■由严重急性呼吸道综合症冠状病毒2(SARS-CoV-2)引起的全球大流行以及对公共卫生的挥之不去的威胁推动了对治疗SARS-CoV-2的有效疗法的寻找。这项研究旨在开发SARS-CoV-2进入的脂质纳米颗粒(LNP)抑制剂,以减少鼻子和上呼吸道的病毒感染。
按照微流体混合方法制备两种类型的LNP制剂。LNP陷阱由DOPC组成,DSPC,胆固醇,和各种刺突蛋白结合配体修饰的DSPE-PEG-COOH,包括ACE2肽,重组人ACE2(rhACE2)或刺突蛋白单克隆抗体(mAb)。LNP-Trim由电离阳离子DLin-MC3-DMA组成,DSPC,胆固醇,和包封siACE2或siTMPRSS2的DMG-PEG脂质。测定两种制剂在气道上皮细胞(Calu-3)中的生物相容性和细胞摄取。使用SARS-CoV-2刺突蛋白结合测定(LNP-Trap)进行活性的功能评估,宿主受体敲低(LNP-Trim),和SARS-CoV-2假病毒中和测定(LNP-Trap和LNP-Trim)。鼻内给药后在小鼠中评估荧光标记的LNP制剂的定位和组织分布。
两种LNP制剂基于在浓度高达1mg/mL的Calu-3细胞中的细胞阻抗和MTT细胞毒性研究是生物相容的。LNP-Trap制剂能够结合刺突蛋白并在Calu-3细胞中抑制假病毒感染达90%。LNP-Trim制剂在mRNA(70%减少)和蛋白质水平(50%减少)减少ACE2和TMPRSS2。用LNP-Trim处理的Calu-3细胞中宿主靶标的抑制导致超过90%的假病毒感染抑制。体内研究表明,在鼻给药后,LNP-Trap和LNP-Trim在鼻腔中的大量保留,全身暴露最少。
■LNP-Trap和LNP-Trim制剂都能够安全有效地抑制气道上皮细胞中的SARS-CoV-2假病毒感染。这些研究为上气道中SARS-CoV-2的局部治疗方法提供了原理证明。
UNASSIGNED: The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the lingering threat to public health has fueled the search for effective therapeutics to treat SARS-CoV-2. This study aimed to develop lipid nanoparticle (LNP) inhibitors of SARS-CoV-2 entry to reduce viral infection in the nose and upper airway.
UNASSIGNED: Two types of LNP formulations were prepared following a microfluidic mixing method. The LNP-Trap consisted of DOPC, DSPC, cholesterol, and DSPE-PEG-COOH modified with various spike protein binding ligands, including ACE2 peptide, recombinant human ACE2 (rhACE2) or monoclonal antibody to spike protein (mAb). The LNP-Trim consisted of ionizing cationic DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG lipids encapsulating siACE2 or siTMPRSS2. Both formulations were assayed for biocompatibility and cell uptake in airway epithelial cells (Calu-3). Functional assessment of activity was performed using SARS-CoV-2 spike protein binding assays (LNP-Trap), host receptor knockdown (LNP-Trim), and SARS-CoV-2 pseudovirus neutralization assay (LNP-Trap and LNP-Trim). Localization and tissue distribution of fluorescently labeled LNP formulations were assessed in mice following intranasal administration.
UNASSIGNED: Both LNP formulations were biocompatible based on cell impedance and MTT cytotoxicity studies in Calu-3 cells at concentrations as high as 1 mg/mL. LNP-Trap formulations were able to bind spike protein and inhibit pseudovirus infection by 90% in Calu-3 cells. LNP-Trim formulations reduced ACE2 and TMPRSS2 at the mRNA (70% reduction) and protein level (50% reduction). The suppression of host targets in Calu-3 cells treated with LNP-Trim resulted in over 90% inhibition of pseudovirus infection. In vivo studies demonstrated substantial retention of LNP-Trap and LNP-Trim in the nasal cavity following nasal administration with minimal systemic exposure.
UNASSIGNED: Both LNP-Trap and LNP-Trim formulations were able to safely and effectively inhibit SARS-CoV-2 pseudoviral infection in airway epithelial cells. These studies provide proof-of-principle for a localized treatment approach for SARS-CoV-2 in the upper airway.