Background The outbreak of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has greatly threatened public health.Recent studies have revealed that the spike receptor-binding domain(RBD)of SARS-CoV-2 is a...Background The outbreak of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has greatly threatened public health.Recent studies have revealed that the spike receptor-binding domain(RBD)of SARS-CoV-2 is a potent target for vaccine development.However,adjuvants are usually required to strengthen the immunogenicity of recombinant antigens.Different types of adjuvants can elicit different immune responses.Methods We developed an RBD recombinant protein vaccine with a polyriboinosinic acid–polyribocytidylic acid[poly(I:C)]adjuvant to evoke a strong immune response.The delivery of poly(I:C)was optimized in two steps.First,poly(I:C)was complexed with a cationic polymer,poly-l-lysine(PLL),to form poly(I:C)–PLL,a polyplex core.Thereafter,it was loaded into five different lipid shells(group II,III-1,2-distearoyl-sn-glycero-3-phosphocholine[DSPC],III-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine[DOPE],IV-DOPE,and IV-DSPC).We performed an enzyme-linked immunosorbent assay and enzyme-linked immunosorbent spot assay to compare the ability of the five lipopolyplex adjuvants to enhance the immunogenicity of the SARS-CoV-2 RBD protein,including humoral and cellular immune responses.Finally,the adjuvant with the highest immunogenicity was selected to verify the protective immunity of the vaccine through animal challenge experiments.Results Recombinant RBD protein has low immunogenicity.The different adjuvants we developed enhanced the immunogenicity of the RBD protein in different ways.Among the lipopolyplexes,those containing DOPE(III-DOPE and IV-DOPE)elicited RBD-specific immunoglobulin G antibody responses,and adjuvants with four components elicited better RBD-specific immunoglobulin G antibody responses than those containing three components(P<0.05).The IC50 and IC90 titers indicated that the IV-DOPE lipopolyplex had the greatest neutralization ability,with IC50 titers of 1/117,490.Furthermore,in the challenge study,IV-DOPE lipopolyplex protected mice from SARS-CoV-2 infection.On the fourth day after infectio展开更多
RNA interference is a promising therapeutical approach presently hindered by delivery concerns such as rapid RNA degradation and targeting of individual tissues.Injectable hydrogels are one potentially simple and dire...RNA interference is a promising therapeutical approach presently hindered by delivery concerns such as rapid RNA degradation and targeting of individual tissues.Injectable hydrogels are one potentially simple and direct route towards overcoming these barriers.Here we report on the utility of a combination of a mildly modified form of the clinically utilised fibrin hydrogel with Invivofectamine^(■)3.0,a lipid nonviral transfection vector,for local and sustained release.PEGylation of fibrin allowed for controlled release of small interfering RNA(siRNA)-lipopolyplexes for at least 10 days and greatly increased the stability of fibrin in vitro and in vivo.A 3D cell culture model and a release study showed transfection efficacy of siRNA-lipopolyplexes was retained for a minimum of 7 days.Injection in conjunction with PEGylated-fibrinogen significantly increased retention of siRNA-lipopolyplexes in mouse skeletal muscle and enhanced knockdown of myostatin mRNA that correlated with muscle growth.Thus,the increased efficacy observed here for the combination of a lipid nanoparticle,the only type of nonviral vector approved for the clinic,with fibrin,might allow for more rapid translation of injectable hydrogel-based RNA interference.展开更多
目的:为降低聚阳离子基因载体polyplex的正电荷和毒性,在其表面构建中性磷脂膜制备lipopolyplex,并测定lipopolyplex对小鼠结肠癌细胞CT26和人乳腺癌细胞MCF-7的细胞毒性。方法:采用PEI25KDa与DNA复合制备polyplex,在polyplex体系中加...目的:为降低聚阳离子基因载体polyplex的正电荷和毒性,在其表面构建中性磷脂膜制备lipopolyplex,并测定lipopolyplex对小鼠结肠癌细胞CT26和人乳腺癌细胞MCF-7的细胞毒性。方法:采用PEI25KDa与DNA复合制备polyplex,在polyplex体系中加入中性脂质体和SADGE制备lipopolyplex。采用琼脂糖凝胶电泳考察lipopolyplex对质粒DNA的包裹能力;采用激光粒度仪和zeta电位分析仪测定lipopolyplex的粒径与zeta电位;采用透射电镜观察lipopolyplex的形态;采用CCK-8试剂盒考察lipopolyplex对CT26和MCF-7的细胞毒性。结果:琼脂糖凝胶电泳显示lipopolyplex可以完全包裹质粒DNA;lipopolyplex的粒径在200 nm左右,电位在-20 m V左右;透射电镜下为较为规则的球状颗粒;lipopolyplex在CT26和MCF-7细胞中的毒性明显低于聚阳离子基因载体polyplex。结论:在polyplex表面成功构建中性磷脂膜制备的lipopolyplex,可以完全的包裹DNA并且细胞毒性明显低于polyplex,在基因输送载体领域具有潜在应用价值。展开更多
文摘Background The outbreak of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has greatly threatened public health.Recent studies have revealed that the spike receptor-binding domain(RBD)of SARS-CoV-2 is a potent target for vaccine development.However,adjuvants are usually required to strengthen the immunogenicity of recombinant antigens.Different types of adjuvants can elicit different immune responses.Methods We developed an RBD recombinant protein vaccine with a polyriboinosinic acid–polyribocytidylic acid[poly(I:C)]adjuvant to evoke a strong immune response.The delivery of poly(I:C)was optimized in two steps.First,poly(I:C)was complexed with a cationic polymer,poly-l-lysine(PLL),to form poly(I:C)–PLL,a polyplex core.Thereafter,it was loaded into five different lipid shells(group II,III-1,2-distearoyl-sn-glycero-3-phosphocholine[DSPC],III-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine[DOPE],IV-DOPE,and IV-DSPC).We performed an enzyme-linked immunosorbent assay and enzyme-linked immunosorbent spot assay to compare the ability of the five lipopolyplex adjuvants to enhance the immunogenicity of the SARS-CoV-2 RBD protein,including humoral and cellular immune responses.Finally,the adjuvant with the highest immunogenicity was selected to verify the protective immunity of the vaccine through animal challenge experiments.Results Recombinant RBD protein has low immunogenicity.The different adjuvants we developed enhanced the immunogenicity of the RBD protein in different ways.Among the lipopolyplexes,those containing DOPE(III-DOPE and IV-DOPE)elicited RBD-specific immunoglobulin G antibody responses,and adjuvants with four components elicited better RBD-specific immunoglobulin G antibody responses than those containing three components(P<0.05).The IC50 and IC90 titers indicated that the IV-DOPE lipopolyplex had the greatest neutralization ability,with IC50 titers of 1/117,490.Furthermore,in the challenge study,IV-DOPE lipopolyplex protected mice from SARS-CoV-2 infection.On the fourth day after infectio
基金supported by the National Research Foundation of South Africa(93550)International Centre for Genetic Engineering and Biotechnology(CRP/ZAF14-01)the South African Medical Research Council(416007).
文摘RNA interference is a promising therapeutical approach presently hindered by delivery concerns such as rapid RNA degradation and targeting of individual tissues.Injectable hydrogels are one potentially simple and direct route towards overcoming these barriers.Here we report on the utility of a combination of a mildly modified form of the clinically utilised fibrin hydrogel with Invivofectamine^(■)3.0,a lipid nonviral transfection vector,for local and sustained release.PEGylation of fibrin allowed for controlled release of small interfering RNA(siRNA)-lipopolyplexes for at least 10 days and greatly increased the stability of fibrin in vitro and in vivo.A 3D cell culture model and a release study showed transfection efficacy of siRNA-lipopolyplexes was retained for a minimum of 7 days.Injection in conjunction with PEGylated-fibrinogen significantly increased retention of siRNA-lipopolyplexes in mouse skeletal muscle and enhanced knockdown of myostatin mRNA that correlated with muscle growth.Thus,the increased efficacy observed here for the combination of a lipid nanoparticle,the only type of nonviral vector approved for the clinic,with fibrin,might allow for more rapid translation of injectable hydrogel-based RNA interference.
文摘目的:为降低聚阳离子基因载体polyplex的正电荷和毒性,在其表面构建中性磷脂膜制备lipopolyplex,并测定lipopolyplex对小鼠结肠癌细胞CT26和人乳腺癌细胞MCF-7的细胞毒性。方法:采用PEI25KDa与DNA复合制备polyplex,在polyplex体系中加入中性脂质体和SADGE制备lipopolyplex。采用琼脂糖凝胶电泳考察lipopolyplex对质粒DNA的包裹能力;采用激光粒度仪和zeta电位分析仪测定lipopolyplex的粒径与zeta电位;采用透射电镜观察lipopolyplex的形态;采用CCK-8试剂盒考察lipopolyplex对CT26和MCF-7的细胞毒性。结果:琼脂糖凝胶电泳显示lipopolyplex可以完全包裹质粒DNA;lipopolyplex的粒径在200 nm左右,电位在-20 m V左右;透射电镜下为较为规则的球状颗粒;lipopolyplex在CT26和MCF-7细胞中的毒性明显低于聚阳离子基因载体polyplex。结论:在polyplex表面成功构建中性磷脂膜制备的lipopolyplex,可以完全的包裹DNA并且细胞毒性明显低于polyplex,在基因输送载体领域具有潜在应用价值。
