masked the protein surface more effectively, providing better protection against enzymatic degradation and reduction of immunogenicity. As the promising next generation PEGylation agents, brush structural PEGs (poly(poly(ethylene glycol) methyl ether acrylate)s (poly(PEGMA))s with bio-reactive chain end groups are expected to have similar properties to branched PEGs.Poly(ethylene glycol)(PEG) hydrogels are water-swellable, non-toxic, non-immunogenic, and already approved by the US Food and Drug Administration for various clinical uses. Consequently, PEG hydrogels have attracted extensive interest in biomedical science in areas such as the controlled release of biomolecules, wound healing, drug delivery, and as scaffolds for regenerative medicine. PEG is often called a 'stealth material' as it is relatively inert in physiological media. PEG has also been used successfully to render surface resistance to biomolecule adsorption. Poly(poly(ethylene glycol) acrylate)s ((polyPEG-A)s) have also been synthesized to generate bioconjugates either using in situ polymerization or postpolymerization modification via living radical polymerization. Hydrogels that biodegrade can be advantageous for bio-applications] as they can be degraded in vivo into small polymer fragments suitable for excretion from the body. PEG hydrogels have previously been synthesized using different cross-linking methods (e.g. photo-or thermal-initiation), involving a range of cross-linking agents. However, as expected it is very difficult to avoid the residual cross-linking agent during the gelating process. The residual cross-linking agents can then compromise the biocompatibility of the hydrogels making them unacceptable for biomedical or pharmaceutical applications. Therefore the generation of hydrogels using biocompatible cross-linking agents is desirable. The common groups used to confer biodegradability on synthetic polymers are ester and acetal linkages and disulfide bonds. Disulfide bonds biodegrade in the presence of glutathione (the most abundant intracellular thiol (0.2-10mM) in most mammalian and many prokaryotic cells). Disulfide linkages in proteins are essential in the presentation of tertiary structure and are formed between thiol groups of cysteine residues.Besides of the biotin and avidin system, the cyclodextrin-polymer hybrid materials have attracted a lot of interest, as cyclodextrin can be utilized as a biodegradable building block, as either a carrier (e.g., for hydrophobic drugs) or as a multifunctional unit for attachment to other groups through its multiple hydroxyl groups. Several previous studies have used the formation of cyclodextrin inclusion complexes with hydrophobic molecules (such as cholesterol) in aqueous solution to build up supramolecular structures and to form cross-linked networks. In this present work, biodegradable, cyclodextrin-polymer structures have been constructed from star polymer architectures and inclusion complexes. As these structures are noncross-linked, they can be readily characterized by conventional methods.Base on the above discussion, we attempted to take the advantages of RAFT polymerization to form poly(PEGMS)s and then the polymers been conbined with biomolecules such as protein, biotin and chesteriol to form bioconjugates. The polymer-protein conjugates were analysed by its bioactivitis, and the others biomolecules were crosslinked to form biodegradable hydrogels.First of all, a series of well-defined thiazolidine-2-thione-functionalized poly(PEGMA)s, with different structure and different molecular weight of were successfully prepared using the RAFT polymerization method. These poly(PEGMA)s were subsequently conjugated to protein (lysozyme) through amide linkages. The polymer structure and the molecular weight of the polymers were found to influence the number of polymer chains attached to the protein and hence reduced the protein bioactivity caused by conjugation. Higher molecular weight and longer side-chains are of some advantages to retain the polymer-protein conjugates' bioactivity. The mid-functionalized polymer-protein conjugations show much more protein bioactivity compare to their linear counterpart-protein conjugation indicating that the mid-functionalized poly(PEGMA)s exhibited a promising balance between the protein bioactivity (up to32.3%) and the protein protection by the covalent linkage with polymer.And then, We have demonstrated the preparation of biodegradable PEG hydrogels cross-linked with biotin-avidin association. The utilization of a protein-based, biotin-avidin cross-linking approach obviates the need for potentially toxic chemical cross-linking agents, therefore it may be particularly useful in biomedical or pharmaceutical applications. Finally, we have successfully synthesized a trifunctional RAFT agent and used it to synthesize three-armed polymers of polyPEG-A using a "core first" methodology. The radical polymerization of three-armed star polymers was found to be well-controlled by the RAFT mechanism. The trithiocarbonate groups at the termini of the star-arms were subjected to aminolysis generating PDS end-groups. And the cholesterol were then attached to the arm-termini at high yields. Finally, the cholesterol terminal polymers were utilized to form supramolecular structures vis complexation with cyclodextrin forming inclusion complexes. Thus, in this work we have successfully produced biodegradable polymer hydrogels that can easily be adapted to form cross-linked functional gels.