Preparation of biodegradable sound-sensitive agent and application of biodegradable sound-sensitive agent in acoustic immunotherapy
By converting Cu2O nanocubes into Cu3P nanocubes, the problem of non-specific degradation of Cu2O in sonotherapy was solved, achieving tumor-specific degradation and enhanced sonodynamic immunotherapy effects, activating anti-tumor immune responses, enhancing tumor treatment efficacy, and avoiding long-term toxicity.
Patent Information
- Application Number
- CN202511447681.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-11
- Publication Date
- 2026-02-27
AI Technical Summary
Cu2O nanomaterials are easily degraded in acidic tumor microenvironments and also degrade under normal physiological conditions, which limits their application in acoustic therapy. At the same time, their wide bandgap structure and rapid electron-hole pair recombination limit their acoustic potential.
Cu2O nanocubes were transformed into Cu3P nanocubes through an in-situ phosphating strategy, endowing them with tumor-specific degradation properties. Furthermore, the combined use of Celastrol and αPD-L1 loading enhanced the efficacy of sonodynamic immunotherapy.
Cu3P nanocubes degrade in tumor cells, activating copper death, increasing PD-L1 expression, activating the cGAS-STING pathway, achieving a cascade amplification of anti-tumor immune responses, enhancing the therapeutic effect of tumors, and avoiding long-term toxicity through tumor-specific drug release and rapid in vivo clearance.
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Figure CN121573653A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine, and relates to preparation of a biodegradable sonosensitizer and application thereof in acoustic immunotherapy, and provides a preparation method of a Cu3P biodegradable sonosensitizer and application thereof in acoustic immunotherapy. BACKGROUND
[0002] In recent years, immune checkpoint inhibitors (ICIs) represented by lung cancer immunotherapy have become a major breakthrough in this field. They have become a new treatment method for patients with driver gene-negative, advanced or locally advanced lung cancer and patients with resistance to targeted therapy. Unlike other anti-tumor methods, immunotherapy focuses on activating the body's own immune system to create an unfavorable "soil" for tumor growth, indirectly eliminating cancer cells. Programmed death ligand 1 (PD-L1) inhibitor is a representative immune checkpoint inhibitor in lung cancer treatment. However, due to insufficient PD-L1 expression and immune response dysfunction in the tumor microenvironment (TME), a large part of patients still show poor response to immune checkpoint blockade (ICB) mediated treatment. In addition, patients receiving αPD-L1 monotherapy often have varying degrees of drug resistance or immune-related adverse reactions, ultimately affecting treatment effect. Although enhancing anti-PD-LI treatment combined with different treatment strategies (such as sonodynamic therapy (SDT), chemodynamic therapy (CDT) and phototherapy) for lung cancer has shown efficacy, new strategies are still needed that can directly increase the level of tumor-infiltrating lymphocytes in the TME, convert "cold" tumors to "hot" tumors, and improve the potential response to ICB.
[0003] Recently, the emergence of programmed cell death mechanisms such as ferroptosis, pyroptosis and copper death provides new treatment strategies to overcome apoptosis resistance and activate immunotherapy. Copper death is a unique form of copper-induced cell death, and its mechanism involves excessive copper ions (Cu + ) hindering mitochondrial metabolic activity. In addition, copper death can induce immunogenic cell death (ICD) of tumor cells, release damage-associated molecular patterns, promote maturation of dendritic cells (DC) and infiltration of cytotoxic T lymphocytes. In addition to inducing ICD, copper death can increase the expression of PD-LI in tumor cells, activate the cGAS-STING pathway, and achieve cascade amplification of anti-tumor immune response.
[0004] Due to its narrow band gap and Cu +Mediated Fenton-like reaction, semiconductor oxide Cu2O has the ability as a sonosensitizer and nano-enzyme In addition, Cu2O has good degradation characteristics, which makes Cu2O have copper oxidation induction ability. On this basis, Cu2O-based nanomaterials are expected to combine copper hyperplasia-mediated tumor treatment with SDT and CDT, realize the cascade amplification of ROS and the copper hyperplasia effect, and finally activate a strong adaptive immune response. Unfortunately, the characteristics of Cu2O that is easy to oxidize and degrade is a double-edged sword. It not only can degrade in acidic TME, but also can degrade under normal physiological conditions, leading to copper death of normal cells. In addition, the wide bandgap structure and fast electron-hole pair recombination of Cu2O-based nanomaterials limit their potential application as sonosensitizers in SDT. SUMMARY
[0005] The present application is directed to the above technical problems, and in view of the defects of the current Cu2O-based nanomaterials in the application of sonosensitization therapy, the present application first reports an in-situ phosphidation strategy for Cu2O nanocubes to regulate the degradation behavior of Cu2O nanocubes. Compared with Cu2O nanocubes, the obtained Cu3P nanocubes can only degrade in tumor cells but not in normal cells, thereby endowing Cu3P nanocubes with TME-responsive degradation characteristics.
[0006] In the first aspect of the present application, a preparation method of Cu3P nanocubes is provided, comprising the following steps:
[0007] (a) Under stirring conditions, CuSO4 solution and NaOH solution are sequentially added to a sodium citrate solution, and finally an antioxidant is added and stirring is continued for a certain period of time; after 24 h, the precipitate is collected by centrifugation and washed with deionized water for multiple times to obtain Cu2O;
[0008] (b) After Cu2O and sodium phosphate are fully ground and mixed at a mass ratio of 1:3-10, annealing is carried out at 200-300 ℃ under the protection of flowing inert gas for 2-4 h; the collected bulk product is fully ground and washed with deionized water and ethanol alternately to obtain Cu3P nanocubes.
[0009] The preferred process conditions of the above two steps are as follows:
[0010] In step (a), the concentration of the sodium citrate solution is 6.5×10 -4 g / mL, the concentration of the CuSO4 solution is 1.2 mol / L, the concentration of the NaOH solution is 4.8 mol / L, and the concentration of the antioxidant is 1.1 mol / L; the volume ratio among the sodium citrate solution, the CuSO4 solution, the NaOH, and the antioxidant is 400:1:1:1; when adding NaOH or the antioxidant, both are added after the previous substance is added and stirred for 5-6 minutes.
[0011] The antioxidant is selected from any one of ascorbic acid, glutathione, cysteine, uric acid, sulfite, catechin.
[0012] The centrifugal collection condition is 12000g, 5 min.
[0013] In step (b), Cu2O and sodium phosphate are in a mass ratio of 1:5, and the inert gas is selected from argon.
[0014] In the second aspect of the present application, a Cu3P nanocube is provided, which is prepared by the method described above.
[0015] By detecting the tumor-specific degradation characteristics of Cu3P nanocubes, the potential clinical translation advantages of Cu3P can be highlighted: first, the prepared Cu3P nanocubes have a narrower band gap than Cu2O nanocubes, showing higher sonodynamic activity; second, the tumor-specific release of Cu ions can trigger the cascade amplification of ROS production, inducing tumor-specific copper death, leading to severe cell damage and activating the ICD effect; third, the copper death effect induced by Cu3P not only increases the expression of PD-LI in tumor cells, thereby sensitizing ICB-mediated tumor treatment, but also activates the cGAS-STING pathway, achieving cascade amplification of anti-tumor immune response; fourth, the response degradation characteristics of TME make Cu3P nanocubes an ideal drug-loaded nanoplatform, which realizes tumor-specific drug release by loading lung cancer first-line drug Celastrol (Cel), further enhancing the anti-tumor effect. These favorable characteristics enable the Cu3P-Cel combined with alpha PD-L1 copper death-enhanced ultrasound immunotherapy to achieve satisfactory therapeutic effect on primary and distant tumors. In addition, since Cu3P nanocubes can be degraded into Cu and P ions and rapidly cleared by the liver and kidneys, unlike many other non-biodegradable inorganic nanomaterials, there should be no concern about long-term toxicity when Cu3P is used in vivo.
[0016] Based on the above research, the third aspect of the present application provides the use of Cu3P nanocubes as a sonosensitizer. The results of the kinetic performance evaluation show that the sonodynamic performance and chemical kinetic activity of Cu3P are significantly better than those of Cu2O nanoparticles, and at the same time, Cu3P can also efficiently consume GSH.
[0017] Further, the sonosensitizer is used as a drug carrier to load a first-line drug for malignant tumors. In the specific embodiments of the present application, Cu3P-Cel is taken as an example, and the results show that a large amount of active oxygen can be generated under US irradiation, and Cu3P-Cel can completely kill tumor cells.
[0018] In a fourth aspect, the present invention provides a sonoimmunotherapy agent comprising the aforementioned sonosensitive agent Cu3P nanocubes and a loaded drug.
[0019] Preferably, the acoustic immunotherapy agent also includes αPD-L1. Experimental results showed that Cu3P-Cel + αPD-L1 exhibited the best tumor therapeutic effect, completely inhibiting tumor growth; Cu3P-Cel + αPD-L1 prolonged the survival time of mice; and Cu3P-Cel + αPD-L1 did not show significant long-term in vivo toxicity. Furthermore, under US irradiation, Cu3P-Cel + αPD-L1 strongly promoted dendritic cell maturation, producing large amounts of CD4+. + CD8 + T cells thus trigger a powerful immunotherapy.
[0020] Furthermore, in a fifth aspect, the present invention provides a sonoimmunotherapy agent for pancreatic cancer, which is composed of Cu3P-Celastrol sonosensitive agent and αPD-L1.
[0021] The treatment method for Cu3P-Celastrol sound sensitizer is as follows: Celastrol is dissolved in ethanol, Cu3PNPs solution and Cel solution are mixed at a mass ratio of Cu3P to Celastrol of 1:2 and magnetically stirred overnight; the precipitate is collected by centrifugation and washed repeatedly with ethanol to remove excess Celastrol, thus obtaining Cu3P-Celastrol sound sensitizer.
[0022] The beneficial protections and effects of this invention are as follows:
[0023] This invention overcomes the lack of tumor-specific degradation characteristics of Cu2O nanocubes by employing an in-situ phosphating strategy, and improves their sonodynamic and chemodynamic properties. Furthermore, by loading Cel and combining it with αPD-L1, it achieves powerful sonodynamic immunotherapy, aiming to solve the problems of low ROS yield and easy consumption of ROS by endogenous GSH in current nanosonic sensitizers, and enhance the sonodynamic tumor treatment effect through effective regulation of the tumor microenvironment. Attached Figure Description
[0024] Figure 1 The detection results for Cu3P are shown: a) TEM image of Cu3P; b) HRTEM image of Cu3P.
[0025] Figure 2 The results of the acoustic and dynamic performance tests of Cu3P are shown: a) Cu3P's... 1 O2 generation rate test; b) Cu2O and Cu3P 1c) Comparison of O2 generation rates; c) Comparison of ESR spectra of Cu3P.
[0026] Figure 3 The results of the chemical kinetic activity detection of Cu3P are shown: a) the •OH generation rate test of Cu3P; b) the comparison of the •OH generation rates of Cu2O and Cu3P; c) the comparison of ESR spectra of Cu3P.
[0027] Figure 4 The tumor cell killing effects of Cu3P and Cu3P-Cel are shown: a) confocal images of reactive oxygen species generated by Cu3P and Cu3P-Cel; b) and c) Cu3P and Cu3P-Cel under US irradiation (50 kHz, 3.0 W cm⁻¹). -2 Comparison of cell survival rates (5 min) or without irradiation.
[0028] Figure 5 The in vivo tumor-suppressing effect of Cu3P-Cel was demonstrated: ab) changes in tumor volume after intravenous injection of Cu3P-Cel; c) survival time of mice after treatment; d) changes in body weight of mice during tumor treatment.
[0029] Figure 6 The study demonstrated the immunotherapeutic effects of Cu3P-Cel + αPD-L1: a) changes in dendritic cell maturation in lymphoid tissue after different treatment modalities; b) changes in T cell content in the primary tumor. Detailed Implementation
[0030] The following embodiments and experimental examples further illustrate the present invention and should not be construed as limiting the invention. The embodiments do not include a detailed description of conventional methods, which are well known to those skilled in the art and described in numerous publications.
[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention, and the preferred embodiments and materials described in the specific implementation are for illustrative purposes only.
[0032] Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.
[0033] Example 1: Preparation method of heterojunction acoustic sensitizer (Cu3P) and drug-loaded complex:
[0034] a) 0.26 g of sodium citrate was added to 400 mL of deionized water and stirred until completely dissolved. 1 mL of CuSO4 solution (1.2 M) was added, and stirring was continued for 5 min. Then 1 mL of NaOH solution (4.8 M) was added, and stirring was continued for 5 min. Finally, 1 mL of ascorbic acid (1.1 M) was added, and stirring was continued for 5 min. After 24 h, the precipitate was collected by centrifugation (12000 g, 5 min) and washed with deionized water 3 times to obtain Cu2O.
[0035] b) 0.3 g of Cu2O and 1.5 g of sodium phosphate were thoroughly mixed in a mortar and annealed at 200 or 300 °C under flowing argon for 3 h. The collected bulk product was thoroughly ground with a mortar and pestle and then washed with deionized water and ethanol alternately to obtain Cu3P cubes. Electron microscopy detection results showed that the obtained Cu3P particle size distribution was 20 nm, and under high magnification electron microscopy observation, it had obvious lattice fringes.
[0036] c) Cel was dissolved in ethanol, and the Cu3P NPs solution was magnetically stirred with the Cel solution (Cu3P: Cel = 1:2 (mass ratio)) overnight. The precipitate was collected by centrifugation and washed with ethanol multiple times to remove excess Cel to obtain Cu3P-Cel.
[0037] Example 2 Testing of the sonodynamic performance, chemical kinetic activity and GSH consumption performance of Cu3P sonosensitizers
[0038] a) The Cu3P sonosensitizers prepared in the present application can generate a large amount of singlet oxygen (1O2) under low-intensity ultrasound 1 O2), by using 1,3-diphenyl isobenzofuran (DPBF) as an 1 O2probe, the 1 O2generation efficiency of Cu3P sonosensitizers under ultrasound irradiation was detected to evaluate their sonodynamic performance.
[0039] b) The Cu3P sonosensitizers prepared in the present application can undergo Fenton reaction under acidic conditions to generate a large amount of hydroxyl radicals (·OH), by using 3,3',5,5'-tetramethylbenzidine (TMB) as an ·OH probe, the chemical kinetic performance of Cu3P sonosensitizers was evaluated.
[0040] c) The Cu3P sonosensitizers prepared in the present application can consume GSH, by using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a GSH probe, the ability of Cu3P sonosensitizers to consume GSH was evaluated.
[0041] Over time, Cu3P generates 1The amount of O2 gradually decreases, and the variation trend of singlet oxygen at different time periods is the same, which is low at first and then high and tends to be horizontal with the increase of wavelength Figure 2 a); the comparison of the fitting line and ESR spectrum shows that the sonodynamic performance of Cu3P is obviously better than that of Cu2O nanoparticles Figure 2 b, 2c).
[0042] The chemical kinetics results show that the •OH production rate of Cu3P gradually increases with the increase of concentration Figure 3 a); the comparison of the fitting line and ESR spectrum shows that the chemical kinetics activity of Cu3P is obviously better than that of Cu2O nanoparticles Figure 3 b, 3c); at the same time, Cu3P can also efficiently consume GSH.
[0043] Example 3 In vitro sonodynamic therapy of Cu3P-Cel sonosensitizer
[0044] The present application detects the cell survival rate of cells treated by Cu3P and US irradiation by MTT reagent. Human lung cancer cells (A549) are seeded in a 96-well plate at a density of 5000 per well, cultured for 24 hours, then different concentrations (0, 6, 12, 18, 24, 30 μg / mL) of Cu3P-Cel are added for 4 hours, and then irradiated by US (50 kHz, 3.0 W / cm 2 ) for 5 minutes, and then the in vitro sonodynamic therapy effect of Cu3P-Cel is detected by MTT reagent.
[0045] The results are shown in Figure 4 , Cu3P-Cel can generate a large amount of active oxygen under US irradiation Figure 4 a); under US irradiation, Cu3P and Cu3P-Cel both show tumor cell killing effect, and with the increase of drug concentration, Cu3P-Cel gradually shows stronger cell killing effect, and even can completely kill tumor cells Figure 4 b, 4c).
[0046] Example 4 In vivo sonodynamic therapy of Cu3P-Cel sonosensitizer
[0047] 3-5 weeks old female mice are implanted with 100 μL (200 million) of mouse lung cancer cells (LLC) subcutaneously in the left and right armpit, and when the tumor volume grows to 100 mm 3 , the mice are divided into 6 groups (5 in each group): (1) normal saline, (2) US irradiation alone (50 kHz, 3.0 W / cm 2, 5 min), (3) Cu3P (30 μg / mL), (4) Cu3P + US (30 μg / mL), (5) Cu3P-Cel + US (30 μg / mL), (6) Cu3P-Cel + US + αPD-L1 (30 μg / mL) + US irradiation (50 kHz, 3.0 W / cm 2 (5 min). The tumor size was measured every other day and the weight of the mice was recorded daily to evaluate the in vivo sonodynamic therapeutic efficiency of Cu3P-Cel sonosensitive agent.
[0048] Figure 5 The study demonstrated the in vivo tumor-suppressive effect of Cu3P-Cel: compared to Cu3P alone, Cu3P-Cel + αPD-L1 exhibited the best tumor therapeutic effect under US irradiation, completely inhibiting tumor growth. Figure 5 a, 5b); Cu3P-Cel + αPD-L1 can prolong the survival time of mice ( Figure 5 c); meanwhile, Cu3P-Cel + αPD-L1 did not show significant long-term in vivo toxicity ( Figure 5 d).
[0049] Example 5: In vivo immunotherapy with Cu3P-Cel sound-sensitizing agent
[0050] 100 μL (2 million cells) of mouse lung cancer cells (LLC) were subcutaneously implanted into the left and right axillae of 3-5 week old female mice. The tumors were allowed to grow to 100 mm². 3 The mice were divided into 6 groups (n=5 per group): (1) saline, (2) US irradiation alone (50 kHz, 3.0 W / cm²). 2 , 5 min), (3) Cu3P (30 μg / mL), (4) Cu3P + US (30 μg / mL), (5) Cu3P-Cel + US (30 μg / mL), (6) Cu3P-Cel + US + αPD-L1 (30 μg / mL) + US irradiation (50 kHz, 3.0 W / cm 2 (5 min). One day after treatment, the mice were euthanized, and their lymphoid tissue and primary tumor were removed for immunological testing.
[0051] Figure 6 The immunotherapeutic effect of Cu3P-Cel + αPD-L1 was demonstrated: under US irradiation, Cu3P-Cel + αPD-L1 could strongly promote the maturation of dendritic cells ( Figure 6 a) Generates a large amount of CD4 + CD8 +T cells to thereby cause a strong immunotherapy Figure 6 b, 6c).
[0052] The unexplained part of the present invention is the same as the prior art or realized by the prior art. The applicant declares that the present invention is illustrated by the above examples to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. The skilled in the art should understand that any improvement of the present invention, equivalent replacement of each raw material of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc. fall within the scope of protection and disclosure of the present invention.
Claims
1. A method for preparing Cu3P nanocubes, characterized in that, Includes the following steps: (a) Under stirring conditions, CuSO4 solution and NaOH solution were added to sodium citrate solution in sequence, and finally an antioxidant was added and stirring was continued for a certain period of time; after 24 h, the precipitate was collected by centrifugation and washed several times with deionized water to obtain Cu2O; (b) After thoroughly grinding and mixing Cu2O and sodium phosphate at a mass ratio of 1:3~10, anneal at 200~300 °C under flowing inert gas protection for 2~4 h; after thoroughly grinding the collected bulk product, wash it alternately with deionized water and ethanol to obtain Cu3P nanocubes.
2. The method for preparing Cu3P nanocubes according to claim 1, characterized in that: in, In step (a), the concentration of the sodium citrate solution is 6.5 × 10⁻⁶. -4 The concentration of CuSO4 solution is 1.2 mol / L, g / mL. The concentration of the NaOH solution is 4.8 mol / L, and the concentration of the antioxidant is 1.1 mol / L. The volume ratio of sodium citrate solution, CuSO4 solution, NaOH, and oxidant is 400:1:1:1; The centrifugation collection conditions were 12000g for 5 min; When adding NaOH or antioxidants, add them after the former has been added and stirred for 5-6 minutes; The antioxidant is selected from any one of ascorbic acid, glutathione, cysteine, uric acid, sulfite, and catechin.
3. The method for preparing Cu3P nanocubes according to claim 1, characterized in that: in, In step (b), Cu2O and sodium phosphate are mixed in a mass ratio of 1:5, and the inert gas is selected from argon.
4. A Cu3P nanocube, characterized in that, It is prepared by the method described in any one of claims 1 to 3.
5. The application of the Cu3P nanocubes according to claim 4 as a sound-sensing agent.
6. The application according to claim 5, characterized in that, The sonosensitive agent is used as a drug carrier to load first-line clinical drugs for malignant tumors.
7. A sound immunotherapy agent, characterized in that, It consists of the acoustic sensitizer as described in claim 5 and the loaded drug.
8. The acoustic immunotherapy agent according to claim 7, characterized in that, It also includes αPD-L1.
9. A sonoimmunotherapy agent for pancreatic cancer, characterized in that, It is composed of Cu3P-Celastrol acoustic sensor and αPD-L1.
10. The pancreatic cancer acoustic immunotherapy agent according to claim 9, characterized in that, The treatment method for Cu3P-Celastrol sound sensitizer is as follows: Dissolve Celastrol in ethanol, mix Cu3P NPs solution and Cel solution at a mass ratio of Cu3P to Celastrol of 1:2, and stir magnetically overnight; collect the precipitate by centrifugation, wash repeatedly with ethanol to remove excess Celastrol, and obtain Cu3P-Celastrol sound sensitizer.