Metformin-loaded mesenchymal stem cell-derived exosomes modulate CD4+ T-cell subset frequencies in PBMCs from patients with RRMS in vitro

Relapsing-remitting multiple sclerosis (RRMS) is marked by a profound imbalance within the CD​4+ T cell compartment, characterized by an increase in pro-inflammatory effector subsets and a concomitant reduction in regulatory T cells, which together sustain neuroinflammation and drive disease progression. Despite its promising immunomodulatory properties, the clinical translation of metformin for RRMS is limited by its rapid clearance and poor bioavailability. Exosomes secreted by mesenchymal stem cells (MSCs) constitute a novel drug delivery system with intrinsic immunomodulatory properties and the capacity to enhance drug stability. This in vitro study evaluated the immunomodulatory effects of human adipose-derived MSC (hADMSC) exosomes loaded with metformin (Exo-Met) on the frequencies of CD4+ T cell subsets in peripheral blood mononuclear cells (PBMCs) isolated from patients with RRMS. PBMCs from patients with RRMS and healthy controls were treated with exosomes, metformin, Exo-Met, or IFN-β. Frequencies of IFN-γ​​+ CD4​​+ T, IL-17​​+ CD4+ T, and FoxP3​+ CD25+ CD4+ T cells were analyzed by flow cytometry at 24 and 72 h. At baseline, the frequencies of IFN-γ​+ CD4​+ T and IL-17+ CD4+ T cells were significantly higher in RRMS patients compared to healthy controls. Concomitantly, a lower frequency of FoxP3​+ CD25+ CD4​+ T cells was found in the patient group. Treatment with exosomes elicited a partial immunomodulatory effect on the frequencies of CD4+ T cell subsets. In contrast, metformin significantly reduced the frequency of IFN-γ​+ CD4+ T cells within 24 h and IL-17+ CD4+ T cells by 72 h, bringing both to levels comparable to those of healthy controls. Nevertheless, FoxP3​+ CD25+ CD4​+ T cell frequencies in the RRMS group remained significantly below healthy control levels even after 72 h. Compared with the single treatments, Exo-Met demonstrated enhanced, multifaceted immunomodulatory effects. It induced a rapid reduction of IL-17+ CD4+ T cells within 24 h and a significant decline in IFN-γ​+ CD4+ T cells by 72 h. Furthermore, Exo-Met rapidly enriched the FoxP3​+ CD25+ CD4+ T cells, bringing their frequencies toward levels comparable to healthy controls within 24 h. The results demonstrate that Exo-Met exerts a dual immunomodulatory effect in vitro in PBMCs from patients with RRMS, simultaneously reducing pathogenic pro-inflammatory CD4+ T cell subsets while promoting the regulatory subset, thereby representing a promising nanotherapeutic strategy that warrants further preclinical and clinical investigation.