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.
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)
出处
《中国药学杂志》
CAS
CSCD
北大核心
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)。
