Characterization of carbosilane dendrimers as effective carriers of siRNA to HIV-infected lymphocytes.
One of the primary limitations of RNA interference as a technique for gene regulation is effective delivery of siRNA into the target cells. Dendrimers are nanoparticles that are increasingly being used as oligonucleotide and drug delivery vehicles. We have developed amino-terminated carbosilane dendrimers (CBS) as a means to protect and transport siRNA. Initially, stability studies showed that CBS bind siRNA via electrostatic interactions. Dendrimer-bound siRNA was found to be resistant to degradation by RNase. Cytotoxicity assays of CBS/siRNA dendriplexes with peripheral blood mononuclear cells (PBMC) and the lymphocytic cell line SupT1 revealed a maximum safe dendrimer concentration of 25 microg/ml. Next, utilizing flow cytometry and confocal microscopy, lymphocytes were seen to be successfully transfected by fluorochrome-labeled siRNA either naked or complexed with CBS. Dendriplexes with +/- charge ratio of 2 were determined to have the highest transfection efficiency while maintaining a low level of toxicity in these systems including hard-to-transfect HIV-infected PBMC. Finally, CBS/siRNA dendriplexes were shown to silence GAPDH expression and reduce HIV replication in SupT1 and PBMC. These results point to the possibility of utilizing dendrimers such as CBS to deliver and transfect siRNA into lymphocytes thus allowing the use of RNA interference as a potential alternative therapy for HIV infection.
Weber
Nick
N
Laboratorio Inmunobiología Molecular, Hospital General Universitario Gregorio Marañón, Madrid, Spain.
Ortega
Paula
P
Clemente
Maria Isabel
MI
Shcharbin
Dzmitry
D
Bryszewska
Maria
M
de la Mata
F Javier
FJ
Gómez
Rafael
R
Muñoz-Fernández
M Angeles
MA
D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D050091:Dendrimers / Q000737:chemistry / Q000494:pharmacology*; D018274:Electroporation; D020868:Gene Silencing; D006678:HIV / Q000187:drug effects*; D015658:HIV Infections / Q000188:drug therapy; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D018613:Microscopy, Confocal; D053758:Nanoparticles / Q000737:chemistry; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics; D012821:Silanes / Q000737:chemistry / Q000494:pharmacology*; D014162:Transfection
D050091:Dendrimers; D034741:RNA, Small Interfering; D012821:Silanes; C504072:carbosilane