Preparation method and application of tumor microenvironment responsive AIE-alginate hydrogel

Abstract

The invention discloses a preparation method and application of tumor microenvironment responsive AIE-alginate hydrogel. The preparation method comprises the following steps: S1, synthesizing CQu; s2, synthesizing AIE loaded flower-like calcium carbonate; and S3, synthesizing the AIE-alginate hydrogel. The AIE-alginate hydrogel can be applied to preparation of medicines for treating tumors, and the AIE-alginate hydrogel is administrated in an injection way during illumination and radiotherapy. The AIE-alginate hydrogel mediated photodynamic therapy is combined with radiotherapy, so that tumor cells can be effectively killed, 'multiple treatment through one-time injection 'is realized, the radiotherapy treatment effect is improved, and the defect that the radiotherapy is not thorough in tumor treatment is overcome.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a preparation method and application of an AIE-alginate hydrogel responsive to a tumor microenvironment. Background Art

[0002] Colorectal cancer is one of the malignant tumors with a high mortality rate. The main treatment for colorectal cancer is surgery, supplemented by chemotherapy and radiotherapy. Among them, the elderly have a higher risk of colorectal cancer, and the risk of surgery is high for them, so conservative treatment is often used. For patients with colorectal metastasis who miss the opportunity for surgery, palliative treatment including adjuvant radiotherapy is used to alleviate tumor progression. Patients who undergo surgical treatment may also have incomplete resection of tumor tissue, leading to tumor recurrence or metastasis. For the above reasons, up to 30% of patients experience disease recurrence. Among the treatments for colorectal cancer, radiotherapy is a non-invasive treatment method, but its radiation dose is often limited to the maximum tolerated dose of the abdomen (50Gy). Therefore, its efficacy is also limited.

[0003] Photodynamic therapy (PDT) has been applied to the clinical treatment of superficial tumors as a new cancer treatment method. Under laser irradiation, toxic reactive oxygen species are generated in tumor tissues through photosensitizers and tumor cell death is induced. More importantly, this non-invasive PDT has been shown to induce immunogenic cell death and stimulate immune responses, thereby inhibiting tumor cell growth. Aggregation-induced luminescence materials have the characteristics of easy structural regulation, high photostability, and efficient photodynamic conversion. They can generate powerful reactive oxygen species to kill cancer cells through PDT at the tumor site, showing important prospects in the treatment of tumors. However, low light penetration leads to limited therapeutic effects of PDT, which in turn limits PDT-driven immunotherapy. The hypoxic microenvironment of the tumor inhibits the effect of PDT, while overexpressed glutathione in the tumor microenvironment scavenges reactive oxygen species, thereby inhibiting tumor killing efficiency. Therefore, there is an urgent need to combine PDT with other treatments to enhance the therapeutic effect.

[0004] In recent years, calcium overload has attracted great attention as an emerging strategy for cancer treatment. In order to maintain physiological activities, including regulating cell membrane function and mediating organelle function, calcium ion concentration is balanced at about 10 -7mol / L. Excessive calcium ions disrupt the intracellular balance and induce cell death. Taking advantage of this property, many studies have been devoted to designing nanomedicines containing calcium ion compositions to release tumor cell-permeable calcium ion production and induce mitochondrial damage, thereby inhibiting tumor progression. At the same time, some calcium ion-based nanomedicines have been reported to promote immune cell infiltration and activate tumor cell immunotherapy. However, because calcium ions are easily excreted into the extracellular environment, thus limiting the efficiency of calcium ion absorption, the tumor treatment effect is not ideal. It has been reported that a large amount of reactive oxygen species can induce mitochondrial dysfunction and impair the mitochondrial calcium ion buffering capacity, opening ion channels that are overloaded with calcium ions. Therefore, the reactive oxygen content in tumor cells can be increased by other therapeutic methods to enhance the anti-tumor efficacy of calcium overload. Summary of the invention

[0005] The present invention proposes a method for preparing an AIE-alginate hydrogel responsive to a tumor microenvironment and its application. AIE-alginate hydrogel-mediated photodynamic therapy combined with radiotherapy can effectively kill tumor cells, achieve "one injection for multiple treatments", improve the therapeutic effect of radiotherapy, and make up for the shortcoming that radiotherapy is not thorough in treating tumors.

[0006] In order to achieve the above purpose, this application adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a tumor microenvironment-responsive AIE-alginate hydrogel, comprising the following steps:

[0008] S1, synthesis of CQu: dissolving 9-ethyl-9H-carbazole-2-carboxaldehyde and N-(2-hydroxyethyl)-4-methylquinoline bromide in anhydrous ethanol, adding piperidine dropwise and reflux the solution under nitrogen for reaction;

[0009] After cooling to room temperature, the precipitate was collected, washed with cold ethanol and dried to obtain a yellow solid as the bromide salt of the product;

[0010] The solid was dissolved in acetone, and then a saturated aqueous solution of ammonium hexafluorophosphate was added, and after stirring, the solution was evaporated to dryness;

[0011] The product was purified by flash silica gel column chromatography, eluting with dichloromethane / methanol in a volume ratio of 20:1 to obtain a red solid, namely CQu;

[0012] S2, synthesis of AIE-loaded flower-like calcium carbonate: calcium acetate / calcium chloride was dissolved in ethanol containing CQu and polyvinyl pyrrolidone to form mixture A, and ammonium bicarbonate was dissolved in anhydrous ethanol to form mixture B;

[0013] The mixture A and the mixture B were placed in a sealed container, kept at 30-50° C. overnight, centrifuged and purified using ethanol to obtain flower-like calcium carbonate loaded with AIE;

[0014] S3: Synthesis of AIE-alginate hydrogel: AIE-loaded flower-like calcium carbonate and calcium alginate were dissolved in deionized water to obtain AIE-alginate hydrogel.

[0015] In the above technical scheme, in S1, 0.2-0.4 g, 0.5-4 mmol, 9-ethyl-9H-carbazole-2-carboxaldehyde and 0.31 g, 0.5-4 mmol, N-(2-hydroxyethyl)-4-methylquinoline bromide are added to every 15 mL of anhydrous ethanol.

[0016] In the above technical solution, in S2, 70-130 mg of calcium acetate or 50-150 mg of calcium chloride is dissolved in every 100 mL of ethanol containing CQu and polyvinyl pyrrolidone.

[0017] In the above technical solution, in S2, the mass percentage of CQu in ethanol is 5-30%, and the mass percentage of polyvinyl pyrrolidone is 5-20%.

[0018] In the above technical solution, in S2, the molar ratio of mixture A to mixture B is 5:1 to 1:5.

[0019] In the above technical solution, in S3, the concentration of AIE-loaded flower-like calcium carbonate dissolved in deionized water is 50-1000 μg / ml, and the concentration of calcium alginate is 5-20 mg / ml.

[0020] In a second aspect, the present invention provides an AIE-alginate hydrogel prepared by the above preparation method.

[0021] In a third aspect, the present invention provides the use of the above-mentioned AIE-alginate hydrogel in the preparation of a drug for treating tumors.

[0022] In the above technical solution, the tumor includes one or more of colorectal cancer, liver cancer, lung cancer, gastric cancer, breast cancer, ovarian cancer, oral cancer, esophageal cancer, bile duct cancer, leukemia and glioma.

[0023] In the above technical solution, the AIE-alginate hydrogel is administered by injection at the same time as light irradiation and radiotherapy.

[0024] The beneficial effects of the present invention are:

[0025] The present invention has developed a flower-shaped calcium carbonate nanosensitizer doped with aggregation-induced emission molecules CQu. In a weakly acidic tumor microenvironment, calcium ions can be released from the material, and then in situ in the tumor area with the sodium alginate injected together to form an alginate hydrogel, in which a high concentration of CQu is fixed to form an AIE-alginate hydrogel. Under laser irradiation, the AIE-alginate hydrogel continuously produces reactive oxygen species, achieving "one injection for multiple treatments". The powerful reactive oxygen species promotes the entry of calcium ions into cells. The reactive oxygen species and calcium ion overload produced can enhance the killing of tumor cells by radiotherapy, and can induce immune responses and inhibit tumor recurrence. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 are characterization diagrams; wherein, (A) transmission electron microscopy images of flower-like calcium carbonate and AIE-loaded flower-like calcium carbonate in buffer solutions of pH=7.4, pH=6.5 and pH=5.5; (B) X-ray diffraction analysis of AIE-loaded flower-like calcium carbonate; (C) PL spectra of CQu, AIE-loaded flower-like calcium carbonate and calcium carbonate; (D) calcium ion and (E) CQu release behavior of AIE-loaded flower-like calcium carbonate at different pH values; (F) images of AIE-alginate hydrogel (1) at pH=6.5, AIE-loaded flower-like calcium carbonate (2) at pH=6.5 and AIE-loaded flower-like calcium carbonate (3) at pH=5.5.

[0027] Figure 2 In vitro photodynamic therapy enhances the radiotherapy effect of AIE-alginate hydrogel on mouse colorectal cancer cell CT 26; (A) Fluo-4 AM detects intracellular calcium ions (scale bar: 50μm); (B) JC-1 fluorescence staining of CT 26 cells (scale bar: 50μm) and (C) flow cytometry test results; (D) Transmission electron microscopy of cells after treatment; (E) DNA double-strand break fluorescence staining (scale bar: 20μm) and linear reactive oxygen species fluorescence staining (scale bar: 20μm), (F) clone formation result picture. *p<0.01, **p<0.005, ***p<0.001; Student t test.

[0028] Figure 3 This is an in vivo test of the anti-tumor effect; (A) Experimental flow chart; (B) Mouse treatment chart. After different treatments; (C) Mouse survival rate and (D) Mouse body weight data. *p<0.01, **p<0.005, ***p<0.001.

[0029] Figure 4is an in vivo immune activation analysis; wherein, (A) a schematic diagram of the process of AIE-alginate hydrogel-mediated photodynamic therapy for radiosensitization for T cell activation for cancer immunotherapy; (B) tumor volume of primary tumors and (C) distant tumors; (D) survival rate of bilateral tumor-bearing mice with various treatments; (E) flow cytometric analysis of CD80+CD86+ cell frequency in lymph nodes; (F) representative flow cytometric graphs representing the frequency of CD4+ or CD8+ tumor cells in tumors; (G) mature DC populations in lymph nodes; (H) quantification of CD8+ T cell frequency in tumors; (I) TNF-α and (J) IFN-γ in serum.

[0030] Figure 5 is an in vivo tumor recurrence inhibition test. (A) Schematic diagram of the treatment procedure in the re-challenge model; (B) Flow cytometry data of cells (CD3+CD8+CD62L-CD44+) in different treatment groups; (C) Representative images of CD8+ immunofluorescence, scale bar: 50μm; (D) is a schematic diagram of immune response. . Data are mean ± SD. *p<0.01, **p<0.005, ***p<0.001. DETAILED DESCRIPTION

[0031] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0032] Example 1

[0033] The specific steps for preparing AIE-alginate hydrogel are:

[0034] First, CQu was synthesized. 9-Ethyl-9H-carbazole-2-carboxaldehyde (0.28 g, 1.28 mmol) and N-(2-hydroxyethyl)-4-methylquinoline bromide (0.31 g, 1.16 mmol) were dissolved in anhydrous ethanol (15 mL). Two drops of piperidine were added and the solution was refluxed under nitrogen for 3 hours to react. After cooling to room temperature, the precipitate was collected, washed with cold ethanol and dried to obtain the bromide salt of the product as a yellow solid. The solid was dissolved in 20 mL of acetone, and then 20 mL of saturated aqueous ammonium hexafluorophosphate solution was added. After stirring for 30 minutes, the solution was evaporated to dryness. The product was purified by flash silica gel column chromatography and eluted with dichloromethane / methanol (20:1) to obtain a red solid, namely CQu.

[0035] Then synthesize the flower-like calcium carbonate loaded with AIE. Dissolve 70 mg of calcium acetate in 100 mL of ethanol containing CQu and polyvinyl pyrrolidone. Next, dissolve ammonium bicarbonate in a beaker filled with anhydrous ethanol. Then put the two mixtures into a sealed container and keep them at 30-50 ° C overnight. Then centrifuge at 3000-7000 rpm and further purify several times with ethanol to obtain the flower-like calcium carbonate loaded with AIE.

[0036] Finally, AIE-loaded flower-like calcium carbonate (500 μg / ml) and calcium alginate (10 mg / ml) were dissolved in deionized water to obtain AIE-alginate hydrogel.

[0037] Example 2

[0038] The specific steps for preparing AIE-alginate hydrogel are:

[0039] First, CQu was synthesized. 9-Ethyl-9H-carbazole-2-carboxaldehyde (0.28 g, 1.28 mmol) and N-(2-hydroxyethyl)-4-methylquinoline bromide (0.31 g, 1.16 mmol) were dissolved in anhydrous ethanol (15 mL). Two drops of piperidine were added and the solution was refluxed under nitrogen for 3 hours to react. After cooling to room temperature, the precipitate was collected, washed with cold ethanol and dried to obtain the bromide salt of the product as a yellow solid. The solid was dissolved in 20 mL of acetone, and then 20 mL of saturated aqueous ammonium hexafluorophosphate solution was added. After stirring for 30 minutes, the solution was evaporated to dryness. The product was purified by flash silica gel column chromatography and eluted with dichloromethane / methanol (20:1) to obtain a red solid, namely CQu.

[0040] Then synthesize the flower-like calcium carbonate loaded with AIE. Dissolve 150 mg of calcium chloride in 100 mL of ethanol containing CQu and polyvinyl pyrrolidone. Next, dissolve sodium bicarbonate in a beaker filled with anhydrous ethanol. Then put the two mixtures into a sealed container and keep them at 30-50 ° C overnight. Then centrifuge at 3000-7000 rpm and further purify several times with ethanol to obtain the flower-like calcium carbonate loaded with AIE.

[0041] Finally, AIE-loaded flower-like calcium carbonate (500 μg / ml) and calcium alginate (10 mg / ml) were dissolved in deionized water to obtain AIE-alginate hydrogel.

[0042] The inventors synthesized AIE-alginate hydrogel according to the steps of Example 1, i.e., flower-like calcium carbonate containing AIE molecules CQu molecules, and its transmission electron microscopy image is as follows: Figure 1 As shown in A. In buffer solutions with different pH values, the AIE-loaded flower-like calcium carbonate exhibits decomposition behavior that changes with pH. Figure 1B is the X-ray diffraction analysis spectrum of AIE-loaded flower-like calcium carbonate, which confirms the presence of a large amount of calcium carbonate. Figure 1 The PL spectrum in C further confirmed the presence of CQu in the AIE-loaded flower-like calcium carbonate. In addition, the release behavior of calcium ions and CQu in buffer solutions with different pH values ​​of the AIE-loaded flower-like calcium carbonate was tested ( Figure 1 D and 1E). Figure 1 As shown in F, it can be seen that the AIE-alginate hydrogel is in a solidified state in a weakly acidic environment.

[0043] The inventors tested the use of AIE-alginate hydrogel-mediated photodynamic therapy to enhance the therapeutic effect of radiotherapy in vitro. First, they verified that under laser irradiation, AIE-loaded flower-like calcium carbonate can release a large amount of calcium ions to induce mitochondrial damage ( Figure 2 A, 2B and 2C). Figure 2 D is a transmission electron microscopy image of cells after AIE-alginate hydrogel-mediated photodynamic therapy, verifying the killing effect of AIE-alginate hydrogel-mediated photodynamic therapy on mitochondria. Figure 2 E is the fluorescent staining of DNA double-strand breaks after different treatments, proving that the DNA damage in the combined treatment group was the most severe. Among them, the fluorescent staining of cell linear reactive oxygen species after different treatments proved that the most linear reactive oxygen species were produced in the combined treatment group. In addition, a clone formation experiment was performed, and the results are shown in Figure 2 As shown in F, it is confirmed that the combined treatment can effectively inhibit the proliferation of tumor cells.

[0044] Next, 25 μL of CT 26 cell suspension (1×10 8 A mouse colorectal orthotopic tumor model was established with 100 cells / mL to evaluate the feasibility of in vivo AIE-alginate hydrogel-mediated photodynamic therapy for enhancing radiotherapy. Seven days after the orthotopic cancer model was established, the mice were assigned to the following treatment groups: (1) control group (no treatment); (2) radiotherapy group alone; (3) AIE-alginate hydrogel group; (4) AIE-alginate hydrogel + light group; (5) AIE-alginate hydrogel + radiotherapy group; (6) AIE-alginate hydrogel + light + radiotherapy group. Among them, radiotherapy is irradiation of the tumor part, using MV energy level radiotherapy rays with a dose of 8Gy, and the light is 660nm white light irradiated at the tumor site. The concentration of AIE-alginate hydrogel is 10mg·kg -1 , for intratumoral injection. AIE-alginate hydrogel injection and laser irradiation were performed under endoscopic visualization ( Figure 3 B). A significant improvement in the survival rate of mice was observed in the synergistic treatment group (AIE-alginate hydrogel + light + radiotherapy group) ( Figure 3C). Rapid tumor progression was observed in mice with orthotopic tumors, which impaired the normal physiological activities of the mice. This led to weight loss in mice in the radiotherapy group and the AIE-alginate hydrogel group ( Figure 3 D), while no weight loss was detected in the AIE-alginate hydrogel + light + radiotherapy group, further supporting the synergistic antitumor activity of this treatment method.

[0045] Next, the inventors tested the in vivo immune response associated with AIE-alginate hydrogel + light + radiotherapy treatment ( Figure 4 A). The immune response induced by the combined strategy to inhibit distant tumor growth was evaluated in a bilateral CT26 tumor-bearing mouse model. Mice in the AIE-alginate hydrogel + light + radiotherapy group showed significant tumor growth inhibition ( Figure 4 B) Figure 4 As shown in C, mice treated with radiotherapy alone or AIE-alginate hydrogel alone did not show significant distant tumor inhibition, and better inhibition was observed in the AIE-alginate hydrogel + light irradiation group and the AIE-alginate hydrogel + radiotherapy group. In sharp contrast, the AIE-alginate hydrogel + light irradiation + radiotherapy group significantly inhibited the growth of distant tumors, showing the potential of AIE-alginate hydrogel-mediated photodynamic therapy to enhance radioimmunotherapy. The AIE-alginate hydrogel + light irradiation + radiotherapy group also showed the highest survival rate at day 60, which was 80% ( Figure 4 D). Dendritic cells are important mediators of the initiation and regulation of adaptive immunity through their ability to activate T cells. Therefore, the in vivo immune responses associated with the developed synergistic therapeutic strategy were evaluated ( Figure 4 A). The best maturation of dendritic cells in the draining lymph nodes was observed in the AIE-alginate hydrogel + light + radiotherapy group, with a peak maturity level of 35.1%, which was 2.9 and 1.8 times higher than that in the radiotherapy alone group and the AIE-alginate hydrogel + radiotherapy group, respectively ( Figure 4 E and 4G). An increase in the CD8+ T cell population was also detected in the AIE-alginate hydrogel+light+radiotherapy group, which is consistent with the highest degree of intratumoral T cell activation ( Figure 4 F and 4H). Compared with the radiotherapy alone group, the serum levels of pro-inflammatory TNF-α and IFN-γ in the AIE-alginate hydrogel + light + radiotherapy group also increased ( Figure 4 I and 4J). Therefore, the AIE-alginate hydrogel + light irradiation + radiotherapy strategy significantly enhanced the maturation of dendritic cells and the activation of T cells, contributing to the enhancement of antitumor immunity.

[0046] The inventors further investigated whether the FA treatment strategy could induce a long-term immune response against tumors. Figure 5Experimental procedure in A. Tumor growth in each treatment group was monitored 7 days after resection. Figure 5 B shows that the AIE-alginate hydrogel + light + radiotherapy group activated effector memory T cells, indicating the immunogenic memory of AIE-alginate hydrogel + light + radiotherapy. Figure 5 Immunofluorescence staining of CD8+ in tumor sections in C verified the promoting effect of AIE-alginate hydrogel+light irradiation+radiotherapy group on CD8+T cell activation.

[0047] Collectively, these results suggest that AIE-alginate hydrogel+light+radiotherapy can effectively stimulate immunogenic memory and thus inhibit tumor recurrence.

[0048] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing a tumor microenvironment-responsive AIE-alginate hydrogel, characterized in that: The following steps are involved: S1, synthesis of CQu: dissolving 9-ethyl-9H-carbazole-2-carboxaldehyde and N-(2-hydroxyethyl)-4-methylquinoline bromide in anhydrous ethanol, adding piperidine dropwise and reflux the solution under nitrogen for reaction; After cooling to room temperature, the precipitate was collected, washed with cold ethanol and dried to obtain a yellow solid as the bromide salt of the product; The solid was dissolved in acetone, and then a saturated aqueous solution of ammonium hexafluorophosphate was added, and after stirring, the solution was evaporated to dryness; The product was purified by flash silica gel column chromatography, eluting with dichloromethane / methanol in a volume ratio of 20:1 to obtain a red solid, namely CQu; S2, synthesis of AIE-loaded flower-like calcium carbonate: calcium acetate / calcium chloride was dissolved in ethanol containing CQu and polyvinyl pyrrolidone to form mixture A, and ammonium bicarbonate was dissolved in anhydrous ethanol to form mixture B; The mixture A and the mixture B were placed in a sealed container, kept at 30-50° C. overnight, centrifuged and purified using ethanol to obtain flower-like calcium carbonate loaded with AIE; S3: Synthesis of AIE-alginate hydrogel: AIE-loaded flower-like calcium carbonate and calcium alginate were dissolved in deionized water to obtain AIE-alginate hydrogel.

2. The preparation method according to claim 1, characterized in that: In S1, 0.2-0.4 g, 0.5-4 mmol, of 9-ethyl-9H-carbazole-2-carboxaldehyde and 0.31 g, 0.5-4 mmol, of N-(2-hydroxyethyl)-4-methylquinoline bromide were added to every 15 mL of anhydrous ethanol.

3. The preparation method according to claim 1, characterized in that: In S2, 70-130 mg of calcium acetate or 50-150 mg of calcium chloride is dissolved in every 100 mL of ethanol containing CQu and polyvinyl pyrrolidone.

4. The preparation method according to claim 1, characterized in that: In S2, the mass percentage of CQu in ethanol is 5-30%, and the mass percentage of polyvinyl pyrrolidone is 5-20%.

5. The preparation method according to claim 1, characterized in that: In S2, the molar ratio of mixture A to mixture B is 5:1 to 1:

5.

6. The preparation method according to claim 1, characterized in that: In S3, the concentration of AIE-loaded flower-like calcium carbonate dissolved in deionized water is 50-1000 μg / ml, and the concentration of calcium alginate is 5-20 mg / ml.

7. AIE-alginate hydrogel prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the AIE-alginate hydrogel according to claim 7 in the preparation of a drug for treating tumors.

9. The use according to claim 8, characterized in that: The tumor includes one or more of colorectal cancer, liver cancer, lung cancer, gastric cancer, breast cancer, ovarian cancer, oral cancer, esophageal cancer, bile duct cancer, leukemia and glioma.

10. The application according to claim 8, characterized in that: The AIE-alginate hydrogel was administered by injection simultaneously with light and radiotherapy.

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