磁性纳米粒子的细胞内吞及基因转染研究

发布时间：2018-09-11 09:03

【摘要】：磁性纳米粒子（magnetic nanoparticles, MNPs）具有广阔的生物医学应用前景，包括磁共振造影、细胞标记、药物/基因载体、肿瘤热疗等。磁性纳米粒子与细胞的相互作用研究是其生物医学应用的基础。近年来，关于纳米粒子的细胞内吞研究取得了许多进展。然而，纳米粒子的尺寸及表面电荷对内吞的影响尚存在广泛争议；相同的细胞对不同理化性质的纳米材料的内吞存在着差异性；不同的应用目的对纳米粒子的内吞的要求也不同；细胞对MNPs的内吞规律在体外和体内也存在较大的差异。因此，本文从以下三方面进一步研究了细胞对MNPs的内吞： ⅰ）人肺腺癌细胞SPC-A1对谷胱甘肽（氧化型谷胱甘肽，Oxidizedglutathione, GSSG）修饰的纳米磁粒MNPs-GSSG的生物相容性及内吞规律研究。研究结果表明MNPs-GSSG生物相容性好，可被SPC-A1细胞高效内吞，且在细胞内能长期滞留。SPC-A1细胞对MNPs-GSSG的内吞是需要能量的、浓度及时间依赖性的内吞。MNPs-GSSG粒子的安全性、细胞内吞的高效性、在细胞内的长期滞留以及细胞内吞量的可控性，对于磁共振造影、细胞标记以及热疗等生物医学应用都具有重要的意义。 ⅱ）SPC-A1及WI-38（人胚肺细胞）对氨基硅烷（γ-氨丙基三乙氧基硅烷， γ-Aminopropyl triethoxysilane, APTES）修饰的纳米磁粒MNPs-APTES的内吞量比较研究。两种细胞对MNPs-APTES的内吞量存在巨大差异，且粒子在SPC-A1细胞中可长时间滞留。这对于癌细胞的体内磁共振造影、细胞示踪及肿瘤热疗具有重要的意义。 ⅲ）SPC-A1对MNPs-GSSG，MNPs-APTES的内吞机制研究。结果表明大小相似的两种粒子的内吞机制不同，这说明表面修饰比粒径大小对内吞机制的影响要大。这提示我们可以通过改变MNPs表面修饰来改变其内吞机制以适应不同的应用目的。近年来非病毒载体由于成本低、制作方便而发展迅速，然而，如何提高非病毒基因的转染效率仍然是基因转染的瓶颈。2002年出现并发展起来的磁转染（Magnetofection）技术能够提高非病毒载体的转染效率。然而，MNPs与非病毒载体之间究竟采用何种方式构建转染载体才能有效提高非病毒载体的转染效率，并没有统一的准则和指导思想。这部分归因于磁转染的机理并不清楚，也就是说构建成的磁转染复合体各组分在细胞内的命运及在转染过程中的作用需要进一步研究、阐释。本文比较了表面不同电荷特性的的MNPs的磁转染性能，发现不论带正电性的聚乙烯亚胺（Polyethylenimine, PEI）纳米磁粒MNPs-PEI，还是带负电性的柠檬酸（citric acid, CA）纳米磁粒MNPs-CA、羧甲基葡聚糖（carboxymethyldextran, CMD）纳米磁粒MNPs-CMD，都可以和转染载体（PEI或脂质体）及pDNA（质粒DNA，plasmid DNA）靠静电自组装形成磁转染复合体（magnetofectins）。静电自组装构建的磁转染复合体能够提高PEI或脂质体的基因表达水平和/或阳性细胞表达率，且缩短了转染时间。然而，磁转染效率具有细胞系依赖性，细胞类别不同，转染效率差异较大。本文重点研究了磁转染复合体的各个组分在细胞内的途径、命运及作用。通过多种分析表征研究发现，MNPs在转染中的作用为将转染复合体拉到细胞表面，并且在进核之前与PEI/pDNA复合体分离；本文证明自由PEI而不是包覆在MNPs上的PEI对转染起到重要的作用。本文提出构建转染复合体的原则为：静电自组装的转染复合体中，，MNPs与PEI/pDNA复合体之间的结合力既要足够稳定能实现磁场力对转染复合体的操纵，又要使得MNPs与PEI/pDNA复合体在细胞内容易分离。磁转染机理的阐释及载体构建原则的提出，对于MNPs的表面修饰及磁转染载体的构建具有指导意义。
[Abstract]:Magnetic nanoparticles (MNPs) have a wide range of biomedical applications, including magnetic resonance imaging, cell markers, drug/gene carriers, tumor hyperthermia and so on.
The interaction between magnetic nanoparticles and cells is the basis of their biomedical applications. In recent years, many advances have been made in the study of endocytosis of nanoparticles. There are differences in the endocytosis of MNPs in vitro and in vivo. Therefore, the endocytosis of MNPs by cells is further studied in the following three aspects:
_) Biocompatibility and endocytosis of human lung adenocarcinoma cell line SPC-A1 to glutathione (oxidized glutathione, GSSG) modified magnetic nanoparticles MNPs-GSSG were studied. The results showed that MNPs-GSSG had good biocompatibility, could be efficiently endocytozed by SPC-A1 cells and could be retained in cells for a long time. Endocytosis is energy-dependent, concentration-and time-dependent. The safety of MNPs-GSSG particles, the high efficiency of endocytosis, the long-term retention in cells and the controllability of endocytosis are of great significance for the biomedical applications such as magnetic resonance imaging, cell markers and hyperthermia.
II) Comparison of endocytosis of SPC-A1 and WI-38 (human embryonic lung cells) MNPs-APTES nanoparticles modified by gamma-aminopropyl triethoxysilane (APTES). The endocytosis of the two kinds of cells to MNPs-APTES was significantly different, and the particles could remain in SPC-A1 cells for a long time. In vivo magnetic resonance imaging, cell tracing and hyperthermia are of great significance.
(iii) The endocytosis mechanism of MNPs-GSSG and MNPs-APTES was studied by SPC-A1. The results showed that the endocytosis mechanism of the two particles with similar size was different, which indicated that the surface modification had greater influence on the endocytosis mechanism than the particle size. This suggested that the endocytosis mechanism could be changed by changing the surface modification of MNPs to adapt to different application purposes.
In recent years, non-viral vectors have developed rapidly because of their low cost and convenient preparation. However, how to improve the transfection efficiency of non-viral genes is still the bottleneck of gene transfection. There is no uniform criterion and guiding ideology on how to construct transfection vectors to effectively improve the transfection efficiency of non-viral vectors. This is partly due to the unclear mechanism of magnetic transfection, that is to say, the fate of each component of the magnetic transfection complex in cells and its role in the transfection process need to be further studied and elucidated. Release.
In this paper, the magnetic transfection properties of MNPs with different surface charge characteristics were compared. It was found that MNPs-CMD with positive polyethylenimine (PEI) nanoparticles, negative citric acid (CA) nanoparticles and carboxymethyldextran (CMD) nanoparticles could be combined with MNPs-PEI with negative citric acid (CA) nanoparticles. Transfection vectors (PEI or liposomes) and pDNA (plasmid DNA, plasmid DNA) form magnetofectins by electrostatic self-assembly. Magnetic transfection complexes constructed by electrostatic self-assembly can improve the gene expression level and/or positive cell expression rate of PEI or liposomes, and shorten the transfection time. However, magnetic transfection efficiency has cell lines. Depending on the cell types, the transfection efficiency varies greatly.
In this paper, we focus on the intracellular pathway, fate and role of the various components of the magneto-transfection complex. It is found that the role of MNPs in transfection is to pull the transfection complex onto the cell surface and separate it from the PEI/pDNA complex before it enters the nucleus. The principle of constructing transfection complexes is that the binding force between MNPs and PEI/pDNA complexes in electrostatically self-assembled transfection complexes should be stable enough to manipulate the transfection complexes by magnetic field, and the MNPs and PEI/pDNA complexes should be easily separated in cells. The explanations and the principles of vector construction are of guiding significance to the surface modification of MNPs and the construction of magnetic transfection vector.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：TB383.1;R318.0

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