Photo-thermal and tumor microenvironment dual-response two-dimensional nano-drug and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses a photo-thermal and tumor microenvironment dual-response two-dimensional nano-drug and a preparation method thereof. The preparation method comprises the following steps: preparing a black phosphorus two-dimensional nanosheet; the preparation method comprises the following steps: mixing a black phosphorus two-dimensional nanosheet with a first solvent, then adding a bivalent copper salt to enable copper ions to be fully loaded on the black phosphorus two-dimensional nanosheet, forming monovalent copper ions under the action of black phosphorus, and carrying out primary centrifugation to remove redundant copper ions; adding a second solvent, mixing with a dopamine solution, finally adding alkali liquor, stirring in a dark place, and carrying out secondary centrifugation, so as to obtain the two-dimensional nano-drug. The preparation method has the characteristics of simplicity in operation, mild preparation conditions, strong controllability and high repeatability, is easy for large-scale production, and is beneficial to reducing the cost and improving the feasibility of clinical application. By utilizing the method, the tumor microenvironment-light control double-response two-dimensional nano-drug with diagnosis and treatment integration can be prepared.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a two-dimensional nanomedicine with dual responses to photothermal and tumor microenvironment and a preparation method thereof. Background Art

[0002] Breast cancer is called "the number one killer of women's health" by the medical community, and it seriously threatens women's physical and mental health. Traditional treatments for breast cancer include surgical resection, chemotherapy, radiotherapy, etc. These treatments can alleviate and inhibit the development of breast cancer to a certain extent. However, due to the complexity, diversity and heterogeneity of breast cancer, a single clinical treatment model has poor efficacy for breast cancer, and the treatment process often causes serious side effects and complications. Therefore, the existing single treatment model is difficult to meet the treatment needs of breast cancer, and it is urgent to explore and develop new synergistic treatment strategies.

[0003] In recent years, new treatment methods such as chemodynamic therapy (CDT), photothermal therapy (PTT) and copper death have been widely studied. Chemodynamic and photothermal therapies have the advantages of being non-invasive, spatiotemporally controllable and low toxic, which have greatly promoted the vigorous development of multimodal nanomedicine research. Among them, PTT can exert a hyperthermia effect to ablate tumor cells, and local hyperthermia can preferentially enhance the permeability and fluidity of cell membranes, thereby increasing the cell's uptake of drugs; chemodynamics can catalyze the overexpressed hydrogen peroxide (H2O2) in the lesions to produce reactive oxygen species, which can kill tumors efficiently and specifically; copper death, as a novel cell death mechanism, has a dual action pathway. On the one hand, Cu + After release, it can act as a Fenton-like reaction catalyst, reacting with overexpressed H2O2 in the tumor to produce high concentrations of ·OH free radicals, killing cancer cells. + It can also trigger the aggregation of sulfonylation (DLAT, PDHA1) and inhibit the TCA cycle, thereby inducing copper death of cancer cells. Photothermal / chemodynamic / copper death synergistic therapy can overcome the shortcomings of single treatment and improve the efficiency and safety of tumor treatment. However, the current defects of insufficient accumulation of drugs in the lesion site, difficulty in responding to microenvironment controlled release of drugs, and lack of effective follow-up methods to observe the efficacy seriously hinder the application of synergistic treatment systems.

[0004] Specifically, the current treatment of breast cancer still has the following problems and technical difficulties: (1) Clinically, tumor imaging has long been plagued by technical bottlenecks such as "not being able to see deep" and "not being able to see clearly" with ultrasound technology. (2) Traditional tumor treatment methods, such as surgery, chemotherapy, and radiotherapy, often have problems such as large damage to surrounding normal tissues, serious side effects, and easy development of drug resistance. Especially for malignant tumors such as breast cancer, the effect of a single treatment is not ideal, and the recurrence rate is high. (3) In the field of photothermal therapy, although commonly used two-dimensional nanomedicines have excellent photothermal properties, they have poor stability under physiological conditions and are easily reacted with oxygen and water, which limits their wide application in clinical practice. (4) Free copper ions are highly toxic, and traditional nanomedicines usually lack responsiveness and cannot accurately deliver copper ions to tumor cells, resulting in the current copper death therapy having the defects of causing potential cell damage, lack of targeting, and insufficient efficacy. (5) Existing combined treatment strategies are difficult to efficiently load copper ions, especially monovalent copper ions that are key to inducing copper death, and are unable to achieve efficient enrichment in the lesion area and precise in situ release of copper ions in the tumor, as well as provide an effective imaging follow-up model during the treatment process, resulting in unsatisfactory effects of existing photothermal / chemodynamic / copper death induction therapies. (6) The traditional preparation process of responsive nanomaterials is complicated and expensive.

[0005] Therefore, there is an urgent need to provide a two-dimensional nanomedicine that can accurately deliver copper ions to tumor cells and achieve good photothermal / chemodynamic / copper death-inducing therapy effects. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a two-dimensional nanomedicine with dual response to photothermal and tumor microenvironment and a preparation method thereof. The two-dimensional nanomedicine provided by the present invention can accurately deliver copper ions to tumor cells and achieve good photothermal / chemodynamic / copper death induction therapy effects.

[0007] The present invention provides a method for preparing a two-dimensional nanomedicine with dual responses to photothermal and tumor microenvironment.

[0008] Specifically, a method for preparing a two-dimensional nanomedicine with dual responses to photothermal and tumor microenvironment comprises the following steps:

[0009] (1) Preparation of black phosphorus two-dimensional nanosheets;

[0010] (2) The black phosphorus two-dimensional nanosheets prepared in step (1) are mixed with a first solvent, and then a divalent copper salt is added to fully load the copper ions on the black phosphorus two-dimensional nanosheets, and monovalent copper ions are formed under the action of black phosphorus; excess copper ions are removed by centrifugation; a second solvent is added and mixed with a dopamine solution, and finally an alkali solution is added, stirred in the dark, and centrifuged again to obtain a two-dimensional nanomedicine that is dual-responsive to light, heat, and tumor microenvironment.

[0011] Preferably, in step (1), the specific process of preparing black phosphorus two-dimensional nanosheets is: mixing and grinding black phosphorus powder with a third solvent to obtain black phosphorus micropowder; mixing the black phosphorus micropowder with a fourth solvent to obtain a black phosphorus micropowder dispersion; subjecting the black phosphorus micropowder dispersion to ice bath probe ultrasonic treatment to obtain a black phosphorus two-dimensional thin sheet dispersion; subjecting the black phosphorus two-dimensional thin sheet dispersion to water bath ultrasonic treatment to obtain a black phosphorus nanosheet dispersion; and finally subjecting the black phosphorus nanosheet dispersion to differential centrifugation treatment to obtain black phosphorus two-dimensional nanosheets.

[0012] Preferably, the third solvent and the fourth solvent are independently selected from one of isopropanol, N-methylpyrrolidone (NMP) and ethanol.

[0013] Preferably, the mass concentration of the black phosphorus powder in the black phosphorus powder dispersion is 1-10 mg / mL, such as 5 mg / mL.

[0014] Preferably, the process of the ice bath probe ultrasonic treatment is to make the ultrasonic wave of the ice bath probe generate directional bubbles in the vertical direction to cut the black phosphorus powder into thin sheets. This process can effectively convert the three-dimensional black phosphorus powder into black phosphorus two-dimensional nanosheets with large lateral size and thickness.

[0015] Preferably, the power of the ice bath probe ultrasonic treatment is 100-500W, and the time of the ice bath probe ultrasonic treatment is 2-8 hours.

[0016] Preferably, the power of the water bath ultrasonic treatment is 100-500 W, the temperature of the water bath ultrasonic treatment is 1-15° C., and the time of the water bath ultrasonic treatment is 5-10 hours. As the water bath ultrasonic treatment proceeds, the size and thickness of the black phosphorus two-dimensional flakes can be greatly reduced and peeled off to obtain black phosphorus nanosheets.

[0017] Preferably, the differential centrifugation process is: firstly centrifuge the black phosphorus nanosheet dispersion at a centrifugal speed of 2-5Krpm to obtain a supernatant, and then centrifuge the supernatant at a centrifugal speed of 8-12Krpm to obtain a precipitate, namely the black phosphorus two-dimensional nanosheet. The differential centrifugation can make the size and thickness of the ultrasonically exfoliated black phosphorus nanosheet more uniform, and further obtain the black phosphorus two-dimensional nanosheet.

[0018] Preferably, in step (2), the divalent copper salt is one of copper chloride, copper sulfate and copper nitrate.

[0019] Preferably, in step (2), the mass ratio of copper in the divalent copper salt to the mass ratio of the black phosphorus two-dimensional nanosheets is 1:(3-30).

[0020] Preferably, in step (2), the first solvent and the second solvent are ethanol solutions, and the mass fraction of the ethanol solution is 10%-100%.

[0021] Preferably, in step (2), the rotation speed of the first centrifugation is 12000-18000 rpm.

[0022] Preferably, in step (2), the concentration of the dopamine aqueous solution is 50-200 mg / mL; further preferably, the concentration of the dopamine aqueous solution is 100-180 mg / mL; more preferably, the concentration of the dopamine aqueous solution is 120-180 mg / mL.

[0023] Preferably, in step (2), the mass ratio of dopamine in the dopamine aqueous solution to the mass ratio of the black phosphorus two-dimensional nanosheets is (1-3):1; preferably, the mass ratio of dopamine in the dopamine aqueous solution to the mass ratio of the black phosphorus two-dimensional nanosheets is (1-2):1, such as 1.5:1.

[0024] Preferably, in step (2), the alkali solution is a sodium hydroxide solution, and the concentration of the alkali solution is 10-25 mg / mL. By adding the alkali solution to create alkaline conditions, not only can the self-polymerization of dopamine to form polydopamine be promoted, but the phenolic hydroxyl and amine functional groups in the dopamine molecules are more easily deprotonated, thereby increasing their reactivity, so that the dopamine molecules can react with the electrophilic black phosphorus two-dimensional nanosheets through OH and NH bonds, thereby achieving polymerization.

[0025] Preferably, in step (2), the time of stirring in the dark is 2-8 h; more preferably, the time of stirring in the dark is 2.5-6 h.

[0026] Preferably, in step (2), the rotation speed of the secondary centrifugation is 10000-12000 rpm, and the time of the secondary centrifugation is 3-10 min.

[0027] The present invention also provides a two-dimensional nanomedicine with dual responses to photothermal and tumor microenvironment.

[0028] Specifically, a two-dimensional nanomedicine with dual responses to photothermal and tumor microenvironment is prepared by the above preparation method, wherein the two-dimensional nanomedicine includes black phosphorus two-dimensional nanosheets and monovalent copper ions and dopamine attached to the black phosphorus two-dimensional nanosheets; the particle size of the two-dimensional nanomedicine is 50-300nm.

[0029] Preferably, the black phosphorus two-dimensional nanosheet has a diameter of 100-300 nm and a thickness of 2-5 nm.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The preparation method provided by the present invention first prepares black phosphorus two-dimensional nanosheets by liquid phase ultrasonic exfoliation; then copper ions are loaded onto the black phosphorus two-dimensional nanosheets through stirring and adsorption centrifugation; further, in order to reduce its inherent toxicity, the self-polymerization of dopamine monomers is utilized under alkaline conditions to form a polydopamine shell containing a large number of active groups on the copper ion-loaded black phosphorus two-dimensional nanosheets. The preparation method has the characteristics of simple operation, mild preparation conditions, strong controllability, and high repeatability. It is easy to mass produce, which is conducive to reducing costs and improving the feasibility of clinical application. The method can be used to prepare a two-dimensional nanomedicine with integrated diagnosis and treatment of tumor microenvironment-light control dual response.

[0032] (2) The novel two-dimensional nanomedicine provided by the present invention, which has both exogenous light and endogenous tumor microenvironment response, can be used for efficient treatment of breast cancer in a coordinated manner of photothermal / chemodynamic / copper death, and can achieve efficient integrated diagnosis and treatment of breast cancer by enhancing photoacoustic signals for imaging follow-up. The two-dimensional nanomedicine efficiently loads copper ions, can accurately deliver copper ions to tumor cells, and achieve good photothermal / chemodynamic / copper death induction therapy effects.

[0033] (3) The present invention utilizes black-scale nanosheets to efficiently load copper ions, and increases the proportion of monovalent copper ions through redox reactions, which is greatly beneficial to the induction of copper death; in addition, the black-scale nanosheets loaded with copper ions are modified with dopamine to improve the drug light response efficiency while preventing copper ion leakage and improving safety; more importantly, the two-dimensional nanomedicine can respond to light and the acidic microenvironment of the tumor and accurately release copper ions at the tumor site; the released monovalent copper ions, on the one hand, react with endogenous H2O2 in the body to mediate the Fenton reaction to catalyze the synthesis of the most active hydroxyl radicals, thereby causing oxidative damage to tumor cells. On the other hand, the released copper ions produce the aggregation of sulfylated (DLAT, PDHA1), interfere with the TCA cycle, and lead to efficient copper death of tumor cells, thereby achieving the positioning and killing of cancer cells, and having important clinical application value.

[0034] (4) The two-dimensional nanomedicine prepared by the present invention comprises a black phosphorus two-dimensional nanosheet and a monovalent copper ion and dopamine attached to the black phosphorus two-dimensional nanosheet. Its morphology is stable and controllable, the copper ions carried can be accurately released, it has good biocompatibility and safety, and excellent photothermal / pH responsiveness. The black phosphorus nanosheet itself has excellent photothermal conversion efficiency. After being modified with dopamine, its photothermal performance is further enhanced, and it can quickly generate heat under near-infrared light irradiation, effectively improving the effect of photothermal therapy.

[0035] (5) The two-dimensional nanomedicine prepared by the present invention has good biocompatibility and safety in the body, and can be biodegraded after the treatment is completed, reducing the long-term impact on the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The black phosphorus two-dimensional nanosheet prepared in the embodiment of the present invention;

[0037] Figure 2 Schematic diagram of the preparation of the two-dimensional nanomedicine BPNs@Cu@PDA in an embodiment of the present invention;

[0038] Figure 3 The two-dimensional nanomedicine BPNs@Cu@PDA prepared in the embodiment of the present invention;

[0039] Figure 4 A schematic diagram of the mechanism of the multifunctional novel integrated diagnosis and treatment two-dimensional drug for the synergistic and efficient treatment of breast cancer in an embodiment of the present invention;

[0040] Figure 5 A transmission electron microscope image of the two-dimensional nanomedicine BPNs@Cu@PDA prepared in an embodiment of the present invention;

[0041] Figure 6 A high-resolution transmission electron microscope image of the two-dimensional nanomedicine BPNs@Cu@PDA prepared in an embodiment of the present invention;

[0042] Figure 7 Mapping image of the two-dimensional nanomedicine BPNs@Cu@PDA prepared in an embodiment of the present invention;

[0043] Figure 8 This is an EDS image of the two-dimensional nanomedicine BPNs@Cu@PDA prepared in an embodiment of the present invention;

[0044] Fig. 9 This is a particle size distribution diagram of the two-dimensional nanomedicine BPNs@Cu@PDA prepared in an embodiment of the present invention;

[0045] Fig.10Potential curves of two-dimensional nanomedicine BPNs, BPNs@Cu and BPNs@Cu@PDA prepared in the embodiments of the present invention;

[0046] Fig.11 This is an XPS spectrum of the Cu2P binding energy in the two-dimensional nanomedicine BPNs@Cu@PDA prepared in an embodiment of the present invention;

[0047] Fig.12 This is an X-ray diffraction pattern of the two-dimensional nanodrug BPNs@Cu@PDA prepared in an embodiment of the present invention;

[0048] Fig.13 This is the XPS spectrum of the two-dimensional nanomedicine BPNs@Cu@PDA prepared in an embodiment of the present invention;

[0049] Fig.14 The temperature evaluation curves of two-dimensional nanodrug BPNs@Cu@PDA solutions with different concentrations under near-infrared laser irradiation;

[0050] Fig.15 The temperature change curve of the two-dimensional nanodrug BPNs@Cu@PDA solution irradiated by near-infrared laser of different powers;

[0051] Fig.16 The temperature rise curve of the two-dimensional nanodrug BPNs@Cu@PDA solution under laser irradiation and the temperature drop curve after the laser is turned off;

[0052] Fig.17 The five temperature rise curves of the two-dimensional nanodrug BPNs@Cu@PDA solution under irradiation and the cooling curve after the laser is turned off;

[0053] Fig.18 This is a graph showing the ROS production (ultraviolet absorption) of different treatment groups detected using TMB indicator;

[0054] Fig.19 This is a graph of ROS production in different treatment groups detected using TMB indicator (microplate reader), data are mean ± SD, (n = 5);

[0055] Fig. 20 Confocal images of ROS staining of 4T1 cells;

[0056] Fig.21 Flow cytometry plots generated for ROS in different treatment groups;

[0057] Fig. 22 The photothermal images of mice in different treatment groups at different time periods;

[0058] Fig.23 For the corresponding Fig. 22 Temperature variation diagram of different temperatures. DETAILED DESCRIPTION

[0059] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0060] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0061] Example

[0062] The embodiment of the present invention provides a method for preparing a two-dimensional nanomedicine BP@Cu@PDA that is dual-responsive to light, heat and tumor microenvironment. Figure 2 As shown, the following steps are included:

[0063] (1) Preparation of black phosphorus two-dimensional nanosheets (BPNs)

[0064] In the experiment, an improved liquid phase method was used to effectively exfoliate and obtain ultra-thin black phosphorus two-dimensional nanosheets. It combines ice bath probe ultrasonic treatment and water bath ultrasonic treatment. The specific method is as follows:

[0065] Step 1: The purchased black phosphorus powder (Nano Beike Biotechnology Co., Ltd.) was ground in an agate mortar with a trace amount of isopropanol solvent for 20 minutes to ensure that large particles were fully crushed into small particles.

[0066] Step 2: Weigh 5 mg of fully ground phosphorus powder, pour it into a 15 mL centrifuge tube, and add a certain amount of isopropanol solvent to make the volume 10 mL to ensure that the final concentration of black phosphorus powder is about 5 mg / mL, and seal it with a sealing film.

[0067] Step 3: Next, the ultrasonic wave of the ice bath probe ultrasound is used to generate directional bubbles in the vertical direction to cut the phosphorus powder distributed around the probe into thin sheets. In this way, the phosphorus powder with a three-dimensional structure can be effectively converted into black phosphorus two-dimensional nanosheets with large lateral size and thickness (the power of the ice bath probe ultrasound is 300W, and the switching cycle is 3 / 3s). Then, after 6 hours of ice bath probe ultrasound, black phosphorus two-dimensional sheets were obtained;

[0068] Step 4: The obtained black phosphorus two-dimensional flake dispersion is transferred to a water bath ultrasonic treatment for 8 hours. With the water bath ultrasonic treatment, the size and thickness of the black phosphorus two-dimensional flakes can be greatly reduced and peeled off to obtain black phosphorus nanosheets (the water bath ultrasonic power is 400 W, and the temperature is maintained at about 10°C through a water circulation system during the ultrasonic process);

[0069] Step 5: In order to ensure that the size and thickness of the ultrasonically peeled phosphorus nanosheets are uniform, the obtained phosphorus dispersion is then subjected to differential centrifugation at a centrifugal speed of 3Krpm, and then the obtained supernatant is further centrifuged at a centrifugal speed of 10Krpm. The obtained precipitate is the black phosphorus two-dimensional nanosheet (BP two-dimensional nanosheet), and its appearance is as follows: Figure 1 shown.

[0070] (2) Preparation of BPNs@Cu@PDA

[0071] Step 1: Take 1 mL of black phosphorus two-dimensional nanosheets (stored in isopropanol solution, 1 mg / mL) and centrifuge at 15000 rpm for 10 min. After centrifugation, discard the supernatant and keep the precipitate. Then add 1 mL of ethanol and mix evenly by ultrasonic blowing.

[0072] Step 2: After adding anhydrous cupric chloride (BP:Cu mass ratio is 3:1), the mixture is stirred for 10 minutes in the dark to allow the copper ions to be fully loaded on the black phosphorus two-dimensional nanosheets.

[0073] Step 3: Centrifuge at 15000 rpm to remove excess copper ions, and wash the precipitate with water three times. Finally, add 1 mL of 80% ethanol solution to the precipitate again and mix it evenly by ultrasonic blowing.

[0074] Step 4: Add 10 μL of dopamine aqueous solution (150 mg / mL), followed by 25 μL of NaOH aqueous solution (20 mg / mL), and stir the mixture in the dark for 4 h.

[0075] Step 5: Centrifuge at 12000 rpm for 5 min. Wash the precipitate with water for 3 times to prepare the two-dimensional nanomedicine BPNs@Cu@PDA (BCP for short). Figure 3 shown.

[0076] The two-dimensional nano drug BCP provided by the present invention can construct a new nano-diagnosis and treatment integrated platform with copper death induction, photothermal therapy and chemodynamic therapy, clarify the synergistic mechanism of multiple combined treatment modes, and solve the shortcomings of poor efficacy of single treatment mode for breast cancer. Figure 4 shown.

[0077] The morphology and performance test of the two-dimensional nanomedicine BCP with dual response to photothermal and tumor microenvironment prepared in the embodiment of the present invention are carried out. The details are as follows:

[0078] (1) Morphological characterization of the two-dimensional nanodrug BP@Cu@PDA that responds to both photothermal and tumor microenvironment

[0079] In transmission electron microscopy (TEM) images, BCPs showed flake-like morphology and uniform size ( Figure 5 ), the scale bar is 200 nm. Dynamic light scattering (DLS) analysis revealed that the average hydrodynamic diameter of BCP is approximately 200 nm ( Fig. 9 ), which is consistent with the particle size measured by TEM. Figure 6 ) image, the lattice spacing of BCP is 0.262 nm, corresponding to the (040) plane of BPNs, and the lattice spacing presents an orderly stripe pattern. X-ray diffraction pattern ( Fig.12 ) further confirmed that the synthesized BCP lattice spacing presented an ordered stripe pattern. In addition, high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) images and corresponding regional elemental mapping images further showed that copper was uniformly distributed throughout the BPNs and the prepared BCP had a 30 nm thick dopamine coating ( Figure 7 , STEM). At the same time, copper successfully loaded onto BCP can be detected by zeta potential (e.g. Fig.10 ), from -28mV (BPNs) to -12mV (BPNs@Cu), which is caused by the loading of positively charged copper ions, and then continued to drop to -26mV (BCP) after dopamine was coated, which is mainly because dopamine can provide more negatively charged particles under alkaline conditions. In addition, EDS ( Figure 8 ) and X-ray photoelectron spectroscopy (XPS, Fig.13 ) analyzed the chemical composition of BCP and found Cu and N elements. That is, after coating with PDA, a new N peak representing PDA amino group and a new Cu2p peak representing copper ions appeared in the XPS spectrum of BCP, further proving that copper ions were loaded on BPNs and the PDA coating was successfully formed. In addition, two new peaks were observed in the XPS spectrum of BCP, located at 530.75eV and 285.35eV, respectively, which were mainly attributed to C=O and C=N, further proving that PDA was successfully coated with BC. In the XPS peak distribution of copper ions ( Fig.11 ), not only two Cu 2+ Satellite peaks (962.5eV and 942.7eV). In addition, a representative peak of monovalent copper ions was found, namely Cu+2p3 / 2 peak (952.2eV) and Cu+2p1 / 2 peak (955.2eV), which further confirmed the successful formation of Cu=O bond, mainly due to Cu 2+ Can undergo redox reaction with P(0) to generate Cu + In conclusion, the zeta potential mapping and XPS results of the synthesized nanocomposites proved that BCP was successfully prepared.

[0080] (2) Photothermal performance of two-dimensional nanomedicine BCP with dual response to photothermal and tumor microenvironment

[0081] In order to verify whether BCP has excellent photothermal effect, an infrared thermal imager was used to monitor the temperature changes after exposure to 808nm laser at different concentrations of BCP or different light powers. A single control variable method was adopted, and photothermal conversion experiments were carried out using different concentrations and different light powers. The experiment found that in sharp contrast to the other experimental groups, the control group (pure deionized water (0ppm)) had a power density of 1W / cm -2 After 10 minutes of 808 nm laser irradiation, the temperature of the synthesized BCP increased not only with the increase of concentration (e.g. Fig.14 ), and as the optical power increases, the temperature of the BCP continues to rise (e.g. Fig.15 ). It shows that the temperature of BCP increases rapidly in a time- and concentration-dependent manner, reaching a maximum value after about 10 minutes of exposure, indicating that BCP has a significant cumulative photothermal effect, which may be attributed to the contribution of two excellent photothermal BPNs and PDA shells. After that, 5 cycles were used to verify the photothermal stability of this material. After 5 rounds of laser on / off cycles, it showed excellent photothermal stability without obvious temperature decay (such as Fig.16 , Fig.17 ). In conclusion, the prepared BCP was found to have high photothermal conversion ability, excellent photothermal stability and thermal accumulation effect, which is helpful to further evaluate its photothermal cytotoxicity in vitro.

[0082] (3) The photothermal / PH responsive performance of the two-dimensional nanodrug BCP with dual responsiveness to photothermal and tumor microenvironment was evaluated in a preliminary experiment. The experimental results were based on the hypothesis that the degradation of dopamine would be triggered and the release of copper ions and ROS would be generated. Based on the principle that TMB can react with the strongly oxidizing ·OH, lose an electron, and generate blue TMB+ (ox-TMB), the UV / visible / near-infrared absorption spectrum shows a clear TMB characteristic absorption peak at about 652nm (TMB absorption peak). 3,3',5,5'-tetramethylbenzidine (TMB for short) was used as a detection reagent for detection. Cu loaded on BPNs 2+ and Cu + The efficiency of generating hydroxyl radicals (·OH) with the help of hydrogen peroxide (H2O2) substrate (e.g. Fig.18). The pH7.4 group showed almost no absorption after 10 minutes of incubation with TMB and H2O2 substrates, while the pH6.5 group and pH7.4+laser group showed moderate absorption at 652nm, indicating that a small amount of OH free radicals were effectively generated. Compared with other groups, under the same conditions, the pH6.5+Laser group had the strongest light absorption at 652nm. This may be due to the dual stimulation of acidic conditions and laser irradiation, which enhanced the cleavage of the Cu=O bond of BCP by heat, and as the dopamine layer gradually depolymerized, a large amount of Cu was loaded on BPNs. 2+ and Cu + Based on Cu 2+ and Cu + The interaction with the hydrogen peroxide (H2O2) substrate enhances the generation of ·OH (e.g. Fig.19 ). Further confocal and flow cytometry detection was performed as follows: 4T1 cells were plated at 1×10 5 The cells were seeded at a density of 10 cells / mL in confocal culture dishes and 6-well plates and cultured for 12 hours. Then, the culture medium in each well was replaced with BCP solution diluted with culture medium. After 4 hours of culture, the cells were illuminated by 808 nm laser at 1.0 W / cm 2 The cells were irradiated with a power density of 10 minutes; the cells were then cultured at 37°C for another 8 hours; finally, the H2DCFH-DA probe and the Hoechest probe (final concentration of 5 μM) were added to the culture medium and incubated for another 30 minutes. After the cells were washed with PBS, they were imaged using a confocal microscope and detected on a flow cytometer. The experiment found that the two-dimensional drug prepared in the embodiment of the present invention has the effect of low pH and photothermal dual response (ultraviolet absorption spectroscopy to determine TMB), and in the confocal (such as Fig. 20 ) and streaming (such as Fig.21 ) was also verified in the experimental verification.

[0083] (4) Photothermal performance test of two-dimensional nanomedicine BCP with dual response to photothermal and tumor microenvironment in mice

[0084] In order to evaluate the accumulation and retention of BCP in the tumor site, 100 μL of BCP dispersion (equivalent to a BCP dose of 5 mg / kg) was intravenously injected into 4T1 tumor-bearing mice. 12 hours after intravenous injection of BCP and saline, the 4T1 tumor-bearing mice were treated with 808 nm laser for 10 minutes. Figure 22-Figure 23 As shown, there was no significant change in the saline injection group (PBS), the light irradiation group (Laser) and the drug group. -2Under laser irradiation, the temperature of the BCP-treated group climbed to 50°C, which well demonstrated that BCP with a size of about 200 nm can effectively achieve passive targeting of tumor tissues through the enhanced permeability and retention (EPR) effect. In addition, BCP has an excellent photothermal conversion efficiency of 1.0 W cm -2 The laser intensity is sufficient to melt the PDA and maintain the temperature at about 50°C throughout the treatment process, thereby preventing any potential harm to the tumor tissue caused by the photothermal treatment. In summary, it is found in the present invention that the prepared BCP has a high photothermal conversion ability and a good accumulation and retention ability of BCP at the tumor site.

[0085] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a two-dimensional nano drug, characterized in that: The following steps are involved: (1) Preparation of black phosphorus two-dimensional nanosheets; (2) The black phosphorus two-dimensional nanosheets prepared in step (1) are mixed with a first solvent, and then a divalent copper salt is added to load copper ions on the black phosphorus two-dimensional nanosheets, and monovalent copper ions are formed under the action of black phosphorus, and then the excess copper ions are removed by centrifugation; a second solvent is added and mixed with a dopamine solution, and finally an alkali solution is added, stirred in the dark, and centrifuged a second time to obtain a two-dimensional nanomedicine.

2. The preparation method according to claim 1, characterized in that: In step (1), the specific process of preparing black phosphorus two-dimensional nanosheets is as follows: black phosphorus powder is mixed and ground with a third solvent to obtain black phosphorus micropowder; the black phosphorus micropowder is mixed with a fourth solvent to obtain a black phosphorus micropowder dispersion; the black phosphorus micropowder dispersion is subjected to ice bath probe ultrasonic treatment to obtain a black phosphorus two-dimensional thin sheet dispersion; the black phosphorus two-dimensional thin sheet dispersion is subjected to water bath ultrasonic treatment to obtain a black phosphorus nanosheet dispersion; and finally the black phosphorus nanosheet dispersion is subjected to differential centrifugation treatment to obtain black phosphorus two-dimensional nanosheets.

3. The preparation method according to claim 2, characterized in that: The mass concentration of the black phosphorus powder in the black phosphorus powder dispersion is 1-10 mg / mL.

4. The preparation method according to claim 2, characterized in that: The process of the ice bath probe ultrasonic treatment is: the ultrasonic wave of the ice bath probe generates directional bubbles in the vertical direction to cut the black phosphorus powder into thin slices; the power of the ice bath probe ultrasonic treatment is 100-500W, and the time of the ice bath probe ultrasonic treatment is 2-8 hours.

5. The preparation method according to claim 2, characterized in that: The power of the water bath ultrasonic treatment is 100-500W, the temperature of the water bath ultrasonic treatment is 1-15°C, and the time of the water bath ultrasonic treatment is 5-10 hours.

6. The preparation method according to claim 2, characterized in that: The differential centrifugation process is as follows: firstly, the black phosphorus nanosheet dispersion is centrifuged at a centrifugal speed of 2-5Krpm to obtain a supernatant, and then the supernatant is centrifuged at a centrifugal speed of 8-12Krpm to obtain a precipitate, namely, the black phosphorus two-dimensional nanosheet.

7. The preparation method according to any one of claims 1 to 6, characterized in that: In step (2), the mass ratio of copper in the divalent copper salt to the mass ratio of the black phosphorus two-dimensional nanosheets is 1:(3-30).

8. The preparation method according to any one of claims 1 to 6, characterized in that: In step (2), the concentration of the dopamine aqueous solution is 50-200 mg / mL; the mass ratio of dopamine in the dopamine aqueous solution to the mass ratio of the black phosphorus two-dimensional nanosheets is (1-3):

1.

9. A two-dimensional nanomedicine, characterized in that: Prepared by the preparation method described in any one of claims 1-8, the two-dimensional nanomedicine comprises black phosphorus two-dimensional nanosheets and monovalent copper ions and dopamine attached to the black phosphorus two-dimensional nanosheets; the particle size of the two-dimensional nanomedicine is 50-300nm.

10. The two-dimensional nano drug according to claim 9, characterized in that: The black phosphorus two-dimensional nanosheet has a diameter of 100-300 nm and a thickness of 2-5 nm.

Images