In Vitro And Vivo Study On RGD-immobilized Chitosan Nanoparticles-mediated VEGF165 Gene In The Treatment Of Limb Ischemia

Background and ObjectiveGene therapy has been developed as a potential cure for the treatment of peripheral arterial diseases(PAD).However,viral vectors such as retrovirus and adenovirus have serious safety concerns such as potential oncogenicity,toxicity and immunogenicity. Non-viral strategies have been developed as an alternative for gene delivery,but also have their limitation in low transfection efficiency and poor target ability.Chitosan(CH) is a polycationic biopolymer,and characterized as cheap, environmental-friendly production,good bacteriostatic effects and high biocompatibility.Studies showed it could serve as a non-virus gene vector.However, the efficiency and target ability still needed to improve.Arg-Gly-Asp(RGD) peptides were found in extracellular matrix,and could promote attachment of cells as it could be recognized by the adhesion receptors on the cell membrane.Endothelia cells from tumor or neo-vasulariztion could specifically express some kind of RGD containing peptides,such asα_vβ₃,and supposed to enhance the affinity between cells to materials.In this study,we tried to investigate the target ability and gene transfer-efficiency of chitosan nanoparticles modified with RGD as a new vector with plasmid DNA in vitro, and the therapy effect with plasmid vascular endothelial growth factor 165(pVEGF165) for angiogenic gene therapy in ischemic limbs of mice.Methods1.Nanoparticles of chitosan(CH) and chitosan linked with RGD(CH-RGD) were made by complex coacervation method,and were labeled with fluorescent isothiocyanate (FITC).With Hy926 cultured cell line system,the adhesion and endocytosis of cells to the compounds were assessed with flow cytometry and confocal laser microscopy, respectively.Using a report gene,pEGFP,CH-RGD-pEGFP nano-gene transfer system was compared to CH-pEGFP nano-gene transfer system and another polycationic polymer,lipofetamine2000,in the transfection efficiency with Hy926 cells in vitro.And then using a therapeutic gene,humen pVEGF₁₆₅,the CH-RGD-pVEGF165 nano-gene transfer system,CH-pVEGF165 nano-gene transfer system and lipofetamine2000-pVEGF165 gene transfer system were transfected with Hy926 cells in vitro,then compared the VEGF expression in the supernatant detected with the ABC-ELISA method and the number of living cells 1 day,3,7 and 14 days after transfection.2.We made mice hindlimb ischemia models and divided them into four groups in random,CH-RGD-pVEGF165 nano-gene system,CH-pVEGF165 nano-gene system, pVEGF165 and equal dose sterilized distilled water were separately intramuscular injected into the ischemic limbs.The gastrocnemius of ischemic limbs were made to paraffin sections for HE stained,PCIMA and VEGF immunohistochemistry stained to observe the capillary density and VEGF expression in 7,14 and 28 days after treatment.Real-Time PCR method and western blot method were also used to observed the VEGF expression in the ischemic limbs after treatment.Results1.In vitro:The adhesion and endocytosis via Chitosan-RGD-nanoparticles were much better than Chitosan-nanoparticles,expressing as more fluorescent positive cells 99.51%vs. 95.57%(p>0.05),and more intensity of fluorescent 132.47 vs.72.28(p<0.05). Report gene system showed CH-RGD-pEGFP had a significant high transfection efficiency compared with CH-pEGFP,35.7%vs.14.3%(p<0.001),and a little high transfection efficiency compared with lipofetamine2000-pEGFP,35.7%vs 31.3%, (p>0.05).The CH-RGD-pVEGF165 also had a significant high VEGF expression in the supernatant compared with CH-pVEGF165 and lipofetamine2000-pVEGF165 in 1 day,3,7 and 14 days after transfection.The cytotoxicitiy of CH-RGD nanoparticles were significant lower than Iipofetamine2000 by caculating the living cells(p<0.01).2.In vivo:The capillary density were significant higher in CH-RGD-pVEGF165 group than the other three groups(p<0.01),and the muscular cells were not severely ischemic changed.Both VEGF mRNA and VEGF expression were significant high in CH-RGD-pVEGF165 group than the other three groups(p<0.01),which were detected by Real-Time PCR method and western blot method separately.CH-RGD-pVEGF165 group had no limb necrosis.Conclusion1.Chitosan nanoparticles modified with RGD could improve the adhesion and endocytosis of cells in vitro,and increase gene transfection efficiency.2.CH-RGD-pVEGF165 nano-gene system had the potential advances of target ability and therapeutic effect in the gene therapy for the treatment of limb ischemia.3.CH-RGD nanoparticles showed low cytotoxicity and high biocompatibility as gene vector in vitro and in vivo.