PCFM Lab of Ministry of Education, School of Materials Science and Engineering, Sun Yat-sen University, Guangzhou 510275, P.R. China.
Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510275, P.R. China.
Sci Adv. 2020 Jul 15;6(29):eabb4005. doi: 10.1126/sciadv.abb4005. eCollection 2020 Jul.
Controlled release of CRISPR-Cas9 ribonucleoprotein (RNP) and codelivery with other drugs remain a challenge. We demonstrate controlled release of CRISPR-Cas9 RNP and codelivery with antitumor photosensitizer chlorin e6 (Ce6) using near-infrared (NIR)- and reducing agent-responsive nanoparticles in a mouse tumor model. Nitrilotriacetic acid-decorated micelles can bind His-tagged Cas9 RNP. Lysosomal escape of nanoparticles was triggered by NIR-induced reactive oxygen species (ROS) generation by Ce6 in tumor cells. Cytoplasmic release of Cas9/single-guide RNA (sgRNA) was achieved by reduction of disulfide bond. Cas9/sgRNA targeted the antioxidant regulator , enhancing tumor cell sensitivity to ROS. Without NIR irradiation, Cas9 was degraded in lysosomes and gene editing failed in normal tissues. The synergistic effects of Ce6 photodynamic therapy and gene editing were confirmed in vivo. Controlled release of CRISPR-Cas9 RNP and codelivery with Ce6 using stimuli-responsive nanoparticles represent a versatile strategy for gene editing with potentially synergistic drug effects.
CRISPR-Cas9 核糖核蛋白（RNP）的控制释放及其与其他药物的共递送仍然是一个挑战。我们在小鼠肿瘤模型中展示了使用近红外（NIR）和还原剂响应纳米粒子对 CRISPR-Cas9 RNP 的控制释放及其与抗肿瘤光敏剂氯乙酮（Ce6）的共递送。氮三乙酸（Nitrilotriacetic acid，NTA）修饰的胶束可以与带有组氨酸标签的 Cas9 RNP 结合。纳米粒子的溶酶体逃逸是由 Ce6 在肿瘤细胞中 NIR 诱导的活性氧（ROS）产生引发的。通过还原二硫键实现 Cas9/sgRNA 的细胞质释放。Cas9/sgRNA 靶向抗氧化调节剂，增强了肿瘤细胞对 ROS 的敏感性。没有 NIR 照射，Cas9 在溶酶体中被降解，正常组织中的基因编辑失败。体内证实了 Ce6 光动力疗法和基因编辑的协同作用。使用刺激响应性纳米粒子控制释放 CRISPR-Cas9 RNP 及其与 Ce6 的共递送，为具有潜在协同药物作用的基因编辑提供了一种多功能策略。
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