Nanoreactor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of nano biology, and discloses a nano reactor and a preparation method and application thereof, and the nano reactor comprises nano particles, cell membrane nano particles wrapping the surfaces of the nano particles, and disulfiram loaded on the surfaces of the cell membrane nano particles; and the nano particles are Cu9S8 nano particles. The Cu9S8 nanoparticles are taken as a core, and cell membrane nanoparticles are wrapped outside the nanoparticles, so that homologous tumor targeting is realized, the blood circulation capability is improved, a nanoreactor can be quickly targeted to a tumor part, and the influence on normal tissues / cells is reduced; the chemotherapeutic drug nano-reactor can be combined with photothermal therapy, Cu < 2 + > is quickly converted into Cu < + > under the assistance of the photothermal therapy so as to be chelated with disulfiram, so that the chemotherapeutic drug can be synthesized in situ in a tumor microenvironment by the nano-reactor, and an excellent anti-tumor effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of nano-biotechnology, and in particular to a nano-reactor and a preparation method and application thereof. Background Art

[0002] In recent years, the incidence and mortality of cancer have increased rapidly. Chemotherapy is currently one of the main methods for clinical treatment of cancer. However, the selectivity and targeting of chemotherapy drugs are still poor, and there are disadvantages such as large side effects, easy to develop drug resistance and systemic toxicity. At present, in situ generation of chemotherapy drugs in the tumor microenvironment is an emerging chemotherapy strategy that can greatly improve the therapeutic effect and reduce adverse side effects. Therefore, it has become a research hotspot in tumor chemotherapy. However, it has been difficult to achieve artificially controllable promotion of low-toxicity prodrugs to be converted into chemotherapy drugs in situ in the tumor to improve their therapeutic effect.

[0003] In order to achieve the best therapeutic effect, the idea of ​​"synergistic therapy" was proposed. By cleverly combining the advantages of multiple treatment methods, the synergistic treatment effect is better than the simple addition of multiple treatment methods. For example, chemotherapy can be used in synergistic treatment of tumors with photodynamic therapy, photothermal therapy, chemodynamic therapy, etc., which not only plays the advantages of each treatment method, but also overcomes chemotherapy resistance. However, the nanomedicines used in the existing synergistic treatment process are not highly targeted to tumors. Therefore, it is urgent to provide a nanomedicine with excellent tumor targeting, so that it can synthesize chemotherapy drugs in situ in the tumor microenvironment, and at the same time achieve a better anti-tumor effect through synergistic treatment. Summary of the invention

[0004] In view of this, the present invention provides a nanoreactor, which has excellent tumor targeting and can synthesize chemotherapy drugs in situ in the tumor microenvironment to achieve excellent anti-tumor effects.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] On the one hand, the present invention provides a nanoreactor, comprising: nanoparticles, cell membrane nanoparticles wrapped on the surface of the nanoparticles, and disulfiram loaded on the surface of the cell membrane nanoparticles; the nanoparticles are Cu9S8 nanoparticles.

[0007] Preferably, the cell membrane nanoparticles are tumor cell membrane nanoparticles modified with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and polyethylene glycol (DSPE-PEG).

[0008] Preferably, the particle size of the nanoreactor is 93-360 nm.

[0009] Preferably, the disulfiram is loaded on the surface of the cell membrane nanoparticles by electrostatic adsorption.

[0010] Preferably, the cell membrane nanoparticles are coated on the surface of the nanoparticles by extrusion.

[0011] Preferably, the mass ratio of the Cu9S8 nanoparticles, the cell membrane nanoparticles and the disulfiram is (0-20):300:(0-10), and the mass of the Cu9S8 nanoparticles and the disulfiram is not 0.

[0012] On the other hand, the present invention also provides a method for preparing the nanoreactor described in any one of the above, comprising the following steps:

[0013] (1) Cu9S8 nanoparticles and cell membrane nanoparticles are mixed and then extruded to obtain Cu9S8@CM nanoparticles;

[0014] (2) dispersing the Cu9S8@CM nanoparticles in a solution to obtain a Cu9S8@CM dispersion;

[0015] (3) The Cu9S8@CM dispersion and the disulfiram solution are mixed to obtain a nanoreactor in the solution.

[0016] Preferably, the concentration of the Cu9S8@CM dispersion in step (2) is 0.3-0.32 mg / mL;

[0017] In step (3), the volume ratio of the Cu9S8@CM dispersion to the disulfiram solution is 999:1, and the concentration of the disulfiram solution is 0-10 mg / mL and is not 0.

[0018] Preferably, the preparation method of the Cu9S8 nanoparticles is as follows: after mixing a polyvinyl pyrrolidone solution and a copper salt, sodium hydroxide is added to adjust the pH value, and then a reducing agent and a sulfur-containing inorganic salt are added to carry out a sulfidation reaction to obtain Cu9S8 nanoparticles.

[0019] Preferably, the mass ratio of the polyvinyl pyrrolidone to the copper salt is 1:0.04-0.08, and the copper salt includes any one or more of copper acetate, copper chloride and copper sulfate.

[0020] Preferably, the temperature of the vulcanization reaction is 60-70° C. and the time is 1-3 hours.

[0021] Preferably, the sulfur-containing inorganic salt includes any one or more of sodium hydrosulfide, sodium sulfide and sodium thiosulfate.

[0022] Furthermore, the present invention also provides a use of the nanoreactor described in any one of the above items or the nanoreactor prepared by any one of the above preparation methods in the preparation of anti-tumor drugs for combined photothermal-chemotherapy treatment.

[0023] The present invention provides a nanoreactor, which has the following beneficial effects compared with the prior art:

[0024] The present invention uses Cu9S8 nanoparticles as the core, and achieves homologous tumor targeting and improved blood circulation capacity by wrapping cell membrane nanoparticles outside the nanoparticles, which enables the nanoreactor to quickly target the tumor site and reduce the impact on normal tissues / cells.

[0025] The chemotherapy drug nanoreactor of the present invention can be combined with photothermal therapy. Under the assistance of photothermal therapy, Cu 2+ Rapid conversion to Cu + Thus, it chelates with disulfiram, so that the nanoreactor can synthesize chemotherapy drugs in situ in the tumor microenvironment and achieve excellent anti-tumor effects.

[0026] When applied to photothermal therapy, the nanoreactor of the present invention can enhance the permeability of cell membranes, increase the accumulation of drugs in cells and reduce the production of heat shock proteins, increase the sensitivity of tumor cells to chemotherapeutic drugs, and enhance the effect of chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0028] Figure 1 This is the electron microscope image of Cu9S8@CM@DSF;

[0029] Figure 2 is the particle size distribution diagram of Cu9S8@CM@DSF;

[0030] Figure 3 UV-visible near-infrared absorption spectra of different samples;

[0031] Figure 4 This is a graph showing the effects of different concentrations of Cu9S8 and DSF on 4T1 cell viability;

[0032] Figure 5 The photothermal heating curves of Cu9S8@CM@DSF with different concentrations;

[0033] Figure 6The uptake of Cu9S8@DSF and Cu9S8@CM@DSF by 4T1 tumor cells at different times;

[0034] Figure 7 This is a diagram showing the killing effect of Cu9S8@CM@DSF on 4T1 tumor cells;

[0035] Figure 8 In vivo fluorescence imaging to monitor the tumor enrichment of Cu9S8@DSF and Cu9S8@CM@DSF;

[0036] Fig. 9 This is the distribution diagram of Cu9S8@DSF and Cu9S8@CM@DSF in isolated mouse organs. DETAILED DESCRIPTION

[0037] The present invention will be described below by specific examples, and it will be appreciated by those skilled in the art that the following specific examples are only for illustrative purposes, and do not limit the scope of the present invention in any way. In addition, in the following examples, unless otherwise specified, the reagents and equipment used are all commercially available. If in the following examples, specific treatment conditions and treatment methods are not clearly described, then conditions and methods known in the art can be used to process.

[0038] In one aspect of the present invention, the present invention proposes a nanoreactor, comprising: nanoparticles, cell membrane nanoparticles wrapped on the surface of the nanoparticles, and disulfiram loaded on the surface of the cell membrane nanoparticles; the nanoparticles are Cu9S8 nanoparticles.

[0039] In the present invention, Cu9S8 nanoparticles are used as the copper source and core of the nanoreactor. By wrapping cell membrane nanoparticles on the surface of the nanoparticles, the ability of homologous tumor targeting and blood circulation improvement can be achieved, so that the nanoreactor can quickly target the tumor site and reduce the impact on normal tissues / cells.

[0040] In some embodiments of the present invention, the cell membrane nanoparticles are coated on the surface of the nanoparticles by extrusion, and the coating thickness is about 11.56 nm; the cell membrane nanoparticles are tumor cell membrane nanoparticles modified by 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and polyethylene glycol (DSPE-PEG), DSPE-PEG is purchased from the market, wherein the molecular weight of polyethylene glycol can be 2000-5000, preferably DSPE-PEG 5000 Modified tumor cell membrane nanoparticles by DSPE-PEG 5000Modification onto tumor cell membrane nanoparticles allows PEGylation of the tumor cell membrane nanoparticles, which not only inherits the homologous targeting of tumor cell membrane nanoparticles, but also improves the circulation ability of the nanoparticles in the blood.

[0041] In some embodiments of the present invention, the particle size of the nanoreactor is 93-360 nm, for example, 93 nm, 100 nm, 200 nm, 300 nm, 360 nm, etc.

[0042] In some embodiments of the present invention, the disulfiram is loaded on the surface of the cell membrane nanoparticles by electrostatic adsorption.

[0043] The chemotherapy drug nanoreactor of the present invention can be combined with photothermal therapy. Under the assistance of photothermal therapy, Cu 2+ Rapid conversion to Cu + Thus, it chelates with disulfiram, so that the nanoreactor can synthesize chemotherapy drugs in situ in the tumor microenvironment and achieve excellent anti-tumor effects.

[0044] In some embodiments of the present invention, the mass ratio of the Cu9S8 nanoparticles, the cell membrane nanoparticles and the disulfiram is (0-20):300:(0-10), and the masses of the Cu9S8 nanoparticles and the disulfiram are not zero.

[0045] It should be noted that the nanoreactor of the present invention exists in the form of a mixed solution of water and ethanol (or PBS buffer and ethanol), and the volume ratio of ethanol to water / PBS buffer is 1:999, that is, ethanol accounts for 0.1% of the volume of the mixed solution. Wherein, water or PBS buffer is used as a dispersion liquid of cell membrane nanoparticles wrapped with nanoparticles, and ethanol is used as a solvent for disulfiram. In the final mixed solution, the concentration of Cu9S8 nanoparticles is 0-20μg / ml and is not 0, for example, it can be 2μg / ml, 5μg / ml, 7.5μg / ml, 10μg / ml, 20μg / ml, etc., the concentration of cell membrane nanoparticles is 0.3mg / mL, and the concentration of disulfiram is 0-10μg / mL and is not 0, for example, it can be 2μg / mL, 4μg / mL, 6μg / mL, 8μg / mL, 10μg / mL, etc.

[0046] In another aspect of the present invention, the present invention provides a method for preparing the nanoreactor described in any one of the above, comprising the following steps:

[0047] (1) Cu9S8 nanoparticles were mixed with cell membrane nanoparticles and then extruded to obtain Cu9S8@CM nanoparticles.

[0048] In some embodiments of the present invention, Cu9S8 nanoparticles are mixed with cell membrane nanoparticles, and then the mixture is extruded through a polycarbonate film (PC film) using an extruder. The pore size of the polycarbonate film can be, for example, 400 nm, and the number of extrusions can be 3 times, 5 times, 7 times, 9 times, etc. There is no special limitation on this and it can be adjusted according to actual conditions.

[0049] In some embodiments of the present invention, the preparation method of the Cu9S8 nanoparticles is as follows: after mixing a polyvinyl pyrrolidone solution and a copper salt, sodium hydroxide is added to adjust the pH value, and then a reducing agent and a sulfur-containing inorganic salt are added to carry out a sulfidation reaction to obtain Cu9S8 nanoparticles. In the preparation method of Cu9S8 nanoparticles, sodium hydroxide reacts with copper ions in the copper salt to generate copper hydroxide, and the copper hydroxide is reduced to cuprous oxide by adding a reducing agent, and the cuprous oxide and the sulfur-containing inorganic salt undergo a sulfidation reaction to form Cu9S8.

[0050] In some embodiments of the present invention, the polyvinyl pyrrolidone may be polyvinyl pyrrolidone K30, and the solvent used to dissolve the polyvinyl pyrrolidone may be water or an organic solvent such as ethanol or ethylene glycol. The polyvinyl pyrrolidone not only acts as a stabilizer, but also helps to form a nanostructure with uniform and controllable size by regulating the growth rate and morphology of the crystal; the mass ratio of the polyvinyl pyrrolidone to the copper salt is 1:0.04-0.08, for example, 1:0.04, 1:0.05, 1:0.06, 1:0.07, 1:0.08, etc. If the amount of copper salt added is too little, the yield of Cu9S8 nanoparticles will be reduced; The copper salt includes any one or more of copper acetate, copper chloride and copper sulfate, which are used as the copper source for synthesizing Cu9S8 nanoparticles; the temperature of the sulfidation reaction is 60-70°C, the time is 1-3h, for example, it can be 60°C, 65°C, 70°C, etc., and the reaction time is 1-3h, for example, it can be 1h, 2h, 3h, etc. If the reaction temperature is too high, the reducing agent may be decomposed and inactivated, and the reducing ability may be reduced. If the reaction time is too low, it will lead to incomplete growth, reduction and sulfidation of copper nanoparticles; the sulfur-containing inorganic salt includes any one or more of sodium hydrosulfide, sodium sulfide and sodium thiosulfate, which are used to sulfide the copper source into Cu9S8.

[0051] In some specific embodiments of the present invention, the preparation method of the Cu9S8 nanoparticles is as follows: 720 mg of polyvinyl pyrrolidone K30 (weight average molecular weight of 44000-54000) is dissolved in 75 mL of water, and 25.5 mg of CuCl2·2H2O is added under stirring, and after 20 minutes, NaOH solution is added to adjust the pH to 9, and stirring is continued for 5 minutes, and then 19.2 mL of hydrazine hydrate is added and reacted for 5 minutes; 1.067 mL of Na2S·9H2O (320 mg / mL) is added at 60°C, and stirred for 2 hours to obtain a product, and the product is centrifuged and washed twice with ethanol to obtain Cu9S8 nanoparticles.

[0052] In some other specific embodiments of the present invention, the preparation method of the Cu9S8 nanoparticles is as follows: 1g of polyvinyl pyrrolidone (PVP, weight-average molecular weight is 29000) is dissolved in 30mL of ethylene glycol, 80mg of cupric acetate is added under stirring, after 2h, NaOH solution is added to adjust the pH to 9, and stirring is continued for 30min, then 264.2mg of ascorbic acid is added, and after reduction at 70°C for 30min, it is washed twice with H2O, dispersed in water, and then continuously sulfurized for 2h with 1mL of NaHS (235mg / ml) to obtain a product, the product is centrifuged and washed twice with deionized water to obtain Cu9S8 nanoparticles.

[0053] In some embodiments of the present invention, the cell membrane nanoparticles are prepared by conventional methods, and the steps are as follows: 4T1 cells are digested with trypsin-diaminetetraacetic acid (Trypsin-EDTA, 0.05%), resuspended with phosphate buffered saline (PBS), and the remaining PBS is removed after centrifugation; the harvested 4T1 cells are treated with RIPA lysis buffer containing 0.1 mol / L phenylmethylsulfonyl fluoride (PMSF) at 4°C for 1 hour, and then ultrasonicated for 2 minutes (2s-on, 5s-off, 20% power) in an ultrasonic cell disruption system with an ice bath to completely lyse the cells; the cell suspension after lysis is centrifuged at low speed for 15 minutes to precipitate intracellular impurities, the supernatant is collected, and ultracentrifuged for 1 hour to obtain cell membrane nanoparticle fragments, and the cell membrane nanoparticle fragments are suspended in PBS after washing with PBS three times, in which tumor cell membrane nanoparticles, phospholipid polyethylene glycol (DSPE-PEG 5000 ) concentration ratio of 1:2, placed in a shaker, mixed at 37 ° C for 2 h, and finally used an extruder to extrude the mixture through a PC membrane with a pore size of 400 nm for 7 times to obtain uniform PEG-modified cell membrane nanoparticles (CM), which were stored at 4 ° C for use.

[0054] (2) Dispersing Cu9S8@CM nanoparticles in a solution to obtain a Cu9S8@CM dispersion.

[0055] In some embodiments of the present invention, the concentration of the Cu9S8@CM dispersion in step (2) is 0.3-0.32 mg / mL; the solution can be water or PBS buffer.

[0056] In the Cu9S8@CM dispersion, the concentration of Cu9S8 nanoparticles is 0-20 μg / ml and is not 0, for example, it can be 2 μg / ml, 5 μg / ml, 7.5 μg / ml, 10 μg / ml, 20 μg / ml, etc. The concentration of cell membrane nanoparticles is 0.3 mg / mL. If the concentration of Cu9S8 nanoparticles is too high, some cell membrane nanoparticles will not be effectively wrapped on the surface of Cu9S8 nanoparticles.

[0057] (3) The Cu9S8@CM dispersion and the disulfiram solution are mixed to obtain a nanoreactor in the solution.

[0058] In some embodiments of the present invention, the concentration of the disulfiram solution is 0-10 mg / mL, for example, it can be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, etc.; the volume ratio of the Cu9S8@CM dispersion and the disulfiram solution is 999:1, for example, when 999 μL of Cu9S8@CM dispersion is added, 1 μL of disulfiram solution needs to be added.

[0059] In some embodiments of the present invention, in the solution, the concentration of Cu9S8 nanoparticles is 0-20 μg / ml and is not 0, for example, it can be 2 μg / ml, 5 μg / ml, 7.5 μg / ml, 10 μg / ml, 20 μg / ml, etc., the concentration of cell membrane nanoparticles is 0.3 mg / mL, and the concentration of disulfiram is 0-10 μg / mL and is not 0, preferably 3.125-100 ng / mL, for example, it can be 3.125 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 80 ng / mL, 100 ng / mL, etc.

[0060] In some embodiments of the present invention, the mixing may be performed in a homogenizer. Since the purpose of the mixing is to evenly mix the Cu9S8@CM dispersion and the disulfiram solution, there is no particular limitation on the mixing time, which can be adjusted according to actual conditions.

[0061] In another aspect of the present invention, the present invention also provides an application of the nanoreactor described in any one of the above or the nanoreactor prepared by any one of the preparation methods described in the above in the photothermal-chemotherapy combined treatment of anti-tumor drugs.

[0062] The technical scheme of the present invention will be described clearly and completely below in conjunction with the accompanying drawings and embodiments. The embodiments of the present application are only used as examples, and all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0063] Example 1

[0064] This embodiment provides a method for preparing a nanoreactor, and the specific steps are as follows:

[0065] (1) Preparation of Cu9S8 nanoparticles: 720 mg of polyvinylpyrrolidone K30 (weight average molecular weight of 44000-54000) was dissolved in 75 mL of water, and 25.5 mg of CuCl2·2H2O was added under stirring. After 20 minutes, NaOH solution was added to adjust the pH to 9, and the mixture was stirred for 5 minutes. Then, 19.2 mL of hydrazine hydrate was added to react for 5 minutes. 1.067 mL of Na2S·9H2O (320 mg / mL) was added at 60° C. and stirred for 2 hours to obtain a product. The product was centrifuged and washed twice with ethanol to obtain Cu9S8 nanoparticles.

[0066] (2) Preparation of cell membrane nanoparticles: 4T1 cells were digested with trypsin-EDTA (0.05%), resuspended with phosphate buffered saline (PBS), and the remaining PBS was removed after centrifugation; the harvested 4T1 cells were treated with RIPA lysis buffer containing 0.1 M phenylmethylsulfonyl fluoride at 4°C for 1 h, and then sonicated for 2 min (2s-on, 5s-off, 20% power) in an ultrasonic cell disruption system with an ice bath to completely lyse the cells; the lysed cell suspension was centrifuged at low speed (500g) for 15 min to precipitate intracellular impurities, the supernatant was collected, and ultracentrifuged (100000g) for 1 h to obtain cell membrane nanoparticle fragments, which were washed with PBS three times and then suspended in PBS, in which tumor cell membrane nanoparticles, DSPE-PEG 5000 After mixing at a concentration ratio of 1:2, place in a shaker and mix at 37°C for 2 h. Finally, use an extruder to extrude the mixture through a PC membrane with a pore size of 400 nm for 7 times to obtain uniform PEG-modified cell membrane nanoparticles (CM), which are stored at 4°C for later use:

[0067] (3) Cu9S8 nanoparticles and cell membrane nanoparticles were mixed at a mass ratio of 7.5:300, and then the mixture was extruded through a polycarbonate film with a pore size of 400 nm using an extruder for 7 times to obtain Cu9S8@CM nanoparticles;

[0068] (4) The Cu9S8@CM nanoparticles were dispersed in water to obtain a Cu9S8@CM dispersion with a concentration of 0.3075 mg / mL. The Cu9S8@CM dispersion and a disulfiram (DSF) ethanol solution with a concentration of 10 mg / mL were mixed in a ratio of 999 μL:1 μL, and the mixture was placed in a mixer and mixed for 4 h to obtain a nanoreactor Cu9S8@CM@DSF in the solution. In the solution containing the nanoreactor, the concentration of Cu9S8 nanoparticles was 7.5 μg / mL, the concentration of cell membrane nanoparticles was 0.3 mg / mL, and the concentration of DSF was 10 μg / mL.

[0069] like Figure 1 The electron microscope image of Cu9S8@CM@DSF prepared in this example is shown. Figure 1 It can be seen that Cu9S8@CM@DSF presents a hollow spherical morphology with a diameter of 178.79 nm, and the two edge lines prove that the cell membrane nanoparticles are successfully encapsulated in the nanoparticles, and the encapsulation thickness of the cell membrane nanoparticles is 11.56 nm.

[0070] like Figure 2 , which is a particle size distribution diagram of Cu9S8@CM@DSF prepared in this example. Cu9S8@CM@DSF was dispersed in phosphate buffer, and its hydrodynamic diameter was measured by dynamic light scattering. The results showed that its particle size ranged from 93 to 360 nm, with an average diameter of 181.63 nm.

[0071] like Figure 3 As shown in A, it is a UV-visible near infrared absorption spectrum of DSF, Cu9S8, CM, Cu9S8@CM, and Cu9S8@CM@DSF in Example 1 detected by UV-NIR-visible spectrophotometer. It can be seen from the figure that Cu9S8 has UV absorption in the NIR-II region (1000-1200nm); after being wrapped by cell membrane nanoparticles (CM), it shows a characteristic peak of CM at about 205nm, proving that Cu9S8 is successfully wrapped by CM; after loading DSF, the UV absorption peak of CM at 205nm is enhanced, and the characteristic absorption peak of DSF is shown at about 216nm, proving that DSF is successfully loaded on Cu9S8@CM.

[0072] Comparative Example 1

[0073] The present invention provides a method for preparing a nanoreactor, and the specific steps are as follows:

[0074] (1) Preparation of Cu9S8 nanoparticles: 720 mg of polyvinylpyrrolidone K30 (weight average molecular weight of 44000-54000) was dissolved in 75 mL of water, and 25.5 mg of CuCl2·2H2O was added under stirring. After 20 minutes, NaOH solution was added to adjust the pH to 9, and the mixture was stirred for 5 minutes. Then, 19.2 mL of hydrazine hydrate was added to react for 5 minutes. 1.067 mL of Na2S·9H2O (320 mg / mL) was added at 60° C. and stirred for 2 hours to obtain a product. The product was centrifuged and washed twice with ethanol to obtain Cu9S8 nanoparticles.

[0075] (2) Cu9S8 nanoparticles were dispersed in water to obtain a Cu9S8 solution with a concentration of 7.5 μg / mL. The Cu9S8 solution and a disulfiram (DSF) ethanol solution with a concentration of 10 mg / mL were mixed in a ratio of 999 μL:1 μL, and the mixture was placed in a mixer and mixed for 4 h to obtain Cu9S8@DSF in the solution. In the solution containing Cu9S8@DSF, the concentration of Cu9S8 was 7.5 μg / mL, and the concentration of DSF was 10 μg / mL.

[0076] Test Example 1

[0077] In vitro, the physiological environment (pH = 7.4) and the lysosome environment (pH = 5.0) in tumor cells were simulated to detect the ultraviolet absorption of Cu9S8@DSF of Comparative Example 1 and Cu9S8@CM@DSF of Example 1. The results are as follows: Figure 3 As shown in B, the Cu9S8@DSF curve represents the ultraviolet absorption of Cu9S8@DSF in a simulated physiological environment (pH = 7.4) in vitro, the Cu9S8@CM@DSF curve represents the ultraviolet absorption of Cu9S8@CM@DSF in a simulated physiological environment (pH = 7.4) in vitro, and the Degraded curve represents the ultraviolet absorption of Cu9S8@CM@DSF in the lysosome environment (pH = 5.0) in tumor cells.

[0078] from Figure 3As can be seen from B, Cu9S8 and DSF can form Cu9S8@DSF through chelation at pH = 7.4, and its characteristic absorption peak is about 450nm. When simulating the physiological environment, Cu9S8@CM@DSF did not show the characteristic absorption peak of Cu9S8@DSF at 450nm because CM successfully isolated Cu9S8 from DSF. When simulating the lysosome environment in tumor cells, the absorption peak of CM at about 205nm decreased, proving that CM was destroyed, and the characteristic absorption peak of DSF reappeared at about 216nm. At the same time, new absorption peaks were shown at about 260nm and 423nm, proving that DSF was released and successfully synthesized with Cu9S8 to form the chemotherapy drug Cu9S8@DSF, and the absorption peaks originally located at 275nm and 450nm were blue-shifted.

[0079] Test Example 2

[0080] 4T1 tumor cells were seeded in 96-well plates at 1×10 5 The cells were cultured at 5% CO2 and 37°C for 12 h, and then treated with fresh 1640 medium containing different DSF concentrations (0, 1, 2, 4, 6, 8 μg / mL) and fresh 1640 medium containing different Cu9S8 concentrations (0, 2.5, 5, 7.5, 10, 20 μg / mL) for 24 h. The effects of different concentrations of DSF and Cu9S8 on 4T1 cell viability were determined by standard CCK-8 method. The results are shown in Figure 4 shown.

[0081] Depend on Figure 4 It can be seen that within the concentration range of 0-8ug / mL, any concentration of DSF will not reduce the viability of 4T1 cells, and DSF has no killing effect on 4T1 cells; Cu9S8 has no significant effect on the viability of 4T1 cells within the concentration range of 0-7.5μg / mL, and has the characteristics of high safety.

[0082] Test Example 3

[0083] The effect of the concentration of Cu9S8 nanoparticles in Cu9S8@CM@DSF on the photothermal heating curve was detected. 100uL Cu9S8@CM@DSF containing different concentrations of Cu9S8 nanoparticles was placed in a centrifuge tube and exposed to NIR-Ⅱ at 1064nm (0.7W / cm 2 ) laser, the solution temperature was detected by infrared thermal imager, with deionized water as control. The results are as follows Figure 5 shown.

[0084] Depend on Figure 5It can be seen that under NIR-Ⅱ illumination, when the concentration of Cu9S8 nanoparticles in Cu9S8@CM@DSF is 7.5 μg / mL, the temperature can be rapidly increased and stabilized in a suitable range below 45°C, meeting the temperature conditions required for mild photothermal treatment.

[0085] Test Example 4

[0086] 4T1 cells were seeded in 15 mm glass-bottom culture dishes at a rate of 1 × 10 4 After incubation for 12 h under standard conditions, the cells were treated with fresh culture medium containing Cu9S8@DSF of Comparative Example 1 and Cu9S8@CM@DSF of Example 1 labeled with Cy5.5 for 4 h. At 1 h and 4 h of co-incubation, the cells in the confocal dish were rinsed with PBS, fixed with paraformaldehyde for 15 min, and the cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Finally, the uptake of nanoparticles by tumor cells was recorded using a confocal microscope. The results are shown in FIG. Figure 6 shown.

[0087] from Figure 6 It can be seen that compared with Cu9S8@DSF, Cu9S8@CM@DSF can be quickly taken up into tumor cells.

[0088] Test Example 5

[0089] The standard CCK-8 method was used to determine the effect of Cu9S8@CM@DSF on cell viability. 4T1 tumor cells were seeded in 96-well plates, with 1×10 5 The cells were cultured at 5% CO2 and 37°C for 12 h, and then treated with fresh 1640 medium containing Cu9S8@CM@DSF with different DSF concentrations (0, 3.125, 6.25, 12.5, 25, and 50 ng / mL). After 4 h of treatment, the cells were exposed to laser irradiation for 10 min (1064 nm, 0.7 W / cm 2 ), and then cultured for another 24 hours. Finally, after washing with PBS solution, the culture medium in each well was replaced with 1640 culture medium containing CCK-8 solution (100μL, 10% CCK-8), and incubated for another 15 minutes. The light absorption at a wavelength of 450nm was measured with an ELISA reader to evaluate the viability of the 4T1 tumor cells after treatment. The experiment set up an illumination group and a non-illumination group. The results are shown in Figure 7 shown.

[0090] from Figure 7 It can be seen that when combined with NIR-Ⅱ mild photothermal treatment, the killing effect of Cu9S8@CM@DSF on 4T1 tumor cells was significantly enhanced, proving that mild photothermal treatment can accelerate the in situ synthesis of the chemotherapy drug Cu9S8@DSF and enhance the efficacy of chemotherapy.

[0091] It should be noted that Test Example 2 is a cell experiment, using a higher concentration of DSF solution, the purpose of which is to verify whether DSF can kill tumor cells. The results show that the concentration of 0-8ug / mL DSF solution has no effect on tumor cells. Test Example 5 is the optimal concentration of DSF in the Cu9S8@CM@DSF reactor determined after testing. The test results show that when the DSF concentration is 50ng / mL, Cu9S8@CM@DSF has the best killing effect on tumor cells.

[0092] Test Example 6

[0093] The Cu9S8@DSF chemotherapy drug of Comparative Example 1 and the Cu9S8@CM@DSF nanoreactor of Example 1 were labeled with Cy 5.5 dye, respectively, and injected into 4T1 tumor-bearing BALB / C mice through the tail vein. The distribution of Cu9S8@DSF and Cu9S8@CM@DSF in the mice at different time points was recorded. Then, the mice were killed after 24 hours, and the heart, liver, spleen, lung, kidney, and tumor were removed. In vitro fluorescence imaging was performed using a small animal in vivo imaging system. The results are shown in FIG. Figure 8 and Fig. 9 shown.

[0094] from Figure 8 and Fig. 9 It can be seen that compared with Cu9S8@DSF chemotherapy drugs, the Cu9S8@CM@DSF nanoreactor can target the tumor site very quickly, thereby generating chemotherapy drugs in situ at the tumor site, and at 24 hours, there is little or even no accumulation in other organs, showing an excellent tumor targeting effect.

[0095] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A nanoreactor, characterized in that: include: Nanoparticles, cell membrane nanoparticles coated on the surface of the nanoparticles, and disulfiram loaded on the surface of the cell membrane nanoparticles; The nanoparticles are Cu9S8 nanoparticles.

2. The nanoreactor according to claim 1, characterized in that: The cell membrane nanoparticles are tumor cell membrane nanoparticles modified with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and polyethylene glycol; The particle size of the nanoreactor is 93-360nm.

3. The nanoreactor according to claim 1 or 2, characterized in that: The disulfiram is loaded on the surface of the cell membrane nanoparticles by electrostatic adsorption; The cell membrane nanoparticles are wrapped on the surface of the nanoparticles by an extrusion method.

4. The nanoreactor according to claim 1, characterized in that: The mass ratio of the Cu9S8 nanoparticles, the cell membrane nanoparticles and the disulfiram is (0-20):300:(0-10), and the masses of the Cu9S8 nanoparticles and the disulfiram are not zero.

5. A method for preparing a nanoreactor according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Cu9S8 nanoparticles and cell membrane nanoparticles are mixed and then extruded to obtain Cu9S8@CM nanoparticles; (2) dispersing the Cu9S8@CM nanoparticles in a solution to obtain a Cu9S8@CM dispersion; (3) The Cu9S8@CM dispersion and the disulfiram solution are mixed to obtain a nanoreactor in the solution.

6. The method for preparing a nanoreactor according to claim 5, characterized in that: The concentration of the Cu9S8@CM dispersion in step (2) is 0.3-0.32 mg / mL; In step (3), the volume ratio of the Cu9S8@CM dispersion to the disulfiram solution is 999:1, and the concentration of the disulfiram solution is 0-10 mg / mL and is not 0.

7. The method for preparing a nanoreactor according to claim 5, characterized in that: The preparation method of the Cu9S8 nanoparticles is as follows: after mixing a polyvinyl pyrrolidone solution and a copper salt, sodium hydroxide is added to adjust the pH value, and then a reducing agent and a sulfur-containing inorganic salt are added to carry out a sulfurization reaction to obtain Cu9S8 nanoparticles.

8. The method for preparing a nanoreactor according to claim 7, characterized in that: The mass ratio of the polyvinyl pyrrolidone to the copper salt is 1:0.04-0.08, and the copper salt includes any one or more of copper acetate, copper chloride and copper sulfate.

9. The method for preparing a nanoreactor according to claim 7 or 8, characterized in that: The temperature of the vulcanization reaction is 60-70°C and the time is 1-3h; The sulfur-containing inorganic salt includes any one or more of sodium hydrosulfide, sodium sulfide and sodium thiosulfate.

10. Use of the nanoreactor according to any one of claims 1 to 4 or the nanoreactor prepared by the preparation method according to any one of claims 5 to 9 in the preparation of anti-tumor drugs for combined photothermal-chemotherapy treatment.

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