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<p>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. <i>E. coli</i> was made competent using CaCl<sub>2</sub> or CaCl<sub>2</sub>–MgCl<sub>2</sub>. 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&#xa0;provided&#xa0;more efficient transformation&#xa0;compared with transformation using heat shock (i.e.&#xa0;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 × 10<sup>7</sup> CFU/µg on CaCl<sub>2</sub>–MgCl<sub>2</sub>-made competent bacteria) and direct electrospray (i.e. mean ± SD 8.79 (0.12) × 10<sup>9</sup> CFU/µg on CaCl<sub>2</sub>-made competent bacteria), respectively. Furthermore, the use of MgCl<sub>2</sub>–CaCl<sub>2</sub> for making the bacteria competent proved more efficient than CaCl<sub>2</sub> alone in the transformation process. The findings highlight electrospray as a cost-effective alternative for bacterial transformation technology.</p>

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

  • 期刊论文

  • 发表日期：

    2025年7月16日

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  • Mohsen Abedi Ostad,
  • Fatemeh Oroojalian,
  • Elham Sharifian,
  • Amir Amani,
  • Roghaye Arezumand

摘要

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.