摘要
目的探讨二硫键优化透明质酸介导的肝癌靶向纳米探针的构建方法和理化性质,并评价对肝癌细胞治疗作用的体外生物学特性及MRI的可行性。方法利用点击化学反应将含二硫键的端炔基聚己内酯(alkyne-SS-PCL)与端叠氮基透明质酸(HA-N3)合成透明质酸-二硫键-聚己内酯(HA-SS-PCL),再通过透析方法将阿霉素(DOX)及超顺磁性氧化铁(SPIO)包载于HA-SS-PCL内核中,制成透明质酸-二硫键-聚己内酯@阿霉素/超顺磁性氧化铁(HA-SS-PCL@DOX/SPIO),测量其粒径、DOX及SPIO负载率。利用四唑盐比色法(MTT),以碱性磷酸盐(PBS)为对照组,评价HA-SS-PCL对人肝癌细胞HepG2及正常肝细胞LO2的安全性,透明质酸-聚己内酯@阿霉素/超顺磁性氧化铁(HA-PCL@DOX/SPIO)及HA-SS-PCL@DOX/SPIO对人肝癌细胞HepG2的细胞毒性。采用免疫荧光及流式细胞术观察HA-PCL@DOX/SPIO及HA-SS-PCL@DOX/SPIO组人肝癌HepG2细胞表面CD44受体表达情况。通过体外MRI成像,以PBS为对照组,观察SPIO浓度为10、20、40、80μg/ml时,HA-PCL@DOX/SPIO及HA-SS-PCL@DOX/SPIO纳米探针T2WI信号强度变化情况。两组间计量资料的比较采用t检验,多组间计量资料的比较采用单因素方差分析。结果成功构建纳米探针HA-SS-PCL@DOX/SPIO,其粒径为(126.9±6.3)nm,呈大小均一的球形结构,该纳米探针对DOX及SPIO负载率分别为61.4%、58.7%。MTT试验显示,HA-SS-PCL即使在浓度为500μg/ml时,HepG2及LO2细胞存活率仍>80%;当DOX浓度为10.00μg/ml时,HA-PCL@DOX/SPIO组HepG2细胞存活率为66.6%,HA-SS-PCL@DOX/SPIO组HepG2细胞存活率为55.2%。免疫荧光及流式细胞检测HA-PCL@DOX/SPIO及HA-SS-PCL@DOX/SPIO组均具有较强的荧光,后者荧光强度更强,分别为139.70±8.52、245.06±13.21。体外MRI成像示HA-PCL@DOX/SPIO及HA-SS-PCL@DOX/SPIO较对照组其T2信号强度均有明显下降(F值分别为613.591、569.234,P均<0.01),后者下降率更为显著。结论经二硫键优化透明质酸介导的肝癌靶向纳米
Objective To explore the construction method and physicochemical properties of disulfide-bonded hyaluronic acid-functionalized sodium-meter probe for hepatocellular carcinoma,and its biological evaluation in vitro and feasibility of MRI. Methods Synthesis of hyaluronic acid-disulfide-bonded-poly ε-caprolactone (HA-SS-PCL) by disulfide-bonded alkynyl-terminated polycaprolacton (alkyne-SS-PCL) and azido-terminated hyaluronic acid (HA-N3) by clicking chemical reaction,then doxorubicin (DOX) and superparamagnetic iron oxide (SPIO) were encapsulated in HA-SS-PCL core by dialysis method.HA-SS-PCL@DOX/SPIO was prepared and its particle size,DOX and SPIO loading rate were measured. With PBS as control group,the safety of HA-SS-PCL on human hepatocellular carcinoma cells HepG2 and normal liver cells LO2 was evaluated by the methylthiazolyl tetrazolium (MTT) assay, and the cytotoxicity of HA-PCL@DOX/SPIO and HA-SS-PCL@DOX/SPIO on human hepatocellular carcinoma cells HepG2 was evaluated. Immunofluorescence and flow cytometry were used to observe the expression of CD44 receptor on the surface of HepG2 cells in HA-PCL@DOX/SPIO and HA-SS-PCL@DOX/SPIO groups.Through in vitro MRI, PBS was used as the control group to observe the changes of T2 signal intensity of HA-PCL@DOX/SPIO and HA-SS-PCL@DOX/SPIO groups when SPIO concentration was 10,20,40,80 μg/ml. One way ANOVA test and t test were used. Results HA-SS-PCL@DOX/SPIO sodium-meter probes were successfully constructed. The particle size of HA-SS-PCL@DOX/SPIO was (126.9±6.3) nm,and they were spherical with uniform size. The loading rates of DOX and SPIO were 61.4% and 58.7%. MTT assay showed that the survival rate of HepG2 and LO2 cells was more than 80% even at 500 μg/ml of HA-SS-PCL,66.6% in HA-PCL@DOX/SPIO group and 55.2% in HA-SS-PCL@DOX/SPIO group. Immunofluorescence and flow cytometry showed that HA-PCL@DOX/SPIO and HA-SS-PCL@DOX/SPIO groups all have strong fluorescence,and the latter has stronger fluorescence intensity the former fluorescence intensity was 139.70±8.5
作者
刘国顺
杨惠康
李雯曦
熊炜烽
黎蕾
谌丹丹
郭媛
江新青
Liu Guoshun;Yang Huikang;Li Wenxi;Xiong Weifeng;Li Lei;Chen Dandan;Guo Yuan;Jiang Xinqing(Department of Radiology, Guangzhou First People's Hospital, the Second Affiliated Hospital of South China University of Technology, Guangzhou 510180,China;Department of Geriatrics, Guangzhou First People's Hospital, the Second Affiliated Hospital of South China University of Technology, Guangzhou 510180,China;Department of Radiology, the 184th Hospital of PLA, Yingtan 335000,China)
出处
《中华放射学杂志》
CAS
CSCD
北大核心
2019年第7期603-608,共6页
Chinese Journal of Radiology
基金
国家自然科学基金(81271665).
关键词
磁共振成像
二硫键
透明质酸
纳米探针
Magnetic resonance imaging
Disulfide
Hyaluronicacid
Sodium-meter probe
