【摘要】：磁性納米粒子（magnetic nanoparticles, MNPs）具有廣闊的生物醫(yī)學應用前景，包括磁共振造影、細胞標記、藥物/基因載體、腫瘤熱療等。 磁性納米粒子與細胞的相互作用研究是其生物醫(yī)學應用的基礎。近年來，關于納米粒子的細胞內吞研究取得了許多進展。然而，納米粒子的尺寸及表面電荷對內吞的影響尚存在廣泛爭議；相同的細胞對不同理化性質的納米材料的內吞存在著差異性；不同的應用目的對納米粒子的內吞的要求也不同；細胞對MNPs的內吞規(guī)律在體外和體內也存在較大的差異。因此，本文從以下三方面進一步研究了細胞對MNPs的內吞： �。┤朔蜗侔┘毎鸖PC-A1對谷胱甘肽（氧化型谷胱甘肽，Oxidizedglutathione, GSSG）修飾的納米磁粒MNPs-GSSG的生物相容性及內吞規(guī)律研究。研究結果表明MNPs-GSSG生物相容性好，可被SPC-A1細胞高效內吞，且在細胞內能長期滯留。SPC-A1細胞對MNPs-GSSG的內吞是需要能量的、濃度及時間依賴性的內吞。MNPs-GSSG粒子的安全性、細胞內吞的高效性、在細胞內的長期滯留以及細胞內吞量的可控性，對于磁共振造影、細胞標記以及熱療等生物醫(yī)學應用都具有重要的意義。 ⅱ）SPC-A1及WI-38（人胚肺細胞）對氨基硅烷（γ-氨丙基三乙氧基硅烷， γ-Aminopropyl triethoxysilane, APTES）修飾的納米磁粒MNPs-APTES的內吞量比較研究。兩種細胞對MNPs-APTES的內吞量存在巨大差異，且粒子在SPC-A1細胞中可長時間滯留。這對于癌細胞的體內磁共振造影、細胞示蹤及腫瘤熱療具有重要的意義。 ⅲ）SPC-A1對MNPs-GSSG，MNPs-APTES的內吞機制研究。結果表明大小相似的兩種粒子的內吞機制不同，這說明表面修飾比粒徑大小對內吞機制的影響要大。這提示我們可以通過改變MNPs表面修飾來改變其內吞機制以適應不同的應用目的。 近年來非病毒載體由于成本低、制作方便而發(fā)展迅速，然而，如何提高非病毒基因的轉染效率仍然是基因轉染的瓶頸。2002年出現并發(fā)展起來的磁轉染（Magnetofection）技術能夠提高非病毒載體的轉染效率。然而，MNPs與非病毒載體之間究竟采用何種方式構建轉染載體才能有效提高非病毒載體的轉染效率，并沒有統(tǒng)一的準則和指導思想。這部分歸因于磁轉染的機理并不清楚，也就是說構建成的磁轉染復合體各組分在細胞內的命運及在轉染過程中的作用需要進一步研究、闡釋。 本文比較了表面不同電荷特性的的MNPs的磁轉染性能，發(fā)現不論帶正電性的聚乙烯亞胺（Polyethylenimine, PEI）納米磁粒MNPs-PEI，還是帶負電性的檸檬酸（citric acid, CA）納米磁粒MNPs-CA、羧甲基葡聚糖（carboxymethyldextran, CMD）納米磁粒MNPs-CMD，都可以和轉染載體（PEI或脂質體）及pDNA（質粒DNA，plasmid DNA）靠靜電自組裝形成磁轉染復合體（magnetofectins）。靜電自組裝構建的磁轉染復合體能夠提高PEI或脂質體的基因表達水平和/或陽性細胞表達率，且縮短了轉染時間。然而，磁轉染效率具有細胞系依賴性，細胞類別不同，轉染效率差異較大。 本文重點研究了磁轉染復合體的各個組分在細胞內的途徑、命運及作用。通過多種分析表征研究發(fā)現，MNPs在轉染中的作用為將轉染復合體拉到細胞表面，并且在進核之前與PEI/pDNA復合體分離；本文證明自由PEI而不是包覆在MNPs上的PEI對轉染起到重要的作用。本文提出構建轉染復合體的原則為：靜電自組裝的轉染復合體中，，MNPs與PEI/pDNA復合體之間的結合力既要足夠穩(wěn)定能實現磁場力對轉染復合體的操縱，又要使得MNPs與PEI/pDNA復合體在細胞內容易分離。磁轉染機理的闡釋及載體構建原則的提出，對于MNPs的表面修飾及磁轉染載體的構建具有指導意義。
[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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：TB383.1;R318.0

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1 李新新;侯森;馮喜增;;無機納米粒子作為基因載體的研究進展[J];生命科學;2008年03期

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