海藻酸—磷脂微囊复合水凝胶的制备、表征及包载siRNA的应用研究

发布时间：2018-11-13 08:28

【摘要】：癌症这类威胁人类生命健康的疾病一直都是医学界的一大难题。目前,化学疗法仍然是常用治疗癌症的手段,而传统的化疗存在毒副作用大、选择性差、治疗效果差等缺陷,因此限制了其应用前景和发展潜能。为此,人们期望利用纳米尺寸颗粒或药物递送体固有的表面效应和小尺寸效应增加抗肿瘤化疗药物的生物稳定性和生物利用度。基于纳米抗肿瘤化疗药物的生物活性取决于使用的纳米材质的化学结构和物理性能的特点,通过优化纳米药物载体性能提高药效,或通过分子修饰载体以获得纳米药物递送体主动靶向递送和定位释药的能力。基因治疗是当今治疗恶性肿瘤、遗传性疾病及后天疾病较为理想的手段,siRNA能够降解同源序列的m RNA,特异抑制癌细胞相关基因的表达,从而抑制癌细胞的生长繁殖、侵袭和扩散。但目前可用于将外源性基因导入病变特定部位的技术仍然有限,为了进一步实现基因治疗的目的,必须首先解决基因传递的问题。因此,基因治疗的关键在于获得能够有效包裹基因药物、携载其通过细胞膜并传递进入细胞核使其表达的载体。脂质体因具有无毒性、靶向性、生物相容性、缓释性等诸多优点备受重视,是目前最具有潜力的药物、基因、蛋白等的递送系统之一,但它的应用范围因热力学不稳定性而受到限制。为此,通过对微囊进行物理化学修饰,调整尺寸大小等各种途径提高它在体内外的稳定性,开发出了各种较为稳定的脂质体,例如:隐形脂质体、固体脂质纳米颗粒等,尽管得到了一些改进,但其稳定性问题仍然存在。本文首先是在磷脂微囊胶体中加入含有二价阳离子的电解质溶液(氯化锰、氯化钙、氯化镁),确保胶体微囊处于较好的稳定状态,然后将稳定磷脂微囊体系与海藻酸钠(Sodium alginate,SA)结合形成新型水凝胶,克服了磷脂微囊在液体中的热力学不稳定性,并且利用该材料包载鱼精蛋白-siRNA,并表征其形貌和包封。本研究共分为三章,其主要内容如下:第一章概述了基因、药物治疗面临的困难和存在的缺陷,目前常用的各类基因、药物载体,药物的主动靶向性和被动靶向性以及智能响应性释放的手段。第二章采用薄膜分散法合成磷脂微囊,在磷脂微囊胶体中加入含有二价阳离子的电解质溶液(氯化锰、氯化钙、氯化镁),确保胶体微囊处于较好的稳定状态。然后将稳定的磷脂微囊体系与SA结合形成复合水凝胶,对该材料进行了动态光散射和扫描电镜表征,溶胀率的测定及毒理分析。第三章利用磷脂微囊包载鱼精蛋白-siRNA,测定其粒径和电位,并进行稳定性考察、透射电镜表征,然后与SA结合形成水凝胶。研究结果如下:1.磷脂微囊中加入电解质溶液,测定其粒径及Zeta电位值,结果表明,加入MnCl2、CaCl2溶液,微囊胶体的粒径没有明显的变化。但加入某些浓度MgCl2电解质,其粒径明显变大。当体系中电解质溶液的浓度分别为6.00 mmol·L-1 MnCl2、12.50 mmol·L-1CaCl2、27.00 mmol·L-1MgCl2时,其Zeta电位值均可表现出体系具有较好的稳定性,Zeta电位值分别为53.11±2.14 mV、54.85±3.65 mV、53.52±1.05 mV。2.将氯化锰、氯化钙、氯化镁磷脂微囊胶体分别与SA混合,氯化镁与SA形成水凝胶极弱,氯化钙与SA形成水凝胶能力强于氯化锰。对氯化锰、氯化钙磷脂微囊与SA形成的水凝胶进行扫描电镜(Scanning electron microscope,SEM)表征、溶胀率测定。结果表明,利用PS-Ca2+-SA这种交联键合方式可以将微囊固定化,且形貌规整,结构稳定,细胞毒性基本为零,具有良好的细胞相容性和生物安全性。而氯化锰与SA形成的水凝胶不能将微囊捕获。3.聚丙烯酰胺凝胶电泳确定鱼精蛋白-siRNA最佳复合质量比为4:1,用磷脂微囊包载鱼精蛋白-siRNA复合物,测定其粒径和Zeta电位分别为124.0 nm、43.42 mV,透射电镜表征其形貌和包封情况。结果表明磷脂微囊可以包载鱼精蛋白-siRNA,其粒径较空白磷脂微囊略微增大,然后将其与SA结合形成水凝胶。
[Abstract]:Cancer, a disease that threatens the health of human life, has been a major problem in the medical world. At present, chemical therapy is still a common method for treating cancer, and the traditional chemotherapy has the defects of large toxic and side effect, poor selectivity, poor treatment effect and so on, thus limiting the application prospect and the development potential. To this end, it is desirable to increase the biological stability and bioavailability of the anti-tumor chemotherapeutic agent with the inherent surface effects and small size effects of the nano-sized particles or drug delivery body. the biological activity of the drug based on the nano anti-tumor chemotherapy depends on the chemical structure and the physical property of the nano material used, and the drug effect is improved by optimizing the performance of the nano medicine carrier, or by a molecular modification vector to obtain the ability of the nanodrug delivery body to actively target delivery and position release. Gene therapy is an ideal means for the treatment of malignant tumors, genetic diseases and acquired diseases. The siRNA can degrade the m-RNA of the homologous sequence and specifically inhibit the expression of the cancer cell-related genes, thereby inhibiting the growth and reproduction, invasion and diffusion of cancer cells. However, the technique currently available to introduce an exogenous gene into a specific site of the lesion is still limited, and in order to further achieve the purpose of gene therapy, it is necessary to first address the problem of gene transfer. Therefore, the key to gene therapy is to obtain a vector capable of effectively wrapping a gene drug, carrying it through the cell membrane and transferring it into the nucleus to express it. The liposome is one of the most promising drug, gene, protein and other delivery systems due to many advantages, such as non-toxicity, targeting, biocompatibility, and slow release, but its application range is limited by thermodynamic instability. To this end, through the physical-chemical modification of the micro-capsule, the size and the like are adjusted to improve the stability of the liposome outside the body, and various more stable liposomes are developed, for example, the invisible liposome, the solid lipid nanoparticles, and the like, but its stability problem is still present. The method comprises the following steps of: adding an electrolyte solution (manganese chloride, calcium chloride and magnesium chloride) containing a divalent cation in a phospholipid microcapsule colloid, ensuring that the colloid micro-capsule is in a better stable state, and combining the stable phospholipid microcapsule system with sodium alginate (SA) to form a novel hydrogel, The thermodynamic instability of the phospholipid microcapsule in the liquid is overcome, and the protamine-siRNA is loaded with the material, and the morphology and the encapsulation are characterized. This study is divided into three chapters. The main contents of this study are as follows: The first chapter provides an overview of the difficulties and defects of the gene and drug treatment, the various types of genes commonly used, the drug carrier, the active targeting and the passive targeting of the drugs, and the means of intelligent response release. In the second chapter, the membrane dispersion method is used to synthesize the phospholipid microcapsule, and the electrolyte solution containing the divalent cation (manganese chloride, calcium chloride and magnesium chloride) is added to the phospholipid microcapsule colloid to ensure that the colloid micro-capsule is in a better stable state. and then the stable phospholipid microcapsule system and SA are combined to form a composite hydrogel, and the material is subjected to dynamic light scattering and scanning electron microscopy, and the swelling ratio is determined and the toxicological analysis is carried out. The third chapter uses the phospholipid microcapsule to carry the protamine-siRNA to determine its particle size and potential, and conduct the stability study, the transmission electron microscope characterization, and then combine with SA to form the hydrogel. The results of the study are as follows: 1. The particle size and Zeta potential of the microcapsule were measured by adding the electrolyte solution to the phospholipid microcapsule. The results showed that the particle size of the micro-encapsulated colloid was not changed obviously with the addition of MnCl2 and CaCl2 solution. but some concentration of mgcl2 electrolyte is added, and the particle size of the mgcl2 electrolyte is obviously changed. When the concentration of the electrolyte solution in the system is 6.00 mmol 路 L-1MnCl2, 12.50 mmol 路 L-1CaCl2, and 27.00 mmol 路 L-1MgCl2, the Zeta potential value of the system can show that the system has good stability. The Zeta potential value is 53. 11-2.14mV, 54. 85-3.65 mV, 53. 52-1. 05mV. 2, respectively. the colloid of the manganese chloride, the calcium chloride and the magnesium chloride phospholipid is respectively mixed with the SA, and the magnesium chloride and the SA form the hydrogel very weak; and the calcium chloride and the SA form the hydrogel with the capability of being stronger than that of the manganese chloride. Scanning electron microscopy (SEM) was used to characterize the water-gel formed by the micro-capsule of manganese chloride, calcium chloride and the SA, and the swelling rate was determined. The results show that the micro-capsule can be immobilized by using PS-Ca2 +-SA, and the morphology is regular, the structure is stable, the cytotoxicity is basically zero, and it has good cell compatibility and biological safety. and the hydrogel which is formed by manganese chloride and the sa cannot capture the microcapsules. The optimal composite mass ratio of protamine-siRNA was 4: 1, and the particle size and Zeta potential were 124.0nm, 432.42mV, respectively. The results showed that the phospholipid microcapsules can be encapsulated with protamine-siRNA, whose particle size is slightly larger than that of the blank phospholipid microcapsule, and then it is combined with SA to form a hydrogel.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O648.17

【参考文献】