Particle size influences fibronectin internalization and degradation by fibroblasts |
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Authors: | Peter Bozavikov Dhaarmini RajshankarWilson Lee Christopher A. McCulloch |
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Affiliation: | Matrix Dynamics Group, University of Toronto, Toronto, ON |
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Abstract: | The application of nanotechnology for drug targeting underlines the importance of controlling the kinetics and cellular sites of delivery for optimal therapeutic outcomes. Here we examined the effect of particle size on internalization and degradation of surface-bound fibronectin by fibroblasts using polystyrene nanoparticles (NPs; 51 nm) and microparticles (MPs; 1 μm). Fibronectin was strongly bound by NPs and MPs as assessed by immuno-dot blot analysis (5.1 ±0.4×10– 5 pg fibronectin per μm2 of NP surface; 4.2±±0.3×10–5 pg fibronectin per μm2 of MP surface; p>0.2). We estimated that ~193 fibronectin molecules bound to a MP compared with 0.6 fibronectin molecules per NP, indicating that ~40% of nanoparticles were not bound by fibronectin. One hour after incubation, fibronectin-coated NPs and MPs were rapidly internalized by Rat-2 fibroblasts. MPs and NPs were engulfed partly by receptor-mediated endocytosis as indicated by decreased uptake when incubated at 4 °C, or by depletion of ATP with sodium azide. Pulse-chase experiments showed minimal exocytosis of NPs and MPs. Internalization of NPs and MPs was inhibited by jasplakinolide, whereas internalization of MPs but not NPs was inhibited by latrunculin B and by integrin-blocking antibodies. Extraction of plasma membrane cholesterol with methyl β-cyclodextrin inhibited internalization of fibronectin-coated NPs but not MPs. Biotinylated fibronectin internalized by cells was extensively degraded on MPs but not NPs. Particle size affects actin and clathrin-dependent internalization mechanisms leading to fibronectin degradation on MPs but not NPs. Thus either prolonged, controlled release or an immediate delivery of drugs can be achieved by adjusting the particle size along with matrix proteins such as FN. |
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Keywords: | BSA, bovine serum albumin DAPI, 4&prime ,6-diamidino-2-phenylindole DMEM, Dulbecco?s Modified Eagle?s medium DMSO, dimethyl sulfoxide ECM, extracellular matrix GAPDH, Glyceraldehyde 3-phosphate dehydrogenase FITC, fluorescein isothiocyanate FN, fibronectin mAb, monoclonal antibody MP, microparticle NP, nanoparticle pAb, polyclonal antibody PLL, poly-l-lysine PBS, phosphate buffered saline TBST, Tris-buffered-saline with 0.1% Tween-20 |
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