DC-SIGN靶向的载铜绿假单胞菌DNA疫苗纳米粒的构建及免疫效力评价  

Construction and immunogenicity evaluation of DC-SIGN-targeted Pseudomonas aeruginosa DNA vaccine nanoparticle

作　　者：江晓烽 张娅婷 赵轩 田林霞 余娴[1] JIANG Xiao-feng;ZHANG Ya-ting;ZHAO Xuan;TIAN Lin-xia;YU Xian(PhaseⅠClinical Trial Center of Chongqing Medical University Affiliated Second Hospital,Chongqing 400010,China)

机构地区：[1]重庆医科大学附属第二医院Ⅰ期临床试验中心,重庆400010

出　　处：《中国药理学通报》2024年第11期2184-2192,共9页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 82072327);重庆市自然科学基金面上项目(No CSTB2022NSCQ-MSX0987);重庆医科大学附属第二医院“宽仁英才”项目。

摘　　要：目的构建一种靶向树突状细胞(dendritic cells,DC)乳-N-岩藻糖戊糖(lacto-N-fucopentoseⅢ,Lewis X)修饰的载铜绿假单胞菌(Pseudomonas aeruginosa,PA)PcrV和OprF联合DNA疫苗的PLGA纳米粒,为预防PA临床感染提供新思路。方法利用双乳化-溶剂挥发法制备载PcrV和OprF联合DNA的PLGA纳米粒(PLGA+PcrV/OprF)或载pEGFP的PLGA纳米粒(PLGA+pEGFP);在此基础上,利用酰胺缩合反应将DC-SIGN靶向配体Lewis X连接至PLGA纳米粒表面,制备Lewis X修饰的PLGA+PcrV/OprF(Lewis X-PLGA+PcrV/OprF)、Lewis X修饰的PLGA-pEGFP(Lewis X-PLGA+pEGFP);以水化直径、Zeta电位、包封率与载药量为指标对Lewis X-PLGA+PcrV/OprF进行表征分析;用CCK-8考察其细胞毒性;通过Lewis X-PLGA+pEGFP体外转染进行DC靶向验证;进一步通过Lewis X-PLGA+PcrV/OprF溶酶体逃逸评价Lewis X修饰的携载DNA的PLGA纳米粒的体外靶向性能;通过检测该纳米粒的淋巴细胞增殖水平、体液免疫水平和免疫保护水平,评价其免疫效力。结果制备的Lewis X-PLGA+PcrV/OprF水化直径为(201.17±1.6)nm,包封率为(85.72±5.3)%,Zeta电位为+(31.17±1.8)mV;Lewis X-PLGA+PcrV/OprF在DC2.4中的细胞毒性试验显示细胞存活率均在85%以上;荧光显微镜观察Lewis X-PLGA+pEGFP体外转染结果表明,DC2.4更能摄取表达Lewis X-PLGA+pEGFP,具有DC-SIGN特异性靶向性能;激光共聚焦观察溶酶体逃逸结果表明,Lewis X-PLGA+PcrV/OprF发生溶酶体逃逸后有更多的DNA进入细胞质;体内免疫结果显示,靶向DNA疫苗的淋巴细胞增殖水平和抗体滴度水平显著增加,进一步提高了感染急性肺炎小鼠的生存率,减少了小鼠肺部细菌负荷。结论成功构建DC-SIGN靶向的载PA DNA疫苗纳米粒Lewis X-PLGA;促进了其携载的DNA转染进入DC;促进了更多PA DNA疫苗内吞进入DC溶酶体,逃逸出更多PA DNA至细胞质,从而引起体内显著的免疫应答,增强了疫苗保护效力。Aim To construct a DC-targeted modified(lacto-N-fucop-entoseⅢ,Lewis X)Pseudomonas aeruginosa(PA)DNA vaccine PLGA nanoparticle(Lewis X-PLGA)loading PA PcrV and OprF genes combination,and provide a new idea for the prevention of PA clinical infection.Methods The PLGA nanoparticles loading PA PcrV and OprF combined DNA(PLGA+PcrV/OprF)or loading pEGFP(PLGA+pEGFP)were prepared by double emulsification-solvent evaporation method.On this basis,the DC-SIGN-targeted ligand Lewis X was connected to the surface of PLGA nanoparticles by amide condensation reaction.Lewis X-modified PLGA-PcrV/OprF(Lewis X-PLGA+PcrV/OprF)and Lewis X-modified PLGA-pEGFP(Lewis X-PLGA+pEGFP)were prepared.Particle size,Zeta potential,encapsulation rate and drug loading were evaluated to characterize Lewis X-PLGA+PcrV/OprF.The cytotoxicity of Lewis X-PLGA+PcrV/OprF was investigated by CCK-8.DC targeting of Lewis X-PLGA+pEGFP was validated in vitro transfection.Further,Lewis X-PLGA+PcrV/OprF lysosome escape was used to evaluate the targeting performance of Lewis X-modified PLGA nanoparticles loading DNA in vitro.The immunoefficacy of the nanoparticles was evaluated by detecting the level of lymphocyte proliferation,humoral immunity and immune protection.Results The diameter of Lewis X-PLGA+PcrV/OprF was(201.17±1.6)nm.The encapsulation efficiency of Lewis X-PLGA+PcrV/OprF was(85.72±5.3)%.The Zeta potential of Lewis X-PLGA+PcrV/OprF was+(31.17±1.8)mV.In the DC2.4 cytotoxicity test,the cell survival rates were above 85%.The results of fluorescence microscopy after Lewis X-PLGA+pEGFP transfection in vitro showed that Lewis X-PLGA+pEGFP was more readily taken up by DC2.4,and Lewis X-PLGA+pEGFP had a DC-SIGN specific targeting performance.The CLSM’s observation of Lewis X-PLGA+Pcrv/OprF showed that more DNA escaped from the lysosome into the cytoplasm.The results suggested that Lewis X-PLGA had DC2.4 targeting performance.Because DC2.4 cells endocyted more Lewis X-PLGA+Pcrv/OprF into the lysosome,the amount of DNA carried by the nanoparticles escapi

关 键 词：DC-SIGN靶向 铜绿假单胞菌 PLGA 溶酶体 DNA疫苗 纳米粒 

分 类 号：R392[医药卫生—免疫学]