Objectives In the last decade, it has been demonstrated that solid lipid nanoparticles (SLN) are pharmaceutical nanocarriers capable to incorporate biologically active substances. The aim of the present study is to test the safety and the internalization efficiency of SLN in airway epithelial cells in vitro, in order to use these nanoparticles as carriers for anti-oxidant natural substances such as resveratrol or lycopene in future studies. Methods Fluorescently labelled SLN were formulated according to the melt-emulsification method, by using Gelucire® 50/13 as lipid and 6-coumarin (6-COUM) as dye 1 . Starting by an initial dose of 6 mg of 6-COUM, the resulting particles were found around 235 nm in mean size and quite spherical in morphology as shown by TEM microphotograph (Fig. 1). Moreover, a core-shell structure is well evidenced according to that reported for these nanocarriers 2 . Thus, the 4 nm wide dark circular line around the SLN can be reasonably ascribed to the surfactant monolayer shell surrounding the SLN. To study the uptake of the particles, airway epithelial cells H441 were incubated with three doses (0.2, 1, 10 g/ml) of freshly prepared SLN carrying the green fluorophore 6-coumarin for 4, 8 or 24h; then cells were treated or not treated with trypan blue (0.04%) to quench the fluorescence of the membrane-associated but not internalized nanoparticles, and then analyzed to flow cytometry. We also evaluated the safety and citotoxicity of the SLN by incubating cells with the same doses and for the same times used in the uptake experiments and performing MTT assay and evaluating the viability of the treated cells by propidium iodide exclusion assay by flow-cytometry. Results Flow cytometry analysis demonstrated that even at the lowest dose (0.2 g/ml) and after only 4 hrs of incubation, the cells were highly positive for the 6-COUM fluorescent signal (~80%) and the percentage increased to 100% with the other doses. We also observed the persistence of 6-coumarin signal in cells up to 10 days, 14 days and 16 days with 0.2, 1, and 10 g/ml respectively, after 4 hrs of incubation. Both MTT assay and propidium iodide exclusion assay demonstrated a very low and not significant cytotoxicity at the three tested doses. Conclusions We have found optimal and safe doses of SLN to obtain an high rate of internalization in an airway epithelial cell line; in the next future we will load these SLN with anti-oxidant natural substances to asses their efficacy in vitro as a potential therapy for airway chronic inflammatory diseases such as asthma or COPD. Acknowledgments: Stefano Castellani is a researcher funded by the project “Futureinresearch” of the Regione Puglia.

Safety and uptake of solid lipid nanoparticles by airway epithelial cells: an in vitro study

MANDRACCHIA, DELIA;
2016-01-01

Abstract

Objectives In the last decade, it has been demonstrated that solid lipid nanoparticles (SLN) are pharmaceutical nanocarriers capable to incorporate biologically active substances. The aim of the present study is to test the safety and the internalization efficiency of SLN in airway epithelial cells in vitro, in order to use these nanoparticles as carriers for anti-oxidant natural substances such as resveratrol or lycopene in future studies. Methods Fluorescently labelled SLN were formulated according to the melt-emulsification method, by using Gelucire® 50/13 as lipid and 6-coumarin (6-COUM) as dye 1 . Starting by an initial dose of 6 mg of 6-COUM, the resulting particles were found around 235 nm in mean size and quite spherical in morphology as shown by TEM microphotograph (Fig. 1). Moreover, a core-shell structure is well evidenced according to that reported for these nanocarriers 2 . Thus, the 4 nm wide dark circular line around the SLN can be reasonably ascribed to the surfactant monolayer shell surrounding the SLN. To study the uptake of the particles, airway epithelial cells H441 were incubated with three doses (0.2, 1, 10 g/ml) of freshly prepared SLN carrying the green fluorophore 6-coumarin for 4, 8 or 24h; then cells were treated or not treated with trypan blue (0.04%) to quench the fluorescence of the membrane-associated but not internalized nanoparticles, and then analyzed to flow cytometry. We also evaluated the safety and citotoxicity of the SLN by incubating cells with the same doses and for the same times used in the uptake experiments and performing MTT assay and evaluating the viability of the treated cells by propidium iodide exclusion assay by flow-cytometry. Results Flow cytometry analysis demonstrated that even at the lowest dose (0.2 g/ml) and after only 4 hrs of incubation, the cells were highly positive for the 6-COUM fluorescent signal (~80%) and the percentage increased to 100% with the other doses. We also observed the persistence of 6-coumarin signal in cells up to 10 days, 14 days and 16 days with 0.2, 1, and 10 g/ml respectively, after 4 hrs of incubation. Both MTT assay and propidium iodide exclusion assay demonstrated a very low and not significant cytotoxicity at the three tested doses. Conclusions We have found optimal and safe doses of SLN to obtain an high rate of internalization in an airway epithelial cell line; in the next future we will load these SLN with anti-oxidant natural substances to asses their efficacy in vitro as a potential therapy for airway chronic inflammatory diseases such as asthma or COPD. Acknowledgments: Stefano Castellani is a researcher funded by the project “Futureinresearch” of the Regione Puglia.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11379/529831
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