具標靶化之超順磁氧化鐵奈米粒子MnMEIO-silane-NH2-mPEG之合成、特性探討及其在磁振造影上之應用

Abstract

本研究成功設計具專一性標靶化之影像對比劑，以超順磁氧化鐵奈米粒子為核心，並利用聚乙二醇 (mPEG) 與silane作為表面修飾之材料，藉由表面修飾mPEG以提高對比劑之水溶性及生物相容性。在設計的環節中，利用silane與mPEG間之-NH2官能基作為生物探針與螢光發光團鍵結處，使超順磁氧化鐵奈米粒子兼具磁振造影與光學影像雙重功能之特性。此外，位於奈米粒子最外層mPEG亦能提供遮蔽正電荷 (-NH2 + H+  -NH3+) 之效果，進而減少對比劑與腫瘤細胞表面之負電荷產生靜電吸引力。 本研究成功合成對比劑 (MnMEIO-silane-NH2-mPEG) ，具備良好水溶性與超順磁之特性。此對比劑具有非常高的弛緩率 (r1及r2) ，分別為42.1 ± 1.7及293.5 ± 6.2 mM-1s-1，能有效地提高磁振造影之對比度。此外，不論在不同的pH值環境或室溫放置數週，其對比劑之水合粒徑呈現穩定而無聚集之現象，弛緩率 (r1及r2) 也無明顯變化，表示本研究合成之對比劑具備優越之穩定性。 體內及體外實驗結果證實，將Erbitux單株抗體鍵結在對比劑上 (MnMEIO-silane-NH2-(Erb)-mPEG) 可專一性標靶EGFR表現型腫瘤細胞。此外，本研究嘗試將MUC4單株抗體鍵結在對比劑 (MnMEIO-silane-NH2-(MUC4)-mPEG) 上，實驗結果亦證實此對比劑具備高專一性標靶mucin4表現型腫瘤細胞。因此，證實本研究所合成之對比劑 (MnMEIO-silane-NH2-mPEG) 實屬一良好平台，可鍵結不同生物探針進行不同腫瘤細胞之診斷與追蹤。本研究更進一步利用雙抗抗體 (Bispecific antibody) 作為生物探針，強化對比劑標靶之效果。實驗結果證實，對比劑MnMEIO-silane-NH2-(Bis)-mPEG可有效標靶Her2/neu及EGFR表現型腫瘤細胞，具有取代現行臨床使用之標靶抗體Herceptin之潛力。同時，目前相關研究中，尚未將雙抗抗體應用於診斷腫瘤細胞技術上，故本研究在這方面可說是一大突破。 由各項分析結果顯示，本研究成功設計具專一性標靶化之影像對比劑，除了具備高生物相容性與穩定性外，同時也兼具磁振造影與光學影像雙重功能之特性。

A new multifunctional nanoparticles (MnMEIO-silane-NH2-mPEG NPs), consisting of a manganese-doped iron oxide nanoparticle core (MnMEIO), a copolymer shell of silane and amine-functionalized poly(ethylene glycol), were successfully developed in this study for improving the solubility and biocompatibility of the contrast agent. There are two key features in MnMEIO-silane-NH2-mPEG, one is -NH2 functional group between silane and mPEG which can be used as a binding site for bio-probes and NIRF dyes result in the MR-optical dual modalities of nanoparticles. The other is the flexible PEG, which masks the non-conjugated reactive amine groups (-NH2 + H+  -NH3+) and reduces nonspecific binding of MnMEIO-silane-NH2-mPEG to cells. The nanoparticles (MnMEIO-silane-NH2-mPEG) are provided with the properties of water solubility and superparamagnetism in this study. The relaxivities (r1 and r2) of the MnMEIO-silane-NH2-mPEG NPs measured at 20 MHz and 37.0 ± 0.1 °C were 42.1 ± 1.7 and 293.5 ± 6.2 mM-1s-1, respectively. This indicates that the nanoparticles can efficiently improve the imaging contrast. Furthermore, the DLS data indicate no significant variation in hydrodynamic size of MnMEIO-silane-NH2-mPEG NPs across a wide pH range (pH 4-10), reassuring high colloidal stability of MnMEIO-silane-NH2-mPEG NPs under physiological conditions. Finally, no significant variation in hydrodynamic size and relaxivity of MnMEIO-silane-NH2-mPEG NPs were observed over a long time period. The in vitro and in vivo results experimentally demonstrated that the anti-EGFR antibody conjugated nanoparticles (MnMEIO-silane-NH2-(Erb)-mPEG NPs) could significantly reduce nonspecific binding and increase the imaging specificity to EGFR-overexpressing tumors. Besides, the anti-mucin4 antibody (MUC4) were used to forming the nanoparticles (MnMEIO-silane-NH2-(MUC4)-mPEG NPs) for diagnosis of mucin4-expressing pancreatic tumors. Besed on the results, MnMEIO-silane-NH2-mPEG NPs is a well-established platform to combine different targeting moieties for diagnostic and therapeutic use of various tumors. Furthermore, a multifunctional nanoparticles (MnMEIO-silane-NH2-(Bis)-mPEG NPs) which covalently conjugated anti-Her2/neu and anti-EGFR receptors bispecific antibody (Bis) were also developed for recognizing the tumors expressing both Her2/neu and/or EGFR. For Her2/neu-overexpressing tumors, MnMEIO-silane-NH2-(Bis)-mPEG can be a potential targeting agent for Herceptin replacement. For the current tumor diagnosis associated studies, this study which applied bispecific antibosies as a targeting moiety is the first approach in tumoral diagnosis area. These experimental results demonstrated that nanoparticles conjugated with various monoclonal antibody could specifically and effectively target to tumors. As a results, a MR-optical dual modality contrast agent system with high biocompatibility and stability is performed for the design and development of the next-generation nanoscale diagnostic and therapeutic modalities.