Soluble Flt-1 gene delivery using PEI-g-PEG-RGD conjugate for anti-angiogenesis

Abstract

Angiogenesis is defined as the formation of new blood vessels from pre-existing microvessels. In particular, tumor growth and metastasis was found to be dependent on angiogenesis and hence, anti-angiogenic therapy has become a promising strategy for cancer treatment. Anti-angiogenic therapy is used to inhibit tumor growth and metastasis by destroying neighboring blood vessels that supply tumor cells with oxygen and nutrients and also provide an exit route for tumors to enter the bloodstream. An angiogenic factor, VEGF, stimulates endothelial cell proliferation, migration and tube formation via the interaction with VEGF receptors, Flt-1 (fms-like tyrosine kinase-1) and / or flk-1/KDR (fetal liver kinase-1/kinase domain). The intervention to block VEGF action has been accomplished by a variety of methods including antibodies directed against its cognate receptors. One novel method to inhibit the angiogenic action of VEGF is the administration of soluble Flt-1 (sFlt-1), a potent, and selective inhibitor of VEGF. We have previously reported an angiogenic endothelial cell-targeted polymeric gene delivery system, PEI-g-PEG-RGD, developed by incorporating the avb5 integrin-binding RGD peptide, ACDCRGDCFC (single-letter amino acid code), into the cationic polymer, polyethyleneimine (PEI) via hydrophilic polyethylene glycol (PEG) spacer. These findings predicted that the gene carrier, PEI-g-PEG-RGD, might deliver genes coding sFlt-1 into an angiogenic endothelial cells expressing the avb5 integrins on their surface with a site-specific manner. In this study, we constructed the therapeutic gene coding sFlt-1 (pCMVsFlt-1) and evaluated the effect of PEI-g-PEG-RGD / therapeutic gene for an ant-iangiogenic therapy. To confirm the efficacy of the transgene, the transfected sFlt-1 should suppress the VEGF-driven proliferation of endothelial cells. An endothelial inhibition assay was performed using CADMECs. The PEI-g-PEG-RGD/pCMV-sFlt-1 complex inhibited the CADMEC proliferation by 63% compared with non-treated control thus confirming the PEI-g-PEG-RGD-mediated secretion of functionally active soluble Flt-1. In this report, we have used a genetic approach for expression of sFlt-1 that is an endogenously expressed, potent VEGF antagonist. We have evaluated the function of therapeutic gene carrier with an inhibition of endothelial cell proliferation assay. The complex of therapeutic gene and PEI-g-PEG-RGD conjugate efficiently inhibits proliferation of endothelial cells by blocking the binding of VEGF to the membrane bound Flt-1 receptor. The use of a non-viral gene carrier to deliver an antiangiogenic gene can demonstrate a low continuous dosage through repeated injections which can be applied for future clinical use.