以原子轉移自由基聚合形成嵌段共聚物於聚二甲基矽氧烷表面之結構設計與抗菌能力之研究

Architecture design and antibacterial efficacy of dual functional block copolymer brushes on poly(dimethylsiloxane) surfaces via surface-initiated atom transfer radical polymerization

陳子媞(Zi-Ti Chen)

指導教授 ： 張哲政

國立臺灣大學/理學院/化學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202002476

摘要

聚二甲基矽氧烷 (PDMS) 為導管或藥物傳遞的介質，其表面具有疏水性，致使細菌容易貼附，而造成感染問題。許多團隊致力於研究PDMS的表面修飾，藉由改變其表面化學性質，並在表面連結接觸性殺菌劑 (例如甲基丙烯酸二甲基氨基乙酯 (DMA) 之四級銨鹽類qDMA+) 的高分子或排除性抗菌劑 (例如具親水性之兩性離子 [2-(甲基丙烯酰基氧基)乙基]二甲基-(3-磺酸丙基)氢氧化銨 (SB)) 的高分子等，而達到抗菌功效。本研究使用硫醇丙基三甲氧基矽烷 (MTS) 進行表面改質，並導入2-溴-2-甲基丙酸烯丙酯 (ABrMP)，以引發原子轉移自由基聚合 (SI-ATRP)，使接觸性和排除性抗菌劑得以分別鍵結於改質之PDMS介質表面，並用以製備雙重性能之雙層高分子共聚物，目的乃著重於研究高分子結構與抗菌效果之關係。該效果之建立，乃藉由化學分析電子能譜術 (XPS)、反射式傅立葉轉換紅外線光譜術 (ATR-FTIR)、接觸角 (CA) 量測等，鑑定MTS、ABrMP、和抗菌高分子在改質之聚二甲基矽氧烷樣品表面的鍵結與結構，並透過細菌貼附實驗，及雷射掃描共軛焦顯微術，觀測貼附於樣品表面之染色細菌，瞭解樣品抗菌效能及活菌與死菌之分佈，且以塗抹法(Spread plate method) 檢測總菌落數，而獲知抗菌高分子在改質樣品表面的抗菌殺菌能力。基於XPS元素和氧化態分析，本研究開發了高分子在改質表面的抗菌鑑測方法，乃使用各聚合物鏈之單體數 (n) 和聚合物鏈結到PDMS改質樣品表面之Si位點的接枝百分比 (GP) 為參數，以呈現DMA或SB單體所組成之單層高分子或雙層高分子共聚物的抗菌殺菌性。以大腸桿菌接種4小時為例，研究結果顯示，PDMS改質表面上鏈結接觸性殺菌劑之單層高分子材料PDMS-qDMA+ (nqDMA+ = 26.7; GPqDMA+/Si = 1.61%) 呈現極為有效的抗菌活性，塗抹法顯示該材料表面無任何細菌生存。以掃描式電子顯微鏡 (SEM) 觀察細菌在該表面的形貌顯示，接種後，PDMS-qDMA+表面上的大腸桿菌乃通過一系列過程被消滅，這過程包括 (1) 膜擴展，(2) 膜穿孔，(3) 細胞內質滲出，(4) 胞體損傷，(5) 細胞變形，(6) 細胞破壞，(7) 細胞破碎，和(8) 細胞裂解。文獻查考顯示，本研究乃是首次觀察到細菌附著在抗菌材料表面上之膜擴展導致滅菌的現象。相比之下，PDMS改質表面上鏈結排除性抗菌劑之單層高分子材料PDMS-SB的聚合物鏈長較短 (nSB = 7.95)，且接枝百分比較低 (GPSB/Si = 0.436%)。根據弗洛里半徑 (Flory radius) 計算而得的嫁接參數(grafting parameter) 顯示，大腸桿菌細胞可能吸附在SB單層高分子中。當qDMA+ 鏈結在PDMS-SB表面，可提高所製得之PDMS-SB-qDMA+ 雙層高分子共聚物的抗菌活性，乃因為qDMA+以長鏈 (nqDMA+ = 22.9) 嫁接到SB層。另外，將長的SB聚合物鏈 (nSB = 55.7) 接枝到qDMA+長鏈 (nqDMA+ = 26.7) 上，因兩者的協同作用，使所製得之雙層高分子共聚物PDMS-qDMA+-SB表面具有極強的殺菌作用。在該共聚物表面上，細菌細胞幾乎不會通過SB的兩性離子層，而該層之下的qDMA+ 層則強烈吸引著細胞，致使其細胞膜被極大化地擴展，使細胞的雙末端同步發生裂解。就目前所知，細胞之雙末端同步裂解機制，乃是在本研究中被首次發現。本研究因此在雙層高分子共聚物抗菌材料的設計、結構、和細菌在材料的裂解機制上，有重要貢獻。

關鍵字

抗菌 ； 雙層抗菌高分子 ； 共聚物 ； 鍵結結構 ； 表面引發原子轉移自由基聚合方法 ； 細菌裂解機制

並列摘要

Bacterial biofilm formation causes severe safety problems on PDMS biomedical devices such as blood pumps, catheter, and drug delivery systems. In order to curtail the problems, the PDMS surface was modified for antibacterial functionality through (i) surface activation via silanization with (3-mercaptopropyl)trimethoxysilane (MTS) to form PDMS-S, (ii) uv-initiated thiol-ene reaction with allyl 2-bromo-2-methylpropionate (ABrMP) to form initiated PDMS-Br, and then (iii) surface mono- or dual-functionalization via grafting by atom transfer radical polymerization (ATRP) of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SB) polymer chains to form PDMS-SB or 2-(dimethylamino)ethyl methacrylate (DMA) polymer chains to form PDMS-DMA. Further quaternization of DMA on PDMS produced bactericidal qDMA+ polymers. X-ray photoelectron spectroscopy (XPS), attenuated total reflection-Fourier transform infrared spectroscopy (FTIR), and contact angle (CA) measurements confirmed the modification. In addition, bacterial-killing efficacy tests, using E. coli as an example, with spread plate method and LIVE/DEAD staining with confocal laser scanning microscopy (CLSM) were performed so as to understand the dependence of the antibacterial capacity of the modified PDMS surfaces upon the grafting structure of the polymers. Based on XPS elemental and oxidation-state analyses, a characterization method was developed to determine the bonding/grafting structure of MTS, ABrMP, and the polymer chains on PDMS, including the number of DMA, qDMA+, or SB monomers (nDMA or nSB) per polymer chain bonded to the PDMS surface and the grafting percentage (GP) of the polymer chains to the Si site of PDMS. The resulting PDMS-qDMA+ (nqDMA+ = 26.7; GPqDMA+/Si = 1.61%) exhibited potent antibacterial activity against E. coli inoculum of 4 hr, which showed nil bacteria by spread plate method. SEM micrographs revealed that after the inoculation, E. coli on PDMS-qDMA+ were disrupted via a series of processes including (1) membrane outspread, (2) membrane perforation, (3) exudation of intracellular components, (4) body scathe, (5) cell deformation, (6) cell corruption, (7) cell fragmentation, and (8) cell lysis. To the best of our knowledge, this was the first observation about membrane outspread of bacteria adhering to the antibacterial surface. In comparison, the bacteria repellent PDMS-SB surface had short polymer chains (nSB = 7.95) and low grafting percentage (GPSB/Si = 0.436%). Instead of repellence, the grafting parameter calculated based on the Flory radius, indicated that the E. coli cell may adsorb to SB. Dual-functionalization of PDMS through the second-stage ATRP of qDMA+ on PDMS-SB (PDMS-SB-qDMA+) improved the antibacterial activity, because of the long chains of qDMA+ (nqDMA+ = 22.9) grafted to SB. The synergetic attraction achieved through successfully grafting long SB polymer chains (nSB = 55.7) to the long chains of qDMA+ (nqDMA+ = 26.7) made the resulting dual-functionalized PDMS-qDMA+-SB surface extremely bactericidal. This architectured construct of zwitterionic polymers on quaternary ammonium had a SB layer through which the bacterial cell barely diffused, and the interaction of the underlying qDMA+ layer strongly attracted the cell and extensively outspreaded its membrane. A dual-end cell lysis thus took place. The lysis mechanism, to the best of our knowledge, was the first observation of the kind. Overall, PDMS-qDMA+ and PDMS-qDMA+-SB had the greatest bactericidal efficacy of 100%, and PDMS-SB-qDMA+ had ~98%. The antibacterial efficacy may be described based on the grafting parameter and the grafting structure obtained from XPS analyses. The modified PDMS surface having both a high number of monomers per chain and a relatively high grafting percentage exhibited potent bactericidal function.

並列關鍵字

antibacterial ； dual-functionalization ； grafting structure ； block copolymers ； SI-ATRP ； cell lysis mechanism

參考文獻

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