title: Cellular uptake mechanisms of novel anionic siRNA lipoplexes.
abstract: OBJECTIVE
  To investigate cellular uptake pathways of novel anionic siRNA-lipoplexes as a function of formulation composition.
  
  
  METHODS
  Anionic formulations with anionic lipid/Ca(2+)/siRNA ratio of 1.3/2.5/1 (AF1) and 1.3/0.3/1 (AF2) were utilized. Uptake mechanisms were investigated using uptake inhibition and co-localization approaches in breast cancer cells. Actin-mediated uptake was investigated using actin polymerization and rearrangement assays. Silencing efficiency and endosomal escaping capability of lipoplexes were evaluated. The cationic formulation Lipofectamine-2000 was used as a control.
  
  
  RESULTS
  Anionic lipoplexes entered the breast cancer cells via endocytosis specifically via macropinocytosis or via both macropinocytosis and HSPG (heparin sulfate proteoglycans) pathways, depending on the Ca(2+)/siRNA ratio. Additionally, uptake of these lipoplexes was both microtubule and actin dependent. The control cationic lipid-siRNA complexes (Lipofectamine-2000) were internalized via both endocytic (phagocytosis, HSPG) and non-endocytic (membrane fusion) pathways. Their uptake was microtubule independent but actin dependent. Silencing efficiency of the AF2 formulation was negligible mainly due to poor endosomal release (rate-limiting step).
  
  
  CONCLUSIONS
  Formulation composition significantly influences the internalization mechanism of anionic lipoplexes. Uptake mechanism together with formulation bioactivity helped in identification of the rate-limiting steps to efficient siRNA delivery. Such studies are extremely useful for formulation optimization to achieve enhanced intracellular delivery of nucleic acids.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, 69 N Eagleville Rd, Unit 3092, Storrs, Connecticut 06269, USA.
authors.forename: Mamta
authors.initials: M
authors.lastname: Kapoor
authors.forename: Diane J
authors.initials: DJ
authors.lastname: Burgess
mesh_terms: D000199:Actins / Q000378:metabolism; D000838:Anions / Q000737:chemistry*; D001940:Breast / Q000166:cytology / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics; D002118:Calcium / Q000737:chemistry; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D010873:Pinocytosis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics / Q000493:pharmacokinetics*
chemical_list: D000199:Actins; D000838:Anions; D008055:Lipids; C086724:Lipofectamine; D034741:RNA, Small Interfering; D002118:Calcium