| ObjectiveAs a popular two-dimensional nanomaterial in recent years,black phosphorous nanosheets have a band gap tuning range that allows wide absorption in the visible,infrared and ultraviolet regions,so they have good photothermal and photodynamic effects.Moreover,black phosphorous nanosheets have a higher specific surface area and are negatively charged,so small and positively charged molecular drugs can be more encapsulated by electrostatic interaction.In this study,it is planned to use phosphorus nanosheets as a carrier,and loading doxorubicin(DOX)to form a nano-drug carrier system.Synergistic chemotherapy,photothermal,and photodynamic therapy are combined to kill tumor cells and enhance the anti-tumor effect.MethodsIn this study,black phosphorus nanoparticles(BPs)were prepared by the liquid phase stripping method,and doxorubicin and electrostatic adsorption were loaded on the surface of BP to construct a BP-DOX nano-drug delivery system.And its particle size,potential,drug release,photothermal properties,etc.were characterized.At the same time,the anti-tumor effect of the nano drug delivery system was evaluated through in vivo and in vitro experiments.1.Construction of nano-black phosphorus drug delivery system and analysis of its propertiesIn this part,black phosphorus nanoparticles(BPs)were prepared by the liquid phase stripping method,and related characterization was performed.Then,doxorubicin was loaded onto the surface of BP by electrostatic adsorption to construct a BP-DOX nano-drug delivery system.The ratio of drug to carrier and the factors of drug loading time in the preparation were investigated.And the performance of the drug loading system was investigated.The drug release ability of the drug-carrying system was investigated mainly through in vitro drug release experiments;the photothermal and photodynamic properties of the drug-carrying system were investigated through photothermal and photodynamic studies;the safety performance of the nanosystem was investigated through hemolysis experiments.2.Anti-tumor effect of black phosphorus nano drug delivery system in vitroTaking Hela cells as the research object,using the CCK-8 cell viability kit,the cytotoxicity of the nano drug delivery system was investigated.The integrity of the cell membrane was tested by examining the cell morphology.Through the study of different endocytosis mechanisms,the mechanism of cellular uptake of the nano drug-carrying system is studied.The content of reactive oxygen species in the cell and the effect of oxidative stress on the nano drug delivery system after entering the cell are used as indicators to explore its toxicity characteristics and process.Finally,the drug-loading system loaded with doxorubicin on the tumor cell killing ability was evaluated.3.Anti-tumor effect of black phosphorus nano drug delivery system in vivoThe BALB/c nude mice were used as experimental subjects to verify the anti-tumor effect of the nano drug delivery system in vivo.Hela cells were planted subcutaneously in nude mice.After administration,808nm laser was used to irradiate tumor tissue to monitor tumor temperature changes.At the same time,tumor volume was measured at intervals.After 12 days,blood,tumor tissue,heart,liver,spleen,and lungs were dissected and collected.Kidney,to investigate the effect of nanoparticles on liver and kidney function in nude mice.Results1.In this study,the BPs prepared by the liquid phase stripping method had an average particle size of 168.4 nm,a polydispersity index of 0.199;the encapsulation rate and drug loading rate were used as indicators to evaluate the drug loading of nanosheets.The results show that when the mass ratio of DOX to carrier is 4:1,the drug loading rate is 73.23%,which is the best ratio.Secondly,the drug release ability of the drug-carrying system was measured.The results showed that the black phosphorous nano-drug delivery system has good drug-releasing performance under acidic conditions.After 808nm laser irradiation,the photothermal effect further promotes the accelerated release of the nano-drug delivery system.The photothermal performance of the black phosphorous nanometer drug-loading system was studied,and it was found that the photothermal effect and photostability of BP-DOX at the same concentration of black phosphorous were similar to that of black phosphorous,and the photothermal performance of the nanoparticles was not affected by the drug loading.Research on the photodynamic properties of the black phosphorous nano-drug-loaded system found that the photodynamic properties of the black phosphorous nano-drug-loaded system did not change due to the drug loading.2.Hela cells were used to investigate the in vitro anti-tumor performance of the black phosphorus nano drug delivery system.First,the CCK-8 experiment shows that the black phosphorus nano-drug carrier system has no obvious cytotoxicity within the range of 10μg·mL-1 incubation for 24 hours and is considered non-toxic.Secondly,by adding five kinds of endocytosis inhibitors to explore the internalization mechanism of the black phosphorus nano drug delivery system,the data shows that the black phosphorus nano drug delivery system enters the cell mainly through pinocytosis,which is a way of active transport.Need to consume a lot of energy.Then,by measuring the content of active oxygen in the cell,it shows that the content of active oxygen in the cell is significantly increased after the black phosphorous nanometer drug-carrying system is treated with light.By comparing the cell viability of Hela cells before and after administration and before and after light,it is found that the black phosphorus nano drug-carrying system has the best anti-tumor effect in vitro under 808nm light.3.In vivo experiments use BALB/c mice to study the anti-tumor effect of the black phosphorus nano-drug delivery system and its biological safety.First,the photo thermal performance of the black phosphorus nano drug delivery system was studied in vivo,and it was found that the tumor temperature in the mice given the black phosphorus nano drug delivery system increased faster than that of the PBS group and the DOX group.Anatomy of the mice 12 days after the administration revealed that the tumor volume of the mice given the black phosphorus nano drug delivery system was the smallest.The tumor inhibition rate was as high as 81.47%.The HE stained sections of tumor tissues showed that the tumor cells of mice given black phosphorus nano drug delivery system were destroyed and fragmented.TUNEL cell apoptosis staining can also show that the tumor tissues of mice given black phosphorus nano drug delivery system have obvious apoptosis.Then we evaluated the in vivo safety of the BP-DOX nano drug delivery system.The results showed that there was no significant difference between the four groups in the renal function indexes of ALB,BUN and CRE in nude mice;There is a significant difference between the normal group,indicating that the treatment may affect liver function.The stained sections of various organs showed no obvious tissue damage.The above shows that there is no obvious physical damage except for the effect on the liver function of nude mice.ConclusionUsing black phosphorus nanosheets as a drug-carrying system to load doxorubicin through electrostatic action,on the one hand,it can rely on the photothermal and photodynamic properties of black phosphorus nanoparticles,and use the photothermal effect and the reactive oxygen species generated to inhibit tumor cells.On the other hand,by loading the chemotherapeutic drug doxorubicin to accelerate its release in the acidic microenvironment of the tumor,the passive targeting function of the drug-loading system is achieved,so that the doxorubicin can be effectively enriched in the tumor tissue to achieve better The therapeutic effect and lower toxic and side effects of this kind of chemotherapeutic drugs targeting tumor tissues cooperate with the photothermal and photodynamic therapy of nanodrug carrier to have more excellent tumor treatment effects. |
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