Pyroelectric nanoparticles based on black phospho-Fe < 3 + > complex as well as preparation method and application of pyroelectric nanoparticles

Abstract

The invention discloses a pyroelectric nano-particle based on a black phosphoryl-Fe < 3 + > complex as well as a preparation method and application of the pyroelectric nano-particle. The pyroelectric nanoparticle comprises a cell membrane and a black phosphorus-Fe < 3 + > complex wrapped in the cell membrane. The preparation method comprises the following steps: uniformly shaking the cell membrane and the black phosphoryl-Fe < 3 + > complex in an air bath, and extruding by using a liposome extruder. Under laser on / off irradiation, the prepared nano-particles are triggered by temperature change to generate pyroelectric catalysis for water decomposition in tumor intercellular fluid, aggregation of the nano-drug at a tumor site is improved, tumor cells are further efficiently killed, and in the catalysis process, along with generation of oxygen and ROS, polarization of tumor-related macrophages is further induced, so that the tumor-related macrophages are effectively killed, and the tumor-related macrophages are effectively killed. Anti-tumor immune response is activated, and tumor recurrence and metastasis are inhibited.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and in particular to a method for treating tumors based on black phosphorus-Fe 3+ Pyroelectric nanoparticles of complexes and preparation method and application thereof. Background Art

[0002] Traditional photocatalytic processes are usually limited by the penetration depth in biological tissues and cannot achieve efficient catalysis in vivo. As an emerging catalytic strategy, pyroelectrocatalysis can effectively absorb photons in a short period of time and quickly convert them into heat energy, which in turn causes temperature changes and separates electrons and holes, thereby releasing the charges bound to its surface, causing redox reactions, and achieving efficient catalysis of tumor interstitial fluid water decomposition, enhancing the accumulation of nanomedicines at the tumor site. In addition, reactive oxygen free radicals (ROS) are generated during the catalytic process, further blocking the unlimited proliferation of tumor cells and activating the body's immune response. Therefore, pyroelectrocatalysis can improve the therapeutic effect of tumors by enhancing the accumulation of nanomedicines in tumors.

[0003] At present, pyroelectric catalysis shows great application potential in various medical fields. In addition to high catalytic activity, pyroelectric materials can also achieve surface charge polarization under temperature changes. Positive and negative charges can catalyze substrate redox reactions or intervene in biological reaction processes, promote the production of effector molecules, and have high biosafety. They are widely used in biomedical fields, such as decontamination, sterilization and tumor treatment. Summary of the invention

[0004] In view of the above background technology, the present invention provides a black phosphorus-Fe 3+ Pyroelectric nanoparticles of complexes and preparation method and application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In one aspect, the present invention provides a black phosphorus-Fe 3+ The pyroelectric nanoparticles of the complex include cell membrane and black phosphorus-Fe 3+ Complex.

[0007] As a preferred embodiment, the cell membrane is a tumor cell membrane; in the technical solution of the present invention, the tumor cell membrane specifically includes breast cancer cell membrane, cervical cancer cell membrane and pancreatic cancer cell membrane.

[0008] As a preferred embodiment, the black phosphorus-Fe 3+ The preparation method of the complex comprises the following steps:

[0009] The black phosphorus nanosheets were surface treated to release coordination sites; Fe 3+ The black phosphorus-Fe 3+ In the technical solution of the present invention, Fe 3+ The successful coordination with black phosphorus nanosheets can be characterized by XPS;

[0010] Preferably, the Fe 3+ The source is soluble ferric iron salts, such as FeCl3, Fe2(SO4)3 and Fe(NO3)3;

[0011] Preferably, the Fe 3+ The mass of Fe in the source is 0.1% to 10% of the mass of the black phosphorus nanosheets;

[0012] Preferably, the temperature of the coordination reaction is 60-70°C, the time of the coordination reaction is 12-24h, the coordination reaction is carried out under stirring conditions, and the coordination reaction is carried out under light-proof conditions; in some specific embodiments, the coordination reaction further includes post-treatments of centrifugation, washing and vacuum drying;

[0013] Preferably, the surface treatment is to heat and stir the black phosphorus nanosheets in an alkaline solution;

[0014] In some specific embodiments, the alkaline solution is a mixed solution of ammonia water and N-methylpyrrolidone (NMP); in the technical solution of the present invention, the alkaline environment can enhance the deprotonation of the ligand, making it easier to coordinate with the metal ion;

[0015] Preferably, the heating temperature is 40 to 80° C.; the heating time is 2 to 4 hours;

[0016] Preferably, the black phosphorus nanosheets are prepared by a probe ultrasonic method of black phosphorus crystals;

[0017] Preferably, the black phosphorus crystals are prepared by a solvothermal method of red phosphorus; the solvent of the solvothermal method is ethylenediamine; the temperature of the solvothermal method is 140 to 180°C; the time of the solvothermal method is 12 to 24 hours; in some specific embodiments, the solvothermal method further includes post-treatments of centrifugation, washing and drying.

[0018] As a preferred embodiment, the black phosphorus-Fe 3+ The mass ratio of the complex to the cell membrane is 2 to 8:1.

[0019] In another aspect, the present invention provides a method for preparing the above-mentioned pyroelectric nanoparticles, comprising the following steps:

[0020] Cell membrane and black phosphorus-Fe 3+The complex is shaken in an air bath and extruded using a liposome extruder to obtain the pyroelectric nanoparticles.

[0021] As a preferred embodiment, the temperature of the air bath is 36.5 to 38° C.; the time of the air bath is 12 to 24 hours.

[0022] As a preferred embodiment, the extrusion is repeated 10 to 30 times.

[0023] In some specific embodiments, the extrusion further comprises an operation of obtaining the pyroelectric nanoparticles by centrifugation.

[0024] In another aspect, the present invention provides use of the above-mentioned pyroelectric nanoparticles in the preparation of anti-tumor drugs.

[0025] In the technical solution of the present invention, the tumor includes breast cancer, cervical cancer, pancreatic cancer and the like.

[0026] The above technical solution has the following beneficial effects:

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] 1. The present invention provides a black phosphorus-Fe 3+ The pyroelectric nanoparticles of the complex use black phosphorus (BP), a typical pyroelectric material, as a carrier and combine it with Fe 3+ Coordination to form black phosphorus-Fe 3+ The pyroelectric complex is then coated with cell membrane to form nanoparticles; among them, black phosphorus-Fe 3+ The complex undergoes separation of positive and negative charges under temperature changes: the positive charge catalyzes the decomposition of water in the tumor interstitial fluid to produce oxygen, enhancing the intratumoral accumulation of nanomedicines and achieving tumor cell killing; the negative charge reacts with oxygen to generate ROS, activating the body's immune response and further inhibiting tumor recurrence and metastasis;

[0029] 2. The pyroelectric nanoparticles provided by the present invention can trigger pyroelectric catalysis in vivo through temperature changes under the stimulation of laser on / off;

[0030] 3. The pyroelectric nanoparticles provided by the present invention achieve excellent therapeutic effects and high biosafety in the treatment of tumor-bearing mice through the combined effects of nano-drug efficient intratumor targeting and immunity, and have the potential for clinical transformation;

[0031] 4. The pyroelectric nanoparticles provided by the present invention can kill tumor cells through pyroelectric catalysis, broaden the biological application of pyroelectric materials, and provide a new strategy for anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1The black phosphorus-Fe prepared in Example 1 of the present invention 3+ XPS pattern of the complex;

[0033] Figure 2 This is a TEM image of the nanoparticles prepared in Example 2 of the present invention;

[0034] Figure 3 This is a graph showing the ability of the nanoparticles prepared in Example 3 of the present invention to catalyze ROS generation;

[0035] Figure 4 This is a graph showing the ability of black phosphorus in Example 1 of the present invention to generate oxygen by pyroelectrocatalytic water decomposition under temperature changes;

[0036] Figure 5 BP-Fe in Example 1 of the present invention 3+ Macrophage polarization images after @M and BP@M were incubated with M2 phenotype tumor-associated macrophages under laser irradiation;

[0037] Figure 6 This is the cell homologous targeting map after being treated with the nanoparticles prepared in Example 2 of the present invention;

[0038] Figure 7 This is a live-death picture of cells treated with nanoparticles prepared in Example 2 of the present invention;

[0039] Figure 8 is the tumor inhibition rate of tumor-bearing mice treated with the nanoparticles prepared in Example 3 of the present invention;

[0040] Fig. 9 The weight change of tumor-bearing mice after being treated with the nanoparticles prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0041] The following embodiments are only some embodiments of the present invention, rather than all embodiments. Therefore, the detailed description in the embodiments of the present invention provided below is not intended to limit the scope of the present invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the protection scope of the present invention.

[0042] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0043] Example 1

[0044] Red phosphorus (2.5 g) was added to 15 mL of ethylenediamine, and the mixture was stirred and heated at 140°C for 12 h in a 50 mL polytetrafluoroethylene high pressure reactor; after cooling naturally to room temperature, the precipitate was collected by centrifugation, washed with deionized water and ethanol for 3 times, and dried in a vacuum drying oven at 60°C for 10 h to obtain black phosphorus (BP) crystals;

[0045] 10 mg of black phosphorus crystals were dispersed in 10 mL of N-methylpyrrolidone (NMP) and ultrasonically disrupted by a sonic tip at 1200 W for 20 h (on / off cycle: 2 s / 4 s) to prepare black phosphorus (BP) nanosheets; an aqueous ammonia solution (1.0 M, 100 μL) was added and stirred at 60 °C for 2 h to release the coordination sites on the surface of the BP nanosheets;

[0046] FeCl3·6H2O aqueous solution (5 mL, containing 50 μg FeCl3·6H2O) was added dropwise, stirred at 300 rpm for 12 h at 60 °C in the dark, and the product was centrifuged and washed with ethanol and water to remove unreacted ions to obtain black phosphorus-Fe 3+ Complex (BP-Fe 3+ );

[0047] Black phosphorus-Fe 3+ The complex (0.2 g) was mixed with 1 mL of 100 mg / mL Hela cervical cancer cell membrane solution in an air bath at 38°C overnight, and then extruded 20 times using a liposome extruder and centrifuged at 10,000 rpm for 1 hour to obtain a precipitate, which was a black phosphorus-Fe 3+ The pyroelectric nanoparticles of the complex (BP-Fe 3+ @M).

[0048] The black phosphorus-Fe prepared in this example 3+ The XPS pattern of the complex is shown in Figure 1 , the characteristic peak at 133.0 eV is attributed to Fe 3+ The coordination with BP nanosheets confirms that black phosphorus-Fe 3+ Complex.

[0049] Example 2

[0050] Red phosphorus (5 g) was added to 30 mL of ethylenediamine, and the mixture was heated at 140°C for 20 h in a 100 mL polytetrafluoroethylene high-pressure reactor after stirring; after cooling naturally to room temperature, the precipitate was collected by centrifugation, washed with deionized water and ethanol for 3 times, and dried in a vacuum drying oven at 60°C for 10 h to obtain black phosphorus crystals;

[0051] 50 mg of black phosphorus crystals were dispersed in 50 mL of N-methylpyrrolidone (NMP) and ultrasonically disrupted by a sonic tip at 1200 W for 30 h (on / off cycle: 2 s / 4 s) to prepare black phosphorus nanosheets; an aqueous ammonia solution (2.0 M, 200 μL) was added and stirred at 60 °C for 2 h to release the coordination sites on the surface of the BP nanosheets;

[0052] FeCl3·6H2O aqueous solution (12 mL, containing 0.6 mg FeCl3·6H2O) was added dropwise, stirred at 65°C and 300 rpm for 12 h in the dark, and the product was centrifuged and washed with ethanol and water to remove unreacted ions to obtain black phosphorus-Fe 3+ Complex (BP-Fe 3+ );

[0053] Black phosphorus-Fe 3+ The complex (0.2 g) was mixed with 1 mL of 50 mg / mL Hela cervical cancer cell membrane solution in an air bath at 38°C overnight, and then extruded 20 times using a liposome extruder and centrifuged at 10,000 rpm for 1 hour to obtain a precipitate, which was the black phosphorus-Fe 3+ The pyroelectric nanoparticles of the complex (BP-Fe 3+ @M).

[0054] The black phosphorus-Fe prepared in this example 3+ The complex pyroelectric nanoparticles, transmission electron microscopy (TEM) image is shown in Figure 2 ,from Figure 2 It can be observed that black phosphorus-Fe 3+ The complex completely coated the tumor cell membrane.

[0055] Example 3

[0056] Red phosphorus (1.5 g) was added to 10 mL of ethylenediamine, and the mixture was heated at 140°C for 24 h in a 100 mL polytetrafluoroethylene high-pressure reactor after stirring; after cooling naturally to room temperature, the precipitate was collected by centrifugation, washed with deionized water and ethanol for 3 times, and dried in a vacuum drying oven at 60°C for 10 h to obtain black phosphorus crystals;

[0057] 12 mg of black phosphorus crystals were dispersed in 20 mL of N-methylpyrrolidone (NMP) and ultrasonically disrupted by a sonic tip at 1200 W for 25 h (on / off cycle: 2 s / 4 s) to prepare black phosphorus nanosheets; an aqueous ammonia solution (1.0 M, 100 μL) was added and stirred at 60 °C for 2 h to release the coordination sites on the surface of the BP nanosheets;

[0058] FeCl3·6H2O aqueous solution (5 mL, containing 0.5 mg FeCl3·6H2O) was added dropwise, stirred at 300 rpm for 12 h at 70 °C in the dark, and the product was centrifuged and washed with ethanol and water to remove unreacted ions to obtain black phosphorus-Fe 3+ Complex (BP-Fe 3+ );

[0059] Black phosphorus-Fe 3+ The complex (0.2 g) was mixed with 1 mL of 25 mg / mL Hela cervical cancer cell membrane solution in an air bath at 38°C overnight, and then extruded 20 times using a liposome extruder and centrifuged at 10,000 rpm for 1 hour to obtain a precipitate, which was the black phosphorus-Fe 3+ The pyroelectric nanoparticles of the complex (BP-Fe 3+ @M).

[0060] This example tests the ability of the nanoparticles to catalyze the generation of ROS through a methylene blue degradation experiment. Figure 3 ; In the figure, MB represents methylene blue solution, BP@M+Laser and BP-Fe 3+ @M+Laser respectively represent the degradation of MB after adding corresponding nanoparticles to MB and irradiating with 808nm laser (L). The preparation process of BP@M nanoparticles is the same as that of BP-Fe in this example. 3+ @M nanoparticles, the difference is that no coordination reaction is carried out, and the tumor cell membrane is coated with black phosphorus nanosheets. As can be seen from the figure, the pyroelectric nanoparticles (BP-Fe 3+ The characteristic absorption peak of methylene blue in the BP@M+Laser group was significantly decreased compared with that in the BP@M+Laser group, which confirmed that the nanoparticles had a good ability to catalyze the generation of ROS.

[0061] Performance Test:

[0062] (1) Ability to catalyze water decomposition to produce oxygen:

[0063] The BP nanosheets in Example 1 were placed in a 15-65°C hot and cold circulating water bath, and the cycle was performed every 10 minutes. The oxygen production was detected by gas chromatography. The test results are shown in Figure 4 ;from Figure 4 It can be seen that with the increase in the number of thermal cycles, the oxygen production gradually increases, and the total oxygen production per gram of catalyst reaches 0.89 mmol after 30 hot and cold cycles.

[0064] (2)BP-Fe 3+ @M+Laser induces polarization of M2 macrophages:

[0065] M2 macrophages were incubated with the nanoparticles prepared in Example 1 overnight, and the expression levels of CD86 (a marker for M1 macrophages) and CD 206 (a marker for M2 macrophages) were detected by flow cytometry. Figure 5 As shown, compared with the saline group, BP-Fe 3+ In the M+Laser group (+Laser means that the group was irradiated with 808nm laser during incubation), the expression of CD86 increased by 6.35 times, confirming that BP-Fe 3+ @MThe ROS generated during the thermoelectrocatalytic process further induced the polarization of M2 macrophages to M1 macrophages. Figure 5 In the example, the preparation method of BP@M is the same as that of Example 1, except that the tumor cell membrane is coated with black phosphorus nanosheets.

[0066] (3) Active targeting:

[0067] The black phosphorus-Fe prepared in Example 2 3+ The pyroelectric nanoparticles of the complex were dyed red with rhodamine B and incubated with human renal epithelial (293T) cells and cervical cancer (Hela) cells. The fluorescence images are shown in Figure 6 .from Figure 6 It can be seen that the bright red signal observed in cervical cancer cells (Hela) is stronger than that in human renal epithelial cells. The reason is that the pyroelectric nanoparticles use the cervical cancer cell membrane, and the membrane protein on its surface can recognize and actively target HeLa cells to promote cell uptake. From this test, it can be seen that the pyroelectric nanoparticles prepared by the present invention have active targeting ability and can aggregate at tumor cells.

[0068] (4) Cytotoxicity to tumor cells:

[0069] The nanoparticles prepared in Example 2 were co-incubated with tumor cells for 24 hours and stimulated by laser thermal cycling 3 times, and then cell live and dead staining was performed. Figure 7 Shown: BP-Fe 3+ @The cells in group M were completely apoptotic after laser thermal stimulation (BP-Fe 3+ @M+Laser), proving that the nanoparticles prepared by the present invention have strong killing effect on tumor cells.

[0070] (5) Therapeutic effect on tumor-bearing mice:

[0071] The tumor-bearing mice were divided into 5 groups, 6 mice in each group, and treated with normal saline (500 μL), BP-Fe 3+ Nanoparticle solution (0.2 mg / mL, 500 μL) BP-Fe prepared in Example 3 3+The nanoparticle solution (0.2 mg / mL, 500 μL) was injected into the tail vein of tumor-bearing mice, and the tumor site was irradiated with 808 nm laser (1.5 W / cm 2 , 3 minutes), and the tumor volume and mouse weight changes were monitored every two days. The tumor volume of mice after different treatments is shown in Figure 8 As can be seen from the figure, BP-Fe 3+ Laser group (BP-Fe 3+ +Laser) had a lower tumor volume than BP-Fe 3+ No laser group (BP-Fe 3+ ), indicating that BP-Fe 3+ It can kill tumor cells efficiently through pyroelectric catalysis and stop the cell growth cycle. 3+ @M plus laser group (BP-Fe 3+ @M+Laser) has a higher tumor inhibition rate than BP-Fe 3+ Laser group (BP-Fe 3+ +Laser), which proves that the encapsulation of tumor cell membrane increases the targeting effect of drugs on tumor sites, thereby increasing the accumulation of drugs in tumors and improving the treatment efficiency. 3+ @M plus laser treatment group (BP-Fe 3+ @M+Laser) had a significantly higher inhibitory effect on tumor volume than all control groups, indicating that the nanomedicine can kill tumor cells and achieve efficient tumor treatment. There was no significant difference in the weight of mice in different treatment groups within 14 days of treatment ( Fig. 9 ), proving that the nanomedicine has high biosafety and great potential for clinical application. Figure 8 and Fig. 9 Medium, BP-Fe 3+ The method for preparing nanoparticles is the same as that in Example 3, except that the tumor cell membrane is not coated.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A black phosphorus-Fe 3+ The pyroelectric nanoparticles of the complex are characterized by: The pyroelectric nanoparticles include a cell membrane and black phosphorus-Fe 3+ Complex.

2. The pyroelectric nanoparticles according to claim 1, characterized in that: The cell membrane is a tumor cell membrane.

3. The pyroelectric nanoparticles according to claim 1, characterized in that: The black phosphorus-Fe 3+ The preparation method of the complex comprises the following steps: The black phosphorus nanosheets were surface treated to release coordination sites; Fe 3+ The black phosphorus-Fe 3+ Complexes; Preferably, the Fe 3+ The mass of Fe in the source is 0.1% to 10% of the mass of the black phosphorus nanosheets; Preferably, the Fe 3+ The source is a soluble ferric iron salt; Preferably, the temperature of the coordination reaction is 60-70° C., and the time of the coordination reaction is 12-24 hours.

4. The pyroelectric nanoparticles according to claim 3, characterized in that: The surface treatment is to heat and stir the black phosphorus nanosheets in an alkaline solution; Preferably, the heating temperature is 40-80° C.; and the heating time is 2-4 hours.

5. The pyroelectric nanoparticles according to claim 3, characterized in that: The black phosphorus nanosheets are prepared by a probe ultrasonic method of black phosphorus crystals; Preferably, the black phosphorus crystals are prepared by a solvothermal method of red phosphorus; Preferably, the solvent of the solvothermal reaction is ethylenediamine; the temperature of the solvothermal reaction is 140 to 180° C.; and the time of the solvothermal reaction is 12 to 24 hours.

6. The pyroelectric nanoparticles according to claim 1, characterized in that: The black phosphorus-Fe 3+ The mass ratio of the complex to the cell membrane is 2 to 8:

1.

7. The method for preparing pyroelectric nanoparticles according to any one of claims 1 to 6, characterized in that: The following steps are involved: Cell membrane and black phosphorus-Fe 3+ The complex is shaken in an air bath and extruded using a liposome extruder to obtain the pyroelectric nanoparticles.

8. The preparation method according to claim 7, characterized in that: The temperature of the air bath is 36.5-38° C.; the time of the air bath is 12-24 hours.

9. The preparation method according to claim 7, characterized in that: The extrusion is repeated 10 to 30 times.

10. Use of the pyroelectric nanoparticles according to claim 7 in the preparation of anti-tumor drugs.

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