Purpose. The purpose of this work was to characterize the process of endocytosis, exocytosis, and intracellular retention of poly (D,L-lactide-co-glycolide) nanoparticles in vitro using human arterial vascular smooth muscle cells (VSMCs).
Methods. Nanoparticles containing bovine serum albumin (BSA) as a model protein and 6-coumarin as a fluorescent marker were formulated by a double emulsion-solvent evaporation technique. The endocytosis and exocytosis of nanoparticles in VSMCs were studied using confocal microscopy and their intracellular uptake and retention were determined quantitatively using high-performance liquid chromatography.
Results. Cellular uptake of nanoparticles (mean particle size 97 ± 3 nm) was a concentration-, time-, and energy-dependent endocytic process. Confocal microscopy demonstrated that nanoparticles were internalized rapidly, with nanoparticles seen inside the cells as early as within 1 min after incubation. The nanoparticle uptake increased with incubation time in the presence of nanoparticles in the medium; however, once the extracellular nanoparticle concentration gradient was removed, exocytosis of nanoparticles occurred with about 65% of the internalized fraction undergoing exocytosis in 30 min. Exocytosis of nanoparticles was slower than the exocytosis of a fluid phase marker, Lucifer yellow. Furthermore, the exocytosis of nanoparticles was reduced after the treatment of cells with the combination of sodium azide and deoxyglucose, suggesting that exocytosis of nanoparticles is an energy-dependent process. The nanoparticle retention increased with increasing nanoparticle dose in the medium but the effect was relatively less significant with the increase in incubation time. Interestingly, the exocytosis of nanoparticles was almost completely inhibited when the medium was depleted of serum. Further studies suggest that the addition of BSA in the serum free medium with or without platelet derived growth factor (PDGF) induced exocytosis of nanoparticles. The above result suggests that the protein in the medium is either adsorbed onto nanoparticles and/or carried along with nanoparticles inside the cells, which probably interacts with the exocytic pathway and leads to greater exocytosis of nanoparticles.
Conclusions. The study demonstrated that endocytosis and exocytosis of nanoparticles are dynamic and energy-dependent processes. Better understanding of the mechanisms of endocytosis and exocytosis, studies determining the effect of nanoparticle formulation and composition that may affect both the processes, and characterization of intracellular distribution of nanoparticles with surface modifications would be useful in exploring nanoparticles for intracellular delivery of therapeutic agents.