高效、安全的树形高分子基因及药物载体的制备与应用

发布时间：2018-05-10 14:56

本文选题：高分子 + 树形高分子　；参考：《华东师范大学》2014年博士论文

【摘要】：树形高分子是一类具有树枝状结构的合成大分子。这类分子具有一些特有的性质如良好的单分散性、确定的尺寸、可控的表面官能团、良好的水溶性及生物相容性等。这些性质使得树形高分子在很多领域取得了广泛的应用。特别是在生物医学应用方面,树形高分子被广泛应用为基因及药物载体。但树形高分子距离临床应用需求仍有相当的差距,其作为基因或药物载体时存在转染或运载效率不高、生物相容性差等缺点。如何制备兼具高效、安全等属性的树形高分子基因及药物载体是当前研究的难点。本论文旨在设计高效、安全的树形高分子基因及药物载体。采用如下策略：(1)通过还原敏感的双功能交联剂将低代数树形高分子交联,从而制备可降解的纳米聚集体；(2)在低代数树形高分子表面进行氟化修饰,通过自组装形成纳米囊泡结构；(3)通过氢键调控树形高分子的基因转染效率；(4)将树形高分子与环糊精共价连接,提高树形高分子的生物相容性及载药效率。本论文的主要研究方法及结论总结如下： (1)使用含二硫键的双功能交联剂对低代数树形高分子进行化学交联,制备可降解的纳米聚集体,并将之用作为基因载体。这种基因载体可以有效地结合核酸并形成200nm左右的复合物。所形成的复合物具有高的细胞摄入能力,且该聚集体材料能够在细胞内降解。该方法制备的聚集体材料在多种细胞中的转染效率远高于低代数和高代数树形高分子,与分子量25kDa的支化聚乙烯亚胺(bPEI25kD)相当。另外,该聚集体具有良好的生物相容性,其细胞毒性远低于高代数树形高分子和bPEI25kD。在某些细胞上,其细胞毒性甚至低于低代数树形高分子。进一步研究发现,在低代数树形高分子表面直接引入可进行化学交联的巯基也可大幅提高其基因转染效率。采用这种化学交联的方式也可以提高其它高分子材料的基因转染效率。 (2)以低代数树形高分子和七氟丁酸酐为原料,通过简单的化学反应及提纯方法获得了氟化修饰的低代数树形高分子,并将之用作为基因载体。这类基因载体在多种细胞上都表现出高基因转染效率,远高于低代数和高代数树形高分子以及一些商业化的转染试剂,与当前最佳的商业化转染试剂Lipofectamine2000效率相当。这类转染载体在多细胞球体模型中表现出比Lipofectamine2000更佳的转染效率。即使在低核酸浓度条件下,其转染效率及在多细胞球体中的穿透深度均优于Lipofectamine2000在高核酸浓度条件下的水平。此外,随着修饰到树形高分子表面的七氟丁酸基团的数量增加,这类材料基因转染时所需的氮磷比迅速降低。这些氟化修饰的低代数树形高分子材料的分子量远低于常规的阳离子高分子基因载体,且具有较强的自组装能力,可在水溶液中自组装成100-200nm的囊泡结构,与脂质体类基因载体的行为相似。细胞毒性实验表明,氟化修饰的低代数树形高分子具有良好的生物相容性,细胞毒性远低于氟化修饰的高代数树形高分子和bPEI25kD等载体。 (3)在阳离子树形高分子表面共价连接了一系列核酸碱基衍生物,并将之用作为基因载体。这类高分子材料可以有效地结合核酸形成稳定的复合物。共价连接核酸碱基衍生物之后,树形高分子材料的转染效率大幅提升,与多种商业化的转染试剂相当,而且转染不同的核酸时,表现出稳定的转染效率。这类载体的转染效率与所修饰核酸碱基衍生物中的氢键供受体情况密切相关,能与核酸碱基形成互补氢键的修饰产物表现出更高的转染效率。在树形高分子表面修饰核酸碱基衍生物不仅未带来额外的细胞毒性,而且降低了阳离子树形高分子自身的细胞毒性。因此,氢键调控方法可以发展成为一种制备高效、安全的高分子基因载体的新方法。 (4)在树形高分子表面共价连接了α-,β-,γ-环糊精,并将之用作为药物载体。借助二维核磁共振技术初步研究了以上三种共价连接产物对五种客体分子的装载行为。客体分子在连接产物中的定位或分布取决于客体分子的尺寸,带电状态,疏水性及环糊精的空腔尺寸。将环糊精共价连接到树形高分子表面可改善树形高分子的药物装载能力,扩大树形高分子装载药物的适用范围,提高树形高分子的生物相容性并提高其细胞摄入能力等,为临床组合治疗提供了新的材料设计策略。本论文通过几种新的化学修饰策略来制备高效、安全的高分子基因和药物载体,在提高载体效率及生物相容性两方面取得了双赢。拓展了树形高分子的生物医学应用范围。后续的研究我们将进一步评估这些高分子基因及药物载体的体内输送效率及治疗效果,为树形高分子进入临床基因治疗和药物治疗奠定良好的材料学基础。
[Abstract]:Tree - shaped macromolecule is a kind of synthetic macromolecule with dendritic structure . This kind of molecule has some special properties such as good monodisperse property , definite size , controllable surface functional group , good water solubility and biocompatibility , etc . These properties make the tree - shaped macromolecule widely used in many fields . Especially in biomedical applications , the tree - shaped macromolecule is widely used as gene and drug carrier .

The purpose of this thesis is to design efficient and safe tree - shaped polymer gene and drug carrier . The following strategies are adopted : ( 1 ) the low - algebraic arborescent polymer is cross - linked by reduction - sensitive bifunctional cross - linking agent , thus preparing degradable nano aggregate ;
( 2 ) performing fluorination modification on the surface of a low - algebraic tree - shaped polymer , and forming a nano - vesicle structure by self - assembly ;
( 3 ) regulating the gene transfection efficiency of the dendritic polymer through hydrogen bonds ;
( 4 ) covalently linking the tree - shaped polymer and the cyclodextrin to improve the biocompatibility and the drug loading efficiency of the tree - shaped macromolecule .

in addition , that aggregate material prepared by the method has high cell uptake capability , and the aggregate material can be degraded in the cell .

( 2 ) The low - algebra tree - shaped polymer and heptafluorobutyric anhydride are used as raw materials to obtain fluorinated modified low - algebraic arborescent polymers through simple chemical reaction and purification methods . The transfection efficiency and the penetration depth in the multi - cell sphere are much better than that of the Lipofectamine 2000 under the condition of low nucleic acid concentration .

( 3 ) A series of nucleic acid base derivatives are covalently linked to the surface of a cationic tree polymer and used as a gene carrier . The high molecular material can effectively combine nucleic acid to form stable complexes . The transfection efficiency of the dendritic polymer material is greatly improved after covalent attachment of nucleic acid base derivatives , and the modified products with complementary hydrogen bonds with the nucleic acid bases show higher transfection efficiency . Therefore , the hydrogen bond regulating method can be developed into a novel method for preparing high efficiency and safe polymer gene carriers .

( 4 ) Co - covalently linking 伪 - , 尾 - , 纬 - cyclodextrin on the surface of tree - shaped polymer as a drug carrier . The localization or distribution of the above three covalently linked products to five guest molecules is investigated by means of two - dimensional nuclear magnetic resonance technology . The localization or distribution of the object molecules in the connection products depends on the size , charged state , hydrophobicity and the cavity size of the cyclodextrin . The covalent attachment of the cyclodextrin to the surface of the tree - shaped polymer can improve the drug loading capacity of the tree - shaped macromolecule , enlarge the application range of the tree - shaped macromolecule , improve the biocompatibility of the tree - shaped macromolecule and improve the cell uptake ability , and provide a new material design strategy for clinical combination therapy .

In this paper , several new chemical modification strategies are adopted to prepare high - efficiency , safe polymer gene and drug carrier , which has achieved win - win in improving the efficiency and biocompatibility of the carrier . The research on the efficiency and the therapeutic effect of these high - molecular genes and drug carriers will be further evaluated , which lays a good foundation for the entry of tree - shaped macromolecules into clinical gene therapy and drug therapy .

【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R94

【共引文献】