In Situ Induction Of Nucleolin Cluster By DNA-Au Nanodevices On Tumor Cell Surface For Regulating Apoptosis

Cell surface receptors are molecules that sense extracellular signal ligands and mediate transmembrane signal transduction to manipulate downstream intracellular signals and further affect the physiological and pathological states of cells.Cell behaviors such as growth factor signal transduction,immune system function,cell communication,cell adhesion,cell migration,cell activation,and apoptosis are all regulated by the cell surface receptor clusters.As a typical receptor,nucleolin is involved in many signaling pathways related to cell proliferation,cell cycle,apoptosis and overexpression on the cell surface of tumor cells.It is also closely related to the occurrence and development of tumors and has become an important target for cancer diagnosis and treatment.The regulation of apoptosis by the cluster of nucleolin on the cell surface may provide a new way for tumor treatment,and is expected to provide a basis for the research and development of anti-tumor drug targets.It has been reported that a number of strategies involving molecular nanomodels,magnetic drives,polymers,and DNA nanostructures have been developed to control cell surface receptor aggregation and thus regulate cell fate.Now receptor aggregation regulation mostly depends on the external stimulus,such as light,heat,magnetic fields,etc.,which may cause damage in normal tissue.Therefore,it is of great significance to develop novel receptor aggregation strategies that can effectively kill tumor cells while reducing the damage to normal tissues.Tumor microenvironment(TME),as an important marker to distinguish tumor cells from normal cells,has gradually received clinical attention.Abnormal factors in the tumor microenvironment,such as hypoxia,high concentration of ATP,low extracellular p H,and abnormal expression of tumor-associated enzymes,are thought to be characteristics of cancer,which also play an important role in receptor aggregation.Therefore,in situ application of the tumor microenvironment to trigger receptor aggregation promises to provide a new and powerful means for targeted therapy.Gold nanoparticles(Au NPs)exhibit the advantages of high stability,easy modification,good biocompatibility,large specific surface area,and unique local surface plasmonic resonance(LSPR)absorption and scattering.Meanwhile,DNA nanomaterials display good biocompatibility and editable ability.Therefore,in this thesis,Au NPsAS1411-apt nanodevice and AI-Au intelligent nanodevice were designed and constructed for targeting cancer cell surface by the specific binding between the AS1411 aptamer and nucleolin.Most importantly,the as-prepared nanodevice can induce the clusters of nucleolin on the surface of cells to promote apoptosis of cancer cells.The contents of the research are as follows:1.In situ activation of nucleolin clusters triggered by the high ATP concentrations on the surface of tumor cells for cell apoptosisIn this study,Au NPs-AS1411-apt nanodevices were constructed by connecting NCL aptamer AS1411 to targeted cancer cells and ATP splitting aptamer to Au NPs using Au-S bonds.By exploiting the specific binding of a high concentration ATP on the surface of tumor cells to its cleaved aptamer,Au NPs cross-linked to induce the aggregation of NCL receptors on tumor cell surface,thereby enabling apoptosis regulation.Au NPs-AS1411-apt was successfully constructed and characterized using UV-Vis absorption spectroscopy and zeta potential.In addition,the results of UV-Vis absorption spectrum,LSPR scattering spectrum and dark field microscopic(DFM)imaging demonstrated that ATP could induce the aggregation of Au NPs-AS1411-apt.Subsequently,the stability experiments have shown that Au NPs-AS1411-apt has good serum stability and nuclease stability.Finally,cytotoxicity experiments demonstrated that Au NPs-AS1411-apt had good biocompatibility with normal L-02 cells and displayed the highest cytotoxicity on MCF-7 cells,which was enhanced with the increase of ATP concentration.CalceinAM/PI staining showed that the Au NPs-AS1411-apt nanodevice inhibited MCF-7 cells more significantly with the increasing ATP concentration.Nuclear staining also confirmed its apoptotic effect.All the above results indicated that Au NPs-AS1411-apt had good stability and suppression of cell activity.In addition,we also investigated the Cyt-c/caspase-3 apoptosis pathway,which suggested that the Au NPs-AS1411-apt nanodevice-induced apoptosis may have an endogenous mitochondrial pathway.2.In situ activation of nucleolin clusters triggered by the tumor extracellular acidic environment on the tumor cell surface for cell apoptosisWe further modified the NCL aptamer AS1411 and acid-responsive DNA sequence(I-strand)onto Au NPs surface to construct AI-Au smart nanodevices targeting tumor cell surface.The slightly acidic environment of tumor cells was used to trigger the I-strand on Au NPs surface to form an i-Motif structure,making two or more adjacent AI-Au smart nanodevices on the cell surface to form cross-linking or aggregation states,thereby in situ inducing the aggregation of NCL receptors and triggering the apoptosis of tumor cells.LSPR absorption and scattering spectra,hydration particle size,and SEM results showed that when the p H of solution decreased gradually(7.4,6.5,6.0,5.0),the aggregation degree of AI-Au intelligent nanodevices gradually increased.DFM imaging confirmed that AI-Au nanodevice mediated nucleolin cross-linking at the cell surface,resulting in approximately 60% cytotoxic effects.Experiments such as Calcein-AM/PI staining,nuclear dye staining,and flow cytometry demonstrated that as acidity increased,cell apoptosis became more evident.Immunofluorescence imaging further confirmed the Cytc/caspase-3 apoptotic pathway induced by the AI-Au nanodevice.By taking advantage of the unique properties of cancer cells’ slightly acidic microenvironment,the proposed strategy is able to achieve specific cancer cell apoptosis by in situ activation of tumor cell membrane receptor aggregation.In summary,Au NPs-AS1411-apt nanodevice and AI-Au smart nanodevice were developed on the cell membrane by using the surface microenvironment of tumor cells(high concentration of ATP and slightly acidic environment)to in situ induce nucleolin clusters on the cell surface and achieve specific tumor cell apoptosis study.This strategy avoided the damage to normal cells,was simple to operate and highly versatile.More importantly,it provided a basis for the research and development of new drugs and was expected to become a promising new means of cancer treatment.