A radioactive hydrogel based on the crosslinking of sodium alginate and metal ions, its preparation method and application

Abstract

The present invention belongs to the field of tumor drugs, and discloses a radioactive hydrogel based on the crosslinking of sodium alginate and metal ions, and its preparation method and application. The preparation method of the radioactive hydrogel comprises the following steps: mixing lutetium chloride and metal ions to obtain a metal ion mixed solution, and then incorporating <supgt;177< / supgt;Lu radioactive isotope into the obtained metal ion mixed solution to obtain a metal ion crosslinking agent; injecting the metal ion crosslinking agent into the sodium alginate solution under vortex conditions, and centrifuging to discard the supernatant to obtain the radioactive hydrogel. The radioactive hydrogel (<supgt;177< / supgt;Lu-RH) provided by the present invention has good tumor colonization ability, can be directly injected into the tumor to achieve local radiotherapy, has good radioactive stability, and can stably stay at the tumor site for more than 120 hours, ensuring the stability of the radioactive label during the treatment process.

Description

Technical Field

[0001] The present invention belongs to the field of tumor drugs, and particularly relates to a radioactive hydrogel based on the crosslinking of sodium alginate and metal ions, and its preparation method and application. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors. Although patients over 50 years old still account for the vast majority of colorectal cancer cases, the proportion of young patients is increasing significantly, and their conditions are more aggressive, which is closely related to unhealthy lifestyles. The rectum and sigmoid colon are the most common sites of colorectal cancer. At present, the main clinical treatment method is surgical resection, but the resulting permanent fistulas often seriously reduce the quality of life of patients. Therefore, most colorectal cancer patients have a strong desire to preserve their anus. However, the pathological complete remission rate (pCR) of existing clinical neoadjuvant therapies is relatively low, and immunotherapies such as programmed death receptor 1 (αPD-1), anti-programmed death ligand 1 (αPD-L1), and cytotoxic T lymphocyte-associated protein-4 (CTLA-4) are only effective for colorectal cancer tumors with high microsatellite instability (MSI), but this only accounts for 4%-5% of CRC patients. Therefore, it is necessary to develop a new combined treatment strategy to replace surgical treatment or provide more favorable conditions for preserving the anus during surgical resection.

[0003] Radiotherapy is a treatment method that uses external ionizing radiation or radioactive isotopes to kill tumors and is widely used in the field of cancer treatment. In radioisotope therapy (RIT), ensuring the long-term and stable retention of radioactive isotopes at the tumor site is the key to ensuring their efficacy. Clinically, directly implanting radioactive isotopes into tumors for brachytherapy has been successfully applied to the treatment of prostate cancer and liver cancer. 177 Lu, as a metal radioactive isotope, has stable chemical properties, a short radiation range, and a moderate half-life, making it an ideal radioactive drug. However, 177 The clinical application of Lu usually requires combination with specific ligands such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or other functional carriers. This not only increases the treatment cost and more stringent constraints but also poses a potential risk of off-target effects. Therefore, there is an urgent need for a radiotherapy reagent with low treatment cost, wide applicability, and good tumor suppression effect. Summary of the Invention

[0004] In order to overcome the deficiencies in the prior art, the present invention provides a radioactive hydrogel based on the crosslinking of sodium alginate and metal ions, and its preparation method and application. By incorporating radioactive isotopes into the hydrogel 177Lu is made to continuously release β-rays after being injected into the tumor site, achieving an effective local killing effect. In addition, propionate, as a metabolite of Roseburia minuta (R.m), has the effect of inducing apoptosis in colorectal tumor cells. By adding sodium propionate (SP) to improve the radioactive hydrogel, 177 177 Lu-RH@SP is formed, which binds to anti-programmed death ligand 1 (αPD-L1). By promoting the maturation of dendritic cells (DC cells), reversing T cell exhaustion, inhibiting the activity of histone deacetylase (HDAC), and promoting the degradation of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) and other mechanisms, it induces apoptosis of tumor cells and enhances the anti-tumor immune response.

[0005] To achieve the above object, the present invention is implemented by the following technical solutions:

[0006] A preparation method of a radioactive hydrogel based on the cross-linking of sodium alginate and metal ions, comprising the following steps: mixing a lutetium chloride solution and a calcium chloride solution to obtain a metal ion mixed solution, and then incorporating the radioactive isotope 177 Lu into the obtained metal ion mixed solution to obtain a metal ion cross-linking agent; injecting the metal ion cross-linking agent into the sodium alginate solution under vortex conditions, and centrifuging to discard the supernatant to obtain the radioactive hydrogel.

[0007] Further, the preparation method of the metal ion cross-linking agent includes: mixing a lutetium chloride solution and a calcium chloride solution to obtain a metal ion cross-linking agent, and the volume ratio of the lutetium chloride solution to the calcium chloride solution is 1:0.8 - 1.2.

[0008] Further, the molar concentration of the lutetium chloride solution is 4 - 6 mM, and the molar concentration of the calcium chloride solution is 8 - 12 mM.

[0009] Further, the concentration of the sodium alginate solution is 0.8 - 1.2 w / v%.

[0010] Further, the 177 Ratio of the Lu radioactive isotope to the metal ion mixed solution is 80 - 120 mCi:50 μL.

[0011] Further, the centrifugation conditions are centrifuging at 2500 - 3500 rpm for 20 - 40 minutes.

[0012] The present invention also provides a radioactive hydrogel prepared by the above preparation method.

[0013] The present invention also provides the application of the described radioactive hydrogel in the preparation of tumor drugs.

[0014] Further, the tumor drugs include the above-mentioned radioactive hydrogel and sodium propionate, and the tumor is a low rectal tumor.

[0015] Furthermore, the drug is used to inhibit tumor growth, inhibit tumor cell proliferation, reduce tumor malignancy and invasiveness, and promote tumor cell apoptosis.

[0016] The present invention also provides a method for improving the above-mentioned radioactive hydrogel based on the cross-linking of sodium alginate and metal ions. The improvement measure is to pre-dissolve 2.5 mg / mL of sodium propionate in the sodium alginate solution.

[0017] The present invention also provides the application of the above-mentioned radioactive hydrogel based on the cross-linking of sodium alginate and metal ions in the preparation of drugs for low rectal cancer. The drugs for low rectal cancer are injected into the tumor, continuously release β-rays at the tumor site, and achieve an effective local killing effect. At the same time, combined with anti-programmed death ligand 1 (αPD-L1) treatment, it has the effects of promoting the maturation of dendritic cells (DC cells), inducing tumor cell apoptosis and enhancing the anti-tumor immune response.

[0018] Furthermore, the dosage form of the drugs for low rectal cancer is an injection, and the administration method is to use an insulin syringe to inject in situ into the low rectal tumor.

[0019] Beneficial effects

[0020] Sodium alginate, as a biocompatible and biodegradable biomaterial, has been widely used in the medical field. Generally, the carboxyl groups present in alginate can form stable complexes with metal ions through coordination. By adjusting the concentration of sodium alginate and the amount of metal ions, the viscosity and strength of the obtained hydrogel can be adjusted, laying a foundation for the subsequent preparation of radioactive hydrogels with excellent properties.

[0021] In addition, the occurrence and development of tumors are closely related to the imbalance of the gut microbiota, and the metabolites derived from the gut microbiota are the key hubs connecting the gut microbiota and cancer progression. These microbe-derived metabolites can accumulate in the tumor microenvironment (TEM) and regulate related signaling pathways by acting as ligands for specific receptors and regulators of certain protein activities, thus affecting the development process of tumors. Among them, sodium propionate, as a metabolite of Roseburia minuta (R.m), plays a crucial role in promoting tumor cell apoptosis, and it is positively correlated with a good treatment response. It can promote the maturation of dendritic cells (DC cells) and coordinate the proteasome degradation in colorectal cancer cells, thereby promoting the apoptosis of tumor cells.

[0022] The present invention provides a radioactive hydrogel based on the cross-linking of sodium alginate and metal ions ( 177(Lu-RH) has good tumor internal colonization ability, can be directly injected into the tumor for local radiotherapy, and has good radioactive stability, can stably retain at the tumor site for more than 120 hours, ensuring the stability of the radioactive label during the treatment process.

[0023] The present invention improves the radioactive hydrogel ( 177 Lu-RH@SP) by adding sodium propionate (SP), which can significantly inhibit tumor growth. 177 The sodium propionate released by Lu-RH@SP can induce apoptosis of tumor cells. By upregulating the HECT domain E3 ubiquitin protein ligase 2 (HECTD2), it targets histone lysine N-methyltransferase 2 (EHMT2), and then coordinates proteasomal degradation, resulting in changes in the expression of proteins related to tumor cell apoptosis, such as an increase in the level of tumor necrosis factor α-induced protein 1 (TNFAIP1), a decrease in the expression of histone lysine N-methyltransferase 2 (EHMT2), and an increase in activated caspase 3 (Cleaved-Caspase 3), ultimately promoting tumor cell apoptosis. At the same time, the present invention incorporates the radioactive isotope 177 Lu into the hydrogel, so that after injection into the tumor site, it continuously releases β-rays to directly kill tumor cells in the tumor tissue, achieving an effective local killing effect. The above two mechanisms work synergistically to significantly inhibit or even eradicate the growth and metastasis of the primary tumor.

[0024] The radioactive hydrogel involved in the present invention 177 When Lu-RH@SP is used in combination with αPD-L1, it can induce tumor cell apoptosis and enhance the anti-tumor immune response by reversing T cell exhaustion, inhibiting the activity of histone deacetylase (HDAC), promoting the degradation of histone lysine N-methyltransferase 2 (EHMT2), etc., thereby improving the treatment effect.

[0025] Based on the anal-preserving needs of patients with low rectal cancer, the present invention designs a radioactive hydrogel ( 177 Lu-RH) crosslinked by sodium alginate and metal ions, innovatively combines the intestinal microbiota, brachytherapy and tumor immunotherapy, opens up a new way for the treatment of low rectal cancer, has great potential in improving the treatment effect, improving the prognosis and quality of life of patients, reducing the side effects of traditional treatment methods, etc., and shows good application prospects in the field of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a flow chart of the present invention;

[0027] Figure 2 is the preparation and characterization of the radioactive hydrogel ( 177 Lu-RH); (a) The formation of the radioactive hydrogel involves sodium alginate and Ca2+ , Lu 3+ and trace amounts of 177 Lu 3+ cross-linking schematic diagram; (b) Photograph of radioactive hydrogel ( 177 Lu-RH); (c) 177 Radiostability of Lu-RH in phosphate-buffered saline (PBS, pH = 7.4) and fetal bovine serum (FBS); (d) Transmission electron microscope (TEM) image of radioactive hydrogel ( 177 Lu-RH) (scale bar = 100 μm); (e) In-situ injection of 177 Lu-RH for SPECT-CT imaging; (f) Representative hematoxylin and eosin (H&E) staining images of major organs collected from mice treated with 177 Lu-RH, including liver, spleen, kidney, lung, and heart (scale bar = 50 μm);

[0028] Figure 3 For tumor treatment using radioactive hydrogel ( 177 Lu-RH); (a) Schematic diagram of the experimental design and timeline of the CT26 in-situ tumor model; (b) Re-grouping of experimental mice according to the average fluorescence intensity of tumors in the control group; The near-complete remission (nCR) group was defined as ≤20% of the intensity of the control group; The partial remission (PR) group was defined as ≥70% of the intensity of the control group (n = 5); (c) Photographs of the rectal lengths of mice in the healthy group, control group, near-complete remission (nCR) group, and partial remission (PR) group; (d) Statistical analysis of the colon lengths (cm) of each group (n = 5); (e-f) Tumor weights (e) and tumor volumes (mm 3 ) (f) of mice in different groups; (g) Body weight curves of mice in different treatment groups; (h-i) Flow cytometry data (h) and statistical values (i) of dendritic cell (DC cell) maturity in different treatment groups (n = 4); Error bars represent mean ± standard deviation (n = 4); P-values in d, f, g, i were calculated by one-way analysis of variance (ANOVA); (*P < 0.05, **P < 0.01, ***P < 0.001, ****P <0.0001); (j) Flow cytometry analysis of in-vivo dendritic cell maturation;

[0029] Figure 416S rDNA and targeted metabolomics analysis for the near-complete remission (nCR) group and partial remission (PR) group; (a-c) Observed Sobs index (a), Chao index (b), and ACE index (c) to depict the community richness of the near-complete remission (nCR) group and partial remission (PR) group, with a total of eight samples (n = 8); (d) Alpha diversity of fecal microbial communities shown by the Shannon-Wiener index (Shannon index) and Simpson diversity index (Simpson index); (e) Beta diversity of fecal microbial communities shown by the principal coordinate analysis (PCoA) plot; (f) Non-metric multidimensional scaling (NMSD) analysis of the healthy group, near-complete remission (nCR) group, and partial remission (PR) group (n = 8); (g) Non-metric multidimensional scaling (NMDS) analysis of the near-complete remission (nCR) group and partial remission (PR) group (n = 8); (h) Gut microbiota health index (GMHI) and dysregulation index (MDI); (i) Venn diagram of the near-complete remission (nCR) group and partial remission (PR) group at the OTU level; (j) t-test bar chart at the genus level; (k) Statistical table of individual genus difference tests; The error bars in the above charts represent the mean ± standard deviation (n = 8); (l) Acetic acid, propionic acid, butyric acid, and valeric acid contents (μg / mL) in the healthy group, near-complete remission (nCR) group, and partial remission (PR) group; The error bars represent the mean ± standard deviation (n = 5); P values were calculated by one-way analysis of variance (ANOVA) in a, b, c, d, h, k, l; (*P < 0.05, **P < 0.01, ***P < 0.001);

[0030] Figure 5 Improvement of radioactive hydrogels ( 177 Lu-RH@SP) and their application in tumor treatment; (a) Schematic diagram of the synthesis of propionate-containing radioactive hydrogels ( 177 Lu-RH@SP); (b) Schematic diagram of the experimental design and timeline; (c) Propionate-containing radioactive hydrogels ( 177 Lu-RH@SP) release propionic acid over time; (d) Bioluminescence of mice in different treatment groups (n = 3); (e) Tumor photos of mice in different treatment groups; (f-g) Tumor weights (f) and tumor volumes (mm 3)(g); (h) Rectal lengths (cm) of mice in different treatment groups; error bars represent mean ± s.d (n = 5); (i) Representative immunohistochemical (IHC) staining images of Ki-67 in mice (= 50 µm); (j) Expressions of tumor necrosis factor α-induced protein 1 (TNFAIP1), cleaved caspase 3, and euchromatic histone-lysine N-methyltransferase 2 (EHMT2-specific antibody) in tumor tissues of mice; (k-l) Micrographs (k) and quantitative analysis (l) of TUNEL staining in tumors of mice in different treatment groups; error bars represent mean ± s.d (n = 3); P values were calculated using one-way ANOVA test for f, g, h, l (*P < 0.05, **P < 0.01, ***P < 0.001);

[0031] Figure 6 For radioimmunotherapy of tumors based on 177 Lu-RH@SP; (a) Schematic diagram of experimental design and timeline; (b) 177 Mechanism of action of Lu-RH@SP in the treatment of low rectal cancer; (c) Bioluminescence of mice under different treatment methods; (d-e) Tumor volumes (mm 3 )(d) and tumor weights (e) of mice in different treatment groups; (f-g) Statistical values (f) and photographs (g) of rectal lengths (cm) of mice in different treatment groups; error bars represent mean ± s.d (n = 5); (h-i) Flow cytometry data (h) and statistical values (i); error bars represent mean ± s.d (n = 4); (j-k) Flow cytometry data (j) and statistical values (k) of T cell exhaustion in different treatment groups; error bars represent mean ± s.d (n = 5); P values were calculated using one-way ANOVA test for g, h, i, k (*P < 0.05, **P < 0.01, ***P < 0.001). Detailed implementation manners

[0032] The present invention will be further described below with reference to the accompanying drawings. The following examples are only used to more clearly illustrate the technical solutions of the present invention and cannot be used to limit the protection scope of the present invention.

[0033] Example 1

[0034] The embodiment of the present invention provides a preparation method of a radioactive hydrogel ( 177 Lu-RH) based on the crosslinking of sodium alginate and metal ions.

[0035] 1. Experimental materials

[0036] Sodium alginate, anhydrous calcium chloride, anhydrous lutetium chloride, and sodium propionate were all purchased from Maclin, 177LuCl 3 (ITGISotope Technologies Garching GmbH), PBS, fetal bovine serum, CT26 cells, insulin syringe, hematoxylin, eosin, scanning electron microscopy (SEM), single photon emission computed tomography (SPECT).

[0037] 2. Experimental Methods

[0038] 2.1 Preparation of radioactive hydrogel ( 177 Lu-RH)

[0039] Anhydrous lutetium chloride with a concentration of 5 mM and anhydrous calcium chloride with a volume ratio of 1:1 were mixed to obtain a metal ion mixed solution, and then radioactive isotopes were added to the obtained metal ion mixed solution at a ratio of 100 mCi / 50 μL. 177 Lu, a metal ion crosslinker was obtained. The metal ion crosslinker was injected into a 1% (w / v) sodium alginate solution under vortex conditions to form a uniform hydrogel, and then centrifuged at a speed of 3000 rpm for 30 min. After the centrifugation, the supernatant was discarded to obtain a prefabricated radioactive hydrogel ( 177 Lu-RH), such as Figure 2 As shown in ab.

[0040] 2.2 Radioactivity stability determination

[0041] The synthesized radioactive hydrogel was placed in phosphate buffered saline (PBS, pH=7.2) and 10% fetal bovine serum (FBS), respectively, and centrifuged at 3000 rpm to precipitate the hydrogel at the bottom of the EP tube. The hydrogel was incubated at 37°C and the radioactivity in the supernatant was measured every 24 hours using a radioactivity meter. 177 Lu radioactivity was continued for 120 h to evaluate its radiostability.

[0042] 2.3 Radioactive hydrogel characterization

[0043] Freeze-dried radioactive hydrogel ( 177 The hydrogel structure was observed using scanning electron microscopy (SEM).

[0044] 2.4 Detection of in situ retention of radioactive hydrogels

[0045] After establishing the mouse CT26 low-position colorectal tumor model, 50 μL containing 100 μCi 177 Lu radioactive hydrogel ( 177 Lu-RH) was injected into the tumor, and its retention in the tumor was observed using single photon emission computed tomography (SPECT) imaging.

[0046] 2.5 Evaluation of the biosafety of radioactive hydrogel

[0047] After establishing a mouse CT26 low rectal tumor model, 50 μL of radioactive hydrogel containing 100 μCi 177 Lu ( 177 Lu-RH) was injected into the tumor. After the mice were euthanized, their main organs were taken for hematoxylin and eosin (H&E) staining.

[0048] 3. Experimental results

[0049] 3.1 Radioactive stability

[0050] As Figure 2 shown in 177 Figure c, the

[0051] Lu radioactive hydrogel can stably retain in the tumor site for more than 120 hours, ensuring the stability of the radioactive label during the treatment process.

[0052] After freeze-drying the hydrogel, it was observed under a scanning electron microscope (SEM) that the hydrogel exhibited a typical network structure, indicating that the radioactive hydrogel was successfully cross-linked, as Figure 2 shown in

[0053] Figure d.

[0054] Figure 2 The results in 177 Figure e show that

[0055] Lu-RH was fixed in the tumor and remained stable at the tumor site for two half-lives (~322 h), and no radioactive leakage was observed.

[0056] The experimental results are as Figure 2 shown in

[0057] Example 2

[0058] This example evaluates the effect of 177 Lu-RH in treating low rectal tumors in mice.

[0059] 1. Experimental materials

[0060] CT26 cells expressing luciferase, anti-mouse PD-L1 antibody (Bio X Cell, USA), CD11c, CD80, CD86 dyes, fecal DNA kit (Omega, D4015), IVIS spectrum imaging system (IVIS spectrum, Perkinelmer), flow cytometer (BD FACSVerse), stereomicroscope, microinjector.

[0061] 2. Experimental methods

[0062] 2.1 Mouse model of low rectal cancer and treatment

[0063] Under a stereomicroscope, a mouse model of low rectal cancer was established. Using a microinjector (1×10 5 ), CT26 cells expressing luciferase were injected into the rectal terminal mucosa of BALB / C mice, and the injection volume was about 10 μL.

[0064] On the 7th day after tumor inoculation, 50 μL of radioactive hydrogel ( 177 Lu-RH) was injected into the tumor by intratumoral injection, combined with 100 μg / mL αPD-L1 intravenous injection for treatment. The control group was not treated, as Figure 3 shown in a.

[0065] 2.2 Detection and analysis of tumor growth

[0066] The growth of mouse tumors was regularly monitored using an IVIS spectrum imaging system. Mice were imaged at time points such as day 0, day 7, day 14, and day 21 after treatment, and the fluorescence intensity data of the tumor site were recorded. The mice were euthanized 21 days after tumor implantation, and the tumor weight, volume were measured, and the change in rectal length was observed.

[0067] 2.3 Flow cytometry to detect the maturation of dendritic cells

[0068] After the experiment, the mice were sacrificed, and single-cell suspensions were prepared from the mesenteric lymph nodes of the mice. After staining with specific antibodies (CD11c, CD80, CD86), flow cytometry analysis was performed to detect the maturation of dendritic cells.

[0069] 2.4 16S rDNA intestinal microbial diversity sequencing

[0070] Collect the intestinal contents of mice and perform 16S rDNA microbial diversity and targeted metabolomics sequencing on them. Use a fecal DNA kit (Omega, D4015) to extract total genomic DNA from the intestinal contents of mice. After measuring the purity using spectroscopic analysis technology (Nano Drop), perform 16S rDNA sequencing (Illumina MiSeq PE300 platform) to evaluate the diversity of the medical microbiota, analyze the composition of its microbiota. Sequencing data processing and bioinformatics analysis are carried out on the large biological cloud platform. The content of sequencing data processing and bioinformatics analysis includes α-diversity (Sobs, Chao, ACE, Shannon-Wiener index (Shannon), Simpson diversity index (Simpson index)) and β-diversity (principal coordinate analysis (PCoA), non-metric multidimensional scaling (NMDS) analysis), and display the similarities and differences in species composition between the two groups through a Venn diagram (Venn diagram). Analyze the differences in microbial species and abundances at the genus level using a community histogram and t-test.

[0071] 3. Experimental results

[0072] 3.1 Effect of radioactive hydrogel on treating low rectal tumors in mice

[0073] Different mice 177 showed different responses to Lu-RH treatment. According to the ratio of the average fluorescence intensity of the tumor site to that of the control group, the experimental mice were re-grouped. Those with an average fluorescence intensity less than 20% of the control group were classified into the near-complete remission (nCR) group, and those greater than or equal to 70% were classified into the partial remission (PR) group, as Figure 3 shown in b. Analysis found that the rectal length of mice in the partial remission (nCR) group was similar to that of healthy mice, only slightly shorter, while the rectal lengths of mice in the partial remission (PR) group and the control group were significantly shortened, as Figure 3 shown in c-d, suggesting that tumor infiltration triggered a severe inflammatory response; at the same time, the tumor burden of mice in the near-complete remission (nCR) group was significantly reduced, and the tumors of some individuals completely regressed. The tumor weight ( Figure 3 e) and size ( Figure 3 f) also decreased significantly, in sharp contrast to the partial remission (PR) group. And the difference in body weight change ( Figure 3 g) further verified the obvious difference in treatment effects between different groups from the side, suggesting 177 that Lu-RH had a significant treatment effect on mice in the near-complete remission group.

[0074] 3.2 Detection of dendritic cell maturation by flow cytometry

[0075] Flow cytometry results showed that the proportion of mature dendritic cells (DCs) in the near-complete remission (nCR) treatment group increased to 34%, showing a significant advantage compared with 23% in the partial remission (PR) treatment group ( Figure 3 h-j). This indicates that there are indeed certain factors in the near-complete remission (nCR) group that contribute to enhancing the anti-tumor immune response. In contrast, there seem to be factors in the partial remission (PR) group that can effectively inhibit the anti-tumor immune response, resulting in relatively poor treatment effects in this group, a lower proportion of mature dendritic cells, and a more obvious state of tumor immunosuppression.

[0076] 3.3 16S rDNA gut microbiota diversity sequencing

[0077] In the analysis of the α-diversity of the gut microbiota, it was found that the Sobs, Chao, and ACE indices of the near-complete remission (nCR) group were significantly higher than those of the partial remission (PR) group, indicating that the near-complete remission (nCR) group showed higher community richness than the partial remission (PR) group ( Figure 4 a-c). The Shannon-Wiener index (Shannon index) and Simpson diversity index (Simpson index) showed differences in community diversity between the two groups, with the community diversity of the near-complete remission (nCR) group being significantly higher than that of the partial remission (PR) group ( Figure 4 d).

[0078] Subsequently, we used the tePCoA plot (unweighted UniFrac principal coordinate analysis plot) based on 16S rDNA sequence data to perform post-processing clustering analysis on the samples. First, principal coordinate analysis at the OTU level showed significant differences between the gut microbiota of healthy mice and tumor-bearing mice ( Figure 4 e). Further analysis found that non-metric multidimensional scaling analysis at the OTU level also indicated obvious differences in community composition between the near-complete remission (nCR) group and the partial remission (PR) group ( Figure 4 f). At the same time, Figure 4 The OTU non-metric multidimensional scaling (NMDS) analysis in g also obtained corroborating results, further confirming the significant differences in the sample community structure between the two groups.

[0079] The differences between the gut microbiota health index (GMHI) and the microbial dysbiosis index (MDI) directly reflect that the gut microbiota health status of the near-complete remission (nCR) group is much better than that of the partial remission (PR) group ( Figure 4 h).

[0080] Analysis by Venn diagram revealed that there were a certain number of unique sub-species units (OTUs) between the near-complete remission (nCR) group and the partial remission (PR) group. The near-complete remission (nCR) group had 309 unique sub-species units, and the partial remission (PR) group had 233 sub-species units ( Figure 4 i), which suggested that there were significant differences in the species composition of the gut microbiota between the two groups of mice. At the genus level, the abundance of Helicobacter was significantly higher in the partial remission (PR) group than in the near-complete remission (nCR) group, while the abundances of Venkonia, Odoribacter, and Roseburia were significantly higher in the near-complete remission (nCR) group than in the partial remission (PR) group ( Figure 4 j). These microorganisms can produce metabolites such as short-chain fatty acids (SCFAs), which have a positive effect on maintaining the balance of the gut microbial community, gut immune regulation, and defense. In addition, there were statistically significant differences in the abundances of the marginal bacteria between the two groups, and the difference test was confirmed ( Figure 4 k). The results of non-targeted metabolomics showed that the levels of short-chain fatty acids such as acetic acid, propionic acid, butyric acid, and valeric acid in the near-complete remission (nCR) group were significantly higher than those in the partial remission (PR) group, similar to those in healthy mice ( Figure 4 l).

[0081] Overall, the effect of radioactive hydrogel in treating tumors may be related to the changes in the gut microbiota and metabolites. The better therapeutic effect of the near-complete remission (nCR) group may benefit from its healthier gut microbiota, higher levels of beneficial microorganisms and their metabolites (such as SCFAs), while the partial remission (PR) group has an imbalance in the microbial community and relatively fewer beneficial microorganisms. This provides clues from the perspective of gut microbiota for further studying the mechanism of radioactive hydrogel in treating tumors, suggesting that the therapeutic effect of radioactive hydrogel can be optimized by regulating the gut microbiota.

[0082] Example 3

[0083] Based on the study of gut microbial metabolites in Example 2, this example modified the radioactive hydrogel ( 177 Lu-RH) and evaluated the efficacy of its combined immunotherapy for low rectal cancer in mice:

[0084] 1. Experimental materials

[0085] Sodium propionate, sodium alginate, lutetium chloride anhydrous, and calcium chloride anhydrous were all purchased from Maclin, 177 LuCl 3(ITGISotope TechnologiES Garching GmbH), PBS, dialysis bag, CT26 cells expressing luciferase, anti-mouse PD-L1 antibody (Bio X Cell, USA), Roseburia minuta (R.m), tumor necrosis factor α-induced protein 1 (TNFAIP1), cleaved caspase 3, specific antibody against euchromatic histone-lysine N-methyltransferase 2 (EHMT2), CD11c, CD80, CD86 dyes, microsyringe, gavage needle, insulin syringe, ruler, stereomicroscope, IVIS spectrum imaging system (IVIS spectrum, Perkin elmer), flow cytometer (BD FACSVerse).

[0086] 2. Experimental methods

[0087] 2.1 Synthesis and characterization of the modified radioactive hydrogel

[0088] The synthesis method is similar to 177 Lu-RH, but sodium propionate needs to be dissolved in 1% (w / v) sodium alginate solution at a concentration of 2.5 mg / mL in advance, and then mixed with the metal ion cross-linking agent to form a hydrogel, obtaining 177 Lu-RH@SP, as shown in Figure 5 a.

[0089] The SP release kinetics in PBS at different pH values (pH = 5.6, pH = 7.4) was evaluated by the dialysis bag method. The samples were incubated at a constant temperature of 37 °C, and the concentration of propionate released in the dialysis bag was measured every 2 days for 10 days to monitor its release characteristics.

[0090] 2.2 177 Effect of combined treatment with Lu - RH@SP on tumor growth of low rectal cancer in mice

[0091] Mice from the same source as in the previous experiment were selected, and an in-situ injection tumor model was established using the CT26 mouse colorectal cancer cell line. They were randomly divided into 5 groups, namely the control group (G1), R.m (G2), 177 Lu-RH + αPD-L1 (G3), R.m + 177 Lu-RH + αPD-L1 (G4), 177 Lu-RH@SP + αPD-L1 (G5), as shown in Figure 5 b. Three days after establishing the tumor model, some experimental group mice were gavaged with Roseburia minuta (groups G2 and G4). After the tumor was established, the corresponding experimental group mice were injected with in-situ hydrogel (groups G3, G4, and G5 were injected with 177 Lu-RH or177 (G3, G4, and G5 groups) were treated with Lu-RH@SP) and αPD-L1 via tail vein injection. During the entire experiment, the growth of tumors in mice was regularly monitored using an IVIS spectral imaging system, and data such as the fluorescence intensity at the tumor site were recorded. After the treatment ended, the mice were sacrificed, and samples of tumors, rectum, and mesenteric lymph nodes were collected. The tumor samples were weighed and their volumes were measured. The length of the rectum was measured using a ruler, and immunohistochemical analysis was performed on the tumor tissues, focusing on detecting key indicators such as Ki-67 positive cells, so as to comprehensively evaluate the growth characteristics of tumors and the treatment effects.

[0092] 2.3 Verification of the effect of SP release on tumor cells

[0093] Tumor tissues from two groups of mice, G1 (control group) and G5 ( 177 treatment group with Lu-RH@SP + αPD-L1), were collected, with a sample size of n = 4 for each group. The collected tumor tissues were lysed to extract the proteins therein, and then protein immunoblotting technology was used, and specific antibodies were selected to detect the expression levels of proteins such as tumor necrosis factor α-induced protein 1 (TNFAIP1), cleaved caspase 3 (Cleaved-Caspase 3), and euchromatic histone-lysine N-methyltransferase 2 (EHMT2). At the same time, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed on the tumor tissues. The stained tissue sections were observed under a microscope, and the number and distribution of apoptotic cells were counted to evaluate the differences in the degree of apoptosis between different groups.

[0094] 2.4 Verification 177 Whether Lu-RH@SP can replace R.m in the treatment of low rectal cancer in mice

[0095] To verify 177 whether Lu-RH@SP can completely replace R.m, we conducted a new experiment in which R.m was not orally administered to the mice. Mice from the same source as before were selected, and a tumor model was established by in-situ injection using the CT26 murine colorectal cancer cell line. Subsequently, the mice were randomly divided into 5 groups, namely the control group (G1), Hyd@SP (G2), Hyd@SP + αPD-L1 (G3), 177 Lu-RH@SP (G4), 177 Lu-RH@SP + αPD-L1 (G5), and the grouping and treatment conditions are as shown in Figure 6 a - b. During the entire experiment, the growth of tumors in mice was regularly monitored using an IVIS spectral imaging system, as shown in Figure 6As shown in Figure c. After the experiment, the mice were sacrificed, and samples such as tumors, rectums, and mesenteric lymph nodes were collected. The tumor samples were weighed and measured for volume to evaluate tumor growth; the rectum length was measured to evaluate the impact of the tumor on the rectum and the protective effect of the treatment on the rectum; the mesenteric lymph nodes were sorted by flow cytometry to detect the expression of dendritic cell surface maturation markers such as CD11c, CD80, and CD86 and the expression of T cell exhaustion markers such as PD-1 and Tim-3 in the mouse tumor tissue, so as to comprehensively analyze 177 the feasibility and potential advantages of Lu - RH@SP replacing R.m.

[0096] 3. Experimental results

[0097] 3.1 177 Release characteristics of Lu - RH@SP

[0098] Release kinetics evaluation found that within 10 days, it could stably release SP under physiological conditions, and the free SP could reach 900 μg / mL after 10 days. As Figure 5 shown in Figure c, this indicates that the radioactive hydrogel has good SP release performance and can continuously provide a certain concentration of SP in vivo, laying a foundation for subsequent studies on its therapeutic effect on tumors.

[0099] 3.2 177 Effect of Lu-RH@SP combination therapy on the growth of low rectal cancer tumors in mice

[0100] In group G5 ( 177 Lu-RH@SP + αPD - L1), the tumor burden of the mice was significantly reduced, and the tumors of some mice even completely regressed, showing obvious differences compared with other groups. As Figure 5 shown in Figures d - g. In group G1, the tumors continued to grow, and the tumor volume and weight increased continuously; in group G3 ( 177 Lu-RH + αPD-L1) and group G4 (R.m + 177 Lu-RH + αPD-L1), the tumor growth was also inhibited to a certain extent, but the effect was not as good as that of group G5, indicating that 177 the combination of Lu-RH@SP and immunotherapy has a good effect on inhibiting tumor growth.

[0101] The rectum length of the mice in group G1 was significantly shortened due to tumor infiltration, and the rectum lengths of groups G2, G3, and G4 were also affected to varying degrees. In contrast, the rectum length of the mice in group G5 was the longest. As Figure 5 shown in Figure h, this indicates that the treatment has a good protective effect on the rectum and the degree of tumor invasion of the rectum is relatively light.

[0102] Immunohistochemical analysis showed that ( Figure 5i), The Ki-67 positive cells in the tumor tissues of G5 group mice were significantly reduced, indicating that 177 Lu-RH@SP can inhibit the proliferation of tumor cells, reduce the malignancy and invasiveness of tumors, and play an important role in the local control of tumor tissues.

[0103] 3.3 Verify the effect of SP released on tumor cells

[0104] The results of Western blot analysis showed that ( Figure 5 j), The tumor necrosis factor α-induced protein 1 (TNFAIP1) and cleaved caspase 3 (Cleaved-Caspase 3) in the tumor tissues of G5 group mice were significantly increased, and the expression of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) was down-regulated. The up-regulation of tumor necrosis factor α-induced protein 1 (TNFAIP1) and cleaved caspase 3 (Cleaved-Caspase 3) indicates that the apoptosis-related signaling pathway is activated, promoting the apoptosis process of tumor cells; while the down-regulation of EHMT2 expression may be related to the apoptosis induction mechanism of tumor cells, and its expression change is involved in 177 the regulation of SP released by Lu-RH@SP on tumor cell apoptosis.

[0105] The results of TUNEL staining showed that ( Figure 5 k), The cell damage in G5 group was the most obvious, apoptosis occurred widely, and a large number of tumor cells showed apoptotic characteristics, such as chromatin condensation and margination of the nucleus. This further confirmed that 177 SP released by Lu-RH@SP can induce tumor cell apoptosis, visually demonstrating its killing effect on tumor cells at the cellular morphological level, which is mutually confirmed with the Western blot results, jointly indicating 177 the important role of Lu-RH@SP in promoting tumor cell apoptosis.

[0106] 3.4 Verification 177 whether Lu-RH@SP can replace R.m in the treatment of low rectal cancer in mice

[0107] 177 The tumor size ( Figure 6 d), weight ( Figure 6 e) of mice in the Lu-RH@SP+αPD-L1 group (G5) were significantly reduced, and the treatment effect on the rectal length was significant ( Figure 6 f-g), indicating that this combined treatment regimen has obvious effects in inhibiting tumor growth and protecting the rectum. In contrast, the tumor growth in the control group (G1) was not controlled; the Hyd@SP group (G2) and 177The Lu-RH@SP group (G4) had a less effective treatment outcome compared to the combination treatment group, indicating that αPD-L1 played an important synergistic role in combination therapy.

[0108] Flow cytometry analysis revealed that ( Figure 6 h-i), 177 treatment with the Lu-RH@SP + αPD-L1 group (G5) significantly enhanced dendritic cell maturation, with increased expression of mature markers on the surface of dendritic cells, more mature cell morphology and function, and the ability to better activate T cell immune responses and regulate the tumor microenvironment.

[0109] Meanwhile, the exhaustion of T cells in the mesenteric lymph nodes of mice in the G5 group was significantly reversed. As Figure 6 shown in j-k, the expression of T cell exhaustion markers decreased, and T cell function was restored, indicating that 177 the combined use of Lu-RH@SP and αPD-L1 could regulate the tumor immune microenvironment, enhance the effect of tumor immunotherapy, and exert a synergistic anti-tumor effect.

[0110] In summary, 177 after the SP in Lu-RH@SP is released into the tumor microenvironment and taken up by tumor cells, it inhibits the activity of histone deacetylase (HDAC), leading to the degradation of EHMT2 and the upregulation of TNFAIP1, inducing apoptosis of tumor cells. Meanwhile, 177 Lu 3+ emitted β radiation directly kills tumor cells within the tumor tissue. The two mechanisms synergistically inhibit or eradicate the growth and metastasis of primary tumors. And when combined with αPD-L1, 177 Lu-RH@SP can promote the maturation of dendritic cells (DC cells), reverse T cell exhaustion, further enhance the tumor immune response, and amplify the overall anti-tumor effect. These series of results indicate that 177 the combined immunotherapy with Lu-RH@SP has significant advantages in the treatment of murine low rectal cancer, providing strong experimental evidence for its further clinical application.

[0111] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principles of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Application of radioactive hydrogel based on cross-linking of sodium alginate and metal ions in the preparation of drugs for treating low rectal cancer, characterized in that: The preparation method of the radioactive hydrogel comprises the following steps: mixing lutetium chloride and metal ions to obtain a metal ion mixed solution, and then adding a radioactive isotope into the metal ion mixed solution. 177 Lu obtains a metal ion crosslinker; dissolving sodium propionate in a sodium alginate solution, injecting the metal ion crosslinker into the sodium alginate solution under vortex conditions, and obtaining a radioactive hydrogel after centrifugation and discarding the supernatant; the preparation method of the metal ion mixed solution comprises: mixing a lutetium chloride solution and a calcium chloride solution to obtain a metal ion mixed solution, wherein the volume ratio of the lutetium chloride solution to the calcium chloride solution is 1:0.8-1.

2.

2. The use according to claim 1, characterized in that: The molar concentration of the lutetium chloride solution is 4-6 mM, and the molar concentration of the calcium chloride solution is 8-12 mM.

3. The use according to claim 1, characterized in that: The concentration of the sodium alginate solution is 0.8-1.2 w / v%.

4. The use according to claim 1, characterized in that: Said 177 The ratio of Lu radioisotope and metal ion mixed solution is 80~120 mCi:50 μL.

5. The use according to claim 1, characterized in that The centrifugation condition is 2500-3500 rpm for 20-40 minutes.

6. The use according to claim 1, characterized in that: The drug is used to inhibit tumor growth.

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