靶向氧化低密度脂蛋白纳米探针活体检测小鼠腹主动脉粥样斑块的MRI研究

摘要

目的 构建新型抗氧化低密度脂蛋白纳米铁(anti-Ox-LDL-MNPs)探针,探讨该探针检测ApoE-/-小鼠粥样斑块的价值.方法 采用化学连接剂将Ox-LDL单克隆抗体与二巯基丁二酸(DMSA)修饰的磁赤铁矿纳米粒子(MNPs)相结合,构建具有免疫活性的分子探针.通过ELISA、透射电子显微镜(TEM)等方法检测其免疫活性及形态.ApoE-/-小鼠高脂饮食8周,尾静脉注射Ox-LDL靶向及对照探针(IgG-MNPs),分别在注射前及注射后24h行MRI,检查后处死小鼠取腹主动脉行病理学检查.行配对t检验分析数据.结果 Anti-Ox-LDL-MNPs和IgG-MNPs探针磁饱和强度高,分布均匀,生物活性较强,水合粒径分别为(21.5±3.3) nm及(22.3±4.1) nm.2组ApoE-/-小鼠腹主动脉平均信噪比(CNR)在探针注射前分别为20.89±1.50和20.84±5.47,注射后24 h anti-Ox-LDL-MNPs探针组小鼠CNR显著下降(7.30±1.19;t 5.373,P＜0.01;n=5),IgG-MNPs探针组CNR略有下降(18.66±2.84;t=2.620,P＞0.05;n=5).免疫组织化学检查和普鲁士蓝染色证实Ox-LDL靶向探针在ApoE-/-小鼠腹主动脉壁斑块内沉积,与巨噬细胞共定位.结论 靶向Ox-LDL纳米铁MRI探针能活体检测动脉粥样硬化斑块Ox-LDL.%Objective To synthesize,characterize and test the efficacy of maghemite nanoparticles (MNPs) linked with antibodies against oxidized low-density lipoprotein (Ox-LDL) (anti-Ox-LDL-MNPs) and to evaluate its efficiency as in vivo MRI contrast agent in ApoE-/-mice.Methods Anti-human Ox-LDL monoclonal antibody and nonspecific IgG antibody were prepared by grafting antibody on the surface of 2,3-dimercaptosuccinnic acid (DMSA)-coated MNPs using the linker of (1-(3-dimethylaminopropyl)-3-ethylcarbodimide) hydrochloride(EDC).The morphology and activity of anti-Ox-LDL-MNPs were evaluated by transmission electron microscopy (TEM),and ELISA.ApoE-/-mice were divided into anti-Ox-LDL-MNPs group and IgG-MNPs group (n=5 in each group) and imaged at 7.0 T MR prior to contrast administration and 24 h after injection of 30 mg Fe/kg.MRI was performed using black-blood T2 weighted multi-spin multiecho sequence.Prussian blue,CD68 and Ox-LDL immunohistological stains were undertaken for the detection of iron,macrophages and Ox-LDL in excised mouse aorta.Paired t test was used to analyze the data.Results Anti-Ox-LDL-MNPs or IgG-MNPs had similar hydrated diameters ((21.5±3.3) nm and (22.3±4.1) nm).Significant modulation of the MR signal was observed with anti-Ox-LDL-MNPs (20.89±1.50 vs 7.30± 1.19; t =5.373,P＜0.01),minor MR signal modulation was observed following administration of IgG-MNPs (20.84±5.47 vs 18.66±2.84; t =2.620,P＞0.05).Immunohistochemistry confirmed the co-localization of the macrophages and iron oxide nanoparticles after anti-Ox-LDL-MNPs administration.Conclusions Ox-LDL targeting MNPs nanoparticles may be used to directly detect Ox-LDL and image atherosclerotic lesions within 24 h after nanoparticle administration.It may be a strategy for the evaluation of atherosclerotic plaques in vivo.