title: Role of endocytic uptake in transfection efficiency of solid lipid nanoparticles-based nonviral vectors.
abstract: 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: Pharmacokinetics, Nanotechnology and Gene Therapy Group, Pharmacy Faculty, University of the Basque Country UPV/EHU, Vitoria-Gasteiz, Spain; Centro de Investigación Lascaray Ikergunea, Universidad del País Vasco UPV/EHU, Vitoria-Gasteiz, Spain.
authors: Aritz Perez
authors: AP
authors: Ruiz de Garibay
authors: María Ángeles
authors: MÁ
authors: Solinís Aspiazu
authors: Alicia
authors: A
authors: Rodríguez Gascón
authors: Haleh
authors: H
authors: Ganjian
authors: Renate
authors: R
authors: Fuchs
mesh_terms: D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D011992:Endosomes / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry; D053758:Nanoparticles / Q000737:chemistry; D010316:Particle Size; D055672:Static Electricity; D014162:Transfection / Q000379:methods*
chemical_list: D008055:Lipids; C485184:enhanced green fluorescent protein; D000643:Ammonium Chloride; D049452:Green Fluorescent Proteins
keywords: endocytosis; lysosome; nonviral vector; solid lipid nanoparticles; transfection