αvβ3 Integrin Recycling Governs the Intracellular Trafficking and Efflux of cRGDfK-Modified PLGA Nanoparticles

Background Elucidating the cellular transport mechanisms of active-targeting nanocarriers is critical for optimizing their therapeutic efficacy. Here, cRGDfK-modified poly(lactic-co-glycolic acid) nanoparticles (cRGDfK-PLGA-NPs) were developed as an αvβ3 integrin-targeted delivery platform, and their cellular uptake, intracellular trafficking, and efflux dynamics were systematically characterized in HeLa cells. Results Confocal laser scanning microscopy and HPLC analysis demonstrated significantly enhanced cellular uptake of cRGDfK-PLGA-NPs compared with unmodified nanoparticles, predominantly mediated through αvβ3 integrin-dependent endocytosis via clathrin-mediated pathways and macropinocytosis. Following internalization, cRGDfK-PLGA-NPs were rapidly delivered to early endosomes (EE), followed by redistribution to the endocytic recycling compartment (ERC) and progressive accumulation in late endosomes (LE). Real-time and fixed-cell colocalization analyses identified three distinct intracellular trafficking routes: the EE–LE pathway, the EE–ERC–slow efflux pathway, and the EE–fast efflux pathway. Efflux profiling revealed a biphasic kinetic pattern consisting of an initial rapid phase (~ 5 min) followed by a slower phase (~ 10 min). To elucidate the mechanistic basis of this behavior, αvβ3 integrin trafficking was quantified using an ELISA-based assay, which revealed cyclic endocytosis–recycling dynamics with a periodicity of approximately 5 min. Comparative analysis with a cRGDfK fluorescent probe showed that its rapid efflux closely mirrored receptor recycling kinetics, indicating that the fast efflux of cRGDfK-PLGA-NPs is driven by αvβ3 integrin-mediated recycling, whereas the slower efflux phase is likely associated with ERC-dependent transport. Conclusions This study uncovers a previously underappreciated coupling between receptor recycling dynamics and nanoparticle intracellular fate, establishing αvβ3 integrin trafficking as a key determinant of active-targeting nanocarrier transport. These findings provide mechanistic guidance for the rational engineering of active-targeting nanomedicines and offer new design principles for improving intracellular retention and therapeutic performance.