Potential Method of Autophagy Imaging with Cationized Gelatin Nanospheres Incorporating Molecular Beacon.

Abstract

The objective of this research is to develop an imaging method with cationized gelatin nanospheres incorporating molecular beacon (cGNSMB) to visualize an autophagy activity in living cells. Cationized gelatin nanospheres (cGNS) were prepared by the conventional coacervation method, and then molecular beacon (MB) was incorporated into them. The cGNSMB prepared were internalized into cells at a high efficiency. In this study, a starvation medium of serum and amino acids-free was used to induce autophagy. The autophagy activity was confirmed by an immunofluorescence staining for microtubule-associated proteins light chain 3B (LC3B) of an autophagy specific protein. With the autophagy induction time, the number of LC3 fluorescent dots increased, which indicated an increased autophagy activity. As the autophagy-related genes, sequestosome 1 (SQSTM1) and cathepsin F (CTSF), which up-regulate after autophagy induction, were chosen as the targets of cGNSMB. The fluorescence intensity of cGNSMB targeting to SQSTM1 and CTSF increased with the starvation treatment time, which well corresponded with the gene expression results. When applied to cells in different autophagy conditions, the cGNSMB visualized the autophagy activity corresponding with the autophagy condition of cells. From the results obtained, it was concluded that the cGNSMB provide a promising method to visualize the autophagy of cells. The advantage of cGNSMB visualization is to obtain the temporal and spatial information without destroying sample cells.