Lipid nanoparticles(LNPs)are critical for the delivery of drugs and nucleic acids.However,current mRNA-LNP formulations require stringent freezing for storage,which limits their global distribution.Our previous studie...Lipid nanoparticles(LNPs)are critical for the delivery of drugs and nucleic acids.However,current mRNA-LNP formulations require stringent freezing for storage,which limits their global distribution.Our previous studies demonstrated that optimizing the lipid type or molar ratio of Comirnaty-type mRNA-LNPs could enhance their lyophilization stability,thus improving their long-term storage stability under mild conditions.This study aims to enhance the storage stability of Spikevax-type mRNA-LNPs by optimizing lipid compositions and utilizing lyophilization for storage at 4�C.Fifteen mRNA-LNP formulations were evaluated for their physicochemical properties and transfection efficiency(TE)in human embryonic kidney(HEK)-293T cells using the I-optimal design of mixture experiments.Mathematical models were developed to predict the relationships among encapsulation efficiency,transfection performance and lipid ratios.The optimized mRNA-LNP formulation(N4),with a 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)-to-cholesterol ratio of 0.36,exhibited superior stability and TE after lyophilization.N4 outperformed the original Spikevax formulation in several cell lines,including eye-derived ARPE-19 cells and lung-derived A549 cells.In vivo,N4 demonstrated high TE in the spleen of C57BL/6 mice both before and after lyophilization,with no signals observed in the kidneys,heart or eyes.These findings suggest that the optimized N4 formulation offers a robust,stable and efficient delivery system for gene therapy and vaccines,potentially overcoming the storage limitations of current Spikevax-type mRNA-LNPs and broadening their therapeutic applications.展开更多
Objective:The development of gene carriers for efficient gene delivery into cells has attracted growing attention in recent years.The aim of this study was to achieve a better outcome of AAV-293 cells transfection by ...Objective:The development of gene carriers for efficient gene delivery into cells has attracted growing attention in recent years.The aim of this study was to achieve a better outcome of AAV-293 cells transfection by plasmid DNA.Methods:We studied the optimal condition for higher efficiency of cationic lipid-mediated cell transfection.Four experimental groups were set.Plasmid DNA and liposome were mixed in each groups at different ratios(μg:μL),1:2.5,1:3.5,1:4.0 and 1:5.0,respectively.LacZ gene functioned as reporter gene,measuring the transfection efficiency of the four groups using the method of X-gal staining.Results:When the ratio was 1:3.5,the cell transfection rate was the highest.While the ratio of 1:2.5 recommended by product manual achieve the lowest transfection rate.Their difference had statistical significance.Conclusion:In order to obtain a higher transfection efficiency,optimization on conditions of the ratio of plasmid DNA to liposome is necessary in cell transfection.展开更多
Three cationic lipids with lysylated（1）, histidylated（2）, and arginylated（3） headgroups and cholesterol hydrophobic moiety were synthesized. The average sizes of liposomes and lipoplexes were around 100 and 160 ...Three cationic lipids with lysylated（1）, histidylated（2）, and arginylated（3） headgroups and cholesterol hydrophobic moiety were synthesized. The average sizes of liposomes and lipoplexes were around 100 and 160 nm, respectively. The gene transfection efficiency of the three lipoplexes loaded with pGL3 or pORF-LacZ was compared on 293T cells in the presence or the absence of serum. The transfection efficiency of the three lipoplexes in a serum-free medium was 2 to 3-fold higher than that of dioleoyl-trimethylammonium propane（DOTAP）. In the presence of serum, however, most of the lipoplexes showed lower transfection activities; only lipoplex 3 retained its high transfection efficiency.展开更多
Suspension cells play a crucial role in many biological processes. However, compared to adherent cells, it is particularly challenging to introduce exogenous genes into suspension cells to regulate their biological fu...Suspension cells play a crucial role in many biological processes. However, compared to adherent cells, it is particularly challenging to introduce exogenous genes into suspension cells to regulate their biological functions with non-viral gene vectors, mainly due to the low cellular uptake and endosomal escape of polyplexes. Herein, to improve the interactions of polyplexes with cellular membranes, we design and synthesize highly branched poly(β-amino ester)(HPAE) via an “A2 + B4 + C2” Michael addition strategy.Results show that branching significantly increases DNA condensation of HPAE, cellular uptake and endosomal escape of HPAE/DNA polyplexes. In mast cells(MCs), HPAE exhibits up to 80-fold higher gene transfection efficiency compared to the corresponding linear poly(β-amino ester)(LPAE) and the leading commercial gene transfection reagents PEI25k, jetPEI, and Lipofectamine 3000, without causing obvious cytotoxicity. Our study establishes a reliable non-viral platform for efficient gene transfection of suspension cells.展开更多
Advantages and disadvantages of four transfection methods of DNA vaccines into the body were introduced in this study, including direct transfection, transfection of bacterial vectors, cationic lipofection and cationi...Advantages and disadvantages of four transfection methods of DNA vaccines into the body were introduced in this study, including direct transfection, transfection of bacterial vectors, cationic lipofection and cationic polymer transfection. Cationic polymer had the characteristics of high transfection efficiency, simple operation, wide application, good repeatability and low cell virulence, which could be expected as a new kind of effi- cient transfection reagents. In practical application, various transfection methods have their own advantages and disadvantages, so the best choice should be made in the design process of vaccines based on targets, test cost, using objects and its convenience.展开更多
The recent commercialization of gene products has sparked significant interest in gene therapy,necessitating efficient and precise gene delivery via various vectors.Currently,viral vectors and lipid-based nanocarriers...The recent commercialization of gene products has sparked significant interest in gene therapy,necessitating efficient and precise gene delivery via various vectors.Currently,viral vectors and lipid-based nanocarriers are the predominant choices and have been extensively investigated and reviewed.Beyond these vectors,polymeric nanocarriers also hold the promise in therapeutic gene delivery owing to their versatile functionalities,such as improving the stability,cellar uptake and endosomal escape of nucleic acid drugs,along with precise delivery to targeted tissues.This review presents a brief overview of the status quo of the emerging polymeric nanocarriers for therapeutic gene delivery,focusing on key cationic polymers,nanocarrier types,and preparation methods.It also highlights targeted diseases,strategies to improve delivery efficiency,and potential future directions in this research area.The review is hoped to inspire the development,optimization,and clinical translation of highly efficient polymeric nanocarriers for therapeutic gene delivery.展开更多
A biodegradable gene transfer vector, poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine] has been developed by thermal polycondensation of aspartic acid and lysine, and branch poly(ethylenimine) (Mw less than ...A biodegradable gene transfer vector, poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine] has been developed by thermal polycondensation of aspartic acid and lysine, and branch poly(ethylenimine) (Mw less than 600) was grafted to the backbone. The polymer was characterized by 1H NMR. It appeared lower cytotoxity compared to poly(ethylenimine) (25KDa), which was quantified by MTT assay. Electrophoresis indicated that the polymer could retardate DNA at N/P ratio 1.2-1.8 (w/w). Transfection efficiency of the complexes was studied in NT2 cell lines. It was 1.5 fold higher than molecular weight PEI (Mw = 25KDa).展开更多
OBJECTIVE To investigate the effect of polyethylene imine glycol （PEI-PEG）/siRNA nanocomposites in the in vitro transfection of human gastric cancer SGC7901 cell lines and the down-regulation of gene expression of t...OBJECTIVE To investigate the effect of polyethylene imine glycol （PEI-PEG）/siRNA nanocomposites in the in vitro transfection of human gastric cancer SGC7901 cell lines and the down-regulation of gene expression of the adherence factor CD44v6. METHODS PEI-PEG/siRNA nanoparticles, in different N/P ratios, were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the control group. The transfection efficiencies were observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay （mononuclear cell direct cytotoxicity assay）, and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. Based on the different N/P ratios, PEI-PEG/siRNA composites were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the controls. The transfection efficiency was observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. RESULTS After transfection, the transfection efficiency of the PEI-PEG/siRNA nanocomposites increased incrementally in N/P ratio value. The transfection efficiency improved with an increase in N/P ratio. When the N/P value was 15, fluorescence became more intense in the PEI-PEG/siRNA group than in the Lipo2000/siRNA group. At the same time, cell viability was （80.4 ± 5.6）% in the MTT reduction assay, which was similar to that in the Lipo2000/siRNA group. The results of Western blot analysis showed that the expression level of CD44v6 protein decreased to （59.7 ± 3.0）% after siRNA-CD44v6 was inhibited. CONCLUSION PEI-PEG could effectively form the nanocomposite in combination with siRNA, be transfected into the SGC7901 gastric cancer cell lines and inhibit CD44v6 protein expression. Moreover, as a genetic carrier, PEI-PEG copolymer has greater advantages, including high transfection e. ciency, less cytotoxicity and an easily alterable vector structure.展开更多
A series of novel water soluble chitosan derivatives as gene vectors was synthesized. The delivery systems were tested for their ability to form complexes with plasmid DNA by utilizing agarose gel electrophoresis, par...A series of novel water soluble chitosan derivatives as gene vectors was synthesized. The delivery systems were tested for their ability to form complexes with plasmid DNA by utilizing agarose gel electrophoresis, particle size analysis, zeta potential measurement and scanning electron microscopy. Furthermore, cytotoxicity of chitosan derivatives and transfection efficiency of polyplexes were evaluated in vitro. The experimental results showed that the novel chitosan derivatives had lower cytotoxicity, good DNA condensation, and higher transfection efficiencies compared to chitosan in both 293T and HeLa cell lines. It was indicated that these chitosan derivatives were promising candidates for non-viral gene vectors.展开更多
The work described the synthesis and evaluation of PEI-g-comb-PEG-transferrin as a potential system for gene therapy in vitro. The MW of PEG was 10KDa, and PEI was 2KDa. Its structure was identified by NMR, FT-IR and ...The work described the synthesis and evaluation of PEI-g-comb-PEG-transferrin as a potential system for gene therapy in vitro. The MW of PEG was 10KDa, and PEI was 2KDa. Its structure was identified by NMR, FT-IR and TGA spectroscopy. MTT assay found that at concentration up to 4000 n mol/L of the polymer, cell viability was over 85%. The bio-character of polymer/DNA complex was characterized by agarose gel electrophoresis, ethidium bromide exclusion and zeta-potential assay. The polymer could retardate DNA at N/P ratio 3.0-3.5 （mol/mol）. The particle size of the polymer/DNA complex was less than 300 nm. Transfection efficiency of the complex was studied in COS7 and NT2 cell lines.展开更多
Protoplast-based transient gene expression system has been widely used in plant genome editing because of its simple operation and less time-consuming.In order to establish a universal protoplast-based transient trans...Protoplast-based transient gene expression system has been widely used in plant genome editing because of its simple operation and less time-consuming.In order to establish a universal protoplast-based transient transfection system for verifying activities of genome editing vectors containing targets in Brassica,we systematically optimized factors affecting protoplast isolation and transient gene expression.We established an efficient protoplast-based transient gene expression system(PTGE)in Chinese cabbage,achieving high protoplast yield of 4.9×10^(5)·g^(-1)FW,viability over 95%,and transfection efficiency of 76%.We showed for the first time that pretreatment of protoplasts with a hypotonic MMG could significantly enhance the transfection efficiency.Furthermore,protoplasts incubated at 37℃ for 6 min improved the transfection efficiency to 86%.We also demonstrated that PTGE worked well(more than 50%transfection efficiency)in multiple Brassica species including cabbage,Pak Choi,Chinese kale,and turnip.Finally,PTGE was used for validating the activities of CRISPR/Cas9 vectors containing targets in Chinese cabbage,cabbage,and pak choi,demonstrating the broad applicability of the established PTGE for genome editing in Brassica crops.展开更多
Incorporating functional ligands and biodegradable bonds into biocompatible low-molecular-weight(LMW)polymers,such as 1.8 kDa poly(ethylenimine)(PEI1.8 k),is a common strategy to improve the properties of LMW polymers...Incorporating functional ligands and biodegradable bonds into biocompatible low-molecular-weight(LMW)polymers,such as 1.8 kDa poly(ethylenimine)(PEI1.8 k),is a common strategy to improve the properties of LMW polymers including biosafety and delivery efficacy.This study demonstrates the hypothesis that introducing different functional ligands and linked reductive disulfides in PEI 1.8k will achieve superior siRNA transfection efficiency.By incorporating PEI-X(X represents cholesterol(Ch),heptafluorobutyric anhydride(HFBA,F)and 4-carboxyphenylboronic acid(PBA))functional ligands into PEI 1.8k and subsequently crosslinking with each other via disulfide bond links,reductive-responsive PEI-X-SS-X-PEI copolymers were constructed to enhance the cellular transfection via the synergistic effect of the high affinity of Ch,F and PBA to cell membranes and the disulfide reduction triggered intracellular disassembly of micelles and subsequent siRNA release.Extraordinarily,ternary Ch-SS-F-SS-PBA micelles exhibited the strongest siRNA transfection efficiencies in in vitro cell experiments and in vivo animal experiments due to the coordination of enhanced serum stability,promoted cell uptake and endosomal escape,and cell targeting ability.This strategy of constructed multifunctional polymer here we called"building-block crosslinking"showed a simple and smart way to synthesize new materials.Also this strategy of constructing ligands-directed reduction-sensitive micelles improves the transfection efficiency of LMW PEI and provides a valuable insight to develop novel gene delivery systems.展开更多
A polymeric polyethylenimine(PEI)-based prodrug of anticancer doxorubicin(DOX)(PEI-hyd-DOX) was designed by attaching DOX to PEI via an acid-labile hydrazone bond, for the achievement of biocontrollable gene and drug ...A polymeric polyethylenimine(PEI)-based prodrug of anticancer doxorubicin(DOX)(PEI-hyd-DOX) was designed by attaching DOX to PEI via an acid-labile hydrazone bond, for the achievement of biocontrollable gene and drug co-delivery in response to the intracellular acid microenvironments in the late endosome/lysosome compartments. The cytotoxicity of PEI-hyd-DOX was evaluated by the MTT assay and the cellular uptake was monitored using confocal laser scanning microscopy. The polymeric prodrug can respond with a high sensitivity to the specific acid condition inside cells, thus permitting the precise biocontrol over intracellular drug liberation with high drug efficacy. The chemical attachment of drug molecules also led to the relatively reduced toxicity and the enhanced transfection efficiency compared with parent PEI. The resulting data adumbrated the potential of PEI-hyd-DOX to co-deliver DOX and therapeutic gene for the combination of chemotherapy and gene therapy.展开更多
Gene therapy has drawn great attention in the treatments of many diseases,especially for cardiovascular diseases.However,the development of gene carriers with low cytotoxicity and multitargeting function is still a ch...Gene therapy has drawn great attention in the treatments of many diseases,especially for cardiovascular diseases.However,the development of gene carriers with low cytotoxicity and multitargeting function is still a challenge.Herein,the multitargeting REDV-G-TATG-NLS peptide was conjugated to amphiphilic cationic copolymer poly(e-caprolactone-co-3(S)-methyl-morpho-line-2,5-dione)-g-polyethyleneimine(PCLMD-g-PEI)via a heterobifunctional orthopyridyl disulfde-poly(ethylene glycol)-N-hydroxysuccinimide(OPSS-PEG-NHS)linker to prepare PCLMD-g-PEI-PEG-REDV-G-TAT-G-NLS copolymers with the aim to develop the gene carriers with low cytotoxicity and high transfection efficiency.The multitargeting micelles were prepared from PCLMD-g-PEI-PEG-REDV-G-TAT-G-NLS copolymers by self-assembly method and used to load pEGFP-ZNF580 plasmids(pDNA)to form gene complexes for enhancing the proliferation and migration of endothelial cells(ECs).The loading pDNA capacity was proved by agarose gel electrophoresis assay.These multitargeting gene com-plexes exhibited low cytotoxicity by 3-(4,-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide assay.The high internalization efficiency of these gene complexes was confirmed by flow cytometry.The results of in vitro transfection demonstrated that these multitargeting gene complexes possessed relatively high transfection effi-ciency.The rapid migration of ECs transfected by these gene complexes was verified by wound healing assay.Owing to ECs-targeting ability,cell-penetrating ability and nuclear targeting capacity of REDV-G-TAT-G-NLS pep-tide,the multitargeting polycationic gene carrier with low cytotoxicity and high transfection efficiency has great potential in gene therapy.展开更多
基金supported by the Zhejiang Provincial Natural Science Foundation of China(LQ24H120006)the Wenzhou Municipal Science and Technology Bureau(Y2023799)+3 种基金the Startup Foundation for Scientific Research,Eye Hospital,Wenzhou Medical University(KYQD20211204)the National Natural Science Foundation of China(52250710155)the National Key Research and Development Program of China(2021YFA1101200,2022YFA1105501)the Project of State Key Laboratory of Ophthalmology,Optometry and Visual Science,the Wenzhou Medical University(J02-20210201).
文摘Lipid nanoparticles(LNPs)are critical for the delivery of drugs and nucleic acids.However,current mRNA-LNP formulations require stringent freezing for storage,which limits their global distribution.Our previous studies demonstrated that optimizing the lipid type or molar ratio of Comirnaty-type mRNA-LNPs could enhance their lyophilization stability,thus improving their long-term storage stability under mild conditions.This study aims to enhance the storage stability of Spikevax-type mRNA-LNPs by optimizing lipid compositions and utilizing lyophilization for storage at 4�C.Fifteen mRNA-LNP formulations were evaluated for their physicochemical properties and transfection efficiency(TE)in human embryonic kidney(HEK)-293T cells using the I-optimal design of mixture experiments.Mathematical models were developed to predict the relationships among encapsulation efficiency,transfection performance and lipid ratios.The optimized mRNA-LNP formulation(N4),with a 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)-to-cholesterol ratio of 0.36,exhibited superior stability and TE after lyophilization.N4 outperformed the original Spikevax formulation in several cell lines,including eye-derived ARPE-19 cells and lung-derived A549 cells.In vivo,N4 demonstrated high TE in the spleen of C57BL/6 mice both before and after lyophilization,with no signals observed in the kidneys,heart or eyes.These findings suggest that the optimized N4 formulation offers a robust,stable and efficient delivery system for gene therapy and vaccines,potentially overcoming the storage limitations of current Spikevax-type mRNA-LNPs and broadening their therapeutic applications.
文摘Objective:The development of gene carriers for efficient gene delivery into cells has attracted growing attention in recent years.The aim of this study was to achieve a better outcome of AAV-293 cells transfection by plasmid DNA.Methods:We studied the optimal condition for higher efficiency of cationic lipid-mediated cell transfection.Four experimental groups were set.Plasmid DNA and liposome were mixed in each groups at different ratios(μg:μL),1:2.5,1:3.5,1:4.0 and 1:5.0,respectively.LacZ gene functioned as reporter gene,measuring the transfection efficiency of the four groups using the method of X-gal staining.Results:When the ratio was 1:3.5,the cell transfection rate was the highest.While the ratio of 1:2.5 recommended by product manual achieve the lowest transfection rate.Their difference had statistical significance.Conclusion:In order to obtain a higher transfection efficiency,optimization on conditions of the ratio of plasmid DNA to liposome is necessary in cell transfection.
基金Supported by the National Basic Research Program of China(No.2005CB623903).
文摘Three cationic lipids with lysylated（1）, histidylated（2）, and arginylated（3） headgroups and cholesterol hydrophobic moiety were synthesized. The average sizes of liposomes and lipoplexes were around 100 and 160 nm, respectively. The gene transfection efficiency of the three lipoplexes loaded with pGL3 or pORF-LacZ was compared on 293T cells in the presence or the absence of serum. The transfection efficiency of the three lipoplexes in a serum-free medium was 2 to 3-fold higher than that of dioleoyl-trimethylammonium propane（DOTAP）. In the presence of serum, however, most of the lipoplexes showed lower transfection activities; only lipoplex 3 retained its high transfection efficiency.
基金funded by National Natural Science Foundation of China (NSFC, No. 51903202)the Innovation Capability Support Program of Shaanxi (No. 2022TD-48)the Key R&D Program of Shaanxi Province (No. 2020GXLH-Y-016)。
文摘Suspension cells play a crucial role in many biological processes. However, compared to adherent cells, it is particularly challenging to introduce exogenous genes into suspension cells to regulate their biological functions with non-viral gene vectors, mainly due to the low cellular uptake and endosomal escape of polyplexes. Herein, to improve the interactions of polyplexes with cellular membranes, we design and synthesize highly branched poly(β-amino ester)(HPAE) via an “A2 + B4 + C2” Michael addition strategy.Results show that branching significantly increases DNA condensation of HPAE, cellular uptake and endosomal escape of HPAE/DNA polyplexes. In mast cells(MCs), HPAE exhibits up to 80-fold higher gene transfection efficiency compared to the corresponding linear poly(β-amino ester)(LPAE) and the leading commercial gene transfection reagents PEI25k, jetPEI, and Lipofectamine 3000, without causing obvious cytotoxicity. Our study establishes a reliable non-viral platform for efficient gene transfection of suspension cells.
基金supported by Project of Zhejiang Province(2006C12086)
文摘Advantages and disadvantages of four transfection methods of DNA vaccines into the body were introduced in this study, including direct transfection, transfection of bacterial vectors, cationic lipofection and cationic polymer transfection. Cationic polymer had the characteristics of high transfection efficiency, simple operation, wide application, good repeatability and low cell virulence, which could be expected as a new kind of effi- cient transfection reagents. In practical application, various transfection methods have their own advantages and disadvantages, so the best choice should be made in the design process of vaccines based on targets, test cost, using objects and its convenience.
基金supported by National Natural Science Foundation of China(82104082)Natural Science Foundation of Qinghai Province(2024-ZJ-911).
文摘The recent commercialization of gene products has sparked significant interest in gene therapy,necessitating efficient and precise gene delivery via various vectors.Currently,viral vectors and lipid-based nanocarriers are the predominant choices and have been extensively investigated and reviewed.Beyond these vectors,polymeric nanocarriers also hold the promise in therapeutic gene delivery owing to their versatile functionalities,such as improving the stability,cellar uptake and endosomal escape of nucleic acid drugs,along with precise delivery to targeted tissues.This review presents a brief overview of the status quo of the emerging polymeric nanocarriers for therapeutic gene delivery,focusing on key cationic polymers,nanocarrier types,and preparation methods.It also highlights targeted diseases,strategies to improve delivery efficiency,and potential future directions in this research area.The review is hoped to inspire the development,optimization,and clinical translation of highly efficient polymeric nanocarriers for therapeutic gene delivery.
文摘A biodegradable gene transfer vector, poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine] has been developed by thermal polycondensation of aspartic acid and lysine, and branch poly(ethylenimine) (Mw less than 600) was grafted to the backbone. The polymer was characterized by 1H NMR. It appeared lower cytotoxity compared to poly(ethylenimine) (25KDa), which was quantified by MTT assay. Electrophoresis indicated that the polymer could retardate DNA at N/P ratio 1.2-1.8 (w/w). Transfection efficiency of the complexes was studied in NT2 cell lines. It was 1.5 fold higher than molecular weight PEI (Mw = 25KDa).
基金This work was supported by the following funds： National Natural Science Foundation of China （No.30670951） Guangdong Provincial+5 种基金 Natural Science Foundation （No.06021322） Fund of Guangzhou Municipal Scientific Problem-Solving Program （No. 2003 Z 3-E0381） Fund of Guangdong Provincial Scientific Problem-Solving Program （No.2005 B31211002） Guangdong Provincial Government and Ministry of Education Project com- bining project initiation, study and research （No.2009B090300277）.
文摘OBJECTIVE To investigate the effect of polyethylene imine glycol （PEI-PEG）/siRNA nanocomposites in the in vitro transfection of human gastric cancer SGC7901 cell lines and the down-regulation of gene expression of the adherence factor CD44v6. METHODS PEI-PEG/siRNA nanoparticles, in different N/P ratios, were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the control group. The transfection efficiencies were observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay （mononuclear cell direct cytotoxicity assay）, and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. Based on the different N/P ratios, PEI-PEG/siRNA composites were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the controls. The transfection efficiency was observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. RESULTS After transfection, the transfection efficiency of the PEI-PEG/siRNA nanocomposites increased incrementally in N/P ratio value. The transfection efficiency improved with an increase in N/P ratio. When the N/P value was 15, fluorescence became more intense in the PEI-PEG/siRNA group than in the Lipo2000/siRNA group. At the same time, cell viability was （80.4 ± 5.6）% in the MTT reduction assay, which was similar to that in the Lipo2000/siRNA group. The results of Western blot analysis showed that the expression level of CD44v6 protein decreased to （59.7 ± 3.0）% after siRNA-CD44v6 was inhibited. CONCLUSION PEI-PEG could effectively form the nanocomposite in combination with siRNA, be transfected into the SGC7901 gastric cancer cell lines and inhibit CD44v6 protein expression. Moreover, as a genetic carrier, PEI-PEG copolymer has greater advantages, including high transfection e. ciency, less cytotoxicity and an easily alterable vector structure.
基金Funded by the National Natural Science Foundation of China(Nos.21204071,51273156)the Natural Science Foundation of Hubei Province(2014CFB833)the Innovation Research Fund of Wuhan university of Technology(20121049720006)
文摘A series of novel water soluble chitosan derivatives as gene vectors was synthesized. The delivery systems were tested for their ability to form complexes with plasmid DNA by utilizing agarose gel electrophoresis, particle size analysis, zeta potential measurement and scanning electron microscopy. Furthermore, cytotoxicity of chitosan derivatives and transfection efficiency of polyplexes were evaluated in vitro. The experimental results showed that the novel chitosan derivatives had lower cytotoxicity, good DNA condensation, and higher transfection efficiencies compared to chitosan in both 293T and HeLa cell lines. It was indicated that these chitosan derivatives were promising candidates for non-viral gene vectors.
文摘The work described the synthesis and evaluation of PEI-g-comb-PEG-transferrin as a potential system for gene therapy in vitro. The MW of PEG was 10KDa, and PEI was 2KDa. Its structure was identified by NMR, FT-IR and TGA spectroscopy. MTT assay found that at concentration up to 4000 n mol/L of the polymer, cell viability was over 85%. The bio-character of polymer/DNA complex was characterized by agarose gel electrophoresis, ethidium bromide exclusion and zeta-potential assay. The polymer could retardate DNA at N/P ratio 3.0-3.5 （mol/mol）. The particle size of the polymer/DNA complex was less than 300 nm. Transfection efficiency of the complex was studied in COS7 and NT2 cell lines.
基金financially supported by the Key project of National Natural Science Foundation of China (Grant No.32330096)Innovative Research Group Project of Hebei Natural Science Foundation (Grant No.C2024204246)+3 种基金S&T Program of Hebei (Grant Nos.21372901D23567601H)Natural Science Foundation of Hebei (Grant No.C2023204119)the Starting Grant from Hebei Agricultural University (Grant No.YJ201958)。
文摘Protoplast-based transient gene expression system has been widely used in plant genome editing because of its simple operation and less time-consuming.In order to establish a universal protoplast-based transient transfection system for verifying activities of genome editing vectors containing targets in Brassica,we systematically optimized factors affecting protoplast isolation and transient gene expression.We established an efficient protoplast-based transient gene expression system(PTGE)in Chinese cabbage,achieving high protoplast yield of 4.9×10^(5)·g^(-1)FW,viability over 95%,and transfection efficiency of 76%.We showed for the first time that pretreatment of protoplasts with a hypotonic MMG could significantly enhance the transfection efficiency.Furthermore,protoplasts incubated at 37℃ for 6 min improved the transfection efficiency to 86%.We also demonstrated that PTGE worked well(more than 50%transfection efficiency)in multiple Brassica species including cabbage,Pak Choi,Chinese kale,and turnip.Finally,PTGE was used for validating the activities of CRISPR/Cas9 vectors containing targets in Chinese cabbage,cabbage,and pak choi,demonstrating the broad applicability of the established PTGE for genome editing in Brassica crops.
基金supported by the National Natural Science Foundation of China(81903556)the Natural Science Fund for Colleges and Universities in Jiangsu Province(19KJB350004)supported by the National Health and Medical Research Council(NHMRC)Early Career Fellowship(1112258)of Australia。
文摘Incorporating functional ligands and biodegradable bonds into biocompatible low-molecular-weight(LMW)polymers,such as 1.8 kDa poly(ethylenimine)(PEI1.8 k),is a common strategy to improve the properties of LMW polymers including biosafety and delivery efficacy.This study demonstrates the hypothesis that introducing different functional ligands and linked reductive disulfides in PEI 1.8k will achieve superior siRNA transfection efficiency.By incorporating PEI-X(X represents cholesterol(Ch),heptafluorobutyric anhydride(HFBA,F)and 4-carboxyphenylboronic acid(PBA))functional ligands into PEI 1.8k and subsequently crosslinking with each other via disulfide bond links,reductive-responsive PEI-X-SS-X-PEI copolymers were constructed to enhance the cellular transfection via the synergistic effect of the high affinity of Ch,F and PBA to cell membranes and the disulfide reduction triggered intracellular disassembly of micelles and subsequent siRNA release.Extraordinarily,ternary Ch-SS-F-SS-PBA micelles exhibited the strongest siRNA transfection efficiencies in in vitro cell experiments and in vivo animal experiments due to the coordination of enhanced serum stability,promoted cell uptake and endosomal escape,and cell targeting ability.This strategy of constructed multifunctional polymer here we called"building-block crosslinking"showed a simple and smart way to synthesize new materials.Also this strategy of constructing ligands-directed reduction-sensitive micelles improves the transfection efficiency of LMW PEI and provides a valuable insight to develop novel gene delivery systems.
基金supported by the National Natural Science Foundation of China (21374085, 21174110, 51233003)the Natural Science Foundation of Hubei Province of China (2014CFB697)the Fundamental Research Funds for the Central Universities (2042014kf0193)
文摘A polymeric polyethylenimine(PEI)-based prodrug of anticancer doxorubicin(DOX)(PEI-hyd-DOX) was designed by attaching DOX to PEI via an acid-labile hydrazone bond, for the achievement of biocontrollable gene and drug co-delivery in response to the intracellular acid microenvironments in the late endosome/lysosome compartments. The cytotoxicity of PEI-hyd-DOX was evaluated by the MTT assay and the cellular uptake was monitored using confocal laser scanning microscopy. The polymeric prodrug can respond with a high sensitivity to the specific acid condition inside cells, thus permitting the precise biocontrol over intracellular drug liberation with high drug efficacy. The chemical attachment of drug molecules also led to the relatively reduced toxicity and the enhanced transfection efficiency compared with parent PEI. The resulting data adumbrated the potential of PEI-hyd-DOX to co-deliver DOX and therapeutic gene for the combination of chemotherapy and gene therapy.
基金This project was supported by the National Natural Science Foundation of China(Grant Nos.51673145,51873149,21875157 and 51963018)the National Key Research and Development Program of China(Grant No.2016YFC1100300)the International Science and Technology Cooperation Program of China(Grant No.2013DFG52040).
文摘Gene therapy has drawn great attention in the treatments of many diseases,especially for cardiovascular diseases.However,the development of gene carriers with low cytotoxicity and multitargeting function is still a challenge.Herein,the multitargeting REDV-G-TATG-NLS peptide was conjugated to amphiphilic cationic copolymer poly(e-caprolactone-co-3(S)-methyl-morpho-line-2,5-dione)-g-polyethyleneimine(PCLMD-g-PEI)via a heterobifunctional orthopyridyl disulfde-poly(ethylene glycol)-N-hydroxysuccinimide(OPSS-PEG-NHS)linker to prepare PCLMD-g-PEI-PEG-REDV-G-TAT-G-NLS copolymers with the aim to develop the gene carriers with low cytotoxicity and high transfection efficiency.The multitargeting micelles were prepared from PCLMD-g-PEI-PEG-REDV-G-TAT-G-NLS copolymers by self-assembly method and used to load pEGFP-ZNF580 plasmids(pDNA)to form gene complexes for enhancing the proliferation and migration of endothelial cells(ECs).The loading pDNA capacity was proved by agarose gel electrophoresis assay.These multitargeting gene com-plexes exhibited low cytotoxicity by 3-(4,-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide assay.The high internalization efficiency of these gene complexes was confirmed by flow cytometry.The results of in vitro transfection demonstrated that these multitargeting gene complexes possessed relatively high transfection effi-ciency.The rapid migration of ECs transfected by these gene complexes was verified by wound healing assay.Owing to ECs-targeting ability,cell-penetrating ability and nuclear targeting capacity of REDV-G-TAT-G-NLS pep-tide,the multitargeting polycationic gene carrier with low cytotoxicity and high transfection efficiency has great potential in gene therapy.
