Class A Scavenger Receptor-Mediated Phagocytosis of Bovine Milk Exosomes in Murine Bone Marrow-Derived Macrophages

Abstract Objectives Bovine milk exosomes (BMEs) are promising candidates for delivering drugs to brain tumors because they are scalable, bioavailable after oral administration, and cross the blood-brain barrier. The use of BMEs in drug delivery is limited by their rapid elimination by macrophages. The objectives of this study were to identify the BME transporter and assess BME transport kinetics in murine bone marrow-derived macrophages (BMDMs) as a first step toward developing strategies that decrease the elimination of drug-loaded BMEs. Methods BMEs were isolated by differential centrifugation from skim milk. For transport studies, proteins and lipids on the BME surface were labeled with HiLyte™ Fluor 750 hydrazide and PKH26, respectively, and RNAs in BMEs were labeled with Exo-Red; unlabeled BMEs were used to assess background noise. Bone marrow cells were isolated from the femur and tibia of both C57BL/6J and scavenger receptor A-I/II knockout mice and differentiated ex vivo into BMDMs for use in transport studies. ANOVA, Dunnett's and Kruskal-Wallis test, Dunn's post-hoc test, unpaired t-test, and two-tailed Mann–Whitney U tests were used for statistical analysis; P < 0.05 was considered statistically significant. Results The uptake of BME was not saturated at the highest concentration used (4.3 × 1011 BMEs/mL) and the longest incubation time (53 h) tested. Chemical inhibition of phagocytosis by cytochalasin D led to a 69 ± 18% decrease in BME uptake compared to solvent controls (P ˂ 0.05), whereas inhibition of macropinocytosis, caveolar-dependent endocytosis, and endocytosis of clathrin-coated vesicles had no significant effect on BME uptake (11%-33% decrease compared to controls; P > 0.05). Treatment with inhibitors of class A scavenger receptor (CASR), fucoidan and dextran sulfate caused a 70 ± 8.1% and 70 ± 18% decrease (P ˂ 0.05), respectively, in BME uptake. The role of CASR in BME uptake was confirmed by using a genetics approach: the uptake of BMEs by BMDMs from scavenger receptor A-I/II knockout mice decreased by 58 ± 23% compared to BMDMs from wild-type mice (P ˂ 0.05). Conclusions BME uptake is facilitated by CASR in BMDMs and uptake cannot be saturated under physiological conditions. Funding Sources NIH 1P20GM104320, and NIFA 2016–67,001-25,301 and 2020–67,017-30,834, USDA Hatch-1,011,996, and USDA W4002 (all to J.Z.). J.Z is a consultant for PureTech Health, Inc.