【作者】 胡楚；

【导师】 卓仁禧； 钟振林；

【作者基本信息】 武汉大学 ， 高分子化学与物理， 2010， 博士

【摘要】 基因治疗概念的提出和研究的进展给用传统医疗手段难以治疗的疾病如遗传病、癌症等的根治带来了希望。然而,治疗性外源基因本身并不能有效进入细胞,缺乏高效的基因传递系统成为限制基因治疗临床应用的最大障碍之一。本文第一章对基因传递的各种方法作了较系统的评述,并着重介绍了无机纳米材料作为基因载体的研究进展。用于基因治疗的理想载体必须同时具有优异的基因传递效率、良好的生物安全性、便于批量制备等性能,有时还需要具有对特定细胞或组织的靶向性。大多数无机纳米粒子生物相容性良好、毒性很低,并且制备尺寸和结构可控的无机纳米粒子的技术已经比较成熟,能实现批量生产。无机纳米粒子的表面反应活性高、可接枝的密度大,易于同时与多个靶向分子作用。表面经过适当修饰的无机纳米粒子能包载基因并通过细胞的内吞作用介导基因进入细胞,从而实现基因转染。因此,以易于合成的、结构和尺寸确定的无机纳米粒子为平台,在其表面进行合理的功能化,有可能得到性能优异的新型基因载体。我们选用最常见的纳米二氧化硅和纳米金粒子作为平台,因为这两种无机纳米粒子都具备制备简单、粒径可控、易于功能化、毒性较低等特点。将聚乙烯亚胺(PEI)通过共价键接枝到纳米粒子表面,成功合成了PEI修饰的纳米二氧化硅粒子和纳米金粒子,体外实验结果表明它们都可以用作高效低毒的基因载体。在第二章中,我们先通过反相微乳液法制备了表面巯基修饰的二氧化硅纳米粒子SiNP-SH,再利用能连接胺基与巯基的偶联剂SPDP将PEI25k以S-S键接到SiNP上,成功制备了PEI25k修饰的二氧化硅纳米粒子SiNP-SS-PEI25k;我们利用Stober法制备了未经修饰的二氧化硅纳米粒子,并通过带有环氧基的硅烷偶联剂将PEI键接到SiNP上,成功制备了低分子量PEI800修饰的二氧化硅纳米粒子SiNP-PEI800。透射电镜显示两种方法制备的SiNP粒径都在100nm左右。MTT法测定结果显示SiNP-SS-PEI25k对HeLa细胞的毒性远低于PEI25k,其半数抑制浓度是PEI25k的15倍,而SiNP-PEI800的毒性则略低于PEI800。凝胶电泳显示SiNP-SS-PEI25k在低N／P即N／P=2时即能与DNA完全复合,而SiNP-PEI800仅能在高的N／P即N／P=10时与DNA充分复合,因而证明了SiNP-SS-PEI25k的复合能力明显高于SiNP-PEI800。两种二氧化硅纳米粒子均能在转染N／P的范围内对DNA起到保护其不受核酸降解酶降解的作用。HeLa细胞的转染实验结果证明在无血清培养基中,SiNP-SS-PEI25k的转染效率要略低于PEI25k; SiNP-PEI800的转染效率不高,但仍比未经修饰的PEI800的转染效率提高了约2个数量级；在含10%血清的培养基中,SiNP-SS-PEI25k的转染效率可达到PEI25k转染效率的6倍,SiNP-PEI800的转染效率则不受血清的影响而降低,几乎与无血清时相同。在第三章中,我们在硼氢化钠还原氯金酸的反应中加入巯基化的低分子量PEI(PEI 800 Da),通过改变PEI与Au的投料摩尔比,成功制备了三种不同PEI接枝密度的金纳米粒子(GNP-PEI800)。透射电镜观察金纳米粒子的粒径在6 nm左右,分散均匀,无聚集倾向。MTT法测定结果显示低PEI接枝密度的金纳米粒子具有良好的体外细胞相容性,对COS-7细胞的毒性远远低于PEI25k,与PEI800的毒性相近；高PEI接枝密度的金纳米粒子毒性较大,但其半数抑制浓度仍是PEI25k的四倍。凝胶电泳实验证明金纳米粒子在较低N／P(N／P=4)时就能完全阻滞DNA在电场中的移动。在合适的N／P比范围内,GNP-PEI800能与pGL3质粒形成直径约为200nm、表面电势约为+30mV的复合物微粒。该复合物能保护DNA不受核酸降解酶的作用。在无血清培养基中,GNP-PEI800的转染效率达到了PEI25k效率的量级,比未经修饰PEI800的效率高出了近4个数量级。GNP-PEI800的转染效率随PEI接枝密度的增加而升高,高PEI接枝密度的金纳米粒子的转染效率还会随血清浓度的增加而进一步提高：在含10%血清的培养基中,其最高转染效率比PEI25k高出了约2个数量级,而较低PEI接枝密度的金纳米粒子则能保持转染效率不变。细胞示踪实验证明了COS-7细胞对高PEI接枝密度金纳米粒子的摄取量很高,这也可能是高PEI接枝密度金纳米粒子细胞毒性较大的原因。第四章的工作中我们进一步研究了金纳米粒子上PEI的接枝密度、PEI的分子量及巯基含量、以及金纳米粒子的粒径等因素与基因传递性能之间的构效关系。通过改变还原剂的种类和浓度,我们制备了表面被PEI 1800Da修饰的不同粒径的金纳米粒子(GNP-PEI1800)。透射电镜结果显示,硼氢化钠还原制备的GNP-PEI1800的粒径约为6nm,而不同浓度的柠檬酸钠还原的金纳米粒子的粒径分别为19nm、49nm、98nm。根据计算可知,随着金纳米粒子的粒径增加,GNP上所能接上的PEI的量急剧减少,当粒径超过19nm后,由于GNP-PEI1800上PEI的量过少,难以对DNA进行有效的缩合。MTT法测定结果显示接枝密度较低的金纳米粒子毒性与PEI1800Da相近,而接枝密度高的金纳米粒子的毒性也低于PEI25k。凝胶电泳实验证明GNP-PEI1800的复合能力随SH含量和接枝密度的增加而增强：最高接枝密度和SH含量的GNP-PEI1800能在极低的N／P即N／P=0.5时与DNA完全地复合,而最低接枝密度和SH含量的GNP-PEI1800则仅能在N／P=6时使DNA全部滞留在孔中。在当N／P比高于10后,金纳米粒子均可与DNA形成粒径在150nm左右、表面带有正电荷的稳定的复合物。金纳米粒子在转染N／P下均能保护DNA不受核酸降解酶的作用。在无血清培养基中,金纳米粒子在最优N／P下的转染效率都比PEI25k高出一个量级；在含10%血清的培养基中,金纳米粒子的最高转染效率要比PEI25k高出2至3个数量级。与低分子量的GNP-PEI800相比较,虽然细胞毒性稍高,较高分子量的PEI1800修饰的金纳米粒子GNP-PEI1800具有更高的缩合DNA的能力和更高的介导基因转染的效率。更多还原

【Abstract】 The emergence and advances of gene therapy brings expectation of potential cures to diseases such as genetic disorders and cancers that currently lack effective treatments. Therapeutic nucleic acids themselves, however, cannot effectively enter cells. Practical clinical applications of gene therapy have been limited due to lack of efficient transport system.The first chapter of this dissertation gives a systematical yet brief introduction to gene-delivery systems, with special focus on the advances of inorganic nanoparticles for use as gene vectors. An ideal vector for gene therapy must be highly effective, biologically safe, and easy to be prepared in quantity. Many types of inorganic nanoparticles are either biologically inlet or biocompatible. There are well developed procedures for the preparation inorganic nanoparticles with controllable size and structures. The huge area and reactive sites on the surface of inorganic nanoparticles make is feasible to modify the nanoparticle for various functions. Adequately modified inorganic nanoparticles were found to be able to condense nucleic acids and bring them into cell mediating gene transfection. Therefore, reasonable modification of well-defined inorganic nanoparticles is expected to produce novel gene vectors with high efficiency and good biocompatibility.Silica and gold nanoparticles (SiNP and GNP) with controlled size can be easily prepared. They are known to have low toxicity and be readily functionalized on the surface. By covalently attaching polyethylenimine (PEI) on the surface, we have successfully prepared PEI-modified SiNP and GNP, and found them to be very promising gene vectors.In chapter 2, thiol-modified silica nanoparticles (SiNP-SH) were first prepared by reverse microemulsion method. Then, PEI 25 kDa was chemically grafted to the SiNP utilizing SPDP as a coupling reagent that reacts with NH2 groups of PEI and SH groups of silica nanoparticles, affording PEI-modified SiNP (SiNP-PEI25k). Low molecular weight PEI-modified SiNP (SiNP-PEI800) was prepared by another route. First, silica nanoparticles were prepared by Stober method. Then, PEI 800Da was chemically grafted to the SiNP using an expoxy-containing silane coupling reagent that reacts with NH2 groups of PEI and surface OH groups of silica nanoparticles. TEM photos showed that the sizes of the two types of PEI-modified silica nanoparticles are around 100 nm. MTT results shows PEI25k modified SiNP is less toxic than that of pure PEI. Gel retardation assays revealed that SiNP-PEI25k can condense with DNA completely at the relatively low N/P ratio of 2, which means its condense ability is stronger than that of PEI800 modified silica nanoparticles. Both types of the PEI modified silica nanoparticles can provide effective protection of DNA from attack by DNase I. The results of the transfection shows the transfection efficiency of PEI modified silica nanoparticles is lower than that of PEI25K. PEI 800 modified silica nanoparticles SiNP-PEI800 shows moderate efficiency in gene delivery of DNA into HeLa cells in vitro. Even in the presence of 10% serum, PEI modified silica nanoparticles can retain its transfection activity.In chapter 3, gold nanoparticles (GNPs) conjugated with low molecular weight polyethylenimine (PEI 800Da) were prepared and investigated for use as transfection vectors. The polyethylenimine conjugated GNPs (GNP-PEI800s) can retard plasmid DNA completely at N/P ratios above 4 in electrophoresis on agarose gel and they also render effective protection of DNA from attack by DNase. TEM imaging revealed GNP-PEI800s with higher PEI grafting density resulted in more compact and smaller complexes with plasmid DNA, compared to those obtained with lower grafting density ones. These complexes showed high efficiency in gene delivery in monkey kidney cells in vitro. In the absence of serum, GNP-PEI800s can transfect pGL-3 to COS-7 cells 3 to 4 orders more efficient than unmodified PEI800, reaching almost the same magnitude of PEI 25 kDa. More importantly, in contrast to the dramatically lowered efficiency of high molecular weight PEIs such as PEI 25 kDa in the presence of serum, the efficiency of GNP-PEI800s can be retained or even enhanced in serum-containing media. GNP-PEI800 1.3 exhibited transfection efficiency exceeds 60-fold that of PEI 25 kDa in 10% serum medium. All GNP-PEI800s exhibit mild cytotoxicity in comparison with PEI 25 kDa. Chapter 4 presents further investigations of the influence of preparation method, size, chemical composition, grafting density on the properties of GNP-PEIs for gene transfection. PEI with molecular of 1800 Da was used to prepare to PEI-modified GNPs. The GNP sizes were modulated by changing the type and concentration of reduction reagents. TEM photo shows the gold nanoparticles reduced by NaBH4 is around 6 nm and the size of gold nanoparticles reduced by sodium citrate are 19 nm, 49 nm, and 98 nm, respectively. As the increase of size of gold nanoparticles, the grafting density and total PEI content decrease so sharply that they cannot condense the DNA effectively. As a result, the gold nanoparticles used for transfection are all reduced by NaBH4. MTT results showed that the toxicity of gold nanoparticles with low PEI grafting density is as low as that of PEI800. Gold nanoparticles with high PEI grafting density were less toxic than PEI25k. The polyethylenimine conjugated GNP-PEI1800 can retard plasmid DNA completely at N/P ratios above 10 in electrophoresis on agarose gel and they also render effective protection of DNA from attack by DNase. TEM imaging revealed GNP-PEI1800 with higher PEI grafting density resulted in cationic stable complexes with a size of 150 nm. These complexes showed high efficiency in gene delivery in monkey kidney cells in vitro. In the absence of serum, GNP-PEI1800s can transfect pGL3 to COS-7 cells 1 order of magnificent more efficient than PEI25k. More importantly, in contrast to the dramatically lowered efficiency of high molecular weight PEIs such as PEI 25 kDa in the presence of serum, the efficiency of GNP-PEI1800s can be retained or even enhanced in serum-containing media. The highest transfection efficiency achieved by GNP-PEI1800 were around 3 orders of magnificent higher than that of PEI25k in 10% serum medium. The results suggested that GNP-PEI1800 is very promising candidate for in vivo gene delivery.更多还原