Protein therapeutics are an expanding area for research and drug development, and lipid nanoparticles (LNPs) are the most prominent nonviral vehicles for protein delivery. The most common methods for assessing protein delivery by LNPs include assays that measure the total amount of protein taken up by cells and assays that measure the phenotypic changes associated with protein delivery. However, assays for total cellular uptake include large amounts of protein that are trapped in endosomes or are otherwise nonfunctional. Assays for functional delivery are important, but the readouts are indirect and amplified, limiting the quantitative interpretation. Here, we apply an assay for cytosolic delivery, the chloroalkane penetration assay (CAPA), to measure the cytosolic delivery of a (–30) green fluorescent protein (GFP) fused to Cre recombinase (Cre(–30)GFP) fusion protein by LNPs. We compare these data to the data from total cellular uptake and functional delivery assays to provide a richer analysis of uptake and endosomal escape for LNP-mediated protein delivery. We also use CAPA for a screen of a small library of lipidoids, identifying those with a promising ability to deliver Cre(–30)GFP to the cytosol of mammalian cells. With careful controls and optimized conditions, we expect that CAPA will be a useful tool for investigating the rate, efficiency, and mechanisms of LNP-mediated delivery of therapeutic proteins.

中文翻译：

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使用氯烷渗透测定研究脂质纳米颗粒对蛋白质的胞质递送

蛋白质治疗是研究和药物开发的一个不断扩大的领域，脂质纳米粒子（LNP）是最重要的蛋白质递送非病毒载体。评估 LNP 蛋白质递送的最常见方法包括测量细胞摄取的蛋白质总量的测定以及测量与蛋白质递送相关的表型变化的测定。然而，总细胞摄取的测定包括大量被内体捕获或以其他方式无功能的蛋白质。功能传递的测定很重要，但读数是间接的和放大的，限制了定量解释。在这里，我们应用细胞质递送测定，即氯烷渗透测定 (CAPA)，通过以下方法测量与 Cre 重组酶 (Cre(–30)GFP) 融合蛋白融合的 (–30) 绿色荧光蛋白 (GFP) 的细胞质递送： LNP。我们将这些数据与总细胞摄取和功能递送测定的数据进行比较，以提供对 LNP 介导的蛋白质递送的摄取和内体逃逸的更丰富的分析。我们还使用 CAPA 筛选小型类脂质库，识别那些具有将 Cre(–30)GFP 递送至哺乳动物细胞胞浆的有前景能力的类脂质。通过仔细的控制和优化的条件，我们预计 CAPA 将成为研究 LNP 介导的治疗性蛋白质递送的速率、效率和机制的有用工具。