銀粒子/矽片奈米複合物之細胞與基因毒性與機制探討

Abstract

近年來奈米銀粒子(AgNP)已被利用於許多抗菌產品上，例如紡織物、淨水器、體內骨釘等等。許多研究發現AgNP具有良好的抗菌性，但其高活性也會對人體造成傷害。本實驗室開發片狀矽酸鹽黏土(NSP)使奈米銀粒子(AgNP)穩定在NSP表面上，合成出奈米混成材料-奈米矽片銀粒子(AgNP/NSP)。本研究對AgNP/NSP進行一系列毒性相關研究，利用人永生化表皮细胞(HaCaT cells)鑑定其細胞毒性。以MTT assay測試評估不同的AgNP/NSP重量比的細胞毒性，其結果顯示NSP的重量百分比越高，會造成較高的細胞毒性。但根據我們先前的研究，高百分比的NSP，合成出的AgNP/NSP其AgNP粒子較小且抗菌能力較好。綜合分析其對於各種不同格蘭氏陰性與格蘭氏陽性菌的最低抑菌濃度，AgNP/NSP重量百分比7/93在其最低抑菌濃度(AgNP: 3.9 ppm)有最高的HaCaT cells細胞存活率(95%)。 此外，利用四種不同之測試系統對AgNP/NSP的基因毒性做評估，分別為: 體外哺乳類細胞的染色體異常分析法 (In vitro Chromosome aberration test) ，體外彗星測試 (In vitro Comet assay) ，體外微生物基因突變分析 (In vitro Ames test) 和體內微核試驗 (In vivo micronucleus assay) 。由以上基因毒性測試下，AgNP/NSP皆無觀察到染色體異常與DNA的損害。這些結果印證AgNP/NSP為安全奈米混合材料可發展於生醫應用上。由TEM的觀察，我們發現AgNP/NSP低毒性機制來自於NSP的高表面積，其高表面積可阻止AgNP穿透細胞膜，並降低基因毒性。綜合以上各點，AgNP/NSP是安全且有效的抗菌劑適合發展生醫應用。

In recent years, silver nanoparticles (AgNP) has been used in many antibacterial products, such as drinking water purification and bone cement or treatment of many skin conditions and the use as antibacterial textile. Since nanomaterials and nanotechnologies are expected to yield numerous health and health care advances, tighter the regulation of nanotechnology has been demanded. Silver nanoparticles (AgNP) are known for excellent bactericidal properties, but there are the concerns of the possible adversity toward normal cells. We have developed the one-nanometer-thick silicate platelets (NSP) from the natural silicate clays and employed them as the high surface support for enhancing antimicrobial efficacy but mitigating the toxicity of normal cells. The NSP nanoplatelets were in the geometric dimension of ca. 80 × 80 × 1 nm3 and possessing of intensive ionic charges on the local surface. The synthesis of the nanohybrids of AgNPs immobilized on NSP (AgNP/NSP) were proven to have the enhanced antimicrobial properties against the growth of bacteria including Gram-positive and Gram-negative. In this study, we synthesized AgNP/NSP nanohybrids in three different weight ratios (1/99, 7/93 and 15/85) and particle size in the range of 3.8-9.3 nm in diameter. Cytotoxicity by using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and genotoxicity by the methods of chromosomal aberration, comet, Ames and micronucleus assays were determined. Overall, AgNP/NSP showed none of significant cytotoxicity nor DNA damage at the bactericidal dose. By comparison, the synthesized AgNP in polymeric organic dispersants were shown high cytotoxicity and genotoxicity by the same tests. Due to the high surface support of the NSP, NSP prevents the immobilized AgNP enter cell membrane, which lower the risk associated in AgNP toxicity. Hence, the newly developed AgNP/NSP nanohybrids demonstrated low toxicity based on the cytotoxic and genotoxic tests and suitability for potential uses in biomedical areas.