The Preparation Of Fructose-modified Copper-based Nanomaterials And Their Biological Effects

Objective: Osteosarcoma is the most common orthopaedic malignancy and poses a serious economic burden and health threat to patients and society.The neoadjuvant chemotherapy regimen of preoperative chemotherapy +surgical resection of the tumour + postoperative chemotherapy has significantly improved patient outcomes and 5-year event-free survival rates,of which chemotherapy is a key factor,but with the consequent emergence of tumour cell resistance,it has become a hot clinical issue that needs to be addressed.Infections caused by pathogenic bacteria can have a serious impact on the body’s function,either alone or in association with other diseases,and the chronic misuse of antibiotics has led to increased bacterial resistance,making effective antimicrobial resistance increasingly challenging.The emergence and rapid development of biomedical materials has gradually provided new ideas and solutions for biomedical diagnosis and treatment,among which nanomaterials with high specific surface area have been widely studied and applied.In this study,copper-based nanomaterials are proposed to be prepared based on a hydrothermal reaction and applied in antitumour and antibacterial studies.The anti-tumour and antibacterial activities of copper-based nanomaterials will be explored while their mechanisms of action will be investigated in depth,and the biological activities of copper-based nanomaterials will be evaluated comprehensively.Methods:(1)A mixture of water and ethanol was used as the reaction system,and different ratios of water and ethanol were adjusted to add fructose 1,6-diphosphate(FDP)solution and copper chloride solution,which were mixed with magnetic stirring and transferred to the reaction kettle and placed in an oven,and the reaction time and reaction temperature were adjusted for hydrothermal reaction to prepare copper-based nanomaterials.Fructose-modified cuprous chloride nanoparticles(Fru-Cu Cl NPs)and copper hydrogen phosphate nanosheets self-assembled microspheres(CPNSMSs)were produced,respectively.(2)Taking the preparation of copper hydrogen phosphate nanosheet self-assembled microspheres as an example,the reaction was terminated at different time points during the hydrothermal reaction to obtain a series of products,and the products were characterized by scanning electron microscopy to explore the formation mechanism of copper-based nanomaterials.(3)The morphology,structure,dimensions,phases and chemical properties of the copper-based nanomaterials were carefully characterized by scanning electron microscopy,transmission electron microscopy,selected area electron diffraction,elemental mapping,X-ray powder diffraction analysis,X-ray photoelectron spectroscopy,fourier transform infrared spectroscopy and thermogravimetric analysis.(4)Doxorubicin(DOX)was loaded onto fructose-modified cuprous chloride nanoparticles by physical adsorption and its drug loading rate was analyzed by UV-visible photometry.Human osteosarcoma cells(MG63)and doxorubicin-resistant human osteosarcoma cells(MG63/DXR)were selected to evaluate the antitumor activity of fructose-modified copper chloride nanoparticles loaded with adriamycin by MTT assay and live-dead cell staining,and further explore the antitumor mechanism of fructosemodified copper chloride nanoparticles loaded with adriamycin by Western blot and RNA-Seq.(5)Typical Gram-negative bacteria(Escherichia coli,E.coli)and Gram-positive bacteria(Staphylococcus aureus,S.aureus)were selected to evaluate the antibacterial activity of the copper hydrogen phosphate nanosheets self-assembled microspheres.To further explore the antibacterial mechanism,hydroxyl radicals(· OH)were measured by electron spin resonance(ESR)and 3,3’,5,5’-Tetramethylbenzidine(TMB)colourimetric method to investigate the antibacterial mechanism of the copper hydrogen phosphate nanosheets self-assembled microspheres.Results:(1)By optimizing the substrate concentration and hydrothermal reaction conditions of the reaction system,two homogeneous,stable and well-dispersed materials named as fructose-modified cuprous chloride nanoparticles(Fru-Cu Cl NPs)and copper hydrogen phosphate nanosheet self-assembled microspheres(CP-NSMSs)were fabricated,respectively.(2)Taking the preparation of copper hydrogen phosphate nanosheet self-assembled microspheres as an example,the formation mechanism of copper-based nanomaterials was revealed by scanning electron microscopy characterization of the products at different time points:(i)hydrothermal reaction of FDP to produce phosphate and organic small molecule fructose;(ii)reaction of phosphate ions with copper ions to produce amorphous copper phosphate-organic molecule hybrid microspheres;(iii)in situ formation of nanoflowers within the microspheres,while the amorphous copper phosphate phase crystallizes into a copper hydrogen phosphate phase;(iv)continued growth and assembly of copper hydrogen phosphate nanosheets to form copper hydrogen phosphate nanosheet self-assembled microspheres.(3)MTT assay and live-dead cell assays suggested that the doxorubicinloaded fructose-modified copper chloride nanoparticles possessed excellent osteosarcoma cell killing activity,and the Western blot results suggested that the tumor cells highly expressed the fructose transporter membrane protein GLUT5,suggesting that the doxorubicin-loaded fructose-modified copper chloride nanoparticles could target and enrich in tumor cells,reverse tumor drug resistance to a certain extent,and synergistically exert their anti-tumor effects.The RNA-Seq results suggested that doxorubicin-loaded fructosemodified copper chloride nanoparticles reversed drug resistance by activating MAPK signaling pathway as well as enhancing ROS effectmediated apoptosis.(4)The copper hydrogen phosphate nanosheets self-assembled microspheres exhibited excellent anti-E.coli and anti-S.aureus activities.Further mechanistic studies suggest that copper in the copper hydrogen phosphate nanosheets self-assembled microspheres possesses peroxidaselike activity,which generates reactive oxygen hydroxyl radicals through redox reactions with hydrogen peroxide,consumes the reducing agent glutathione and damages bacterial cell membranes to exert antibacterial effects.Conclusion: Copper-based nanomaterials were successfully prepared by initiating hydrothermal reactions with the addition of fructose 1,6-diphosphate(FDP)solution and copper chloride solution using water and ethanol as the reaction system.The copper-based nanomaterials in this study possess excellent drug-carrying properties,antitumor effects and antibacterial activities.It provides a new feasible idea for the treatment of clinical osteosarcoma,the problem of drug resistance of chemotherapeutic drugs and the problem of bacterial infection,and a new feasible solution for the design,drugcarrying delivery and targeted delivery of nanomaterials in basic research,which has strong research significance and scientific value.