奈米材料毒性評估測試—天然脫層矽片之細胞毒性與基因毒性

A General Methodology for Testing Nanomaterial Toxicology — Cytotoxicity and Genotoxicity of the Exfoliated Silicate Platelets from the Natural Clays

李佩如(Pei-Ru Li)

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/碩士(2009年)

https://doi.org/10.6342/NTU.2009.00086

摘要

近年來奈米科技的進步對科學與工業進展均造成很大的衝擊。但製造新材料之發明對環境及人體安全性問題及新材料的研究應該更受重視。本篇論文是利用本實驗室發明之奈米尺寸之矽片(Nanoscale Silicate Platelet；NSP)做毒性探討。奈米尺寸之矽片新材料簡稱奈米矽片，是由天然黏土脫層而得，可視為溫和的矽酸鹽礦物。然而，奈米矽片對於環境是獨特且未知的，其因獨立片狀的脫層結構而具高的表面積。再者，奈米矽片具有高的視徑比和特殊的幾何形狀，它的毒性在學術上是被感興趣的。奈米矽片的尺寸為80 × 80 × 1 nm3，藉由脫層後得到之奈米矽片，其表面擁有獨特的離子電荷和抗菌的能力。此論文中，我們利用掃描式電子顯微鏡去觀察奈米矽片對中國倉鼠卵巢癌細胞(CHO cells)的作用。另外，利用三種不同之測試系統對新穎材料奈米矽片的基因毒性做評估，分別為：體外彗星測試(In vitro Comet assay)，體內微核試驗(In vivo micronucleus assay)和微生物基因突變分析(Ames test)。由彗星測試得知，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，DNA無損害。微核試驗顯示餵食小鼠濃度為500 mg/kg b. w.之奈米矽片對囓齒類動物周邊血液所引發的微核數與控制組相比無顯著之差異。經由微生物基因突變分析之結果顯示，濃度為1000 μg/plate之奈米矽片對沙門菌突變菌係之基因毒性與控制組相比無顯著之增加。由上述三種不同基因毒性測試呈現奈米矽片無致突變的影響。利用MTT assay和LDH assay測試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；培養24小時後，有劑量效應的細胞毒性。急性口服毒性測試中，對Sprague-Dawley 雄鼠及雌鼠的半數致死量(LD50)皆大於5,700 mg/kg。這些結果印證奈米矽片為安全奈米材料可發展於生醫應用上。

關鍵字

奈米矽片 ； 細胞毒性 ； 基因毒性 ； 奈米材料 ； 急性口服毒性

並列摘要

Recent advances in nanotechnology may largely impact science advance and industrial progresses. However, the creation of new material might also affect the society and environmental pollutions due to their unknown properties. Safety issues and the detailed understandings on the newly developed materials ought to be the concerns and responsibilities of the scientists. In this thesis, the toxicity concerns on the nanometer-sized silicate platelets (NSP) that were developed in our research group are addressed. The NSP material is first time prepared from the exfoliation of the natural clays which is commonly considered as a benign silicate mineral. However, the NSP material is unique and unknown to the environment, with the exfoliated structure of individual platelets rather than in an isomerism form but lamellar platelet alignment. Especially the layered silicates are totally delaminated into highly surfaced silicates in thin platelet forms. Its toxicity is academically interesting because their difference in high aspect ratio and geometric shape. Furthermore, the NSP possessing ionic charges on the surface of silicate plates with average geometric dimension of ca. 80 × 80 × 1 nm3 has demonstrated its potential antimicrobial behavior. With these reasons in mind, this study has centered on the chemistry of NSP interacting with the incubated CHO cells and the physical examination of their interacting morphologies by scanning electron microscope. The genotoxic effect of NSP was examined by using three different test systems: the Comet assay test in Chinese Hamster Qvary (CHO) cells in vitro, micronucleus (MN) assay in vivo and the Salmonella gene mutation assay (Ames test) in strain TA98, TA100, TA102, TA1535 and TA1537. It is importantly noted that the results from the Comet assay showed none of DNA damage after 24 h of incubation with 1000 μg/mL of NSP concentration. The MN test indicated no significant micronucleus induction by NSP in the CHO cells at any concentrations tested. With five different strains of Salmonella typhimurium, there are none of mutation found when exposing with NSP. No mutagenic effect was observed in three different genetic toxicity test systems. The cytotoxicity was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release, and shown a low cytotoxicity effect on Hs68 cells. The CHO cells below a concentration of 1000 μg/mL at 12 h incubation and dose dependent cytotoxicity effect at 24 h incubation and LDH assay also showed low damage on Hs68 cells and CHO cells below a concentration of 1000 μg/mL at 24 h incubation. Investigation on potential acute oral toxicity in rats showed that the median lethal dose (LD50) of NSP by oral treatment was greater than 5,700 mg/kg for both male and female Sprague-Dawley (SD) rats. These results have implicated that NSP is a promising new silicate materials for using as a labeling reagent and carries for biomedical applications.

並列關鍵字

nanoscale silicate platelets ； cytotoxicity ； genotoxicity ； nanomaterials ； acute oral toxicity

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