Department of Chemistry, Sharif University of Technology, Tehran, Iran.
Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
Sci Rep. 2020 Nov 26;10(1):20672. doi: 10.1038/s41598-020-77809-1.
Evaluation of the effect of different parameters for designing a non-viral vector in gene delivery systems has great importance. In this manner, 2D crystals, precisely layered double hydroxides, have attracted the attention of scientists due to their significant adjustability and low-toxicity and low-cost preparation procedure. In this work, the relationship between different physicochemical properties of LDH, including pH, size, zeta potential, and synthesis procedure, was investigated and optimized for CRISPR/Cas9 delivery and reverse fluorescence response to the EGFP. In this manner, ZnAl LDH and ZnAl HMTA LDH were synthesized and characterized and applied in the HEK-293 cell line to deliver CRISPR/Cas9. The results were optimized by different characterizations as well as Gel Electrophoresis and showed acceptable binding ability with the DNA that could be considered as a promising and also new gold-standard for the delivery of CRISPR/Cas9. Also, the relationship of the presence of tertiary amines (in this case, hexamethylenetetramine (HMTA) as the templates) in the structure of the ZnAl LDH, as well as the gene delivery application, was evaluated. The results showed more than 79% of relative cell viability in most of the weight ratios of LDH to CRISPR/Cas9; fully quenching the fluorescence intensity of the EGFP/LDH in the presence of 15 µg mL of the protoporphyrins along with the detection limit of below 2.1 µg mL, the transfection efficiency of around 33% of the GFP positive cell for ZnAl LDH and more than 38% for the ZnAl LDH in the presence of its tertiary amine template.
评价设计非病毒载体基因传递系统中不同参数的效果具有重要意义。在这种情况下，二维晶体，即精确分层的层状双氢氧化物，由于其显著的可调节性、低毒性和低成本制备程序而引起了科学家的关注。在这项工作中，研究并优化了 LDH 的不同物理化学性质（包括 pH 值、尺寸、zeta 电位和合成程序）之间的关系，以用于 CRISPR/Cas9 传递和对 EGFP 的反向荧光响应。以这种方式合成并表征了 ZnAl LDH 和 ZnAl HMTA LDH，并将其应用于 HEK-293 细胞系中传递 CRISPR/Cas9。通过不同的特性以及凝胶电泳对结果进行了优化，并显示出与 DNA 具有可接受的结合能力，这可以被认为是 CRISPR/Cas9 传递的有前途的新金标准。此外，还评估了 ZnAl LDH 结构中叔胺（在这种情况下，六亚甲基四胺 (HMTA) 作为模板）的存在以及基因传递应用之间的关系。结果表明，在 LDH 与 CRISPR/Cas9 的大多数重量比下，细胞活力超过 79%；在存在 15 µg mL 原卟啉啉的情况下，完全猝灭 EGFP/LDH 的荧光强度，检测限低于 2.1 µg mL，GFP 阳性细胞的转染效率约为 33% ZnAl LDH 和在存在其叔胺模板的情况下，超过 38% ZnAl LDH。
