Convenient Monitoring System of Intracellular microRNA Expression during Adipogenesis via Mechanical Stimulus‐Induced Exocytosis of Lipovesicular miRNA Beacon

Abstract

Noninvasive investigation of microRNAs (miRNAs) expression, which is deeply related to biological phenomena such as stem cell differentiation, in culture soup is particularly useful for monitoring of stem cell differentiation without phototoxicity of living cells, especially when cell morphologies remain unchanged during differentiation. However, real-time detection of miRNA in culture soup is not recommended because of insufficient miRNA amounts in culture soup. In this study, a convenient method is introduced for real-time assessing intracellular miRNA in culture soup by using lipovesicular miRNA beacon (Lipo-mB) and mechanical stimulus-mediated exocytosis. Pipetting-harvest of culture soup induces exocytosis-secretion of fluorescence signal of Lipo-mB from cytoplasm into culture soup. To demonstrate this method, Lipo-mB is applied for monitoring of adipogenesis by analyzing the expression levels of various intracellular miRNAs, which are related to adipogenesis regulators. The fluorescence intensity profile of the culture soup is correlated with the quantitative reverse-transcription-polymerase chain reaction data and absorbance of Oil Red O staining. These results demonstrate that Lipo-mB can successfully monitor stem cell differentiation by sensing changes in miRNA expression from culture soup of living cells. Lipo-mB can be further developed as an accurate sensing system for analyzing subtle differences in genotype, even when changes in phenotype cannot be observed.

Nanoemulsion-Assisted siRNA Delivery to Modulate the Nervous Tumor Microenvironment in the Treatment of Pancreatic Cancer

“…We also found that the siRNA and PCX fluorescent signals were mostly localized at the tumor periphery in the EXO-1-treated animals (Figure H). The tumor accumulation and penetration of P@P EPs decreased significantly when we pretreated them with EXO-1, suggesting the importance of exocytosis in the observed findings . We propose that after endocytic vesicles pinch off from the plasma membrane and fuse with the endosome, P@P EPs can enter the endoplasmic reticulum or Golgi apparatus and may then leave the cell via vesicles related to the conventional secretion system.…”

Sensitive Plasmonic Detection of miR-10b in Biological Samples Using Enzyme-Assisted Target Recycling and Developed LSPR Probe

“…First, this system was applied to the cell-culture soup to verify the effectiveness of the real miRNA. An analysis of the miRNA in the culture soup or exosomes can be conducted to easily determine the intracellular miRNA expression levels in the cellular state. , To investigate the sensor performance in the biological sample, the Hs746t cell line was chosen based on their miR-10b expression level. As shown in Figure b, a distinguishable LSPR peak shift was observed for the miR-10b sensing system that was complementary to miR-10b in the culture soup of the Hs746T cells.…”

DNA-Based Nanostructures for Live-Cell Analysis

“…In one example, fluorophore-labeled recognition strands hybridized to short quencher-labeled strands were encapsulated in a liposomal core for microRNA imaging. 99 In another example, molecular beacons were encapsulated within a PLGA core for intracellular mRNA detection. 63 Other nanoparticles have also been explored; for example, in one study aptamer−switch probes for ATP detection were fixed in the porous network of polyacrylamide nanoparticles and delivered into cells.…”

“…This strategy is limited to the use of nanoparticles with dynamic, porous, or polymeric cores, most commonly liposome- or PLGA-based structures. In one example, fluorophore-labeled recognition strands hybridized to short quencher-labeled strands were encapsulated in a liposomal core for microRNA imaging . In another example, molecular beacons were encapsulated within a PLGA core for intracellular mRNA detection .…”