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Res."],"published-print":{"date-parts":[[2025,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Metastatic colorectal cancer (CRC) has the dismal 5-year survival rate of only 14%, and immunotherapies fail to improve the patient outcome. One reason for the poor response rate is the slightly acidic (~\u20096.5) immunosuppressive microenvironment. Interleukin 12 (IL-12) is a highly potent pro-inflammatory cytokine that can stimulate tumor immune cells and reverse immunosuppression by inducing interferon gamma (IFN-\u03b3) expression. However, its clinical applications are hindered by systemic side effects. In this study, we developed pH-responsive polymeric nanoparticles (NPs) encapsulating IL-12 to enhance its therapeutic efficacy into the tumor microenvironment (TME). IL-12-loaded pH-responsive NPs induced antitumoral pro-inflammatory response in macrophages at pH\u2009~\u20096.5, determined by increased IFN-\u03b3 levels and nitric oxide (NO) release, without affecting metabolic activity. In contrast, IL-12-loaded pH non-responsive PLGA NPs showed much lower macrophage activation. To validate the specificity and efficacy in a complex immune-rich microenvironment, we developed a novel CRC 3D immuno-spheroid by incorporating human monocyte-derived macrophages with tumor cells in collagen, mimicking CRC spatial organization and extracellular matrix. The interaction of IL-12 pH-responsive NPs induced macrophage polarization, by providing a reduction of M2-like markers (CD14\u2009+\u2009CD163+) while increasing pro-inflammatory M1-like counterparts (CD14\u2009+\u2009CD86+). Moreover, IL-12 pH-responsive NPs increased IFN-\u03b3 levels and reduced anti-inflammatory IL-10 secretion. Overall, this study provides two major findings (1) a pH-responsive NP system to effectively deliver IL-12 to the TME and reprogram local macrophages into pro-inflammatory phenotype; (2) a macrophage-enriched human 3D immuno-spheroid in vitro system as a tool to test the effectivity of immunomodulatory NPs.<\/jats:p>","DOI":"10.1007\/s13346-025-01896-8","type":"journal-article","created":{"date-parts":[[2025,6,16]],"date-time":"2025-06-16T15:29:49Z","timestamp":1750087789000},"page":"4775-4794","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Immunostimulatory effects of IL-12 targeted pH-responsive nanoparticles in macrophage-enriched 3D immuno-spheroids in vitro model"],"prefix":"10.1007","volume":"15","author":[{"given":"Maria Jos\u00e9","family":"Silveira","sequence":"first","affiliation":[]},{"given":"Cl\u00e1udia","family":"Martins","sequence":"additional","affiliation":[]},{"given":"Ana P.","family":"Cardoso","sequence":"additional","affiliation":[]},{"given":"Marc J. 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