转铁蛋白导向脂质核磁造影剂纳米粒子(TfNIR-LipNBD-Ma...●

转铁蛋白导向脂质核磁造影剂纳米粒子(TfNIR-LipNBD-Ma...● NIRNBD转铁蛋白导向脂质核磁造影剂纳米粒子(Tf-Lip- Magnevist)—肿瘤靶向磁共振造影剂 ，111112王秩秋，单良，王颂平， Alexandrv Korotcov，梁兴杰 (1. Molecular Imaging Laboratory, Department of Radiology, Howard University, Washington DC;2.中国国家纳米科学中心，北京 100080) 摘要:造影剂辅助的核磁共振成像是目前肿瘤诊断的最好方法之一。但是由于核磁 共振成像内在的低灵敏性以及造影剂的非特异性，导致肿瘤早期诊断较为困难。本文将一 种新的肿瘤靶向核磁造影剂纳米粒子应用于早期肿瘤的影像诊断。这种新的肿瘤靶向核磁 造影剂纳米粒子由配体转铁蛋白(Tf)，纳米水平的正电脂质体 (Lip)载体和临床常用的造影 NIRNBD剂Magnevist(Tf-Lip-Magnevist)三部分构成。 ……… 关键词:核磁共振;肿瘤诊断;转铁蛋白;纳米粒子;靶向性;光学成像 中图分类号:Q6-33 基金项目:NIH/NCRR/RCMI 2G12RR003048, USAMRMC W81XWH-05-1-0291, and NIH 5U54CA091431, 中国科学院王宽诚科研奖金 通讯作者:王秩秋， pwang@howard.edu; 0 引 言 核磁共振成像因其具无创、快速、高解析率、高对比度等特点，在临床上广为使用。特别在肿瘤的诊断中，该技术利用病变组织和正常组织物理特性的不同，获得结构、 [1]功能影像，已经成为原发肿瘤和肿瘤转移早期诊断中不可或缺的重要依据。肿瘤的形成是长时间、多因素控制、多步骤、多基因突变的复杂变化过程。大多数恶性肿瘤都是单克隆起源，呈现无控制性生长。……… 1 材料与方法 NIRNBD1.1 双重标记靶向纳米核磁造影剂Tf-Lip-Magnevist的制备 阳离子脂质体1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)和1,2-dioleoyl- 3- trimethylammonium-propane (DOTAP)按1:1 的比例(重量)混合溶解在氯仿溶液中(Lip)，或在DOTAP:DOPE中再加入……… 1.2 细胞培养和动物模型 我们用人乳腺癌MDA-MB-231-luc (Caliper Life Sciences, Hopkinton, MA) 培养细胞和动物模型来测试我们新构件的靶向纳米核磁造影剂的有效性。MDA-MB-231-luc细胞转染了北美萤火虫荧光素酶基因，表达高水平的转铁蛋白受体。……… 1.3 共焦显微镜观察 MDA-MB-231-luc细胞培养在8孔细胞培养板上，培养24小时达到40%到50%后，吸 NIRNBDNBD去原培养基，加入150 ml含25µL探针(Tf-Lip-Magnevist, Tf-Lip-dye或 单独Alexa 680)的无血清无抗生素培养基，……… NIRNBD1.4 靶向纳米造影剂Tf-Lip-Magnevist体外靶向特异性影像分析 细胞培养在10 cm直径的培养皿中至细胞占据80%面积，吸去培养液，加入3 ml含 NIRNBDNBDNBD200 µL各种不同探针(Tf-Lip-Magnevist, Tf-Lip-dye, Lip-dye, Magnevist 或Alexa 680)的培养基，培养60分钟。用PBS溶液洗3次后，收集细胞并调节至相同数目。经离心后，……… NIRNBD1.5 靶向纳米造影剂Tf-Lip-Magnevist体内靶向特异性及有效性影像分析 NIRNBD为了评价Tf-Lip-Magnevist的肿瘤靶向性，在MDA-MB-231-luc肿瘤模型，首先用Xenogen IVIS 200光学呈像仪进行光学成像。成像和信号的定量由Living Image (Caliper Life Science, Hopkinton, MA)软件控制。……… 2 结 果 NIRNBD2.1 Tf-Lip-Magnevist的细胞内吞共焦显微镜观察 NIRNBDNIRNBD细胞和Tf-Lip-Magnevist或Magnevist分别培养5分钟至2小时。Tf-Lip- NIRNBDMagnevist孵育的细胞显示很强的Tf近红外荧光信号(图1, A, D1)和绿色Lip信号(图1 B, E1)。在和Magnevist孵育的细胞中检测不到任何荧光信号(图1 D2, E2)。……… 图1 NIRNBD2.2 Tf-Lip-Magnevist介导的肿瘤影像对比度增强 NIR为了研究探针是否在体内能够特异性的富集在肿瘤，我们首先进行了以Tf肿瘤内积 NIRNBD聚为目标的光学成像。在静脉注射探针Tf-Lip-Magnevist后，近红外荧光信号在10分钟时便清晰可辨，在90分钟到2小时荧光的强度达到最大(图2)。……… 图2 3 讨 论 肿瘤细胞生长异常迅速，不仅在形态学上与正常细胞大不相同，而且在细胞表面蛋白空间分布及绝对数量上也与正常细胞存在巨大差异。这种特殊的病理生理微环境差异为肿瘤的早期诊断和靶向治疗提供了很大空间。……… 致谢:感谢中国国家纳米科学中心孟幻博士对文稿的编辑整理 参考文献: 1. Gillies RJ, Bhujwalla ZM, Evelhoch J, Garwood M, Neeman M, Robinson SP, Sotak CH, Van Der SB. Applications of magnetic resonance in model systems:tumor biology and physiology. Neoplasia, 2000,2:139-151 2. Pautler RG. Mouse MRI: concepts and applications in physiology. Physiol, 2004,19:168- 175 3. Hornak JP. The basics of MRI. 4. Persigehl T, Heindel W, Bremer C. MR and optical approaches to molecular imaging. Abdom Imaging, 2005,30:342-354 5. Artemov D. Molecular magnetic resonance imaging with targeted contrast agent. J Cell Biochem, 2003,90:518-524 6. Massoud TF, Gambhir SS. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Gene Dev, 2003,17:545-580 ……… NIRNBDTransferrin Liposome Nanoparticle (Tf-Lip-Magnevist) – A Tumor Targeting MRI Contrast Agent 11111,2Paul C Wang, Liang Shan, Songping Wang, Alexandru Korotcov, Xingjie Liang. (1. Molecular Imaging Laboratory, Department of Radiology, Howard University, Washington DC，US;2. Laboratory of Nanomedicine and Nanobiology, National Center for Nanoscience Technology of China, Beijing 100080, China) Abstract :Magnetic resonance imaging with contrast agent enhancement is one of the best imaging method for tumor diagnosis. However, due to the low sensitivity and nonspecific deposition of contrast agent in tissue, early detection of tumor by MRI is still challenging. In this paper, the authors present a new targeted nanosized MRI contrast agent for early detection of tumor. This targeted MRI contrast agent nanoparticle is composed of three components: transferrin (Tf) as ligand, cationic liposome as carrier, and a clinically used MRI contrast agent, NIRNBDMagnevist, as payload (Tf-Lip-Magnevist). ……… Key Words: Magnetic resonance imaging; Tumor diagnosis; Transferrin; Nanoparticle; Targeting property; Optical imaging This work was supported by grants from NIH/NCRR/RCMI 2G12RR003048, USAMRMC W81XWH-05-1-0291, and NIH 5U54CA091431 Corresponding author: WANG Paul C, E-mail:pwang@howard.edu; NIRNBDNIR图1. Tf-Lip-Magnevist的细胞内吞饮共焦显微镜观察和光学成像。细胞与Tf- NBDNIRNBDLip-Magnevist或Magnevist分别共浴1小时。共焦显微镜观察表明Tf (A) 和 Lip (B) 共同存在于胞浆(C)。在细胞清洗，收集，离心，沉淀去除上清液后光学影像检测 NIRNBD(D-E)，同样表明细胞内Tf (D1) 和 Lip (E1)荧光信号。胞核用DAPI反染。 Magnevist 孕育的细胞团块则无红色(D2)或绿色(E2)的荧光 NIRFig.1 Confocal and optical detection of the reporters in cells incubated with the probe Tf- NBDLip-CA or Magnevist alone for one hour. Panels A-C are representative confocal microscopic NIRNBDimages, showing cytoplasmic distribution of Tf (A) and Lip (B) and their co-localization NIR(C). Panels D-E are optical images of the cell pellets. Strong fluorescent signals of Tf (D, lane NBDNIRNBD1) and Lip (E, lane 1) are detected in cells incubated with Tf-Lip-CA, but not in cells incubated with Magnevist alone (D and E, lane 2) NIRNBDNIRNBD图2. Tf-Lip-Magnevist的光学影像肿瘤检测。图示Tf-Lip-Magnevist经尾静脉 注入后不同时间，裸鼠肿瘤模型影像。在一定时间内肿瘤内荧光信号逐渐增强。背景荧光 信号则由强变弱(A-E)。大的肿瘤内荧光信号(左侧)明显强于小的肿瘤内荧光信号(右 2侧)。 E显示肿瘤及对侧肌肉内荧光信号随时间的变化(荧光强度单位，p/s/cm/sr) NIRNBDFig.2 Optical imaging of the tumors following i.v. administration of the Tf-Lip-CA, NIRshowing preferential accumulation of the fluorescent signal of Tf in tumors (panels A-D). The fluorescence signal was detectable as early as 10 min and reached a maximum at about 2 h and then deceased gradually. Panel E shows the plot of time versus signal intensity obtained from the 2tumor and the contralateral muscle. The signal intensity is expressed as p/s/cm/sr