Magnetic Graphene for High-Performance Isolation of Bone Marrow Mesenchymal Stem Cell-Derived Exosomes via Ca2+-Dependent Reversible Recognition

Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) have emerged as a promising therapeutic modality, owing to their potent immunomodulatory and tissue repair capabilities. However, the lack of high-efficiency and high-purity isolation strategies remains a major barrier to the clinical translation of BMSC-Exos. Herein, we rationally designed a high-performance TIM4@MGO magnetic bead system through the functionalization of magnetic graphene oxide (MGO) with T cell immunoglobulin and mucin domain-containing protein 4 (TIM4), which enables the rapid and efficient isolation of exosomes within only 30 min. First, in the presence of Ca²⁺, TIM4 immobilized on the surface of magnetic beads specifically recognizes and binds to phosphatidylserine (PS) exposed on the exosomal membrane, enabling the selective capture of exosomes. Moreover, the large specific surface area of MGO substantially increases the loading density of TIM4, thereby markedly enhancing the binding capacity and recovery efficiency of the exosomes. Subsequently, chelation of Ca²⁺ by EDTA induces charge reversal of the TIM4 domain, triggering highly reversible dissociation of the TIM4–PS interaction. This thus allows for the efficient release of exosomes under mild physiological conditions while preserving their structural and functional integrity. Additionally, TIM4@MGO retained over 84% of the initial isolation efficiency even after three consecutive reuse cycles, demonstrating exceptional reusability in practical applications. In vitro experiments confirmed that BMSC-Exos isolated via TIM4@MGO markedly promoted L929 cell proliferation and accelerated scratch wound healing. Proteomic profiling further revealed that BMSC-Exos isolated by TIM4@MGO were significantly enriched in proteins associated with extracellular matrix remodeling and the TNF signaling pathway, implying their potential role in fibrosis regulation. In a mouse pulmonary fibrosis model, compared with those isolated by ultracentrifugation and polyethylene glycol precipitation methods, TIM4@MGO-isolated BMSC-Exos exerted superior therapeutic efficacy in mitigating lung tissue structural damage and attenuating fibrotic progression. Collectively, this study establishes a high-efficiency, high-purity, and reusable exosome isolation technique, offering robust technical support for both fundamental research and the clinical translation of BMSC-Exo therapeutics.