A 131i-labeled iron tannic acid nanoparticle hyaluronic acid phenylboronic acid radioactive hydrogel, preparation method and application thereof

Abstract

The application discloses a kind of 131 I labeled iron tannic acid nanoparticle hyaluronic acid phenylboronic acid radioactive hydrogel and preparation method and application;The hydrogel provided by the application can construct local physical limited radioactive "reservoir" in tumor, significantly improve the retention and stability of nuclide in tumor, reduce dissociation and diffusion escape, reduce the risk of system exposure, and improve the potential of treatment window of local radio delivery.The system of the application has good biocompatibility, and shows a synergistic effect of inhibiting tumor proliferation and inducing in situ vaccine-like immune microenvironment regulation in an immune-competent tumor model, providing a new strategy for combined application with immune checkpoint blockade therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering materials, specifically to a ¹³¹I-labeled iron tannic acid nanoparticle hyaluronic acid phenylboronic acid radioactive hydrogel, its preparation method, and its application. Background Technology

[0002] Malignant tumors are characterized by their aggressive growth, high recurrence rate, and tendency to metastasize, making them a serious threat to human health (Sung H et al., CA: A Cancer Journal for Clinicians, 2021, 71(3): 209-249). Current cancer treatments mainly include surgical resection, radiotherapy, chemotherapy, targeted therapy, and immunotherapy (Miller KD et al., CA: A Cancer Journal for Clinicians, 2022, 72(5): 409-436). Although these treatments have achieved certain clinical efficacy, some solid tumors still face challenges such as local recurrence, high risk of metastasis, insensitivity to treatment, or dosage limitations leading to insufficient efficacy. Furthermore, systemic therapy often involves varying degrees of toxic side effects (Citrin DE, New England Journal of Medicine, 2017, 377(11):1065-1075). Therefore, developing novel treatment strategies and delivery systems that balance efficacy and safety and are suitable for local treatment of solid tumors is of great significance.

[0003] Radionuclide therapy utilizes the continuous release of ionizing radiation such as beta rays from radioisotopes to cause DNA damage and induce cell death in tumor cells. It offers advantages such as sustained action, local irradiation capability, and compatibility with other treatment modalities, and has shown potential for application in various cancer treatments in recent years (Sgouros G et al., Nature Reviews DrugDiscovery, 2020, 19(9): 589-608). Especially in the treatment of solid tumors, the direct introduction of radionuclides into tumor tissue through local delivery methods such as intratumoral injection is expected to increase the local absorbed dose and reduce systemic exposure (Marcu L et al., Critical Reviews in Oncology / Hematology, 2018, 131: 39-47). However, the efficacy and safety of radionuclide therapy are highly dependent on the retention and targeting ability of the radionuclide in the tumor: on the one hand, the short retention time of the radionuclide or its carrier in the tumor, its diffusion and escape or entry into the circulatory system, can lead to difficulty in maintaining the local effective dose and a decrease in the therapeutic effect; on the other hand, radionuclide escape may cause increased radiation exposure to non-target organs, bringing the risk of systemic toxicity (Melero I et al., Cancer Cell, 2021, 39(10): 1319-1322). Radionuclides, represented by iodine isotopes, may be exchanged or delabeled in the physiological environment, resulting in non-targeted distribution, thereby affecting the therapeutic window (Weber WA et al., Nature Reviews Clinical Oncology, 2022, 19(8): 534-550).

[0004] Hydrogels are widely used in local drug delivery and tissue engineering due to their high water content, good tissue compatibility, and ability to be injected into the tumor for in-situ shaping (Li J et al., Nature Reviews Materials, 2016, 1(12): 16071). Injectable hydrogels can form a "drug reservoir" at the tumor site through intratumoral injection, enabling sustained release and continuous action of functional components, and to a certain extent, playing a physical confinement role, thereby reducing diffusion and escape (Zhang Y et al., Biomaterials, 2021, 277: 121094). Hyaluronic acid (HA), as a natural extracellular matrix component, has good biocompatibility, degradability and tumor targeting (it can specifically bind to the CD44 receptor highly expressed on the surface of tumor cells), making it an ideal matrix material for constructing injectable hydrogels (Burdick JA et al., Advanced Materials, 2011, 23(12): H41-H56; Mattheolabakis G et al., Journal of Controlled Release, 2015, 213: 124-135). Iron-tannic acid nanoparticles (FETA) are a typical metal-polyphenol network material. Their abundant polyphenol structures can efficiently load radionuclides and also serve as dynamic crosslinking sites to participate in the formation of gel networks (Ejima H et al., Science, 2013, 341(6142): 154-157; Guo Jet al., Angewandte Chemie International Edition, 2014, 53(22): 5546-5551). Summary of the Invention

[0005] This invention aims to provide a radionuclide delivery system suitable for intratumoral injection in solid tumors. Under physiological conditions, it can rapidly form a stable hydrogel and construct a local physical confinement environment, thereby improving the retention and stability of radionuclides within the tumor, reducing nuclide dissociation and diffusion, thus lowering the risk of system exposure and improving the efficacy and safety of local radiotherapy. Furthermore, if the system has imaging visualization potential, it will be more conducive to the assessment and monitoring of intratumoral distribution and retention.

[0006] To overcome the problems of easy escape of radionuclides during local delivery, insufficient intratumoral retention, and the resulting systemic exposure risks in existing technologies, the primary objective of this invention is to provide a ¹³¹I-FETA-HA / Gel radioactive hydrogel reservoir. This material is composed of 131I-labeled iron-tannic acid nanoparticles (131I-FETA) and hyaluronic acid modified with phenylboronic acid groups (HA-PBA): hyaluronic acid (HA) introduces phenylboronic acid (PBA) groups into its side chain via amidation coupling, forming HA-PBA, which provides crosslinking sites for subsequent hydrogel network construction; the FETA nanoparticles are loaded with 131I nuclides using the Iodo-Gen method, resulting in 131I-FETA with high labeling efficiency and good radiostability. When 131I-FETA and HA-PBA are mixed under physiological conditions, the PBA groups on the side chain of HA-PBA undergo borate ester crosslinking and related interactions with the ortho-bisphenol / cis-diol groups in the tannic acid polyphenol structure on the FETA surface, forming an injectable radioactive hydrogel. This hydrogel constructs a locally physically confined radioactive "reservoir" within the tumor, thereby improving the retention and stability of the radionuclide within the tumor, reducing dissociation and diffusion, lowering the risk of systemic exposure, and enhancing the efficacy and safety of local radiotherapy.

[0007] This invention provides a ¹³¹I-FETA-HA / Gel hydrogel reservoir, the hydrogel reservoir being shown in formula (I) below:

[0008] The crosslinking mechanism of Formula (I) ¹³¹I-FETA-HA / Gel hydrogel reservoir; In Formula (I), the degree of polymerization n is 100-250 (In this invention, hyaluronic acid with a molecular weight of 40-100 kDa is selected as the starting material, thereby controlling the degree of polymerization n of the hydrogel skeleton within the range of 100-250).

[0009] Furthermore, the HA-PBA is prepared by an amidation coupling reaction of HA and PBA.

[0010] This invention also provides a method for preparing the above-mentioned ¹³¹I-FETA-HA / Gel hydrogel reservoir, comprising the following steps: (1) Preparation of iron-tannic acid nanoparticles (FETA): Tannic acid (TA) solution and anhydrous ferric chloride (FeCl3) solution were mixed at room temperature and stirred for 4-6 hours to form FETA dispersion. FETA was obtained by dialysis purification. (2) Preparation of phenylboronic acid modified hyaluronic acid (HA-PBA): HA and PBA were dissolved in water and dimethyl sulfoxide (DMSO) respectively at room temperature, then mixed and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBt) were added as catalysts. The pH was adjusted to 3.5~5 and amidation coupling reaction was carried out for 48 hours. After dialysis purification and freeze drying, HA-PBA was obtained. (3)¹³¹I labeling: Take the FETA dispersion obtained in step (1), mix it with Na¹³¹I, add it to a reaction tube pre-coated with Iodo-Gen, react for 20~40 minutes, and then purify by ultrafiltration and centrifugation to obtain ¹³¹I-FETA; (4) Formation of hydrogel: The HA-PBA obtained in step (2) is dissolved in deionized water and mixed with the ¹³¹I-FETA solution obtained in step (3). After a borate ester crosslinking reaction, the radioactive hydrogel reservoir is obtained.

[0011] Preferably, in step (1), the molar ratio of TA to FeCl3 is 1:(1.3~1.8); the concentration of the TA solution is 2%~6% (w / v), and the concentration of the FeCl3 solution is 0.4%~0.8% (w / v); the dialysis is performed using a dialysis bag with a molecular weight cutoff of 2000~6000 Da, the dialysis time is 1~2 days, and the water is changed every 2~4 hours; the equivalent concentration of TA in the purified FETA dispersion is 1%~3% (w / v).

[0012] Preferably, in step (2), the molecular weight of HA is 40~100 kDa; the concentration of the HA solution is 0.3%~1% (w / v); the molar ratio of HA, PBA, EDC, and HOBt is 1:(1.0~1.3):(0.8~1.2):(0.8~1.2); the dialysis uses a dialysis bag with a molecular weight cutoff of 2000~6000 Da, the dialysis time is 3~5 days, and the water is changed every 6~12 hours; the freeze-drying temperature is -80℃, and the freeze-drying time is 3~5 days.

[0013] Preferably, the amount of Iodo-Gen used in step (3) is 0.5~1.0 mg, and the amount of Na¹³¹I used is 0.8~1.5 mCi per 1 mL FETA; the ultrafiltration centrifugation uses an ultrafiltration tube with a molecular weight cutoff of 10 kDa, a centrifugation speed of 5000 rpm, a centrifugation time of 20~30 minutes, and repeats the centrifugation until the lower clear liquid is free of radioactivity.

[0014] Preferably, the concentration of the HA-PBA solution in step (4) is 1.5%~2.5% (w / v); the equivalent concentration of the ¹³¹I-FETA solution, calculated as TA, is 1%~3% (w / v); the volume ratio of the HA-PBA solution to the ¹³¹I-FETA solution is (2.5~3):1; n(HA-PBA) is calculated as the number of moles of hyaluronic acid repeat disaccharide units in HA-PBA, and n(Fe) is calculated as the number of moles of iron in FETA, and the molar ratio of HA-PBA to FETA is (5~8):1.

[0015] The present invention also provides the use of the above-described ¹³¹I-FETA-HA / Gel hydrogel reservoir in the preparation of a medicament for intratumoral injection to treat tumors.

[0016] The present invention has, but is not limited to, the following beneficial technical effects: (1) This invention constructs a hydrogel with excellent injectability and tissue adhesion by building iron-tannic acid nanoparticles (FETA) and phenylboronic acid-modified hyaluronic acid (HA-PBA) and utilizing the dynamic covalent bond (boronic acid ester bond) that is rapidly formed between PBA and the o-dihydroxyl group of TA under physiological or slightly acidic conditions. This hydrogel can form a gel without the addition of any strong oxidant or strong base, which greatly protects the activity of the loading group. At the same time, the HA backbone can act as a natural ligand to specifically bind to the CD44 receptor that is highly expressed on the surface of tumor cells, realizing the bio-anchoring of the hydrogel in the tumor stroma, effectively resisting tissue fluid flushing, and providing an ideal spatial confinement environment for the local storage of radionuclides. After intratumoral injection, ¹³¹I-FETA-HA / Gel significantly prolongs the intratumoral retention time of the radionuclide, reduces diffusion and escape to surrounding normal tissues and the circulatory system, and reduces the risk of systemic exposure.

[0017] (2) This invention fully utilizes the abundant polyphenol structure on the tannic acid (TA) molecule, eliminating the need for complex chemical chelating agents or cumbersome skeleton modifications. It achieves efficient and stable labeling of radioactive ¹³¹I via a mild solid-phase oxidation method (such as the Iodo-Gen method). The labeling rate can reach approximately 75-85%, and it exhibits extremely high radiostability and retention rate under physiological conditions (such as in a serum environment) (stability >85% after 14 days in serum). This effectively avoids premature dissociation of free iodine, ensures the integrity of the radioactive reservoir, resolves the contradiction between insufficient retention and system exposure often caused by conventional nanomedicines, and broadens the therapeutic window of the local radiopharmaceutical delivery system.

[0018] (3) The carrier materials used in this invention, hyaluronic acid and iron-tannic acid nanoparticles, are both natural or biomimetic materials with good biocompatibility, and their degradation products are safe and non-toxic. In vivo safety evaluation showed that no pathological damage was found in the H&E staining sections of the major organs (heart, liver, spleen, lung, and kidney) of the mice in the treatment group, and the key indicators of blood routine were within the normal range. There was no obvious bone marrow suppression or systemic toxicity, indicating that the system has good biocompatibility and in vivo safety.

[0019] (4) The radioactive hydrogel of this invention effectively integrates multiple functions such as image guidance, physical killing, and immune regulation, realizing "therapeutic integration". On the one hand, the system has excellent single-photon emission computed tomography (SPECT / CT) imaging capabilities, which can realize non-invasive and real-time monitoring of the distribution range, boundary, and retention dynamics of the radioactive hydrogel within the tumor; on the other hand, the β rays continuously emitted by ¹³¹I precisely kill tumor cells and effectively inhibit tumor proliferation (significantly downregulate Ki67 expression), while reshaping the local tumor microenvironment. This continuous local irradiation successfully triggered an in situ vaccine-like (ISV-like) immune response, induced adaptive upregulation of PD-L1 in tumor tissue, and promoted the reversal of the tumor phenotype from "immune cold" to "immune hot". Attached Figure Description

[0020] To clearly illustrate the specific embodiments of the present invention and certain detection techniques used in the experiments, the implementation schemes and the techniques used will be described below, mainly through the accompanying drawings.

[0021] Figure 1 This is a schematic diagram illustrating the preparation and crosslinking mechanism of the ¹³¹I-labeled iron-tannic acid nanoparticle / hyaluronic acid-phenylboronic acid radioactive hydrogel reservoir (¹³¹I-FETA-HA / Gel) described in this invention.

[0022] Figure 2 This is a schematic diagram of the synthesis process of iodine-labeled iron-tannic acid nanoparticles (I-FETA) and phenylboronic acid-modified hyaluronic acid (HA-PBA) as described in this invention.

[0023] Figure 3 The images show the 1H NMR spectra of HA and HA-PBA obtained in Example 1 (A) and a schematic diagram of the gelation process of mixing ¹³¹I-FETA and HA-PBA (B).

[0024] Figure 4 The graph shows the relationship between iodine ion labeling efficiency and reaction time in Example 3 (A), and the results of the radiostability test of ¹³¹I-FETA and ¹³¹I-FETA-HA / Gel in serum (B).

[0025] Figure 5The images show the in vivo evaluation results of CT26 tumor-bearing mice after intratumoral injection of different preparations in Example 4. Among them, A is an actual picture of the tumor dissected on the 7th day after drug administration, B is a statistical comparison of tumor weight in each group, C is a live fluorescence imaging image of mice before and after treatment, D is a curve of tumor volume change within 7 days of treatment, E is H&E staining image of the main organs (heart, liver, spleen, lung, kidney) of mice in each group on the 7th day after drug administration, F is a SPECT / CT imaging image 72 hours after ¹³¹I-FETA-HA / Gel treatment, and G is an immunohistochemical and immunofluorescence staining image of tumor tissue after ¹³¹I-FETA-HA / Gel treatment.

[0026] Specific implementation methods The specific embodiments of the present invention are explained with the aid of examples. Except for the detection technology, which does not limit the present invention in any way, some of the solutions in the described embodiments are part of the embodiments of the invention. All embodiments obtained by ordinary people in the art without creative results are within the protection scope of the present invention. Example

[0027] like Figure 1 As shown, this invention provides a method for preparing a ¹³¹I-FETA-HA / Gel hydrogel reservoir, comprising the following steps: (1) Preparation of iron-tannic acid nanoparticles (FETA): Tannic acid (TA) solution and anhydrous ferric chloride (FeCl3) solution were mixed at room temperature and stirred to form FETA dispersion. FETA was obtained by dialysis purification.

[0028] (2) Preparation of phenylboronic acid modified hyaluronic acid (HA-PBA): HA and PBA were dissolved in water and dimethyl sulfoxide (DMSO) respectively at room temperature, then mixed and a catalyst was added. The pH was adjusted and an amidation coupling reaction was carried out. After dialysis purification and freeze drying, HA-PBA was obtained.

[0029] (3) Preparation of ¹³¹I-labeled iron-tannic acid nanoparticles (¹³¹I-FETA): Take the FETA dispersion obtained in step (1), mix it with Na¹³¹I, add it to a reaction tube pre-coated with Iodo-Gen, and purify it by ultrafiltration and centrifugation to obtain ¹³¹I-FETA.

[0030] (4) Preparation of radioactive hydrogel (¹³¹I-FETA-HA / Gel): Dissolve the HA-PBA obtained in step (2) in deionized water, mix it with the ¹³¹I-FETA solution obtained in step (3), and obtain the radioactive hydrogel reservoir through borate ester crosslinking reaction.

[0031] The room temperature mentioned in step (1) is usually between 25 and 30°C.

[0032] Step (1) The molar ratio of TA to FeCl3 is 1:1.5; The concentration of the TA solution in step (1) is 4% (w / v); the concentration of the FeCl3 solution is 0.6% (w / v). In step (1), the dialysis is performed using a dialysis bag with a molecular weight cutoff of 2000-6000 Da. The water is changed every 4 hours, and the dialysis is performed for a total of 2 days.

[0033] The room temperature mentioned in step (2) is usually between 25 and 30°C.

[0034] The molecular weight of HA in step (2) is 40 to 100 kDa.

[0035] The molar ratio of HA, PBA, EDC and HOBt in step (2) is 1:1.2:1.0:1.0.

[0036] The concentration of the HA solution in step (2) is 0.5% (w / v).

[0037] In step (2), the dialysis is performed using a dialysis bag with a molecular weight cutoff of 2000 to 6000 Da. The water is changed every 8 hours, and the dialysis is performed for a total of 4 days.

[0038] The freeze-drying temperature in step (2) is -80℃, and the drying time is 5 days.

[0039] The amounts of Iodo-Gen and Na¹³¹I in step (3) are: 0.5 mg of Iodo-Gen, 1 mCi of Na¹³¹I (based on 1 mL FETA), and a reaction time of 30 min.

[0040] In step (3), the ultrafiltration centrifugation is carried out using an ultrafiltration tube with a molecular weight cutoff of 10 kDa, centrifuged at 5000 rpm for 20 min, and the centrifugation is repeated until the lower clear liquid is free of radioactivity.

[0041] The concentration of the HA-PBA solution in step (4) is 2% (w / v); the equivalent concentration of the ¹³¹I-FETA solution, calculated as TA, is 2% (w / v).

[0042] In step (4), the volume ratio of the HA-PBA solution to the ¹³¹I-FETA solution is 3:1.

[0043] The specific synthesis process of the ¹³¹I-labeled hydrogel reservoir described in this invention is as follows: Figure 2 As shown.

[0044] The above-mentioned ¹³¹I-FETA-HA / Gel hydrogel reservoir is used in biomedical engineering.

[0045] The application is: the application of the aforementioned ¹³¹I-FETA-HA / Gel hydrogel reservoir in the field of tumor treatment.

[0046] Example 2 (1) Preparation of iron-tannic acid nanoparticles (FETA): At room temperature, 4 g of tannic acid (TA) was dissolved in 100 mL of deionized water to prepare a 4% (w / v) TA solution; 0.6 g of anhydrous ferric chloride (FeCl3) was dissolved in 100 mL of deionized water to prepare a 0.6% (w / v) FeCl3 solution. The FeCl3 solution was slowly added dropwise to the TA solution while stirring rapidly, and the reaction was carried out for 5 hours to form a FETA dispersion. After the reaction was completed, the dispersion was transferred to a dialysis bag with a molecular weight cutoff of 2000~6000 Da and dialyzed in deionized water for 2 days, with the water changed every 4 hours, to obtain a purified FETA dispersion. The equivalent concentration of TA in the purified dispersion was determined to be 2% (w / v).

[0047] (2) Preparation of phenylboronic acid-modified hyaluronic acid (HA-PBA): At room temperature, 0.5 g of hyaluronic acid (HA) was dissolved in 100 mL of deionized water to prepare a 0.5% (w / v) HA solution; 0.215 g of 2-aminophenylboronic acid (PBA) was dissolved in 5 mL of dimethyl sulfoxide (DMSO). The HA solution and PBA solution were mixed, and then 0.17 g of 1-hydroxybenzotriazole (HOBt) and 0.24 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were added as catalysts. The pH was adjusted to 4.0 with HCl, and the amidation coupling reaction was carried out for 48 hours. After the reaction, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 2000~6000 Da and dialyzed in deionized water for 4 days, with the water changed every 8 hours. After dialysis, the solution was freeze-dried at -80℃ for 4 days to obtain HA-PBA.

[0048] (3) Preparation of ¹³¹I-labeled iron-tannic acid nanoparticles (¹³¹I-FETA): Take 1 mL of the purified FETA dispersion obtained in step (1) above, mix it with 1 mCi Na¹³¹I, and add it to a reaction tube pre-coated with 0.5 mg Iodo-Gen. React at room temperature for 30 minutes. After the reaction is complete, transfer the reaction solution to an ultrafiltration tube with a molecular weight cutoff of 10 kDa, centrifuge at 5000 rpm for 25 minutes, discard the lower supernatant, add 1 mL of deionized water and mix well. Repeat centrifugation until the lower supernatant is free of radioactivity to obtain the ¹³¹I-FETA dispersion.

[0049] (4) Preparation of radioactive hydrogel (¹³¹I-FETA-HA / Gel): Dissolve 0.2 g of HA-PBA obtained in step (2) in 10 mL of deionized water to prepare a 2% (w / v) HA-PBA solution. Take 3 mL of HA-PBA solution and mix it evenly with 1 mL of ¹³¹I-FETA dispersion obtained in step (3) (equivalent concentration of 2% (w / v) based on TA). After borate ester crosslinking reaction, the radioactive hydrogel reservoir is obtained.

[0050] The results are as follows Figure 3 As shown, the 1H NMR spectrum results confirm the successful synthesis of HA-PBA, and the mixture of HA-PBA and FETA can effectively form a gel.

[0051] Example 3 Evaluation of labeling efficiency and radiostability of ¹³¹I-FETA-HA / Gel hydrogel The beta rays produced by iodine-131 can penetrate tumor tissue and induce DNA double-strand breaks, thereby effectively inhibiting tumor proliferation. 131 The labeling and radiochemical purity of iodine directly determine the antitumor effect and systemic toxicity of the hydrogel. In vitro, the loading conditions were first optimized using non-radioactive NaI. 1 mL of the FETA dispersion prepared in Example 1 was added to 1 mg of NaI (dissolved in 0.1 mL of deionized water), thoroughly mixed, and transferred to a reaction tube pre-coated with 0.5 mg of Iodo-Gen. The reaction was carried out at room temperature for 30 min. After the reaction, the reaction solution was transferred to an ultrafiltration tube and centrifuged. The supernatant was collected, and the iodine labeling rate was determined by detecting the iodine ion content in the supernatant. Iodine ion standard solutions were used for analysis by ion chromatography to obtain a standard curve related to iodine ions. By adjusting the reaction time of the hydrogel and NaI in the Iodo-Gen reaction tube (at different time points), the change curve of the iodine labeling rate over time was determined. Finally, the optimal labeling reaction time and labeling rate were selected for subsequent... 131 The labeling of I created the conditions. Experimental results are as follows: Figure 4 As shown in Figure A, the labeling efficiency of iodide ions gradually increases with time, reaching approximately 80% at a reaction time of 30 min. Subsequently, the labeling efficiency of iodide ions does not change significantly with further time, indicating that the labeling rate reaches its optimal level at this point. After confirming the optimal reaction time, radioactive Na₂O₃ was used... 131 I-FETA was labeled and mixed with HA-PBA to obtain ¹³¹I-FETA-HA / Gel hydrogel. The ¹³¹I-FETA dispersion (ungelatinized) and ¹³¹I-FETA-HA / Gel hydrogel were placed in fetal bovine serum, and the stability of iodide ions was observed. The results are as follows: Figure 4 As shown in Figure B, a very small amount of [amount missing] was detected in the ¹³¹I-FETA-HA / Gel hydrogel group as time increased. 131 I slowly dissociates from the hydrogel matrix, but by day 14, its radioactivity retention rate is still as high as about 85%, indicating good stability.

[0052] Example 4 Male BALB / c mice aged 6-8 weeks were subcutaneously inoculated with CT26 colon cancer cells on their right back to establish a CT26 tumor-bearing mouse model. When the tumor volume grew to approximately 100-150 mm³, the tumor-bearing mice were randomly divided into three groups: a blank control group (PBS), a ¹³¹I-FETA group, and a ¹³¹I-FETA-HA / Gel group. Each group received a single injection of the corresponding formulation (¹³¹I activity 500 uCi, 50 μL) into the tumor. The general condition of the mice was observed after administration, and tumor volume and mouse weight were measured daily for 7 consecutive days. The tumor volume was calculated using the formula: V = (length × width²) / 2. SPECT / CT imaging was performed 72 hours after administration. In the ¹³¹I-FETA-HA / Gel group, the intratumoral radioactivity signal was still clearly visible at 168 hours (7 days). On day 3 of treatment, a random number of mice were euthanized, and major organs (heart, liver, spleen, lung, and kidney) and tumor tissue were collected. The radioactivity intensity in each sample was quantitatively measured using a gamma counter. On day 7 of treatment, the remaining mice in each group were sacrificed, tumor tissue was collected and weighed, and major organs were subjected to H&E staining to observe pathological changes. Tumor tissue from each group of mice was subjected to Ki67 immunohistochemical staining and PD-L1 immunofluorescence staining to observe tumor cell proliferation activity and changes in the immune microenvironment. The gamma counter results showed that on day 3 of treatment, most of the ¹³¹I in the ¹³¹I-FETA-HA / Gel group remained confined within the tumor tissue, with only a small amount of residual radioactivity detected in the liver and kidneys. This indicates that a very small portion of the dissociated radionuclide was excreted through normal hepatic and renal metabolic pathways, without causing severe systemic accumulation. Figure 5 As shown in A and 5B, on day 7 after drug administration, the tumor size and weight of mice in the blank group significantly increased; while the tumor size and weight of mice in the ¹³¹I-FETA-HA / Gel group were significantly lower than those in the other two groups. Figure 5 As shown in C and 5D, the tumor volume in the control group mice continued to grow rapidly during the treatment period, while the tumor volume in the ¹³¹I-FETA-HA / Gel group mice remained small throughout the treatment period. The results indicate that intratumoral injection of ¹³¹I-FETA-HA / Gel can significantly inhibit tumor growth. Figure 5As shown in Figure E, on day 7 of treatment, no significant pathological damage was observed in the H&E stained sections of major organs such as the heart, liver, spleen, lungs, and kidneys of mice in the ¹³¹I-FETA-HA / Gel group, while kidney and lung damage were observed in the ¹³¹I-FETA group. Ki67 immunohistochemical staining results showed that the proportion of Ki67-positive cells in the tumor tissue of the ¹³¹I-FETA-HA / Gel group was significantly lower than that in the control group, indicating that the radioactive hydrogel effectively inhibited tumor cell proliferation. PD-L1 immunofluorescence staining results showed that the PD-L1 expression level in the tumor tissue of the ¹³¹I-FETA-HA / Gel group was significantly upregulated, indicating that local radiotherapy triggered an immune response in the tumor microenvironment and induced adaptive PD-L1 expression, providing a basis for combined immune checkpoint blockade therapy.

Claims

1. A ¹³¹I-labeled iron-tannic acid nanoparticle / hyaluronic acid-phenylboronic acid radioactive hydrogel; characterized in that... The structure of the hydrogel is shown in formula (I): Formula (1).

2. A method for preparing a ¹³¹I-labeled iron-tannic acid nanoparticle / hyaluronic acid-phenylboronic acid radioactive hydrogel, characterized in that: The hydrogel is formed by a borate ester crosslinking reaction between ¹³¹I-labeled iron-tannic acid nanoparticles ¹³¹I-FETA and hyaluronic acid HA-PBA modified with phenylboronic acid groups; wherein, the phenylboronic acid groups of the HA-PBA side chains crosslink with the polyphenol structure on the surface of the ¹³¹I-FETA to form a hydrogel network.

3. The preparation method according to claim 2, characterized in that: The HA-PBA is prepared by amidation coupling reaction of hyaluronic acid and phenylboronic acid; FETA is formed by coordination self-assembly of tannic acid and anhydrous ferric chloride.

4. A preparation method as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Preparation of iron-tannic acid nanoparticles FETA: At room temperature, TA solution was mixed with anhydrous ferric chloride solution and stirred for 4-6 hours to form FETA dispersion. FETA was obtained by dialysis purification. (2) Preparation of phenylboronic acid modified hyaluronic acid HA-PBA: HA and PBA were dissolved in water and dimethyl sulfoxide (DMSO) respectively at room temperature, then mixed and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBt) were added as catalysts. The pH was adjusted to 3.5-5 and amidation coupling reaction was carried out for 48 hours. After dialysis purification and freeze drying, HA-PBA was obtained. (3)¹³¹I labeling: Take the FETA dispersion obtained in step (1), mix it with Na¹³¹I solution, add it to a reaction tube pre-coated with Iodo-Gen, react for 20~40 minutes, and purify by ultrafiltration and centrifugation to obtain ¹³¹I-FETA; (4) Formation of hydrogel: The HA-PBA obtained in step (2) is dissolved in deionized water and mixed with the ¹³¹I-FETA solution obtained in step (3). The radioactive hydrogel is obtained by borate ester crosslinking reaction.

5. The preparation method according to claim 4, characterized in that: In step (1), the concentration of the TA solution is 2%~6% (w / v), the concentration of the anhydrous ferric chloride solution is 0.4%~0.8% (w / v), and the equivalent concentration of TA in the FETA dispersion is 1%~3% (w / v). In step (2), the concentration of the HA solution is 0.3%~1% (w / v). In step (4), the concentration of the solution after the HA-PBA is dissolved in water is 1.5%~2.5% (w / v), and the equivalent concentration of the ¹³¹I-FETA solution, calculated as TA, is 1%~3% (w / v).

6. The preparation method according to claim 4, characterized in that, In step (1), the molar ratio of TA to anhydrous ferric chloride is 1:(1.3~1.8). In step (2), the molar ratio of HA, PBA, EDC, and HOBt is 1:(1.0~1.3):(0.8~1.2):(0.8~1.2). In step (4), the number of moles of HA repeating disaccharide units in HA-PBA is n(HA-PBA), and the number of moles of iron in FETA is n(Fe). The molar ratio of HA-PBA to FETA is (5~8):

1.

7. The preparation method according to claim 4, characterized in that, In step (4), the volume ratio of the HA-PBA solution dissolved in water to the ¹³¹I-FETA solution is (2.5~3):

1.

8. The preparation method according to claim 4, characterized in that, In step (1), the dialysis uses a dialysis bag with a molecular weight cutoff of 2000~6000 Da, the dialysis time is 1~2 days, and the water is changed every 2~4 hours; In step (2), the molecular weight of HA is 40~100 kDa, the dialysis uses a dialysis bag with a molecular weight cutoff of 2000~6000 Da, the dialysis time is 3~5 days, the water is changed every 6~12 hours, the freeze-drying temperature is -80℃, and the freeze-drying time is 3~5 days; In step (3), the amount of Iodo-Gen used is 0.5~1.0 mg, the amount of Na¹³¹I used is 0.8~1.5 mCi per 1 mL FETA, the ultrafiltration centrifugation uses an ultrafiltration tube with a molecular weight cutoff of 10 kDa, the centrifugation speed is 5000 rpm, the centrifugation time is 20~30 minutes, and the centrifugation is repeated until the lower clear liquid is free of radioactivity.

9. The use of the ¹³¹I-labeled iron-tannic acid nanoparticles / hyaluronic acid-phenylboronic acid radioactive hydrogel of claim 1 in the preparation of a medicament for intratumoral injection to treat tumors.

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