A Ternary Synergistic eNOS Gene Delivery System Based on Calcium Ion and L-Arginine for Accelerating Angiogenesis by Maximizing NO Production.

Purpose: This study aimed to construct a delivery system based on L-arginine-modified calcium phosphate (CaP) to load eNOS plasmids (peNOS), which could amply nitric oxide (NO) to repair endothelial damage, promote angiogenic activities and alleviate inflammation. Methods: pDNA-loaded CaP nanocomplex (CaP/pDNA) were prepared by co-precipitation method, subsequently modified by L-arginine. The gene transfection efficiency, pro-angiogenic and anti-inflammatory ability were investigated in vivo and in vitro. The therapeutic effect on ischemic hindlimb in vivo was assessed. Results: L-arginine modification augmented the transfection efficiency of CaP/peNOS to elevate the eNOS expression, and then served as NO substrate catalyzed by eNOS. At the same time, calcium ions produced by degradation of CaP carriers enhanced the activity of eNOS. In vitro experiments, the loading capability and transfection performance of R(L)-CaP were confirmed to be superior to that of CaP. Additionally, HUVECs treated with R(L)-CaP/peNOS showed the strongest NO release, cell migration, tube formation and the lowest inflammatory levels compared to the CaP/peNOS and R(D)-CaP/peNOS groups. We also demonstrated the advantages of R(L)-CaP/peNOS in increasing blood reperfusion in hindlimb ischemia mice by accelerating angiogenesis and reducing inflammation, which can be attributed to the highest eNOS-derived NO production. Conclusion: The combination strategy of peNOS transfection, L-arginine supplement and calcium ions addition is a promising therapeutic approach for certain vascular diseases, based on the synergistic NO production.