抗蛋白质非特异性吸附材料内在机理和特性的研究

发布时间：2018-10-12 20:25

【摘要】：抗蛋白质非特异性吸附材料可以有效减少材料表面的蛋白质吸附从而提高材料的生物相容性。传统的聚乙二醇(PEG)材料在复杂的生物环境中,其长效性和稳定性非常有限,已有研究发现PEG分子能够激活部分人体的免疫,PEG修饰的蛋白质药物活性急剧下降。而两性离子材料如聚甲基丙烯酰氧基乙基磷酰胆碱(pMPC).聚磺基甜菜碱甲基丙烯酸酯(pSBMA)和聚羧基甜菜碱甲基丙烯酸酯(pCBMA)越来越多地被证实比PEG有更加长效的生物相容性。因此研究PEG以及两性离子材料抗蛋白质非特异性吸附的机理不仅有助于更加了解材料抗蛋白质吸附的机制,优先选择合适的抗蛋白质非特异性材料,更有助于设计并发现更优秀的抗蛋白质非特异性吸附材料。本论文围绕抗蛋白质非特异性吸附材料的内在机理和特性,首先创建了低场核磁方法学考察PEG和具有代表性的两性离子聚合物pSBMA不同的水合能力,以及大分子PEG与蛋白质的作用情况；进一步探索溶液状态下长短链PEG以及pSBMA对比PEG和蛋白质不同的作用特性；最后设计蛋白质在水凝胶中的扩散直接体现PEG和SBMA与蛋白质不同的作用力。主要内容和结论包括以下六个部分： 1、利用低场核磁采集不同浓度的PEG水溶液CPMG序列反演得到T2横向弛豫时间,定量计算PEG紧密结合的水分子量,结果表明一个EG单元结合一个水分子,并得到DSC方法验证,同时跟踪聚合物的溶解过程中自身链段的物理行为。 2、利用低场核磁T2反演技术定量研究pSBMA和PEG不同的水合能力以及材料周围水分子的状态。证明pSBMA每个单元比PEG结合更多的水(SB-8个,EG-1个),同时pSBMA的水合层水分子排列比PEG更加紧密,而饱和水层形成后pSBMA周围的水分子比PEG周围的水分子更自由。 3、利用低场核磁T2反演技术定量研究不同分子量的PEG与蛋白质的相互作用,发现PEG与蛋白质在溶液中存在较强的相互作用,且该相互作用和PEG分子量以及蛋白质类型相关,并定量计算PEG与蛋白质的结合常数在104到105M-1。 4、通过荧光、高场核磁共振、原子力显微镜进一步研究不同分子量PEG与蛋白质相互作用的方式及对结合区域的影响。验证了PEG和蛋白质的相互作用与分子量密切相关,小分子量PEG (400Da)由于较高的亲水性和较短的分子链,无法与蛋白质形成多点接触,从而急剧降低了和蛋白质分子的相互作用力。 5、通过荧光、高场核磁共振、原子力显微镜对比研究大分子量的pSBMA和PEG与蛋白质分子不同的相互作用。证明pSBMA和蛋白质的相互作用不明显,而PEG和蛋白质之间的疏水相互作用有可能导致蛋白质分子结构的变化。 6、研究荧光标记蛋白质在PEG和SBMA不同配比的水凝胶中的扩散行为,发现蛋白质分子在PEG水凝胶中扩散的速度明显低于在SBMA水凝胶中的扩散速度,进一步证明了PEG和蛋白质存在较大的相互作用,而SBMA与蛋白质几乎没有相互作用。
[Abstract]:Anti-protein non-specific adsorption materials can effectively reduce the surface protein adsorption and improve the biocompatibility of the materials. The long effect and stability of traditional polyethylene glycol (PEG) materials in complex biological environments are very limited. It has been found that PEG molecules can activate part of human immunity, and the drug activity of PEG modified proteins is decreased sharply. Amphoteric materials such as polymethacryloxy ethyl phosphatidylcholine (pMPC).) Polysulfonbetaine methacrylate (pSBMA) and polycarboxybetaine methacrylate (pCBMA) have more and more long-lasting biocompatibility than PEG. Therefore, the study of the mechanism of anti-protein nonspecific adsorption by PEG and amphoteric ion materials is not only helpful to understand the mechanism of anti-protein adsorption, but also to select suitable anti-protein non-specific materials. It is also helpful to design and find better anti-protein non-specific adsorption materials. Based on the intrinsic mechanism and properties of non-specific anti-protein adsorption materials, a low-field NMR methodology was established to investigate the different hydration capacities of PEG and the representative amphoteric polymer pSBMA. And the interaction of macromolecular PEG with protein, and further explore the different interaction characteristics of long-chain PEG and pSBMA compared with PEG and protein under the condition of solution. Finally, the diffusion of proteins in hydrogels was designed to directly reflect the different interaction forces between PEG and SBMA. The main contents and conclusions include the following six parts: 1. The T 2 transverse relaxation time is obtained by using the CPMG sequence of PEG aqueous solution with different concentrations collected by low field NMR, and the tightly bound water molecular weight of PEG is calculated quantitatively. The results show that a EG unit is combined with a water molecule and verified by DSC method. At the same time, the physical behavior of the chain segments in the dissolution process of the polymer was tracked. 2. The low field NMR T2 inversion technique was used to quantitatively study the different hydration capacities of pSBMA and PEG and the states of water molecules around the materials. It is proved that each unit of pSBMA binds more water than PEG (SB-8, EG-1), and that the water molecules in the hydration layer of pSBMA are arranged more closely than PEG. However, after the formation of saturated water layer, the water molecules around pSBMA are freer than those around PEG. 3. The interaction between PEG with different molecular weight and protein was quantitatively studied by using T2 inversion technique of low field nuclear magnetic field. It was found that there was a strong interaction between PEG and protein in solution, and the interaction was related to the molecular weight of PEG and the type of protein. The binding constants of PEG and protein were calculated quantitatively from 104 to 105M-1.4. High field nuclear magnetic resonance (HNMR) and atomic force microscopy (AFM) were used to study the interaction between PEG and protein with different molecular weight and its effect on the binding region. It is proved that the interaction between PEG and protein is closely related to the molecular weight. Because of its high hydrophilicity and short molecular chain, small molecular weight PEG (400Da) cannot form multi-point contact with protein. Therefore, the interaction force with protein molecules was reduced sharply. 5. The different interactions between pSBMA and PEG with large molecular weight were studied by fluorescence, high field nuclear magnetic resonance and atomic force microscopy. It proves that the interaction between pSBMA and protein is not obvious. The hydrophobic interaction between PEG and protein may lead to the change of protein molecular structure. 6. The diffusion behavior of fluorescent labeled proteins in hydrogels with different ratios of PEG and SBMA was studied. It is found that the diffusion rate of protein molecules in PEG hydrogels is obviously lower than that in SBMA hydrogels, which further proves that there is a large interaction between PEG and proteins, while there is almost no interaction between SBMA and proteins.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08;TQ424

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