The Study Of Black Phosphorus Nanosheets On Cellular Biological Effects And Photothermal Cancer Therapy

ObjectiveBlack phosphorus(BP)nanomaterials because of its unique physical and optical properties,has been widely studied in biomedical fields such as drug delivery,biological sensing,biological imaging and cancer therapy.With the development of biomedical applications,more and more attentions have been paid to the safety of black phosphorus nanomaterials.However,its toxicity characteristics have not been elaborated in depth,the researches on security controllability,environmental intervention and potential biological threats are still in infancy.Therefore,it is of great value and significance to comprehensively,systematically and accurately evaluate the safety of black phosphorus nanomaterials,clarify its toxicity characteristics.Enable it to focus on the target sites effectively,avoiding contact with normal tissues as much as possible.Now,this paper will combine the conventional analysis methods and the mass spectrometry-based metabolomics assay platform to evaluate the cellular biological effects of black phosphorus nanosheets(BPs).And further to prepare the nanoparticles with active tumor targeting ability by cell membrane biomimetic technology for the photothermal cancer therapy.It provided further understanding for the safe use of black phosphorus nanomaterials.Methods1.The cellular biological effects of black phosphorus nanosheets by routine methods.In this study,BPs was prepared by liquid exfoliation method,the morphology,thickness and Raman spectrum of BPs were characterized by transmission electron microscope,atomic force microscope and Raman spectrometer.The average particle size and potential of BPs were further studied by nanometer particle size analyzer.Healthy BALB/c mice as animal model,the peripheral blood cells count,blood biochemical indicators,oxidative stress and pathology of mice were used to evaluate the biological effects in vivo of BP nanosheets.Human lung cancer cells(A549),human normal lung epithelial cells(BEAS-2B),human liver cancer cells(HepG2)and normal liver cells(LO2)as in vitro models.Cell morphology,cell uptake,cell activity,cell membrane integrity,intracellular reactive oxygen species generation,oxidative stress related enzymes and proteins were used as the evaluation indexes to evaluate the cellular biological effects of BPs.2.Metabolomics of normal/cancer cells with black phosphorus nanosheets.Normal cells(BEAS-2B,LO2)and cancer cells(HepG2 and A549)were used as cell models.Non-target metabolomics analysis platform based on ultra-high-performance liquid chromatography(UPLC)and time of flight quadrupole mass spectrometry(Q-TOF-MS)combined with pattern recognition and metabolic pathway analysis were used to study the internal and external metabolic characteristics of normal/cancer cells after BPs intervention.The cells were lysed by repeated freezing and thawing with liquid nitrogen,and the metabolites were enriched with organic solvents.ACQUITY UPLC system was used to collect data,ACQUITY HSS T3 chromatographic column(100 mm × 2.1 mm,1.8 μm)was used for sample separation.Electrospray ionization(ESI),and the positive and negative ions were detected respectively.Multivariate statistical variables(PCA,PLS-DA,OPLS-DA)were used to analyze and process the data to obtain the differential metabolic phenotypes of the cells.Metabolomics Pathway Analysis was used to obtain metabolic pathways,the related biological events were reviewed through the literature.3.Study on photothermal antitumor effect of RGD peptide modified erythrocyte membrane coated black phosphorus nanosheets.RGD peptide was modified on erythrocyte membrane by lipid chimeric method,the modified erythrocyte membrane was coated on the surface of black phosphorus nanosheets by co-ultrasonic method.Transmission electron microscope was used to observed the morphology of RGD-RBC@BP nanoparticles.The combination of RGD peptide,erythrocyte membrane and black phosphorus nanosheets were further determined by flow cytometry and laser confocal microscopy.The protein characteristics of nanoparticles were observed by sodium lauryl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The particles sizes were determined by dynamic light scattering and the photothermal performance was observed by 808 nm laser irradiation.In vitro antitumor effects of nanoparticles were estimated by cell uptake,generation of intracellular reactive oxygen species,cell cytotoxicity and apoptosis.In vivo pharmacokinetic experiments and tissue distribution experiments to observed the blood circulation and tumor targeting respectively.Animal model of subcutaneous cervical tumor was used to evaluate the anti-tumor effect of photothermal in vivo,its safety was evaluated by observing the liver and kidney function and pathological sections of main organs.Results1.The cellular biological effects of black phosphorus nanosheets by routine methods.In this paper,the average particle size of BPs which was prepared by liquid exfoliation method was 224.2 nm,the Zeta potential was-25.2 mV,and the thickness was 5.46 nm.A single intravenous injection of 5 mg·kg-1 BPs in healthy female BALB/c mice was used to detect the changes of body weight,hematological index,blood biochemical index,H&E and TUNEL staining of tissue sections after 1,2 and 6 days of administration.The results showed that BPs induced acute inflammation,hemolysis,liver and kidney dysfunction in healthy mice.The activities of lipid peroxidation and superoxide dismutase in liver were decreased,and the liver cells were apoptotic.BPs accumulation in lung tissue,lung tissue cell apoptosis.The above results indicated that a single intravenous injection of 5 mg·kg-1 BPs would cause damage to mice,and liver and lung were the main target organs.In vitro cell experiments showed that BPs could inhibit the growth of BEAS-2B,LO2,A549 and HepG2 cells,and this inhibition was concentration-dependent.BPs induced the increase of intracellular reactive oxygen species and activated the Nrf2/HO-1 signaling pathway,leading to the increase of malondialdehyde level and Nrf2 mRNA and protein expression.Total glutathione levels and mRNA,protein expressions of HO-1 were decreased.In vitro safety evaluation of BPs,it was found that BPs can cause damage to normal cells and inhibit the growth of cancer cells within a certain time and concentration range.2.Metabolomics of normal/cancer cells with black phosphorus nanosheets.The changes of metabolites in A549,BEAS-2B,HepG2 and LO2 cells were interfered by BPs at specific concentrations(10 μg·mL-1)had been observed by metabolomic analysis platform based on LC-MS/MS.The interfering biological metabolites were mainly focus on nucleotides,amino acids,sugars and phospholipids.The intervention of BPs in BEAS-2B cells mainly involved in glutathione metabolism,pantothenic acid and CoA biosynthesis,glutamate metabolism,phospholipid biosynthesis,acetone aldehyde degradation,etc.The intervention of BPs in LO2 cells mainly involved in pyruvaldehyde degradation,arachidonic acid metabolism,glutathione metabolism,pyruvate metabolism and alanine metabolism.The intervention of BPs in A549 cells mainly involved in phospholipid biosynthesis,branch chain fatty acid oxidation and arachidonic acid metabolism and in HepG2 cells mainly involved in purine metabolism,glutathione metabolism,glutamate metabolism,pyruvaldehyde degradation,arachidonic acid metabolism and pyruvate metabolism.This indicated that BPs interfered with the normal metabolic balance of A549,BEAS-2B,HepG2 and LO2 cells.3.Study on photothermal antitumor effect of RGD peptide modified erythrocyte membrane coated black phosphorus nanosheets.RGD peptide could be modified on the phospholipid bilayer of erythrocyte membrane by lipid chimeric method,then the modified erythrocyte membrane was coated on the surface of black phosphorus nanosheets with ultrasonic method.The prepared RGD-RBC@BP nanoparticles had a particle size of 185.7 nm and the protein characteristics of erythrocyte membrane.Photothermal properties were not affected by the modification of cell membrane.In vivo experiments showed that the blood circulation time of RGD-RBC@BP nanoparticles was 1.50 times longer than traditional PEG-modified black phosphorus nanosheets.The distribution in tumor tissues was also 2.60 times higher than PEGylated black phosphorus nanosheets.In vitro and in vivo experiments had proved that it has a good photothermal effect on the treatment of cervical cancer.The tumor inhibition rate in vivo was 73.27%and the administration method was safe.ConclusionBlack phosphorus nanosheets not only could cause intracellular and extracellular metabolic interference in normal cells(BEAS-2B,LO2)and cancer cells(A549,HepG2),but also caused cell apoptosis due to induce oxidative stress.For the application of black phosphorus nanosheets in vivo,the potential metabolic interferences and damages should be considered,the contact with normal cells and tissues should be avoided or reduced as much as possible to avoid the metabolic disorders of cells.RGD peptide-specific targeting of tumor angio-integrin-coated black phosphorus nanosheets had the advantages of long blood circulation,strong tumor targeting ability and effective accumulation in tumor sites,which could be used in the photothermal treatment of cervical cancer.This study provided a further understanding of the safe use of black phosphorus nanomaterials.