RNAi-mediated gene knockdown and anti-angiogenic therapy of RCCs using a cyclic RGD-modified liposomal-siRNA system

Abstract

Angiogenesis is one of crucial processes associated with tumor growth and development, and consequently a prime target for cancer therapy. Although tumor endothelial cells (TECs) play a key role in pathological angiogenesis, investigating phenotypical changes in neovessels when a gene expression in TEC is suppressed is a difficult task. Small interfering RNA (siRNA) represents a potential agent due to its ability to silence a gene of interest. We previously developed a system for in vivo siRNA delivery to cancer cells that involves a liposomal-delivery system, a MEND that contains a unique pH-sensitive cationic lipid, YSK05 (YSK-MEND). In the present study, we report on the development of a system that permits the delivery of siRNA to TECs by combining the YSK-MEND and a ligand that is specific to TECs. Cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) is a well-known ligand to α V β 3 integrin, which is selectively expressed at high levels in TECs.We incorporated cRGD into the YSK-MEND (RGD-MEND) to achieve an efficient gene silencing in TECs. Quantitative RT-PCR and the 5' rapid amplification of cDNA ends PCR indicated that the intravenous injection of RGD-MEND at a dose of 4.0 mg/kg induced a significant RNAi-mediated gene reduction in TEC but not in endothelial cells of other organs. Finally, we evaluated the therapeutic potency of the RGD-MEND encapsulating siRNA against vascular endothelial growth factor receptor 2. A substantial delay in tumor growth was observed after three sequential RGD-MEND injections on alternate days. In conclusion, the RGD-MEND represents a new approach for the characterization of TECs and for us in anti-angiogenic therapy.

Going beyond the liver: Progress and challenges of targeted delivery of siRNA therapeutics

“…The increased expression of α V β 3 integrin on tumor endothelial cells was exploited for delivery of siRNA through a cationic lipid system with cyclic RGD grafted onto its surface [128]. In vitro, the integrin presence was essential for successful uptake and gene silencing, and in vivo, the targeted system resulted in siRNA-mediated gene silencing in tumor endothelial cells, but not in other endothelia.…”

Remodeling of the Extracellular Matrix by Endothelial Cell-Targeting siRNA Improves the EPR-Based Delivery of 100 nm Particles

“…Thus, the prepared RGD-MEND was able to deliver siRNA specifically to TECs. 15 When the LNPs were fluorescently labeled, a lipophilic dye, DiI or DiD was added to the lipid mixture prior to the first dilution. The recovery rate and encapsulation efficiency of siRNA were determined by RiboGreen.…”

“…We previously confirmed that an RGD-MEND encapsulating si-VR2 caused a delay in tumor growth by decreasing the density of microvessels in tumor tissue. 15 On the other hand, a continuous treatment (3 separate injections of the RGD-MEND was started prior to the first injection of DOX-LNP) and the results indicated a more moderate therapeutic effect (not data shown). This can be attributed to short-lived silencing by the RGD-MEND (< 72 hours).…”

“…14 Owing to these functional devices, the RGD-MEND had the ability to inhibit a TEC gene at a dose of 0.75 mg siRNA/kg. 11,15 We investigated the alteration in the intratumoral distribution of nano drug delivery systems (DDSs) and the tumor microenvironment after vasculature maturation via the inhibition of VEGFR2 on TECs by the RGD-MEND. Our results suggest that vasculature leakiness as the result of immature vessels is not necessarily required for the extravasation of LNPs, at least in hypervascular cancer, and that carefully controlling the tumor microenvironment, including ECMs, has the potential for maximizing the therapeutic effect of nanoparticles.…”

Efficient Packaging of Plasmid DNA Using a pH Sensitive Cationic Lipid for Delivery to Hepatocytes

“…7) The YSK-MEND effectively delivered siRNA to the liver, 8) tumors 9) and tumor endothelial cells. 10) This type of YSK-MEND would also be predicted to have potential for pDNA delivery.…”