Fabrication and Application of a Dual-Layer Polymeric Microneedle Patch for Immunotherapy: Controlled Nitric Oxide Release via Cerium -based Metal-Organic Framework to Induce Immunogenic Cell Death

Triple-negative breast cancer (TNBC) poses a significant clinical challenge due to its high invasiveness and recurrence rate. The cellular heterogeneity and metastatic potential of TNBC often render traditional surgical resection and chemotherapy ineffective, leading to treatment failure and relapse. In recent years, immunotherapy strategies that induce immunogenic cell death (ICD) through high-dose nitric oxide (NO) have emerged as promising approaches. In this study, we combined immunotherapy with microneedle (MN) patch technology to deliver therapeutic agents locally and non-invasively to the tumor site, aiming to enhance therapeutic efficacy while minimizing systemic side effects.A bilayer MN patch was fabricated using gelatin and methacrylated gelatin (GelMA), where GelMA is synthesized by modifying gelatin with methacrylic anhydride (MA). The backing layer of the patch was composed of a mixture of carboxymethyl cellulose (CMC) and polyvinylpyrrolidone (PVP). By compartmentalizing different therapeutic agents within distinct microneedle layers, a synergistic therapeutic effect was achieved.The successful synthesis of S-nitrosoglutathione (GSNO) and cerium-based metal-organic framework (Ce-MOF) was confirmed by Fourier-transform infrared spectroscopy (FTIR). The morphology, crystalline structure, and dispersion stability of Ce-MOF were further characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), dynamic light scattering (DLS), and zeta potential analysis. GSNO, NH₂-UiO-66(Ce), and doxorubicin (DOX) were separately loaded into the bilayer MN patch, and their drug release behaviors were evaluated. The gelatin layer facilitated rapid drug release, while the photo-crosslinkable GelMA layer enabled sustained release. Ce-MOF catalyzed NO release from GSNO in a concentration- and time-dependent manner, maintaining stable catalytic activity for at least four weeks. The bilayer design further enhanced NO release efficiency by spatially separating Ce-MOF and GSNO.Biocompatibility tests confirmed that both the microneedle patch and Ce-MOF exhibited excellent cytocompatibility. In 4T1 breast cancer cells, Ce-MOF effectively catalyzed NO generation from GSNO, significantly increasing intracellular NO levels. Co-treatment with DOX further amplified reactive oxygen species (ROS) production. The triple-combination group (MOF+GSNO+DOX) demonstrated the most pronounced cytotoxic and apoptotic effects, along with robust induction of damage-associated molecular patterns (DAMPs), including calreticulin (CRT) translocation to the cell membrane and high-mobility group box 1 (HMGB-1) release from the nucleus, indicating strong ICD induction.In summary, this study successfully developed a bilayer MN patch capable of synergistically inducing ROS and DAMPs release. In vivo studies using a mouse tumor model further validated that the triple-combination treatment effectively suppressed tumor growth and significantly enhanced CD4⁺ and CD8⁺ T cell infiltration in the spleen. Immunohistochemical analysis of tumor sections further revealed that MN-patch treatment promoted M1 macrophage polarization, dendritic cell maturation, and the accumulation of CD4⁺ and CD8⁺ T cells within the tumor, demonstrating that this strategy can effectively enhance intratumoral effector immune responses.