DNA Nanoribbon Mediated In Situ Synthesis Of Metal Nanoclusters In Cells And Fluorescence Imaging

Cancer is one of the most life-threaten diseases today,therefore its accurate diagnosis is vital for human health and life.In recent years,the application of nanotechnology and biocompatible nanomaterials has facilitated cancer diagnosis.Researchers have taken advantage of the unique microenvironment of tumours（low p H,high GSH content and ROS levels）to self-assemble in situ fluorescent metal nanoclusters to localize tumour cells,meanwhile,DNA nanostructures with good modifiability can be used as templates in the diagnosis and treatment of tumour cells.However,the use of DNA Nanoribbons（DNRs）as templates for the synthesis of DNA Nanoribbons/Metal Nanoclusters（DNRs/MNCs）in the tumour microenvironment for cancer diagnosis has not yet been reported.In this experiment,DNR/GNCs complexes（DNA Nanoribbons/Gold Nanoclusters）and PS-DNR/AgNCs complexes（Phosphorothioate Modified DNA Nanoribbons/Silver Nanoclusters）were synthesized by in situ self-assembly using the tumour microenvironment,and their chemical and biological characteristics were studied.Laser confocal microscopy results indicated that the two complexes could form selective imaging for tumour cells with enhanced optical properties than conventional metal nanoclusters.Both complexes had good biocompactibility as verified by cytotoxic test.The main experimental results are as follows.（1）In situ synthesis and fluorescence imaging of DNR/GNCs complexesUsing DNR as a template,the DNR/GNCs complexes was synthesized in situ in cancer cells by adding HAu Cl₄ solution.Chemical characterization of DNR/GNCs complexes was performed after lysis of MCF 7 cell lines.The excitation wavelength,emission wavelength,and UV absorption peak of DNR/GNCs complexes were determined to be 455 nm,530 nm,and 450 nm by means of fluorescence and UV absorption spectroscopy experiments.Atomic force microscopy（AFM）and transmission electron microscopy（TEM）studies showed that the height and width of the DNR/GNCs complexes were 4.29 nm and 2.43 nm,respectively.The presence of Au⁰,Au⁺and Au³⁺with a molar ratio of 1:2:8 in the DNR/GNCs complexes was validated by high-resolution transmission electron microscopy（high-resolution TEM）,X-ray diffraction（XRD）and X-ray photoelectron spectroscopy（XPS）.The above characterisation demonstrated the successful preparation of DNR/GNCs complexes.In three selected cancer cell lines（MCF 7,HCT 116 and Hep G2）,DNR/GNCs complexes presented higher fluorescence intensity than that of the control samples（NC,DNR,GNCs,GNCs-ds DNA）as observed by laser confocal microscopy,indicating the successful in-situ synthesis of fluorescent DNR/GNCs complexes in the cancer cells.Among these three cancer cell lines,MCF 7 cell lines showed the strongest fluorescence intensity,demonstrating that DNR/GNCs complexes were selective to the tumour microenvironment,with MCF7 being the favorable cell.Results of fluorescence intensity and long-term cell tracking experiments demonstrated that the DNR/GNCs complexes had a long retention time and could be used as a stable biofluorescent labelling probe for cellular localisation.cells containing DNR/GNCs complexes were proved to be safe by the Cytotoxicity assays.（2）In situ synthesis and fluorescence imaging of PS-DNR/GNCs complexesThe PS-DNR/AgNCs complexes was synthesized in situ by adding Ag（GSH）⁺complexes in cancer cell lines using PS-DNR as a template.The fluorescence spectra and UV absorption spectra of PS-DNR/AgNCs complexes were recorded after lysis of MCF 7 cell lines lines,and the excitation wavelengths were determined to be 285 nm,365 nm and 440 nm,emission wavelength 535 nm and UV absorption peak 410 nm.TEM revealed that PS-DNR had a width of 1.92 nm.The results of high-resolution TEM and XRD showed that both Agand Ag₂O were present in the PS-DNR/AgNCs complexes;the results of energy dispersive XPS showed that the ratio of PS-DNR monomer to Agin the PS-DNR/AgNCs complexes was9.5:1（208\22=9.5）.The above characterisation demonstrated the successful preparation of PS-DNR/AgNCs complexes.The fluorescence intensity of the PS-DNR/AgNCs complexes in MCF 7 cell lines was higher than that of the control（NC,AgNCs）under laser confocal microscopy,suggesting that the fluorescent PS-DNR/AgNCs complexes has been synthesized in situ in MCF 7 cell lines.The MCF 7 cell lines containing PS-DNR/AgNCs were incubated,and the evolution of fluorescence was followed by laser confocal microscopy.The results showed that PS-DNR enhanced the fluorescence of AgNCs and prolonged the retention time of AgNCs in cell lines.Cytotoxicity assay indicated that PS-DNR/AgNCs complexes was not toxic to cell lines.Fluorescence intensity analysis of 968 compound after treatment of cell lines showed that968 compound could inhibit the intracellular p H induced the preparation of PS-DNR/AgNCs complexes.