Alamoudi Abdullah A, Méndez Paula A, Workman David, Schätzlein Andreas G, Uchegbu Ijeoma F
UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK.
Department of Pharmaceutics, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
Biomedicines. 2022 Sep 3;10(9):2182. doi: 10.3390/biomedicines10092182.
Therapeutic gene silencing in the brain is usually achieved using highly invasive intracranial administration methods and/or comparatively toxic vectors. In this work, we use a relatively biocompatible vector: poly(ethylene glycol) star-shaped polymer capped with amine groups (4APPA) via the nose to brain route. 4APPA complexes anti- itchy E3 ubiquitin protein ligase (anti-ITCH) siRNA to form positively charged (zeta potential +15 ± 5 mV) 150 nm nanoparticles. The siRNA-4APPA polyplexes demonstrated low cellular toxicity (IC50 = 13.92 ± 6 mg mL) in the A431 cell line and were three orders of magnitude less toxic than Lipofectamine 2000 (IC50 = 0.033 ± 0.04 mg mL) in this cell line. Cell association and uptake of fluorescently labelled siRNA bound to siRNA-4APPA nanoparticles was demonstrated using fluorescent activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), respectively. Gene silencing of the ITCH gene was observed in vitro in the A431 cell line (65% down regulation when compared to the use of anti-ITCH siRNA alone). On intranasal dosing with fluorescently labelled siRNA-4APPA polyplexes, fluorescence was seen in the cells of the olfactory bulb, cerebral cortex and mid-brain regions. Finally, down regulation of ITCH was seen in the brain cells (54 ± 13% ITCH remaining compared to untreated controls) in a healthy rat model, following intranasal dosing of siRNA-4APPA nanoparticles (0.15 mg kg siRNA twice daily for 3 days). Gene silencing in the brain may be achieved by intranasal administration of siRNA- poly(ethylene glycol) based polyplexes.
大脑中的治疗性基因沉默通常通过高度侵入性的颅内给药方法和/或毒性相对较大的载体来实现。在这项研究中，我们使用了一种相对生物相容性较好的载体：通过鼻脑途径给药的胺基封端的聚乙二醇星形聚合物（4APPA）。4APPA与抗瘙痒E3泛素蛋白连接酶（抗ITCH）siRNA形成带正电荷（ζ电位为+15±5 mV）的150 nm纳米颗粒。siRNA-4APPA多聚体在A431细胞系中表现出低细胞毒性（IC50 = 13.92±6 mg/mL），并且在该细胞系中其毒性比Lipofectamine 2000（IC50 = 0.033±0.04 mg/mL）低三个数量级。分别使用荧光激活细胞分选（FACS）和共聚焦激光扫描显微镜（CLSM）证实了与siRNA-4APPA纳米颗粒结合的荧光标记siRNA的细胞结合和摄取。在A431细胞系中体外观察到ITCH基因的基因沉默（与单独使用抗ITCH siRNA相比下调65%）。在用荧光标记的siRNA-4APPA多聚体进行鼻内给药时，在嗅球、大脑皮层和中脑区域的细胞中观察到荧光。最后，在健康大鼠模型中，在鼻内给药siRNA-4APPA纳米颗粒（0.15 mg/kg siRNA，每日两次，共3天）后，在脑细胞中观察到ITCH下调（与未处理的对照组相比，ITCH残留54±13%）。通过鼻内给药基于聚乙二醇的siRNA多聚体可以实现大脑中的基因沉默。
