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.

基因工程的血管内皮细胞已在动物模型中获得令人满意的缺血治疗。然而，其临床过程仍然受到转染结构毒性和单一治疗途径的障碍的严重限制。为此，在绿色过程的启发下，我们创新地将“绿色”概念引入基因传递。“绿色”是指通过简单，经济和无毒的过程充分利用载体和基因。在此，提出了“生物活性载体”与“ CO ₂修饰”的组合策略。基于从ZNF580到内皮型一氧化氮合酶（eNOS）的级联表达，我们设计了基于L-精氨酸作为生物活性载体的阳离子肽，以递送pZNF580。此外，CO ₂进行鼓泡以赋予生物活性载体以pH敏感的纳米炸弹效应。载体通过纳米炸弹作用显着增强了内/溶酶体逃逸，从而实现了高pZNF580的传递和表达。反过来，载体在完成基因传递后将“增强的载体”身份改变为NO底物，并进一步被转染触发的eNOS过表达催化，这通过其协同促进作用大大放大了NO的产生。这种简单的设计获得了ZNF580（介导血管生成）和NO（介导血管生成和抗炎）水平的双高结果。在严重的后肢缺血（CLI）小鼠中，该策略同时促进了血管生成并减轻了炎症，从而共同恢复了血流并拯救了肢体。此外，它对肌肉组织无毒。多路径实现了显着的CLI处理。综上所述，我们在CO之间建立了积极的反馈通过绿色设计和过程增强了₂型生物活性载体和靶基因的表达。通过简化基因复合物的输入，可以最大化和扩大基因疗法的输出，从而确保高转染，简单的结构和低毒性。我们认为，这种绿色策略将为基因传递提供新的观点。