A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene delivery system.
A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene delivery system.
复制标题
非病毒PHEMA+壳聚糖纳米层介导的高效基因输送系统。
DOI：
10.2147/ijn.s43168
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发表时间：
2013
影响因子：
8
通讯作者：
Singh SR
中科院分区：
文献类型：
--
作者：
Eroglu E;Tiwari PM;Waffo AB;Miller ME;Vig K;Dennis VA;Singh SR
由于细胞膜屏障的存在，DNA进入真核细胞的转运是最小的，这限制了DNA疫苗、基因沉默和基因治疗的应用。几种可用的转染试剂和技术已被用来规避这个问题。或者，非病毒纳米级载体已被证明绕过真核细胞膜。在目前的工作中，我们开发了一种独特的纳米材料，pHEMA+壳聚糖纳米球（PCNS），它由聚（2-羟乙基甲基丙烯酸酯）纳米球包围壳聚糖阳离子壳，我们用它来封装呼吸道合胞病毒（RSV）-F基因构建体（DNA疫苗的模型）。这种新的纳米材料能够在不使用商业转染试剂的情况下转染各种真核细胞系。使用透射电子显微镜（TEM），荧光激活细胞分选（FACS），和免疫荧光，我们清楚地表明，带正电荷的PCNS能够结合到带负电荷的细胞膜，并采取了内吞作用，在Cos-7细胞。使用定量聚合酶链反应（qPCR），我们还评估了与PCNS和不使用脂质体为基础的转染介质，在Cos-7，HEp-2，和Vero细胞的转染效率。为了评估PCNS在体内的转染效率，将这些含有RSV-F基因的新型纳米材料肌内注射到BALB/c小鼠中，导致转基因的高拷贝数。在这项研究中，我们报告，第一次，应用PCNS作为纳米载体的基因传递在体外和体内。
The transport of DNA into eukaryotic cells is minimal because of the cell membrane barrier, and this limits the application of DNA vaccines, gene silencing, and gene therapy. Several available transfection reagents and techniques have been used to circumvent this problem. Alternatively, nonviral nanoscale vectors have been shown to bypass the eukaryotic cell membrane. In the present work, we developed a unique nanomaterial, pHEMA+chitosan nanospheres (PCNSs), which consisted of poly(2-hydroxyethyl methacrylate) nanospheres surrounded by a chitosan cationic shell, and we used this for encapsulation of a respiratory syncytial virus (RSV)-F gene construct (a model for a DNA vaccine). The new nanomaterial was capable of transfecting various eukaryotic cell lines without the use of a commercial transfection reagent. Using transmission electron microscopy, (TEM), fluorescence activated cell sorting (FACS), and immunofluorescence, we clearly demonstrated that the positively charged PCNSs were able to bind to the negatively charged cell membrane and were taken up by endocytosis, in Cos-7 cells. Using quantitative polymerase chain reaction (qPCR), we also evaluated the efficiency of transfection achieved with PCNSs and without the use of a liposomal-based transfection mediator, in Cos-7, HEp-2, and Vero cells. To assess the transfection efficiency of the PCNSs in vivo, these novel nanomaterials containing RSV-F gene were injected intramuscularly into BALB/c mice, resulting in high copy number of the transgene. In this study, we report, for the first time, the application of the PCNSs as a nanovehicle for gene delivery in vitro and in vivo.