Critical evaluation of different lysosomal labeling methods used to analyze RNA nanocarrier trafficking in cells
Date
2021
Authors
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Publisher
University of Delaware
Abstract
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 RNA (ribonucleic acid)) or nucleus (for DNA (deoxyribonucleic acid)). This process is poorly understood and remains an area of active research towards 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 (LysoTracker Red, transient LAMP1-mGFP (lysosomal associated membrane protein 1-mutant green fluorescent protein) transfection, and stable lentiviral LAMP1-mGFP transfection) on cellular metabolic activity and nanocarrier uptake in U87 human glioblastoma cells using polyethylenimine (PEI)/RNA polyplexes as a model nanocarrier. We also examined the extent of nanocarrier/label colocalization. Transient transfection induces cytotoxicity as evidenced by reduced metabolic activity relative to untransfected U87s and U87s labeled with LysoTracker, while U87s stably expressing LAMP1-mGFP exhibit increased metabolic activity. Congruently, confocal imaging demonstrates that U87s stably transduced with LAMP1-mGFP exhibit increased polyplex uptake compared to U87s labeled with LysoTracker Red. These findings indicate that lysosomal labels can profoundly alter cellular function and cell-nanocarrier interactions, presenting critical new considerations for researchers investigating nanoparticle endo/lysosomal trafficking and escape.
Description
Keywords
Nanomedicine, Nucleic acid, Polyplex, RNAi, Trafficking, Uptake