Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study.
Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing.
Asgeirsdóttir
Sigridur A
SA
Department of Pathology & Medical Biology, Medical Biology Section, Laboratory for Endothelial Biomedicine & Vascular Drug Targeting Research, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. s.a.asgeirsdottir@med.umcg.nl
Talman
Eduard G
EG
de Graaf
Inge A
IA
Kamps
Jan A A M
JA
Satchell
Simon C
SC
Mathieson
Peter W
PW
Ruiters
Marcel H J
MH
Molema
Grietje
G
D000368:Aged; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015703:Antigens, CD / Q000235:genetics*; D001665:Binding Sites; D001692:Biological Transport; D015820:Cadherins / Q000235:genetics* / Q000276:immunology; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000276:immunology / Q000378:metabolism*; D007668:Kidney / Q000276:immunology / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D010316:Particle Size; D011726:Pyridinium Compounds / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D046509:Tissue Culture Techniques; D014162:Transfection; D000099152:Cadherin 5
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015820:Cadherins; D011726:Pyridinium Compounds; D034741:RNA, Small Interfering; D000099152:Cadherin 5; C532953:1-methyl-4-(9-dioleyl)methylpyridinium