The extracellular matrix (ECM) plays a pivotal role in regulating immune cell behavior and modulating the biological response to nanomaterials (NMs). Understanding how immune cells interact with nanomaterials (NMs) in physiologically relevant environments is critical for the rational design of safe and effective biomedical applications. While conventional in vitro models typically expose cells to NMs in suspension, in vivo these materials frequently interact with ECM components, altering both matrix properties and cellular uptake mechanisms. This critical ECM–NM interface is often overlooked in nanosafety assessments and nanomedicine design. In this study, supported by the PRIN 2022 PNRR program, we developed ECM-mimetic like matrices to investigate the microenvironmental influence on macrophage responses to polystyrene nanoparticles (NPs). The synthetic substrates were engineered via electrospinning using biocompatible polymers blended with gelatin and hyaluronic acid (HA)—two key ECM components commonly found in hydrogels and biomedical gels. These matrices recapitulate essential structural and biochemical features of native ECM. These matrices were pre-loaded with polystyrene nanoparticles (NPs) and used to study the response of THP-1-derived macrophage-like cells. The results showed efficient cell adhesion to all matrix types, particularly gelatin-rich ones, with no significant cytotoxicity up to 48 hours. Importantly, gelatin-containing matrices adsorbed significantly more NPs compared to controls, influencing NP availability and cell interaction. Fluorescence microscopy and flow cytometry confirmed NP internalization by macrophages in contact with the substrates. Notably, in this ECM-like configuration, cell viability was affected by the presence of NPs. Furthermore, we investigated the macrophage polarization in pro- or anti-inflammatory phenotypes through flow cytometry analysis. Our findings underscore the importance of using engineered ECM-inspired systems to better replicate the in vivo microenvironment in nanotoxicology and immunoengineering studies. Understanding how ECM properties affect NM bioavailability and immune cell behavior is crucial for advancing research in nanotoxicology, immunomodulation, and the development of next-generation therapeutic systems.