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.

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The objective of this research is to design a reverse transfection system with cationized gelatin nanospheres (cGNS) incorporating a molecular beacon (MB) to visualize a cell function. The cGNS were prepared by the conventional coacervation method. The MB as an imaging probe was incorporated into the cGNS to prepare imaging complexes (cGNS MB ). The conventional transfection of 2D culture was performed by incubating MC3T3 cells in the medium containing cGNS MB . The reverse transfection was done by incubating cells on the substrate which had been precoated with both gelatin and cGNS MB . Significantly higher internalization efficiency and fluorescence intensity of cGNS MB were observed in the reverse transfection system than in the conventional one. To apply this system for visualization of 3D cell aggregate, gelatin microspheres (GMS) were prepared, while cGNS MB were bound on the GMS to prepare the GMS-cGNS MB of a cell scaffold. Then the cells were incubated with GMS-cGNS MB to form 3D cell aggregates. On the other hand, as a control, the conventional transfection of 3D culture was performed by incubating the cell aggregates formed with the medium containing cGNS MB . Homogeneous fluorescence of MB from the inside to the outside of aggregates was observed for the reverse transfection group. However, for the conventional transfection, the fluorescence was observed only around the surface of cell aggregates. It is concluded that the reverse transfection system with cGNS incorporating MB is promising to visualize the cell function of a higher transfection efficiency for the 2D culture and in a homogeneous manner for the 3D culture.