To further enhance the transfection efficiency of a micelleplex system based on monomethoxy poly（ethylene glycol）-block-poly（e-caprolactone）-block-poly（L-lysine） （MPEG-b-PCL-b-PLL）, cholesterol （Chol） moieti...To further enhance the transfection efficiency of a micelleplex system based on monomethoxy poly（ethylene glycol）-block-poly（e-caprolactone）-block-poly（L-lysine） （MPEG-b-PCL-b-PLL）, cholesterol （Chol） moieties are attached to the e-termini of PLL segments to obtain MPEG-b-PCL-b-PLL/Chol. The structure and morphology of the copolymer are studied by IH-NMR, TEM and DLS （dynamic light scattering）. The cytotoxicity, cell uptake, endosomal release and mRNA knockdown are studied by MTT （3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide） assay, flow cytometry, CLSM （confocal laser scanning microscopy） and RT-PCR （real-time polymerase chain reaction）. The results show that compared to their precursor MPEG-b-PCL-b-PLL, the cholesterol-grafted copolymer shows significantly lower toxicity, more rapid cellular endocytosis and endosome escape, and consequently displays enhanced siRNA transfection efficiency even at a lower N/P ratio. These improvements are ascribed to enhanced interaction of the cholesterol moieties with both cellular membrane and endosomal membrane. Moreover, effect of the PLL block length is examined. The final conclusion is that long enough PLL segments and incorporation of proper fraction of cholesterol onto the PLL segments benefit the enhancement of siRNA transfection efficiency.展开更多
Cyclin-dependent kinases 4 and 6 inhibitors(CDK4/6i)have been demonstrated to trigger antitumor immunity for tumor regression.However,the therapeutic performance of CDK4/6i-meadiated cancer immunotherapy was impaired ...Cyclin-dependent kinases 4 and 6 inhibitors(CDK4/6i)have been demonstrated to trigger antitumor immunity for tumor regression.However,the therapeutic performance of CDK4/6i-meadiated cancer immunotherapy was impaired by the immunosuppressive tumor microenvironment(ITM)due to overexpression of programmed death ligand 1(PD-L1)on the surface of cancer cell membrane.To improve the immunotherapeutic performance of CDK4/6i,we herein developed endosomal acidactivatable micelleplex for si RNA delivery and PD-L1 knockdown in the tumor cells in vitro and in vivo.We further demonstrated that the combination of PD-L1 knockdown and CDK4/6 inhibition facilitated intratumoral infiltration of cytotoxic T lymphocytes(CTLs),and elicited protective immune response and efficiently suppressed tumor growth in vivo.This study revealed the importance of molecular design of the micelleplex for highly efficient si RNA delivery,which might provide a novel insight for RNAi-based cancer immunotherapy.展开更多
基金supported by the National Natural Science Foundation of China (No. 21004062)"100 Talents Program" of the Chinese Academy of Sciences (No. KGCX2-YW-802)the Ministry of Science and Technology ofChina ("973 Project", No. 2009CB930102)
文摘To further enhance the transfection efficiency of a micelleplex system based on monomethoxy poly（ethylene glycol）-block-poly（e-caprolactone）-block-poly（L-lysine） （MPEG-b-PCL-b-PLL）, cholesterol （Chol） moieties are attached to the e-termini of PLL segments to obtain MPEG-b-PCL-b-PLL/Chol. The structure and morphology of the copolymer are studied by IH-NMR, TEM and DLS （dynamic light scattering）. The cytotoxicity, cell uptake, endosomal release and mRNA knockdown are studied by MTT （3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide） assay, flow cytometry, CLSM （confocal laser scanning microscopy） and RT-PCR （real-time polymerase chain reaction）. The results show that compared to their precursor MPEG-b-PCL-b-PLL, the cholesterol-grafted copolymer shows significantly lower toxicity, more rapid cellular endocytosis and endosome escape, and consequently displays enhanced siRNA transfection efficiency even at a lower N/P ratio. These improvements are ascribed to enhanced interaction of the cholesterol moieties with both cellular membrane and endosomal membrane. Moreover, effect of the PLL block length is examined. The final conclusion is that long enough PLL segments and incorporation of proper fraction of cholesterol onto the PLL segments benefit the enhancement of siRNA transfection efficiency.
基金financially supported by the National Natural Science Foundation of China(Nos.51873228 and 31671024)Basic Research Program of Shenzhen(No.JCYJ20180227175420974)+1 种基金Science and Technology Development Fund,Macao SAR(No.083/2017/A2)Open Research Fund of State Key Laboratory of Polymer Physics and Chemistry,Changchun Institute of Applied Chemistry,Chinese Academy of Sciences。
文摘Cyclin-dependent kinases 4 and 6 inhibitors(CDK4/6i)have been demonstrated to trigger antitumor immunity for tumor regression.However,the therapeutic performance of CDK4/6i-meadiated cancer immunotherapy was impaired by the immunosuppressive tumor microenvironment(ITM)due to overexpression of programmed death ligand 1(PD-L1)on the surface of cancer cell membrane.To improve the immunotherapeutic performance of CDK4/6i,we herein developed endosomal acidactivatable micelleplex for si RNA delivery and PD-L1 knockdown in the tumor cells in vitro and in vivo.We further demonstrated that the combination of PD-L1 knockdown and CDK4/6 inhibition facilitated intratumoral infiltration of cytotoxic T lymphocytes(CTLs),and elicited protective immune response and efficiently suppressed tumor growth in vivo.This study revealed the importance of molecular design of the micelleplex for highly efficient si RNA delivery,which might provide a novel insight for RNAi-based cancer immunotherapy.
