脫層黏土之抑菌功能與生物界面作用機制探討

Mechanistic Studies on Antimicrobial Property and Bio-Interaction of Exfoliated Nanoscale Silicate Platelets

魏郡萩(Jiun-Chiou Wei)

指導教授 ： 林江珍

國立臺灣大學/工學院/高分子科學與工程學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.10892

摘要

奈米技術發展數十年，各式各樣的奈米材料已被發現與製備包括天然的無機矽酸鹽層黏土。為了延伸應用無機矽酸鹽層黏土，將其用於蛋白質固定化、抗菌劑與奈米銀粒子/矽酸鹽層黏土複合材之探討。首先，發現牛血清蛋白 (BSA) 與矽酸鹽層黏土可經由直接插層法或逐步插層法製得。逐步插層法為藉由離子交換反應將聚醚胺 (poly-amine) 插層撐開層距隨後可允許蛋白質進入層間。為克服層狀黏土之立體障礙，更進一步發展脫層型奈米矽片用以直接吸附蛋白質，經由逐步程序使生物性巨分子進入層間或以脫層型奈米矽片直接吸附固定於表面，已被成功製備。
延伸脫層型奈米矽片之研究，發現奈米矽片具有抵抗多重種類之微生物，包括耐甲氧西林金黃色葡萄球菌及耐銀離子大腸桿菌，由SEM證實奈米矽片會經由物理捕抓方式貼附於細菌表面，更以電子順磁共振光譜證實奈米矽片經光激發可產生1O2進而達到殺菌效果。另外，利用體外彗星測試 (In vitro Comet assay) 評估奈米矽片的基因毒性得知，濃度為1000 μg/mL之奈米矽片與中國倉鼠卵巢癌細胞培養24小時後不會造成DNA損害。利用MTT assay評估奈米矽片的細胞毒性，濃度為1000 μg/mL之奈米矽片和中國倉鼠卵巢癌細胞培養12小時後，顯示為低毒性。
更進一步設計新型奈米複合材料-奈米矽片銀，藉由控制奈米矽片與銀之比例，獲得不同尺寸3.6‒35 nm之奈米銀粒子 (AgNP)。將不同比例之奈米矽片銀水溶液進行抗菌測試，發現抗菌能力受銀粒子大小及奈米矽片含量影響。且奈米矽片使銀粒子固定在表面而可能不經由穿透細胞即造成死亡。

關鍵字

抗菌 ； 奈米矽片 ； 活性氧化物 ； 電子順磁共振光譜 ； 細胞毒性 ； 基因毒性 ； 銀奈米粒子

並列摘要

Nanotechnology has been developed for decades and various nanomaterials are synthesized including the materials derived from the natural inorganic silicate clays. To extend the applications of nanomaterials, the inorganic silicate clays were used in protein immobilize, antimicrobial agent and silver nanoparticle/silicate clay nanohybrids.
In the first part of this study, the hybrids of the model bovine serum albumin (BSA) protein and layered clay were prepared from the direct and stepwise intercalation. The stepwise intercalation involves the uses of the polyamine salts to expand silicate space and subsequently embedding BSA into layered clays. To overcome the steric hindrance of the narrow spacing in the layered clays, the randomized nanoscale silicate platelets (NSP) was used to direct the adsorption of proteins on the surface. Both techniques of embedding large molecules into the silicate gallery and absorbing onto the exposed NSP surface have been successfully developed.
We further observed the unexpected antimicrobial behavior for NSP, prepared from the exfoliation of natural clays. A broad spectrum of microorganisms including methicillin-resistant Staphylococcus aureus (MRSA) and silver ion resistance Escherichia coli (E. coli) are tested. The NSP enabling of physically adhering onto to microbe surfaces and resulting antimicrobial ability is attributed to the singlet oxygen (1O2) generation tested for the photoexcited NSP using spectroscopic methods (EPR spin-trapping). The material safety on toxicity was investigated by using the Comet assay test in vitro and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The Comet assay showed no DNA damage after incubation with 1000 μg/mL of NSP and MTT assay showed a low cytotoxicity below 1000 μg/mL and a dose-dependent effect.
Further manipulating the function of silicate clays, we found that the silver nanoparticles (AgNP) could be tailored by using NSP as the supports. The amount of adding the thin silicate platelets at the composition ratio from 0.5/99.5 to 50/50 of Ag/silicate can largely affect the formation of the AgNP size ranging from 3.6 nm to 35 nm. The evaluation for antibacterial activity has shown a silver size-dependent effect on controlling the growth of dermal pathogens. The antimicrobial efficacy can be corresponded to the size of AgNP and the amount of NSP presence.

並列關鍵字

Antimicrobial ； nanosilicate platelets ； Reactive oxygen species ； Electron paramagnetic resonance ； cytotoxicity ； genotoxicity ； silver nanoparticles

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