中华放射学杂志
中華放射學雜誌
중화방사학잡지
Chinese Journal of Radiology
2014年
5期
363-368
,共6页
杨蕊梦%李楠楠%张黎明%王黎%傅超萍%黄丹萍%魏新华%赖丽莎%江新青
楊蕊夢%李楠楠%張黎明%王黎%傅超萍%黃丹萍%魏新華%賴麗莎%江新青
양예몽%리남남%장려명%왕려%부초평%황단평%위신화%뢰려사%강신청
透明质酸%分子探针%磁共振成像
透明質痠%分子探針%磁共振成像
투명질산%분자탐침%자공진성상
Hyaluronic acid%Molecular probes%Magnetic resonance imaging
目的:探讨超顺磁性氧化铁-透明质酸( SPIO-HA) MR分子探针的构建方法和理化性状,及其对肿瘤CD44受体高表达肿瘤细胞的体外靶向作用。方法利用3-氨基丙基三甲氧基硅烷对经典共沉淀法合成的超顺磁性氧化铁( SPIO)进行修饰,制成SPIO-NH2。同时制备低分子量透明质酸( HA),与SPIO-NH2反应,制成SPIO-HA。以未修饰的SPIO为对照组,SPIO-HA为实验组,测量粒径、Zeta电位值及SPIO-HA表面HA的含量,并应用3.0 T MR扫描仪对铁浓度分别为0.09、0.18、0.27、0.36和0.45 mmol/L的上述2组溶液进行T2弛豫率测量。评价SPIO-HA的安全性,采用免疫荧光法观察人肝癌HepG2细胞表面CD44受体表达情况,采用普鲁士蓝染色观察SPIO-HA纳米颗粒对人肝癌HepG2细胞的靶向作用,采用异硫氰酸荧光素( FITC)对2组纳米微粒进行标记,经荧光成像和流式细胞术观察HepG2细胞对FITC-SPIO-HA及FITC-SPIO纳米颗粒吸收情况。行MRI体外观察2组MR分子探针与HepG2细胞孵育后T2值的差异,并采用单因素方差分析比较空白对照组、SPIO-HA组及SPIO组T2信号值的差异。结果成功构建肿瘤CD44受体靶向性SPIO-HA MR分子探针。 SPIO-HA及SPIO粒径分别为18.2和22.4 nm,水合粒径分别为91.1和103.2 nm,Zeta电位分别为(-45.00±0.86)和(-18.50±0.73) mV。 SPIO-HA 表面HA 的质量分数为24%。 SPIO 和SPIO-NH2-HA的弛豫率分别为0.191×106和0.212×106 M-1· s-1。即使Fe浓度>100μg/ml,SPIO及SPIO-HA与2种细胞( HepG2及HL7702细胞)孵育后,其细胞存活率均保持在80%以上;免疫荧光法检测HepG2人肝癌细胞有较强的CD44受体阳性染色。经普鲁士蓝染色后,SPIO-HA组HepG2细胞内可见大量蓝染颗粒。 SPIO组细胞内未见明确蓝染颗粒。2组FITC标记的纳米微粒分别与HepG2细胞孵育后,SPIO-HA-FITC组HepG2细胞内及细胞膜上均可见绿色荧光, SPIO-FITC组未见明确荧光染色。体外MR成像,空白对照组、SPIO组和SPIO-HA组T2值分别为(115.20±0.36)、(115.07±0.81)和(21.67±0.21)ms,差异有统计学意义(F=31703.339,P<0.01)。结论利用HA构建的MR分子探针SPIO-HA,理化性质优良,对人肝癌细胞HepG2具有较好的靶向性。
目的:探討超順磁性氧化鐵-透明質痠( SPIO-HA) MR分子探針的構建方法和理化性狀,及其對腫瘤CD44受體高錶達腫瘤細胞的體外靶嚮作用。方法利用3-氨基丙基三甲氧基硅烷對經典共沉澱法閤成的超順磁性氧化鐵( SPIO)進行脩飾,製成SPIO-NH2。同時製備低分子量透明質痠( HA),與SPIO-NH2反應,製成SPIO-HA。以未脩飾的SPIO為對照組,SPIO-HA為實驗組,測量粒徑、Zeta電位值及SPIO-HA錶麵HA的含量,併應用3.0 T MR掃描儀對鐵濃度分彆為0.09、0.18、0.27、0.36和0.45 mmol/L的上述2組溶液進行T2弛豫率測量。評價SPIO-HA的安全性,採用免疫熒光法觀察人肝癌HepG2細胞錶麵CD44受體錶達情況,採用普魯士藍染色觀察SPIO-HA納米顆粒對人肝癌HepG2細胞的靶嚮作用,採用異硫氰痠熒光素( FITC)對2組納米微粒進行標記,經熒光成像和流式細胞術觀察HepG2細胞對FITC-SPIO-HA及FITC-SPIO納米顆粒吸收情況。行MRI體外觀察2組MR分子探針與HepG2細胞孵育後T2值的差異,併採用單因素方差分析比較空白對照組、SPIO-HA組及SPIO組T2信號值的差異。結果成功構建腫瘤CD44受體靶嚮性SPIO-HA MR分子探針。 SPIO-HA及SPIO粒徑分彆為18.2和22.4 nm,水閤粒徑分彆為91.1和103.2 nm,Zeta電位分彆為(-45.00±0.86)和(-18.50±0.73) mV。 SPIO-HA 錶麵HA 的質量分數為24%。 SPIO 和SPIO-NH2-HA的弛豫率分彆為0.191×106和0.212×106 M-1· s-1。即使Fe濃度>100μg/ml,SPIO及SPIO-HA與2種細胞( HepG2及HL7702細胞)孵育後,其細胞存活率均保持在80%以上;免疫熒光法檢測HepG2人肝癌細胞有較彊的CD44受體暘性染色。經普魯士藍染色後,SPIO-HA組HepG2細胞內可見大量藍染顆粒。 SPIO組細胞內未見明確藍染顆粒。2組FITC標記的納米微粒分彆與HepG2細胞孵育後,SPIO-HA-FITC組HepG2細胞內及細胞膜上均可見綠色熒光, SPIO-FITC組未見明確熒光染色。體外MR成像,空白對照組、SPIO組和SPIO-HA組T2值分彆為(115.20±0.36)、(115.07±0.81)和(21.67±0.21)ms,差異有統計學意義(F=31703.339,P<0.01)。結論利用HA構建的MR分子探針SPIO-HA,理化性質優良,對人肝癌細胞HepG2具有較好的靶嚮性。
목적:탐토초순자성양화철-투명질산( SPIO-HA) MR분자탐침적구건방법화이화성상,급기대종류CD44수체고표체종류세포적체외파향작용。방법이용3-안기병기삼갑양기규완대경전공침정법합성적초순자성양화철( SPIO)진행수식,제성SPIO-NH2。동시제비저분자량투명질산( HA),여SPIO-NH2반응,제성SPIO-HA。이미수식적SPIO위대조조,SPIO-HA위실험조,측량립경、Zeta전위치급SPIO-HA표면HA적함량,병응용3.0 T MR소묘의대철농도분별위0.09、0.18、0.27、0.36화0.45 mmol/L적상술2조용액진행T2이예솔측량。평개SPIO-HA적안전성,채용면역형광법관찰인간암HepG2세포표면CD44수체표체정황,채용보로사람염색관찰SPIO-HA납미과립대인간암HepG2세포적파향작용,채용이류청산형광소( FITC)대2조납미미립진행표기,경형광성상화류식세포술관찰HepG2세포대FITC-SPIO-HA급FITC-SPIO납미과립흡수정황。행MRI체외관찰2조MR분자탐침여HepG2세포부육후T2치적차이,병채용단인소방차분석비교공백대조조、SPIO-HA조급SPIO조T2신호치적차이。결과성공구건종류CD44수체파향성SPIO-HA MR분자탐침。 SPIO-HA급SPIO립경분별위18.2화22.4 nm,수합립경분별위91.1화103.2 nm,Zeta전위분별위(-45.00±0.86)화(-18.50±0.73) mV。 SPIO-HA 표면HA 적질량분수위24%。 SPIO 화SPIO-NH2-HA적이예솔분별위0.191×106화0.212×106 M-1· s-1。즉사Fe농도>100μg/ml,SPIO급SPIO-HA여2충세포( HepG2급HL7702세포)부육후,기세포존활솔균보지재80%이상;면역형광법검측HepG2인간암세포유교강적CD44수체양성염색。경보로사람염색후,SPIO-HA조HepG2세포내가견대량람염과립。 SPIO조세포내미견명학람염과립。2조FITC표기적납미미립분별여HepG2세포부육후,SPIO-HA-FITC조HepG2세포내급세포막상균가견록색형광, SPIO-FITC조미견명학형광염색。체외MR성상,공백대조조、SPIO조화SPIO-HA조T2치분별위(115.20±0.36)、(115.07±0.81)화(21.67±0.21)ms,차이유통계학의의(F=31703.339,P<0.01)。결론이용HA구건적MR분자탐침SPIO-HA,이화성질우량,대인간암세포HepG2구유교호적파향성。
Objective To explore a promising system for tumor CD 44 receptor-targeted imaging and to investigate their physic-chemical properties and targeting effect on CD 44 abundant cancer cells in vitro.Methods The superparamagnetic iron oxide ( SPIO) nanoparticles were prepared by a coprecipitation in alkaline media starting from a mixed of the ferrous and ferric solution.And then the surface of the SPIO nanoparticles were modified with APTMS by a reaction with the hydroxyl groups.Finally, the hyaluronan-modified SPIO ( SPIO-HA) nanoparticles were prepared.Control and experimental groups were established after adding SPIO or SPIO-HA as agents respectively.Transmission electron microscopy ( TEM) and particle size analyzer were used to measure these nanoparticle sizes and the hydrodynamic diameters.Thermogravimetric analysis ( TGA) was carried out to evaluate the HA-content on the surface of SPIO-HA.The MRI T2 ralaxivities (1/T2 ) of the two groups at different Fe concentrations (0.09, 0.18, 0.27, 0.36, 0.45 mmol/L ) were measured on a 3.0T MR system.HepG2 cells and HL7702 cells were used for assessment of cells viability by methyl thiazolyl tetrazolium ( MTT ) assay.Prussian blue staining , immunoassay fluorescence image and flow cytometry were carried out to determine the targeted cellular uptake of SPIO-HA nanoparticles.MRI were performed to show the MR T 2 value changes after incubating with HepG2 cancer cells by using T 2 WI sequences at a clinical 3.0 T MR system.One-way analysis of variance was performed to determine significant changes in MR T 2 values of blank control , SPIO-HA and SPIO groups.Results The SPIO-HA and SPIO NPs were fairly homogeneous with an average core size of 18.2 and 22.4 nm, hydrodynamic diameter of 91.1 and 103.2 nm, Zeta potential of (-45.00 ±0.86) mV and (-18.50 ±0.73) mV, and magnetic relaxivity of 0.212 ×106 M-1 · s-1 and 0.191 ×106 M-1 · s-1.Based on the TGA data , HA accounted for 24%weight of each SPIO-HA.The internalization of the SPIO-HA was confirmed by prussian blue staining , while the cells showed no obvious blue stains with SPIO , incubation of SPIO-HA with tumor cells led to blue color inside the cells.After that, we examined cancer cell binding of FITC-SPIO-HA by immunoassay fluorescence image and flow cytometry.The green fluorescence resulting from FITC-SPIO-HA was observed inside the cells in both the cytoplasm and the plasmalemma.Tumor cells treated with SPIO-HA exhibited higher fluorescence signals with 7.97-fold enhancement observed for HepG 2 cells over control particles.In vitro MR, mean T2 values of blank control , SPIO and SPIO-HA groups were ( 115.20 ±0.36 ), ( 115.07 ±0.81 ) and ( 21.67 ±0.21 ) ms, respectively.There was significant difference among those three groups (F=31 703.339,P<0.01), MR T2 values of HepG2 cells treated with the SPIO-HA NPs were lower than blank and SPIO group.In comparison, SPIO did not generate any MRI signal changes compared with blank group.Conclusion The tumor CD44 receptor-targeted MR molecular probe SPIO-HA had a good physic-chemical property and well targeted HepG2 cells.