奈米物質 (Nanoparticles) 因其本身的成分、大小、型態、溶解度、電位或表面化學鍵結等特性的不同而具有各種物理化學特性，這種的多樣性使得奈米物質展現出有別於塊材的特色，因而大量的被開發及應用於各種領域中，而隨著發展與應用層面擴大，奈米物質將無可避免的被釋放在環境當中，成為潛在的環境風險因子，因此目前迫切需要發展能夠快速且高通量的奈米毒性篩選模式以篩選出更安全的奈米物質。近年有部分文獻探討如何開發建立奈米物質高通量篩選的平台，包含藉由體外測試使用適當的高通量方法來評估細胞損傷反應並預測其在體內的不良結果，在動物或整個生物體中進行研究。過去研究顯示，奈米物質會導致細胞毒性，包含氧化壓力 (Oxidative stress) 以及自體吞噬 (Autophagy) ，而其中自體吞噬作用已被認為是奈米物質誘導毒性的主要機轉之一，研究顯示奈米物質暴露會藉由自體吞噬作用的失衡導致細胞損傷。故本研究旨在藉由斑馬魚胚胎模式建立自體吞噬高通量奈米毒性篩選模式。
首先本研究使用螢光標記之奈米物質，包括奈米銀和奈米金來探討奈米物質主要累積的標的器官，實驗結果顯示奈米銀和奈米金能夠累積於多種器官當中，包含:頭部、腸道、胃與肝臟。並且我們發現奈米物質會導致肝臟損傷或發育受阻，故肝臟是奈米物質毒性的關鍵標的器官。我們進一步使用斑馬魚肝細胞株確認奈米物質會誘發氧化壓力與自體吞噬活性的相關蛋白表達，確認自體吞噬相關蛋白質是適用於做為奈米毒性的生物標記。在奈米物質篩選之斑馬魚胚胎高通量毒性測試策略的暴露條件上，我們發現去離子水相較於標準魚類胚胎培養水而言更能夠保留奈米物質的原始特性，針對胚胎高通量上機部分，經過不同微孔盤測試後，使用 3D 列印製作出的插件並與 96 孔微孔盤配合使用可以達到最佳的胚胎定位與定向，最後考量胚胎特性，使用Image J軟體開發的分析系統進行的數據分析較符合胚胎實驗結果。最後藉由上述建立之奈米物質斑馬魚胚胎高通量毒性測試再次評估適宜胚胎模式的生物標記，結果顯示自體吞噬相關蛋白，包含Microtubule-associated protein 1 light chain 3 (LC3) 與Sequestosome 1 (p62/SQSTM1) 是能夠精準預測奈米物質導致動物死亡的良好生物標記。綜合上述，本研究成功建立一套適用於斑馬魚胚胎檢測奈米物質毒性的高通量篩選策略，自體吞噬相關的生物標記能反應奈米物質導致的毒性，此系統預期能夠在未來應用於奈米物質用途的監管決策和風險評估。

Nanoparticles, with their diverse physicochemical properties—such as variations in composition, size, shape, solubility, charge, and surface chemistry—exhibit unique characteristics distinct from bulk materials, leading to widespread use across various fields. Their environmental release, however, poses potential risks, emphasizing the need for rapid, high-throughput nanotoxicity screening models. This study aims to develop such a model based on autophagy using the zebrafish embryo model. We found that silver and gold nanoparticles accumulate in various organs, notably the liver, causing damage and developmental arrest, thus making the liver a key target for toxicity. Analysis with zebrafish liver cell lines confirmed that nanoparticles induce oxidative stress and autophagy-related protein expression, establishing these proteins as suitable biomarkers. We optimized exposure conditions for high-throughput testing, discovering that deionized water better preserves nanoparticle properties. The use of 3D-printed inserts with 96-well plates achieved optimal embryo positioning. Data analysis with ImageJ software aligned well with experimental results. The established screening system allowed us to re-evaluate biomarkers, confirming that autophagy-related proteins, including LC3 and p62/SQSTM1, are effective for predicting nanoparticle-induced mortality. This study successfully established a set of high-throughput screening strategies suitable for detecting nanoparticle toxicity in zebrafish embryos. The trial results indicate that these biomarkers reflect nanoparticle toxicity to varying degrees, with autophagy showing a particularly high correlation. These strategies can enhance the accuracy and reliability of nanomaterial safety assessments and can be applied in future regulatory decisions and risk assessments for nanoparticle applications.

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