In vivo delivery of an exogenous molecule into murine T lymphocytes using a lymphatic drug delivery system combined with sonoporation

Physical delivery of exogenous molecules into lymphocytes is extremely challenging because conven- tional methods have notable limitations. Here, we evaluated the potential use of acoustic liposomes (ALs) and sonoporation to deliver exogenous molecules into lymphocytes within a lymph node (LN). MXH10/ Mo-lpr/lpr (MXH10/Mo/lpr) mice, which show systemic LN swelling, were used as the model system. After direct injection into the subiliac LN, a solution containing both ALs and TOTO-3 fluorophores (molecular weight: 1355) was able to reach the downstream proper axillary LN (PALN) via the lymphatic vessels (LVs). This led to the accumulation of a high concentration of TOTO-3 fluorophores and ALs in the lymphatic sinuses of the PALN, where a large number of lymphocytes were densely packed. Exposure of the PALN to >1.93 W/cm2 of970-kHz ultrasound allowed the solution to extravasate into the parenchyma and reach the large number of lymphocytes in the sinuses. Flow cytometric analysis showed that TOTO-3 molecules were delivered into 0.49 ± 0.23% of CD8þ7AAD? cytotoxic T lymphocytes. Furthermore, there was no evidence of tissue damage. Thus, direct administration of drugs into LVs combined with sono- poration can improve the delivery of exogenous molecules into primary lymphocytes. This technique could become a novel approach to immunotherapy. ©