Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, Florida 32610, USA.
Pharm Res. 2011 Apr;28(4):720-30. doi: 10.1007/s11095-010-0353-x. Epub 2011 Jan 19.
To develop an efficient and safe strategy to introduce a therapeutic gene into target cells in vivo for cancer therapy. The overall efficiency is based on proper selection of the delivery vector and expressed protein.
A plasmid coding for a specific cytotoxic fusion peptide, p14ARF-TAT, was evaluated in a xenograft mouse tumor model. The expressed peptide consisted of three domains, a secretory signal, a membrane permeability segment and a cytotoxic fragment. Gene expression was verified in U87-MG cells by Western blot and cytotoxicity confirmed with CyQuant assay. To improve the delivery, a FGF2 targeting peptide, MQLPLATC, was incorporated into the vector, which was evaluated using a luciferase-expressing plasmid.
The luciferase activity in vitro was two-fold higher with the targeted formulations, and cytotoxicity was three-fold higher with expression of the p14ARF-TAT protein. A murine xenograph model of human glioma (U87-MG cells) tumors was used to address in vivo activity. FGF2-targeted lipoplexes demonstrated increased tumor volume reduction as compared to non-targeted formulations. RT-PCR and Western blot of tumor homogenizes indicated p14ARF-TAT expression in tumors along with other tissues.
p14ARF-TAT was cytotoxic and is a promising approach when combined with an efficient targeting.
开发一种高效、安全的策略，将治疗基因导入体内的靶细胞用于癌症治疗。整体效率基于对递送载体和表达蛋白的正确选择。
在异种移植小鼠肿瘤模型中评估了一种编码特定细胞毒性融合肽 p14ARF-TAT 的质粒。表达的肽由三个结构域组成，一个分泌信号、一个膜通透性片段和一个细胞毒性片段。通过 Western blot 在 U87-MG 细胞中验证基因表达，并通过 CyQuant 测定法确认细胞毒性。为了提高递送效率，将 FGF2 靶向肽 MQLPLATC 整合到载体中，并用表达荧光素酶的质粒进行评估。
体外的荧光素酶活性靶向制剂提高了两倍，p14ARF-TAT 蛋白表达的细胞毒性提高了三倍。用人神经胶质瘤（U87-MG 细胞）肿瘤的小鼠异种移植模型来解决体内活性问题。与非靶向制剂相比，FGF2 靶向脂质体显示出肿瘤体积减少的增加。肿瘤匀浆的 RT-PCR 和 Western blot 分析表明，p14ARF-TAT 在肿瘤及其他组织中表达。
p14ARF-TAT 具有细胞毒性，与高效靶向结合是一种很有前途的方法。
