Preparation And Cell Imaging Of Rotenone Conjugated Gold Nanoclusters

The ability to visualize the uptake and biodistribution of pesticide inside cells contributes to understanding the action mechanism of pesticide. In this paper, the conjugates were synthesized through pesticide conjugated fluorescent gold nanoclusters. We also investigated its fluorescence imaging, cell cytotoxicity and cell uptake in Sf9 cells.Au-BSA(2.16 ± 0.40 nm) were prepared by using one-pot method while BSA acted as both a reductant and a stabilizer. Au-Gly-R( 2.93 ± 0.49 nm) conjugates were the first synthesized through glycine(Gly) and rotenone(R) functionalized Au-BSA. Photoluminescence emission spectra of Au-Gly-R conjugates showed the maximum emission peak at 496 nm when excited at 424 nm. The successful covalent linkage of Gly and R with Au-BSA was confirmed by using UV-visible and Fourier transform infrared transmission spectroscopy(FTIR). Thermogravimetric analysis(TGA) demonstrated the percent of Gly and R molecules in Au-Gly-R was 12.48%. The viability of Sf9 cells maintained above 80% when they had been incubated with Au-BSA, Au-R and Au-Gly-R up to the final gold concentration at 25 ?g m L-1 for 24 h. So we chose the final gold concentration of 25 ?g m L-1 to do other related biological experiments. In vitro cytotoxicity studies showed that 25 ?g m L-1 gold concentration of Au-Gly-R was low toxicity to cells and it was further employed as optical probe for active imaging of Sf9 cells in vitro. The cells treated with Au-BSA and Au-R did not exhibit the fluorescence under confocal laser scanning microscopy(CLSM). In contrast, strong blue fluorescence was observed in the Sf9 cell cytoplasm treated with Au-Gly-R. The dramatic difference demonstrated that the cellular uptake of Au-Gly-R was more than Au-BSA and Au-R in Sf9 cells and the presence of Gly on the surface of Au-BSA increased their cellular uptake by the Sf9 cells which were glycine dependent substantially. ICP-MS was performed after the incubation of Sf9 cells with Au-BSA, Au-R and Au-Gly-R at 25 ?g m L-1 gold concentration for 24 h. When Sf9 cells were incubated with Au-Gly-R, the gold content was 2.2 and 2.6 times higher than that when Sf9 cells were incubated with Au-BSA and Au-R, respectively. The results agreed with the fluorescent images examined by LSCM showing the enhanced cellular uptake of Au-Gly-R after Au-BSA modified by Gly. These results demonstrated the efficient internalization of Au-Gly-R in Sf9 cells.GSH acted as both a reductant and a stabilizer, and Au-GSH could be obtained under alkaline conditions. Au-GSH-R conjugates were synthesized through rotenone functionalized Au-GSH. Photoluminescence emission spectra of Au-GSH conjugates showed the maximum emission peak at 610 nm when excited at 500 nm. The successful covalent linkage of R with Au-GSH was confirmed by using UV-visible and FTIR. We incubated Sf9 cells with Au-GSH and Au-GSH-R at 25 ?g m L-1 gold concentration for 24 h, respectively. The cells were then analyzed using photoacoustic imaging and fluorescence imaging system. The cells treated with Au-GSH could generate photoacoustic signal and exhibit the fluorescence. In contrast, no photoacoustic signal and fluorescence was observed in the Sf9 cell treated with Au-GSH-R, which might be attributed to the weakened fluorescence intensity of Au-GSH-R. The results demonstrated that Au-GSH could be absorbed by the Sf9 cells as fluorescent probes.In this paper, the uptake and biodistribution of Au-Gly-R inside Sf9 cells was investigated. The visualization results demonstrated that the presence of Gly on the surface of Au NCs increased their cellular uptake by the Sf9 cells. In addition, The results of photoacoustic imaging demonstrated that Au-GSH could be absorbed by the Sf9 cells. All the results supported the conclusion that the Au NCs conjugates have great potential for cell imaging in insect cytology. And amino acid functionalized gold nanoclusters could increase the cellular uptake of pesticide conjugates, which contributes to understanding the action mechanism of pesticide in insect cell and providing a scientific basis for the theory of guided pesticides.