The preparation of superparamagnetic Fe3O4/SiO2­-polyethyleneimine composite particles and their applications in gene delivery

doi:10.3969/j.issn.2095-4344.2014.16.018

Abstract

Abstract:

BACKGROUND: It is vital to choose the appropriate carrier with low toxicity and high gene transfection efficiency in gene therapy, which is harmless to human body and environment.

OBJECTIVE: To prepare superparamagnetic Fe₃O₄/SiO₂-polyethyleneimine (PEI) composite particles.

METHODS: Fe₃O₄ nanoparticles were prepared via an emulsion solvent evaporation method and superparamagnetic Fe₃O₄/SiO₂ core shell microspheres were prepared successfully subsequently via a modified stober method. The microspheres were further modified with PEI to obtain superparamagnetic Fe₃O₄/SiO₂-PEI composite particles. The structures and properties of resultant composite particles microspheres were characterized by transmission electron microscopy, zeta potential and vibrating sample magnetometer. Superparamagnetic Fe₃O₄/SiO₂-PEI composite particles were mixed with plasmid DNA at different mass ratios (29∶1, 39∶1, 49∶1, 59∶1, 68∶1, 78∶1, 88∶1). The in vitro gene transfection ability was evaluated by Hela cells with the transfection of plasmid DNA encoded with green fluorescent protein and the transfection efficiency was determined by confocal fluorescence microscopy.

RESULTS AND CONCLUSION: We successfully synthesized the Fe₃O₄/SiO₂-PEI composite particles with good dispersibility and even size distribution (about 100 nm). The surface charge was 21.07 mV, and the saturation magnetization was 28.05 emu/g that meant superparamagnetism. When the mass ratio was 59∶1, all the plasmid DNA was adherent to the Fe₃O₄/SiO₂-PEI composite particles; when the mass ratio was > 59∶1, there were excessive Fe₃O₄/SiO₂-PEI composite particles. Therefore, the mass ratio of 59:1 could lead to a better outcome for HeLa cell transfection. These results indicate that the Fe₃O₄/SiO₂-PEI composite particles can dramatically improve the transfection efficiency of plasmid DNA compared with PEI.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: microspheres, green fluorescent proteins, transfection

CLC Number:

R318

Zhang Qing-yun, Li Rong-rong, Deng Gui-ru, Zhu Hui, Yang Xiao-ying. The preparation of superparamagnetic Fe₃O₄/SiO₂-polyethyleneimine composite particles and their applications in gene delivery[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(16): 2570-2575.

Figures/Tables 2

2.1 Fe3O4/SiO2核壳型微球的制备及表征如图1所示，采用化学沉积法合成Fe3O4纳米粒子，平均粒径在20 nm以下；通过乳化溶剂挥发法制备了Fe3O4纳米粒子聚集体，该聚集体的粒径在80 nm以下；经吐温-80修饰该Fe3O4纳米粒子聚集体后，采用改进的stober法，在碱性环境下催化正硅酸四乙酯水解制备 Fe3O4/SiO2核壳结构微球，该微球的分散性良好，粒径均在100 nm以下，透射电镜表示二氧化硅成功的包裹在Fe3O4聚集体表面。由于SiO2的具有良好的生物稳定性和亲水性，使得复合微球的生物稳定性和相容性变的更好，整个壳层不容易被破坏，同时也更易于修饰。 Fe3O4/SiO2微球经丁二酸酐修饰后后，得到羧基化的Fe3O4/SiO2，Zeta电位检测表明其表面电荷为-29.04 mV，与阳离子聚合物聚乙酰亚胺反应后得到的 Fe3O4/SiO2-聚乙酰亚胺复合微球的Zeta电位为+21.07 mV，这说明阳离子聚合物聚乙酰亚胺与Fe3O4/SiO2微球表面的羧基阴离子(-COO-)通过静电引力相互作用，聚乙酰亚胺已经成功的修饰到Fe3O4/SiO2微球的氧化硅壳层表面上。在微球表面引入亲水性阳离子聚合物聚乙酰亚胺不仅提供了运输和解离基因的能力，也提高了在水中的稳定性。纳米粒子容易发生聚集和团聚，难以形成稳定的分散体系，磁性纳米粒子还具备强烈的各向异性的偶极距相互作用，更容易团聚。因此，制备分散性良好的磁性纳米粒子是磁性纳米材料研究领域不可避免的话题。克服这一问题常采用纳米粒子包裹修饰，这样就有效阻止了粒子之问的团聚，增加了分散稳定性。经典的stober法是用于制备无机-无机复合材料的方法之一。在醇溶剂中，将SiO2包裹在一定量的金属粒子表面，利用氨水作催化剂及形貌控制剂，催化TEOS发生水解和缩合反应，生成的二氧化硅沉积在粒子表面，得到核壳结构的复合粒子。通过改变反应氨水、种子和TEOS含量可以改变复合颗粒的形貌及尺寸。早在1992年，Hmori等[31]就利用TEOS的水解在锭子状α-Fe2O3表面覆盖了一层均匀厚度的二氧化硅。此法制得的二氧化硅磁性复合微球兼具磁性纳米粒子优异的磁性和二氧化硅的生物相容性和稳定性，在生物学和药学方面有着广泛的应用。而包裹上二氧化硅的最大优点是能够很容易地在二氧化硅表面连接上所需要的基团。实验通过活化羧基Fe3O4/SiO2微球与聚乙酰亚胺发生酰胺化反应，从而成功地将聚乙酰亚胺修饰到Fe3O4/SiO2微球上，微球带正电荷，为其通过静电作用吸附pDNA进而将其转运到靶向部位并成功表达相应产物奠定了基础。 2.2 磁性复合微球的磁性能的测试图2磁滞回线所示，Fe3O4纳米粒子的饱和磁化强度为61.48 emu/g，SiO2包裹后磁性纳米粒子的饱和磁化强度下降为30.48 emu/g；Fe3O4/SiO2-聚乙酰亚胺的饱和磁化强度为28.05 emu/g。3种粒子的矫顽力基本保持一致，均趋近于零，几乎没有磁滞，表明其均具有较好的超顺磁性。这个研究结果和以往的文献结果类似，当包裹上非磁性材料后，Fe3O4磁性纳米粒子的饱和磁化强度均表现出下降。 2.3 体外转染实验 2.3.1 电泳实验由图3显示：随着聚合物微粒与pDNA质量比的不断增加，越来越多的pDNA质粒被吸附在Fe3O4/SiO2-聚乙酰亚胺复合微球上，此时pDNA质粒过量，当质量比达到59∶1时，几乎所有的pDNA质粒都被留在凝胶中如第5孔，表明在此条件下几乎所有的pDNA质粒都被吸附在Fe3O4/SiO2-聚乙酰亚胺复合微球上；当质量比大于59∶1时，Fe3O4/SiO2-聚乙酰亚胺复合微球过量，这说明二者存在强烈而有效的作用力。由此可见，聚合物微粒与pDNA的质量比为59∶1时结果较好，在此条件下，所有的pDNA质粒正好被吸附在所有Fe3O4/SiO2-聚乙酰亚胺复合微球上，二者均无过量，因此将使用这一比例计算负载量以指导转染实验。 2.3.2 转染实验通过Fe3O4/SiO2-聚乙酰亚胺复合微球与聚乙酰亚胺对HeLa细胞绿色荧光蛋白基因转染能力的比较，可清晰地看出图4A中无载体转染的绿色荧光蛋白，几乎无绿色存在；图4B显示Fe3O4/SiO-聚乙酰亚胺复合微球转染绿色荧光蛋白的图像，绿色荧光明显，细胞存活状况良好；图4C显示聚乙酰亚胺转染的绿色荧光蛋白，能看到少量绿色荧光，但细胞凋亡明显。由此说明当Fe3O4/SiO2-聚乙酰亚胺复合微球与pDNA 质粒的质量比为410∶1时Fe3O4/SiO2-聚乙酰亚胺复合微球有好的转染效率，这可能是由于Fe3O4/SiO2-聚乙酰亚胺复合微球表面剩余的正电荷与绿色荧光蛋白表面的负电荷之间的静电作用使之形成更为紧密的复合物，这些结果显示Fe3O4/SiO2-聚乙酰亚胺复合微球能使pDNA的转染效率显著增强，并且Fe3O4/SiO2-聚乙酰亚胺复合微球具有一定的生物安全性。同时与聚乙酰亚胺相比，Fe3O4-SiO2-聚乙酰亚胺复合微球可显著提高pDNA的转染效率。"

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The preparation of superparamagnetic Fe₃O₄/SiO₂-polyethyleneimine composite particles and their applications in gene delivery

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