Gene silencing activity of siRNA polyplexes based on thiolated N,N,N-trimethylated chitosan.
N,N,N-Trimethylated chitosan (TMC) is a biodegradable polymer emerging as a promising nonviral vector for nucleic acid and protein delivery. In the present study, we investigated whether the introduction of thiol groups in TMC enhances the extracellular stability of the complexes based on this polymer and promotes the intracellular release of siRNA. The gene silencing activity and the cellular cytotoxicity of polyplexes based on thiolated TMC were compared with those based on the nonthiolated counterpart and the regularly used lipidic transfection agent Lipofectamine. Incubation of H1299 human lung cancer cells expressing firefly luciferase with siRNA/thiolated TMC polyplexes resulted in 60-80% gene silencing activity, whereas complexes based on nonthiolated TMC showed less silencing (40%). The silencing activity of the complexes based on Lipofectamine 2000 was about 60-70%. Importantly, the TMC-SH polyplexes retained their silencing activity in the presence of hyaluronic acid, while nonthiolated TMC polyplexes hardly showed any silencing activity, demonstrating their stability against competing anionic macromolecules. Under the experimental conditions tested, the cytotoxicity of the thiolated and nonthiolated siRNA complexes was lower than those based on Lipofectamine. Given the good extracellular stability and good silencing activity, it is concluded that polyplexes based on TMC-SH are attractive systems for further in vivo evaluations.
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
Amir K
AK
Varkouhi
Rolf J
RJ
Verheul
Raymond M
RM
Schiffelers
Twan
T
Lammers
Gert
G
Storm
Wim E
WE
Hennink
D002236:Carbohydrate Conformation; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D048271:Chitosan / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology*; D016503:Drug Delivery Systems / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D015870:Gene Expression; D020868:Gene Silencing / Q000187:drug effects*; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D008055:Lipids / Q000494:pharmacology; D008156:Luciferases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
D008055:Lipids; C086724:Lipofectamine; D034741:RNA, Small Interfering; D013438:Sulfhydryl Compounds; D006820:Hyaluronic Acid; D048271:Chitosan; D008156:Luciferases