{"title":"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。","authors":"Zhila Osmani, Muhammad Amirul Islam, Feng Wang, Sabrina Rodrigues Meira, Marianna Kulka","doi":"10.1186/s13007-024-01289-x","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":20100,"journal":{"name":"Plant Methods","volume":null,"pages":null},"PeriodicalIF":4.7000,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11523603/pdf/","citationCount":"0","resultStr":"{\"title\":\"Optimization of a rapid, sensitive, and high throughput molecular sensor to measure canola protoplast respiratory metabolism as a means of screening nanomaterial cytotoxicity.\",\"authors\":\"Zhila Osmani, Muhammad Amirul Islam, Feng Wang, Sabrina Rodrigues Meira, Marianna Kulka\",\"doi\":\"10.1186/s13007-024-01289-x\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":20100,\"journal\":{\"name\":\"Plant Methods\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.7000,\"publicationDate\":\"2024-10-30\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11523603/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Plant Methods\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1186/s13007-024-01289-x\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Plant Methods","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1186/s13007-024-01289-x","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Optimization of a rapid, sensitive, and high throughput molecular sensor to measure canola protoplast respiratory metabolism as a means of screening nanomaterial cytotoxicity.
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.
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.