Confocal microscopy for the analysis of siRNA delivery by polymeric nanoparticles.
Clinical applications of genetic therapies, including delivery of short, interfering RNAs (siRNAs) for RNA interference (RNAi), are limited due to the difficulty of delivering nucleic acids to specific cells of interest while at the same time minimizing toxicity and immunogenicity. The use of cationic polymers to deliver nucleic acid therapeutics has the potential to address these complex issues but is currently limited by low-delivery efficiencies. Although cell culture studies have shown that some polymers can be used to deliver siRNAs and achieve silencing, it is still not clear what physical or chemical properties are needed to ensure that the polymers form active polymer-siRNA complexes. In this study, we used multicolor fluorescence confocal microscopy to analyze the cellular uptake of siRNAs delivered by novel propargyl glycolide polymeric nanoparticles (NPs). Delivery by these vehicles was compared with delivery by linear polyethyleneimine (LPEI) and Lipofectamine 2000 (LF2K), which are both known as effective delivery vehicles for siRNAs. Our results showed that when LF2K and LPEI were used, large quantities of siRNA were delivered rapidly, presumably overwhelming the basal levels of mRNA to initiate silencing. In contrast, our novel polymeric NPs showed delivery of siRNAs but at concentrations that were initially too low to achieve silencing. Nonetheless, the exceptionally low cytotoxicity of our NPs, and the simplicity with which they can be modified, makes them good candidates for further study to optimize their delivery profiles and, in turn, achieve efficient silencing.
Portis
Amanda M
AM
Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan 48824, USA.
Carballo
Georgina
G
Baker
Gregory L
GL
Chan
Christina
C
Walton
S Patrick
SP
D045744:Cell Line, Tumor; D018014:Gene Transfer Techniques / Q000295:instrumentation*; D006801:Humans; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D018613:Microscopy, Confocal; D053758:Nanoparticles / Q000737:chemistry*; D011094:Polyethyleneimine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics
D008055:Lipids; C086724:Lipofectamine; D011108:Polymers; D034741:RNA, Small Interfering; D011094:Polyethyleneimine
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