Non-viral gene delivery is constrained by the dwell time that most synthetic nucleic acid nanocarriers spend inside endosomal compartments. In order to overcome this endosomal-release bottleneck, methods are required that measure nanocarrier uptake kinetics and transfection efficiency simultaneously. Here, we employ live-cell imaging on single-cell arrays (LISCA) to study the delivery-time distribution of lipid-based mRNA complexes under varied serum conditions. By fitting a translation-maturation model to hundreds of individual eGFP reporter fluorescence time courses, the protein expression onset times and the expression rates after transfection are determined. Using this approach, we find that delivery timing and protein expression rates are not intrinsically correlated at the single-cell level, even though population-averaged values of both parameters conjointly change as a function of increasing external serum protein fraction. Lipofectamine-mediated delivery showed decreased transfection efficiency and longer delivery times with increasing serum protein concentration. This is in contrast to ionizable lipid nanoparticle (i-LNP)-mediated transfer, which showed increased efficiency and faster uptake in the presence of serum. In conclusion, the interdependences of single-cell expression rates and onset timing provide additional clues on uptake and release mechanisms, which are useful for improving nucleic acid delivery.

中文翻译：

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基于脂质的转染中mRNA传递时间和蛋白质表达的相关性。

非病毒基因的传递受到大多数合成核酸纳米载体在内体区室中停留的停留时间的限制。为了克服这种内体释放瓶颈，需要同时测量纳米载体摄取动力学和转染效率的方法。在这里，我们采用单细胞阵列（LISCA）上的活细胞成像来研究在各种血清条件下基于脂质的mRNA复合物的递送时间分布。通过将翻译成熟度模型拟合到数百个单独的eGFP报告基因荧光时间过程，可以确定蛋白质表达开始时间和转染后的表达率。使用这种方法，我们发现在单细胞水平上，递送时机和蛋白质表达率并没有内在相关性，即使这两个参数的总体平均值随外部血清蛋白分数的增加而共同变化。脂质体介导的脂质转染显示随着血清蛋白浓度的增加，转染效率降低，交货时间延长。这与可电离的脂质纳米颗粒（i-LNP）介导的转移相反，后者在血清存在下显示出更高的效率和更快的摄取。总之，单细胞表达率和发作时间的相互依赖提供了关于摄取和释放机制的其他线索，这对于改善核酸的传递是有用的。脂质体介导的脂质转染显示随着血清蛋白浓度的增加，转染效率降低，交货时间延长。这与可电离的脂质纳米颗粒（i-LNP）介导的转移相反，后者在血清存在下显示出更高的效率和更快的摄取。总之，单细胞表达率和发作时间的相互依赖提供了关于摄取和释放机制的其他线索，这对于改善核酸的传递是有用的。脂质体介导的脂质转染显示随着血清蛋白浓度的增加，转染效率降低，交货时间延长。这与可电离的脂质纳米颗粒（i-LNP）介导的转移相反，后者在血清存在下显示出更高的效率和更快的摄取。总之，单细胞表达率和发作时间的相互依赖提供了关于摄取和释放机制的其他线索，这对于改善核酸的传递是有用的。