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.

A Reverse Transfection System with Cationized Gelatin Nanospheres Incorporating Molecular Beacon as a Tool to Visualize Cell Function

“…Thus, controlled release of MB is regulated only by changing the degradation nature of cGNS. cGNS as the carrier material to internalize MB for the visualization of cell functions has been reported. − ,, Recently, it was reported that PLGA nanoparticles associated with Ki67 MB were available to detect the Ki67 mRNA. The PLGA nanoparticles are promising as the carrier of MB, although the degraded products may be cytotoxic compared with cGNS .…”

“…Another benefit of gelatin nanospheres as a carrier material enables the imaging probe of MB to achieve the controlled release and prolong the imaging time of MB. It is reported that incorporation into cGNS enables MB to visualize cell functions, such as cell proliferation, 22,23 autophagy, 24 and energy metabolism, for a long time. 25 For the conventional transfection method, first cells are seeded on the culture dish, following by incubating in the culture medium containing the transfection complex.…”

“…Another benefit of gelatin nanospheres as a carrier material enables the imaging probe of MB to achieve the controlled release and prolong the imaging time of MB. It is reported that incorporation into cGNS enables MB to visualize cell functions, such as cell proliferation, , autophagy, and energy metabolism, for a long time …”

Live Imaging of Monocyte/Macrophage Differentiation with Cationized Gelatin Nanospheres Incorporating a Molecular Beacon

“…This indicates that MB of a negative charge is efficiently delivered to cells by using cGNS. Previous studies demonstrate that cGNS MB was able to visualize cellular functions such as apoptosis, cell proliferation, and autophagy . cGNS MB can also visualize the differentiation of monocytes into macrophages.…”

“…For example, the chemical modification of cholesterol, fatty acids, and polyamines has been reported. Cationized gelatin prepared by the chemical introduction of polyamines has been used as the starting material of nanospheres for the intracellular delivery of nucleic acids, such as pDNA, siRNA, , and MB. , The cationized gelatin can escape the lysosomal degradation by the proton sponge effect. , In this study, the nanospheres prepared from cationized gelatin (cGNS) are used as the intracellular delivery carrier of MB.…”