狂犬病毒糖蛋白修饰外泌体构建神经靶向磁共振分子探针的实验研究  

Engineering rabies virus glycoprotein modified exosome as a nerve-targeting magnetic resonance imaging molecular probe

作　　者：张帆 刘晨鹭[1] 严彩英 童明敏[1] 陈双庆[1] Zhang Fan;Liu Chenlu;Yan Caiying;Tong Mingmin;Chen Shuangqing(Department of Medical Imaging,Affiliated Suzhou Hospital of Nanjing Medical University,Suzhou 215000,China)

机构地区：[1]南京医科大学附属苏州医院医学影像科,苏州215000

出　　处：《中华生物医学工程杂志》2023年第1期1-7,共7页Chinese Journal of Biomedical Engineering

基　　金：江苏省自然科学基金(BK20161231)。

摘　　要：目的利用狂犬病毒糖蛋白(RVG)修饰的外泌体与超顺磁性氧化铁纳米颗粒(SPION)构建神经靶向分子探针RVG-Exo-SPION,研究其表征及体外磁共振成像(MRI)效果。方法构建RVG-Lamp2b质粒并转染HEK293细胞,提取膜表达RVG的外泌体,电穿孔与SPION合成磁性分子探针RVG-Exo-SPION。采用免疫印迹、透射电镜分析(TEM)、纳米粒径追踪分析(NTA)及体外毒性分析(CCK-8法)方法分别对分子探针外泌体膜蛋白表达、形状、尺寸和生物相容性进行分析和鉴定,并通过激光共聚焦成像、普鲁士蓝染色和MRI联合评估Neuro-2A细胞对分子探针的摄取能力。结果双酶切法证明目的基因重组至pcDNA3.1质粒中,免疫印迹显示重组质粒被有效转染至HEK293细胞,外泌体标记蛋白CD9和CD63呈高表达。TEM显示修饰和电穿孔后的外泌体保持其正常的形状、尺寸和膜结构,外泌体电穿孔后能见到SPION的存在。NTA显示工程化的外泌体粒径分布窄,粒径分布峰值为140 nm。Neuro-2A细胞与1~100 mg/L不同浓度梯度的RVG-Exo-SPION共同孵育时,细胞的活力差异无统计学意义(均P>0.05)。激光共聚焦显微成像分析显示外泌体能高效地进入细胞,线形图显示实验组荧光强度更高;普鲁士蓝染色验证了RVG-Exo-SPION对Neuro-2A细胞的靶向能力,靶向组的阳性细胞标记率明显高于对照组[(75.8±5.6)%比(19.1±2.5)%),P<0.05];体外MRI显示灰阶值在不同铁浓度(5、10、25、50、100 mg/L)之间有明显区别,且在Fe≥25 mg/L的浓度下易于识别。结论基于RVG修饰外泌体构建的磁性分子探针RVG-Exo-SPION在体外实验中具有较好的稳定性和神经靶向性,为进一步用于体内研究提供了可行性。Objective To construct a nerve-targeting molecular probe,RVG-Exo-SPION,by binding rabies virus glycoprotein(RVG)modified exosome to superparamagnetic iron oxide nanoparticles(SPION),characterize the probe,and validate its performance on magnetic resonance imaging(MRI)in vitro.Methods The RVG-Lamp2b plasmid was constructed and transfected into HEK293 cells.Exosomes expressing RVG on their membrane were extracted,electroporated,and bound with SPION to generate the magnetic molecular probe RVG-Exo-SPION.Western blotting,transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and in vitro cytotoxicity assay(CCK-8 method)were used to analyze or verify the expression,shape,size and biocompatibility of proteins on exosomal membrane of the molecular probe.The uptake of molecular probe by Neuro-2A cells was evaluated by confocal laser scanning microscopy(CLSM),Prussian blue staining and MRI.Results Double enzyme digestion confirmed that the target gene was cloned into the pcDNA3.1 plasmid.Western blotting showed that the recombinant plasmid was effectively transfected into the HEK293 cells as reflected by high expression of exosomal marker proteins CD9 and CD63.TEM showed that the modified and electroporated exosomes maintained their normal shape,size and membrane structure.Presence of SPION could be seen after electroporation of the exosomes.NTA showed that the engineered exosomes had a narrow distribution range of particle size with a peak at 140 nm.Co-incubated with RVG-Exo-SPION at concentrations from 1 to 100 mg/L did not incur significant difference in viability of Neuro-2A cells(all P>0.05).CLSM showed efficient influx of exosomes into the Neuro-2A cells,with higher fluorescence intensity indicated in line graph for the RVG-Exo group compared with the controls.Prussian blue staining verified the Neuro-2A-targeting ability of RVG-Exo-SPION which yielded higher rate of positive cell labeling compared with the control group[(75.8±5.6)%vs(19.1±2.5)%,P<0.05].In vitro MRI showed significant differences

关 键 词：外泌体 狂犬病病毒 糖蛋白 氧化铁磁性纳米颗粒 磁共振成像 药物靶向 分子探针 

分 类 号：Q78[生物学—分子生物学]