Engineering a Dual-Regenerative Cascade: Injectable Hydrogel with Zn2+/l-Arg Cross-Linkers and Macrophage-Targeting Carbon Dots for Synergistic CLI Therapy

Critical limb ischemia (CLI) remains refractory to current revascularization strategies due to its complex pathology. Here, we report an injectable alginate hydrogel that orchestrates a dual-pathway regenerative cascade by simultaneously reprogramming the immune microenvironment and activating direct tissue repair. The system is rationally designed by integrating two functional modules: (i) immunomodulatory carbon dots (PNS-AA@CQDs-COS) that selectively target M1 macrophages and drive them to the M2 phenotype, and (ii) intrinsically bioactive cross-linkers (Zn2+/l-Arg) that serve as sustained-release pro-regenerative signals. Critically, this dual-component architecture enables a synergistic interplay─while the carbon dots establish an anti-inflammatory microenvironment (path 1), the liberated Zn2+ and l-Arg directly activate endothelial cells and muscle satellite cells, thereby initiating coordinated vascular and myogenic regeneration (path 2). In vitro, the hydrogel significantly amplified reparative crosstalk within macrophage-endothelial/myogenic cells’ cocultures. In a murine CLI model, a single injection of the hydrogel resulted in durable recovery of hindlimb perfusion (80.4% by day 14), improved motor function, and enhanced tissue repair. Mechanistic studies revealed that these benefits stem from microenvironment remodeling (M2 polarization and IL-10 upregulation) and modulation of key signaling pathways─suppressing NF-κB while potentiating PI3K/Akt/eNOS. This work presents a material-based strategy that couples immunomodulation with direct cellular activation, offering a synergistic therapeutic platform for CLI and potentially other ischemic diseases.