Department of Biomedical Engineering, University of Delaware, 161 Colburn Lab, Newark, Delaware 19716, United States.
Department of Materials Science & Engineering, University of Delaware, 201 DuPont Hall, Newark, Delaware 19716, United States.
Bioconjug Chem. 2021 Oct 20;32(10):2245-2256. doi: 10.1021/acs.bioconjchem.1c00405. Epub 2021 Sep 20.
The use of nucleic acids to regulate gene expression is a rapidly developing field with immense clinical potential. Nanomaterials are frequently used to deliver nucleic acids into cells as they can overcome the poor cellular uptake and endo/lysosomal degradation of bare nucleic acids. For these nanocarriers to be effective, they must escape endo/lysosomal compartments to deliver their nucleic acid cargo into the cytosol (for ribonucleic acid (RNA)) or nucleus (for deoxyribonucleic acid (DNA)). This process is poorly understood and remains an area of active research toward the goal of developing effective delivery strategies. Fluorescent endo/lysosomal markers are among the most widely employed tools used to evaluate the endosomal escape of nucleic acid nanocarriers. However, the endo/lysosomal labeling method may alter the extent of and route of nanocarrier uptake by cells. The impact of these markers on cellular function and cell-nanocarrier interactions has not been probed in a systematic manner. To investigate this, we compared the effects of several common lysosomal labeling methods, namely, LysoTracker Red (LT Red), transient lysosomal-associated membrane protein 1-mutant green fluorescent protein (LAMP1-mGFP) transfection (Transient GFP), and stable lentiviral LAMP1-mGFP transfection (Stable GFP), on cellular metabolic activity, nanocarrier uptake, nanocarrier/lysosomal label colocalization, and gene silencing potency in U87 glioblastoma and MDA-MB-231 breast cancer cells using polyethyleneimine (PEI)/ribonucleic acid (RNA) polyplexes as a model nanocarrier. In both U87s and MDA-MB-231s, Transient GFP and LT Red labeling reduced metabolic activity relative to untransfected (Parental) cells, while Stable GFP labeling increased metabolic activity. Congruently, flow cytometry indicates Stable GFP cells have greater polyplex uptake than LT Red-labeled cells in both cell lines. Despite these similar trends in uptake, polyplex intracellular trafficking differs in the two cell lines, as confocal imaging revealed greater polyplex/lysosome colocalization in Stable GFP U87 cells than LT Red-labeled U87 cells, while the trend was reversed in MBA-MB-231s. The level of RNA-mediated gene silencing achieved in Parental versus Stable GFP U87 and MDA-MB-231 cells agreed with the observed levels of polyplex/lysosome colocalization, supporting the established concept that endosomal escape is the rate-limiting step for RNA interference. These findings indicate that lysosomal labels can profoundly alter cellular function and cell-nanocarrier interactions, presenting critical new considerations for researchers investigating nanoparticle trafficking.
核酸在基因表达调控中的应用是一个具有巨大临床潜力的快速发展领域。纳米材料常用于将核酸递送入细胞，因为它们可以克服裸露核酸的细胞摄取和内体/溶酶体降解的问题。为了使这些纳米载体有效，它们必须逃离内体/溶酶体室，将其核酸货物递送至细胞质（对于核糖核酸 (RNA)）或细胞核（对于脱氧核糖核酸 (DNA)）。这一过程尚未被充分理解，仍是开发有效递送策略的一个活跃研究领域。荧光内体/溶酶体标记物是评估核酸纳米载体的内体逃逸最广泛使用的工具之一。然而，内体/溶酶体标记方法可能会改变细胞对内体摄取的程度和途径。这些标记物对细胞功能和细胞-纳米载体相互作用的影响尚未以系统的方式进行探究。为了研究这一点，我们比较了几种常见的溶酶体标记方法的效果，即 LysoTracker Red (LT Red)、瞬时溶酶体相关膜蛋白 1 突变型绿色荧光蛋白 (LAMP1-mGFP) 转染 (瞬时 GFP) 和稳定慢病毒 LAMP1-mGFP 转染 (稳定 GFP)，对 U87 神经胶质瘤和 MDA-MB-231 乳腺癌细胞的细胞代谢活性、纳米载体摄取、纳米载体/溶酶体标记物共定位以及聚乙烯亚胺 (PEI)/核糖核酸 (RNA) 多聚体作为模型纳米载体的基因沉默效力的影响。在 U87s 和 MDA-MB-231s 中，瞬时 GFP 和 LT Red 标记均使代谢活性相对于未转染（亲本）细胞降低，而稳定 GFP 标记则增加了代谢活性。同样，流式细胞术表明，在两种细胞系中，稳定 GFP 细胞的多聚体摄取量均大于 LT Red 标记的细胞。尽管摄取有这些相似的趋势，但多聚体细胞内转运在两种细胞系中有所不同，共聚焦成像显示，稳定 GFP U87 细胞中的多聚体/溶酶体共定位大于 LT Red 标记的 U87 细胞，而在 MDA-MB-231s 中则相反。在 Parental 与稳定 GFP U87 和 MDA-MB-231 细胞中实现的 RNA 介导的基因沉默水平与观察到的多聚体/溶酶体共定位水平一致，支持内体逃逸是 RNA 干扰的限速步骤这一既定概念。这些发现表明，溶酶体标记物可以深刻改变细胞功能和细胞-纳米载体相互作用，为研究纳米粒子转运的研究人员提出了重要的新考虑因素。
