基于mPEG-PLGA纳米载体蛋白质内耳递送研究

发布时间：2018-01-21 11:00

本文关键词： 感音神经性聋生物治疗纳米载体乳酸聚乙醇酸-聚乙二醇内耳递送　出处：《中国人民解放军医学院》2014年博士论文　论文类型：学位论文

【摘要】：多种原因如强噪声暴露或者耳毒性药物应用等都能够引起耳蜗内组织结构或者细胞的受损。一般情况下，这种损害过程都开始于耳蜗毛细胞（包括外毛细胞和内毛细胞），随后引起Corti’s器结构的破坏，最终导致听觉损伤。耳聋相关研究认为，听觉损伤最终得以治疗，最根本的方法还是通过外源生物干预治疗使耳蜗毛细胞再生，或者在耳蜗毛细胞损伤的特定病理时期进行相关干预，最终使之得到修复。在各种研究开发的耳聋治疗方案中，生物治疗是最具有应用潜力的。同时，内耳结构内部流动的液体以及相对狭小独立的空间等特点又使得耳蜗可以作为理想的生物治疗体系加以研究应用。科学家们经过对不同类型耳聋发病机制和听觉神经细胞发育的研究和探索，先后发现了一系列针对听功能起保护或修复作用的核酸、多肽和小分子化合物等，这些生物分子可以通过对内耳感觉细胞的修复或再生，实现听功能不同程度的保护或恢复。选择合适的递送载体将目的高效、安全地导入内耳组织和细胞中，是耳聋生物治疗的关键。常用的载体分为两大类：病毒载体及非病毒载体。上世纪九十年代，研究人员首次使用病毒载体将外源基因导入内耳，开创了内耳细胞保护或再生研究中载体技术开发应用的先河。然而，，病毒性载体的缺陷，以及以往报道的致死性个案病例，又让人们开始重新审视应用病毒性载体的安全问题。近年来，对于载体的选择及应用研究，逐渐转向了非病毒性载体，其中，纳米载体应用又成为生物医学领域的发展热点。研究证实，纳米载体能够在生物治疗过程中表现出稳定的表达特性，具备较高的安全性及细胞特异性等优点，为传统的耳病防治和干预研究提供了全新的策略。聚乳酸-羟基乙酸(poly(D,L-lactide-co-gly-colide)，PLGA)是最具代表性的一类纳米类载体系统，为丙交酯和乙交酯的共聚物，具有良好的生物相容性及生物可降解性。经PEG修饰的PLGA纳米载体复合物结构可控，在生理pH值条件下，通过PLGA疏水性核心包载多种多肽、蛋白质类药物，并且依靠该复合物表面的PEG与带有负电荷的细胞膜结合并相互作用，保证了多肽、蛋白质类药物在无明显降解的情况下，通过包吞作用进入细胞内，发挥治疗疾病的作用。本实验第一部分通过对PLGA的表面修饰，成功获得具有缓释功能的PLGA-PEG纳米颗粒递送载体，并且通过双乳法包载BSA蛋白，成功制备mPEG-PLGA-BSA-FITC-NPs，且包载率达到51.2%。第二部分应用已经合成mPEG-PLGA-BSA-FITC-NPs体外转导Hela细胞系，用激光共聚焦荧光显微镜观察细胞内FITC荧光情况，评估mPEG-PLGA-BSA-FITC-NPs体外进入细胞的效率，同时用MTT法检测mPEG-PLGA-BSA-FITC-NPs纳米递送系统对于Hela细胞的毒性并计算细胞存活率。同时，我们首次通过手术手段，应用mPEG-PLGA-BSA-FITC-NPs经完整圆窗膜渗透途径导入活体豚鼠内耳中，用共聚焦显微镜观察并评估mPEG-PLGA-BSA-FITC-NPs在内耳的有效率，验证并突出强调mPEG-PLGA-BSA-FITC-NPs作为一种新型纳米载体在内耳毛细胞再生中的应用潜力。在实验的第三部分，我们开创性的应用脉冲式火花噪声仪对正常小型猪进行噪声暴露，通过对强噪声暴露后的小型猪模型进行听功能评估及耳蜗病理形态学研究，首次成功建立了基于大型哺乳动物的噪声性聋小型猪动物模型，为此后内耳导入研究奠定基础。最后，我们尝试应用mPEG-PLGA-BSA-FITC-NPs进行噪声性聋小型猪动物模型内耳导入研究，以期为mPEG-PLGA-BSA-FITC-NPs纳米载体的内耳应用提供重要的临床前研究数据。本研究创新点： 1．用PEG修饰PLGA纳米颗粒，并成功通过双乳法包载BSA蛋白，最终获的大小约为232±2.6nm、具有良好分散性和缓释特性的纳米颗粒蛋白递送体系； 2.成功将mPEG-PLGA-BSA-FITC-NPs体外导入细胞系，并获得较高安全性和有效性。同时，首次实现mPEG-PLGA-BSA-FITC-NPs在体豚鼠内耳递送，并呈现明显的时间依赖性。 3.首次成功建立小型猪噪声性聋动物模型，分析噪声性聋小型猪听功能及内耳形态特点，填补了大型哺乳动物噪声性聋动物模型的空白。 4.首次尝试应用mPEG-PLGA-BSA-FITC-NPs纳米颗粒进行小型猪噪声性聋动物模型的内耳导入研究，为未来大型哺乳动物听功能干预研究提供了重要的研究基础。
[Abstract]:Various causes , such as strong noise exposure or ototoxic drug use , can cause damage to tissue structures or cells in the cochlea . In general , such damage processes begin with cochlear hair cells ( including outer hair cells and inner hair cells ) , which subsequently cause damage to the Corti ' s device structure and eventually lead to hearing impairment . In the treatment of deafness in various research and development , biotherapy is the most promising . At the same time , the fluid inside the inner ear structure and the relatively narrow and independent space can be used as the ideal biological treatment system . The scientists have discovered a series of nucleic acids , polypeptides and small molecule compounds which have been used to protect or repair different types of deafness , and these biomolecules can protect or restore the hearing function to different degrees by repairing or regenerating the sensory cells of the inner ear . In recent years , researchers have gradually shifted to non - viral vectors by introducing foreign genes into the inner ear using viral vectors . Poly ( D , L - lactide - co - gly - colide ) , poly ( D , L - lactide - co - gly - colide ) ( PLGA ) , is the most representative type of nano - carrier system , which is a copolymer of lactide and glycolide and has good biocompatibility . and the PEG - modified PLGA nano - carrier composite structure is controllable , and under the condition of physiological pH value , a plurality of polypeptides and a protein drug are loaded through a PLGA hydrophobic core , and the PEG on the surface of the compound is combined with the cell membrane with a negative charge and interacts so as to ensure the polypeptide and the protein drug to enter the cell through the endocytosis , so as to play the role of treating diseases . In the first part of the experiment , PLGA - PEG nano - particle delivery vehicle with sustained - release function was successfully obtained by surface modification of PLGA , and the cell survival rate was 51.2 % . Innovation point of this study : 1 . The PLGA nanoparticles were modified with PEG and the BSA protein was successfully prepared by double emulsion method . The final product was 232 卤 2.6 nm , which had good dispersibility and sustained release properties . 2 . The mPEG - PLGA - BSA - FITC - TiO2 was successfully introduced into the cell line in vitro , and high safety and efficacy were obtained . At the same time , the mPEG - PLGA - BSA - FITC - induced drug was first delivered in the inner ear of the guinea pig and showed a significant time - dependence . 3 . The animal model of small pig noise was successfully established for the first time , and the characteristics of hearing function and inner ear morphology were analyzed in order to fill the blank of animal model of noise - induced deafness in large mammals . 4 . In order to provide an important research foundation for the study of auditory function intervention in large mammals in the future , the introduction of mPEG - PLGA - BSA - FITC - TiO2 nanoparticles was first attempted .

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764.43

【共引文献】