用原代大鼠海马神经元为模型,对新型电转染方法Nucleofector^(TM)与脂质体DOTAP和Lipofectaimine^(TM)的转染效率和转染前后细胞存活率进行比较研究,探讨Nucleofector^(TM)的高效性与可靠性。从E18胎鼠海马中取出神经元进行体外培养,并...用原代大鼠海马神经元为模型,对新型电转染方法Nucleofector^(TM)与脂质体DOTAP和Lipofectaimine^(TM)的转染效率和转染前后细胞存活率进行比较研究,探讨Nucleofector^(TM)的高效性与可靠性。从E18胎鼠海马中取出神经元进行体外培养,并用神经微丝(NF)抗体进行免疫细胞化学染色鉴定细胞类型。分别用DOTAP,Lipofectamine^(TM) and Nucleofector^(TM)包裹pCMV-eGFP质粒转染原代大鼠海马神经元。神经元的存活率用流式细胞促检测。实验结果表明:DOTAP和Lipofectamine^(TM)的基因转染效率仅为1.55％和2.45％,而Nucleofector^(TM)的转染效率则超过20％;细胞转染前后的存活率在DOTAP组分别为98.37％和88.35％,Lipofectamine^(TM)组分别为98.37％和90.11％,而在Nucleofector^(TM)组中分别为98.37％和51.82％。上述实验数据表明:Nucleofector^(TM)转染技术能高效并安全地转染原代大鼠海马神经元,但死亡率较高。展开更多
This study aimed to investigate the effects of cationic liposomes containing different cationic lipids （DC-Chol and DOTAP） and different pegylation ratios on siRNA transfection in human U251 glioma cells. The data s...This study aimed to investigate the effects of cationic liposomes containing different cationic lipids （DC-Chol and DOTAP） and different pegylation ratios on siRNA transfection in human U251 glioma cells. The data showed that the transfection efficiency of DOTAP was much higher than that of DC-Chol and PEG at 2 mol% enhanced cellular uptake of siRNA. Cationic liposome-siRNA complexes with particle size around 100 nm were prepared. PEG modification could efficiently stabilize the liposome in the presence of serum, which might protect the siRNA from serum degradation and prolong the circulation time in vivo. Efficient intracellular uptake and lysosome release of siRNA in human U251 glioma cells were observed for pegylated DOTAP-based lipososomes compared with the control transfection reagent lipofectamine 2000. The results demonstrated that this cationic liposome might be a potential vehicle for the in vivo delivery of siRNA.展开更多
Since antigen and adjuvant are rapid clearance in vivo,insufficient delivery to induce dendritic cells(DCs)maturation and cross-presentation,as well as limited migration efficiency of DCs to secondary lymph organs,gre...Since antigen and adjuvant are rapid clearance in vivo,insufficient delivery to induce dendritic cells(DCs)maturation and cross-presentation,as well as limited migration efficiency of DCs to secondary lymph organs,greatly hinders the development of DCs-based immunotherapy.Herein,PCL-PEG-PCL polymersomes(PCEP-PS)as antigen and adjuvants delivery nanoplatforms(IMO-PS)were well-designed,which can electrostatically adsorb OVA antigen on the surface via DOTAP lipid and effectively encapsulate OVA antigen into the inner hydrophilic cavity to achieve both initial antigen exposure as well as slow and sustained antigen release,incorporate MPLA within the lipid layer to ligate with extracellular TLR4 of DCs as well as encapsulate IMQ in the hydrophobic membrane to ligate with intracellular TLR7/8 of DCs for activating synergistic immune responses via different signaling pathways.The IMO-PS significantly improved antigen uptake,promoted DCs maturation and cytokines production.DCs treated with IMO-PS could enhance migration into draining lymphoid nodes,and eventually induced antigen-specific CD8+and CD4+T cell responses and OVA-specific cytotoxic T lymphocyte(CTL)responses.Prophylactic vaccination of EG7-OVA tumor-bearing mice by IMO-PS+DCs significantly extended tumor-free time,effectively suppressed tumor growth,and greatly extended median survival time.The strategy may provide an effective nanoplatform for co-delivery antigen and dual-adjuvants in a spatio-temporally programmed manner for DC-based cancer immunotherapy.展开更多
The extraordinary advantages associated with mRNA vaccines,including their high efficiency,relatively low severity of side effects,and ease of manufacture,have enabled them to be a promising immunotherapy approach aga...The extraordinary advantages associated with mRNA vaccines,including their high efficiency,relatively low severity of side effects,and ease of manufacture,have enabled them to be a promising immunotherapy approach against various infectious diseases and cancers.Nevertheless,most mRNA delivery carriers have many disadvantages,such as high toxicity,poor biocompatibility,and low efficiency in vivo,which have hindered the widespread use of mRNA vaccines.To further characterize and solve these problems and develop a new type of safe and efficient mRNA delivery carrier,a negatively charged SA@DOTAP-mRNA nanovaccine was prepared in this study by coating DOTAP-mRNA with the natural anionic polymer sodium alginate(SA).Intriguingly,the transfection efficiency of SA@DOTAP-mRNA was significantly higher than that of DOTAP-mRNA,which was not due to the increase in cellular uptake but was associated with changes in the endocytosis pathway and the strong lysosome escape ability of SA@DOTAP-mRNA.In addition,we found that SA significantly increased the expression of LUC-mRNA in mice and achieved certain spleen targeting.Finally,we confirmed that SA@DOTAP-mRNA had a stronger antigen-presenting ability in E.G7-OVA tumor-bearing mice,dramatically inducing the proliferation of OVA-specific CLTs and ameliorating the antitumor effect.Therefore,we firmly believe that the coating strategy applied to cationic liposome/mRNA complexes is of potential research value in the field of mRNA delivery and has promising clinical application prospects.展开更多
文摘用原代大鼠海马神经元为模型,对新型电转染方法Nucleofector^(TM)与脂质体DOTAP和Lipofectaimine^(TM)的转染效率和转染前后细胞存活率进行比较研究,探讨Nucleofector^(TM)的高效性与可靠性。从E18胎鼠海马中取出神经元进行体外培养,并用神经微丝(NF)抗体进行免疫细胞化学染色鉴定细胞类型。分别用DOTAP,Lipofectamine^(TM) and Nucleofector^(TM)包裹pCMV-eGFP质粒转染原代大鼠海马神经元。神经元的存活率用流式细胞促检测。实验结果表明:DOTAP和Lipofectamine^(TM)的基因转染效率仅为1.55％和2.45％,而Nucleofector^(TM)的转染效率则超过20％;细胞转染前后的存活率在DOTAP组分别为98.37％和88.35％,Lipofectamine^(TM)组分别为98.37％和90.11％,而在Nucleofector^(TM)组中分别为98.37％和51.82％。上述实验数据表明:Nucleofector^(TM)转染技术能高效并安全地转染原代大鼠海马神经元,但死亡率较高。
基金National Natural Science Foundation of China (Grant No.30701056)Foundation of MOST(973 Program,Grant No.2007CB935801)+1 种基金Beijing Natural Science Foundation of China (Grant No.7083112)Doctoral Fund of Ministry of Education of China(Grant No.20070001813).
文摘This study aimed to investigate the effects of cationic liposomes containing different cationic lipids （DC-Chol and DOTAP） and different pegylation ratios on siRNA transfection in human U251 glioma cells. The data showed that the transfection efficiency of DOTAP was much higher than that of DC-Chol and PEG at 2 mol% enhanced cellular uptake of siRNA. Cationic liposome-siRNA complexes with particle size around 100 nm were prepared. PEG modification could efficiently stabilize the liposome in the presence of serum, which might protect the siRNA from serum degradation and prolong the circulation time in vivo. Efficient intracellular uptake and lysosome release of siRNA in human U251 glioma cells were observed for pegylated DOTAP-based lipososomes compared with the control transfection reagent lipofectamine 2000. The results demonstrated that this cationic liposome might be a potential vehicle for the in vivo delivery of siRNA.
基金financially supported by National Natural Science Foundation of China (Nos. 82072059 and 82172090)CAMS Initiative for Innovative Medicine (No. 2021-I2M-1-058)+1 种基金the Fundamental Research Funds for the Central Universities (Nos. 2019PT320028 and 2019-0831-03)Tianjin Municipal Natural Science Foundation (No. 20JCYBJC00030)
文摘Since antigen and adjuvant are rapid clearance in vivo,insufficient delivery to induce dendritic cells(DCs)maturation and cross-presentation,as well as limited migration efficiency of DCs to secondary lymph organs,greatly hinders the development of DCs-based immunotherapy.Herein,PCL-PEG-PCL polymersomes(PCEP-PS)as antigen and adjuvants delivery nanoplatforms(IMO-PS)were well-designed,which can electrostatically adsorb OVA antigen on the surface via DOTAP lipid and effectively encapsulate OVA antigen into the inner hydrophilic cavity to achieve both initial antigen exposure as well as slow and sustained antigen release,incorporate MPLA within the lipid layer to ligate with extracellular TLR4 of DCs as well as encapsulate IMQ in the hydrophobic membrane to ligate with intracellular TLR7/8 of DCs for activating synergistic immune responses via different signaling pathways.The IMO-PS significantly improved antigen uptake,promoted DCs maturation and cytokines production.DCs treated with IMO-PS could enhance migration into draining lymphoid nodes,and eventually induced antigen-specific CD8+and CD4+T cell responses and OVA-specific cytotoxic T lymphocyte(CTL)responses.Prophylactic vaccination of EG7-OVA tumor-bearing mice by IMO-PS+DCs significantly extended tumor-free time,effectively suppressed tumor growth,and greatly extended median survival time.The strategy may provide an effective nanoplatform for co-delivery antigen and dual-adjuvants in a spatio-temporally programmed manner for DC-based cancer immunotherapy.
基金supported by the National Key Research and Development Program of China(2021YFE0206600)the Sichuan Province Science and Technology Support Program(2021YF-SY0008 and 2020YJ023,China)+1 种基金the Translational Medicine Fund of West China Hospital(CGZH19002,China)the 1.3.5 Project for Disciplines of excellence,West China Hospital,Sichuan University(ZYGD18020/ZYJC18006,China).
文摘The extraordinary advantages associated with mRNA vaccines,including their high efficiency,relatively low severity of side effects,and ease of manufacture,have enabled them to be a promising immunotherapy approach against various infectious diseases and cancers.Nevertheless,most mRNA delivery carriers have many disadvantages,such as high toxicity,poor biocompatibility,and low efficiency in vivo,which have hindered the widespread use of mRNA vaccines.To further characterize and solve these problems and develop a new type of safe and efficient mRNA delivery carrier,a negatively charged SA@DOTAP-mRNA nanovaccine was prepared in this study by coating DOTAP-mRNA with the natural anionic polymer sodium alginate(SA).Intriguingly,the transfection efficiency of SA@DOTAP-mRNA was significantly higher than that of DOTAP-mRNA,which was not due to the increase in cellular uptake but was associated with changes in the endocytosis pathway and the strong lysosome escape ability of SA@DOTAP-mRNA.In addition,we found that SA significantly increased the expression of LUC-mRNA in mice and achieved certain spleen targeting.Finally,we confirmed that SA@DOTAP-mRNA had a stronger antigen-presenting ability in E.G7-OVA tumor-bearing mice,dramatically inducing the proliferation of OVA-specific CLTs and ameliorating the antitumor effect.Therefore,we firmly believe that the coating strategy applied to cationic liposome/mRNA complexes is of potential research value in the field of mRNA delivery and has promising clinical application prospects.
