Ylammonium propane (DOTAP), has regularly been applied as a cationic lipid to get a liposomal delivery system of siRNA by many study groups [14?7]. Among cationic liposomes, DOTAP/Chol liposome is commercially supplied TM as an in vivo transfection reagent (e.g., in vivo MegaFectin from Qbiogene Molecular Biology, in vivo Liposome Transfection Reagent from Sigma-Aldrich), which was demonstrated to possess higher transfection efficiency within the lungs by intravenous injection. Here, we selected chondroitin sulfate C (CS), poly-l-glutamic acid (PGA) and poly-aspartic acid (PAA) as materials for coating cationic DOTAP/Chol lipoplexes of siRNA and evaluated their possible for use as an siRNA delivery vector. Initially, we prepared DOTAP/Chol liposome and measured the particle size and -potential. The liposome size was about 80 nm along with the prospective was + 50 mV. When the liposomes had been mixed with siRNA, the lipoplex size was about 280 nm and the -potential was + 40 mV. Next, we coated the lipoplexes with anionic polymers, CS, PGA and PAA, at a PDE4 Inhibitor Storage & Stability variety of charge ratios (-/ + ), and prepared CS-, PGA- and PAA-coated lipoplexes. With growing amounts of CS, PGA and PAA being added towards the lipoplex, their sizes decreased to 150?00 nm and -potential to a damaging worth (Fig. 1A ). Despite the fact that the sizes of CS-, PGA- and PAA-coated lipoplexes have been smaller sized than that of cationic lipoplex, the anionic polymers may perhaps be able to strongly compact the cationic lipoplex by the electrostatic interaction. The -potentials in the lipoplexes right after the addition of anionic polymers have been pretty much consistently damaging around charge ratios (-/ + ) of 1 in CS, 1.five in PGA and 1.5 in PAA, indicating that nitrogen of cationic lipoplex was fully covered using a sulfate group or a carboxyl group from the anionic polymers. In a earlier study, we p38 MAPK Inhibitor review reported that -potentials on the lipoplexes of pDNA soon after the addition of anionic polymers had been just about consistently unfavorable around charge ratios (-/ + ) of 5.eight in CS and 7 in PGA [5]. The amount of anionic polymer required for covering cationic lipoplex of siRNA was adequate at a reduced level than for the lipoplex of pDNA. For that reason, in subsequent experiments, we decided to use 1 in CS, 1.five in PGA and 1.5 in PAA as optimal charge ratios (-/ + ) for the preparation of anionic polymer-coated lipoplex. three.two. Association of siRNA with all the liposome The association of siRNA with cationic liposome was monitored by gel retardation electrophoresis. Naked siRNA was detected as bands on acrylamide gel. Beyond a charge ratio (-/ + ) of 1/3, no migration of siRNA was observed for cationic lipoplex (Fig. 2A). Having said that, migration of siRNA was observed for CS-, PGA- and PAA-coated lipoplexes at all charge ratios (-/ + ) of anionic polymer/DOTAP when anionic polymers have been added into cationic lipoplex (Fig. 2B), indicating that anionic polymers brought on dissociation of siRNA from lipoplex by competitors for binding to cationic liposome. Previously, we reported that CS and PGA could coat cationic lipoplex of pDNA without releasing pDNA in the cationic lipoplex, and formed steady anionic lipoplexes [5]. In lipoplex of siRNA, the association of cationic liposome with siRNA may well be weaker than that with pDNA.Y. Hattori et al. / Results in Pharma Sciences four (2014) 1?Furthermore, no migration of siRNA-Chol was observed at CS-, PGAand PAA-coated lipoplexes, even at a charge ratio (-/ + ) of 10/1, when anionic polymers had been added into cationic lipoplex of siRNAChol for.
