BACKGROUND: As has been shown for different vector systems, the entry pathway(s) impacts upon the transfection efficiency. The present study aimed to explore the cellular uptake mechanisms of three different vectors based on solid lipid nanoparticles (SLN) in HeLa cells. The use of endocytosis inhibitors that affect specific internalization pathways provides a tool for the study of these routes. METHODS: We prepared three vectors based on solid lipid nanoparticles: without protamine, with protamine, and with protamine and dextran. Uptake, percentage of transfected HeLa cells and enhanced green fluorescent protein (EGFP) production were all analyzed in the presence or absence of different endocytosis inhibitors. In addition, co-localization studies using lysosomal markers were carried out to determine the influence of the trafficking to late endosomal compartments on the transfection capacity of the vectors. RESULTS: Uptake and transfection of each vector was affected differently by each endocytosis inhibitor. Ethylisopropylamiloride (EIPA) did not affect uptake of the DNA-SLN vector, whereas all of the inhibitors affected transfection. In the case of protamine-DNA-SLN and dextran-protamine-DNA-SLN vectors, EIPA affected uptake and dynasore did not decrease transfection. CONCLUSIONS: DNA-SLN vector appear to enter productively by multiple pathways in HeLa cells. By contrast, dynamin does not appear to be essential in the productive entry of protamine-containing vectors. In addition, enhancement of the macropinocytic route increases EGFP production when dextran is added to the vector.
BACKGROUND: As has been shown for different vector systems, the entry pathway(s) impacts upon the transfection efficiency. The present study aimed to explore the cellular uptake mechanisms of three different vectors based on solid lipid nanoparticles (SLN) in HeLa cells. The use of endocytosis inhibitors that affect specific internalization pathways provides a tool for the study of these routes. METHODS: We prepared three vectors based on solid lipid nanoparticles: without protamine, with protamine, and with protamine and dextran. Uptake, percentage of transfected HeLa cells and enhanced green fluorescent protein (EGFP) production were all analyzed in the presence or absence of different endocytosis inhibitors. In addition, co-localization studies using lysosomal markers were carried out to determine the influence of the trafficking to late endosomal compartments on the transfection capacity of the vectors. RESULTS: Uptake and transfection of each vector was affected differently by each endocytosis inhibitor. Ethylisopropylamiloride (EIPA) did not affect uptake of the DNA-SLN vector, whereas all of the inhibitors affected transfection. In the case of protamine-DNA-SLN and dextran-protamine-DNA-SLN vectors, EIPA affected uptake and dynasore did not decrease transfection. CONCLUSIONS: DNA-SLN vector appear to enter productively by multiple pathways in HeLa cells. By contrast, dynamin does not appear to be essential in the productive entry of protamine-containing vectors. In addition, enhancement of the macropinocytic route increases EGFP production when dextran is added to the vector.
Authors: Noha Attia; Mohamed Mashal; Cristina Soto-Sánchez; Gema Martínez-Navarrete; Eduardo Fernández; Santiago Grijalvo; Ramón Eritja; Gustavo Puras; Jose Luis Pedraz Journal: Drug Des Devel Ther Date: 2018-11-16 Impact factor: 4.162