Radioactive hydrogel based on crosslinking of sodium alginate and metal ions as well as preparation method and application of radioactive hydrogel

Abstract

The invention belongs to the field of tumor drugs, and discloses a radioactive hydrogel based on crosslinking of sodium alginate and metal ions and a preparation method and application thereof.The preparation method of the radioactive hydrogel comprises the following steps that lutetium chloride and metal ions are mixed, and a metal ion mixed solution is obtained; doping 177Lu radioactive isotope into the obtained metal ion mixed solution to obtain a metal ion cross-linking agent; and injecting a metal ion cross-linking agent into the sodium alginate solution under a vortex condition, centrifuging, and discarding the supernatant to obtain the radioactive hydrogel. The radioactive hydrogel (177Lu-RH) provided by the invention has good in-tumor colonization capability, can be directly injected into a tumor to realize local radiotherapy, has good radioactive stability, and can be stably retained at a tumor part for more than 120 hours, so that the stability of radioactive labeling in a treatment process is ensured.

Description

Technical Field

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

[0002] Colorectal cancer (CRC) is one of the most common malignancies. Although patients over 50 years old still account for the vast majority of colorectal cancer cases, the proportion of younger patients is increasing significantly, and their disease is more aggressive, which is closely related to an unhealthy lifestyle. The rectum and sigmoid colon are the most common sites of colorectal cancer. The main clinical treatment is surgical resection, but the permanent fistula often seriously reduces the quality of life of patients. Therefore, most patients with colorectal cancer have a strong desire to preserve the anus. However, the pathological complete remission rate (pCR) of existing clinical neoadjuvant therapy is low, and immunotherapy such as programmed death receptor 1 (αPD-1), anti-programmed death ligand 1 (αPD-L1), and cytotoxic T lymphocyte-associated protein-4 (CTLA-4) is 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 anus preservation conditions for surgical resection.

[0003] Radiotherapy is a treatment method that uses external ionizing radiation or radioisotopes 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 radioisotopes at the tumor site is the key to ensuring its effectiveness. In clinical practice, direct implantation of radioisotopes into tumors for brachytherapy has been successfully used in the treatment of prostate cancer and liver cancer. 177 Lu, as a metallic radioisotope, is chemically stable, has a short radiation range and a moderate half-life, making it an ideal radiopharmaceutical. 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 cost of treatment and stricter constraints, but also has the potential risk of off-target effects. Therefore, there is an urgent need for a radiotherapy agent with low treatment cost, wide applicability and good tumor inhibition 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 cross-linking of sodium alginate and metal ions and a preparation method and application thereof, by incorporating radioactive isotopes into the hydrogel. 177Lu, which continuously releases β rays after being injected into the tumor site, achieving effective local killing effect. In addition, propionate, as a metabolite of R. multocida (Rm), has the effect of inducing apoptosis of colorectal tumor cells. Radioactive hydrogels are improved by adding sodium propionate (SP) to form 177 Lu-RH@SP, combined with anti-programmed death ligand 1 (αPD-L1), induces tumor cell apoptosis and enhances anti-tumor immune response by promoting the maturation of dendritic cells (DC cells), reversing T cell exhaustion, inhibiting histone deacetylase (HDAC) activity, and promoting the degradation of histone lysine N-methyltransferase 2 (EHMT2).

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

[0006] A method for preparing a radioactive hydrogel based on cross-linking of sodium alginate and metal ions, comprising the following steps: mixing lutetium chloride and calcium chloride solutions to obtain a metal ion mixed solution, and then doping the obtained metal ion mixed solution with a radioactive isotope. 177 Lu obtains a metal ion crosslinker; injects the metal ion crosslinker into a sodium alginate solution under vortex conditions, and obtains a radioactive hydrogel after centrifugation and discarding the supernatant.

[0007] Furthermore, the preparation method of the metal ion crosslinking agent comprises: mixing a lutetium chloride solution and a calcium chloride solution to obtain the metal ion crosslinking agent, wherein the volume ratio of the lutetium chloride solution to the calcium chloride solution is 1:0.8-1.2.

[0008] Furthermore, 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] Furthermore, the concentration of the sodium alginate solution is 0.8-1.2 w / v%.

[0010] Furthermore, the 177 The ratio of Lu radioisotope and metal ion mixed solution is 80~120 mCi:50μL.

[0011] Furthermore, the centrifugation condition is 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 use of the radioactive hydrogel in preparing tumor drugs.

[0014] Furthermore, the tumor drug includes 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 an improved method for the radioactive hydrogel based on cross-linking of sodium alginate and metal ions, wherein the improved measure is to pre-dissolve 2.5 mg / mL sodium propionate in the sodium alginate solution.

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

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

[0019] Beneficial Effects

[0020] Sodium alginate, as a biocompatible and degradable 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 resulting hydrogel can be adjusted, laying the foundation for the subsequent preparation of radioactive hydrogels with excellent performance.

[0021] In addition, the occurrence and development of tumors are closely related to the imbalance of intestinal flora, and metabolites derived from intestinal flora are the key hubs connecting intestinal flora and cancer progression. These microbial-derived metabolites can accumulate in the tumor microenvironment (TEM), and by acting as ligands for specific receptors and regulators of certain protein activities, they regulate related signaling pathways, thereby affecting the development of tumors. Among them, propionate, as a metabolite of R. multocida (Rm), plays a vital role in promoting tumor cell apoptosis and is positively correlated with a good therapeutic response. It can promote the maturation of dendritic cells (DC cells) and coordinate proteasome degradation in colorectal cancer cells, thereby promoting tumor cell apoptosis.

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

[0023] The radioactive hydrogel ( 177 Lu-RH@SP), which can significantly inhibit tumor growth. 177 Sodium propionate released by Lu-RH@SP can induce apoptosis of tumor cells by upregulating HECT domain E3 ubiquitin protein ligase 2 (HECTD2), targeting histone lysine N-methyltransferase 2 (EHMT2), and then coordinating proteasome degradation, leading to changes in the expression of tumor cell apoptosis-related proteins, such as increased levels of tumor necrosis factor α-induced protein 1 (TNFAIP1), decreased expression of histone lysine N-methyltransferase 2 (EHMT2), and increased activated cysteine ​​protease 3 (Cleaved-Caspase 3), ultimately promoting tumor cell apoptosis. At the same time, the present invention incorporates radioactive isotopes into the hydrogel to 177 Lu, which continuously releases beta rays after being injected into the tumor site 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 primary tumors.

[0024] 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 anti-tumor immune response, thereby improving the therapeutic effect by reversing T cell exhaustion, inhibiting histone deacetylase (HDAC) activity, and promoting the degradation of histone lysine N-methyltransferase 2 (EHMT2).

[0025] Based on the anal preservation needs of patients with low rectal cancer, the present invention designs a radioactive hydrogel based on sodium alginate and metal ion cross-linking ( 177 Lu-RH innovatively combines intestinal microbiota, close-range radiation and tumor immunotherapy, opening up a new avenue for the treatment of low rectal cancer. It has great potential in improving treatment effects, improving patient prognosis and quality of life, and reducing the side effects of traditional treatments, and shows good application prospects in the field of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0027] Figure 2 For radioactive hydrogel ( 177 Preparation and characterization of Lu-RH); (a) The formation of radioactive hydrogel involves sodium alginate and Ca2+ 、Lu 3+ And trace amounts 177 Lu 3+ Schematic diagram of cross-linking; (b) radioactive hydrogel ( 177 Lu-RH) photo; (c) 177 Radiostability of Lu-RH in phosphate buffered saline (PBS, pH = 7.4) and fetal bovine serum (FBS); (d) Radioactive hydrogel ( 177 (e) Transmission electron microscopy (TEM) image of Lu-RH (scale bar = 100 μm); (f) for SPECT-CT imaging 177 Lu-RH was injected in situ; (f) 177 Representative hematoxylin and eosin (H&E)-stained images of major organs collected from Lu-RH-treated mice, including liver, spleen, kidney, lung, and heart (scale bar = 50 μm);

[0028] Figure 3 For the use of radioactive hydrogel ( 177 Lu-RH) tumor treatment; (a) Schematic diagram of the experimental design and timeline of the CT26 orthotopic tumor model; (b) Experimental mice were regrouped according to the mean fluorescence intensity of the tumor 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) Photos of the rectal length of mice in the healthy group, control group, near complete remission (nCR) group, and partial remission (PR) group; (d) Statistical analysis of the colon length (cm) of each group (n=5); (ef) Tumor weight (e) and tumor volume (mm) of mice in different groups 3 ) (f); (g) Body weight curves of mice in different treatment groups; (hi) Flow cytometry data (h) and statistical values ​​(i) of dendritic cell (DC) maturation 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 cytometric analysis of DC maturation in vivo;

[0029] Figure 416S rDNA and targeted metabolomics analysis of the nCR and PR groups; (ac) Observed Sobs index (a), Chao index (b), and ACE index (c) to depict the community richness of the nCR and PR groups, with a total of eight samples (n=8); (d) α diversity of fecal microbial communities displayed by Shannon-Wiener index (Shannon index) and Simpson diversity index (Simpson index); (e) β diversity of fecal microbial communities displayed by principal coordinate analysis (PCoA) plot; (f) non-metric multidimensional scaling (NMSD) analysis of the healthy group, nCR group, and PR group (n=8); (g) analysis of the nCR and PR groups by P < 0.05; (h) P < 0.05; (i) P < 0.05; (ii) P < 0.05; (iii) P < 0.05; (iv) P < 0.05; (v) P < 0.05; (v) P < 0.05; (v) P < 0.05 R) group (n=8) non-metric multidimensional scaling (NMDS) analysis; (h) gut microbiota health index (GMHI) and dysbiosis index (MDI); (i) Venn diagram at the OTU level for the nCR group and the PR group; (j) t-test bar graph at the genus level; (k) statistical table of individual genus difference test; the error bars of the above graphs represent the mean ± standard deviation (n=8); (l) acetic acid, propionic acid, butyric acid and valeric acid contents (μg / mL) in the healthy group, the nCR group and the 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 its application in tumor therapy; (a) propionate-containing radioactive hydrogel ( 177 Schematic diagram of the synthesis of Lu-RH@SP; (a) Schematic diagram of the experimental design and timeline; (c) radioactive hydrogel containing propionate ( 177 Lu-RH@SP) releases propionic acid over time; (d) Bioluminescence of mice in different treatment groups (n=3); (e) Tumor photos of mice in different treatment groups; (fg) Tumor weight (f) and tumor volume (mm) of mice in different treatment groups 3) (g); (h) Rectum length of mice in different treatment groups (cm); Error bars represent mean ± sd (n = 5); (i) Representative mouse Ki-67 immunohistochemistry (IHC) staining images (= 50 µm); (j) Expression of tumor necrosis factor α-induced protein 1 (TNFAIP1), activated caspase 3 (Cleaved-Caspase 3), and histone lysine N-methyltransferase 2 (EHMT2-specific antibody) in mouse tumor tissues; (kl) Microscopic photos (k) and quantitative analysis (l) of TUNEL staining of mouse tumors in different treatment groups; Error bars represent mean ± sd (n = 3); P values ​​were calculated using one-way ANOVA test in f, g, h, and l (*P < 0.05, **P < 0.01, ***P < 0.001);

[0031] Figure 6 Based on 177 Tumor radioimmunotherapy with Lu-RH@SP; (a) Schematic diagram of experimental design and timeline; (b) 177 The mechanism of action of Lu-RH@SP in treating low rectal cancer; (c) Bioluminescence of mice under different treatment methods; (de) Tumor volume (mm) of mice in different treatment groups 3 ) (d) and tumor weight (e); (fg) Statistical values ​​(f) and photos (g) of rectal length (cm) of mice in different treatment groups; error bars represent mean ± SD (n = 5); (hi) flow cytometric data (h) and statistical values ​​(i); error bars represent mean ± SD (n = 4); (jk) flow cytometric data (j) and statistical values ​​(k) of T cell depletion in different treatment groups; error bars represent mean ± SD (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 DESCRIPTION

[0032] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0033] Example 1

[0034] The present invention provides a radioactive hydrogel based on cross-linking of sodium alginate and metal ions ( 177 Preparation method of Lu-RH).

[0035] 1. Experimental Materials

[0036] Sodium alginate, anhydrous calcium chloride, anhydrous lutetium chloride, and sodium propionate were purchased from Maclin. 177LuCl3 (ITGISotope Technologies Garching GmbH), PBS, fetal bovine serum, CT26 cells, insulin syringe, hematoxylin and 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 hydrogels

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

[0048] 3. Experimental results

[0049] 3.1 Radioactive stability

[0050] like Figure 2 As shown in c, 177 Lu radioactive hydrogel can be stably retained at the tumor site for more than 120 hours, ensuring the stability of the radioactive label during treatment.

[0051] 3.2 Radioactive hydrogel characterization

[0052] After freeze-drying the hydrogel, the hydrogel showed a typical network structure under scanning electron microscopy (SEM), indicating that the radioactive hydrogel was successfully cross-linked, e.g. Figure 2 As shown in d.

[0053] 3.3 In situ retention capacity of radioactive hydrogel

[0054] Figure 2 The results show that: 177 Lu-RH was fixed in the tumor and remained stable at the tumor site for two half-lives (~322 h), and no radioactivity leakage was observed.

[0055] 3.4 Biosafety of Radioactive Hydrogel

[0056] The experimental results are as follows Figure 2 As shown in f, this indicates that radioactive hydrogels do not pose a biosafety risk.

[0057] Example 2

[0058] This embodiment 177 The effect of Lu-RH on the treatment of low rectal tumors in mice was evaluated.

[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), stereo microscope, microinjection syringe.

[0061] 2. Experimental Methods

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

[0063] The low rectal cancer mouse model was established under a stereomicroscope. A microinjection syringe (1×10 5 ) CT26 cells expressing luciferase were injected into the terminal rectal mucosa of BALB / C mice with an injection volume of approximately 10 μL.

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

[0065] 2.2 Tumor growth detection and analysis

[0066] The IVIS spectral imaging system was used to regularly monitor the growth of mouse tumors. The mice were imaged and detected at the 0th, 7th, 14th, and 21st days 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 and volume were measured, and the changes in rectal length were observed.

[0067] 2.3 Flow cytometry detection of dendritic cell maturation

[0068] After the experiment, the mice were killed, and the mesenteric lymph nodes of the mice were taken to prepare single-cell suspensions, which were stained with specific antibodies (CD11c, CD80, and CD86) and then analyzed by flow cytometry to detect the maturation of dendritic cells.

[0069] 2.4 16S rDNA intestinal microbial diversity sequencing

[0070] The intestinal contents of mice were collected and subjected to 16S rDNA microbial diversity and targeted metabolomics sequencing. Total genomic DNA was extracted from the intestinal contents of mice using a fecal DNA kit (Omega, D4015). After the purity was determined by spectral analysis technology (Nano Drop), 16S rDNA sequencing (Illumina MiSeq PE300 platform) was performed to evaluate the diversity of the medical microbiome and analyze its composition. Sequencing data processing and bioinformatics analysis were performed on the BigBio Cloud platform. Sequencing data processing and bioinformatics analysis included 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 the similarities and differences in species composition between the two groups were displayed by Venn diagrams. Community histograms and t-tests were used to analyze the differences in microbial species and abundance at the genus level.

[0071] 3. Experimental results

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

[0073] Different mouse pairs 177 There were differences in the response to Lu-RH treatment. The experimental mice were regrouped according to the ratio of the mean fluorescence intensity of the tumor site to that of the control group. The mice with a mean fluorescence intensity less than 20% of the control group were classified as the near complete remission (nCR) group, and those with a mean fluorescence intensity greater than or equal to 70% were classified as the partial remission (PR) group. Figure 3 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 length of mice in the partial remission (PR) group and the control group was significantly shortened, as shown in Figure 3 As shown in cd, it indicates that tumor infiltration triggers severe inflammatory response; at the same time, the tumor burden of mice in the near complete remission (nCR) group was greatly reduced, and some individual tumors completely disappeared, with tumor weight ( Figure 3 e) and size ( Figure 3 f) also significantly reduced, in sharp contrast to the partial remission (PR) group, and the difference in weight change ( Figure 3 g) Further verified the obvious differences in treatment effects between different groups, suggesting 177 Lu-RH had a significant therapeutic effect on mice in the near-complete remission group.

[0074] 3.2 Flow cytometry analysis of dendritic cell maturation

[0075] The results of cell flow cytometry showed that the proportion of mature dendritic cells (DCs) in the near complete remission (nCR) treatment group increased to 34%, which was significantly higher than the 23% in the partial remission (PR) treatment group ( Figure 3 hj). This suggests that there are indeed certain factors in the near-complete remission (nCR) group that help enhance the anti-tumor immune response. In contrast, the partial remission (PR) group seems to have factors that can effectively inhibit the anti-tumor immune response, resulting in a relatively poor treatment effect in this group, a lower proportion of mature dendritic cells, and a more obvious tumor immunosuppression state.

[0076] 3.3 16S rDNA intestinal microbial diversity sequencing

[0077] In the alpha diversity analysis of the intestinal microbial community, it was found that the Sobs, Chao, and ACE indexes 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 a higher community richness than the partial remission (PR) group ( Figure 4 ac). The Shannon-Wiener index (Shannon index) and Simpson diversity index (Simpson index) showed that there were differences in community diversity between the two groups, among which the community diversity of the near-complete remission (nCR) group was significantly higher than that of the partial remission (PR) group ( Figure 3 d).

[0078] Subsequently, we performed post-processing cluster analysis on the samples using tePCoA (unweighted UniFrac principal coordinate analysis) based on 16S rDNA sequence data. First, OTU-level principal coordinate analysis showed significant differences between the intestinal microbiota of healthy mice and tumor-bearing mice ( Figure 4 e). Further analysis found that OTU-level non-metric multidimensional scaling analysis also showed that there were significant 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 non-metric multidimensional scaling (NMDS) analysis of OTUs of g also obtained mutually corroborating results, further confirming the significant differences in the sample community structure between the two groups.

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

[0080] Venn diagram analysis revealed that there were a certain number of unique infraspecies units (OTUs) between the near complete remission (nCR) group and the partial remission (PR) group. There were 309 unique infraspecies units in the near complete remission (nCR) group and 233 unique infraspecies units in the partial remission (PR) group ( Figure 4 i), which suggests that there are significant differences in the species composition of the intestinal microbiota of the two groups of mice. At the genus level, the abundance of Helicobacter in the partial remission (PR) group was significantly higher than that in the near complete remission (nCR) group, while the abundance of Venkenella, Odorobacter, and Roseburia in the near complete remission (nCR) group was significantly higher than that in the partial remission (PR) group ( Figure 4 j), these microorganisms can produce metabolites such as short-chain fatty acids (SCFAs), which play a positive role in maintaining the balance of intestinal microbial communities, intestinal immune regulation and defense. In addition, there was a statistically significant difference in the abundance of 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 acetate, propionate, butyrate and valerate in the near-complete remission (nCR) group were significantly higher than those in the partial remission (PR) group and similar to those in healthy mice ( Figure 4 l).

[0081] In summary, the effect of radioactive hydrogel in treating tumors may be related to changes in intestinal microbial communities and metabolites. The better treatment effect of the near-complete remission (nCR) group may be due to its healthier intestinal microbial community, higher levels of beneficial microorganisms and their metabolites (such as SCFAs), while the partial remission (PR) group has an imbalanced microbial community and relatively fewer beneficial microorganisms. This provides clues from the intestinal microbiome for further research on the mechanism of radioactive hydrogel in treating tumors, suggesting that the therapeutic effect of radioactive hydrogel can be optimized by regulating intestinal microorganisms.

[0082] Example 3

[0083] Based on the study of intestinal microbial metabolites in Example 2, this example studies the radioactive hydrogel ( 177 Improvement of Lu-RH) and evaluation of its combined immunotherapy efficacy against low rectal cancer in mice:

[0084] 1. Experimental Materials

[0085] Sodium propionate, sodium alginate, anhydrous lutetium chloride, and anhydrous calcium chloride were purchased from Maclin. 177LuCl3 (ITGISotope Technologies Garching GmbH), PBS, dialysis bag, CT26 cells expressing luciferase, anti-mouse PD-L1 antibody (Bio X Cell, USA), R. parvum (Rm), tumor necrosis factor α-induced protein 1 (TNFAIP1), activated caspase 3 (Cleaved-Caspase 3), histone lysine N-methyltransferase 2 (EHMT2-specific antibody), CD11c, CD80, CD86 dyes, microsyringe, gavage needle, insulin syringe, ruler, stereomicroscope, IVIS spectrum imaging system (IVIS spectrum, Perkin Elmer), cell flow cytometer (BD FACSVerse).

[0086] 2. Experimental Methods

[0087] 2.1 Synthesis and characterization of modified radioactive hydrogels

[0088] Synthesis method and 177 Lu-RH is similar, 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 a metal ion crosslinker to form a hydrogel. 177 Lu-RH@SP, such as Figure 5 As shown in a.

[0089] The SP release kinetics in PBS with different pH values ​​(pH=5.6, pH=7.4) were evaluated by the dialysis bag method. The samples were incubated at 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 Effects of Lu-RH@SP combined therapy on tumor growth of lower rectal cancer in mice

[0091] The mice from the same source as the previous experiment were selected and the tumor model was established by orthotopic injection of CT26 mouse colorectal cancer cell line. The mice were randomly divided into 5 groups, namely control group (G1), Rm (G2), 177 Lu-RH+αPD-L1(G3),R.m+ 177 Lu-RH+αPD-L1(G4) 177 Lu-RH@SP+αPD-L1(G5) Figure 5 b. Three days after the tumor model was established, mice in some experimental groups were gavaged with Sovilinella parvum (groups G2 and G4). After the tumor was established, mice in the corresponding experimental groups were injected with in situ hydrogel (groups G3, G4, and G5). 177 Lu-RH or177 Lu-RH@SP) and αPD-L1 were injected into the tail vein (groups G3, G4, and G5). During the entire experiment, the IVIS spectral imaging system was used to regularly monitor the growth of mouse tumors and record data such as the fluorescence intensity of the tumor site. After the treatment, the mice were killed, and samples such as tumors, rectums, and mesenteric lymph nodes were collected. The tumor samples were weighed and measured, the length of the rectum was measured with a ruler, and the tumor tissue was immunohistochemically analyzed, focusing on the detection of key indicators such as Ki-67 positive cells, so as to comprehensively evaluate the growth characteristics of the tumor and the treatment effect.

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

[0093] G1 (control group) and G5 ( 177 Tumor tissues of two groups of mice (Lu-RH@SP+αPD-L1 treatment group) were collected, with n=4 samples in each group. The collected tumor tissues were lysed and the proteins were extracted. Then, protein immunoblotting was used, and specific antibodies were used to detect the expression levels of proteins such as tumor necrosis factor α-induced protein 1 (TNFAIP1), activated cysteine ​​protease 3 (Cleaved-Caspase 3), and histone lysine N-methyltransferase 2 (EHMT2). ​​At the same time, the tumor tissues were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and the stained tissue sections were observed under a microscope to count the number and distribution of apoptotic cells and evaluate the differences in the degree of cell apoptosis between different groups.

[0094] 2.4 Verification 177 Can Lu-RH@SP replace Rm in the treatment of low rectal cancer in mice?

[0095] To verify 177 We conducted a new set of experiments to determine whether Lu-RH@SP can completely replace Rm. In this experiment, Rm was not orally administered to mice. The mice from the same source as in the previous experiment were used to establish a tumor model by orthotopic injection of CT26 mouse colorectal cancer cell line. The mice were then randomly divided into 5 groups, namely control group (G1), Hyd@SP (G2), Hyd@SP+αPD-L1 (G3), 177 Lu-RH@SP (G4), 177 Lu - RH@SP+αPD-L1 (G5), group treatment status as follows Figure 6 Throughout the experiment, the IVIS Spectrum Imaging System was used to regularly monitor the tumor growth of mice. Figure 6c. After the experiment, the mice were killed and samples such as tumors, rectum and mesenteric lymph nodes were collected. The tumor samples were weighed and the volume was measured to evaluate the tumor growth; the rectum length was measured to evaluate the effect 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 after different treatments and the expression of T cell exhaustion markers such as PD-1 and Tim-3 in mouse tumor tissues, so as to comprehensively analyze 177 Feasibility and potential advantages of Lu-RH@SP replacing Rm.

[0096] 3. Experimental results

[0097] 3.1 177 Lu - RH@SP release characteristics

[0098] Release kinetics evaluation revealed that it could stably release SP under physiological conditions within 10 days, and the free SP could reach 900 μg / mL after 10 days. Figure 5 As 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 the foundation for subsequent research on its therapeutic effect on tumors.

[0099] 3.2 177 Effects of Lu-RH@SP combined therapy on tumor growth of lower rectal cancer in mice

[0100] G5 Group ( 177 The tumor burden of mice with Lu-RH@SP+αPD-L1 was significantly reduced, and the tumors of some mice even completely disappeared, which was significantly different from the other groups. Figure 5 As shown in dg. The tumor in group G1 continued to grow, and the tumor volume and weight continued to increase; 177 Lu-RH+αPD-L1 and G4 group (R.m+ 177 The growth of the tumor was also inhibited to some extent in the Lu-RH+αPD-L1 group, but the effect was not as good as that in the G5 group. 177 Lu-RH@SP combined with immunotherapy has a good effect in inhibiting tumor growth.

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

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

[0103] 3.3 Verification of the effect of SP release on tumor cells

[0104] The results of Western blotting analysis showed that ( Figure 5 j), the expression of tumor necrosis factor α-induced protein 1 (TNFAIP1) and activated caspase 3 (Cleaved-Caspase 3) in the tumor tissues of mice in the G5 group was significantly increased, and the expression of histone lysine N-methyltransferase 2 (EHMT2) was downregulated. The upregulation of tumor necrosis factor α-induced protein 1 (TNFAIP1) and activated caspase 3 (Cleaved-Caspase 3) indicates that the apoptosis-related signaling pathway is activated, which promotes the apoptosis process of tumor cells; while the downregulation of EHMT2 expression may be related to the apoptosis induction mechanism of tumor cells, and its expression changes are involved in 177 Regulation of tumor cell apoptosis by SP released from Lu-RH@SP.

[0105] The results of TUNEL staining showed that ( Figure 5 k), G5 group cell damage is the most obvious, apoptosis occurs extensively, and a large number of tumor cells show apoptotic characteristics, such as nuclear chromatin condensation and marginalization. This further confirms 177 SP released by Lu-RH@SP can induce apoptosis of tumor cells, which intuitively demonstrates its killing effect on tumor cells from the perspective of cell morphology, which is mutually confirmed by the results of protein immunoblotting, indicating that 177 Lu-RH@SP plays an important role in promoting tumor cell apoptosis.

[0106] 3.4 Verification 177 Can Lu-RH@SP replace Rm in the treatment of low rectal cancer in mice?

[0107] 177 Tumor size of mice in the Lu-RH@SP+αPD-L1 group (G5) ( Figure 6 d) Weight ( Figure 6 e) Rectal length was significantly reduced and the treatment effect was significant ( Figure 6 fg), indicating that the combined treatment regimen was effective in inhibiting tumor growth and protecting the rectum. In contrast, the tumor growth in the control group (G1) was uncontrolled; 177The effect of Lu-RH@SP group (G4) alone was not as good as that of the combined treatment group, indicating that αPD-L1 played an important synergistic role in combined treatment.

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

[0109] At the same time, T cell exhaustion in the mesenteric lymph nodes of mice in the G5 group was significantly reversed. Figure 6 As shown in jk, the expression of T cell exhaustion markers decreased and T cell function was restored, which indicates that 177 The combined use of Lu-RH@SP and αPD-L1 can regulate the tumor immune microenvironment, enhance the effect of tumor immunotherapy, and exert a synergistic anti-tumor effect.

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

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a radioactive hydrogel based on cross-linking of sodium alginate and metal ions, characterized in that: The following steps are involved: Lutetium chloride and metal ions are mixed to obtain a metal ion mixed solution, and then radioactive isotopes are added to the metal ion mixed solution. 177 Lu obtains a metal ion crosslinker; injects the metal ion crosslinker into the sodium alginate solution under vortex conditions, and obtains a radioactive hydrogel after centrifugation and discarding the supernatant.

2. The method for preparing a radioactive hydrogel based on cross-linking of sodium alginate and metal ions according to claim 1, characterized in that: The preparation method of the metal ion mixed solution comprises: mixing a lutetium chloride solution and a calcium chloride solution to obtain the metal ion mixed solution, wherein the volume ratio of the lutetium chloride solution to the calcium chloride solution is 1:0.8-1.

2.

3. The method for preparing a radioactive hydrogel based on cross-linking of sodium alginate and metal ions according to claim 2, 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.

4. The method for preparing radioactive hydrogel based on cross-linking of sodium alginate and metal ions according to claim 1, characterized in that: The concentration of the sodium alginate solution is 0.8-1.2 w / v%.

5. The method for preparing radioactive hydrogel based on cross-linking of sodium alginate and metal ions 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.

6. The method for preparing radioactive hydrogel based on cross-linking of sodium alginate and metal ions according to claim 1, characterized in that: The centrifugation condition is 2500-3500 rpm for 20-40 minutes.

7. A radioactive hydrogel, characterized in that: Prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the radioactive hydrogel according to claim 7 in the preparation of tumor drugs.

9. The use according to claim 8, characterized in that: The tumor drug comprises the radioactive hydrogel according to claim 7 and sodium propionate, and the tumor is a low rectal tumor.

10. The use according to claim 9, characterized in that: The drug is used to inhibit tumor growth, inhibit tumor cell proliferation, reduce tumor malignancy and invasiveness, and promote tumor cell apoptosis.

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