【作者】 龚铖；

【导师】 曾绍群； 张玉慧；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2013， 博士

【摘要】 基因治疗给很多疾病的治疗提供了希望,但是目前依然缺乏安全、高效并能靶向性运输基因到相应细胞中的载体。虽然病毒载体可以有效运输基因到细胞中发挥作用,但其也有一系列的缺点,例如：构建和使用较为复杂,只能携带较小分子量的基因,容易引起炎症反应以及潜在的安全性问题等,这些都严重限制其在生物医学以及临床上的应用。所以,研究者们发展了一系列人工合成的非病毒载体,它们对货物分子尺寸的要求更加宽松,免疫原性更低,而且使用也更加方便。在这些非病毒载体中,阳离子脂质体和高聚物由于其使用简便以及具有相对较高的运输效率而得到了广泛的使用和研究。然而,由于它们具有毒性上的局限,发展新型的能够运输DNA到动物细胞内发挥作用的低毒、高效非病毒载体仍然十分必要。肽型DNA载体是一种新型非病毒DNA载体,与其它非病毒DNA载体(例如：脂质体或者高聚物)相比,它具有以下优点：易于合成,低免疫原性,生物相容性好,在体内容易被降解等。并且,对肽型DNA载体的结构修饰也较容易进行,从而使其具有不同的功能,因此,有可能具有更广泛的应用前景。然而,已有的肽型DNA载体都对细胞或者组织没有靶向性,而靶向性则是基因治疗的前提需求之一。所以,发展能够靶向性运输DNA到相关细胞的肽型载体对于基因治疗十分有意义。细胞穿膜肽是一种被广泛使用的肽型载体。本文从新型细胞穿膜肽的构造和性质分析入手,首先设计并合成了一系列新型含有天然和非天然氨基酸的混合型细胞穿膜肽,对其进入细胞的效率、细胞内分布和抗胰酶降解能力做了检测分析,从中筛选出适合于作为DNA运输载体的性质特征。其次,将靶向恶性肿瘤细胞的叶酸分子加入到筛选出的细胞穿膜肽中,并对其与DNA结合的能力、包裹DNA形成颗粒的性质和携带DNA进入细胞的能力进行了研究。结果表明这种叶酸修饰的细胞穿膜肽不能有效运输DNA到细胞中表达,可能是因为空间位阻过大或者包裹DNA所形成的纳米颗粒所带电荷过低。然后,将一种靶向特定肿瘤细胞的序列作为靶向基团加入到的细胞穿膜肽中,构建了一种新型的肽型载体,并对它与DNA结合的能力、包裹DNA形成颗粒的尺寸和表面电荷、携带DNA进入各种肿瘤细胞的效率、进入细胞的机制和细胞内分布,以及细胞毒性等方面性质进行了系统研究。结果表明这种新型的肽型载体能够有选择性地携带DNA到特定肿瘤细胞系中表达。它具有易于合成、低毒性、低免疫原性、良好的生物相容性、良好的体内降解能力以及能实现特定肿瘤细胞选择性的DNA运输等优点。最后,将一段靶向神经细胞的序列加入到细胞穿膜肽中,构建了另一种新型的肽型载体,并对它与DNA结合的能力、包裹DNA形成颗粒的尺寸和表面电荷、携带DNA进入各种细胞的效率、进入细胞的机制和细胞内分布以及细胞毒性等方面性质进行了系统研究,最终进一步应用到在体运输DNA到鼠脑神经元。结果表明了这种新型的肽型载体不仅能够选择性地运输DNA到神经细胞表达,而且可以有效转染DNA到原代神经元,最重要的是静脉注射后能靶向性运输DNA突破血脑屏障到达小鼠脑部并表达,为构建靶向运输DNA到鼠脑神经元的肽型载体提供了新思路。更多还原

【Abstract】 DNA therapy shows a promising strategy for treating various diseases but still requires efficient and safe systems that can deliver the therapeutic DNA selectively into targeted cells. Viral vectors have been used to transfer genes into cells successfully. However, these vectors have serious disadvantages, such as limited loading capacity, complexity of production, innate immunogenicity, and the risk of inflammatory responses and toxicity, that limit their clinical applications. To avoid these problems, various non-viral carriers have been developed; these carriers display low immunogenicity, relative safety, ease of production, and no cargo size limitation. Of the existing non-viral vectors, cationic lipids and cationic polymers are the most intensively studied and frequently employed. However, they have dose-dependent toxicities in in vivo applications.Cationic peptides have also been explored as gene delivery systems due to several advantages:biodegradability, biocompatibility, less toxicity, and ease of synthesis compared with polymeric carriers. Moreover, the composition of peptides is easy to control. By altering the composition of a given peptide, various functions can be achieved. However, none of these systems has any selectivity toward cells or tissues, which makes it impossible for them to deliver DNA into targeted cells or tissues in vivo and thus seriously limits their applications in gene therapy.Cell-penetrating peptides (CPPs) are one kind of short cationic peptides. In this study, we designed novel CPPs and investigated their characters. A series of heterogeneous oligoarginines consisting of natural and unnatural amino acids were synthesized. Then their uptake efficiencis, intracellular distributions and anti-enzymatic hydrolysis abilities were investigated. From the results we screened out the structure suitable for using as a DNA transport carrier. Then the target motif was added into the CPPs sequence for target DNA delivery. Firstly, the folate acid for tumor targeting was added into the chosen CPP to construct a non-viral peptide based DNA delivery system for tumor targeting DNA delivery. The results showed that although this vector could bind and compack DNA, it could not deliver DNA into the cells. This is possibly because of the steric hindrance or the low zeta potential of the peptide/DNA complex.Next, a tumor targeting sequence was added into the chosen CPP to construct a non-viral peptide-based DNA delivery system which could mediate efficient DNA expression in only selected tumor cells. The cell uptake efficiencies, intracellular distribution, uptake mechanism and cytotoxicity were analyzed. The advantages of this system include its ease of synthesis, low immunogenicity, biocompatibility, low cytotoxicity and selectivity.Finally, a brain targeting sequence was added into the chosen CPP to construct a non-viral peptide-based DNA delivery system which could mediate efficient DNA expression to mice brain after intravenous injection. The cell uptake efficiencies, intracellular distribution, uptake mechanism and cytotoxicity were analyzed. This system could mediate neuronal cell-selective DNA delivery, efficient DNA transfection to primary neurons and targeting DNA delivery into the brain. The advantages of this system include the advantages of peptide carriers mentioned above and brain targeting. The result suggest a new strategy for brain-targeting DNA delivery in vivo.更多还原