Lokras Abhijeet, Thakur Aneesh, Wadhwa Abishek, Thanki Kaushik, Franzyk Henrik, Foged Camilla
Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Front Bioeng Biotechnol. 2021 Jan 14;8:601155. doi: 10.3389/fbioe.2020.601155. eCollection 2020.
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 (L) 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 L content and the L: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 L contents of 15 and 20% (w/w), respectively, and an L: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.
RNA干扰（RNAi）作为一种可逆性沉默任何基因表达的治疗策略，具有前所未有的潜力。RNAi介质即小干扰RNA（siRNA）的治疗性递送可用于治疗以基因过表达为特征的疾病，例如慢性阻塞性肺疾病（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，如肿瘤坏死因子-α（TNF-α）。因此，用siRNA靶向TNF-α是一种有前景的COPD治疗方法。然而，需要一种安全有效的递送系统将TNF-α siRNA递送至难以转染的巨噬细胞的胞质溶胶中。本研究的目的是使用由类脂质转染剂脂质体5（L）和可生物降解聚合物聚（D，L-丙交酯-共-乙交酯）组成的类脂质-聚合物杂化纳米颗粒（LPNs），优化TNF-α siRNA向脂多糖激活的小鼠巨噬细胞系RAW 264.7的细胞内递送。采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关键制剂变量即L含量和L:siRNA比率（w/w）对关键质量属性（CQAs）的影响，随后划定最佳操作空间（OOS）。基于质量目标产品概况确定CQAs，包括尺寸、多分散指数、zeta电位、包封率和载药量，以在活化的RAW 264.7细胞中实现高效且安全的TNF-α基因沉默。鉴定出诱导高效基因沉默和低细胞毒性的制剂，最佳制剂的L含量分别为15%和20%（w/w），L:siRNA重量比为15:1。在OOS内测试的所有制剂在TNF-α-siRNA浓度下介导RAW 264.7细胞中高效且序列特异性的TNF-α基因沉默，该浓度显著低于未包封的TNF-α-siRNA所需的浓度，突出了递送系统的优势。结果还表明，增加siRNA在递送系统中的载药量不一定意味着增强基因沉默。这为进一步开发LPNs作为将TNF-α siRNA递送至巨噬细胞的强大平台技术开辟了新途径，例如在COPD的治疗中。
