壳聚糖纳米粒作为基因治疗载体的研究

发布时间：2018-07-23 14:34

【摘要】：近年来,由于基因治疗在替代功能障碍基因和肿瘤治疗方面的潜在应用前景而备受关注。基因治疗的关键问题之一在于开发安全、高效的基因转染体系。在转基因载体的研究发展过程中,一个明显的趋势是采用非病毒转基因载体,其目的在于降低人体正常细胞致病的风险。 本文选用天然无毒、可生物降解、具有良好生物相容性的带正电荷高分子材料——壳聚糖作为载体,通过纳米生物技术制备了载质粒的壳聚糖-质粒纳米粒。系统地研究了壳聚糖分子量,N/P,pH值,制备方法等因素对壳聚糖-pDNA纳米粒的形态、包埋率、载药率、基因药物在摸拟体内环境下的释放的影响。以优化壳聚糖-pDNA纳米粒的制备条件,探讨制备条件与壳聚糖-pDNA纳米粒的性质相关性,进而揭示壳聚糖-pDNA纳米粒作为基因治疗的载体的机理所在。 壳聚糖的结晶性、力学特性、通透性等都和壳聚糖分子量大小相关,而壳聚糖的许多独特功能只有在分子量降低到一定程度时才表现出来。因此,选择适当的方法对壳聚糖进行降解就显得尤为重要。本文选用超声波技术与H_2O_2氧化降解法相结合的方法对壳聚糖进行降解以获得纯化的不同分子量的壳聚糖。结果表明:在30℃,采用降解体系为4%的壳聚糖的5%乙酸溶液,加入H_2O_2与糖单元摩尔比为1.0,进行超声波辅助降解40min-60min可获得满意的结果。 壳聚糖分子在酸性条件下荷正电,DNA分子在碱性条件下荷负电,我们利用这一特点采用复凝聚法制备基因壳聚糖纳米粒。以纳米粒的平均粒径与形态作为控制指标,进行单因素实验。从纳米粒的粒径分布、形态、包埋率、载药率、基因包埋量和体外释放率等方面考察了纳米粒的基本性质。结果表明:采用复凝聚制备法能够实现壳聚糖对质粒的包埋,包埋率达到83.46%。制备的壳聚糖质粒纳米粒呈亚球型,粒径在300～600nm。壳聚糖质粒纳米粒
[Abstract]:In recent years, gene therapy has attracted much attention because of its potential application in gene substitution for dysfunction and tumor therapy. One of the key problems of gene therapy is to develop a safe and efficient gene transfection system. In the process of research and development of transgenic vectors, there is an obvious trend to adopt non-viral transgenic vectors, which aims to reduce the risk of human normal cell pathogenicity. In this paper, chitosan, a naturally nontoxic, biodegradable and biocompatible positively charged polymer material, was used as carrier to prepare plasmide-loaded chitosan plasmid nanoparticles. The effects of molecular weight of chitosan, pH value of N / P, and preparation method on the morphology, entrapment rate, drug loading rate and drug delivery rate of chitosan pDNA nanoparticles were systematically studied. In order to optimize the preparation conditions of chitosan-pDNA nanoparticles, the relationship between the preparation conditions and the properties of chitosan-pDNA nanoparticles was discussed, and the mechanism of chitosan-pDNA nanoparticles as the carrier of gene therapy was revealed. The crystallinity, mechanical properties and permeability of chitosan are all related to the molecular weight of chitosan. However, many unique functions of chitosan are only shown when the molecular weight decreases to a certain extent. Therefore, it is very important to choose suitable method to degrade chitosan. In this paper, ultrasonic technology combined with H_2O_2 oxidation degradation method was used to degrade chitosan to obtain purified chitosan with different molecular weight. The results showed that at 30 鈩，

本文编号：2139713