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 (cGNSMB). The conventional transfection of 2D culture was performed by incubating MC3T3 cells in the medium containing cGNSMB. The reverse transfection was done by incubating cells on the substrate which had been precoated with both gelatin and cGNSMB. Significantly higher internalization efficiency and fluorescence intensity of cGNSMB 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 cGNSMB were bound on the GMS to prepare the GMS-cGNSMB of a cell scaffold. Then the cells were incubated with GMS-cGNSMB 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 cGNSMB. 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.
Keywords: cationized gelatin nanospheres; cell aggregates; imaging; molecular beacon; transfection technology.