Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

Abstract

Small interfering RNA (siRNA) delivery is a prospective method in gene therapy, but it has application limitations such as negative charge, water solubility and high molecular weight. In this study, a safe and efficient nano-vector, CRS, was designed and synthesized to facilitate siRNA delivery. Physical and chemical properties of VEGF-siRNA/CRS were characterized by methods including scanning electron microscopy (SEM), transmission electron microscopy, zeta potential (ζ) measurement, drug-releasing rate measurement, gel electrophoresis and confocal microscopy. The biological activities were evaluated using cell viability assay, gene-silencing efficacy assay in vitro, real-time polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA) and antitumor tests in vivo. The mean nanoparticle size of VEGF-siRNA/CRS was 121.4±0.3 nm with positive ζ potential of 7.69±4.47 mV. The release rate of VEGF-siRNA from VEGF-siRNA/CRS was 82.50% sustained for 48 h in Tris-ethylenediaminetetraacetic acid buffer (pH 8.0). Real-time polymerase chain reaction was used to analyze the efficiency of the transfection, and the result showed that VEGF mRNA expression had been knocked down by 82.36%. The expression of VEGF protein was also recorded to be downregulated to 14.83% using ELISA. The results of cytotoxicity measured by Cell Counting Kit-8 assay showed that VEGF-siRNA/CRS had significant inhibitory effect on HeLa cells. The results of antitumor assays indicated that VEGF-siRNA/CRS exhibited tumor cell growth inhibition in vivo. The results demonstrated that VEGF-siRNA could be delivered and transported by the designed carrier, while siRNA could be released constantly and led to an increasing gene-silencing effect against VEGF gene. In conclusion, VEGF-siRNA/CRS is a promising carrier for siRNA delivery, and further studies are warranted.

Keywords:

• CRS
• siRNA delivery
• VEGF
• tumor therapy
• antitumor
• biophysical characteristics
• gene silencing
• protein expression

Acknowledgments

This work was supported by the National Natural Science Foundation (81502688), the Basic-Clinical Key Research Grant (16JL72) from Capital Medical University, the Importation and Development of High-Caliber Talents Project of Beijing Municipal Institutions (2013–2015), Natural Science Foundation of Capital Medical University (2015ZR14), China’s 55th Postdoctoral Scientific Research Funds (2014M550768) and the Principal Fund of Capital Medical University (16GLY09). The authors gratefully acknowledge the support from Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, and Beijing Laboratory of Biomedical Materials.

Disclosure

The authors report no conflicts of interest in this work.