Copper-based heterojunction sound-sensitive agent as well as preparation method and application thereof

Abstract

The invention relates to a copper-based heterojunction sound-sensitive agent and a preparation method and application thereof, Cu2O is etched by Se2-to obtain hollow Cu2-xSe, and then graphene quantum dots are loaded on the surface of the Cu2-xSe to obtain the copper-based heterojunction sound-sensitive agent. The copper-based heterojunction sonosensitizer prepared by the preparation method disclosed by the invention not only has efficient sonodynamic and chemical dynamic properties, but also has the capability of consuming glutathione (GSH) in a tumor microenvironment. In addition, the heterojunction sound-sensitive agent can also cause immunotherapy, and strong immune response is caused to achieve the capability of killing tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of sonosensitizers, and particularly relates to a copper-based heterojunction sonosensitizer, a preparation method thereof, and an application thereof. Background Art

[0002] Cancer treatment is an unsolved medical challenge, and recurrence and metastasis remain the main causes of death in cancer patients. Recently, a variety of immune checkpoint inhibitors have been approved by the US Food and Drug Administration (FDA) for clinical use, and the effectiveness of cancer immunotherapy in treating solid tumors such as melanoma, non-small cell lung cancer, and prostate cancer has been demonstrated. By harnessing and training the patient's own immune system to fight cancer cells, cancer immunotherapy has become a promising new method for cancer treatment. However, immunotherapy is still limited by a low response rate (only about 20%) and significant side effects. Immunogenic cell death (ICD) induced by reactive oxygen species (ROS)-mediated tumor treatment strategies such as radiotherapy, photodynamic therapy (PDT), and sonodynamic therapy (SDT) can induce adaptive immunity against dead cell antigens and greatly promote inflammatory cell infiltration. Among these ROS generation treatment modalities, PDT has been approved by the FDA for clinical trials to treat tumors due to its low invasiveness, but the penetration depth of near-infrared laser is limited (<1 cm), making PDT only suitable for the treatment of superficial tumors. Although radiotherapy has deep penetration ability, the damage of ionizing radiation to normal tissues severely restricts the clinical application of radiotherapy. In contrast, SDT based on US-triggered activation of sonosensitizers to generate ROS is the only treatment modality with the largest tissue penetration depth (>10 cm) and low side effects. However, due to inefficient sonosensitizers and complex tumor microenvironment (TME), the insufficient production of ROS limits SDT-induced ICD to a great extent. The wide bandgap and rapid electron-hole pair recombination of inorganic nanomaterials result in low ROS yield. In addition, the ROS generation efficiency induced by oxygen-dependent SDT is severely limited by hypoxia in the TME and overexpression of glutathione (GSH). Therefore, the development of multifunctional sonosensitizers with high-efficient ROS generation and TME regulation capabilities can achieve cascade amplification of ROS generation.

[0003] To overcome the limitations of inorganic sonosensitizers, researchers have proposed the method of constructing heterojunctions. A heterojunction is an interfacial structure composed of two or more different materials, which has unique electronic and optical properties. By constructing heterojunctions, firstly, the separation and migration of carriers in semiconductor sonosensitizers can be optimized. The band bending and charge transfer at the heterojunction interface can promote the effective separation and migration of electrons and holes, reduce the rapid recombination of electron-hole pairs, and thus improve the quantum yield of ROS. Secondly, constructing heterojunctions can enhance the biocompatibility and stability of nanomaterials. By selecting appropriate materials to construct heterojunctions, it can ensure that the sonosensitizer has good biocompatibility and stability, and reduce phototoxicity and side effects. Finally, constructing heterojunctions can regulate the tumor microenvironment, consume reducing substances such as GSH and H2O2 in tumors, generate oxygen and ROS, etc., so as to improve the tumor microenvironment and enhance the SDT treatment effect. Summary of the Invention

[0004] The present invention provides a copper-based heterojunction sonosensitizer, its preparation method and application, aiming to solve the problems such as low ROS yield of current nano sonosensitizers and easy consumption of ROS by endogenous GSH, and enhance the sonodynamic tumor treatment effect through effective regulation of the tumor microenvironment.

[0005] The present invention provides a copper-based heterojunction sonosensitizer, which is obtained by etching Cu2O with Se 2- to obtain hollow Cu 2-x Se, and then loading graphene quantum dots GQD on the surface of Cu 2-x Se, denoted as GQD / Cu 2-x Se.

[0006] The present invention also provides a preparation method of a copper-based heterojunction sonosensitizer, which includes the following steps:

[0007] (1) Prepare Cu2O nanocubic particles, and then dissolve them in deionized water to obtain a Cu2O solution;

[0008] (2) Add the selenium source solution to the above Cu2O solution and stir to obtain hollow Cu 2-x Se;

[0009] (3) Dissolve the hollow Cu 2-x Se and graphene quantum dots in deionized water and stir to obtain a copper-based heterojunction sonosensitizer.

[0010] Preferably, the preparation method of the Cu2O nanocubic particles in step (1) is: dissolve sodium dodecyl sulfate SDS in deionized water and stir, then add copper sulfate solution and stir, then add alkali solution (NaOH) and sodium ascorbate SA and continue to stir, stand for centrifugation, wash and dry to obtain Cu2O nanocubic particles.

[0011] Preferably, the mass-volume ratio of sodium dodecyl sulfate to deionized water is 0.001-0.01 g∶1 mL; the concentration of the copper sulfate solution is 0.1-0.2 M; the molar ratio of sodium dodecyl sulfate, copper sulfate, alkali, and sulfamic acid is 10∶1∶4∶5.

[0012] Preferably, the concentration of the Cu2O solution in step (1) is 0.001-0.002 M.

[0013] Preferably, the volume ratio of the selenium source solution to the Cu2O solution in step (2) is 1-5∶10.

[0014] Preferably, the method for preparing graphene quantum dots in step (3) is as follows: Mix a carbon source with an ethanol solution and phosphoric acid, transfer it to a microwave reaction tube, heat it at 200-300 °C for 10-20 min, and after natural cooling, extract and remove the unreacted carbon source to obtain graphene quantum dots.

[0015] Preferably, the hollow Cu 2-x Se and the mass ratio of graphene quantum dots is 1∶1.

[0016] The present invention also provides an application of a copper-based heterojunction sonosensitizer in the preparation of a drug for sonodynamic cancer treatment.

[0017] Beneficial effects

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] First, due to selenization etching, Cu 2-x Se, which is more stable than Cu2O, can be obtained to prevent cuproptosis in normal tissues. Second, by constructing a heterojunction, the sonodynamic efficiency of the Cu 2-x Se sonosensitizer is enhanced. Then the loaded chemotherapeutic drug GQD will specifically accumulate at the tumor site due to the acid-responsive release of GQD / Cu 2-x Se. Finally, the specifically released Cu 2+ at the tumor site will consume overexpressed GSH. Ultimately, this GQD / Cu 2-x Se heterojunction will specifically achieve a cascade amplification of ROS generation at the tumor site without harming normal tissues. More importantly, the ROS level induced by GQD / Cu 2-x Se is greatly increased through SDT and CDT, which can trigger ICD, and then further induce a strong anti-tumor immune response by promoting DC maturation and activating T lymphocytes. Due to these favorable characteristics, the heterojunction enhances SDT and CDT, and achieves satisfactory therapeutic effects on primary and distant tumors through a cascade reaction of stimulation and immune response. Description of the Drawings

[0020] Figure 1 a) GQD / Cu in 2-x TEM image of Se; b) GQD / Cu 2-x HRTEM image of Se.

[0021] Figure 2 a) GQD / Cu in 2-x Se's 1 O2 production rate test; b) Cu 2-x Se and GQD / Cu 2-x Se's 1 O2 production rate comparison; c) Cu 2-x Se and GQD / Cu 2-x ESR spectrum comparison of Se.

[0022] Figure 3 a) GQD / Cu in 2-x ·OH production rate test of Se; b) Cu 2-x Se and GQD / Cu 2-x ·OH production rate comparison of Se; c) Cu 2-x Se and GQD / Cu 2-x ESR spectrum comparison of Se; d) GQD / Cu 2-x Performance test of Se's consumption of GSH.

[0023] Figure 4 a) Cu in 2-x Se and GQD / Cu 2-x Confocal images of Se's reactive oxygen species production; b) GQD / Cu 2-x Se under US irradiation (50 kHz, 3.0 W cm -2 -2, 5 min) or without irradiation: cell viability comparison.

[0024] Figure 5 a - b) Changes in tumor volume after intravenous injection of GQD / Cu 2-x Se; c) Survival time of the mice after treatment; d) Changes in the body weight of the mice during tumor treatment.

[0025] Figure 6 a) Changes in the maturation of dendritic cells in lymphoid tissues after different treatment methods; b - c) Changes in the content of T cells in the primary tumor. Detailed Implementation Modes

[0026] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0027] Example 1

[0028] This example provides a preparation method of a copper-based heterojunction sonosensitizer:

[0029] a) Dissolve 0.576 g of SDS in 96.1 mL of DI water, stir for 10 min, then add 1 mL of CuSO4·5H2O (0.1 M), stir for 5 min, then add 400 μL of NaOH (1 M), slowly add 2.5 mL of SA (0.2 M) after 30 s, continue to stir for 5 min, and let stand for 10 min. The precipitate is centrifuged at 9500 r / min for 10 min, collected and washed twice with a mixture of ethanol and water (1:1), and then washed with ethanol. Finally, it is dried in vacuo at 60 °C for 12 h to obtain Cu2O nanocubic particles with a size of 86 nm, which are then dissolved in deionized water to obtain a Cu2O solution.

[0030] b) Add 39.48 mg of selenium powder to 9 mL of deionized water and stir for 10 min under a N2 atmosphere. Then add 1 mL of NaBH4

[0031] (1.25 M) and continue to stir for 1 h in a N2 atmosphere to obtain a selenium source solution. Add 6 mL of the selenium source solution to 20 mL of a Cu2O (0.001 M) solution and stir for 4 h, and centrifuge and wash three times to obtain hollow Cu 2-x S e nanocubes.

[0032] c) Mix julolidine (0.05 g) with 9.5 mL of an ethanol solution and 0.5 mL of H3PO4. Transfer the mixture to a microwave reaction tube and heat at 200 °C for 10 min. After natural cooling, use petroleum ether as an extractant to remove the unreacted julolidine to obtain GQD.

[0033] d) Dissolve 10 mg of Cu 2-x Se and 10 mg of GQD in 20 mL of DI water, stir for 24 h and then centrifuge, and wash three times with water to obtain GQD / Cu 2-x Se.

[0034] Testing of the sonodynamic performance, chemodynamic performance and GHS consumption performance of the GQD / Cu 2-x Se sonosensitizer:

[0035] a) The prepared GQD / Cu 2-x Se sonosensitizer can generate a large amount of singlet oxygen ( 1 O2) under low-intensity ultrasound. By using 1,3-diphenylisobenzofuran (DPBF) as the 1 O2 probe, the 2-x O2 generation efficiency of the GQD / Cu 1 Se sonosensitizer under ultrasonic irradiation was detected to evaluate its sonodynamic performance.

[0036] b) The prepared GQD / Cu 2-x Se sonosensitizer can undergo the Fenton reaction under acidic conditions to generate a large amount of hydroxyl radicals (·OH). By using 3,3′,5,5′-tetramethylbenzidine (TMB) as the ·OH probe, the chemodynamic performance of the GQD / Cu 2-x Se sonosensitizer was evaluated.

[0037] c) The prepared GQD / Cu 2-x Se sonosensitizer can consume GSH. By using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) as the GSH probe, the ability of the GQD / Cu 2-x Se sonosensitizer to consume GSH was evaluated.

[0038] GQD / Cu 2-x Se sonosensitizer in vitro sonodynamic and chemodynamic therapy:

[0039] The cell viability of cells treated with GQD / Cu 2-x Se and US irradiation was detected by the MTT method. Mouse breast cancer cells (4T1) were seeded into 96-well plates at a density of 5000 per well and cultured for 24 hours. Then, different concentrations (0, 3.75, 7.5, 15, 30, 60 μg / mL) of GQD / Cu 2-x Se were added and cultured for 4 hours, irradiated with US (50 kHz, 3.0 W cm -2 ) for 5 minutes, and then the in vitro sonodynamic therapy efficacy of GQD / Cu 2-x Se was detected by the standard MTT experiment.

[0040] GQD / Cu 2-x Se sonosensitizer in vivo sonodynamic and chemodynamic therapy:

[0041] 100 μL (2 million) of mouse breast cancer cells (4T1) were subcutaneously implanted at the left and right axillae of 3- to 5-week-old female mice. When the tumor volume grew to 100 mm 3 , the mice were divided into 5 groups (5 mice in each group): (1) normal saline, (2) single US irradiation (50 kHz, 3.0 W cm-2 , 5 min), (3) GQD / Cu 2-x Se (50 μg / mL), (4) Cu 2-x Se + US (50 μg / mL), GQD / Cu 2- x Se (50 μg / mL) + US irradiation (50 kHz, 3.0 W cm -2 , 5 min). The tumor volume was measured every other day and the body weight of the mice was recorded daily to evaluate the in vivo sonodynamic therapy efficiency of the GQD / Cu 2-x Se sonosensitizer.

[0042] GQD / Cu 2-x In vivo immunotherapy of the GQD / Cu

[0043] Female mice at 3 - 5 weeks old were subcutaneously implanted with 100 μL (2 million) of mouse breast cancer cells (4T1) at the left and right axillae. When the tumor volume grew to 100 mm 3 , the mice were divided into 5 groups (5 mice in each group): (1) normal saline, (2) US irradiation alone (50 kHz, 3.0 W cm -2 , 5 min), (3) GQD / Cu 2-x Se (50 μg / mL), (4) Cu 2-x Se + US (50 μg / mL), (5) GQD / Cu 2-x Se (50 μg / mL) + US irradiation (50 kHz, 3.0 W cm -2 , 5 min). One day after treatment, the mice were euthanized, and their lymphoid tissues and primary tumors were removed for immunological tests.

[0044] The results are as follows:

[0045] (1) As Figure 1 can be seen, the particle size distribution of the obtained GQD / Cu 2-x Se was 100 nm. Under high - power electron microscopy observation, it had obvious lattice fringes.

[0046] (2) As Figure 2 can be seen, the sonodynamic performance of GQD / Cu 2-x Se was significantly better than that of Cu 2-x Se nanoparticles.

[0047] (3) As Figure 3 can be seen, the chemodynamic performance of GQD / Cu 2-x Se was significantly better than that of Cu 2-x Se nanoparticles; meanwhile, GQD / Cu 2-x Se could also efficiently consume GSH.

[0048] (4) It can be seen from Figure 4 that GQD / Cu 2-x Se can generate a large amount of reactive oxygen species under US irradiation; under US irradiation, GQD / Cu 2-x Se can completely kill tumor cells.

[0049] (5) It can be seen from Figure 5 that compared with single Cu 2-x Se, under US irradiation, GQD / Cu 2-x Se has the best tumor treatment effect, which can completely inhibit tumor growth; GQD / Cu 2-x Se can extend the survival time of mice; at the same time, GQD / Cu 2- x Se does not show obvious long-term toxicity in vivo.

[0050] (6) It can be seen from Figure 6 that under US irradiation, GQD / Cu 2-x Se can strongly promote the maturation of dendritic cells, generating a large number of CD4 + CD8 + T cells, thus causing a powerful immunotherapy.

Claims

1. A copper-based heterojunction sonosensitizer, characterized in that: Through Se 2- Etching Cu2O to obtain hollow Cu 2-x Se, and then loading graphene quantum dots on the surface of Cu 2-x Se to obtain the product.

2. A preparation method of a copper-based heterojunction sonosensitizer, comprising the following steps: (1) Prepare Cu2O nanocubic particles, and then dissolve them in deionized water to obtain a Cu2O solution; (2) Add the selenium source solution to the above Cu2O solution and stir to obtain hollow Cu 2-x Se; (3) Dissolve hollow Cu 2-x Se and graphene quantum dots in deionized water and stir to obtain a copper-based heterojunction sonosensitizer.

3. The preparation method according to claim 2, characterized in that: The preparation method of the Cu2O nanocubic particles in the step (1) is as follows: dissolve sodium dodecyl sulfate in deionized water and stir, then add a copper sulfate solution and stir, then add an alkali solution and sodium ascorbate and continue to stir, stand for centrifugation, wash and dry to obtain Cu2O nanocubic particles.

4. The preparation method according to claim 3, characterized in that: The mass-volume ratio of the sodium dodecyl sulfate to the deionized water is 0.001-0.01 g: 1 mL; the concentration of the copper sulfate solution is 0.1-0.2 M; the molar ratio of the sodium dodecyl sulfate, copper sulfate, alkali and sodium ascorbate is 10:1:4:

5.

5. The preparation method according to claim 2, characterized in that: The concentration of the Cu2O solution in the step (1) is 0.001-0.002 M.

6. The preparation method according to claim 2, characterized in that: The volume ratio of the selenium source solution to the Cu2O solution in the step (2) is 1-5:

10.

7. The preparation method according to claim 2, characterized in that: The preparation method of the graphene quantum dots in the step (3) is as follows: mix a carbon source with an ethanol solution and phosphoric acid, transfer it to a microwave reaction tube and heat it at 200-300 °C for 10-20 min, and after natural cooling, extract and remove the unreacted carbon source to obtain graphene quantum dots.

8. The preparation method according to claim 2, characterized in that: The hollow Cu in the step (3) 2-x and graphene quantum dots have a mass ratio of 1:

1.

9. An application of the copper-based heterojunction sonosensitizer according to claim 1 in the preparation of a drug for sonodynamic cancer therapy.

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