The modification of siRNA with 3' cholesterol to increase nuclease protection and suppression of native mRNA by select siRNA polyplexes.
Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3' cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC.
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198-6025, USA.
Vishakha V
VV
Ambardekar
Huai-Yun
HY
Han
Michelle L
ML
Varney
Serguei V
SV
Vinogradov
Rakesh K
RK
Singh
Joseph A
JA
Vetro
D000818:Animals; D002478:Cells, Cultured; D002784:Cholesterol / Q000031:analogs & derivatives / Q000737:chemistry*; D005260:Female; D051379:Mice; D000080385:Nanogels; D011092:Polyethylene Glycols / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*
D000080385:Nanogels; D011108:Polymers; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C095164:poly(oxyethylene) cholesteryl ether; C413692:polyethylene glycol polyethyleneimine nanogel; D011092:Polyethylene Glycols; D011094:Polyethyleneimine; D002784:Cholesterol; D012260:Ribonucleases