Molecular weight-dependent cellular distribution and immunoreactivity mechanisms of hyaluronic acid.

HA (Hyaluronic acid, Hyaluronate) exhibits pronounced molecular weight-dependent biological activities; however, the mechanisms linking polymer size to cellular distribution and immune regulation remain incompletely understood. In this study, HA fractions with discrete molecular weight distributions were prepared via acid hydrolysis, with structural integrity validated by NMR and IR analyses. Systematic investigations revealed marked molecular weight-dependent differences in cellular uptake and intracellular localization in macrophages. While low-molecular-weight HA (LMW HA) and high-molecular-weight HA (HMW HA) exhibited minimal cellular internalization and remained predominantly extracellular, medium-molecular-weight HA (MMW HA) underwent efficient endocytosis and preferentially accumulated within lysosomes. Mechanistically, pharmacological perturbation and siRNA-mediated knockdown indicated that MMW HA internalization was largely mediated by a HARE-associated macropinocytosis endocytic pathway. Functional assays and whole-transcriptomic profiling further demonstrated that this intracellular routing was associated with a distinct pro-inflammatory profile, characterized by enhanced IL-1β production together with the activation of TNF-α associated signaling events. In contrast, the extracellularly restricted HMW HA shifted signaling toward interferon-related pathways. Collectively, these findings suggest that HA molecular weight influences not only receptor-mediated internalization but also intracellular trafficking, thereby contributing to distinct immunological outcomes. This work provides mechanistic insight into the size-dependent immunoreactivity of HA and may contribute to the development of HA-based biomaterials with tunable biological functions.