Ultrasonic response type nano copper-based medicine as well as preparation method and application thereof

Abstract

The invention provides an ultrasonic response type nano copper-based medicine as well as a preparation method and application thereof, and belongs to the technical field of biological medicine preparation. The ultrasonic response type nano-copper-based medicine provided by the invention is of a core-shell structure, a shell layer is made of a polylactic acid-glycolic acid copolymer, and a core comprises an ultrasonic response type liquid-gas phase change material and a copper-based medicine. The ultrasonic response type nano-copper-based medicine provided by the invention can be enriched at a tumor part, ultrasonic irradiation is carried out on tumor tissues, so that the ultrasonic response type liquid-gas phase change material is subjected to phase change from a liquid state to a gas state, and a polylactic acid-glycolic acid copolymer shell can be quickly broken; the accurate, efficient and non-destructive release of the copper-based medicine for the diseased region is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine preparation, and in particular relates to an ultrasound-responsive nano-copper-based drug and a preparation method and application thereof. Background Art

[0002] Copper-based drugs not only act as anti-infective agents but also play a vital role in the prevention and treatment of cardiovascular disease and cancer. With the discovery of the mechanism by which copper induces cell death, copper-based drugs have become increasingly widely used in the treatment of cancer. Studies have shown that when intracellular copper levels exceed a certain threshold, cell death is triggered. Therefore, precise regulation of copper levels at the lesion site not only helps improve the therapeutic efficacy of the disease but also protects surrounding normal tissue.

[0003] The rapid development of nanomedicine has made nanocarriers a promising candidate for the precise delivery of copper-based drugs. Nanocarriers can selectively enrich copper-based drugs at the tumor site by adjusting their particle size and leveraging the enhanced permeability and retention effects of tumor tissue, achieving precise delivery to tumor cells. However, during delivery, some copper-based drugs escape from the nanocarriers and are released into normal tissue, causing damage. While enhancing the physical stability of nanocarriers can reduce drug off-target effects, this can also make it difficult for the copper-based drug in the nanocarrier to be released at the lesion site, reducing therapeutic efficacy. Therefore, a method for the precise controlled release of copper-based drugs is urgently needed.

[0004] As research deepens, it has been discovered that various physical and chemical stimuli can cause changes in certain substances, promoting the cleavage and release of copper-based drugs from nanocarriers, such as near-infrared light-responsive, pH-responsive, and X-ray-responsive nanomaterials. However, current stimuli-responsive copper-based drugs still face the following challenges: Near-infrared light has limited penetration. For deep lesions, although nano-copper-based drugs accumulate locally, their release rate is low due to the limited penetration of the light beam. pH-responsive nano-copper-based drugs are easily affected by the body's internal environment during transport, leading to premature release of the copper-based drugs and causing damage to normal tissues. X-rays are absorbed when passing through human tissue, limiting drug release efficiency and increasing damage to normal tissues.

[0005] Therefore, the precise, efficient and non-damaging release of copper-based drugs at the lesion site not only helps to improve the therapeutic effect of the disease, but also protects the surrounding normal tissues. Summary of the Invention

[0006] The present invention aims to provide an ultrasound-responsive nano-copper-based drug, its preparation method, and application. The ultrasound-responsive nano-copper-based drug provided by the present invention can achieve accurate, efficient, and non-destructive release of the copper-based drug at the tumor site.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides an ultrasound-responsive nano-copper-based drug having a core-shell structure, wherein the shell material of the core-shell structure is polylactic acid-glycolic acid copolymer, and the core of the core-shell structure comprises an ultrasound-responsive liquid-gas phase change material and a copper-based drug.

[0009] Preferably, the ultrasonically responsive liquid-gas phase change material includes perfluoropentane and / or perfluorohexane.

[0010] Preferably, the copper-based drug comprises tetracarboxyphenylporphyrin copper and / or copper chloride.

[0011] Preferably, the shell thickness of the ultrasound-responsive nano-copper-based drug is 10 to 15 nm.

[0012] The present invention also provides a method for preparing the ultrasound-responsive nano-copper-based drug described in the above technical solution, comprising the following steps:

[0013] 1) mixing a polylactic acid-co-glycolic acid copolymer solution with a copper-based drug and an ultrasound-responsive liquid-gas phase change material to obtain a mixed solution; the mixing temperature is 0 to 4° C.;

[0014] 2) emulsifying the mixed solution obtained in step 1) to obtain an ultrasound-responsive nano-copper-based drug; the emulsification temperature is 0-4°C.

[0015] Preferably, the mass ratio of the polylactic acid-co-glycolic acid copolymer to the copper-based drug in the polylactic acid-co-glycolic acid copolymer solution is (10-50):(1-5).

[0016] Preferably, the volume ratio of the polylactic acid-glycolic acid copolymer in the polylactic acid-glycolic acid copolymer solution to the ultrasound-responsive liquid-gas phase change material is (10-50) mg:200 μL.

[0017] Preferably, the emulsification is ultrasonic emulsification.

[0018] Preferably, the emulsification includes a first emulsification and a second emulsification performed sequentially; the time of the first emulsification is 5 to 8 minutes; after the first emulsification is completed, an emulsifier is added to perform a second emulsification, and the time of the second emulsification is 3 to 5 minutes.

[0019] The present invention also provides the use of the ultrasound-responsive nano-copper-based drug described in the above technical solution or the ultrasound-responsive nano-copper-based drug obtained by the preparation method described in the above technical solution in the preparation of anti-tumor drugs.

[0020] The present invention provides an ultrasound-responsive nano-copper drug having a core-shell structure, wherein the shell layer of the core-shell structure is made of poly(lactic acid-co-glycolic acid) copolymer, and the core of the core-shell structure comprises an ultrasound-responsive liquid-gas phase change material and a copper-based drug. The ultrasound-responsive nano-copper drug of the present invention has a nanometer size and can selectively accumulate at the tumor site by utilizing the enhanced permeability and retention effect of tumor tissue. Ultrasonic irradiation of tumor tissue causes the ultrasound-responsive liquid-gas phase change material to undergo a phase transition from liquid to gas. When the internal pressure of the shell exceeds the shell's tolerance, the poly(lactic acid-co-glycolic acid) copolymer shell rapidly ruptures, achieving efficient release of the copper-based drug, thereby achieving a good tumor treatment effect. Ultrasonic irradiation of tumor tissue enables precise release of the copper-based drug, reducing the toxic and side effects of the copper-based drug on other normal tissues. Furthermore, the thermal effect generated by ultrasonic irradiation generally does not harm human tissue, enabling non-destructive release. Experimental results show that after ultrasonic irradiation, the ultrasound-responsive nano-copper drug provided by the present invention has a copper-based drug release rate of up to 87.9%, and cell activity is significantly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a dynamic light scattering analysis of the particle size of the ultrasound-responsive nano-copper-based drug in Example 1 of the present invention and the ultrasound-responsive nanoparticles in Comparative Example 1;

[0022] Figure 2 Zeta potential diagrams of the ultrasound-responsive nano-copper-based drug in Example 1 of the present invention and the ultrasound-responsive nanoparticles in Comparative Example 1;

[0023] Figure 3 This is a transmission electron microscope image of the ultrasound-responsive nano-copper-based drug in Example 1 of the present invention;

[0024] Figure 4 The ultraviolet absorption spectra of the ultrasound-responsive nano-copper-based drug in Example 1 of the present invention and the ultrasound-responsive nanoparticles and the copper-based drug in Comparative Example 1 are shown;

[0025] Figure 5 This is a graph showing the change in particle size over time of the ultrasound-responsive nano-copper-based drug in Example 1 of the present invention and the ultrasound-responsive nanoparticles in Comparative Example 1 when stored in the dark at 4° C.;

[0026] Figure 6 Graph showing the release rate of the ultrasound-responsive nano-copper-based drug in Example 1 of the present invention under conditions of ultrasound irradiation and absence of ultrasound irradiation;

[0027] Figure 7 This is a diagram of ultrasound-mediated drug co-localization of the ultrasound-responsive nano-copper-based drug in Example 1 of the present invention with and without ultrasound irradiation;

[0028] Figure 8 This is a graph showing the level of reactive oxygen species generated by ovarian cancer ID8 cells in the presence or absence of ultrasound irradiation by the ultrasound-responsive nano-copper-based drug in Example 1 of the present invention;

[0029] Figure 9 Graph showing the toxic effects of the ultrasound-responsive nano-copper-based drug in Example 1 of the present invention on ovarian cancer ID8 cells at different concentrations and with or without ultrasound irradiation. DETAILED DESCRIPTION

[0030] The present invention provides an ultrasound-responsive nano-copper-based drug having a core-shell structure, wherein the shell material of the core-shell structure is polylactic acid-glycolic acid copolymer, and the core of the core-shell structure comprises an ultrasound-responsive liquid-gas phase change material and a copper-based drug.

[0031] The ultrasound-responsive copper-based nanoparticles provided by the present invention have a core-shell structure, the shell of which is made of poly(lactic acid-co-glycolic acid) copolymer. Using poly(lactic acid-co-glycolic acid) copolymer as a drug-carrying shell not only provides a stable coating for the copper-based drug, preventing it from deviating from its target during delivery, but also ruptures under a specific pressure to release the drug.

[0032] In the present invention, the shell layer is preferably 10 to 15 nm thick, more preferably 10 to 13 nm thick. The present invention uses a polylactic-co-glycolic acid copolymer of the aforementioned thickness as the shell layer to further ensure that it rapidly ruptures under the pressure change caused by the phase transition of the liquid-gas phase change material in the core, thereby releasing the copper-based drug.

[0033] The ultrasound-responsive nano-copper-based medicine provided by the present invention has a core-shell structure, and the core of the core-shell structure comprises an ultrasound-responsive liquid-gas phase change material and a copper-based medicine.

[0034] In the present invention, the ultrasound-responsive liquid-gas phase change material preferably includes perfluoropentane and / or perfluorohexane; in an embodiment of the present invention, the ultrasound-responsive liquid-gas phase change material is perfluoropentane. The present invention utilizes such ultrasound-responsive liquid-gas phase change materials to enable the ultrasound-responsive liquid-gas phase change material in the ultrasound-responsive nano-copper-based drug core to rapidly undergo a liquid-to-gas phase transition when ultrasound irradiates tumor tissue.

[0035] In the present invention, the copper-based drug preferably includes copper tetracarboxyphenylporphyrin and / or copper chloride; in an embodiment of the present invention, the copper-based drug is copper tetracarboxyphenylporphyrin. By selecting the above copper-based drugs, the present invention can exert a better effect on tumor cells.

[0036] In the present invention, the ultrasound-responsive nano-copper-based drug is preferably stored in the dark at 0-4° C. Under the above storage conditions, the ultrasound-responsive nano-copper-based drug can maintain a stable core-shell structure and will not lose efficacy due to premature release of the copper-based drug.

[0037] The ultrasonically responsive nano-copper-based drug provided by the present invention undergoes a phase change in the ultrasonically responsive liquid-gas phase change material under ultrasonic irradiation, which can rapidly rupture the polylactic acid-glycolic acid copolymer shell, thereby achieving accurate, efficient, and damage-free release of the copper-based drug.

[0038] The present invention also provides a method for preparing the ultrasound-responsive nano-copper-based drug described in the above technical solution, comprising the following steps:

[0039] 1) mixing a polylactic acid-co-glycolic acid copolymer solution with a copper-based drug and an ultrasound-responsive liquid-gas phase change material to obtain a mixed solution; the mixing temperature is 0 to 4° C.;

[0040] 2) emulsifying the mixed solution obtained in step 1) to obtain an ultrasound-responsive nano-copper-based drug; the emulsification temperature is 0-4°C.

[0041] The present invention has no particular limitation on the sources of the raw materials, and commercially available products known to those skilled in the art may be used.

[0042] The invention mixes a polylactic acid-glycolic acid copolymer solution with a copper-based drug and an ultrasonic response type liquid-gas phase change material to obtain a mixed solution.

[0043] In the present invention, the polylactic acid-glycolic acid copolymer solution is preferably a dichloromethane solution of polylactic acid-glycolic acid; the concentration of the polylactic acid-glycolic acid copolymer solution is preferably 20-30 g / L, more preferably 25 g / L.

[0044] In the present invention, the copper-based drug is preferably copper tetracarboxyphenylporphyrin and / or copper chloride. In the present invention, the copper-based drug is preferably used in the form of a copper-based drug solution; the concentration of the copper-based drug solution is preferably 2 to 50 g / L, more preferably 10 g / L; the solvent of the copper-based drug solution is preferably dimethyl sulfoxide, alkaline double-distilled water, or dimethylformamide. In an embodiment of the present invention, the copper-based drug solution is a dimethyl sulfoxide solution of copper tetracarboxyphenylporphyrin at a concentration of 10 g / L.

[0045] In the present invention, the ultrasonically responsive liquid-gas phase change material is preferably perfluoro-n-pentane and / or perfluorohexane; in an embodiment of the present invention, the ultrasonically responsive liquid-gas phase change material is perfluoro-n-pentane.

[0046] In the present invention, the mass ratio of the poly(lactic acid-co-glycolic acid) copolymer to the copper-based drug in the poly(lactic acid-co-glycolic acid) copolymer solution is preferably (10-50):(1-5), more preferably (10-20):1. In an embodiment of the present invention, the mass ratio of the poly(lactic acid-co-glycolic acid) copolymer to the copper-based drug is 10:1. By controlling the mass ratio of the poly(lactic acid-co-glycolic acid) copolymer to the copper-based drug within the above range, the poly(lactic acid-co-glycolic acid) copolymer drug-loaded shell can be coated on the copper-based drug.

[0047] In the present invention, the ratio of the mass of the poly(lactic acid-glycolic acid) copolymer in the poly(lactic acid-glycolic acid) copolymer solution to the volume of the ultrasonically responsive liquid-gas phase change material is preferably (10-50) mg:200 μL, more preferably (30-50) mg:200 μL. In an embodiment of the present invention, the ratio of the mass of the poly(lactic acid-glycolic acid) copolymer in the poly(lactic acid-glycolic acid) copolymer solution to the volume of the ultrasonically responsive liquid-gas phase change material is 50 mg:200 μL. By controlling the ratio of the mass of the poly(lactic acid-glycolic acid) copolymer to the volume of the ultrasonically responsive liquid-gas phase change material within the above range, the poly(lactic acid-glycolic acid) copolymer drug-loaded shell can be formed to coat the ultrasonically responsive liquid-gas phase change material, and the thickness of the drug-loaded shell can be controlled.

[0048] In the present invention, the temperature for mixing the polylactic acid-co-glycolic acid copolymer solution with the copper-based drug and the ultrasonically responsive liquid-gas phase change material is 0-4°C, preferably 0°C. The mixing temperature in the present invention is preferably achieved using an ice bath. In the present invention, the mixing of the polylactic acid-co-glycolic acid copolymer solution with the copper-based drug and the ultrasonically responsive liquid-gas phase change material is preferably carried out in the dark. By controlling the mixing conditions, the present invention prevents the ultrasonically responsive liquid-gas phase change material from undergoing a phase change during the mixing process.

[0049] In the present invention, the mixing of the polylactic acid-co-glycolic acid copolymer solution, the copper-based drug and the ultrasonic responsive liquid-gas phase change material is preferably as follows: in an ice bath in the dark, the polylactic acid-co-glycolic acid copolymer solution is first mixed with the copper-based drug solution, and then mixed with the ultrasonic responsive liquid-gas phase change material.

[0050] After obtaining the mixed solution, the present invention emulsifies the mixed solution to obtain an ultrasound-responsive nano-copper-based drug.

[0051] In the present invention, the emulsification is preferably performed in the dark; the emulsification temperature is 0°C to 4°C, preferably 0°C; and the emulsification temperature is preferably achieved in an ice bath. By controlling the emulsification conditions, the present invention can ensure that the ultrasonically responsive liquid-gas phase change material does not undergo phase change.

[0052] In the present invention, the emulsification is preferably ultrasonic emulsification; the ultrasonic emulsification is preferably performed by an ultrasonic cell disruptor; the working parameters of the ultrasonic cell disruptor are preferably 100 to 120 W / cm 2 By controlling the ultrasonic emulsification conditions, the present invention can optimize the emulsification process, improve the emulsification quality and efficiency, and ensure that the ultrasonic responsive liquid-gas phase change material does not undergo phase change.

[0053] In the present invention, the emulsification preferably includes a first emulsification and a second emulsification performed sequentially.

[0054] In the present invention, the first emulsification time is preferably 5 to 8 minutes; in an embodiment of the present invention, the first emulsification time is 6 minutes.

[0055] After the first emulsification is completed, the present invention preferably adds an emulsifier to perform a second emulsification; the time of the second emulsification is preferably 3 to 5 minutes; in an embodiment of the present invention, the time of the second emulsification is 4 minutes.

[0056] In the present invention, the emulsifier is preferably a 4% aqueous solution of polyvinyl alcohol; the volume ratio of the emulsifier to the solvent of the polylactic-co-glycolic acid solution is preferably (4-6):2, more preferably 5.5:2. The present invention can achieve a good emulsification effect by selecting the composition and addition amount of the emulsifier.

[0057] After emulsification is completed, the present invention preferably mixes the emulsion obtained by the emulsification with isopropyl alcohol and stirs them. In the present invention, the concentration of the isopropyl alcohol is preferably 2%, and the volume ratio of the isopropyl alcohol to the solvent of the polylactic acid-co-glycolic acid solution is preferably (5-7):1, more preferably 6:1. In the present invention, the addition of isopropyl alcohol can promote the volatilization of solvents such as dichloromethane; the addition of isopropyl alcohol can also change the surface tension of the emulsion, improve the dispersion effect of the dispersed phase of the emulsion, and ultimately obtain an ultrasound-responsive nano-copper-based drug with uniform particle size.

[0058] In the present invention, the temperature for stirring the emulsion and isopropyl alcohol is preferably 0-4°C; the stirring is preferably carried out in the dark; the stirring time is preferably 2-4 hours, more preferably 3 hours;

[0059] After stirring, the present invention preferably performs solid-liquid separation, washing, and resuspending of the stirred mixed solution in a dark environment to obtain an ultrasound-responsive nano-copper-based drug. In the present invention, the solid-liquid separation method is preferably centrifugation; the centrifugation time is preferably 5 minutes; the centrifugation temperature is preferably 0-4°C; and the centrifugation speed is preferably 12,000 rpm. The washing solvent is preferably pure water pre-cooled to 0-4°C, and the number of washes is preferably three.

[0060] The preparation method provided by the present invention has simple process, safe operation, short cycle and low cost, and can prepare ultrasound-responsive nano copper-based drugs with a core-shell structure. The shell material is polylactic acid-glycolic acid copolymer, and the core includes ultrasound-responsive liquid-gas phase change material and copper-based drugs.

[0061] The present invention also provides the use of the ultrasound-responsive nano-copper-based drug described in the above technical solution or the ultrasound-responsive nano-copper-based drug obtained by the preparation method described in the above technical solution in the preparation of anti-tumor drugs.

[0062] The present invention has no special limitation on the operation of the application, and the application technical solutions well known to those skilled in the art can be adopted.

[0063] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0064] Example 1

[0065] The ultrasound-responsive nano-copper-based drug has a core-shell structure. The shell material is polylactic acid-glycolic acid copolymer, and the core is composed of an ultrasound-responsive liquid-gas phase change material (perfluoropentane) and a copper-based drug (tetracarboxyphenylporphyrin copper).

[0066] The preparation method is:

[0067] 1) Weighing 50 mg of poly(lactic acid-co-glycolic acid) and dissolving it in 2 mL of dichloromethane at room temperature to obtain a poly(lactic acid-co-glycolic acid) solution, placing the poly(lactic acid-co-glycolic acid) solution in an ice-water bath, adding a copper-based drug (protecting from light, weighing 5 mg of copper tetracarboxylphenylporphyrin solid powder, dissolving it in 500 μL of dimethyl sulfoxide at room temperature, and mixing until the powder is completely dissolved), adding an ultrasonically responsive liquid-gas phase change material (200 μL of perfluoropentane), and mixing in an ice-water bath to obtain a mixed solution;

[0068] 2) Use ultrasonic cell disruptor to emulsify the mixed solution for the first time in ice water bath, the parameters are: power density 120W / cm 2 , on 5s, off 5s, the first emulsification time is 6min; then 5.5mL 4% polyvinyl alcohol solution is added, and the mixed solution is emulsified for the second time in an ice water bath using an ultrasonic cell disruptor. The parameters are: power density 120W / cm 2, on 5s, off5s, the second emulsification time is 4min; the emulsion obtained by the second emulsification is transferred into a beaker (wrapped in tin foil to avoid light and placed on ice for pretreatment), 12mL of 2% isopropanol aqueous solution is added, and the mixture is stirred in the dark for 3h under ice bath conditions to obtain a mixed solution after mixing and stirring; the mixed solution after mixing and stirring is placed in an EP tube, centrifuged at 12000rmp and 4°C for 5min, the supernatant is discarded, and the mixture is washed with 1mL of precooled pure water for a total of 3 times until the supernatant is clear and transparent, and resuspended with 1mL of precooled pure water to obtain an ultrasound-responsive nano-copper-based drug, which is stored at low temperature and away from light.

[0069] Comparative Example 1

[0070] The ultrasonically responsive copper-based nanoparticles are of a core-shell structure, wherein the shell material is polylactic acid-glycolic acid copolymer, and the core is an ultrasonically responsive liquid-gas phase change material (perfluoropentane).

[0071] The difference from the preparation method in Example 1 is that the operation of "adding a copper-based drug (5 mg of tetracarboxyphenylporphyrin copper solid powder was weighed in the dark, dissolved in 500 μL of dimethyl sulfoxide at room temperature, and mixed until the powder was completely dissolved)" in Example 1 was replaced by "adding 500 μL of dimethyl sulfoxide", and the obtained nanoparticles were ultrasound-responsive.

[0072] The dynamic light scattering analysis particle size method was used to analyze the particle size of the ultrasound-responsive nano-copper-based drug in Example 1 and the ultrasound-responsive nanoparticles in Comparative Example 1. The dynamic light scattering analysis particle size of the ultrasound-responsive nano-copper-based drug and the ultrasound-responsive nanoparticles was shown in the figure below. Figure 1 shown.

[0073] Figure 1 The CuTCPP-PFP@PLGA in the table represents the ultrasound-responsive nano-copper-based drug in Example 1, and the PFP@PLGA represents the ultrasound-responsive nanoparticles in Comparative Example 1. Figure 1 The average particle size of the ultrasound-responsive copper nanoparticles was 283.8 nm, with a polydispersity index (PdI) of 0.222, less than 0.3, indicating that the particles were uniform. The average particle size of the ultrasound-responsive nanoparticles was 313.9 nm, with a polydispersity index (PdI) of 0.116, less than 0.3, indicating that the particles were uniform. Dynamic light scattering analysis of the particle size of the ultrasound-responsive copper nanoparticles and the ultrasound-responsive nanoparticles indicates that the synthesized copper nanoparticles can be uniformly dispersed in water.

[0074] The Zeta potential of the ultrasound-responsive nano-copper-based drug in Example 1 and the ultrasound-responsive nanoparticles in Comparative Example 1 was detected by electroacoustic technology. The Zeta potential diagrams of the ultrasound-responsive nano-copper-based drug and the ultrasound-responsive nanoparticles were as follows: Figure 2 shown.

[0075] Figure 2 CuTCPP-PFP@PLGA represents the ultrasound-responsive nano-copper-based drug in Example 1, and PFP@PLGA represents the ultrasound-responsive nanoparticles in Comparative Example 1. Figure 2 The surface potential of the ultrasound-responsive copper nanoparticles is -20.7 mV, while the surface potential of the ultrasound-responsive nanoparticles is -16.1 mV. The zeta potential plot shows that the ultrasound-responsive copper nanoparticles have a more negative charge than the ultrasound-responsive nanoparticles.

[0076] The morphology of the ultrasound-responsive nano-copper-based drug in Example 1 was characterized using a transmission electron microscope. The obtained transmission electron microscope image is shown in FIG. Figure 3 shown.

[0077] Figure 3 The particle size of the ultrasound-responsive nano-copper-based drug is about 280 nm, which is consistent with the results of dynamic light scattering analysis. The shell thickness of the ultrasound-responsive nano-copper-based drug is 10 to 15 nm.

[0078] The ultraviolet absorption spectrum detection method was used to detect the ultrasound-responsive nano-copper-based drug in Example 1 and the ultrasound-responsive nanoparticles and copper-based drug in Comparative Example 1. The obtained ultraviolet absorption spectrum is shown in FIG. Figure 4 shown.

[0079] Figure 4 CuTCPP-PFP-PLGA represents the ultrasound-responsive nano-copper-based drug in Example 1, PFP-PLGA represents the ultrasound-responsive nanoparticles in Comparative Example 1, and CuTCPP represents the copper-based drug. Figure 4 The data show that the ultrasound-responsive nanoparticle material has no peak, while the copper-based drug and the ultrasound-responsive nano-copper-based drug have the same ultraviolet absorption peak, which indicates that the ultrasound-responsive nano-copper-based drug nanoparticles successfully encapsulate the copper-based drug, proving that the preparation of the ultrasound-responsive copper-based drug is successful.

[0080] The ultrasound-responsive nano-copper-based drug in Example 1 and the ultrasound-responsive nanoparticles in Application Example 1 were placed in a refrigerator at 4°C and stored away from light. Equal amounts of solution were extracted every other day to measure the particle size. The measurement was continued for seven days. The obtained particle size change over time is shown in the figure below. Figure 5 shown.

[0081] Figure 5CuTCPP-PFP-PLGA represents the ultrasound-responsive nano-copper-based drug in Example 1, and PFP-PLGA represents the ultrasound-responsive nanoparticles in Comparative Example 1. Figure 5 The particle size of ultrasound-responsive nano-copper-based drugs and ultrasound-responsive nanoparticles changed very little at 4°C, indicating that under this storage condition, ultrasound-responsive nano-copper-based drugs can be stored for a long time with good stability.

[0082] The release rate of the ultrasonic responsive nano-copper-based drug in Example 1 under the condition of ultrasonic irradiation was detected. The specific method was as follows: an equal amount of ultrasonic responsive nano-copper-based drug was taken and divided into 3 groups, 115.5 μg in each group, and pure water was added to prepare an ultrasonic responsive nano-copper-based drug solution with a concentration of 5 μg / mL, wherein one group was a blank group, and the other two groups were a control group and an experimental group. The experimental group was subjected to ultrasonic irradiation. After the irradiation, the control group and the experimental group were subjected to high-speed centrifugation to obtain the supernatant. The two groups of supernatants and the blank group were subjected to ultraviolet absorption spectrum detection, and the test results were substituted into the copper-based drug standard curve to obtain the content of copper-based drugs in each group. The control group-blank group and the experimental group-blank group were taken to calculate the relative content of copper-based drugs. The copper-based drug content of the experimental group was 101 μg, and the copper-based drug content of the control group was 3.75 μg. The results of the release rate diagram of the ultrasonic responsive nano-copper-based drug with or without ultrasonic irradiation are as shown in FIG. Figure 6 shown.

[0083] Figure 6 US- represents the control group, and US+ represents the experimental group. Figure 6 The copper-based drug release rate of the control group was 3.25%, and the copper-based drug release rate of the experimental group was 87.42%, indicating that the copper-based drug content in the solution increased significantly after ultrasonic irradiation, proving that the ultrasound-responsive nano-copper-based drug in the present invention can release copper-based drugs under the triggering of ultrasound, and the drug release rate reaches 87.42%.

[0084] The co-localization relationship between the ultrasound-responsive nano-copper-based drug prepared in Example 1 and the copper-based drug was detected under the conditions of ultrasound irradiation and the absence of ultrasound irradiation. The specific method is as follows: first, an appropriate amount of ultrasound-responsive copper-based drug is taken and stained with DiO dye. An equal amount of DiO-stained ultrasound-responsive copper-based drug is added to the cell culture plate. One group is subjected to ultrasound irradiation, while the other group is not subjected to light. The green fluorescence signals of the two groups are detected. The ultrasound-mediated drug co-localization diagram obtained is shown in FIG. Figure 7 shown.

[0085] Figure 7 US- represents no ultrasound irradiation, and US+ represents ultrasound irradiation. Figure 7It shows that without ultrasound irradiation, only the cell nucleus (blue: DAPI) can be observed, and copper-based drugs (green: DiO) cannot be observed; after ultrasound irradiation, the content of copper-based drugs (green: DiO) in the cells increases and is distributed around the cell nucleus (blue: DAPI), proving that ultrasound can promote the entry of copper-based drugs into cells.

[0086] The fluorescence probe method was used to detect the level of reactive oxygen species generated in ovarian cancer ID8 cells using the ultrasound-responsive copper-based drug in Example 1 under conditions with and without ultrasound irradiation. The obtained reactive oxygen species generation level in ovarian cancer ID8 cells is shown in the figure below: Figure 8 shown.

[0087] Figure 8 In the figure, DCFH-DA probes were added to different groups of cells. After treatment with ultrasound, ultrasound-responsive copper-based drugs, and ultrasound-responsive copper-based drugs combined with ultrasound, the cells showed varying degrees of green fluorescence signals. The results showed that the ultrasound-responsive copper-based drug combined with ultrasound irradiation group showed a significant reactive oxygen species green fluorescence signal, while the other groups showed no or weak green fluorescence. This indicates that the simultaneous presence of ultrasound-responsive copper-based drugs and ultrasound can significantly increase reactive oxygen species levels, while ultrasound alone or ultrasound-responsive copper-based drugs alone do not significantly increase reactive oxygen species levels.

[0088] The toxic effect of ultrasound-responsive copper-based drugs on cells was detected. The specific method was as follows: first, the ultrasound-responsive copper-based drug in Example 1 was prepared into a solution with concentrations of 0, 10, 20, 30, and 40 μg / mL using DMEM high-glucose complete medium (DMEM high-glucose basal medium + 10% fetal bovine serum + 1% double antibody (penicillin, streptomycin)), and 100 μL was taken to co-incubate with ovarian cancer ID8 cells. On this basis, ultrasound irradiation and no ultrasound irradiation were performed, and 96-well plates were used for culture with ovarian cancer ID8 cells. The toxic effect of ultrasound-responsive copper-based drugs on ovarian cancer ID8 cells was detected, and the cell activity was detected by cck8. The toxic effect on ovarian cancer ID8 cells was obtained as shown in FIG. Figure 9 shown.

[0089] Figure 9 These are the results of cell activity testing with different concentrations of copper-based drugs under conditions of ultrasound irradiation and without ultrasound irradiation. US- represents no ultrasound irradiation, and US+ represents ultrasound irradiation. Figure 9 This indicates that copper-based drugs have less toxic effects on cells without ultrasonic irradiation, and that ultrasonic irradiation can significantly enhance the killing effect of copper-based drugs on tumor cells.

[0090] In summary, the ultrasound-responsive nano-copper-based drug provided by the present invention has uniform particle size, can be stored for a long time at 4°C with good stability, the copper-based drug is basically not released without ultrasonic irradiation, and has no toxic effect on cells. Ultrasonic irradiation can achieve the release of copper-based drugs at the tumor site, with a drug release rate of up to 87.42%, and a significant killing effect on tumor cells.

[0091] 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An ultrasound-responsive nano-copper-based drug having a core-shell structure, wherein the shell layer of the core-shell structure is made of polylactic acid-glycolic acid copolymer, and the core of the core-shell structure comprises an ultrasound-responsive liquid-gas phase change material and a copper-based drug.

2. The ultrasound-responsive nano-copper-based drug according to claim 1, characterized in that: The ultrasonic responsive liquid-gas phase change material includes perfluoropentane and / or perfluorohexane.

3. The ultrasound-responsive nano-copper-based drug according to claim 1, characterized in that: The copper-based drug includes tetracarboxyphenylporphyrin copper and / or copper chloride.

4. The ultrasound-responsive nano-copper-based drug according to any one of claims 1 to 3, characterized in that: The shell thickness of the ultrasound-responsive nano-copper-based drug is 10-15 nm.

5. The method for preparing the ultrasound-responsive nano-copper-based drug according to any one of claims 1 to 4, comprising the following steps: 1) mixing a polylactic acid-co-glycolic acid copolymer solution with a copper-based drug and an ultrasound-responsive liquid-gas phase change material to obtain a mixed solution; the mixing temperature is 0 to 4° C.; 2) emulsifying the mixed solution obtained in step 1) to obtain an ultrasound-responsive nano-copper-based drug; the emulsification temperature is 0-4°C.

6. The preparation method according to claim 5, characterized in that The mass ratio of the polylactic acid-glycolic acid copolymer to the copper-based drug in the polylactic acid-glycolic acid copolymer solution is (10-50):(1-5).

7. The preparation method according to claim 5, characterized in that The volume ratio of the polylactic acid-glycolic acid copolymer in the polylactic acid-glycolic acid copolymer solution to the ultrasonic responsive liquid-gas phase change material is (10-50) mg:200 μL.

8. The preparation method according to claim 5, characterized in that The emulsification is ultrasonic emulsification.

9. The preparation method according to claim 8, characterized in that The emulsification includes a first emulsification and a second emulsification performed sequentially; the time of the first emulsification is 5 to 8 minutes; after the first emulsification is completed, an emulsifier is added to perform a second emulsification, and the time of the second emulsification is 3 to 5 minutes.

10. Use of the ultrasound-responsive nano-copper-based drug according to any one of claims 1 to 4 or the ultrasound-responsive nano-copper-based drug obtained by the preparation method according to any one of claims 5 to 9 in the preparation of anti-tumor drugs.

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