Intracellular Controlled Release of Molecular Beacon Prolongs the Time Period of mRNA Visualization

The objective of this study is to prepare cationized gelatin nanospheres incorporating molecular beacon (MB) (cGNS_MB) and a cationized gelatin-molecular beacon complex, and evaluate the time period of messenger RNA (mRNA) visualization. The cGNS with different degradabilities were prepared to incorporate MB. There was no difference in the apparent size and zeta potential between the cGNS_MB and the complex, while the MB release from the complex was faster than that from the cGNS_MB. When mouse mesenchymal stem cells were incubated with the complex and cGNS_MB, the amount of MB internalized into cells and cytotoxicity of complex were higher compared with cGNS_MB. However, in the amount range of noncytotoxicity, the amount of MB internalized was at a similar level among them. The intracellular fluorescence of cGNS_MB was observed over 14 days, whereas that of complex disappeared within 5 days. Moreover, the time period of cGNS_MB remaining in the cells prolonged with the increase of glutaraldehyde amount used in cGNS_MB preparation. As the result, it is likely that the intracellular fluorescence was retained at a high level for longer time periods. It is concluded that the intracellular controlled release of MB is promising, to achieve long-term and continuous visualization of mRNA. Impact Statement Molecular beacon (MB) is a versatile activatable imaging probe to detect messenger RNA (mRNA). Cationized gelatin nanospheres incorporating MB (cGNS_MB) with different degradabilities and a cationized gelatin-MB complex were prepared. The intracellular fluorescence of cGNS_MB was observed over 14 days, whereas that of complex disappeared within 5 days. This is because the intracellular decrease of cGNS_MB was slower than that of complex. It is concluded that the intracellular controlled release of MB is promising, to achieve long-term and continuous visualization of mRNA.