Direct electrospraying of chitosan-plasmid nanoparticles enhances transformation efficiency compared to the conventional heat-shock method

IF 3.674 4区 工程技术 Q1 Engineering
Mohsen Abedi Ostad, Fatemeh Oroojalian, Elham Sharifian, Amir Amani, Roghaye Arezumand
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Abstract

Utilizing an effective transformation method is fundamental in genetic and gene delivery studies. In this study, electrospray was evaluated as a simple, cost-effective and highly efficient approach for preparing monodispersed chitosan nanoparticles (CS NPs) carrying plasmid DNA (pDNA) and delivering them to bacteria. CS/pDNA NPs were prepared at three N/P ratios (molar ratio of chitosan nitrogens to DNA phosphates) of 3, 5, and 10. Size of nanoparticles was obtained as 323, 333, and 399 nm, respectively, using DLS. E. coli was made competent using CaCl2 or CaCl2–MgCl2. Then, preformed CS/pDNA NPs, prepared using electrospray, were added to the heat-shocked bacteria. Alternatively, CS and pDNA solutions were mixed and directly electrosprayed on the bacteria. The results showed that direct electrospray of the particles provided more efficient transformation compared with transformation using heat shock (i.e. preformed NPs). Also, N/P ratios of 5 and 3 had maximum transformation efficiency when using heat shock (i.e. mean ± SD 1.23 ± 0.13 × 107 CFU/µg on CaCl2–MgCl2-made competent bacteria) and direct electrospray (i.e. mean ± SD 8.79 (0.12) × 109 CFU/µg on CaCl2-made competent bacteria), respectively. Furthermore, the use of MgCl2–CaCl2 for making the bacteria competent proved more efficient than CaCl2 alone in the transformation process. The findings highlight electrospray as a cost-effective alternative for bacterial transformation technology.

与传统的热冲击法相比,直接电喷涂壳聚糖质粒纳米颗粒提高了转化效率
利用有效的转化方法是遗传和基因传递研究的基础。在本研究中,电喷雾是制备携带质粒DNA (pDNA)的单分散壳聚糖纳米颗粒(CS NPs)并将其递送给细菌的一种简单、经济、高效的方法。CS/pDNA NPs在3个N/P比(壳聚糖氮与DNA磷酸盐的摩尔比)为3、5和10时制备。DLS法得到的纳米颗粒尺寸分别为323、333和399 nm。用CaCl2或CaCl2 - mgcl2对大肠杆菌进行诱导。然后,将电喷雾制备的预制CS/pDNA NPs添加到热休克细菌中。或者,CS和pDNA溶液混合并直接电喷涂在细菌上。结果表明,与热冲击(即预成型NPs)的转化相比,直接电喷雾的转化效率更高。此外,N/P比为5和3时,采用热休克和直接电喷雾的转化效率最高(分别为:对cacl1 - mgcl2制造的感态细菌,平均±SD为1.23±0.13 × 107 CFU/µg);对cacl1制造的感态细菌,平均±SD为8.79 (0.12)× 109 CFU/µg)。此外,在转化过程中,使用MgCl2-CaCl2使细菌胜任被证明比单独使用CaCl2更有效。这一发现强调了电喷雾是一种具有成本效益的细菌转化技术替代方案。
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
Applied Nanoscience
Applied Nanoscience Materials Science-Materials Science (miscellaneous)
CiteScore
7.10
自引率
0.00%
发文量
430
期刊介绍: Applied Nanoscience is a hybrid journal that publishes original articles about state of the art nanoscience and the application of emerging nanotechnologies to areas fundamental to building technologically advanced and sustainable civilization, including areas as diverse as water science, advanced materials, energy, electronics, environmental science and medicine. The journal accepts original and review articles as well as book reviews for publication. All the manuscripts are single-blind peer-reviewed for scientific quality and acceptance.
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