Study Of Layered Double Hydroxide Nanosheets As Transmembrane Carrier For Biomolecules Into Cells

Plant cell membranes are enclosed by dense cell walls,which provide mechanical support for cells,but also hinder the entry of exogenous substances into plant cells.In the study of plant molecular and cellular biology,the process of introducing bioactive molecules is also hindered by plant cell wall.To this end,researchers have developed a variety of techniques for introducing exogenous biological molecules into plant cells.However,these technologies are difficult to be widely used because it is hard to achieve broad-spectrum,high efficiency,low toxicity and economy.In this study,two kinds of hydrotalcite-like nanosheets(Layered Double Hydroxide Nanosheets,LDH-NS)were synthesized by using the method of material science,namely,lactate-intercalated LDH-NS(LDH-Lactate-NS)and acetate-intercalated LDH-NS(LDH-Acetate-NS).The structure,size and surface morphology of LDH-NS were analyzed via a series of characterizations such as atomic force microscope imaging and scanning electron microscope imaging.The X ray diffraction,spectrophotometer and gel electrophoresis imaging were used to explore the ability of LDH-NS to adsorb DNA molecules.The results show that the adsorption capacity of LDH-Lactate-NS nanosheets is higher than that of LDH-Acetate-NS nanosheets,and still maintain a good delaminated state after the adsorption of DNA molecules.In addition,the effect of adsorption time on the ability of LDH-NS to adsorb DNA molecules was analyzed and compared.The results showed that the adsorption rate of LDH-Lactate-NS was higher than that of LDH-Acetate-NS.After 293T cells were treated with cell culture medium containing different concentrations of nanosheets,Hoechst 33342/PI double stained cells were sorted by flow cytometry.It was found that LDH-Lactate-NS and LDH-Acetate-NS nanosheets did not produce serious cytotoxicity,and LDH-Lactate-NS nanosheets had lower cytotoxicity.Guided by the experimental data above,the ability of LDH-Lactate-NS nanosheets as biomolecular carriers to transport DNA molecules into target cells was explored.Firstly,LDH-Lactate-NS@ssDNA-FITC complex was obtained by co-incubation method,and the intracellular signal was detected by laser confocal microscopy after the treatment of cells with the complex.The results showed that LDH-Lactate-NS nanosheets could efficiently transport DNA molecules into 293T cells.In this study,model plant tobacco cells and Arabidopsis thaliana were used to investigate the toxicity of LDH-Lactate-NS on plant cells.Tobacco cells and Arabidopsis thaliana were treated with plant cell culture medium containing different concentrations of nanosheets.It was found that the cytotoxicity of LDH-Lactate-NS nanosheets to tobacco cells and Arabidopsis thaliana was not significant when the concentration of LDH-Lactate-NS nanosheets was lower than 100 ?g/mL.Based on the experimental data above,LDH-Lactate-NS nanosheets coupled with different biological molecules were used to treat tobacco cells and Arabidopsis thaliana,then the laser confocal microscopy was used to detect intracellular signals.The results showed that LDH-Lactate-NS nanosheets could effectively load different biological molecules into intact plant cells.Finally,the transmembrane mechanism of LDH@DNA complex entering into target cells through direct penetration was revealed by means of plant cell biology experiment and dissipative particle dynamics simulation technology,and the effective penetration structure of this complex was found to be as DNA-LDH-DNA.In conclusion,this study confirmed that LDH-Lactate-NS can be used as a biomolecular carrier in plant molecular and cytological research by means of combining material science,plant cytology and computer science,and provided a theoretical basis for further improving the ability of LDH-Lactate-NS to transport biomolecules into intact plant cells.