VEGFR-2靶向超顺磁性氧化铁磁性纳米探针构建及肝癌细胞磁共振分子成像
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摘要
目的制备血管内皮生长因子受体(VEGFR)-2靶向磁共振(MR)分子探针,探讨其对肝癌细胞的特异性靶向作用。方法采用共沉淀法制备超顺磁性氧化铁(SPIO),用壳聚糖对其表面进行修饰,耦联anti-VEGFR2抗体,制备VEGFR-2靶向MR分子探针(anti-VEGFR2-CS@SPION),以未修饰的SPIO纳米粒作为对照组。DLS法测量粒径大小、分布及Zeta电位,3.0T MR检测T2弛豫率。MTT法评价探针的安全性,通过激光共聚焦显微镜检测以及普鲁士蓝染色的方法验证探针与肝癌细胞结合的特异性。3.0T MR观察探针的体外MR成像能力。结果 SPIO和antiVEGFR2-CS@SPION的粒径分别为20.6 nm和38.4 nm;Zeta电位分别为-(20.3±1.32)m V、(3.58±1.28)m V。T2弛豫率分别为0.179×10~6M~(-1)S~(-1)、0.201×106M~(-1)S~(-1)。细胞毒性实验表明探针在高浓度下对细胞没有毒性;激光共聚焦显微镜检测显示,抗体探针与Hep G2细胞特异性结合;antiVEGFR2-CS@SPION与Hep G2细胞孵育后经普鲁士蓝染色,细胞内见较多的蓝染颗粒,而单纯SPIO组细胞内未见蓝色颗粒。体外MR成像显示,anti-VEGFR2-CS@SPION组、单纯SPIO组和空白对照组的T2值分别为(55.6±1.4)ms、(99.8±0.77)ms和(110.8±0.95)ms,差异有统计学意义(F=317.547,P<0.01)。结论壳聚糖修饰和SPIO标记的anti-VEGFR2抗体探针具有良好的生物学特性和体外肝癌细胞MR显像能力。
Objective To prepare a targeting MR molecular probe with vascular endothelial growth factor receptor(VEGFR)-2,to determine the specific targeting function in human hepatoma cell.Methods The superparamagnetic iron oxide nanoparticles was prepared by coprecipitation methods,then the VEGFR-2-loaded MR molecular probe wa S prepared by modifying SPIO with chitosan in surface and coupling anti-VEGFR2. The SPIO nanoparticles with unmodified was treated as control group. The particle size,distributed and Zeta potential were tested by DLS,its T2 relaxivity rate was tested by 3. 0T MR. The security of probe was determined by MTT assay. The combination probe in Hep G2 cell was detected by laser confocal microscopy.Results The particle sizes of SPIO and anti-VEGFR2-CS@ SPION were 20.6nm and 38.4 nm respectiviey,the Zeta potentials were( 20.3±1.32) m V and( 3.58±1.28) m V respectively,and the T2 relaxivity rates were0.179×10~6M~(-1)S~(-1)and 0.201×106M~(-1)S~(-1)respectively. The laser confocal microscopy showed Hep G2 cell specific binding with antibody probe.More blue-stained particles appeared in cells. But only few blue-stained particles appeared in control group. T2 values of anti-VEGFR2-CS@SPION,simple SPIO and empty control groups were( 55.6± 1.4) ms,( 99.8±0.77) ms and( 110.8±0.95) ms respectively,there were significant differences( F = 317.547,P<0.01) between them.Conclusions The anti-VEGFR2 molecular probe modified by chitosan and labeled by SPIO has good biological characteristics and vitro MR imaging for hepatoma cell.
Objective To prepare a targeting MR molecular probe with vascular endothelial growth factor receptor(VEGFR)-2,to determine the specific targeting function in human hepatoma cell.Methods The superparamagnetic iron oxide nanoparticles was prepared by coprecipitation methods,then the VEGFR-2-loaded MR molecular probe wa S prepared by modifying SPIO with chitosan in surface and coupling anti-VEGFR2. The SPIO nanoparticles with unmodified was treated as control group. The particle size,distributed and Zeta potential were tested by DLS,its T2 relaxivity rate was tested by 3. 0T MR. The security of probe was determined by MTT assay. The combination probe in Hep G2 cell was detected by laser confocal microscopy.Results The particle sizes of SPIO and anti-VEGFR2-CS@ SPION were 20.6nm and 38.4 nm respectiviey,the Zeta potentials were( 20.3±1.32) m V and( 3.58±1.28) m V respectively,and the T2 relaxivity rates were0.179×10~6M~(-1)S~(-1)and 0.201×106M~(-1)S~(-1)respectively. The laser confocal microscopy showed Hep G2 cell specific binding with antibody probe.More blue-stained particles appeared in cells. But only few blue-stained particles appeared in control group. T2 values of anti-VEGFR2-CS@SPION,simple SPIO and empty control groups were( 55.6± 1.4) ms,( 99.8±0.77) ms and( 110.8±0.95) ms respectively,there were significant differences( F = 317.547,P<0.01) between them.Conclusions The anti-VEGFR2 molecular probe modified by chitosan and labeled by SPIO has good biological characteristics and vitro MR imaging for hepatoma cell.
引文
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7 Ferrara N,Gerber HP,Le Couter J.The biology of VEGF and its receptors[J].Nat Med,2003;9(6):669-76.
8 Shibuya M.Vascular endothelial growth factor(VEGF)and its receptor(VEGFR)signaling in angiogenesis:a crucial target for anti-and pro-angiogenic therapies[J].Genes Cancer,2011;2(12):1097-105.
9 金光玉,李德奇,夏雷,等.壳聚糖-乳糖酸-超顺磁性氧化铁纳米颗粒的制备及体内磁共振成像中的应用[J].延边大学医学学报,2014;37(4):157-60.
10 Chen HJ,Zhang ZH,Luo LJ,et al.Surface-imprinted chitosan-coated magnetic nanoparticles modified multi-walled carbon nanotubes biosensor for detection of bovine serum albumin[J].Sensors Actuators B Chem,2012;163(1):76-83.
11 杨蕊梦,李楠楠,张黎明,等.肿瘤CD44受体靶向性超顺磁性氧化铁-透明质酸分子探针的构建及体外靶向作用[J].2014;48(5):363-8.
2 Ferrara N,Gerber HP,Le Couter J.The biology of VEGF and its receptors[J].Nat Med,2003;9(6):669-76.
3 Koyama T,Shimura M,Minemoto Y,et al.Evaluation of selective tumor detection by clinical magnetic resonance imaging using antibody-conjugated superparamagnetic iron oxide[J].J Control Rel,2012;159(3):413-8.
4 Wang C,Ravi S,Garapati US,et al.Multifuctional chitosan magnetic-graphene(CMG)nanoparticles:a theranostic platform for tumor-targeted codelivery of drugs,genes and MRI contrast agents[J].J Mater Chem B Mater Biol Med,2013;1(35):4396-405.
5 Barai S,Bandopadhayaya GP,Julka PK,et al.Role of Tc-glucoheptonic acid brain single photon emission computed tomography in differentiation of recurrent brain tumor and post-radiation gliosis[J].Australas Radiol,2004;48(3):296-301.
6 Chung TH,Hsiao JK,Hsu SC,et al.Iron oxide nanoparticle-induced epidermal growth factor receptor expression in human stem cells for tumor therapy[J].ACS Nano,2011;5(12):9807-16.
7 Ferrara N,Gerber HP,Le Couter J.The biology of VEGF and its receptors[J].Nat Med,2003;9(6):669-76.
8 Shibuya M.Vascular endothelial growth factor(VEGF)and its receptor(VEGFR)signaling in angiogenesis:a crucial target for anti-and pro-angiogenic therapies[J].Genes Cancer,2011;2(12):1097-105.
9 金光玉,李德奇,夏雷,等.壳聚糖-乳糖酸-超顺磁性氧化铁纳米颗粒的制备及体内磁共振成像中的应用[J].延边大学医学学报,2014;37(4):157-60.
10 Chen HJ,Zhang ZH,Luo LJ,et al.Surface-imprinted chitosan-coated magnetic nanoparticles modified multi-walled carbon nanotubes biosensor for detection of bovine serum albumin[J].Sensors Actuators B Chem,2012;163(1):76-83.
11 杨蕊梦,李楠楠,张黎明,等.肿瘤CD44受体靶向性超顺磁性氧化铁-透明质酸分子探针的构建及体外靶向作用[J].2014;48(5):363-8.