甘露糖修饰提高瑞喹莫德脂质体对肿瘤的靶向和免疫治疗作用  

Mannose Modification Enhances Tumor Targeting and Immunotherapeutic Effects of Resiquimod Liposomes

作　　者：路岳 袁风娇 郑书慧 宋佳亮 王菲菲 孙若涵 李军 贾殿隆 柳仁民 LU Yue;YUAN Feng-jiao;ZHENG Shu-hui;SONG Jia-liang;WANG Fei-fei;Sun Ruo-han;LI Jun;JIA Dian-long;LIU Ren-min(School of Pharmaceutical Sciences,Liaocheng University,Liaocheng 252059,China;Joint Laboratory for Translational Medicine Research,Liaocheng People′s Hospital,Liaocheng 252000,China)

机构地区：[1]聊城大学药学院,山东聊城252059 [2]聊城市人民医院转化医学研究联合实验室,山东聊城252000

出　　处：《中国药学杂志》2022年第22期1917-1925,共9页Chinese Pharmaceutical Journal

基　　金：国家自然科学基金项目资助(82000066);山东省自然科学基金项目资助(ZR2019PH006);山东省自然科学基金重点项目资助(ZR2020KH019);山东省自然科学基金创新发展联合基金项目资助(ZR2021LSW001)。

摘　　要：目的利用甘露糖(Mannase)修饰瑞喹莫德(R848)脂质体,赋予其对肿瘤相关巨噬细胞(tumor-associated macrophages,TAMs)的主动靶向能力,以提高其对小鼠移植瘤的免疫治疗作用。方法用二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖(Mannase-PEG_(2000)-DSPE)脂材掺入脂质体,并采用pH梯度主动载药法包封R848,制备获得甘露糖修饰的R848脂质体(M-LS/R848)。利用粒度仪、透射电镜等表征其理化性质,透析法分析其释药稳定性。体外考察其对小鼠巨噬细胞RAW264.7的靶向性,体内评估其对小鼠结肠癌细胞MC38移植瘤的靶向性和免疫治疗效果,并利用免疫荧光染色探讨其免疫治疗机制。结果本研究制备的M-LS/R848大小均一,平均粒径为(123.43±2.43)nm,Zeta电位为(-0.33±0.24)mV,为球形或类球形粒子,R848包封率达(85.59±0.37)%,48 h R848释放率为(46.73±0.54)%,稳定性较高。体外靶向实验表明RAW264.7细胞对M-LS/R848的摄取率为(5.29±0.16)%,显著高于LS/R848的摄取率[(1.38±0.15)%]。体内靶向实验表明甘露糖修饰脂质体在MC38肿瘤中的累积显著高于非修饰脂质体。体内抑瘤实验表明M-LS/R848治疗组肿瘤平均体积和重量显著小于LS/R848组,甚至有两只小鼠肿瘤治愈并产生对MC38肿瘤的免疫记忆效应。免疫荧光分析显示M-LS/R848给药组肿瘤组织中M1型巨噬细胞和CD8阳性细胞较LS/R848组明显增多。结论利用甘露糖修饰R848脂质体可有效提高其对肿瘤的靶向性并增强免疫治疗效果。OBJECTIVE To modify the resiquimod(R848)liposomes with mannose to improve its immunotherapeutic effect on mouse tumors through endowing it with active targeting ability to tumor-associated macrophages(TAMs).METHODS The mannose-modified R848 liposomes(M-LS/R848)were prepared by mixing Mannase-PEG_(2000)-DSPE into lipid and encapsulating R848 with a pH gradient driving drug loading method.The physicochemical properties of M-LS/R848 were characterized by laser particle sizer and transmission electron microscope,and the R848 release was analyzed by a dialysis method.In vitro,the targeting of M-LS/R848 to mouse macrophage RAW 264.7 was analysed.In vivo,the tumor-homing and immunotherapy effect of M-LS/R848 were evaluated on MC38 tumor models,and its therapeutic mechanism was investigated by immunofluorescence staining.RESULTS The M-LS/R848 liposomes prepared in this study were uniform spherical particles,with an average diameter of(123.43±2.43)nm and Zeta potential of(-0.33±0.24)mV.The R848 encapsulation rate of M-LS/R848 is(85.59±0.37)%with a release rate of 46.8%in 48 h.In vitro targeting experiments showed that the uptake rate of M-LS/R848 by RAW264.7 cells was(5.29±0.16)%,which significantly higher than that of LS/R848[(1.38±0.15)%].In vivo tumor targeting analysis revealed that the accumulation of mannose-modified liposomes in MC38 tumors was significantly higher than that of non-modified liposomes.The antitumor experiments showed that the average tumor volume and weights of the M-LS/R848-treated group were significantly smaller than that of the LS/R848-treated group,and even two mice were cured and immune to the reinoculation of MC38 tumors.Immunofluorescence analysis revealed that M1-type macrophages and CD8 positive cells in tumor tissues of M-LS/R848-treated group were significantly increased compared with the LS/R848 group.CONCLUSION Modifying R848 liposome with mannose could effectively improve its tumor targeting ability and enhance its immunotherapy effect.

关 键 词：肿瘤免疫治疗 甘露糖修饰 瑞喹莫德脂质体 肿瘤相关巨噬细胞 

分 类 号：R944[医药卫生—药剂学]