ZnPPIX-Loaded Nanoemulsions Reprogram Immunosuppressive Macrophages in Vitro: A Potential Strategy for Glioblastoma Microenvironment Modulation

Background: Glioblastoma (GBM) is a highly malignant primary brain tumor with an overall survival of less than 15 months, despite aggressive treatment. While immunotherapy has shown success in other cancers, it has been largely ineffective in GBM, partly because of the immunosuppressive microenvironment driven by bone marrow-derived macrophages (BMDMs). This immunosuppressive milieu hinders the efficacy of both standard and immune therapies, and highlights the urgent need for novel targeted approaches. Nanomedicine offers promising solutions by enabling selective targeting of tumor-promoting cells, especially in the challenging tumor microenvironment (TME) of GBM. We previously demonstrated that the inhibition of heme-oxygenase-1 (HO-1), an enzyme central to iron metabolism, has the potential to reprogram BMDMs towards a more pro-inflammatory and anti-tumor phenotype. Methods: Using a microfluidic-based approach, we successfully developed an oil-in-water (O/W) nanoemulsion loaded with the HO-1 inhibitor, zinc protoporphyrin IX (NE-ZnPPIX), with optimal characteristics for therapeutic application. The intrinsic fluorescence of ZnPPIX allowed the tracking of its cellular uptake. Functional assays including phenotypic marker analysis and suppression assays were performed to assess the immunomodulatory effects of NE-ZnPPIX on macrophages. Results: Studies on cells derived from GBM patients revealed that NE-ZnPPIX could be selectively internalized by immunosuppressive myeloid cells (particularly BMDMs), malignant cells within the TME, and circulating monocytes while showing minimal uptake by lymphocytes. Our study demonstrated that encapsulated ZnPPIX, similar to free ZnPPIX, effectively reduced the immunosuppressive activity of in vitro-derived macrophages. Furthermore, NE-ZnPPIX showed superior efficacy in modulating macrophage activity by decreasing the expression of CD163, a marker associated with the pro-tumoral immunosuppressive phenotype. Conclusion: Our findings suggest that NE-ZnPPIX may represent a novel and effective strategy for remodeling the immunosuppressive TME of GBM in combination with established immune-stimulating therapies. This approach has the potential to enhance the efficacy of immunotherapy by overcoming the tumor immune evasion mechanisms.