Dynamics of Endocytosis and Exocytosis of Poly(D,L-Lactide-co-Glycolide) Nanoparticles in Vascular Smooth Muscle Cells
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.
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- 1.F. Liu and L. Huang. Development of non-viral vectors for systemic gene delivery. J. Control. Release 78:259-262 (2002).Google Scholar
- 2.M. C. Morris, J. Depollier, J. Mery, F. Heitz, and G. Divita. A peptide carrier for the biologically active proteins into mammalian cells. Nat. Biotechnol. 19:1173-1176 (2001).Google Scholar
- 3.S. R. Schwarze and S. F. Dowdy. In vivo protein transduction: Intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol. Sci. 21:45-48 (2000).Google Scholar
- 4.H. Riezman, P. G. Woodman, G. van Meer, and M. Marsh. Molecular mechanisms of endocytosis. Cell 91:731-738 (1997).Google Scholar
- 5.J. Panyam, W. Z. Zhou, S. Prabha, S. K. Sahoo, and V. Labhasetwar. Rapid endo-lysosomal escape of poly (D,L-lactide-co-glycolide) nanoparticles: Implications for drug and gene delivery. FASEB J. 16:1217-1226 (2002).Google Scholar
- 6.S. Prabha, W. Z. Zhou, J. Panyam, and V. Labhasetwar. Size-dependency of nanoparticle-mediated gene transfection: Studies with fractionated nanoparticles. Int. J. Pharm. 244:105-115 (2002).Google Scholar
- 7.S. K. Sahoo, J. Panyam, S. Prabha, and V. Labhasetwar. Residual polyvinyl alcohol associated with poly (D,L-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake. J. Control. Release 82:105-114 (2002).Google Scholar
- 8.J. M. Anderson and M. S. Shive. Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv. Drug Deliv. Rev. 28:5-24 (1997).Google Scholar
- 9.V. Labhasetwar. Nanoparticles for drug delivery. Pharm. News 4:28-31 (1997).Google Scholar
- 10.J. Panyam and V. Labhasetwar. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Del. Rev. (2002) in press.Google Scholar
- 11.V. Labhasetwar, C. Song, and R. J. Levy. Nanoparticle drug delivery for restenosis. Adv. Drug Deliv. Rev. 24:63-85 (1997).Google Scholar
- 12.V. Labhasetwar, C. Song, W. Humphrey, R. Shebuski, and R. J. Levy. Arterial uptake of biodegradable nanoparticles: Effect of surface modifications. J. Pharm. Sci. 87:1229-1234 (1998).Google Scholar
- 13.E. R. O'Brien, C. E. Alpers, D. K. Stewart, M. Ferguson, N. Tran, D. Gordon, E. P. Benditt, T. Hinohara, J. B. Simpson, and S. M. Schwartz. Proliferation in primary and restenotic coronary atherectomy tissue. Implications for antiproliferative therapy. Circ. Res. 73:223-231 (1993).Google Scholar
- 14.J. Davda and V. Labhasetwar. Characterization of nanoparticle uptake by endothelial cells. Int. J. Pharm. 233:51-59 (2002).Google Scholar
- 15.J. Panyam, J. Lof, E. O'Leary, and V. Labhasetwar. Efficiency of Dispatch® and Infiltrator® cardiac infusion catheters in arterial localization of nanoparticles in a porcine coronary model of restenosis. J. Drug Target. 10:515-523 (2002).Google Scholar
- 16.V. P. Torchilin, R. Rammohan, V. Weissig, and T. S. Levchenko. TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors. Proc. Natl. Acad. Sci. USA 98:8786-8791 (2001).Google Scholar
- 17.K. A. Foster, M. Yazdanian, and K. L. Audus. Microparticulate uptake mechanisms of in-vitro cell culture models of the respiratory epithelium. J. Pharm. Pharmacol. 53:57-66 (2001).Google Scholar
- 18.H. Suh, B. Jeong, F. Liu, and S. W. Kim. Cellular uptake study of biodegradable nanoparticles in vascular smooth muscle cells. Pharm. Res. 15:1495-1498 (1998).Google Scholar
- 19.M. R. Bennet, D. F. Gibson, S. M. Schwartz, and J. F. Tait. Binding and phagocytosis of apoptotic vascular smooth muscle cells is mediated in part by exposure of phosphatidylserine. Circ. Res. 77:1136-1142 (1995).Google Scholar
- 20.A. Rupper and J. Cardelli. Regulation of phagocytosis and endo-phagosomal trafficking pathways in Dictyostelium discoideum. Biochim. Biophys. Acta 1525:205-216 (2001).Google Scholar
- 21.A. Catizone, A. L. Medolago Albani, F. Reola, and T. Alescio. A quantitative assessment of non specific pinocytosis by human endothelial cells surviving in vitro. Cell Mol. Biol. 39:155-169 (1993).Google Scholar
- 22.E. M. Gibbs and G. E. Lienhard. Fluid-phase endocytosis by isolated rat adipocytes. J. Cell. Physiol. 121:569-575 (1984).Google Scholar
- 23.B. Goud, C. Jouanne, and J. C. Antoine. Reversible pinocytosis of horse radish peroxidase in lymphoid cells. Exp. Cell Res. 153:218-235 (1984).Google Scholar
- 24.J. Gruenberg. The endocytic pathway: a mosaic of domains. Nat. Rev. Mol. Cell Biol. 2:721-730 (2001).Google Scholar
- 25.J. A. Swanson, B. D. Yirinec, and S. C. Silverstein. Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages. J. Cell Biol. 100:851-859 (1985).Google Scholar
- 26.M. Colin, M. Maurice, G. Trugnan, M. Kornprobst, H. R.P., A. Knight, R. G. Cooper, A. D. Miller, J. Capeau, C. Coutelle, and M. C. Brahimi-Horn. Cell delivery, intracellular trafficking and expression of an integrin-mediated gene transfer vector in tracheal epithelial cells. Gene Ther. 7:139-152 (2000).Google Scholar
- 27.M. J. Buckmaster, D. Lo Braico Jr., A. L. Ferris, and B. Storrie. Retention of pinocytized solute by CHO cell lysosomes correlates with molecular weight. Cell Biol. Int. Rep. 11:501-507 (1987).Google Scholar
- 28.H. Tomoda, Y. Kishimoto, and Y. C. Lee. Temperature effect on endocytosis and exocytosis by rabbit alveolar macrophages. J. Biol. Chem. 264:15445-15450 (1989).Google Scholar
- 29.M. Teresa Girao da Cruz, S. Simoes, P. P. C. Pires, S. Nir, and M. C. Pedroso de Lima. Kinetic analysis of the initial steps involved in lipoplex-cell interactions: Effect of various factors that influence transfection activity. Biochim. Biophys. Acta 1510:136-151 (2001).Google Scholar
- 30.I. Fishbein, M. Chorny, S. Banau, A. Levitzki, H. D. Danenberg, J. Gao, X. Chen, E. Moerman, I. Gati, V. Goldwasser, and G. Golomb. Formulation and delivery mode affect disposition and activity of tryphostin-loaded nanoparticles in the rat carotid model. Arterioscler. Thromb. Vasc. Biol. 21:1434-1439 (2001).Google Scholar
- 31.L. A. Guzman, V. Labhasetwar, C. Song, Y. Jang, A. M. Lincoff, R. Levy, and E. J. Topol. Local intraluminal infusion of biodegradable polymeric nanoparticles. A novel approach for prolonged drug delivery after balloon angioplasty. Circulation 94:1441-1448 (1996).Google Scholar
- 32.K. Furumoto, K. Ogawara, M. Yoshida, Y. Takakura, M. Hashida, K. Higaki, and T. Kimura. Biliary excretion of polystyrene microspheres depends on the type of receptor-mediated uptake in rat liver. Biochim. Biophys. Acta 1526:221-226 (2001).Google Scholar