Internalization of benzylisoquinoline alkaloids by resting and activated bone marrow-derived mast cells utilizes energy-dependent mechanisms

Abstract

Abstract Objective and design Drug delivery to inflammatory cells is dependent upon poorly understood, complex endocytic processes. Berberine (BBR), a benzylisoquinoline alkaloid, binds to heparin and targets glycosaminoglycan-rich granules in mast cells (MC), but the mechanism of BBR internalization is unknown. Methods BMMC were treated with various concentrations of BBR for different amounts of time and BBR internalization was assessed by flow cytometry and fluorescence microscopy. BMMC were pretreated with endocytic inhibitors or a growth factor (IL-3) prior to BBR exposure to access mechanisms of its internalization. Results After 24 h, 48 ± 0.8% of BMMC internalized BBR and this process was dependent upon temperature and the presence of glucose in the medium. Methanol fixation reduced BBR internalization, suggesting the involvement of an energy-dependent active transport mechanism. To determine mode of internalization, BBR was encapsulated into Lipofectamine TM lipoplexes since these are known to circumvent classical endocytic pathways. Incorporating BBR into lipoplexes decreased BBR internalization by 26% and 10% (10 μg/ml and 100 μg/ml Lipo-BBR respectively) by BMMC. BBR endocytosis was significantly reduced by Latrunculin B (88%), Cytochalasin B (87%), Chloroquine (86.5%) and 3-methyladenine (91%), indicating that actin polymerization, lysosomal pH and lysosomal self-degradation via the autophagy pathway was involved. In contrast, IL-3 treatment significantly enhanced BBR endocytosis (54% by 40 ng/ml IL-3) suggesting that IL-3 signaling pathways play a role in internalization. Conclusions Our data suggests that internalization of BBR by resting and IL-3-activated BMMC utilizes an energy-dependent pathway that is dependent upon glucose metabolism and temperature. Furthermore, this process requires actin polymerization and lysosomal trafficking. These data suggest internalization of benzylisoquinoline compounds is an active and complex process.