Engineering a Dual-Regenerative Cascade: Injectable Hydrogel with Zn²⁺/l-Arg Cross-Linkers and Macrophage-Targeting...

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.