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, Fe 3 O 4 NPs were incorporated into the Met-GA suspension. This combination leverages Fe 3 O 4 NPs-mediated photothermal triggering to achieve in situ gelation while enabling multitiered immune cell reprogramming, resulting in a multifunctional hydrogel designated as Fe 3 O 4 NPs@Met-GA-H. This Fe 3 O 4 NPs@Met-GA-H not only induced immunogenic cell death (ICD), as evidenced by 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 Fe 3 O 4 NPs@Met-GA-H demonstrated robust immunotherapeutic efficacy in vivo 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. Schematic illustration of Fe 3 O 4 NPs@Met-GA-H-mediated immunomodulation and tumor suppression. This combinatorial strategy achieves robust immunogenic cell death (ICD) with concomitant DAMPs release, polarization shift from immunosuppressive M2 to tumoricidal M1 TAMs phenotype, and significant suppression of PD-L1, establishing a self-reinforcing immunosurveillance loop. • Metformin glycyrrhetinic acid salts self-assemble into a binary injectable hydrogel that is carrier-free and achieves 100% theoretical drug loading. • The incorporation of Fe 3 O 4 NPs not only enables in situ gelation but also mediates photothermally triggered on-demand pulsatile drug release. • The injectable hydrogel simultaneously induces immunogenic cell death, promotes macrophage repolarization, and downregulates PD-L1 expression. • All components comprising the gel are clinically validated, significantly facilitating its clinical translation.