Dynamics of Endocytosis and Exocytosis of Poly(D,L-Lactide-co-Glycolide) Nanoparticles in Vascular Smooth Muscle Cells

Summary:

The formulation of macromolecular agents such as proteins, oligonucleotides, and DNA in drug delivery systems has drawn interest because of their effectiveness in treating diseases. However, the effectiveness of such systems is severely limited because the macromolecules often need to enter cells in order to access their sites of action. They are hindered by inefficient uptake and their susceptibility to degradation during an endocytosis based uptake process. While other systems such as viruses and protein transduction domains have been used to successfully enter cells, they are able to carry only a few types of therapeutics. Moreover, some of these systems are limited by toxicity and immunogenicity concerns.

The authors of this paper have previously shown that biodegradable poly-PLGA nanoparticles can escape degradation and deliver therapeutics into the cytoplasm. They are also able to release the agent in a sustained manner. However, their internalization and retention inside the cell has not been studied in detail. Therefore, in this paper, the authors characterize endocytosis and exocytosis of PLGA nanoparticles in human arterial vascular smooth muscles cells (VSMCs).

Dose dependent and time dependent cellular uptake of nanoparticles were studied along with the process of exocytosis under normal and metabolically inhibited conditions. BSA was used as a macromolecule model and 6-coumarin as a fluorescent marker. These were loaded onto the nanoparticles using a double emulsion-solvent evaporation technique. The dye was shown to stay within the nanoparticle throughout the intake and study process. Cells were plated with two different media: regular growth medium and serum-free medium, both containing nanoparticles. In order to study dose-dependent nanoparticle uptake, the cells were incubated with different concentrations of the nanoparticle suspension for 1 hour. To study time-dependent nanoparticle uptake, cells were incubated at a given nanoparticle concentration for different time periods. Exocytosis under normal conditions was studied by initially incubating the cells with nanoparticles for a given time period in the two media types. The uninternalized nanoparticles were then washed off and the cells were incubated in growth medium without nanoparticles. Cells were removed and lysed at varying time intervals to determine the fraction of nanoparticles retained. In order to study the metabolic inhibition of nanoparticle exocytosis, cells were first incubated with nanoparticles for a given time period, then washed and incubated in regular medium containing sodium azide and deoxyglucose. These molecules inhibit the production of ATP and thus reduce the ability of a cell to perform exocytosis. Again, cell lysates were studied to determine nanoparticle concentration. Finally, Lucifer yellow, a fluid phase marker was used as a control for exocytosis. Cell lysates were processed to determine the nanoparticle levels through high-performance liquid chromatography. These processes were also visualized using confocal laser scanning microscopy.

The uptake of nanoparticles was shown to be both dose and time dependent. In the dose-dependent study, uptake was linear at lower doses (10-100g), tapering off at higher doses (500-1000g). The results of the time-dependent study showed that uptake began as early as 10-30 seconds (Figure 3), was rapid during the first two hours and reached saturation in 4-6 hours. Figure 2 displays a graph of the uptake. The exocytosis study showed that a majority (65%) of the internalized nanoparticles were exocytosed immediately after the removal of nanoparticles from the external growth media. Furthermore, exocytosis was inhibited in the presence of metabolic inhibitors, confirming that endocytosis was the mode of uptake. Interestingly, the exocytosis was almost completely inhibited when cells were places in serum-free media whereas the addition of BSA to the media induced exocytosis. The authors suggest that this indicated the protein is either adsorbed or carried along with the nanoparticles into the cells and interact with the exocytic pathway, leading to the increased exocytosis.

Conclusion:

Overall, the study shows that the nanoparticles are internalized rapidly in a saturable, energy-dependent process. However, a majority of the internalized particles are exocytosed immediately when the concentration gradient across the cell membrane is removed. Nonetheless, the authors speculate that this may not be a problem in vivo because the concentration of nanoparticles will not fall as rapidly in the extracellular matrix. This would allow the cells to reach a mass transport equilibrium and thus retain more nanoparticles. Among other things, the mechanisms of endocytosis and exocytosis and the effect of nanoparticle formulation and composition on the processes need to be explored before further conclusions can be drawn.