Fabrication Of HA/PEI-Functionalized Carbon Dots For Biomedicalapplications

Objective:Carbon dots,as a new type of carbon nano material,gain a considerable attention among people over the years.With fairly good biocompatibility,chemical stability and excellent fluorescence property,carbon dots are hence widely applied in the field of bioimaging,gene transfer,bio-sensors and so on.Based on the former research of our group,with hyaluronic acid and polyethyleneimine as precursors,they synthesized by hydrothermal carbonization a novel fluorescent carbon nanoparticles—HA-PEI CDs(HP-CDs)and added new functions to carbon dots apart from their superior physical and chemical properties.This subject systematically characterized the basic performance about HP-CDs,investigated its potential applications in bioimaging and gene transfer as well as its influence on cancer development.Methods:1.Hyaluronic acid and polyethyleneimine were used as raw materials to synthesize HP-CDs by hydrothermal carbonization method.Physical properties of HP-CDs were characterized by high-resolution transmission electron microscopy and X-ray diffraction.Their chemical properties were revealed by X-ray photoelectron spectroscopy and Fourier transformation infrared spectrometry while optical properties were demonstrated by ultraviolet visible spectrophotometer and fluorophotometer.2.By assay of CCK-8 and hemolysis,the biocompatibility of HP-CDs was detected.Laser scanning confocal microscope tested HP-CDs’ capability as fluorescent probes applied in bioimaging.3.Nucleic acid wrapping capability of HP-CDs with differential quality was demonstrated by gel retardation.HP-CDs’ protection ability for RNA was observed upon agarose gel electrophoresis.Laser scanning confocal microscope was used to demenstrating the transfection efficiency of HP-CDs with red fluorescent protein plasmid.4.The Human Protein Atlas was searched while He La and MCF-7 cells’ differential expression of CD44 were detected by q-PCR in order to ensure the cell line of high and low expression of CD44.CD44 m RNA expressing variations of the said which HP-CDs caused were revealed by q-PCR.By flow cytometry and q-PCR were used to analyze if HP-CDs target to cancer cells through hyaluronic acid receptor CD44-mediated endocytosis,which meanwhile revealed the targeting capability of HP-CDs.HP-CD’s influence upon emigration and invasion of cancer cells was demonstrated by Transwell,scratch test and westernblot.Results:1.Preparation and physical,chemical as well as optical properties of HP-CDs.(1)HP-CDs achieved its best carbonization effects and fluorescent properties when hyaluronic acid and polyethyleneimine were proportioned as 7:4 and heated for 4hours at 180℃.(2)HP-CDs had homogeneous dispersity and an appearance of almost a standard sphere with the size of about 2.25 nm,the phase structure of which was also amorphous.(3)HP-CDs,mainly consisted of carbon,oxgen and nitrogen,contained hydrophilic groups including hydroxyl,carboxy and amide and thus possessed extremely good water solubility.(4)HP-CDs presented excellent fluorescent property and optical stability.2.Biocompatibility and bioimaging capability of HP-CDs.(1)HP-CDs of different concentration(50-400μg/m L)proved no significant toxicity to He La,VSMC cells which had a survival rate of above 90%.Cells activity still remained above 90% after 300μg/m L HP-CDs co-cultured by 72 hours with the said cells.(2)Similarly,HP-CDs of all different concentrions(50-400μg/m L)caused no apparent hemolysis.(3)HP-CDs did not change the morphology of He La,instead they went into cytoplasm and emitted strongly bright green fluorescence under blue light excitation.3.Gene condensation and transfer capability of HP-CDs.(1)HP-CDs completely neutralized the negative charge of nucleic acid when they were 3 times the mass of targeted gene.Along with the increase of HP-CDs,the extent that HP-CDs/gene complexes transferring to cathod was strengthened and the growing bright fluorescence was visible under ultraviolet light.(2)HP-CDs protected RNA by reducing its degration.(3)HP-CDs,as effective spontaneous green fluorescent gene transfection agents,had successfully transferred red fluorescent protein plasmid.4.HP-CDs targeted to tumor cells and influenced the emigration and invation of them.(1)Based on searching of The Human Protein Atlas and the conclusion that the relative expression of CD44 in He La was about 122 times than that of MCF-7,He La was selected as CD44-high expression cell line while the opposite was MCF-7.The following experiments were conducted to examine the difference on the above-mentioned cell lines by HP-CDs.(2)HP-CDs caused obvious rise of CD44 m RNA expression level of the above two cell lines,among which the CD44 expression level of He La remained the most prominent,which also suggested that HP-CDs had the capability of binding CD44.(3)For He La,the uptake of HP-CDs showed more than that for MCF-7.By a mean of HA pretreatment,accumulation of HP-CDs for these two cells decreased differently,among which the HP-CDs accumulation in He La reduced more notably.These results further illustrated the special target between HP-CDs and CD44.(4)HP-CDs concentration-dependently inhibited the emigration and invasion of cancer cells,and down-regulated the expressions of MMP-2 and MMP-9.Conclusion:1.HP-CDs were successfully synthesized by simple hydrothermal carbonization of hyaluronic acid and polyethyleneimine.HP-CDs had excellent dispersity,good solubility,small uniform particles,phase structure of amorphous body as well as super fluorescent properties.2.HP-CDs had good biocompatibility and can be used for cell imaging.3.HP-CDs exhibited excellent capabilities of transmitting and protecting gene.4.HP-CDs can be intake into cells through hyaluronic acid receptor-mediated endocytosis,which demonstrated the capability of tumor targeting and inhibited emigration and invasion of tumor cells.