Critical Evaluation of Different Lysosomal Labeling Methods Used to Analyze RNA Nanocarrier Trafficking in Cells.

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.

Polymer nanocarriers for MicroRNA delivery.

Abnormal expression of microRNAs (miRNAs), which are highlyconserved noncoding RNAs that regulate the expression of various genes post transcriptionally to control cellular functions, has been associated with the development of many diseases. In some cases, disease-promoting miRNAs are upregulated, while in other instances disease-suppressive miRNAs are downregulated. To alleviate this imbalanced miRNA expression, either antagomiRs or miRNA mimics can be delivered to cells to inhibit or promote miRNA expression, respectively. Unfortunately, the clinical translation of bare antagomiRs and miRNA mimics has been challenging because nucleic acids are susceptible to nuclease degradation, display unfavorable pharmacokinetics, and cannot passively enter cells. This review emphasizes the challenges associated with miRNA mimic delivery and then discusses the design and implementation of polymer nanocarriers to overcome these challenges. Preclinical efforts are summarized, and a forward-looking perspective on the future clinical translation of polymer nanomaterials as miRNA delivery vehicles is provided.