以巯基壳聚糖为载体材料加载基因经口服给药的探索

发布时间：2018-05-01 01:07

本文选题：巯基壳聚糖 + 纳米-微球　；参考：《华侨大学》2016年硕士论文

【摘要】：巯基壳聚糖(Thiolated chitosan,TCS)非病毒载体具有免疫原性低等优点,口服纳米基因药物的有效性已得到大量实验证实。本文通过离子交联法制备不同粒径、电位的TCS纳米粒,同时构建一种壳-核结构的TCS纳米-微球新型载体。系统研究了TCS-pDNA纳米粒及TCS-pDNA纳米-微球加载基因药物经口服吸收的稳定性,探讨了以TCS为载体材料加载基因药物口服吸收和表达。通过效应法优化TCS纳米粒的制备工艺,结果表明,当TCS浓度为1-4mg·mL-1,多聚磷酸钠浓度为1-2 mg·mL-1,转速为1000 rpm,溶液的pH为3.5-5.5,可制备得到粒径在100-500 nm,电位在+20-50 mV,形状规则,分散良好的TCS纳米粒。进一步优化表明,当外壳聚已内酯(PCL)质量分数为1-5%,内核TCS质量10-60 mg,转速3000-5000 rpm时,可制备出规则圆形、空心、表面光滑的TCS纳米-微球。以pDNA为模型药物,制备得到了TCS-pDNA纳米粒及纳米-微球。研究了TCS-pDNA纳米粒在DNAse-I酶以及模拟的人工胃液与肠液中的稳定性。结果表明,不同电位的TCS-pDNA纳米粒能很好的对抗DNAse-I的降解,免受模拟人工胃液与肠液的影响,稳定性良好,且大粒径纳米粒稳定性明显优于小粒径。TCS-pDNA纳米-微球能很好的保护pDNA免受胃肠液的破坏与降解,稳定性优于单纯的TCS-pDNA纳米粒子。通过建立Caco-2细胞模型,研究了不同粒径的TCS-pDNA纳米粒在Caco-2细胞中的转染效率,结果表明,不同粒径的TCS-pDNA都能转染Caco-2细胞,但小粒径的TCS-pDNA的转染效率较大粒径的高,且转染效率呈时间依赖性。通过建立小鼠肠襻模型,研究不同粒径的TCS-pDNA纳米粒及纳米-微球在肠襻组织的转染效率,给药2 d后,肠中能检测到转基因EGFP的表达,且小粒径TCS-pDNA纳米粒转染效率较高,TCS-p DNA纳米-微球组的转染效率明显高于TCS-pDNA纳米粒组。研究结果表明以PCL为壳制备的壳-核结构的TCS-pDNA纳米-微球可提高基因口服吸收表达效率。通过加强纳米基因药物生物药剂学方面的研究,为今后促进口服纳米基因药物的剂型设计和临床应用奠定基础。
[Abstract]:Thiolated chitosanine (TCSs) non-viral vectors have the advantages of low immunogenicity. The effectiveness of oral nano-gene drugs has been confirmed by a large number of experiments. In this paper, TCS nanoparticles with different particle size and potential were prepared by ion crosslinking method, and a novel TCS nanometer-microsphere carrier with shell-core structure was constructed at the same time. The stability of oral absorption of TCS-pDNA nanoparticles and TCS-pDNA nanometer-microspheres loaded gene drugs was systematically studied, and the oral absorption and expression of TCS loaded gene drugs were discussed. The preparation process of TCS nanoparticles was optimized by the effect method. The results showed that when the concentration of TCS was 1-4mg mL-1, the concentration of sodium polyphosphate was 1-2 mg mL -1, the rotational speed was 1000 rpm, the pH of the solution was 3.5-5.5, the particle size was 100-500 nm, the potential was 20-50 MV, and the shape was regular. Well dispersed TCS nanoparticles. Further optimization shows that when the mass fraction of the shell polycaprolactone is 1-5, the core TCS mass is 10-60 mg and the rotational speed is 3000-5000 rpm, the regular circular, hollow and smooth TCS nanometer-microspheres can be prepared. TCS-pDNA nanoparticles and nano-microspheres were prepared using pDNA as model drug. The stability of TCS-pDNA nanoparticles in DNAse-I enzyme and simulated gastric juice and intestinal fluid was studied. The results showed that the TCS-pDNA nanoparticles with different potentials could resist the degradation of DNAse-I, avoid the influence of simulated artificial gastric juice and intestinal fluid, and have good stability. The stability of large size nanoparticles is better than that of small particle size. TCS-pDNA nanometer-microspheres can protect pDNA from the destruction and degradation of gastrointestinal fluid, and the stability is better than that of simple TCS-pDNA nanoparticles. The transfection efficiency of TCS-pDNA nanoparticles with different diameters in Caco-2 cells was studied by establishing a Caco-2 cell model. The results showed that TCS-pDNA with different particle sizes could be transfected into Caco-2 cells, but the transfection efficiency of TCS-pDNA with small size was higher than that of Caco-2 cells. The transfection efficiency was time dependent. The transfection efficiency of different sizes of TCS-pDNA nanoparticles and nanometer-microspheres in intestinal haptic tissues was studied by establishing a mouse intestinal loop model. After 2 days of administration, the expression of transgenic EGFP could be detected in the intestine. The transfection efficiency of small diameter TCS-pDNA nanoparticles was higher than that of TCS-pDNA nanoparticles group. The results showed that TCS-pDNA nanometer-microspheres with PCL as shell could improve the efficiency of gene absorption and expression. Through strengthening the research on biomedicine of nanogene drugs, it will lay a foundation for the formulation design and clinical application of oral nano-gene drugs in the future.
【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TQ460.6

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