Genetically Altered Bovine Milk Exosomes (BMEs) Evade Elimination by Murine Bone Marrow-Derived Macrophages (BMDMs)

OBJECTIVES: To develop BMEs that evade elimination by BMDMs. METHODS: Bovine mammary alveolar MAC-T cells secrete MEs and were used to engineer MEs that express proprietary protein features suspected to decrease elimination by BMDMs, denoted UNL1 and UNL2. MAC-T cells were transformed by using lentiviral vectors. BMEs featuring UNL1 or UNL2 were isolated from MAC-T cell culture media supernatant using polyethylene glycol (PEG) precipitation after removing cell debris by using a 0.22-μm filter. The BMEs were labeled using a carbonyl-reactive fluorescent dye and purified by ultracentrifugation. Primary BMDMs was isolated from mouse hind legs (C57BL/6J, aged 8–10 weeks) and seeded in 96-well plates for assessing BME uptake at a physiological concentration (1010 BMEs/mL). Uptake was compared to unmodified BMEs and normalized for BMDM density. Time points were compared pairwise by using t-test, and P < 0.05 was considered significant. RESULTS: The uptake of BME UNL1 by BMDMs was reduced by 37%, 42%, 48% and 47% as compared to unmodified BMEs after 12 h, 24 h, 36 h and 48h, respectively in culture dishes (P < .05; n = 5). Data are preliminary (n = 3), yet encouraging, for BME UNL2: The uptake of BME UNL2 was reduced by 41%, 44%, 46% and 46% compared to unmodified BMEs after 12 h, 24 h, 36 h and 48 h, respectively. CONCLUSIONS: The elimination of BMEs UNL1 and UNL2 is significantly reduced compared unmodified BMEs in BMDM cultures. This is of great importance when using BMEs for delivering therapeutics. FUNDING SOURCES: NIH P20GM104320, NIFA2016-67,001-25,301 and 2022–67,021-36,407), USDA Hatch and W-4002, and the SynGAP Research Fund (all to J. Z.). J.Z. serves as consultant for PureTech Health, Inc.