Characterization of iRGD-Ligand Modified Arginine-Histidine-Rich Peptides for Nucleic Acid Therapeutics Delivery to αvβ3 Integrin-Expressing Cancer Cells.

Anna Egorova,Sergei Selkov,Alexander Selutin,Marianna Maretina,Anton Kiselev,Vladislav Baranov

doi:10.3390/ph13100300

Abstract

Efficient and specific delivery of nucleic acid (NA) therapeutics to tumor cells is extremely important for cancer gene therapy. Various therapeutic strategies include delivery of DNA-therapeutics such as immunostimulatory or suicide genes and delivery of siRNA-therapeutics able to silence expression of cancer-related genes. Peptides are a promising class of non-viral vehicles which are biodegradable and can efficiently condense, protect and specifically deliver NA to the cells. Here we designed arginine-histidine-rich peptide carriers consisting of an iRGD ligand to target αvβ3 integrins and studied them as vehicles for DNA and siRNA delivery to cancer cells. Combination of iRGD-modified and unmodified arginine–histidine-rich peptides during NA complexation resulted in carriers with different ligand contents. The NA-binding and protecting properties in vitro transfection efficiency and cytotoxicity of the DNA- and siRNA-polyplexes were studied and the most efficient carrier RGD1 was determined. The ability of the peptides to mediate specific intracellular uptake was confirmed inhuman cervical carcinoma (HeLa), human kidney (293T) and human pancreatic (PANC-1) cell lines with different αvβ3 integrins surface expression. By means of RGD1 carrier, efficient delivery of the herpes simplex virus (HSV-1) thymidine kinase gene to PANC-1 cells was demonstrated. Subsequent ganciclovir treatment led to a reduction of PANC-1 cells’ viability by up to 54%. Efficient RNAi-mediated down-regulation of GFP and VEGFA gene expression was achieved in MDA-MB-231-GFP+ breast cancer and EA.hy926 endothelial cells, respectively, by means of RGD1/siRNA polyplexes. Here we demonstrated that the peptide carrier RGD1 can be considered as promising candidate for development of NA therapeutics delivery systems useful in cancer gene therapy.

Highlights

Gene therapy is an attractive approach to treat diseases by compensating for gene defects or regulating gene expression by silencing unwanted genes
We designed arginine-histidine-rich peptide carriers consisting of an iRGD ligand to target αvβ3 integrins and studied them as vehicles for DNA and siRNA delivery to cancer cells
DNA- and siRNA carriers were developed by cyclization of a linear iRGD ligand sequence conjugated with arginine-rich peptide

Summary

Introduction

Gene therapy is an attractive approach to treat diseases by compensating for gene defects or regulating gene expression by silencing unwanted genes. The significant research area in gene therapy is related to the development of strategies for cancer treatment [1]. An alternative approach—cancer gene therapy uses various therapeutic strategies that include immunostimulatory gene and vaccine delivery, suicide gene therapy (DNA delivery), anti-angiogenic therapy aimed at silencing of pro-angiogenic gene expression (siRNA delivery) [3,4]. Viral vectors are the most efficient gene delivery vehicles and play an important role in gene therapy [8]. The relatively small capacity for therapeutic NAs, the difficulty in receiving good quality formulations and safety concerns about viruses prompt the development of alternative systems to viral vectors [1]

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Conclusion