Investigation intracellular trafficking of siRNAs following their delivery to cells is of great interest to elucidate dynamics of siRNA in cytoplasm. In this study, we present a novel confocal laser scanning microscopy （CLSM） method to evaluate a novel delivery system of 3＇-peptide-siRNA therapeutic, which was named 3＇-pAs-siRNA/CLD. This method could not only calculate the content of the intracellular 3＇-peptide-siRNA, but also quantify its co-localization with cellular substructure. We observed that 3＇-pAs-siRNA/CLD, which provided the better antitumor capability, also had a better cell uptake, endosome escape and a longer retention time in A375. This novel strategy was proved to be efficient, quantified and visualized, thus making the dynamics research of siRNA in cytoplasm clear and simplified.
Yiping DiaoJing SunMengyi YangBo XuLihe ZhangZhenjun Yang
In this paper,a series of peptide-siRNA conjugates with phosphodiester unit as the linker targeting to Cdc2 gene were synthesized by solid phase stepwise strategy.The conjugation of peptides at either 3’-terminus of siCdc2 bring no change to the classical A-form of RNA duplex,but slightly compromise the thermodynamic stability.Peptide conjugation at the 3’-terminus of sense strand could improve the serum stability obviously,however,the opposite peptide conjugation at the 3’-terminus of antisense strand shows no such influence.According to the results of artificial silencing activity assay system,peptide conjugation at 3’-terminus of antisense strand slightly weakens the silencing activity of siCdc2.But sense strand peptide conjugation exhibits similar silencing activity as native siCdc2,meanwhile,it could mitigate the unwanted off-target effect of sense strand targeting to its own mRNA.
WANG XiaoFengHUANG YeLIU YangCHEN YueJIN HongWeiZHENG YiDU QuanYANG ZhenJunZHANG LiHe
In this study,a series of 5′-phosphorylated siRNAs with D-/L-isonucleotide(isoNA)or locked nucleic acid(LNA)incorporated at the 5′-terminus were synthesized.It was found that after incorporating either isoNA or LNA at the 5′-terminus of the antisense strand or sense strand,the silencing activity of modified strands has been inhibited,which cannot be recovered by phosphorylation at the 5′-terminus;however,the silencing activity of unmodified strand to its own target was increased.This work indicates that the isoNA and LNA modification at 5′-terminus can interfere with the strand selection during the RISC assembling process,and the disturbance of the 5′-phosporylation should not be the only viable mechanism.
A disulfide-modified nucleoside was designed and synthesized. After loading the modified nucleoside on controlled pore glass （CPG）, solid phase synthesis strategy was used to prepare peptide-oligonucleotide conjugates （N-3＇） containing disulfide bond unit. The 3＇-sense strand peptide-siRNA conjugate （VII） maintained good gene silencing activity, while that of the 3＇-antisense strand conjugate decreased somewhat. And the sense strand off-target effect of VII decreased remarkably.
Cationic lipids have been applied to siRNA delivery for tumor therapeutics. However, the excess positive charges of these nanoplexes may lead to high cytotoxicity and nonnegligible immunogenicity both in vitro and in vivo, which limited the applications of gene drugs. We constructed multi-component lipoplex to delivery 3＇,3＂-bis-peptide-siRNA conjugate （pp-siRNA） by the treatment of melanoma. Based on the previous studies that the gemini lipid （CLD） encapsulated pp-siRNA, a novel neutral cytosin-l-yl- lipid （DNCA） was considered to replace a certain ration of CLD by hydrogen bonds and ~t-n stacking for reducing the cytotoxicity. It similarly retained in both the loading efficiency and targeted mRNA inhibition when DNCA was accounted for 40% in the lipoplex, with lower toxicity. Moreover, CLD/DNCA/pp-siRNA nanoplex could be uptake in A375 cells and internalized mainly by macropinocytosis and caveolin-mediated endocytosis. Besides, 90% CLD/DNCA/pp-siRNA nanoplexes presented the highest efficient knockdown for the mutant B-RAF mRNA （-80%）. All the results demonstrated that the mixed cationic and neutral lipids could efficiently realize the delivery of pp-siRNA and had potential application for cancer therapy.
Inspired by the influence of chemical structure of end groups on the phase transition temperature of thermoresponsive polymers,we demonstrated a strategy to control the multi-responsiveness of polymer assemblies via subtle modification of end groups of thermoresponsive polymer segments and revealed its potential application for drug delivery.By developing polymer assemblies composed of poly(aliphatic ester) as the inner core and thermoresponsive polyphosphoester as the outer shell,we showed that end groups of thermoresponsive polyphosphoester segments controlled the surface property of assemblies and further determined the stimuli-responsive behavior.The phase-transition temperatures of the unmodified polymer assemblies are tightly controlled by their surface properties due to the hydrophilic to hydrophobic transitions of end groups in response to an environmental stimulus (e.g.pH or light irradiation).External control over these surface properties can by asserted by adjusting the chemical structure and composition of the terminal groups of the thermoresponsive polyphosphoesters.
Weiwei LiuYucai WangYang LiFeng WangXianzhu YangTianmeng SunJinzhi DuJun Wang
