A cationic cholesterol based nanocarrier for the delivery of p53-EGFP-C3 plasmid to cancer cells.
A cationic cholesterol based nanocarrier for the delivery of p53-EGFP-C3 plasmid to cancer cells.
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DOI：
10.1016/j.biomaterials.2013.10.062
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发表时间：
2014
期刊：
影响因子：
14
通讯作者：
Dr. Santoshi Misra;S. Naz;P. Kondaiah;S. Bhattacharya
中科院分区：
文献类型：
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作者：
Dr. Santoshi Misra;S. Naz;P. Kondaiah;S. Bhattacharya
P53蛋白介导的抗肿瘤策略由于缺乏合适的递送剂而受到限制，这些递送剂具有免疫原性不明显、血清配伍以及早期和容易检测到转基因细胞群的特点。为了克服这些问题，我们构建了一种P53-EGFP-C3融合载体，它可以表达易于检测到的绿色荧光蛋白(GFP)，并可以评估P53介导的抗肿瘤活性。将阳离子胆固醇Gemini(Chol-5L)与天然脂类药物(摩尔比为1：4)混合，通过各种物理方法表征，形成了纳米脂质体。将所制备的克隆在HeLa和另外两个具有不同P53状态的细胞系H1299(P53−/−)和HEK293T(P53+/+)中检测绿色荧光蛋白和功能性P53的表达。用RT-PCR、Western blotting、流式细胞仪、四甲基偶氮唑蓝、台盼蓝等方法筛选细胞，并在荧光显微镜下观察。DNA片段化、细胞周期分析、Annexin-V染色和PARP裂解实验表明，P53-EGFP-C3融合蛋白可诱导癌细胞凋亡。无论所研究的细胞系，Chol-5LD的转染率和凋亡诱导效率均显着高于商品化试剂LipofeTamine2000和Effectene。此外，通过H&E染色观察到裸鼠移植瘤的体积明显减少，肿瘤通过细胞凋亡而减少。
The p53 protein mediated anti-tumor strategy is limited due to the lack of suitable delivery agent with insignificant immunogenic response, serum compatibility, and early and easy detection of the transfected cell population. To overcome these problems, we generated a p53-EGFP-C3 fusion construct which expressed easily detectable green fluorescence protein (GFP) and allowed an estimation of p53 mediated anti-tumor activity. A mixture of cationic cholesterol gemini (Chol-5L) with natural lipid, DOPE (molar ratio 1:4), acronymed as Chol-5LD, formed a nano-liposome as characterized by various physical methods. The prepared clone was evaluated for the expression of GFP and functional p53 in HeLa and two additional cell lines with varied p53 status namely, H1299 (p53−/−) and HEK293T (p53+/+). Transfected cells were screened using RT-PCR, Western blotting, FACS analysis, MTT, Trypan blue assay and visualized under a fluorescence microscope. The p53-EGFP-C3 fusion protein induced apoptosis in cancer cells as evident from DNA fragmentation, cell cycle analysis, Annexin-V staining and PARP cleavage assays. The transfection and apoptosis induction efficiency of Chol-5LD was significantly higher than commercial reagents Lipofectamine2000 and Effectene irrespective of the cell lines examined. Further it significantly decreases the xenograft tumor volume in nude mice tumorsviaapoptosis as observed in H&E staining.