Electrosprayed Alginate Nanoparticles as CRISPR Plasmid DNA Delivery Carrier: Preparation, Optimization, and Characterization
Electrosprayed Alginate Nanoparticles as CRISPR Plasmid DNA Delivery Carrier: Preparation, Optimization, and Characterization
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DOI：
10.3390/ph13080158
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发表时间：
2020-08-01
期刊：
影响因子：
4.6
通讯作者：
Doolaanea, Abd Almonem
中科院分区：
文献类型：
--
作者：
Alallam, Batoul;Altahhan, Sara;Doolaanea, Abd Almonem
随着CRISPR/CRISPR相关蛋白（Cas）系统的出现，治疗性基因编辑变得更加可行。然而，成功实施基于CRISPR/Cas9的治疗需要CRISPR组分的安全有效的体内递送，这仍然具有挑战性。本研究采用电喷雾技术成功制备、优化和表征了载有两种CRISPR质粒的藻酸盐纳米颗粒（ALG NPs）。该递送系统的目的是编辑另一质粒（绿色荧光蛋白（GFP））中的靶基因。评价了配方和工艺变量的影响。CRISPR ALG NP分别显示228 nm和-4.42 mV的平均尺寸和ζ电位。在保持有效载荷完整性的同时实现了超过99.0%的封装效率。ALG NP中CRISPR质粒的存在通过衰减全反射-傅里叶变换红外光谱法证实。测试表明，纳米颗粒是细胞相容的，并成功地将Cas9转基因引入HepG 2细胞中。纳米颗粒转染的HepG 2能够通过在GFP基因中引入双链断裂（DSB）来编辑其靶质粒，表明海藻酸钠纳米颗粒包裹的CRISPR质粒具有生物活性。这表明该方法适用于体外或离体生物医学应用。对这些纳米颗粒的进一步研究可能会产生适合于CRISPR/Cas9系统体内递送的纳米载体。
Therapeutic gene editing is becoming more feasible with the emergence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system. However, the successful implementation of CRISPR/Cas9-based therapeutics requires a safe and efficient in vivo delivery of the CRISPR components, which remains challenging. This study presents successful preparation, optimization, and characterization of alginate nanoparticles (ALG NPs), loaded with two CRISPR plasmids, using electrospray technique. The aim of this delivery system is to edit a target gene in another plasmid (green fluorescent protein (GFP)). The effect of formulation and process variables were evaluated. CRISPR ALG NPs showed mean size and zeta potential of 228 nm and -4.42 mV, respectively. Over 99.0% encapsulation efficiency was achieved while preserving payload integrity. The presence of CRISPR plasmids in the ALG NPs was confirmed by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy. The tests revealed that the nanoparticles were cytocompatible and successfully introduced the Cas9 transgene in HepG2 cells. Nanoparticles-transfected HepG2 was able to edit its target plasmid by introducing double-strand break (DSB) in GFP gene, indicating the bioactivity of CRISPR plasmids encapsulated in alginate nanoparticles. This suggests that this method is suitable for biomedical application in vitro or ex vivo. Future investigation of theses nanoparticles might result in nanocarrier suitable for in vivo delivery of CRISPR/Cas9 system.