Gadolinium-based sulfonamide complex with radiotherapy sensitization effect as well as preparation and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a gadolinium-based sulfonamide complex with a radiotherapy sensitization effect as well as preparation and application of the gadolinium-based sulfonamide complex. The preparation method comprises the following steps: dispersing sulfonamides and PVP in a mixed solution of ethanol and ethylene glycol, and stirring at room temperature to obtain a reaction solution A; dropwise adding Gd (NO3) 3.6 H2O into the reaction solution A, and continuously stirring to obtain a reaction solution B; and carrying out solvothermal reaction on the reaction solution B to obtain the gadolinium-based sulfonamide complex. The gadolinium-based sulfonamide complex prepared by the invention can increase the radiotherapy sensitivity by adjusting the tumor acidic microenvironment. Experiments prove that the gadolinium-based sulfonamide complex can effectively enhance the radiotherapy treatment effect, promote tumor cell apoptosis and slow down the tumor growth speed when being used in cooperation with radiotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a gadolinium-based sulfonamide complex with radiotherapy sensitization effect and a preparation method and application thereof. Background Art

[0002] Radiosensitizers refer to chemical substances or drugs that can enhance the biological effects of radiation when used simultaneously with radiotherapy. Since radiotherapy will inevitably cause radiation damage to adjacent normal tissues, radiosensitizers can effectively reduce the irradiation dose while achieving the same radiotherapy effect, so they have broad application prospects in improving the efficacy of tumor radiotherapy. The mechanism of action of radiosensitizers is mainly to change the radiosensitivity of cells by modifying the intrinsic factors that affect radiosensitivity, thereby killing as many tumor cells as possible, while not affecting or damaging normal cells less, and having no or little toxicity and side effects on the human body. At present, known radiosensitizers include DNA precursor base analogs, electrophilic radiosensitizers, hypoxic cell radiosensitizers, bioreduction compounds, radiation damage repair inhibitors, thiol inhibitors, oxygen utilization inhibitors, oxygen-like compounds, cytotoxic sensitizers, targeted radiosensitizers, Chinese herbal medicine extracts, etc. These radiosensitizers enhance the effect of radiotherapy through various mechanisms, but there are also some defects and challenges, such as dose-limiting adverse reactions, limited clinical effects, insufficient specificity, immunogenicity, and stability issues. Therefore, further research and development are needed to improve its efficacy and safety.

[0003] Changes in the tumor microenvironment, especially the acidic tumor microenvironment, are one of the important reasons for radiotherapy resistance, and the process of radiotherapy is often accompanied by increased acidification. The acidic tumor microenvironment is considered to be one of the representative signs of tumors. Numerous clinical studies have shown that the acidic tumor microenvironment leads to a poor prognosis and is associated with increased metastatic potential and chemotherapy and radiotherapy resistance. Compared with normal cells, tumor cells have an increased intracellular pH (pHi) and a decreased extracellular pH (pHe). Using microelectrode method, magnetic resonance imaging, fluorescence spectroscopy and other methods to measure the pH inside and outside tumor cells, it can be found that the acidity of tumor tissue is much higher than that of normal tissue. This is because the metabolism of tumor tissue is more dependent on anaerobic glycolysis, and its metabolites lactate and H +Together with carbon dioxide produced by respiration, it causes the acidification of the tumor microenvironment. Tumor cells cannot survive at a pHi level that is too low for a long time, so they transport acidic substances to the outside of the cell or transport extracellular alkaline substances into the cell through a series of transport proteins. Data recording the impact of the acidic tumor microenvironment on treatment or the impact of treatment on the acidic tumor microenvironment began to appear in the 1980s. By targeting and regulating the pH of the tumor microenvironment, tumor cell proliferation can be inhibited to a certain extent, tumor invasive ability can be weakened, and tumor resistance to radiotherapy and chemotherapy can be reduced. That is, regulating the acidic tumor microenvironment may be one of the key strategies to improve radiotherapy sensitivity and efficacy.

[0004] In recent years, the therapeutic strategy of changing the acidic microenvironment of tumors is mainly to affect the expression and function of membrane protein transporters, neutralize H + and interfere with tumor glucose metabolism pathways. Carbonic anhydrase (CA) is a H + / HCO3 - Bidirectional transporters have become promising targets for cancer therapy. Among them, carbonic anhydrase 9 (CAIX) is widely expressed in a variety of tumor cells and is one of the main factors of extracellular acidosis. Like other carbonic anhydrases, the extracellular catalytic domain of CA IX exposed to the cell can catalyze the hydration reaction of CO2 and cooperate with bicarbonate transporters to transfer HCO3 - Intracellular alkaline microenvironment is maintained, which is conducive to tumor proliferation. + It exacerbates extracellular acidification, which is beneficial to tumor invasion and metastasis. At the same time, CAIX also has a mucin domain (PG) that is different from other carbonic anhydrases. PG participates in the cell adhesion process and plays an indispensable role in the metastasis of cancer cells. Therefore, interfering with the expression and function of CA IX is beneficial to improving the acidic microenvironment of tumors, and using it as a target in tumor diagnosis and treatment has great development prospects.

[0005] At present, there are relatively few studies on nano-radiotherapy sensitizers that regulate the acidity of the tumor microenvironment, and some existing technologies have reported on other types of radiotherapy sensitizers. For example, patent CN117838874B discloses a preparation method and application of a gadolinium-platinum radiotherapy sensitizer. The invention uses the regulatory properties of gadolinium ions on the growth of platinum nanoparticles to synthesize a gadolinium-platinum radiotherapy sensitizer with MRI capabilities. The gadolinium-platinum radiotherapy sensitizer has CAT activity, which can catalyze the decomposition of H2O2 to produce O2, relieve hypoxia inhibition at the tumor site, and enhance radiotherapy sensitivity; at the same time, the gadolinium-platinum radiotherapy sensitizer also has POD activity, which can catalyze the decomposition of H2O2 to produce ·OH, enhance the efficacy of radiotherapy, and promote tumor cell apoptosis.

[0006] Amorphous infinite coordination polymers (ICPs) have attracted wide attention in tumor treatment due to their good thermal stability, good water solubility, mild and easy preparation conditions. Compared with traditional carrier-free nanomedicines, ICPs are only composed of metal ions and drugs, which are easier to enter the clinic and have a very bright future in the process of tumor treatment. The construction of a multifunctional treatment system with multiple uses and the synergistic treatment strategy between multiple tumor treatment methods have also become one of the research hotspots of researchers. Summary of the invention

[0007] In view of this, the present invention provides a gadolinium sulfonamide complex with radiosensitization effect and its preparation and application. The gadolinium sulfonamide complex has radiosensitization effect, which increases radiosensitivity by regulating the acidic microenvironment of the tumor and promotes tumor cell apoptosis in coordination with radiotherapy.

[0008] One of the purposes of the present invention is to provide a method for preparing a gadolinium-based sulfonamide complex having a radiotherapy sensitization effect.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] The preparation method of a gadolinium-based sulfonamide complex having a radiotherapy sensitization effect comprises the following steps:

[0011] (1) dispersing sulfonamide drugs and PVP in a mixed solution of ethanol and ethylene glycol, stirring at room temperature, to obtain a reaction solution A;

[0012] (2) adding Gd(NO3)3·6H2O dropwise to the reaction solution A obtained in step (1), and continuing stirring to obtain reaction solution B;

[0013] (3) The reaction solution B obtained in step (2) is subjected to a solvothermal reaction to obtain a gadolinium-based sulfonamide complex.

[0014] Furthermore, in step (1), the volume ratio of ethanol to ethylene glycol in the mixed solution of ethanol and ethylene glycol is 1:0 to 13:7, preferably 12:7.

[0015] Furthermore, the molar ratio of the sulfonamide drug to the Gd(NO3)3·6H2O is 1:1; the molar ratio of the sulfonamide drug to the PVP is 1:0 to 1:10, preferably 1:0, 1:1, 1:2.5, 1:5 or 1:10. Wherein, PVP can be 0, and the molar amount of PVP is calculated based on the molecular weight of its monomer.

[0016] Furthermore, the sulfonamide drugs include sulfonamide, sulfadiazine, and acetazolamide.

[0017] Furthermore, in step (1), the stirring time is 25 to 40 minutes; and / or in step (2), the stirring time is 8 to 20 minutes.

[0018] Preferably, in step (1), the stirring time is 30 minutes.

[0019] Preferably, in step (2), the stirring time is 10 minutes.

[0020] Furthermore, in step (3), the reaction temperature is 150-250° C., and the reaction time is 8-12 h.

[0021] Preferably, in step (3), the reaction temperature is 200° C. and the reaction time is 10 h.

[0022] Furthermore, the solvent thermal reaction product is centrifuged, washed and redispersed to obtain a gadolinium-based sulfonamide complex.

[0023] Preferably, the washing comprises washing twice with ethanol and washing once with ddH2O.

[0024] Preferably, the redispersion solvent is water.

[0025] The second object of the present invention is to provide a gadolinium sulfonamide complex prepared by the above-mentioned preparation method.

[0026] To achieve the above object, the present invention adopts the following technical solutions:

[0027] The gadolinium sulfonamide complex is prepared by the above-mentioned preparation method.

[0028] Furthermore, the particle size of the gadolinium sulfonamide complex is 30-2000nm.

[0029] Preferably, the gadolinium sulfonamide complex is a PVP-modified gadolinium sulfonamide complex, and its particle size is 30-30 nm, more preferably 50 nm.

[0030] The third object of the present invention is to provide a use of the aforementioned gadolinium sulfonamide complex in the preparation of anti-tumor drugs.

[0031] To achieve the above object, the present invention adopts the following technical solutions:

[0032] The use of the aforementioned gadolinium sulfonamide complex in the preparation of anti-tumor drugs.

[0033] Furthermore, the anti-tumor effect includes promoting tumor cell apoptosis, slowing down tumor growth rate and / or reducing pH in tumor cells.

[0034] Furthermore, the tumor includes triple-negative breast cancer.

[0035] Furthermore, the tumor cells include 4T1 cells.

[0036] Furthermore, the application includes using the anti-tumor drug in combination with radiotherapy to treat tumors.

[0037] A fourth object of the present invention is to provide an application of the aforementioned gadolinium-based sulfonamide complex in the preparation of a nano-radiotherapy sensitizer for regulating the acidity of the tumor microenvironment.

[0038] To achieve the above object, the present invention adopts the following technical solutions:

[0039] The use of the aforementioned gadolinium-based sulfonamide complex in the preparation of a nano-radiotherapy sensitizer for regulating the acidity of the tumor microenvironment.

[0040] Furthermore, the tumor includes triple-negative breast cancer.

[0041] Furthermore, the regulation of the acidity of the tumor microenvironment includes lowering the pH in tumor cells, thereby downregulating the pentose phosphate pathway, reducing the expression of G6PD protein and the production of NADPH, reducing the consumption of ROS, and promoting tumor cell apoptosis in coordination with radiotherapy.

[0042] The beneficial effects of the present invention are:

[0043] The present invention synthesizes a gadolinium-based sulfonamide complex, and the preparation method is simple to operate and low in cost. The nanomaterial can effectively regulate the pH inside and outside tumor cells, increase the acidity inside tumor cells by inhibiting the expression of carbonic anhydrase 9 (CAIX), thereby downregulating the pentose phosphate pathway, reducing the production of NADPH, and reducing the consumption of ROS, thereby synergizing radiotherapy to achieve an effective tumor killing effect. At the same time, cell and animal experiments show that the nanomaterial has an excellent radiotherapy sensitization ratio, enhances the therapeutic effect of radiotherapy, promotes tumor cell apoptosis, slows down tumor growth rate, and effectively reduces the lung metastasis of in situ tumors. The present invention provides a new solution for improving the survival period and quality of life of triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is the transmission electron microscope image of PGA;

[0045] Figure 2 is the element surface distribution diagram of PGA, where Figure 2 -A is the HAADF graph of PGA, Figure 2 -B is the distribution diagram of Gd element in PGA, Figure 2 -C is the distribution diagram of S element in PGA, Figure 2 -D is the distribution diagram of C elements in PGA, Figure 2 -E is the distribution diagram of N elements in PGA, Figure 2 -F is the distribution diagram of O element in PGA, Figure 2 -G is the combined graph of C, N, O, S, and Gd in PGA;

[0046] Figure 3 FT-IR images of PGA, AZ and PVP;

[0047] Figure 4 This is a graph showing the tumor growth curve of orthotopic 4T1 tumor-bearing mice under different treatments;

[0048] Figure 5 HE images of lung metastasis in orthotopic 4T1 tumor-bearing mice under different treatments;

[0049] Figure 6 This is a graph showing the cytotoxicity test results of PGA on 4T1 cells and HUVEC cells;

[0050] Figure 7 is a graph showing the standard curve of BCECF AM and the changes in intracellular pH after PGA treatment, where: Figure 7 -A is the standard curve of BCECF AM, Figure 7 -B is a graph showing the changes in intracellular pH after PGA treatment;

[0051] Figure 8 This is the measurement result diagram of the relevant indicators of PGA lowering PPP, among which, Figure 8 -A is a graph showing the expression of G6PD after PGA treatment. Figure 8 -B is a graph of the NADPH / NADP+ ratio of 4T1 cells after different treatments. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0053] In the embodiments of the present invention, experimental methods without specifying specific conditions are usually conducted under conventional conditions or under conditions recommended by the manufacturer.

[0054] In the examples of the present invention, acetazolamide and Gd(NO3)3·6H2O were purchased from Ladin; and the BCECF AM probe was purchased from Bio-Tech.

[0055] Example 1. Preparation of Gadolinium Sulfonamide Complexes

[0056] (1) Dissolve 37.9 mg (0.22 mmol) of sulfonamide in a mixed solution of 12 mL of ethanol and 7 mL of ethylene glycol, referred to as reaction solution A, and stir at room temperature for 30 min;

[0057] (2) 1 mL of Gd(NO3)3·6H2O (100 mg, 0.22 mmol) was added dropwise to reaction solution A and stirred for 10 min to obtain reaction solution B;

[0058] (3) Transfer reaction solution B to a reactor and react at 200°C for 10 h;

[0059] (4) The product obtained in step (3) was centrifuged, washed twice with ethanol, washed once with ddH2O, and redispersed in water to obtain Gd-sulf. Transmission electron microscopy showed that the size of the nanoparticles was 800-1500 nm.

[0060] Example 2. Preparation of Gadolinium Sulfonamide Complexes

[0061] (1) Dissolve 55 mg (0.22 mmol) of sulfadiazine in a mixed solution of 12 mL of ethanol and 7 mL of ethylene glycol, referred to as reaction solution A, and stir at room temperature for 30 min;

[0062] (2) 1 mL of Gd(NO3)3·6H2O (100 mg, 0.22 mmol) was added dropwise to reaction solution A and stirred for 10 min to obtain reaction solution B;

[0063] (3) Transfer reaction solution B to a reactor and react at 200°C for 10 h;

[0064] (4) The product obtained in step (3) was centrifuged, washed twice with ethanol, washed once with ddH2O, and redispersed in water to obtain Gd-SD. Transmission electron microscopy showed that the size of the nanoparticles was 1000-2000 nm.

[0065] Example 3. Preparation of Gadolinium Sulfonamide Complexes

[0066] (1) 49 mg (0.22 mmol) of acetazolamide was dissolved in a mixed solution of 12 mL of ethanol and 7 mL of ethylene glycol, referred to as reaction solution A, and stirred at room temperature for 30 min;

[0067] (2) 1 mL of Gd(NO3)3·6H2O (100 mg, 0.22 mmol) was added dropwise to reaction solution A and stirred for 10 min to obtain reaction solution B;

[0068] (3) Transfer reaction solution B to a reactor and react at 200°C for 10 h;

[0069] (4) The product obtained in step (3) was centrifuged, washed twice with ethanol, washed once with ddH2O, and redispersed in water to obtain Gd-AZ. Transmission electron microscopy showed that the size of the nanoparticles was 500-1000 nm.

[0070] Example 4. Preparation of PVP-modified gadolinium-based sulfonamide complexes

[0071] (1) A mixed solution of 49 mg (0.22 mmol) of acetazolamide and 121 mg of PVP dissolved in 12 mL of ethanol and 7 mL of ethylene glycol was referred to as reaction solution A and stirred at room temperature for 30 min;

[0072] (2) 1 mL of Gd(NO3)3·6H2O (100 mg, 0.22 mmol) was added dropwise to reaction solution A and stirred for 10 min to obtain reaction solution B;

[0073] (3) Transfer reaction solution B to a reactor and react at 200°C for 10 h;

[0074] (4) The product obtained in step (3) was centrifuged, washed twice with ethanol, washed once with ddH2O, and redispersed in water to obtain PVP-modified gadolinium sulfonamide complex (PGA for short). Transmission electron microscopy showed that the nanoparticles were about 50 nm in size and were spherical particles with rough surfaces. Figure 1 , Figure 2 .

[0075] Example 5. Animal testing

[0076] 4T1 cells were collected in logarithmic phase and injected into the fourth mammary gland of Balb / c mice at a rate of 1×10 5 / mouse, when the tumor volume reaches 50mm 3 Afterwards, the tumor-bearing mice were randomly divided into four groups: 1) Control, 2) PGA, 3) X-ray, and 4) PGA+X-ray, with 5 mice in each group. Groups 1) and 3) were injected with 100 μL of normal saline as control, groups 2) and 4) were injected with 100 μL of PGA (100 mg / kg) twice through the tail vein, and groups 3) and 4) were irradiated with 4 Gy 24 hours later. The tumor volume was measured every two days and calculated according to the formula 1 / 2 length × width 2 The volume was calculated. Lung tissue was obtained after treatment to verify the inhibitory effects of different treatments on lung metastasis.

[0077] The test results are as follows Figure 4-5 As shown. Figure 4 It can be seen that both PGA and X-ray can inhibit tumor growth to a certain extent, and the combined treatment has the best tumor inhibition effect. The results show that the gadolinium-based sulfonamide complex nanomaterial of the present invention can be used for the synergistic treatment of regulating the acidic microenvironment of tumors and radiotherapy, and tumor growth is significantly inhibited, and its effect is better than that of single treatment. Figure 5 The combined therapy was shown to be effective in reducing lung metastases from primary breast cancer.

[0078] Example 6. Cytotoxicity Measurement

[0079] 4T1 cells and HUVEC cells in the logarithmic growth phase were collected and plated on 96-well plates, with 2000 cells per well, and incubated overnight at 37°C, 5% CO2 incubator. After gradient addition of PGA for 48 hours, the medium in the wells was discarded, and 100 μL of the prepared 10% MTT solution (DMEM basal medium configuration) was added, and incubated in a 37°C incubator for 4 hours, the supernatant was carefully discarded, and 100 μL of DMSO was added to fully dissolve it, and its absorbance (A570) was detected at 570 nm by a microplate reader.

[0080] The test results are as follows Figure 6 As shown in the figure, after PGA treatment, the cell viability of 4T1 cells decreased significantly. However, at the same material concentration, no significant decrease in cell viability was found in HUVEC cells, which means that the nanomaterial has less toxicity on normal cells.

[0081] Example 7. Measurement of intracellular pH

[0082] 4T1 cells in the logarithmic growth phase were collected and plated in 96-well plates, with 2000 cells per well, and incubated overnight at 37°C, 5% CO2 incubator. After gradient addition of PGA for 48 hours, the medium in the wells was discarded, and BCECF AM probe was added at a final concentration of 5 μM, and incubated at 37°C for 30 minutes. + After washing twice with buffer, a buffer containing 5 μM nigericin was added and the cells were detected using a fluorescence microplate reader.

[0083] The results are as follows Figure 7 As shown, PGA treatment alone can reduce the intracellular pH of 4T1 cells in a concentration-dependent manner; and combined with radiotherapy further aggravates the acidification of 4T1 cells (intracellular pH downregulation).

[0084] Example 8. Measurement of intracellular pentose phosphate pathway related indicators

[0085] 4T1 cells in the logarithmic growth phase were collected and plated in six-well plates, with 1*10 5 Cells were incubated overnight and divided into the following four groups: Control group, PGA group, X-ray group and PGA+X-ray group. The amount of PGA given was 200 μg / mL, the irradiation dose was 6 Gy, and the combined treatment group was irradiated by an irradiator 4 hours after PGA administration. After continuing to culture for 48 hours, the cell pellet was collected and used for WB to detect the expression level of G6PD protein.

[0086] 4T1 cells in the logarithmic growth phase were collected and plated in six-well plates, with 1*10 5 Cells were incubated overnight and treated as above. 1 h after irradiation, the culture medium in the wells was aspirated and 200 μL of NADP was added. + / NADPH extract, and gently blow to promote cell lysis, centrifuge at 12000rpm and 4℃ for 10min, then aspirate the supernatant, and aspirate 50μL into a 96-well plate as a test sample. Pipette 100μL of the test sample into a centrifuge tube, heat it in a 60℃ metal bath for 30min, aspirate 50μL into a 96-well plate, add 100μL G6PD working solution (G6PD diluted 50 times with reaction buffer) to each test sample, incubate at 37℃ in the dark for 10min; add 10μL of color development solution to each well, mix well, incubate at 37℃ in the dark for 20min, and measure the absorbance at 450nm with an enzyme marker.

[0087] The results are as follows Figure 8 As shown, PGA treatment could reduce the expression of G6PD protein and the NADPH / NADP+ ratio, indicating that PPP was downregulated after PGA treatment.

[0088] The above results show that the PVP-modified gadolinium sulfonamide complex synthesized by the present invention has the ability to regulate the acidic microenvironment of tumors and thus affect sugar metabolism. The nanomaterial increases the acidity in tumor cells by inhibiting the expression of CAIX, thereby downregulating the pentose phosphate pathway, reducing the production of NADPH, and reducing the consumption of ROS, thereby synergizing with radiotherapy to achieve an effective tumor killing effect. In addition, the nanomaterial also has a good radiotherapy sensitization effect and can promote tumor cell apoptosis in synergistic fashion with radiotherapy.

Claims

1. A method for preparing a gadolinium-based sulfonamide complex having a radiosensitizing effect, characterized in that: The steps include: (1) dispersing sulfonamide drugs and PVP in a mixed solution of ethanol and ethylene glycol, stirring at room temperature, to obtain a reaction solution A; (2) adding Gd(NO3)3·6H2O dropwise to the reaction solution A obtained in step (1), and continuing stirring to obtain reaction solution B; (3) The reaction solution B obtained in step (2) is subjected to a solvothermal reaction to obtain a gadolinium-based sulfonamide complex.

2. The preparation method according to claim 1, characterized in that: In step (1), the volume ratio of ethanol to ethylene glycol in the mixed solution of ethanol and ethylene glycol is 1:0 to 13:

7.

3. The preparation method according to claim 1, characterized in that: The molar ratio of the sulfonamide drug to the Gd(NO3)3·6H2O is 1:1; the molar ratio of the sulfonamide drug to the PVP is 1:0-1:

10.

4. The preparation method according to claim 1, characterized in that: The sulfonamide drugs include sulfonamide, sulfadiazine, and acetazolamide.

5. The preparation method according to claim 1, characterized in that: In step (1), the stirring time is 25 to 40 minutes; And / or in step (2), the stirring time is 8 to 20 minutes.

6. The preparation method according to claim 1, characterized in that: In step (3), the reaction temperature is 150-250° C. and the reaction time is 8-12 h.

7. A gadolinium sulfonamide complex prepared by the preparation method according to any one of claims 1 to 6.

8. The gadolinium sulfonamide complex according to claim 7, characterized in that: The particle size of the gadolinium sulfonamide complex is 30-2000nm.

9. Use of the gadolinium sulfonamide complex according to any one of claims 7 to 8 in the preparation of antitumor drugs.

10. Use of the gadolinium-based sulfonamide complex according to any one of claims 7 to 8 in the preparation of a nano-radiotherapy sensitizer for regulating the acidity of the tumor microenvironment.

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