Gupta Ajay Kumar, Gupta Mona, Yarwood Stephen J, Curtis Adam S G
Centre for Cell Engineering, IBLS, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
J Control Release. 2004 Mar 5;95(2):197-207. doi: 10.1016/j.jconrel.2003.11.006.
The aim of present study was to prepare nanometer sized particles of gelatin via water-in-oil microemulsion system for drug and gene delivery applications. In this study, cross-linked gelatin nanoparticles encapsulating a fluorescent marker molecule fluorescein isothiocyanate-dextran (FITC-Dex, Mol. Wt. 19.3kDa) have been prepared, characterized and their influence on human fibroblasts has been assessed in terms of cell adhesion, cytotoxicity, light microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and observation of cytoskeleton organisation. Gelatin nanoparticles were prepared inside the aqueous cores of sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/n-hexane reverse micelles. Transmission electron microscopy image showed that the particles are spherical in shape with size of 37+/-0.84 nm diameter. The release of FITC-Dex from the nanoparticles in phosphate buffer saline (pH 7.4) is found to increase with time and about 80% of the encapsulated dye is released in 6 h. Cell adhesion studies with human fibroblasts have shown that gelatin nanoparticles do not affect the number of cells adhered to glass as compared to control cells with no particles. Standard cell viability assay demonstrated that cells incubated with gelatin nanoparticles remained more than 100% viable at concentration as high as 500 microg/ml. From SEM image, it was observed that the nanoparticles were internalised and the fibroblasts exhibited vacuoles in the cell body with cell membrane abnormalities. Endocytosis of nanoparticles was confirmed from TEM studies and it resulted in disruption of F-actin and beta-tubulin cytoskeleton. These studies show that the gelatin nanoparticles prepared by water-in-oil microemulsion systems are endocytosed by the fibroblasts without being toxic to cells even at high concentration of nanoparticles.
本研究的目的是通过油包水微乳液体系制备用于药物和基因递送应用的纳米级明胶颗粒。在本研究中，制备了包裹荧光标记分子异硫氰酸荧光素 - 葡聚糖（FITC - Dex，分子量19.3 kDa）的交联明胶纳米颗粒，对其进行了表征，并从细胞粘附、细胞毒性、光学显微镜、扫描电子显微镜（SEM）、透射电子显微镜（TEM）以及细胞骨架组织观察等方面评估了它们对人成纤维细胞的影响。明胶纳米颗粒在双（2 - 乙基己基）磺基琥珀酸钠（AOT）/正己烷反胶束的水核内制备。透射电子显微镜图像显示颗粒呈球形，直径大小为37±0.84 nm。发现在磷酸盐缓冲盐水（pH 7.4）中，FITC - Dex从纳米颗粒中的释放随时间增加，约80%的包封染料在6小时内释放。用人成纤维细胞进行的细胞粘附研究表明，与没有颗粒的对照细胞相比，明胶纳米颗粒不影响粘附在玻璃上的细胞数量。标准细胞活力测定表明，在高达500 μg/ml的浓度下，与明胶纳米颗粒一起孵育的细胞存活率仍超过100%。从SEM图像观察到纳米颗粒被内化，成纤维细胞在细胞体内呈现液泡且细胞膜异常。TEM研究证实了纳米颗粒的内吞作用，并且它导致F - 肌动蛋白和β - 微管蛋白细胞骨架的破坏。这些研究表明，通过油包水微乳液体系制备的明胶纳米颗粒即使在高浓度下也能被成纤维细胞内吞且对细胞无毒。
