Metformin glycyrrhetinic acid binary injectable hydrogel for synergistic tumor immunotherapy via spatiotemporal microenvironment remodeling

The immunosuppressive tumor microenvironment, orchestrated by tumor cells through multifaceted immune evasion mechanisms, presents a significant challenge to the coordinated regulation of immunocytes. To overcome these limitations, we synthesized metformin glycyrrhetinic acid salt (Met-GA), which exhibits a 189-fold increase in anti-tumor potency compared to free metformin. An injectable Met-GA hydrogel (Met-GA-H) was formed through a "heating-cooling" cycling. To enable in situ gelation at the tumor site, Fe₃O₄ NPs were incorporated into the Met-GA suspension. This combination leverages Fe₃O₄ NPs-mediated photothermal triggering to achieve in situ gelation while enabling multitiered immune cell reprogramming, resulting in a multifunctional hydrogel designated as Fe₃O₄ NPs@Met-GA-H. This Fe₃O₄ NPs@Met-GA-H not only induced immunogenic cell death (ICD), as evidenced by a 4.7-fold increase in extracellular ATP, a 1.5-fold upregulation of calreticulin (CRT) exposure, and a 1.5-fold downregulation of high-mobility group box 1 (HMGB1), but also promoted M1 macrophage polarization (2.3-fold increase compared to control) and downregulated PD-L1 expression (2.8-fold decrease compared to control). It enables synergistic modulation of tumor cells, macrophages, and T lymphocytes within a unified platform. Quantitative biodistribution analysis showed a 13.0-fold higher tumor retention on day 7 post-administration compared to the free drug. The Fe₃O₄ NPs@Met-GA-H demonstrated robust immunotherapeutic efficacy <i>in vivo</i> through sustained ICD activation, macrophage reprogramming, immune checkpoint reactivation, and enhanced infiltration of cytotoxic T lymphocytes. This work establishes a regulatory-compliant precision immunotherapy paradigm based on the rational design of FDA-approved components, achieving synchronized spatiotemporal control over tumor-immune interactions.