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.

Efficient siRNA delivery and gene silencing using a lipopolypeptide hybrid vector mediated by a caveolae-mediated and temperature-dependent endocytic pathway

“…These factors including positive surface charge and small particle size must have contributed to the increased transfection efficiency. Our results were supported by other studies indicating that the amount of positive charges on the cell surface and the size of the vector/oligonucleotide complex played important roles in determining successful gene delivery [41–43]. A positively charged structure is desirable since it can preferentially adhere to the negatively charged cell surface receptors, leading to endocytosis.…”

Virus Mimetic Shell-Sheddable Chitosan Micelles for siVEGF Delivery and FRET-Traceable Acid-Triggered Release

“…Therefore, clinical attentions have been largely focused on anti-angiogenesis by inhibiting VEGF to achieve anti-tumor treatment . A number of studies − have shown that small interfering RNA (siRNA) targeting VEGF (siVEGF) has great potential in the treatment of tumors by blocking VEGF genes. However, it is challenging to efficiently deliver siRNA to tumors via systemic administration, with the barriers including poor stability, low cellular uptake, endosomal degradation, and off-target effects.…”