X of siRNA, the association of cationic liposome with siRNA could possibly be weaker than that with pDNA.Y. Hattori et al. / Final results in Pharma Sciences 4 (2014) 1?In addition, no migration of siRNA-Chol was observed at CS-, PGAand PAA-coated lipoplexes, even at a charge ratio (-/ + ) of 10/1, when anionic polymers were added into cationic lipoplex of siRNAChol formed at a charge ratio (-/ + ) of 1/4 (Fig. 2B). From these final results, we confirmed that CS, PGA and PAA could coat cationic lipoplex without the need of releasing siRNA-Chol from the cationic lipoplex, and formed steady anionic lipoplexes. When anionic polymer-coated lipoplexes of siRNA-Chol had been prepared at charge ratios (-/ + ) of 1 in CS, 1.five in PGA and 1.5 in PAA, the sizes and -potentials of CS-, PGA- and PAA-coated lipoplexes had been 299, 233 and 235 nm, and -22.eight, -36.7 and -54.three mV, respectively (Supplemental Table S1). In subsequent experiments, we decided to work with anionic polymer-coated lipoplexes of siRNA and siRNA-Chol for comparison of transfection activity and biodistribution. three.three. In vitro transfection efficiency Generally, in cationic lipoplexes, strong electrostatic interaction using a negatively charged cellular membrane can contribute to high siRNA transfer by means of endocytosis. To investigate no matter whether anionic polymer-coated lipoplexes could be taken up effectively by cells and induce gene suppression by siRNA, we examined the gene knockdown impact using a luciferase assay program with MCF-7-Luc cells. Cationic lipoplex of Luc siRNA or Luc siRNA-Chol exhibited moderate suppression of luciferase activity; however, coating of anionic polymers on the cationic lipoplex caused disappearance of gene knockdown efficacy by cationic lipoplex (Fig. 3A and B), suggesting that negatively charged lipoplexes had been not taken up by the cells because they repulsed the cellular membrane electrostatically. 3.four. Interaction with erythrocytes Cationic lipoplex frequently result in the agglutination of erythrocytes by the sturdy affinity of positively charged lipoplex to the cellular membrane. To investigate no matter whether polymer coatings for cationic lipoplex could avert agglutination with erythrocytes, we observed the agglutination of anionic polymer-coated lipoplex with erythrocytes by microscopy (Fig. four). CS-, PGA- and PAA-coated lipoplexes of siRNA or siRNA-Chol showed no agglutination, despite the fact that cationic lipoplexes did.71989-18-9 web This result indicated that the negatively charged surface of anionic polymer-coated lipoplexes could protect against the agglutination with erythrocytes.4-Fluoropicolinaldehyde site 3.PMID:23927631 five. Biodistribution of siRNA immediately after injection of lipoplex We intravenously injected anionic polymer-coated lipoplexes of Cy5.5-siRNA or Cy5.5-siRNA-Chol into mice, and observed the biodistribution of siRNA at 1 h following the injection by fluorescent microscopy. When naked siRNA and siRNA-Chol were injected, the accumulations had been strongly observed only in the kidneys (Figs. five and six), indicating that naked siRNA was promptly eliminated from the body by filtration inside the kidneys. For siRNA lipoplex, cationic lipoplex was largely accumulated inside the lungs. CS, PGA and PAA coatings of cationic lipoplex decreased the accumulation of siRNA in the lungs and improved it in the liver as well as the kidneys (Fig. five). To confirm irrespective of whether siRNA observed in the kidneys was siRNA or lipoplex of siRNA, we prepared cationic and PGA-coated lipoplexes employing rhodamine-labeled liposome and Cy5.5siRNA, along with the localizations of siRNA and liposome following intravenous injection had been observed by fluores.
