Experimental Study Of Chiral Au/GQDs/Cys Self-assembled Particles For The Treatment Of Squamous Cell Carcinoma Of The Tongue

There are still many challenges to realize the treatment of tongue squamous cell carcinoma(Tongue Squamous Cell Carcinoma,TSCC)with high efficiency and low side effects.Chiral self-assembled inorganic nanomaterials have attracted attention in tumor therapy due to their unique size and surface morphology as well as the unique selectivity of chiral enantiomers in organisms.However,little is known about the therapeutic effects of chiral inorganic self-assembled materials with complex structures with abundant contact sites on tumor cells.In this study,by effectively constructing gold-graphene quantum dots-cysteine self-assembled particles(Au/GQDs/Cys,AGC),we explored its therapeutic effect on TSCC and initially explored its potential mechanism.The specific research contents are as follows:Research purposes:1.Using chloroauric acid,graphene quantum dots(Graphene Quantum Dots,GQDs),chiral cysteine(L/D-cysteine,L/D-Cys)to generate AGC chiral nanoparticles(Nanoparticles,NPs),by adding the surfactant Cetyltrimethyl Ammonium Bromide(CTAB)to increase the electrostatic repulsion on the surface of the nanosheets,the AGC NPs self-assemble into a chiral self-assembled particle with a complex structure and Characterize its physical and chemical properties and structural properties.Further,by changing the content and reaction conditions of GQDs and chiral cysteine ligands,the morphology and size of chiral AGC self-assembly particles were artificially regulated,and a chiral AGC self-assembly system was constructed,using Graph Theory(GT)The method was used to evaluate the structural complexity of AGC,and a chiral AGC self-assembled particle with high structural complexity and suitable size was screened out for the treatment of tongue squamous cell carcinoma.2.Characterize the optical activity and chiral properties of chiral AGC,use density functional theory(DFT)and molecular dynamics(Molecular Dynamics,MD)to simulate and calculate,build a structural model,and explain the source of its chirality.At the same time,its colloidal stability,chemical stability,and cytotoxicity were studied to provide an experimental basis for the application of chiral AGC in the treatment of tongue squamous cell carcinoma.3.Using a variety of cell identification techniques to explore the ability of chiral AGC to induce apoptosis in tongue squamous cell carcinoma Cal-27 cells and to explore its potential mechanism.4.To establish a tumor model of tongue squamous cell carcinoma in nude mice,and to intervene by orthotopic injection of chiral AGC to further verify its ability to treat tongue squamous cell carcinoma and its biological safety.Research methods:1.Using the self-assembly method to synthesize the chiral AGC self-assembly system,through transmission electron microscope(Transmission Electron Microscope,TEM),ultraviolet-visible absorption spectrum(Ultraviolet-Visible Absorbance Spectroscopy,UV-vis),fluorescence spectrum(Fluorescence Spectrum,FL),scanning electron microscope(Scanning Electron Microscope,SEM),energy spectrometer(Energy Dispersive Spectrometer,EDS),Zeta potential(Zeta potential),X-ray diffraction(X-ray Diffraction,XRD),X-ray photoelectron spectroscopy(X-ray Photoelectron Spectroscopy,XPS),Confocal Laser Scanning Microscope(CLSM),Circular Dichroism(CD),Circularly Polarized Luminescence(CPL)morphology and other characterization methods,The morphology,size,material structure,elemental composition,surface charge,optical activity,and chiral properties of the chiral AGC self-assembly system were characterized.According to the summarized rules,multiscale and multi-morphological chiral AGC self-assembled particles were prepared,and the complexity factor(Complexity Index,CI)was calculated by GT method,and the degree of structural complexity was quantitatively evaluated.2.By dispersing AGC in various polar or non-polar solvents such as methanol,dimethyl sulfoxide,toluene,and different p H solutions,the chemical and colloidal stability of AGC was detected.Use DFT and MD to simulate the assembly behavior of the chiral AGC self-assembly system,analyze the interaction between components such as coordination bonds,hydrogen bonds,electrostatic repulsion,etc.,and explore its stable combination mode and chiral source.3.Evaluate the cytotoxicity and Killing and apoptotic effects on tongue squamous cell carcinoma Cal-27 cells.4.Subcutaneously inject Cal-27 cells into nude mice to establish tumor models,and intervene by orthotopic injection of Saline,L-AGC,and D-AGC in different groups of tumors.During the intervention period,the growth of nude mice,the change of tumor volume and the final tumor mass were recorded,the morphology of tumor cells was observed by HE staining and the apoptotic body produced by tunel fluorescence experiment was evaluated.Finally,the main organs of nude mice(heart,Liver,spleen,lung,and kidney)were stained with HE,and their cytological morphology was observed to evaluate the biological safety of the chiral AGC selfassembly system.Research result:1.The characterization results of TEM,UV-vis,SEM,Zeta potential,XRD,XPS,CD,and CPL indicated that chiral AGC self-assembled particles were successfully prepared.In addition,using the rules found in the preparation process,further prepared chiral AGC self-assembled particle systems with particle sizes of 652.5 nm,787.5 nm,1020 nm,2081.9 nm,2675 nm,and 7.2 μm from submicron to micron,And use the method of graph theory to evaluate the complexity of its structure.The calculated CI values are 20.5,40.17,50.5,81.2,83,and 36 respectively.The chiral AGC self-assembled particles with complex morphology provide a research basis for the effective treatment of tongue squamous cell carcinoma.2.The results of dispersion experiments of chiral AGC self-assembled particles in different polar or non-polar solvents and various p H solutions prove their good colloidal and chemical stability.The CLSM and FL characterization results show that there is a large Stokes shift between the optimal excitation wavelength and the maximum emission wavelength of AGC.CD and CPL results show that AGC has multiple optical activities and strong chiral optical response.The maximum intensity of chiral asymmetry factor g-factor is 2.9×10-3,and the maximum intensity of chiral luminescence asymmetry factor glum is 0.06.DFT and MD successfully simulated the self-assembly process and crystal structure of chiral AGC and explained that the chirality comes from the distortion of the gold atom plane in the unit cell.3.The CCK-8 experiment verified that the chiral AGC self-assembly system had good biological safety and showed selectivity to Cal-27 cells.The concentration of150 μg/m L was selected and cultured for 48 h as the subsequent reaction conditions.The results of Hoechst33258 apoptosis staining experiment and FITC/PI cell flow cytometry test showed that AGC has an excellent ability to induce apoptosis of Cal-27 cells,and the apoptosis rate of 1 μm L-AGC was as high as 60%,which was significantly higher than that of other sizes of hands.sex AGC.The results of Western Blot experiments showed that chiral AGC-induced apoptosis of tongue squamous cell carcinoma cells Cal-27 was related to the increased expression of apoptosis-related proteins Caspase-3 and Caspase-9.4.The tongue squamous cell carcinoma tumor model of nude mice was successfully established.The weight change of nude mice and the HE staining results of important organs(heart,liver,spleen,lung,kidney)of nude mice during chiral AGC intervention showed that chiral AGC has superior biosafety.At the same time,the volume change record of the tumor body and the results of HE staining and tunel fluorescence staining verified that chiral AGC can effectively treat TSCC.The tumor inhibition rate of L-AGC was as high as 39.0%,and that of D-AGC was 15.2%.Enantioselective,and the mechanism for the treatment of TSCC is the induction of tumor cell apoptosis.Research conclusion: The chiral AGC self-assembly system not only exhibits excellent chemical stability and colloidal stability,but also has certain chiral fluorescence activity and biological safety,and AGC with a particle size of 1 μm has shown anti-inflammatory effects on tongue squamous cell carcinoma cells.Its specificity and enantioselectivity can efficiently induce apoptosis of tongue squamous cell carcinoma Cal-27 cells,and it has a good ability to inhibit TSCC in nude mouse tumor models.The underlying mechanism is related to the increase of apoptosisrelated protein Caspase-9 is related to the expression level of Caspase-3.