Optimization of a rapid, sensitive, and high throughput molecular sensor to measure canola protoplast respiratory metabolism as a means of screening nanomaterial cytotoxicity.

IF 4.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Zhila Osmani, Muhammad Amirul Islam, Feng Wang, Sabrina Rodrigues Meira, Marianna Kulka
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Abstract

Nanomaterial-mediated plant genetic engineering holds promise for developing new crop cultivars but can be hindered by nanomaterial toxicity to protoplasts. We present a fast, high-throughput method for assessing protoplast viability using resazurin, a non-toxic dye converted to highly fluorescent resorufin during respiration. Protoplasts isolated from hypocotyl canola (Brassica napus L.) were evaluated at varying temperatures (4, 10, 20, 30 ˚C) and time intervals (1-24 h). Optimal conditions for detecting protoplast viability were identified as 20,000 cells incubated with 40 µM resazurin at room temperature for 3 h. The assay was applied to evaluate the cytotoxicity of silver nanospheres, silica nanospheres, cholesteryl-butyrate nanoemulsion, and lipid nanoparticles. The cholesteryl-butyrate nanoemulsion and lipid nanoparticles exhibited toxicity across all tested concentrations (5-500 ng/ml), except at 5 ng/ml. Silver nanospheres were toxic across all tested concentrations (5-500 ng/ml) and sizes (20-100 nm), except for the larger size (100 nm) at 5 ng/ml. Silica nanospheres showed no toxicity at 5 ng/ml across all tested sizes (12-230 nm). Our results highlight that nanoparticle size and concentration significantly impact protoplast toxicity. Overall, the results showed that the resazurin assay is a precise, rapid, and scalable tool for screening nanomaterial cytotoxicity, enabling more accurate evaluations before using nanomaterials in genetic engineering.

优化测量油菜原生质体呼吸代谢的快速、灵敏和高通量分子传感器,作为筛选纳米材料细胞毒性的一种手段。
纳米材料介导的植物基因工程有望开发出新的作物栽培品种,但纳米材料对原生质体的毒性可能会阻碍其发展。我们提出了一种快速、高通量评估原生质体存活率的方法,该方法使用的是一种在呼吸过程中转化为高荧光resorufin的无毒染料resazurin。在不同温度(4、10、20、30 ˚C)和时间间隔(1-24 h)下对从油菜(Brassica napus L.)下胚轴分离的原生质体进行了评估。检测原生质体活力的最佳条件被确定为室温下 20,000 个细胞与 40 µM 的利马唑啉孵育 3 小时。除 5 纳克/毫升外,胆固醇丁酸酯纳米乳液和脂质纳米颗粒在所有测试浓度(5-500 纳克/毫升)下均表现出毒性。银纳米球在所有测试浓度(5-500 纳克/毫升)和尺寸(20-100 纳米)下都具有毒性,但在 5 纳克/毫升时尺寸较大(100 纳米)的银纳米球除外。二氧化硅纳米球在 5 纳克/毫升浓度下对所有测试尺寸(12-230 纳米)均无毒性。我们的结果突出表明,纳米粒子的尺寸和浓度对原生质体的毒性有显著影响。总之,研究结果表明,resazurin 试验是一种精确、快速、可扩展的纳米材料细胞毒性筛选工具,可在基因工程中使用纳米材料之前进行更准确的评估。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Plant Methods
Plant Methods 生物-植物科学
CiteScore
9.20
自引率
3.90%
发文量
121
审稿时长
2 months
期刊介绍: Plant Methods is an open access, peer-reviewed, online journal for the plant research community that encompasses all aspects of technological innovation in the plant sciences. There is no doubt that we have entered an exciting new era in plant biology. The completion of the Arabidopsis genome sequence, and the rapid progress being made in other plant genomics projects are providing unparalleled opportunities for progress in all areas of plant science. Nevertheless, enormous challenges lie ahead if we are to understand the function of every gene in the genome, and how the individual parts work together to make the whole organism. Achieving these goals will require an unprecedented collaborative effort, combining high-throughput, system-wide technologies with more focused approaches that integrate traditional disciplines such as cell biology, biochemistry and molecular genetics. Technological innovation is probably the most important catalyst for progress in any scientific discipline. Plant Methods’ goal is to stimulate the development and adoption of new and improved techniques and research tools and, where appropriate, to promote consistency of methodologies for better integration of data from different laboratories.
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