RNA interference (RNAi) has an unprecedented potential as a therapeutic strategy for reversibly silencing the expression of any gene. Therapeutic delivery of the RNAi mediator, i.e., small interfering RNA (siRNA), can be used to address diseases characterized by gene overexpression, for example inflammatory conditions like chronic obstructive pulmonary disease (COPD). Macrophages play a key role in COPD pathogenesis and are recruited to the airways and lung parenchyma, where they release proinflammatory cytokines, e.g., tumor necrosis factor-alpha (TNF-α). Hence, targeting TNF-α with siRNA is a promising therapeutic approach for COPD management. However, a safe and effective delivery system is required for delivery of TNF-α siRNA into the cytosol of hard-to-transfect macrophages. The purpose of this study was to optimize the intracellular delivery of TNF-α siRNA to the lipopolysaccharide-activated murine macrophage cell line RAW 264.7 using lipidoid-polymer hybrid nanoparticles (LPNs) composed of the lipid-like transfection agent lipidoid 5 (L5) and the biodegradable polymer poly (D,L-lactide-co-glycolide). Applying a quality-by-design approach, the influence of critical formulation variables, i.e., the L5 content and the L5:siRNA ratio (w/w), on critical quality attributes (CQAs) was investigated systematically using risk assessment and design of experiments, followed by delineation of an optimal operating space (OOS). The CQAs were identified based on the quality target product profile and included size, polydispersity index, zeta potential, encapsulation efficiency and loading for achieving efficient and safe TNF-α gene silencing in activated RAW 264.7 cells. Formulations inducing efficient gene silencing and low cytotoxicity were identified, and the optimal formulations displayed L5 contents of 15 and 20% (w/w), respectively, and an L5:siRNA weight ratio of 15:1. All tested formulations within the OOS mediated efficient and sequence-specific TNF-α gene silencing in RAW 264.7 cells at TNF-α-siRNA concentrations, which were significantly lower than the concentrations required of non-encapsulated TNF-α-siRNA, highlighting the benefit of the delivery system. The results also demonstrate that increasing the loading of siRNA into the delivery system does not necessarily imply enhanced gene silencing. This opens new avenues for further exploitation of LPNs as a robust platform technology for delivering TNF-α siRNA to macrophages, e.g., in the management of COPD.

中文翻译：

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使用类脂聚合物混合纳米颗粒优化治疗性抗炎 TNF-α siRNA 到活化巨噬细胞的细胞内递送

RNA 干扰 (RNAi) 作为一种治疗策略具有前所未有的潜力，可以可逆地沉默任何基因的表达。RNAi 介质，即小干扰 RNA (siRNA) 的治疗性递送可用于治疗以基因过度表达为特征的疾病，例如慢性阻塞性肺病 (COPD) 等炎症性疾病。巨噬细胞在 COPD 发病机制中起关键作用，并被募集到气道和肺实质，在那里它们释放促炎细胞因子，例如肿瘤坏死因子-α (TNF-α)。因此，用 siRNA 靶向 TNF-α 是一种很有前途的 COPD 治疗方法。然而，需要一个安全有效的传递系统来将 TNF-α siRNA 传递到难以转染的巨噬细胞的细胞质中。本研究的目的是使用由类脂质转染剂类脂质 5 (L5) 和类脂质聚合物混合纳米粒子 (LPN) 优化向脂多糖激活的鼠巨噬细胞系 RAW 264.7 的细胞内递送 TNF-α siRNA 和可生物降解的聚合物聚（D,L-丙交酯-共-乙交酯）。应用质量源于设计的方法，使用风险评估和实验设计系统地研究了关键配方变量，即 L5 含量和 L5:siRNA 比率 (w/w) 对关键质量属性 (CQA) 的影响，然后是划定最佳操作空间 (OOS)。CQAs 是根据质量目标产品概况确定的，包括尺寸、多分散指数、zeta 电位、在活化的 RAW 264.7 细胞中实现高效和安全的 TNF-α 基因沉默的封装效率和负载。确定了诱导有效基因沉默和低细胞毒性的制剂，最佳制剂显示 L5 含量分别为 15% 和 20% (w/w)，L5:siRNA 重量比为 15:1。在 TNF-α-siRNA 浓度下，OOS 中的所有测试制剂均介导了 RAW 264.7 细胞中有效且序列特异性的 TNF-α 基因沉默，该浓度显着低于非包封 TNF-α-siRNA 所需的浓度，突出了其益处的交付系统。结果还表明，增加 siRNA 在递送系统中的加载并不一定意味着增强的基因沉默。