“Green process” inspires gene delivery: Establishing positive feedback between CO2-enhanced bioactive carrier and gene expression to maximize ECs outputs for multi-pathways CLI therapy

Abstract Gene-engineered vascular endothelial cells have acquired satisfactory ischemia therapy in animal models. However, its clinical process is still severely limited by the hurdle of transfection-structure-toxicity and single therapeutic pathway. To this end, inspired by green process, we innovatively introduced “green” concept into gene delivery. “Green” means fully utilizing carrier and gene via simple, economic and nontoxic process. Herein, the combined strategy of “bioactive carrier” and “CO2-modification” was proposed. On the basis of the cascaded expression from ZNF580 to endothelial nitric oxide synthase (eNOS), we designed cationic peptide based on L-arginine as bioactive carrier to deliver pZNF580. Furthermore, CO2-bubbling was performed to endow bioactive carrier with pH-sensitive nanobomb effect. The carrier dramatically enhanced endo/lysosomal escape via nanobomb effect for high pZNF580 delivery and expression. In turn, carrier changed “enhanced vehicle” identity into NO substrate after completing gene delivery and further was catalyzed by transfection-triggered eNOS overexpression, which greatly amplified NO generation via their synergistic promotion. This simple design obtained dual-high results of ZNF580 (to mediate angiogenesis) and NO (to mediate angiogenesis and anti-inflammation) level. In critical hind limb ischemia (CLI) mice, the strategy simultaneously promoted angiogenesis and mitigated inflammation, which co-worked to recover blood flow and rescue limb. Besides, it was nontoxic to muscle tissue. The multi-pathways achieved significant CLI treatment. Taken together, we established a positive feedback between CO2-enhanced bioactive carrier and target-gene expression via green design and process. This can maximize and expand the output of gene therapy through simplifying the input of gene complexes, thus guaranteeing high transfection, simple structure and low toxicity. We believe that this green strategy will provide a novel viewpoint for gene delivery.