磷酸胆碱化壳聚糖衍生物的合成、表征及生物学评价

发布时间：2018-08-14 11:57

【摘要】：本研究从细胞膜仿生的角度出发，设计合成一种新的壳聚糖磷酰化衍生物，以磷酰胺键的方式将细胞膜结构单元磷酸胆碱基团偶联到壳聚糖骨架上，研究其合成方法、生物学性能及其纳米化方法。为了合成磷酸胆碱化壳聚糖衍生物，我们研究比较了三种壳聚糖的均相磷酸胆碱化方法：(A)采用新型高极性六氟异丙醇作反应介质，基于Antherton-Todd反应实现壳聚糖的直接磷酰化；(B)利用N-邻苯二甲酰化壳聚糖为中间体，以二氯磷酰胆碱为磷酰化试剂；(C)利用6-O-三苯基甲醚化壳聚糖为中间体，基于Antherton-Todd反应实现磷酰化。研究表明合成路线(C)适合用来均相合成磷酸胆碱化壳聚糖衍生物。应用合成路线(C)，，通过改变投料比，基于Antherton-Todd反应合成了三种不同取代度的水溶性磷酸胆碱化壳聚糖衍生物。NMR和FTIR谱图上对应-N+(CH3)3基团吸收峰的出现表明磷酸胆碱基团成功偶联到壳聚糖骨架的氨基上，根据1H NMR谱图的峰强度比计算出三种壳聚糖衍生物的取代度分别为16%、27%和42%。GPC数据显示，与壳聚糖相比，磷酸胆碱化壳聚糖衍生物的分子量有所降低，分子量分布有所拓宽；XRD、TGA、DSC和水溶性实验表明，磷酸胆碱化壳聚糖衍生物的结晶性能和热稳定性均有不同程度地下降，但是其在水中的溶解性能得到了很大地提升，三种取代度的衍生物均可溶于pH=1-12的水溶液中。细胞毒性实验表明，3T3细胞与磷酸胆碱化壳聚糖衍生物共培养的相对增殖率在80%-110%之间，细胞毒性为0级或1级，属于无细胞毒性范畴；血液相容性实验表明，引入磷酸胆碱基团可以延缓衍生物的凝血时间，而且可以有效抑制血小板在其上的黏附与激活；与牛血清白蛋白(BSA)的相互作用表明，磷酸胆碱基团的引入可以有效抑制壳聚糖衍生物与BSA之间的相互作用，减小蛋白质的构象改变，这对避免激活因蛋白质构象改变而导致的不良生物反应具有重要意义。磷酸胆碱化壳聚糖衍生物可自组装形成纳米粒子。研究表明，磷酸胆碱化壳聚糖衍生物仍可与三聚磷酸钠进行离子交联形成纳米粒子，这些纳米粒子呈现规则的球形结构，粒径在60-120nm之间，Zeta电位介于18-28mV；同时，磷酸胆碱化壳聚糖衍生物具有两亲性，可在中性水溶液中自组装成具有疏水核亲水壳的纳米胶束，由低到高，三种取代度衍生物的临界胶束浓度分别为0.129mg/mL，0.201mg/mL，0.256mg/mL。所形成的纳米胶束粒径范围在70-110nm之间，Zeta电位接近于0，介于0-4mV之间。这两类纳米粒子有望应用于药物/基因载体。
[Abstract]:In this study, a new phosphorylation derivative of chitosan was designed and synthesized from the point of view of cell membrane bionics. The phosphorylcholine group of cell membrane structure was coupled to the chitosan skeleton by phosphoramide bond, and the synthesis method of the derivative was studied. Biological properties and nanocrystalline methods. In order to synthesize chitosan derivative of choline phosphate, we studied and compared three homogenous choline phosphate methods: (A) used a new high polarity hexafluoroisopropanol as the reaction medium, and realized the direct phosphorylation of chitosan based on Antherton-Todd reaction; (B) used N-phthalic chitosan as intermediate and; (C) as phosphorylation reagent. 6-O- triphenylmethyl ether chitosan was used as intermediate to realize phosphorylation based on Antherton-Todd reaction. The results show that the synthetic route (C) is suitable for homogeneous synthesis of choline phosphate chitosan derivatives. By changing the feed ratio by using the synthetic route (C), Based on Antherton-Todd reaction, three kinds of water-soluble choline phosphate chitosan derivatives were synthesized. NMR and FTIR spectra showed that the corresponding absorption peaks of -N (CH3) _ 3 group were successfully coupled to the amino group of chitosan skeleton. According to the peak intensity ratio of 1H NMR spectrum, the substitution degree of the three chitosan derivatives is 16% and 42%.GPC data show that compared with chitosan, the molecular weight of chitosan derivative of choline phosphate is lower and the molecular weight distribution is wider. DSC and water solubility tests showed that the crystallization and thermal stability of choline phosphate chitosan derivatives decreased in varying degrees, but their solubility in water was greatly improved. All three derivatives of degree of substitution are soluble in the aqueous solution of pH=1-12. The results of cytotoxicity test showed that the relative proliferation rate was between 80% and 110%, and the cytotoxicity was grade 0 or grade 1, which belonged to the category of no cytotoxicity. The introduction of choline phosphate groups can delay the coagulation time of the derivatives and inhibit the adhesion and activation of platelets on them, and the interaction with bovine serum albumin (BSA) (BSA) suggests that, The introduction of choline phosphate group can effectively inhibit the interaction between chitosan derivatives and BSA and reduce the conformation change of protein, which is important for avoiding the activation of adverse biological reactions caused by protein conformation changes. Chitosan derivatives of choline phosphate can be self-assembled to form nanoparticles. The results show that the chitosan derivatives of choline phosphate can still be crosslinked with sodium tripolyphosphate to form nanoparticles. These nanoparticles have regular spherical structure and the potential of Zeta between 60-120nm is 18-28mV, at the same time, The chitosan derivatives of choline phosphate have amphiphilic properties and can self-assemble into nanomicelles with hydrophobic core hydrophilic shells in neutral aqueous solution. The critical micelle concentrations of the three derivatives are 0.129 mg / mL ~ 0.201 mg / mL ~ (-1) ~ 0.256 mg 路m ~ (-1) 路m ~ (-1), respectively, and the critical micelle concentrations of the three derivatives are 0.129 mg / mL ~ (-1) ~ 0.201 mg / mL ~ (-1) ~ 0.256 mg / m ~ (-1). The particle size range of the resulting micelles is close to 0 between 70-110nm and 0-4mV. These two kinds of nanoparticles are expected to be used in drug / gene carriers.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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