Wan Yu, Dai Wei, Nevagi Reshma J, Toth Istvan, Moyle Peter M
School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia 4072, Queensland, Australia; School of Pharmacy, The University of Queensland, Woolloongabba 4102, Queensland, Australia.
School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia 4072, Queensland, Australia.
Acta Biomater. 2017 Sep 1;59:257-268. doi: 10.1016/j.actbio.2017.06.032. Epub 2017 Jun 24.
The development of carriers for the delivery of oligonucleotide therapeutics is essential for the successful translation of gene therapies to the clinic. In the present study, a delivery system, which combines the fusogenic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) with a well-defined synthetic multifunctional peptide, was produced and optimized for gene delivery, with the aim to develop an efficient gene delivery platform for breast cancer cells. For this purpose, a breast cancer-specific cell targeting peptide (CTP) was incorporated into our leading peptide-based gene delivery system (to generate DEN-K(GALA)-TAT-K(STR)-CTP) to improve its cell-specific internalization, and investigated in combination with a formulation approach (DOPE/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)). DEN-K(GALA)-TAT-K(STR)-CTP alone efficiently complexed with DNA or siRNA, and promoted efficient cellular uptake, but low levels of gene expression. By adding the formulation approach, synergistic improvements in gene expression and silencing were observed compared to the peptide or formulation approaches alone. Of significance, this current system demonstrated more efficient gene knockdown when compared to the leading commercial siRNA delivery agent Lipofectamine® RNAiMAX. The utility of this system was demonstrated through the delivery of BCL2 (B-cell lymphoma 2) siRNA to MCF-7 cells, which led to near complete knockdown of the Bcl-2 protein, and inhibition of MCF-7 cell migration in a wound healing assay. The present peptide/lipid hybrid system is an excellent candidate for the delivery of DNA or siRNA into breast cancer cells.
The identification of safe and effective delivery systems for DNA and siRNA is of great importance. Herein, we developed a well-defined, multifunctional and cell-specific lipidic peptide DEN-K(GALA)-TAT-K(STR)-CTP as a breast cancer cell targeted gene delivery vector. When combined with a lipid component (DOTAP/DOPE), the peptide/lipid hybrid system demonstrated higher gene expression or knockdown levels compared to the peptide or lipid approach alone when used to deliver pDNA or siRNA respectively, indicating synergistic enhancement of gene delivery efficiency. Importantly, this delivery strategy achieved greater knockdown of the Bcl-2 protein when compared to the leading commercial siRNA delivery system Lipofectamine® RNAiMAX, suggesting its potential utility for the targeted treatment of Bcl-2 overexpressing breast cancers.
开发用于递送寡核苷酸疗法的载体对于将基因疗法成功转化至临床至关重要。在本研究中，构建并优化了一种将融合脂质1,2 - 二油酰基 - sn - 甘油 - 3 - 磷酸乙醇胺（DOPE）与一种明确的合成多功能肽相结合的递送系统用于基因递送，旨在为乳腺癌细胞开发一个高效的基因递送平台。为此，将一种乳腺癌特异性细胞靶向肽（CTP）整合到我们基于先导肽的基因递送系统中（以生成DEN - K(GALA) - TAT - K(STR) - CTP）以改善其细胞特异性内化，并与一种配方方法（DOPE/1,2 - 二油酰基 - 3 - 三甲基铵丙烷（DOTAP））联合进行研究。单独的DEN - K(GALA) - TAT - K(STR) - CTP能有效地与DNA或siRNA复合，并促进高效的细胞摄取，但基因表达水平较低。通过添加配方方法，与单独使用肽或配方方法相比，观察到基因表达和沉默有协同改善。重要的是，与领先的商业siRNA递送剂Lipofectamine® RNAiMAX相比，该系统目前显示出更有效的基因敲低。通过将BCL2（B细胞淋巴瘤2）siRNA递送至MCF - 7细胞证明了该系统的实用性，这导致Bcl - 2蛋白几乎完全敲低，并在伤口愈合试验中抑制了MCF - 7细胞迁移。目前的肽/脂质杂合系统是将DNA或siRNA递送至乳腺癌细胞的极佳候选者。
鉴定安全有效的DNA和siRNA递送系统非常重要。在此，我们开发了一种明确的、多功能且细胞特异性的脂质肽DEN - K(GALA) - TAT - K(STR) - CTP作为靶向乳腺癌细胞的基因递送载体。当与脂质成分（DOTAP/DOPE）联合使用时，该肽/脂质杂合系统在分别用于递送pDNA或siRNA时，与单独使用肽或脂质方法相比，显示出更高的基因表达或敲低水平，表明基因递送效率有协同增强。重要的是，与领先的商业siRNA递送系统Lipofectamine® RNAiMAX相比，这种递送策略实现了对Bcl - 2蛋白更大程度的敲低，表明其在靶向治疗Bcl - 2过表达乳腺癌方面的潜在效用。
