Cellular Uptake of Nanoparticles is Regulated by Integrin-Based Adhesion to the Extracellular Matrix

Therapeutic nanoparticle delivery is crucial for a variety of biomedical applications, such as immunizations, gene and drug delivery, tissue engineering, biomedical implant coating, and other regenerative medicine approaches. The uptake of therapeutic nanoparticles occurs through several endocytosis pathways. However, the uptake mechanism of nanoparticles delivered from a substrate is also regulated by cell-substrate interactions and the ability of cells to mechanosense their surrounding extracellular matrix (ECM). These cell-ECM interactions influence integrin signaling, focal adhesion formation, and cytoskeletal rearrangement to impact nanoparticle uptake. In this study, we investigated the role of ECM and ECM-mimetic coatings─collagen I (COL), fibronectin (FN), laminin (LM), hyaluronic acid (HA), and poly-l-lysine (PLL)─on the uptake of poly(lactic-<i>co</i>-glycolic acid) (PLGA) nanoparticles across three distinct cell types: NIH3T3 fibroblasts, primary rat adipose-derived stem cells (ASCs), and RAW264.7 macrophages, which displayed varying levels of integrin-based focal adhesion formation. Using a quartz crystal microbalance with dissipation (QCM-D) and ellipsometry, we thoroughly characterized ECM coatings, showing variations in coating thickness and mechanical properties. FN and COL coatings significantly enhanced cell proliferation, spreading, and focal adhesion formation, correlating with the highest levels of nanoparticle uptake at longer time points. In contrast, HA and LM coatings resulted in reduced cell adhesion and uptake. Consistent with this, cell types with more mature focal adhesions (ASCs, NIH3T3) showed much higher particle uptake in comparison to cells with limited focal adhesion formation (RAW264.7). Live-cell imaging demonstrated dynamic differences in uptake kinetics with LM coatings showing rapid early uptake, while FN and COL promoted sustained uptake over longer durations. Uptake studies using cytochalasin-D revealed that nanoparticle uptake was highly dependent on the actin cytoskeleton, suggesting the involvement of actin-dependent endocytic pathways. Overall, our findings highlight that ECM-dependent regulation of integrin-based adhesions and cytoskeletal organization modulates nanoparticle uptake in a coating- and cell type-dependent manner. These insights provide a foundation for optimizing substrate-based nanoparticle delivery platforms in regenerative medicine and therapeutic applications.