Centre for Cellular and Molecular Biology, Council for Scientific and Industrial Research, Uppal Road, Hyderabad 500007, Andhra Pradesh, India.
Biomaterials. 2012 Mar;33(8):2570-82. doi: 10.1016/j.biomaterials.2011.12.003. Epub 2011 Dec 20.
Cell-penetrating peptide (CPP)-based delivery systems represent a strategy that facilitates DNA import efficiently and non-specifically into cells. To introduce specificity, we devised an approach that combines a cell-penetrating peptide, TAT-Mu (TM) and a targeting ligand, an HER2 antibody mimetic-affibody (AF), designated as TMAF to deliver nucleic acids into the cells. In this study, we synthesized TMAF protein and its truncated versions, i.e. MAF and AF, by expressing the corresponding plasmids in Escherichia coli BL21(DE3)pLysS cells. Purified TMAF binds DNA efficiently and protects plasmid DNA from DNaseI action. Transfection of HER2+ breast cancer cell lines MDA-MB-453, SK-OV-3, SK-BR-3 and an ovarian cancer cell line with plasmid DNA pCMVβ-gal, resulted in enhanced β-galactosidase activity when compared to control MDA-MB-231 cells. Maximal activity observed in MDA-MB-453 cells at DNA:TMAF:Protamine sulphate (PS) corresponding to 1:8:2 charge ratios. Further the observed gene transfection was resistant to serum, sensitive to the presence of free AF and non-toxic. Variants of TMAF although non-toxic, were far less efficient indicating the effective role of the TAT and Mu domains. The observed DNA uptake and reporter gene activity mediated by TMAFin vitro could be linked with the cell-surface density of tyrosine kinase receptor HER2 (ErbB2) levels estimated by Western blot. Further, we confirmed the efficacy of DNA transfer by TMAF protein in xenograft mouse models using MDA-MB-453 cells. Expression of β-galactosidase as the reporter gene, upon intratumoral injection of DNA, in complex with TMAF, lends credence to specific DNA import and distribution within the tumor tissue that was attributed to high HER2 receptor overexpression in MDA-MB-453 cells. Through delivery of anti-TF hshRNA: TMAF: PS complex, we demonstrate specific knockdown of tissue factor (TF) in MDA-MB-453 cells in vitro. Most importantly, in a xenograft mouse model, we observe significant (P<0.05) and specific reduction of tumor volume when anti-TF hshRNA: TMAF: PS complex was injected intratumorally. Collectively our data indicate that AF-based chimeric peptides with nucleic acid binding properties may provide an effective tumor specific strategy to deliver therapeutic nucleic acids.
细胞穿透肽（CPP）为基础的递药系统代表了一种策略，可有效地将 DNA 非特异性地导入细胞。为了引入特异性，我们设计了一种方法，将细胞穿透肽 TAT-Mu（TM）与靶向配体，即 HER2 抗体模拟-affibody（AF）结合，称为 TMAF，用于将核酸递送至细胞内。在这项研究中，我们通过在大肠杆菌 BL21（DE3）pLysS 细胞中表达相应的质粒，合成了 TMAF 蛋白及其截断形式，即 MAF 和 AF。纯化的 TMAF 可有效地结合 DNA，并保护质粒 DNA 免受 DNA 酶 I 的作用。用质粒 DNA pCMVβ-gal 转染 HER2+乳腺癌细胞系 MDA-MB-453、SK-OV-3、SK-BR-3 和卵巢癌细胞系 MDA-MB-231 后，与对照 MDA-MB-231 细胞相比，β-半乳糖苷酶活性增强。在 DNA：TMAF：鱼精蛋白硫酸盐（PS）的电荷比为 1：8：2 时，观察到 MDA-MB-453 细胞中的最大活性。此外，观察到的基因转染对血清有抗性，对游离 AF 的存在敏感，且无毒性。虽然 TMAF 的变体没有毒性，但效率要低得多，这表明 TAT 和 Mu 结构域的有效作用。TMAF 在体外介导的 DNA 摄取和报告基因活性可与通过 Western blot 估计的酪氨酸激酶受体 HER2（ErbB2）水平的细胞表面密度相关联。此外，我们通过使用 MDA-MB-453 细胞的异种移植小鼠模型证实了 TMAF 蛋白在 DNA 转移中的功效。在肿瘤内注射 DNA 与 TMAF 形成复合物后，β-半乳糖苷酶作为报告基因的表达，使我们相信 DNA 能够特异性地导入肿瘤组织并在其中分布，这归因于 MDA-MB-453 细胞中高表达组织因子（TF）受体。通过递送抗 TF hshRNA：TMAF：PS 复合物，我们证明了在 MDA-MB-453 细胞中特异性地敲低组织因子（TF）。最重要的是，在异种移植小鼠模型中，当抗 TF hshRNA：TMAF：PS 复合物被肿瘤内注射时，我们观察到肿瘤体积的显著（P<0.05）和特异性减少。总的来说，我们的数据表明，具有核酸结合特性的基于 AF 的嵌合肽可能为递送至治疗性核酸提供一种有效的肿瘤特异性策略。
