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Application Of Hydrophilic Polymers In Gene Delivery, Protein Immobilization And Protein Crystallization

Posted on:2011-04-09Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y YaoFull Text:PDF
GTID:1101330332472716Subject:Biochemistry and Molecular Biology
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The development of Atom Transfer Radical Polymerization (ATRP) was incredible since its emergence in 1995. ATRP is one of the most important controlled/"living" polymerization techniques and enables preparation of different functional materials. In the biological field, ATRP has been proposed for the polymerization of polymer brushes to modify the surface of biological material, and of biomacromolecular vectors. Especially for polypeptide/protein medicines and gene therapy, the successful design of multifunctional vectors is mostly suitable for ATRP.In this dissertation we synthesized a series of hydrophilic polymers by ATRP. The effects of polymer structures on gene transfection efficiency, protein immobilization on silicon surface and lysozyme crystallization were evaluated.As the concept of gene therapy expanded, the choice of safer and more efficient vectors for delivery of genes is the key to successful gene therapy. Viral vectors are typically very efficient, but the main drawback related to viral vectors is the safety concerns. Synthetic gene-delivery vectors, although safer than recombinant viruses, generally do not possess the required efficacy. In recent years, a variety of effective polymers have been designed specifically for gene delivery, and much has been learned about their structure-function relationships. With the growing understanding of polymer gene-delivery mechanisms and continued efforts of creative polymer chemists, it is likely that polymer-based gene-delivery systems will become an important tool for human gene therapy.First we studied the influence of block sequence of block copolymers on the efficacy of polymer gene vectors. PEGMA, of good biocompatibility and DMAEMA with two amine groups were polymerized by ATRP for its feature of controlled/"living" and low polydispersity, to give the diblock copolymer PDMAEMA-b-poly(PEGMA) and triblock copolymer PDMAEMA-b-poly(PEGMA)-b-PDMAEMA with same molecular weights, same PDMAEMA content and different block sequences. The two block copolymers were used to induce DNA condensation, investigated by agarose gel electrophoresis, atomic force microscopy (AFM), dynamic light scattering (DLS), transfection of 293T cell line and MTT cytotoxicity assay. Significant differences on the assembling configuration and surface charge of polycation/DNA complexes were observed due to block sequences with same N/P ratios, and consequently different 293T cell transfection efficacy and cytotoxicity were obtained.Then the effects of amine group density of polymeric transfectants were evaluated. Polyamines PBAMAM, PBAMAM-b-poly(PEGMA) and PBAMAM-b-PDMAEMA with different amine group densities were synthesized by ATRP for the modification of monomers and studied by agarose gel electrophoresis, AFM and 293T cell transfection. As a result, optimal amine group density is of great importance for the properties of polymeric transfectants.A key focus in protein microarray analysis is the ability to immobilize proteins in their native conformation on substrate surfaces while preserving active sites for functional studies. Several approaches have been developed to immobilize proteins and other biomolecules onto substrate surfaces, using either covalent attachment or noncovalent affinity binding chemistries. In most cases, however, these modes of immobilization are nonspecific, causing the molecules to be randomly orientated on the sufaces. Polymer brushes can be densely prepared by ATRP on biomaterial surfaces, and introduce new properties to the surfaces. Covalent coupling for protein immobilization is an important strategy, and has many advantages:(1) compared with physical adsorption, covalent coupling provides more efficient binding to surfaces and stronger detection signals; (2) covalent coupling is often combined with the protection of the substrate to prevent nonspecific adsorption; (3) covalent coupling allows access to the immobilized protein molecules for small analytes such as ligands and larger analytes such as biomacromolecules or organelles.In the experiment, poly(PEGMA) brushes were synthesized by ATRP to modify the ends of each polymer chain with NHS groups, to give the final surfaces Si-poly(PEGMA)-NHS. After each modification step, the surfaces were characterized by X-ray photoelectron spectroscopy (XPS), AFM and SEM. We compared the ability for protein immobilization of three kinds of silicon surfaces encounter in the process of synthesis:Si-poly(PEGMA), Si-poly(PEGMA)-amine and Si-poly(PEGMA)-NHS. Si-poly(PEGMA) immobilizes proteins by physical adsorption and Si-poly(PEGMA)-amine immobilizes by electrostatic interaction. Si-poly(PEGMA)-NHS which immobilizing proteins by covalent coupling has the greatest performance. Although this modification process has some disadvantages, it provide a new way to synthesize covalent binding surfaces.As we know, protein is a significantly important part of bioactive matters and the targets that most of the medicaments acting on in medical, biology and chemistry fields. So it is becoming significantly important to determine the three dimension structures of proteins. To obtain protein crystals and through the X-ray diffraction is the main route toward structure determination of protein. Crystallization of protein molecules is a multiparameter controlled and complicated process including physical, chemical and biological factors. These parameters are concretely including temperature, time, properties of electrolytes, viscosity, ionic strength, super saturation, purity of proteins, symmetry and stability of protein structure, isoelectric point and so on. To acquire the suitable crystals all the parameters must be taken into account, and choose the best precipitator to improve the crystallization of protein molecule.In biomineralization research field, crystal morphology control of inorganic minerals such as calcium carbonate by using self-assembly of block copolymers, to obtain composite materials with high-performance pattern and hierarchical structures are showed great interest in recent years. These different morphogenesis mechanisms mainly focus on selective polymer adsorption, mesoscopic transformations and higher order assembly. By discussion of the effects of self-assembly of block copolymers on crystallization of protein molecules, it was found that block copolymers can serve as more efficient precipitator candidates in protein crystallization.Herein we prepared a series of polymers, and used these polymers as precipitators in lysozyme crystallization to investigate there self-assembly in solution and the effects of polymers on the morphology of protein crystals. Small Angle X-ray Scattering (SAXS), Dynamic Light Scattering (DLS), Static Light Scattering (SLS) and Transmission Electron Micrograph (TEM) methods were used to investigate the self-assembly of polymers in solution. The effects of the structures and concentration of polymeric precipitators on the lysozyme nucleation, crystal growth and crystal morphology were observed. We found that polymers with PDMAEMA blocks are favorable for nucleation and the self-assembling morphology of polymers significantly affect lysozyme nucleation and crystal growth. The presence of high ionic strength (abundant electrolytes, tacsimate solution) in the mother liquor could destroy the self-assembling processes of polymers, and thus the final morphology of lysozyme crystals did not have significant differences. In contrast, when the tacsimate was excluded from the solutions, different amount of crystal nucleus, crystal sizes and crystal morphologies of lysozyme crystals appeared.In brief, in this doctoral dissertation, hydrophilic polymers were focused on and investigated on their application in gene therapy, protein microarrays and protein crystallization.
Keywords/Search Tags:Atom Transfer Radical Polymerization (ATRP), Gene therapy, polycation/DNA complex, Protein immobilization, Protein crystallization
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