磁性納米粒子的細(xì)胞內(nèi)吞及基因轉(zhuǎn)染研究
發(fā)布時(shí)間：2018-09-11 09:03
【摘要】：磁性納米粒子（magnetic nanoparticles, MNPs）具有廣闊的生物醫(yī)學(xué)應(yīng)用前景，包括磁共振造影、細(xì)胞標(biāo)記、藥物/基因載體、腫瘤熱療等。 磁性納米粒子與細(xì)胞的相互作用研究是其生物醫(yī)學(xué)應(yīng)用的基礎(chǔ)。近年來(lái)，關(guān)于納米粒子的細(xì)胞內(nèi)吞研究取得了許多進(jìn)展。然而，納米粒子的尺寸及表面電荷對(duì)內(nèi)吞的影響尚存在廣泛爭(zhēng)議；相同的細(xì)胞對(duì)不同理化性質(zhì)的納米材料的內(nèi)吞存在著差異性；不同的應(yīng)用目的對(duì)納米粒子的內(nèi)吞的要求也不同；細(xì)胞對(duì)MNPs的內(nèi)吞規(guī)律在體外和體內(nèi)也存在較大的差異。因此，本文從以下三方面進(jìn)一步研究了細(xì)胞對(duì)MNPs的內(nèi)吞： ⅰ）人肺腺癌細(xì)胞SPC-A1對(duì)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(nèi)吞規(guī)律研究。研究結(jié)果表明MNPs-GSSG生物相容性好，可被SPC-A1細(xì)胞高效內(nèi)吞，且在細(xì)胞內(nèi)能長(zhǎng)期滯留。SPC-A1細(xì)胞對(duì)MNPs-GSSG的內(nèi)吞是需要能量的、濃度及時(shí)間依賴性的內(nèi)吞。MNPs-GSSG粒子的安全性、細(xì)胞內(nèi)吞的高效性、在細(xì)胞內(nèi)的長(zhǎng)期滯留以及細(xì)胞內(nèi)吞量的可控性，對(duì)于磁共振造影、細(xì)胞標(biāo)記以及熱療等生物醫(yī)學(xué)應(yīng)用都具有重要的意義。 ⅱ）SPC-A1及WI-38（人胚肺細(xì)胞）對(duì)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(nèi)吞量比較研究。兩種細(xì)胞對(duì)MNPs-APTES的內(nèi)吞量存在巨大差異，且粒子在SPC-A1細(xì)胞中可長(zhǎng)時(shí)間滯留。這對(duì)于癌細(xì)胞的體內(nèi)磁共振造影、細(xì)胞示蹤及腫瘤熱療具有重要的意義。 ⅲ）SPC-A1對(duì)MNPs-GSSG，MNPs-APTES的內(nèi)吞機(jī)制研究。結(jié)果表明大小相似的兩種粒子的內(nèi)吞機(jī)制不同，這說(shuō)明表面修飾比粒徑大小對(duì)內(nèi)吞機(jī)制的影響要大。這提示我們可以通過(guò)改變MNPs表面修飾來(lái)改變其內(nèi)吞機(jī)制以適應(yīng)不同的應(yīng)用目的。 近年來(lái)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(zhuǎn)染效率仍然是基因轉(zhuǎn)染的瓶頸。2002年出現(xiàn)并發(fā)展起來(lái)的磁轉(zhuǎn)染（Magnetofection）技術(shù)能夠提高非病毒載體的轉(zhuǎn)染效率。然而，MNPs與非病毒載體之間究竟采用何種方式構(gòu)建轉(zhuǎn)染載體才能有效提高非病毒載體的轉(zhuǎn)染效率，并沒(méi)有統(tǒng)一的準(zhǔn)則和指導(dǎo)思想。這部分歸因于磁轉(zhuǎn)染的機(jī)理并不清楚，也就是說(shuō)構(gòu)建成的磁轉(zhuǎn)染復(fù)合體各組分在細(xì)胞內(nèi)的命運(yùn)及在轉(zhuǎn)染過(guò)程中的作用需要進(jìn)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(zhuǎn)染性能，發(fā)現(xiàn)不論帶正電性的聚乙烯亞胺（Polyethylenimine, PEI）納米磁粒MNPs-PEI，還是帶負(fù)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(zhuǎn)染載體（PEI或脂質(zhì)體）及pDNA（質(zhì)粒DNA，plasmid DNA）靠靜電自組裝形成磁轉(zhuǎn)染復(fù)合體（magnetofectins）。靜電自組裝構(gòu)建的磁轉(zhuǎn)染復(fù)合體能夠提高PEI或脂質(zhì)體的基因表達(dá)水平和/或陽(yáng)性細(xì)胞表達(dá)率，且縮短了轉(zhuǎn)染時(shí)間。然而，磁轉(zhuǎn)染效率具有細(xì)胞系依賴性，細(xì)胞類別不同，轉(zhuǎn)染效率差異較大。 本文重點(diǎn)研究了磁轉(zhuǎn)染復(fù)合體的各個(gè)組分在細(xì)胞內(nèi)的途徑、命運(yùn)及作用。通過(guò)多種分析表征研究發(fā)現(xiàn)，MNPs在轉(zhuǎn)染中的作用為將轉(zhuǎn)染復(fù)合體拉到細(xì)胞表面，并且在進(jìn)核之前與PEI/pDNA復(fù)合體分離；本文證明自由PEI而不是包覆在MNPs上的PEI對(duì)轉(zhuǎn)染起到重要的作用。本文提出構(gòu)建轉(zhuǎn)染復(fù)合體的原則為：靜電自組裝的轉(zhuǎn)染復(fù)合體中，，MNPs與PEI/pDNA復(fù)合體之間的結(jié)合力既要足夠穩(wěn)定能實(shí)現(xiàn)磁場(chǎng)力對(duì)轉(zhuǎn)染復(fù)合體的操縱，又要使得MNPs與PEI/pDNA復(fù)合體在細(xì)胞內(nèi)容易分離。磁轉(zhuǎn)染機(jī)理的闡釋及載體構(gòu)建原則的提出，對(duì)于MNPs的表面修飾及磁轉(zhuǎn)染載體的構(gòu)建具有指導(dǎo)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(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：TB383.1;R318.0
本文編號(hào)：2236238
[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(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：TB383.1;R318.0
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 李新新;侯森;馮喜增;;無(wú)機(jī)納米粒子作為基因載體的研究進(jìn)展[J];生命科學(xué);2008年03期
本文編號(hào)：2236238
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