Poly-lactic acid nanoparticles (PLA-NP) promote physiological modifications in lung epithelial cells and are internalized by clathrin-coated pits and lipid rafts

Abstract

BackgroundPoly-lactic acid nanoparticles (PLA-NP) are a type of polymeric NP, frequently used as nanomedicines, which have advantages over metallic NP such as the ability to maintain therapeutic drug levels for sustained periods of time. Despite PLA-NP being considered biocompatible, data concerning alterations in cellular physiology are scarce.MethodsWe conducted an extensive evaluation of PLA-NP biocompatibility in human lung epithelial A549 cells using high throughput screening and more complex methodologies. These included measurements of cytotoxicity, cell viability, immunomodulatory potential, and effects upon the cells’ proteome. We used non- and green-fluorescent PLA-NP with 63 and 66 nm diameters, respectively. Cells were exposed with concentrations of 2, 20, 100 and 200 µg/mL, for 24, 48 and 72 h, in most experiments. Moreover, possible endocytic mechanisms of internalization of PLA-NP were investigated, such as those involving caveolae, lipid rafts, macropinocytosis and clathrin-coated pits.ResultsCell viability and proliferation were not altered in response to PLA-NP. Multiplex analysis of secreted mediators revealed a low-level reduction of IL-12p70 and vascular epidermal growth factor (VEGF) in response to PLA-NP, while all other mediators assessed were unaffected. However, changes to the cells’ proteome were observed in response to PLA-NP, and, additionally, the cellular stress marker miR155 was found to reduce. In dual exposures of staurosporine (STS) with PLA-NP, PLA-NP enhanced susceptibility to STS-induced cell death. Finally, PLA-NP were rapidly internalized in association with clathrin-coated pits, and, to a lesser extent, with lipid rafts.ConclusionsThese data demonstrate that PLA-NP are internalized and, in general, tolerated by A549 cells, with no cytotoxicity and no secretion of pro-inflammatory mediators. However, PLA-NP exposure may induce modification of biological functions of A549 cells, which should be considered when designing drug delivery systems. Moreover, the pathways of PLA-NP internalization we detected could contribute to the improvement of selective uptake strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0238-1) contains supplementary material, which is available to authorized users.

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“…Cells were seeded in a 6-well plate (3×10 5 /well) using complete DMEM and allowed to attach overnight. Media was replaced with DMEM containing 5% FBS plus enoxaparin and doxorubicin alone or combined and incubated for 24 h. Staurosporine (STS, 4 μM), a commonly used protein kinase inhibitor and activator of apoptosis, was used as positive control (30,31). STS was dissolved in DMSO and diluted to the final concentration (4 μM) to contain 0.4% DMSO.…”

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“…The intestinal tract is the primary physiological barrier for the oral absorption of multiple substances [ 28 , 29 ]. Lehr et al [ 30 , 31 ] reported that in the inflamed colon, an increased adherence of particles was observed in the thicker mucus layer. A size dependency of the deposition was found, with an increased number of attached particles to the colon found compared with the control group.…”

“…It has been shown that clathrin-mediated endocytosis was an uptake process, which occurs through the formation of clathrin-coated vesicles that form from the plasma membrane [ 26 ]. Clathrin-mediated endocytosis was involved primarily in the internalization of receptors [ 31 , 35 , 36 ]. However, under inflammatory conditions, there are changes in protein complexes consisting of transmembrane proteins that interact in the extracellular environment with adjacent cells and in the intracellular environment with adaptor proteins that link to the cytoskeleton.…”