以新型脂肽为佐剂的LP-LNP构建及其对mRNA疫苗的增效作用  

Construction of LP-LNP with novel lipopeptides as adjuvants and its enhancing effects on mRNA vaccines

作　　者：曹静文 迟宇 李郭成 程浩 邓炎 魏静 朱吉 高英英[1,3] 李海波 CAO Jingwen;CHI Yu;LI Guocheng;CHENG Hao;DENG Yan;WEI Jing;ZHU Ji;GAO Yingying;LI Haibo(Clinical Medical School,Jiamusi University,Jiamusi,Heilongjiang Province,154007;Department of Microbiology and Biochemical Pharmacy,National Engineering Technology Research Center for Immune Biological Products,Faculty of Pharmacy and Laboratory Medicine,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Clinical Laboratory,Banan Hospital Affiliated to Chongqing Medical University,Chongqing,401320,China)

机构地区：[1]佳木斯大学临床医学院,黑龙江佳木斯154007 [2]陆军军医大学(第三军医大学)药学与检验医学系微生物与生化药学教研室,国家免疫生物制品工程技术研究中心,重庆400038 [3]重庆医科大学附属巴南医院检验科,重庆401320

出　　处：《陆军军医大学学报》2024年第17期1925-1933,共9页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(32270988);重庆市自然科学基金重点项目(cstc2020jcyj-zdxmX0027)。

摘　　要：目的构建以新型脂肽为佐剂的递送系统脂肽-脂质纳米颗粒(lipopeptide-lipid nanoparticle,LP-LNP),初步探究其对mRNA疫苗的增效作用。方法设计并合成2种新型脂肽SS-10和SQ18,微流控技术将编码增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)、萤火虫荧光素酶(firefly luciferase,F-luc)和卵清白蛋白(ovalbumin,OVA)的mRNA包载到LP-LNP中,构建以新型脂肽为佐剂的mRNA递送系统;利用动态光散射法表征LP-LNP的粒径、多分散系数;利用HEK-Blue TM mTLR2报告细胞检测其Toll样受体2(Toll-like receptors 2,TLR2)的激活作用,筛选最优脂肽掺入比;将优选的LP-LNP-eGFP-mRNA转染至HEK293T细胞,荧光显微镜观察eGFP的表达;利用活体成像技术考察LP-LNP-F-luc-mRNA在小鼠体内的表达水平;流式细胞术评价LP-LNP-OVA-mRNA免疫小鼠后诱导引流淋巴结中树突状细胞(dendritic cells,DCs)成熟水平以及交叉递呈抗原的能力。结果脂肽SQ18和SS-10分别以0.50%、0.75%的摩尔比掺入后,得到的LP-LNP平均粒径分别为127和121 nm,平均多分散系数分别为0.11和0.08,包封率均在90%以上,且体外安全性好;该配方条件下激活TLR2的能力显著强于阳性对照Pam 2 CSK 4(P<0.01);优选的LP-LNP均可以实现有效的体外转染,其中SQ18以0.50%掺入制备的LP-LNP体内转染效果显著优于传统LNP(P<0.01),且可以显著促进引流淋巴结DCs的成熟以及抗原的交叉递呈(P<0.05)。结论以新型脂肽为佐剂的LP-LNP可以增强mRNA的递送能力,进一步提高mRNA疫苗的免疫效果。Objective To construct lipid nanoparticles(lipopeptide-lipid nanoparticle,LP-LNP)with novel lipopeptides as adjuvants,and initially explore their synergistic effect on mRNA vaccines.Methods Two novel lipopeptides,SS-10 and SQ18,were designed and synthesized.Microfluidic technology was used to encapsulate lipopeptides in different proportions,as well as mRNAs encoding enhanced green fluorescent protein(eGFP),firefly luciferase(F-luc),and ovalbumin(OVA)into lipid nanoparticles to construct an mRNA delivery system with novel lipopeptides as adjuvants(LP-LNP).The particle size and polydispersity coefficient of LP-LNP were measured using dynamic light scattering.The activation effect on Toll-like receptors 2(TLR2)was detected using HEK-Blue TM mTLR2 reporter cells to screen the optimal lipopeptide ratio.The preferred LP-LNP-eGFP-mRNA was transfected into HEK293T cells,and the expression of eGFP was observed under a fluorescence microscope.In vivo imaging was used to investigate the expression level of LP-LNP-F-luc-mRNA in mice.Flow cytometry was used to evaluate the ability of LP-LNP-OVA-mRNA to induce the maturation of dendritic cells(DCs)in draining lymph nodes and cross-presentation of antigens after immunization.Results Lipopeptides SQ18 and SS-10 were incorporated into LNP at 0.50%and 0.75%molar ratios,respectively,to obtain LP-LNP with uniform particle size,high encapsulation efficiency,and good in vitro safety.The ability of this formulation to activate TLR2 was significantly stronger than the positive control Pam 2CSK 4(P<0.01).The preferred LP-LNP obtained effective in vitro transfection,and LP-LNP prepared with SQ18 at 0.50%molar ratio had significantly better in vivo transfection efficiency than traditional LNP(P<0.01),and significantly promoted the maturation of DCs in draining lymph nodes and cross-presentation of antigens(P<0.05).Conclusion LP-LNP with novel lipopeptides as adjuvants can enhance the delivery capacity of mRNA and further improve the immune effect of mRNA vaccines.

关 键 词：脂质纳米颗粒 mRNA疫苗 佐剂 脂肽 

分 类 号：R342.22[医药卫生—基础医学] R914.2R979.5