Ultrasonication-derived CAR-T vesicles preserve antigen-specific cytotoxic function.

Introduction: Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of hematologic malignancies but remains largely ineffective against solid tumors, where stromal barriers and an immunosuppressive microenvironment restrict T-cell infiltration and function. Cell-free vesicular derivatives of therapeutic lymphocytes may help overcome these limitations while preserving antigen specificity. Methods: Artificial vesicles were generated from CAR-T cells (CAR-AVs) by brief ultrasonication followed by differential centrifugation. Their physicochemical and functional properties were evaluated in vitro using scanning electron microscopy, flow cytometry with DiO labeling and recombinant CD19-PE, real-time impedance analysis, and qRT-PCR of apoptosis-associated genes. Results: Scanning electron microscopy revealed nanoscale, membrane-enclosed vesicles with mean ± SD diameters of 132 ± 76 nm for CAR-AVs and 149 ± 63 nm for T cell-derived vesicles (T-AVs). Flow cytometry detected CAR expression on 52.7 ± 20.5% of CAR-AVs, compared with 8.3 ± 6.1% background staining in T-AVs and 64.2 ± 14.4% CD19-PE-positive parental CAR-T cells. At a protein-normalized dose of 15 µg mL-1, CAR-AVs selectively suppressed the proliferation of CD19-expressing tumor cells, reducing the cell index by approximately 26-27% in PC3M(CD19+) cultures and 68% in A431(CD19+) cultures, with minimal effects on antigen-negative counterparts. T-AVs induced only modest, antigen-independent suppression. In antigen-positive models, CAR-AV treatment upregulated Discussion: Ultrasonication-derived CAR-AVs preserve detectable CAR display and retain antigen-associated antitumor activity in solid-tumor models. These findings support CAR-AVs as a promising and potentially scalable cell-free complement to CAR-T therapy, while highlighting the need for particle-resolved quantification, dose-response studies, and