Abstract
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.