Anticancer Activities And Molecular Mechanisms Of Graphene Drug Candidates Targeting DNA Major Grooves

Chemotherapy is the most commonly used treatment of cancer,however,the multidrug resistance and metastatic progression of cancer often lead to the failure of chemotherapy.The development of nanotechnology provides a new platform for chemotherapy,but the construction of such platform is limited to drug deliever.Therefore,nanodrugs still face the disadvantages of high cost and complicated preparation process.Recently,graphene-based nanomaterials have attracted widespread research interests due to their unique ultra-thin atomic layer thickness and large conjugated two-dimensional planar structure.Although a large number of studies have been carried out on the biological effects of various graphene derivatives,no significant anticancer activity has been found.Herein,we explored the broad-spectrum anticancer activity,unique mechanism of action（DNA targeting mechanism）,and the application in tumor therapy of graphene-based nanomaterials.By means of the unique planar structure,low-dimensional effect,and rich surface chemical reactivity of graphene,we developed a large class of novel anticancer drugs.The obtained graphene quantum dots（GQDs）drugs exhibited excellent wide-spectrum anticancer activities,showing significant advantages in overcoming MDR,inhibiting tumor metastasis,and reducing side effects.In addition,we regulatate the optical properties of GQDs through control the nitrogen doping,successfully expanding the absorption of GQDs from visible to the second near-infrared region.By further assembling GQDs on the surface of 2D nanosheets,we achieved the deep-tissue photothermal/chemotherapy combination therapy（PCT）in the NIR-II region at the ultralow power density.This work is divided into five parts:1.Preparation and characterization of DNA major groove targeting GQD candidate anticancer drugs.GQD drugs were synthesized through the acid-catalyzed molecular fusion method using small-molecule julolidine as the precursor.The pharmacophore of GQD drugs is an alkaline nitrogen heterocyclic cluster that surrounds the periphery of the graphene plane frame,which mainly contains six-membered pyridine ring,five-membered pyrrole ring,and benzene ring.Amphiphilic GQD drugs quickly penetrate cell membrane and enter the nucleus,realizing targeted nuclear imaging via the strong affinity between GQDs and DNA.A series of molecular biochemical experiments confirmed that GQD drugs specifically bind to the major groove of DNA.DNA major groove targeting not only endows GQDs with unique anticancer activity,but also makes it have excellent DNA probe function.The fluorescence stability of GQDs are significantly higher than that of commercial small-molecule nucleic acid dyes.2.The broad-spectrum anticancer activity and molecular mechanism of DNA groove targeted GQD candidate drugs.MTT assay showed that GQD drugs possessed broad-spectrum anticancer activity with extremely low IC₅₀,approximately 1.5μg m L^-1（～0.3μM）.GQD drugs also exhibited highly anticancer activity in vivo through intratumoral injection or intravenous injection,which was higher than that of small-molecule anticancer drug DOX.At the molecular level,we demonstrated that GQD drugs could form a stable nano-interface inhibitory layer between DNA and nucleoproteins,leading to the desired multi-target anticancer activity.GQD drugs were high-efficiency dual inhibitors of Topo I and Topo II,which can cause DNA damage,apoptosis,and cell cycle disorders.Based on the WB and RT-PCR results,we then found that GQD drugs cause cell apoptosis through the caspase and Bcl-2 pathway.In vivo fluorescence imaging,histology measurements,blood analysis,and hemolysis assay confirmed that GQD drugs did not cause significant long-term toxicity.3.GQD drugs reversed the MDR of drug-resistant cells and its molecular mechanism.By comparing the cytotoxicity of three groups of drug-resistant cells and drug sensitive cells,it was found that GQD drugs exhibited obvious cytotoxicity for drug-resistant cells.Further study found that GQD drugs at ultralow concentrations can effectively reverse MDR.Specifically,for MCF-7/ADR and HCT-8/PTX drug-resistant cells,GQD drugs decreased the resistance index（RI）of DOX and PTX from 155.95 and88.41 to 0.84 and 3.29,respectively.At the molecular level,we verified that GQD drugs reverse MDR1-mediated drug resistance in a non-traditional way.GQD drugs down-regulated the expression of P-gp in drug-resistant cells by inhibiting the MDR1promoter activity,then realizing the MDR reversal of drug-resistant cells.4.GQD drugs blocked the metastasis of cancer cells and its molecular mechanism.By scratch and transwell assay,we first confirmed that GQD drugs inhibited cell migration and invasion of 4T1 cells.Bioluminescence images revealed that GQD drugs significantly inhibited lung metastasis of 4T1-Luc cells,obtaining an ultrahigh inhibition rate of 84%.In view of the important role of tumor stem cells in promoting cancer cell metastasis,we also studied the molecular mechanism of GQD drugs in inhibiting tumor stem cells.GQD drugs decreased the number of 4T1 cells expressed ALDH from 37.43%to 0.02%,while DOX exhibited no effect on ALDH expression at the same concentrations.5.The preparation of GQDs and GQD-sensitized 2D nanosheet heterojunctions and their application in NIR-II photothermal therapy（PTT）.The NIR-II-responsive GQDs were synthesized through a rapid microwave-assisted method using 1,3,6-trinitropyrene（TNP）and branched polyethylenimine（BPEI）as the precursors.The photothermal conversion efficiency of GQDs at 808 and 1064 nm were calculated to be85.7%and 81.3%,respectively.Based on the excellent NIR-II photothermal conversion properties of GQDs,we further assembled GQDs on the surface of black phosphor（BP）or tungsten disulfide（WS₂）nanosheets,obtaining the GQD/BP and GQD/WS₂heterojunctions.Owing to the excellent PTT effect of our heterojunctions,complete tumor eradication was realized in a deep-tissue tumor model.