Reoxygenating the Tumor Microenvironment with Biomimetic Hemoglobin Nanoparticles Enhances Chemo, CAR-T, and Gene Therapies 2260007

Abstract Introduction Tumor hypoxia diminishes antitumor immunity by stabilizing HIF-1α, promoting M2-like macrophage polarization, and impairing cytotoxic T-cell activity. To reverse these effects, we developed hemoglobin-loaded biomimetic nanoparticles (Hb-BNPs) using a modified nanoprecipitation strategy. The nanoparticles encapsulate hemoglobin within a polycaprolactone matrix and are cloaked with RBC/A549 membranes to provide immune camouflage and sustained oxygen release. This study evaluates their ability to reoxygenate the tumor microenvironment and restores immune and therapeutic responses in non-small-cell lung carcinoma (NSCLC). Methods Hb-BNPs were synthesized via modified nanoprecipitation and membrane cloaking. Physicochemical features were assessed by DLS, TEM. Hypoxia was induced in A549 cells, followed by Hb-BNP treatment. qRT-PCR, paclitaxel IC50 assays, 3D spheroids, LunX CAR-T cytotoxicity, and KANK1-transfection studies evaluated immunologic and therapeutic responses (all n = 3, ANOVA/t). Results Under hypoxia, HIF1A, VEGF, BNIP3, ENO1, HK1, PGK1 were upregulated by 5—8-fold (p < 0.01). After Hb-BNP reoxygenation, these genes were downregulated by 4—6-fold (p < 0.001), and dissolved oxygen increased 5.3-fold (p < 0.001). Oxygen recovery improved the immune function as LunX CAR-T cytotoxicity increased 2.3-fold (p < 0.01), and KANK1-driven transgene expression increased 2.7-fold (p < 0.05). Co-treatment with paclitaxel reduced IC50 from 25,612 ng/mL to 781 ng/mL (p < 0.0001) and enhanced 3D spheroid core cell death 2.4-fold (p < 0.01). Hb-BNPs did not elevate IL-6, TNF-α, or IL-8 in THP-1 macrophages, confirming immune tolerance. Conclusion Hb-BNPs reoxygenate hypoxic tumors, suppress HIF-1α signaling, and restore cytotoxic immune function without inducing inflammation. By normalizing oxygen balance, these biomimetic nanoparticles enhance CAR-T activity, gene transfection, and chemotherapy response, offering a scalable, immune-tolerant platform to overcome hypoxia-driven resistance in solid tumors. Funding Source N/A Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)