釓金屬錯合物及氧化鐵表面修飾做為分子影像之磁振造影對比劑

Abstract

近年來磁振造影(MRI)已被發展至應用於人體到細胞及分子的層級。為了完全開發 MRI 技術應用於細胞、分子與功能性影像，能夠標幟活體細胞的目標化、區域化及數量 化之新一代MRI 對比劑與技術是目前最迫切需要的。因此，目前兩種最主要的MRI 對 比劑是研究與發展的方向：小型分子量之釓金屬錯合物及氧化鐵奈米粒子。高度表現於 人類腫瘤上新穎的MMP-7、Legumain 蛋白酶(protease)或α v β3 受體與腫瘤侵犯及轉移有 關，目標化之MMP-7、Legumain 或cRGD 之胜肽片段可當作標的，目標化至表現這些 蛋白酶或受體類型之腫瘤。本研究之目的為調節不同親脂性之釓金屬錯合物，並將此釓 金屬錯合物鍵結上目標化之MMP-7、Legumain 或cRGD 蛋白酶表現之腫瘤，找出最佳 的脂溶性之釓金屬錯合物，胜肽片段能使釓金屬錯合物目標化沈積於MMP-7 蛋白酶、 Legumain 或cRGD 相關的腫瘤位置。親脂性之釓金屬錯合物能夠安定的沈積於腫瘤細 胞膜表面而應用於MRI 上，可用於標幟與追蹤癌細胞。這些釓金屬錯合物能用於以非 侵入造影追蹤蛋白酶表現。此釓金屬錯合物之物性、化性探討，包括熱力學穩定度、弛 緩率(r1)、內層水分子數、水分子交換速率、轉動相關時間及與蛋白酶或膜蛋白受體作 用之探討是本研究的重點。最後，體外相關實驗與MR 影像也將被研究探討。在氧化鐵 奈米粒子方面，先前研究中有發展出具良好生物相容性的超順磁氧化鐵奈米粒子，其在 磁振造影的顯影上具有較高之靈敏度，無毒性又兼具生物可降解性，我們利用先前的經 驗作為研究T2 磁振造影對比劑的一基本平台。本計劃目的為將超順磁氧化鐵奈米粒子 表面修飾上具生物相容性之高分子聚合物，再鍵結賀癌平(Herceptin)或其他具目標化之 胜肽片段，藉以目標化到腫瘤(乳癌或其他癌症)位置。我們首先合成奈米級之超順磁氧 化鐵粒子及研究其物、化性質，包括氧化鐵奈米粒子粒徑大小、穩定性、磁化率、XRD 晶型研究等，並藉由縮短其橫向弛緩時間(T2)來提高其將來在MRI 上訊號強度的對比。 縱向(T1)及橫向(T2)弛緩率則利用20MHz 弛緩儀在37°C 下測得。最後利用流式細胞儀及 MR 影像來證明已修飾超順磁氧化鐵奈米粒子之效能。

Recently, MRI has been developed to examine living organisms down to the cellular and molecule level. To exploit the advancement of MRI technique for cellular, molecule and functional imaging, there are increasing needs for developing new MRI contrast agents and techniques for cell and molecule labeling to report the localization, movement, mass, and functions of cells in vivo. Therefore, two major classes of contrast agents are available for MRI such as small molecular weight Gd3+ chelates and iron oxide nanoparticles. The purpose of research is tuning the lipophilicity of Gd3+ complexes and conjugating with peptide substrates of MMP-7, Legumain protease or cRGD. MMP-7, Legumain protease and αv β3 receptor are greatly related to tumor invasion and metastasis, and are also highly expressed in majority of human tumors, which make them as the very representative cancer proteases and membrance receptor. To find the optimum lipophilicity of Gd3+ complexes that can be stably incorporated into cell membranes may serve as a useful tool for tumor cell labeling and tracking. Ideally, these Gd3+ chelates should label intact cell membranes noninvasively at low concentrations and with fast kinetics, and should remain on labeled cells over a period of time to allow repetitive imaging. The chemical and physical properties of these Gd3+ complexes will be characterized, including thermodynamic stability constant, relaxivity (r1), the number of inner-sphere water and kinetic parameter (water exchange rate and rotational correlation time). Finally, the MR imaging will be conducted as well. On the other hand, in the case of targeting to tumor cell, the surface of iron oxide nanoparticles modified with dextrin or PEG (polyethylene glycol) and then conjugated with herceptin or targeting peptide will be synthesized. The nano-sized superparamagnetic iron oxide(SPIO) particulates selectively shorten the transverse relaxation time (T2) of nearby water protons and generally produce negative enhancement by decreasing signal intensity. The geometry, structural features, and physical properties of magnetite nanoparticles will be characterized. The longitudinal relaxivity (r1) and transverse relaxivity (r2) will be measured by 20 MHz relaxometer at 37.0 ± 0.1℃. To prove effectiveness