| 摘要: | 金屬有機骨架材料為高度結晶性的孔洞性三維奈米結構，由於其具備良好的耐熱與化學穩定度、可設計調控的孔隙尺度、極佳的比表面積等特性，因此在基礎科學研究及產業發展應用上深具潛力，並且持續獲得相當高度的關注;延續謝發坤老師的化學生物實驗室對於新型式快速合成金屬有機骨架材料配方參數調控的開發能力、及其探討酵素蛋白質包覆在金屬有機骨架材料相關的生化活性具體成果，本論文的研究主題將建立在此現有基礎上，透過整合新興網狀框架化學領域與生物活性體-酵素及細胞-的初步機理研究，希望未來有機會進一步拓展成為實際產業應用相關層面的基石。
本研究主要包含兩大主軸，第一部分著重於金屬有機骨架材料的快速合成開發，秉持綠色化學的理念，有別於傳統熱溶劑法使用大量有機溶劑與調節劑的方式，採用機械力化學法透過微量溶劑輔助研磨設備的協助，鎖定數種孔徑較大的金屬有機骨架材料作為標的物，研析材料相關製備的參數條件，運用物理性球磨頻率碰撞提供化學反應能量的原理，成功地將金屬有機骨架材料的合成步驟與流程時間從過往以時或天為單位，優化縮短成為以分鐘為基準，有利於因應未來工業化量產的效率與產能需求，開發出快速合成金屬有機骨架材料的方法;同時，藉助大孔徑金屬有機骨架材料能於生物相容性的水相合成優勢，亦將有助於酵素分子的包覆及其後續應用，提供更適於其在正常生理環境下的催化表現，並促進反應物與產物在酵素催化反應上的質傳物質效能，更能符合產業應用的期待;第二部分則著重於金屬有機骨架材料與微生物細胞甚至真核細胞酵母菌整合性及保護性的可行性研究，利用水相合成法開發大腸桿菌與金屬有機骨架整合的複合型活體材料，一方面研究大腸桿菌在被包覆的條件下其生化與生長的變化，另方面探討不同金屬有機骨架材料與生醫工業上已被廣泛運用之原核生物-大腸桿菌的生物相容性，並對其抗生素耐受性進行評估，試圖了解金屬有機骨架奈米材料在生物細胞上自組裝形成結晶性結構的差異，透過從表面化學電位角度的切入分析其可能相關的影響，同時，也針對金屬有機骨架材料包覆微生物細胞的抗生素毒殺保護性及其存活能力，並透過體外細胞試驗藉由誘發巨噬細胞活化後釋出的細胞激素進行免疫刺激的初步生物相容性評估，以及包覆大腸桿菌的金屬有機骨架複合材料在不同酸鹼度下與巨噬細胞的相互作用等方面獲得初步的觀察與理解，同時更拓展到真核細胞酵母菌包覆的可行性研究。
綜論上述兩部分的研究，包含金屬有機骨架材料快速合成方法上的拓展，以及藉由此種可控的多樣特性奈米顆粒材料對於生物活體細胞包覆的開創性研究，逐一地探討單晶/多晶顆粒包覆原核細胞大腸桿菌的成型條件、菌體生長活性、抗生素抵抗性、高溫耐熱性、體外免疫原誘發性等，初步完成此等具生物活性複合型孔洞材料的可行性與相容性評估，以利於在細胞治療的運送、保存甚至緩釋型藥物傳輸等醫藥領域能有相對的應用與延伸，冀望作為未來更進一步開發疾病治療、甚至癌症免疫療法的前沿基礎。
;Metal-organic Frameworks (MOFs) are a group of crystalline porous materials composed of metal ions/clusters and organic linkers. Owing to their special characteristics in extraordinary thermo-chemo stability, elastically tunable and adjustable pore size, and ultrahigh specific surface area, MOFs have become a class of potential materials for fundamental research and industrial applications.
Based on the previous studies in Professor Shieh’s MOF-Chemical Biology Lab, this thesis mainly focuses on the following topics. The first part will be related to the synthesis investigation of zirconium-based MOFs (Zr-MOFs) including UiO-66, UiO-67, MOF-801. Compared to the conventional methods for Zr-MOFs, which are constructed in high reaction temperature and time consuming with organic solvent, we have worked out the more efficient approach by using the grinding method under mechanochemistry technology to have a more productive strategy with the possible benefits for either sustainable chemistry or industrial application in the future.
Furthermore, one of the most attractive zinc-based MOFs, Zn-MOF-74 with non-interpenetrating crystallite and large aperture (>14 Å), was utilized to be as a candidate in our study to address the limitation on the restricted aperture size which will inhibit larger substrate penetration and the associated mass transfer issue inside the crosslinked networks due to the fewer degrees of freedom. We successfully encapsulated catalase (CAT) into Zn-MOF-74 crystal (denoted as CAT@Zn-MOF-74) within 10 min at room temperature by using a de novo mild water-based approach. The biocomposite CAT@Zn-MOF-74 exhibited over 3-time better activity in the degradation of H2O2 than that encapsulated into ZIF-90 (zeolitic imidazolate framework-90 with 3.5 Å aperture) owing to the larger aperture and 1D channel in Zn-MOF-74 crystallites even in the presence of proteinase K and urea. In addition, another enzyme, α-chymotrypsin (CHT) was introduced into Zn-MOF-74 (denoted as CHT@Zn-MOF-74) for action against the substrate of L-phenylalanine-p-nitroanilide (HPNA), which is larger than H2O2, to exemplify the substrate-size limitation in Enzyme@MOFs platform. The results showed apparent hydrolysis bioactivity for CHT@Zn-MOF-74 hybrid, however, exhibited undetectable activity for CHT@ZIF-90 due to the smaller aperture barrier for HPNA substrate.
Lastly, we extended the efforts to Cell@MOFs area based on our well-developed de novo technique for Enzyme@MOFs system. As a proof-of-concept, we skillfully devised the plasmid-engineered prokaryotic cell E. coli into a single-crystalline ZIF-90 particle to become the cyborg bacteria (denoted as E. coli@ZIF-90). Selected bacteria are subject to expression of isopropyl-β-D-thiogalactoside (IPTG)-induced green fluorescence protein (GFP) and the kanamycin resistant (kanr). This single cell-inside MOF biocomposite could be free from the attack of physical, chemical, or even biological hazards. The as-synthesized E. coli@ZIF-90 cyborg bacteria exhibited comprehensive size-shielding protection from tested antibiotic hazard.
In addition, release of the E. coli from ZIF-90 preserved its original bioactivity. This characteristic predicts a promising implication in drug delivery and cell therapy, which require the release of active cells on demand. Moreover, pH induced ZIF decomposition provides the chance to liberate the encapsulated active ingredient at acidic tumor sites. In comparison, conventional aggregated-nanocrystalline coating on E. coli (denoted as E. coli⊂ZIF-8) suffered from interfacial defects associated with zeta potential interference between ZIF-8 and E. coli surface. For the preliminary biocompatibility assessment in vitro, a mouse macrophage cell line RAW264.7 was used to evaluate if the biocomposites, especially the lipopolysaccharides (LPS) pyrogens derived from Gram-negative bacterial membrane such as E. coli, can induce the major cytokines index (IL-6, interleukin-6; TNF-α, tumor necrosis factor-α) producing by macrophage cell or not. It demonstrated that E. coli@ZIF-90 significantly prevented the release of inflammatory mediators compared to free E. coli and E. coli⊂ZIF-8. Besides, the phenomenon of the bacteria phagocytosis was observed while E. coli@ZIF-90 was incubated in pH 6.0 culture media, which was simulated to the general acidic microenvironment of tumor sites (~ pH 5.6-6.8), and as well no decomposed in pH 7.0 with the comparison.
Though we give a brief discussion of the synthesis of hierarchical structures, further work could incorporate combinatorial therapeutic agents to target tumor sites, which could potentially increase therapeutic efficacy and reduce side effects. The synthesized composite and synthetic biology investigation here offer new insights into bacteria-mediated tumor therapy, which should become one of the most powerful tools in the battle against cancers. |
