Heterojunction sound-sensitive agent as well as preparation method and application thereof

Abstract

The invention relates to a heterojunction sound-sensitive agent as well as a preparation method and application thereof, Zr-MOF is taken as a template, hollow MnO2 is obtained by an acid etching method, and then carbon dots are loaded on the surface of the MnO2, so that the heterojunction sound-sensitive agent is obtained. The heterojunction sonosensitizer prepared 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 heterojunction sonosensitizer, a preparation method thereof, and an application thereof. Background Art

[0002] Due to their unique physical and chemical properties (such as magnetism, catalytic activity, biocompatibility, etc.), manganese (Mn)-based nanomaterials have been widely studied and applied in the field of nanomedicine. First, Mn-based nanomaterials (such as MnO, MnO2, Mn3O4, etc.) have excellent T1-weighted magnetic resonance imaging performance, which can significantly enhance the imaging contrast of tumor tissues. For example, MnO2 nanoparticles will be reduced to Mn in the tumor microenvironment. 2+ , and the released Mn 2+ can enhance the MRI signal and achieve highly sensitive detection of tumors. Second, Mn-based nanomaterials can be used for sonodynamic therapy (SDT). Mn-based sonosensitizers will generate a large amount of reactive oxygen species (ROS) under the action of ultrasonic waves, inducing apoptosis of tumor cells. At the same time, Mn-based nanomaterials can be used as drug carriers to achieve targeted delivery of drugs. For example, MnO2 nanoparticles can target the tumor microenvironment and decompose under GSH conditions to release the loaded anticancer drugs. Finally, Mn 2+ ions can increase the sensitivity of cyclic GMP-AMP synthase (cGAS) to dsDNA or directly activate cGAS in a DNA-independent manner, promote the generation of cyclic guanosine monophosphate adenosine (cGAMP), activate the interferon regulatory factor (sting) cell signaling pathway, recruit TANK-binding kinase (TBK-1), and ultimately promote the expression of interferon regulatory factor (IRF-3). At the same time, the activation of the STING pathway can stimulate DC maturation, macrophage polarization, and increase the infiltration of T cells and natural killer cells (NK), reverse the immunosuppressive microenvironment, and cause an immune response. Summary of the Invention

[0003] The present invention provides a heterojunction sonosensitizer, a preparation method thereof, and an application thereof, aiming to solve the problems of low ROS production rate 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.

[0004] The present invention provides a heterojunction sonosensitizer, which is obtained by using Zr-MOF as a template and acid etching to obtain hollow MnO2, and then loading carbon dots CD on the surface of MnO2, denoted as CD@H-MnO2.

[0005] The present invention also provides a preparation method of a heterojunction sonosensitizer, including the following steps:

[0006] (1) Prepare Zr-MOF powder, then add the Zr-MOF powder and MnF3 into deionized water, stir, centrifuge and wash, and collect the precipitate; then dissolve the precipitate in a buffer solution with pH 6.5, stir and etch at room temperature, centrifuge and wash, and collect the precipitate to obtain H-MnO2;

[0007] (2) Dissolve polyethyleneimine BPEI, trinitropyrene TNP and methyl-β-cyclodextrin in deionized water and mix well. Then place it in a reaction kettle and react at 200-300 °C for 10-20 h. Collect the product and dialyze to obtain carbon dots CD;

[0008] (3) Dissolve H-MnO2 and carbon dots in deionized water and stir to obtain a heterojunction sonosensitizer.

[0009] Preferably, the preparation method of the Zr-MOF powder in step (1) is: dissolve terephthalic acid H2BDC and zirconium oxychloride octahydrate ZrOCl2·8H2O in N,N-dimethylformamide DMF, stir and sonicate until transparent, then add glacial acetic acid and mix well. Then heat the solution at 80-100 °C for 2-5 h, centrifuge and wash, and collect the precipitate to obtain Zr-MOF powder.

[0010] Preferably, the mass ratio of terephthalic acid to zirconium oxychloride octahydrate is 1-3:1; the mass-volume ratio of terephthalic acid to N,N-dimethylformamide is 10-15 mg:1 mL; the volume ratio of glacial acetic acid to N,N-dimethylformamide is 1:2.

[0011] Preferably, the buffer solution with pH 6.5 in step (1) refers to the buffer solution of acetic acid and sodium acetate.

[0012] Preferably, the mass ratio of polyethyleneimine, trinitropyrene and methyl-β-cyclodextrin in step (2) is 8:1-2:1-2.

[0013] Preferably, the mass ratio of H-MnO2 and carbon dots in step (3) is 1:2.

[0014] The present invention also provides an application of the heterojunction sonosensitizer in the preparation of a drug for sonodynamic cancer therapy.

[0015] Beneficial effects

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] First, H-MnO2 is a nanomaterial that degrades in a GSH-responsive manner, which can prevent damage caused by degradation in normal tissues. Second, the sono-dynamic efficiency of the H-MnO2 sonosensitizer is enhanced by constructing a heterojunction. Then, the loaded chemotherapeutic drug CD will specifically accumulate at the tumor site due to the GSH-responsive release of CD@H-MnO2. These CDs will consume cholesterol, causing cell sclerosis and thus enhancing T cell proliferation. Finally, Mn 3+ and Mn 4+ will consume overexpressed GSH and be reduced to Mn 2+ thereby activating the STING pathway. Ultimately, this CD@H-MnO2 heterojunction will specifically achieve a cascade amplification of ROS generation at the tumor site without damaging normal tissues. More importantly, the ROS level induced by CD@H-MnO2 through SDT is greatly increased, 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 properties, the heterojunction enhances SDT and achieves satisfactory therapeutic effects on primary and distant tumors by stimulating the immune response. Brief Description of the Drawings

[0018] Figure 1 a) TEM image of H-MnO2; b) TEM image of CD@H-MnO2 in

[0019] Figure 2 a) O2 generation rate test of CD@H-MnO2 in 1 ; b) Comparison of O2 generation rates of H-MnO2 and CD@H-MnO2; c) Comparison of ESR spectra of H-MnO2 and CD@H-MnO2 in 1

[0020] Figure 3 a) Oxidase activity test of CD@H-MnO2; b) Comparison of oxidase activities of H-MnO2 and CD@H-MnO2; c) Performance test of CD@H-MnO2 for consuming GSH in

[0021] Figure 4 a) Confocal images of reactive oxygen species generation by H-MnO2 and CD@H-MnO2; b) Comparison of cell viability of CD@H-MnO2 with or without US irradiation (50 kHz, 3.0 W cm -2 , 5 min) in

[0022] Figure 5 a-b) Changes in tumor volume after intravenous injection of CD@H-MnO2; c) Survival time of mice after treatment; d) Changes in body weight of mice during tumor treatment in

[0023] 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 manners

[0024] 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.

[0025] Example 1

[0026] This example provides a preparation method of a heterojunction photosensitizer:

[0027] a) 886.8 mg of H2BDC and 324 mg of ZrOCl2·8H2O are dissolved in 80 mL of DMF, stirred and sonicated until transparent, and then

[0028] 40 mL of glacial acetic acid is added. After thorough mixing, the solution is heated at 90 °C for 4 h, centrifuged and washed three times with DMF and absolute ethanol, and the precipitate is collected to obtain Zr-MOF powder.

[0029] b) 60 mg of Zr-MOF and 20 mg of MnF3 are added to 40 mL of deionized water and stirred for 24 h. Centrifuged and washed three times with deionized water, and the precipitate is collected. Then the precipitate is dissolved in a buffer solution with a pH of 6.5 and etched by stirring at room temperature for 2 h, centrifuged and washed three times with deionized water, and the precipitate is collected to obtain H-MnO2.

[0030] c) 0.8 g of BPEI (M.W. 1800, 99%), 0.1 g of TNP and 0.1 g of methyl-β-cyclodextrin are dissolved in 40 mL of deionized water, thoroughly mixed, placed in a reaction kettle, reacted at 200 °C for 12 h, the product is collected, and dialyzed with a 3500 Da dialysis bag for 7 d to obtain CD.

[0031] d) H-MnO2 and CD are stirred at room temperature for 24 h at a mass ratio of 1:2, centrifuged and washed with water 3 times, and the precipitate is collected to obtain CD@H-MnO2.

[0032] Testing of the sonodynamic performance, oxidase activity and GSH consumption performance of the CD@H-MnO2 photosensitizer:

[0033] a) The prepared CD@H-MnO2 photosensitizer can generate a large amount of singlet oxygen ( 1 O2) under low-intensity ultrasound. By using 1,3-diphenylisobenzofuran (DPBF) as 1The O2 probe detects the 1 O2 generation efficiency of the CD@H-MnO2 sonosensitizer under ultrasonic irradiation to evaluate its sonodynamic performance.

[0034] b) The prepared CD@H-MnO2 sonosensitizer can generate oxidase activity under acidic conditions. By using 3,3′,5,5′-tetramethylbenzidine (TMB) and dihydro-rhodamine 123 (DHR123) as oxidase detection probes, the oxidase activity of the CD@H-MnO2 sonosensitizer is evaluated.

[0035] c) The prepared CD@H-MnO2 sonosensitizer can consume GSH. By using 5,5′-dithiobis(2-nitrobenzoic acid)

[0036] (DTNB) as a GSH probe, the ability of the CD@H-MnO2 sonosensitizer to consume GSH is evaluated.

[0037] In vitro sonodynamic therapy of the CD@H-MnO2 sonosensitizer:

[0038] The cell viability of cells treated with CD@H-MnO2 and US irradiation is detected by CCK8 reagent. Mouse breast cancer cells (4T1) are seeded into 96-well plates at a density of 5000 cells per well and cultured for 24 hours. Then, different concentrations (0, 15, 30, 45, 60, 75 μg / mL) of CD@H-MnO2 are added and cultured for 4 hours. Then, they are irradiated with US (50 kHz, 3.0 W cm -2 ) for 5 minutes, and then the in vitro sonodynamic therapy efficacy of CD@H-MnO2 is detected by CCK8 reagent.

[0039] In vivo sonodynamic therapy of the CD@H-MnO2 sonosensitizer:

[0040] 100 μl (2 million) of mouse breast cancer cells (4T1) are subcutaneously implanted at the left and right axillae of female mice aged 3-5 weeks. When the tumor volume grows to 100 mm 3 , the mice are divided into 6 groups (5 mice in each group): (1) normal saline, (2) single US irradiation (50 kHz, 3.0 W cm -2 , 5 min), (3) H-MnO2 (75 μg / mL), (4) H-MnO2 + US (75 μg / mL), (5) CD@H-MnO2 (75 μg / mL), (6) CD@H-MnO2 (75 μg / mL) + US irradiation (50 kHz, 3.0 W cm -2 , 5 min). The tumor volume is measured every other day and the body weight of the mice is recorded every day to evaluate the in vivo sonodynamic therapy efficiency of the CD@H-MnO2 sonosensitizer.

[0041] In Vivo Immunotherapy of CD@H-MnO2 Sonosensitizer:

[0042] Subcutaneously implant 100 μL (2 million) of mouse breast cancer cells (4T1) into the left and right axillae of female mice aged 3 - 5 weeks. Wait until the tumor volume grows to 100 mm 3 Then, divide the mice into 6 groups (5 mice in each group): (1) Normal saline, (2) US irradiation alone (50 kHz, 3.0 W cm -2 , 5 min), (3) H-MnO2 (75 μg / mL), (4) H-MnO2 + US (75 μg / mL), (5) CD@H-MnO2 (75 μg / mL), (6) CD@H-MnO2 (75 μg / mL) + US irradiation (50 kHz, 3.0 W cm -2 , 5 min). One day after treatment, euthanize the mice and remove their lymphoid tissues and primary tumors for immunological tests respectively.

[0043] The results are as follows:

[0044] (1) As Figure 1 can be seen, the particle size distribution of the obtained CD@H-MnO2 is 230 nm. Under high-power electron microscopy observation, it has obvious CD lattice fringes.

[0045] (2) As Figure 2 can be seen, the sonodynamic performance of CD@H-MnO2 is significantly better than that of H-MnO2 nanoparticles.

[0046] (3) As Figure 3 can be seen, the oxidase activity of CD@H-MnO2 is significantly better than that of H-MnO2 nanoparticles; at the same time, CD@H-MnO2 can also efficiently consume GSH.

[0047] (4) As Figure 4 can be seen, CD@H-MnO2 can generate a large amount of reactive oxygen species under US irradiation; under US irradiation, CD@H-MnO2 can completely kill tumor cells.

[0048] (5) As Figure 5 can be seen, compared with H-MnO2 alone, under US irradiation, CD@H-MnO2 has the best tumor treatment effect, which can completely inhibit tumor growth; CD@H-MnO2 can extend the survival time of mice; at the same time, CD@H-MnO2 does not show obvious long-term toxicity in vivo.

[0049] (6) As Figure 6 can be seen, under US irradiation, CD@H-MnO2 can strongly promote the maturation of dendritic cells and produce a large amount of CD4 +CD8 + T cells thus trigger powerful immunotherapy.

Claims

1. A heterojunction photosensitizer, characterized in that: Hollow MnO2 was obtained using Zr-MOF as a template and by means of acid etching, and then carbon dots were loaded on the surface of MnO2.

2. A method for preparing a heterojunction sonosensitizer, comprising the following steps: (1) Prepare Zr-MOF powder, then add the Zr-MOF powder and MnF3 to deionized water, stir, centrifuge and wash, and collect the precipitate; then dissolve the precipitate in a buffer solution with a pH of 6.5, stir and etch at room temperature, centrifuge and wash, and collect the precipitate to obtain H-MnO2; (2) Dissolve polyethyleneimine, trinitropyrene and methyl-β-cyclodextrin in deionized water and mix well, then place in a reaction kettle and react at 200-300 °C for 10-20 h, collect the product, and dialyze to obtain carbon dots CD; (3) Dissolve H-MnO2 and carbon dots in deionized water and stir to obtain a heterojunction sonosensitizer.

3. The preparation method according to claim 2, characterized in that: The method for preparing the Zr-MOF powder in step (1) is as follows: Dissolve terephthalic acid and zirconium oxychloride octahydrate in N,N-dimethylformamide, stir and sonicate until transparent, then add glacial acetic acid and mix well, and heat the solution at 80-100 °C for 2-5 h, centrifuge and wash, and collect the precipitate to obtain Zr-MOF powder.

4. The preparation method according to claim 3, characterized in that: The mass ratio of terephthalic acid to zirconium oxychloride octahydrate is 1-3:1; the mass-volume ratio of terephthalic acid to N,N-dimethylformamide is 10-15 mg:1 mL; the volume ratio of glacial acetic acid to N,N-dimethylformamide is 1:

2.

5. The preparation method according to claim 2, characterized in that: The buffer solution with a pH of 6.5 in step (1) refers to an acetate and sodium acetate buffer solution.

6. The preparation method according to claim 2, wherein: The mass ratio of polyethyleneimine, trinitropyrene and methyl-β-cyclodextrin in step (2) is 8:1-2:1-2.

7. The preparation method according to claim 2, characterized in that: The mass ratio of H-MnO2 to carbon dots in step (3) is 1:

2.

8. Use of a heterojunction sonosensitizer as described in claim 1 in the preparation of a drug for sonodynamic cancer therapy.

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