Double-lock target control alpha nuclide self-burning molecular cage as well as preparation method and application thereof

Abstract

The invention discloses a double-lock target control alpha nuclide self-burning molecular cage which comprises a pre-targeting delivery system and an active targeting delivery system, the active targeting delivery system comprises an alpha nuclide; the pre-targeting delivery system and the active targeting system release alpha nuclides through a cleavage reaction. Through a pre-targeting-active targeting double-step delivery strategy, the cumulant and the treatment specificity of the alpha nuclide in tumor tissues are remarkably improved, the stability of the alpha nuclide in the delivery process is improved, advanced leakage of the alpha nuclide in blood circulation or normal tissues is avoided, the striking efficiency on the tumor tissues is remarkably improved, and the treatment effect of the alpha nuclide on the tumor tissues is improved. The limitation of a traditional single passive delivery system is broken through.

Description

Technical Field

[0001] This invention belongs to the fields of radiopharmaceutical chemistry and clinical nuclear medicine, specifically relating to a dual-lock target-controlled alpha nuclide self-immolation molecular cage, its preparation method, and its application. Background Technology

[0002] With the continuous development of cancer treatment methods, targeted therapy and Targeted Radionuclide Therapy (TRT) have become research hotspots in recent years. Among them, alpha particle radiation, due to its high linear energy transfer (LET) and short range (only tens to hundreds of micrometers), can cause severe DNA double-strand breaks within a very small area, possessing the unique advantages of efficiently killing tumor cells and reducing damage to surrounding normal tissues. Therefore, alpha emitter-based... 223 Ra, 225 Ac, 211 Treatment strategies such as At (A, etc.) have shown great promise in the treatment of advanced cancers, especially bone metastases.

[0003] Radium-223 ( 223 Ra was approved in 2013 as the first alpha particle radiopharmaceutical that can be used clinically. Its physical half-life is 11.4 days, and it decays to emit high-energy alpha particles (5.8 MeV) with a short range (<100 μm). It belongs to the high energy transfer line density alpha nuclides (80keV / μm) and has great potential for clinical application.

[0004] However, based on 223 The development and clinical application of Ra radiopharmaceuticals still face certain challenges: Firstly, radium, as an alkaline earth element, presents certain challenges. 2+ Chelation reactions in aqueous solutions are difficult, so... 223 Ra-labeled biological vectors are extremely challenging; and 223 Ra decay processes involve daughter nuclides with high recoil capabilities, necessitating carrier immobilization. 223 Ra enables precise tumor targeting. Therefore, how to effectively deliver... 223 Improving the targeting and safety of alpha radionuclide therapy are key issues in its development.

[0005] Currently, the existing technical solutions closest to this invention mainly include the following types: 1) Delivery of hydroxyapatite nanoparticles 223 Ra Hydroxyapatite (HAp) is similar to the natural mineral components in bone tissue and can effectively bind to bone through ion exchange mechanisms.223 Ra has become an ideal carrier material. Existing reports indicate that... 223 Ra was loaded into synthetic HAp nanoparticles and delivered to bone metastases via intravenous injection for targeted radiotherapy. This system demonstrated good tumor suppression and biocompatibility in animal models. However, HAp materials exhibit limited targeting in non-bone tumor treatments, and particle aggregation issues affect their uniform distribution in vivo.

[0006] 2) Delivery of alpha nuclides via liposomes or polymer nanoparticles Liposomes, as flexible nanocarriers, can encapsulate... 223 Ra or its ligand complexes prolong blood circulation time and reduce hepatic uptake. Some studies have employed PEGylated liposomes or polymer nanoparticles for delivery. 223 Ra increases the accumulation at the tumor site. However, liposomes have insufficient stability; the high-energy recoil effect generated after the decay of alpha particles may cause the nuclide to be released from the carrier, reducing the therapeutic effect and increasing the risk of side effects.

[0007] 3) Radioimmunoconjugates (RICs) By coupling alpha nuclides to monoclonal antibodies targeting tumor-specific antigens using chelating agents, radioactive antibody conjugates can be formed, enabling highly selective tumor-targeting killing. 225 Ac、 211 Radioactive isotopes such as At have entered multiple clinical trial phases. However, for... 223 Ra, due to its chemical properties (lack of suitable stabilizing chelating agents) and radioactivity, currently lacks mature RIC products, which limits its application in this area.

[0008] 4) Application in free-form without a carrier 223 Ra Currently used in clinical practice 223 Ra (radium dichloride injection) is injected in free ion form and is primarily used to treat bone metastases through bone-targeted accumulation. While the free form offers the convenience of direct application, it cannot be extended to other types of tumors, and potential off-target toxicity remains a limiting factor.

[0009] In summary, while existing technologies have solved the problem to some extent... 223 Despite the challenges in Ra delivery and application, limitations remain, including insufficient radionuclide immobilization, limited targeting efficiency, and poor adaptability to complex tumor microenvironments. Therefore, the development of next-generation payloads is still necessary. 223 Ra's nanodelivery system aims to improve therapeutic efficacy and safety. Summary of the Invention

[0010] This invention addresses the shortcomings of existing targeted alpha nuclide therapy technologies by proposing a dual-lock target-controlled alpha nuclide self-immolation molecular cage, its preparation method, and its applications, primarily solving the following technical problems: 1. Solve the technical problems of effective fixation and stable targeting of alpha nuclides. In existing technologies, alpha nuclides such as 223 Ra, as an alkaline earth metal ion, is difficult to chelate with ligands, leading to unstable binding with carriers and easy detachment in the bloodstream, causing radionuclide leakage and severe toxicity to normal tissues. This invention significantly improves [the effectiveness of Ra ionization] by constructing a dual structure of a molecular protective layer with a self-ignition mechanism and a nano-hydrogel. 223 The fixed stability of Ra reduces the risk of free nuclide release, enabling stable transport of nuclide in the blood and specific release into tumor tissue.

[0011] 2. Solving the technical problem of off-target migration of daughter radionuclides in the alpha radionuclide decay chain. Alpha nuclides, such as 223 Ra decay produces multiple high-energy progeny nuclides, accompanied by a strong recoil effect, which easily leads to binding breakage and off-target diffusion of nuclides, resulting in severe non-specific radiation damage. To address this technical problem, this invention employs a nanocage structure with embedded self-igniting molecules to achieve synchronous and rapid release after triggering the inverse electron demand Diels-Alder (IEDDA) reaction, effectively locking and controlling the progeny nuclides and reducing off-target radioactive effects.

[0012] 3. Solving technical problems related to insufficient targeting efficiency and dependence on EPR effect. Traditional nanodelivery systems largely rely on the passive EPR effect, which is significantly affected by factors such as tumor type and degree of vascularization, leading to unstable tumor-targeting accumulation effects. This invention innovatively combines a biotin receptor (BR) ligand-mediated active targeting mechanism with IEDDA-triggered covalent localization, forming a "dual-lock" strategy that significantly enhances the specific recognition and drug accumulation capabilities of tumor cells, achieving highly efficient and precise targeted therapy.

[0013] 4. Solving the technical problems of premature drug leakage and uncontrollable release in nanomedicine systems. Current drug delivery systems often suffer from premature drug leakage or uncontrolled drug release in the bloodstream, affecting therapeutic efficacy and increasing side effects. This invention utilizes a 2-hydroxy-5-methyl-1,3-benzenedimethanol backbone, linking the drug / targeting group via carbonate. After IEDDA reaction triggering, a 1:1 synchronous drug release is achieved through an electron cascade elimination reaction, strictly controlling the timing and site of drug release and avoiding premature leakage into the bloodstream.

[0014] 5. Solving the technical problem of limited efficacy of single treatment modalities Traditional alpha-isotope therapy primarily relies on radiation killing, which is prone to decreased efficacy due to immunosuppression and anti-radiation mechanisms. This invention combines alpha-isotope therapy with chemotherapy (Pt... IV The combination of release therapy and immunotherapy (activating anti-tumor immunity through ICD) amplifies the therapeutic effect through multiple synergistic mechanisms. It not only directly kills tumor cells but also activates a sustained anti-tumor immune response, improving the durability and thoroughness of treatment and significantly inhibiting tumor growth, metastasis, and recurrence.

[0015] To address the technical problems existing in the prior art, the present invention proposes the following technical solution: This invention proposes a dual-lock target-controlled alpha nuclide self-immolation molecular cage, comprising a pre-targeted delivery system and an active targeted delivery system. The active targeted delivery system includes an alpha nuclide. The pre-targeted delivery system and the active targeted delivery system release the alpha nuclide through a cleavage reaction. By constructing a novel dual-system delivery platform, the pre-targeted delivery system first delivers the alpha nuclide to the tumor site and improves the tumor microenvironment, while the active targeted delivery system firmly immobilizes the alpha nuclide, preventing its detachment during delivery. The alpha nuclide is only released after the active targeted delivery system reaches the tumor site and undergoes a cleavage reaction with the pre-targeted delivery system, thus achieving highly efficient action of the alpha nuclide at the tumor site.

[0016] Preferably, the cleavage reaction is one of the following: reverse electron demand Diels-Alder reaction, enzyme-responsive cleavage reaction, pH-responsive autolysis reaction, and ROS-responsive autolysis reaction.

[0017] Preferably, the pre-targeted delivery system includes a carrier, a prodrug, a modifying group, and a cleavage group; the prodrug is loaded on the carrier; and the cleavage group is connected to the surface of the carrier through the modifying group.

[0018] Preferably, the active targeted delivery system includes a second carrier, a self-cleaving molecule, and an alpha nuclide; the alpha nuclide is loaded on the second carrier; and the self-cleaving molecule is connected to the surface of the second carrier.

[0019] More preferably, both the first carrier and the second carrier are selected from hydrogel nanoparticles, polypeptide hydrogels, polylactic acid-glycolic acid copolymer (PLGA), silk fibroin nanoparticles, graphene oxide functionalized nanomaterials, and liposomes.

[0020] More preferably, the prodrug is a glutathione depleting agent.

[0021] More preferably, the prodrug is one of oxaliplatin, cisplatin, a small molecule compound modified with a disulfide bridging group, or a ROS-generating agent.

[0022] More preferably, the modifying group is one of hyaluronic acid, RGD peptide, EGFR antibody fragment, PD-L1 monoclonal antibody fragment, folic acid, and small molecule targeting ligand.

[0023] More preferably, the alpha nuclide is 223 Ra, 225 Ac, 211 One of the At types.

[0024] In the above technical solution, the pre-targeted delivery system can functionalize the surface of hydrogel nanoparticles and load them with cisplatin (Pt). IV Prodrugs are pre-accumulated locally in tumors to provide lead signals; active targeted delivery systems effectively immobilize alpha nuclides using molecular cage technology, such as... 223 Ra improves the load delivery stability of alpha nuclides and enables accurate identification and active delivery of pre-enriched regions.

[0025] This invention also provides a method for preparing a dual-lock target-controlled alpha nuclide self-ignition molecular cage, wherein... The preparation method of the pre-targeted delivery system is as follows: Step 1.1: Preparation of carrier one; Step 1.2: Load the prodrug onto the carrier 1 prepared in step 1.1 to prepare a pre-targeted nanosystem precursor; Step 1.3: Couple the modified group with the cleavage group to the surface of the pre-targeted nanosystem precursor prepared in Step 1.2 to prepare the pre-targeted delivery system; The preparation method of the active targeted delivery system is as follows: Step 2.1: Preparation of carrier two; Step 2.2: Load the alpha nuclide onto the carrier two prepared in step 2.1 to prepare the precursor of the active targeting nanosystem; Step 2.3: Preparation of self-cleaving molecules; Step 2.4: The self-cleaving molecule prepared in step 2.3 is reacted and linked with the active targeting nanosystem precursor prepared in step 2.2 to prepare an active targeting delivery system.

[0026] The present invention also provides the application of this drug delivery system in the preparation of radiotargeted drugs for tumors.

[0027] Compared with existing alpha-alpha nuclide delivery technologies (such as single nanoparticle loading, uncontrolled release systems, and conventional surface modification techniques), this invention constructs a novel dual-system delivery platform (a pre-targeting system HAQ@HNPs and an active targeting system). 223 Ra@HNPs), combined with nanogels and self-destructive molecular design, brings the following significant advantages: 1. Improved 223 Ra delivery's targeting and precise release efficiency By pre-regulating the tumor microenvironment (such as reducing intratumoral GSH levels using pre-targeting systems), subsequent [treatment / treatment] was improved. 223 Conditions for Ra release; self-cleavage molecules achieve specific activation and release, avoiding 223 Premature leakage of Ra in the bloodstream or normal tissues significantly improves the effectiveness of targeting tumor tissues.

[0028] 2. Significantly enhanced 223 Stability and security of Ra load systems Using nano-hydrogels as a carrier, combined with the dual encapsulation of self-destructive molecules, effectively prevents... 223 Uncontrolled leakage of Ra reduces the risk of radiation damage to normal tissues; its in vitro stability has been verified. 223 Ra@HNPs maintained >95% radiochemical purity (RCP) within 24 hours under PBS and 10% serum conditions, outperforming the stability of traditional directly loaded nanomaterials.

[0029] 3. The preparation process has been simplified and controllability and repeatability have been improved. The preparation process of the nano-hydrogel is mild (oil-in-water emulsion system + UV crosslinking), which can be mass-produced and the process parameters are controllable, with good particle size uniformity. The self-cleaving molecular design is based on the highly efficient IEDDA reaction, which has the advantages of fast reaction rate and mild reaction conditions, thus improving the consistency and reproducibility of the entire preparation process.

[0030] 4. Reduced the risks associated with radioactive handling and improved the safety of research and clinical translation. Using Ba 2+ As 223 Ra 2+The simulants effectively reduce the risk of radiation exposure for experimenters during physicochemical characterization; while ensuring experimental feasibility, they significantly improve the safety of early-stage research and development, which is beneficial for subsequent scale-up production and preclinical studies.

[0031] 5. It achieves multiple targeting and programmed release, improving treatment efficiency and reducing side effects. The pre-targeting particles actively target tumor cells through HA modification, increasing the initial enrichment rate; subsequently, they are efficiently released locally into the tumor via programmed release of self-cleaving molecules. 223 Ra achieves a local dose amplification effect, significantly improving treatment efficacy and reducing systemic side effects. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the characterization structure of the pre-targeted particles HAQ@HNPs.

[0033] in: Figure 1 a is Pt Ⅳ The 1H-NMR spectrum; Figure 1 b represents HNPs-COOH and Pt Ⅳ @HNPs and HAQ@Pt Ⅳ DLS results for @MPNs; Figure 1 c represents HNPs-COOH and Pt. Ⅳ @HNPs and HAQ@Pt Ⅳ Zeta potentials of @MPNs; Figure 1 d represents the infrared spectra of BCN, HA, and HAQ; Figure 1 e represents the 1H-NMR spectra of HA and HAQ; Figure 1 f represents HAQ and Pt Ⅳ HNPs-COOH, Pt Ⅳ @HNPs and HAQ@Pt Ⅳ The UV-Vis absorption spectrum of @MPNs; Figure 1 g represents HNPs-COOH and Pt Ⅳ @HNPs and HAQ@Pt Ⅳ TEM images of @MPNs; data in the attached figures are mean ± SD.

[0034] Figure 2 This is a schematic diagram of the synthetic route for self-cleaving molecules.

[0035] Figure 3 For active targeting particles 223 Characterization diagram of Ra@HNPs.

[0036] in: Figure 3 a represents the DLS result of Ba@HNPs; Figure 3b represents the Zeta potential of HNPs-NH2, Ba-HNPs, and Ba@HNPs; Figure 3 c is a TEM image of Ba@HNPs; Figure 3 d is the TEM image of Ba@HNPs; Figure 3 e is the TEM elemental spectrum of Ba@HNPs; Figure 3 f represents the UV-Vis absorption spectra of HNPs-NH2, SF molecules, Ba@HNPs, and Ba@HNPs; Figure 3 g represents the UV-Vis absorption spectra of BCN, SF molecules, and mixtures (BCN and SF molecules); Figure 3 h represents the ESI-MS analysis result of SF molecules after the IEEDA reaction; Figure 3 i represents the DLS results of Ba@HNPs in different culture media over 7 days; Figure 3 j is 223 Radiolabeling stability of Ra@HNPs in different culture media at 6 h, 12 h, and 24 h (n = 3). Data are mean ± SD.

[0037] Figure 4 This is a schematic diagram of the in vitro cytotoxicity analysis of HAQ / ²²³Ra@HNPs.

[0038] in: Figure 4 a represents the relative survival rate of B16 / F10 cells after 24 hours of different treatments (n = 4). Figure 4 b to Figure 4 d represents the cell cycle distribution analysis of B16 / F10 cells after different treatments; Figure 4 e and Figure 4 f represents the apoptosis assessment of B16 / F10 cells after different treatments (n = 3). Figure 4 g and Figure 4 h was analyzed by flow cytometry (FCM) of B16 / F10 cells stained with DCFH-DA to reveal the intracellular ROS generation under different treatments (n = 3). Figure 4 i. Glutathione (GSH) levels in B16 / F10 cells after different treatments (n = 3); Figure 4 j represents biotransmission electron microscopy (Bio-TEM) images of primary B16 / F10 cells (control group) and B16 / F10 cells after 24 hours of different treatments. Yellow arrows indicate normal endoplasmic reticulum (ER), and red arrows indicate ER under stress. Data are expressed as mean ± standard deviation, *p < 0.5, **p < 0.01, ***p < 0.001.

[0039] Figure 5This diagram illustrates the targeted precursor study and anti-tumor efficacy evaluation of HAQ / ²²³Ra@HNPs.

[0040] in: Figure 5 a and Figure 5 b shows the biodistribution of free ²²³Ra, ²²³Ra@HNPs, and HAQ / ²²³Ra@HNPs in mice 2 hours and 24 hours after intravenous injection (n = 3). Figure 5 c shows representative bioluminescence images of B16 / F10 tumor-bearing mice at 6 and 24 hours after intravenous injection of HAQ / cy7-²²³Ra@HNPs; Figure 5 d. Ex vivo images of tumors taken out on day 10 in each group (n = 5); Figure 5 e represents the final tumor ex vivo mass statistics (n = 5); Figure 5 f to Figure 5 k represents the individual tumor growth curves of B16 / F10 tumor-bearing mice in each treatment group (n = 5). Figure 5 l represents the change in body weight of mice in each group (n = 5); Figure 5 m represents representative H&E staining pathological images and TUNEL immunohistochemical analysis of tumor tissues after treatment in each group; data are expressed as mean ± standard deviation; *p < 0.5, **p < 0.01, ***p < 0.001; G1: control group; G2: Pt Ⅳ ; G3: free²²³Ra; G4: HAQ@HNPs; G5: ²²³Ra@HNPs; G6: HAQ / ²²³Ra@HNPs.

[0041] Figure 6 This is a schematic diagram illustrating the study of endoplasmic reticulum stress and in vivo immune response induced by HAQ / ²²³Ra@HNPs.

[0042] Figure a shows a heatmap of differentially expressed genes (DEGs) between the control group and the HAQ / ²²³Ra@HNPs treatment group (n = 3); Figure b shows Gene Ontology (GO) enrichment analysis; Figure c shows the GSEA analysis results of the gene set "Innate Immune Escape and Cell-Specific Immune Response"; Figure d shows representative images of CHOP, CALR, and HMGB1 immunofluorescence staining in tumor tissues under different treatments (n = 3); Figures e to l show the results of flow cytometry analysis: splenic DC maturation (e), tumor DC maturation (f), CD4⁺T cells (g), and CD8⁺T cells (h), and their statistical analysis (il) (n = 3); data are expressed as mean ± standard deviation. *p < 0.5, **p < 0.01, ***p < 0.001.

[0043] Figure 7 This is a schematic diagram illustrating the antitumor efficacy evaluation of HAQ / ²²³Ra@HNPs combined with anti-PD-L1 antibody in a metastatic tumor model.

[0044] in: Figure 7 a represents the in vivo administration regimen for combination therapy; Figure 7 b and Figure 7 c shows photographs (b) and quality statistics (c) of primary and distal tumors removed from mice in each treatment group 16 days after treatment (n = 5 per group); G1: Control group; G2: Anti-PD-L1; G3: HAQ@HNPs; G4: HAQ@HNPs + Anti-PD-L1; G5: ²²³Ra@HNPs; G6: ²²³Ra@HNPs + Anti-PD-L1; G7: HAQ / ²²³Ra@HNPs; G8: HAQ / ²²³Ra@HNPs + Anti-PD-L1; Figure 7 d represents the change in mouse body weight after treatment in each group (n = 5); Figure 7 e to Figure 7 m represents the mean tumor growth curve (e) and individual tumor growth curve (fm) for each group of mice (n = 5); np) Flow cytometry analysis results: effector memory T cells (TEM) in the spleen (n), and representative images of CD4⁺ T cells and CD8⁺ T cells in distal tumors of each group (n = 3). Data are expressed as mean ± standard deviation. *p < 0.5, **p < 0.01, ***p < 0.001. Detailed Implementation

[0045] Existing targets 223 While Ra delivery technology has improved the stability and targeting of radionuclide therapy to some extent, it still has the following major drawbacks: 1) Insufficient radionuclide immobilization: Traditional carriers such as hydroxyapatite nanoparticles and liposomes... 223 Ra has limited binding force, and the high-energy recoil effect of alpha particles can easily lead to the escape of nuclides, reducing the therapeutic effect and potentially causing off-target radiation damage.

[0046] 2) Low tumor targeting efficiency: Most vectors mainly rely on passive targeting (EPR effect), which results in low accumulation efficiency in tumor tissues with strong heterogeneity and obvious vascular barriers, thus limiting the therapeutic effect.

[0047] 3) Insufficient biodegradability and biosafety: Some inorganic carriers are difficult to completely degrade and eliminate in the body, and long-term retention may trigger immune responses or chronic toxicity risks.

[0048] 4) Complex preparation and high cost: Some existing delivery systems have complicated preparation processes involving multiple modifications or complex components, resulting in high production costs and limited large-scale applications.

[0049] 5) Poor adaptability: Existing systems are mostly applied to bone metastases, lacking versatility and adaptability for treating other types of solid tumors, thus limiting their application. 223 Ra has the potential for wide-ranging applications.

[0050] The present invention aims to achieve the following technical objectives: 1) By employing a pre-targeting-active targeting two-step delivery strategy, significantly improving... 223 Ra's cumulative amount in tumor tissue and its therapeutic specificity break through the limitations of traditional single passive delivery systems.

[0051] 2) Utilizing molecular cage loading technology to effectively enhance 223 The immobilization force of Ra in the carrier reduces the release of nuclides caused by alpha particle recoil, thus lowering the risk of off-target radiation.

[0052] 3) Achieving alpha-nucleoside targeted internal radiation therapy ( 223 Ra works synergistically with chemotherapy (cisplatin prodrug) to enhance anti-tumor effects and improve treatment outcomes.

[0053] 4) The use of a hydrogel matrix and biodegradable design ensures that the delivery system can be degraded and eliminated in vivo, thus improving biosafety.

[0054] 5) Optimize the preparation process to ensure that the system has high stability, good reproducibility and industrial application potential.

[0055] This invention improves 223 Ra has significant advantages in terms of targeted delivery efficiency, safety, and overall therapeutic effect, and is expected to provide a new and effective treatment strategy for solid tumors. Example 1

[0056] This embodiment discloses a dual-target-controlled alpha nuclide self-immolation molecular cage and its preparation method. The dual-target-controlled alpha nuclide self-immolation molecular cage includes a pre-targeted delivery system (HAQ@HNPs) and an active targeted delivery system (HAQ@HNPs). 223 Ra@HNPs) improves the effectiveness and safety of tumor treatment by precisely regulating the tumor microenvironment and achieving efficient localized release of alpha nuclides. The structure, working principle, and operational relationships of this invention are described in detail below with reference to the accompanying drawings.

[0057] 1.1 Structure and fabrication method of pre-targeted delivery system The pre-targeted delivery system in this embodiment mainly consists of pre-targeted particles, which are hydrogel nanoparticles (HNPs) as the core carrier, loaded with cisplatin (Pt). IV Furthermore, the surface of the hydrogel nanoparticles was functionalized to obtain pre-targeted particles (HAQ@HNPs).

[0058] The specific preparation method of the pre-targeted particles is as follows: 1) Preparation of hydrogel nanoparticles First, polyethylene glycol diacrylate (PEGDA, molecular weight MW 400–600) was used to form an oil-in-water nanoemulsion system with the assistance of a surfactant. Then, under nitrogen protection, the nanoemulsion system was irradiated with UV light (365 nm) and crosslinked in the presence of the photoinitiator 2-hydroxy-2-methylpropiophenone. The reaction was stirred for 3 hours. The resulting blank HNPs were collected by centrifugation (12000 rpm), washed with ethanol and water, and dried to prepare hydrogel nanoparticles (HNPs) with uniform particle size. See [link to previous section]. Figure 1 g.

[0059] It should be noted that, in addition to using hydrogel nanoparticles (HNPs) as carriers, other biocompatible nanomaterials, such as peptide hydrogels, polylactic acid-glycolic acid copolymers (PLGA), silk fibroin nanoparticles, graphene oxide (GO) functionalized nanomaterials, or liposome systems, can all serve as nanodelivery platforms for ²²³Ra loading, achieving similar stable encapsulation and controllable release effects.

[0060] 2) Preparation of pre-targeted nanoparticle precursors Then, through electrostatic adsorption and ester bond linkage, co-incubation was performed in the dark for 4 hours. After the reaction, the product was collected by centrifugation and freeze-dried to achieve the transfer of the prodrug cisplatin (Pt) into the product. IV ) loaded onto the surface of hydrogel nanoparticles (HNPs) to form pre-targeted nanoparticle precursors (Pt) Ⅳ @HNPs), Cisplatin (Pt) IV It can achieve the depletion of glutathione (GSH) in tumors and the pre-regulation of the tumor microenvironment.

[0061] In this step, cisplatin (Pt) IV The preparation method of cisplatin (Pt) involves mixing cisplatin with 30% hydrogen peroxide and stirring the mixture at 60°C for 6 hours. After the reaction, the clarified solution is freeze-dried to obtain a white powder. The obtained white powder is dissolved in dimethyl sulfoxide (DMSO) containing excess succinic anhydride and reacted at room temperature for 12 hours. After the reaction, the clarified solution is freeze-dried again. The obtained powder is then ground to obtain the white solid product cisplatin (Pt). IV ).

[0062] See Figure 1 a, through 1 The characteristic peaks at δ 8.37–8.14 in the ¹H-NMR spectrum confirmed the presence of cisplatin (Pt). IV The amino group in ).

[0063] See Figure 1 b. DLS measurements show that the loaded cisplatin (Pt) IV The particle size increases by about 20 nm after processing.

[0064] See Figure 1 c, the Zeta potential decreased to -14.81 mV.

[0065] See Figure 1 g-2, the pre-targeted particles have a similar particle size to the hydrogel nanoparticles, indicating that cisplatin (Pt) IV It was successfully loaded onto hydrogel nanoparticles.

[0066] 3) Preparation of pre-targeted nanoparticles Finally, hyaluronic acid (HAQ, cleavage group) with a cyclopropane-cyclooctynyl group (BCN group, modification group) was coupled to the pre-targeted particle precursor (Pt) via an esterification reaction promoted by 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC). Ⅳ The surface of the pre-targeted particles (@HNPs) was stirred and reacted overnight under argon protection at room temperature. After the reaction, the particles were collected by centrifugation, washed, and dried to form the final pre-targeted particles (HAQ@HNPs).

[0067] See Figure 1 d, 2127 cm⁻¹ appears in the FTIR spectrum -1 The characteristic peak of C≡C is located at this point.

[0068] See Figure 1 e, 1 The characteristic peaks of the BCN group also appeared in the H-NMR spectrum.

[0069] Both confirm the successful synthesis of pre-targeted particles (HAQ@HNPs).

[0070] See Figure 1 TEM images of g-3 show that the pre-targeted particles (HAQ@HNPs) are uniformly spherical with a particle size of about 300 nm, slightly smaller than the 350 nm measured by DLS, which is attributed to the adsorption effect of the hydrogel system.

[0071] See Figure 1 f. In the UV-Vis absorption spectrum, the redshift absorption peak at 292 nm of the pre-targeted particles (HAQ@HNPs) further verifies the successful modification of HAQ.

[0072] Therefore, by using pre-targeted particle precursors (Pt) Ⅳ Modifying hyaluronic acid (HAQ) with cyclopropane-cyclooctylene (BCN) groups onto @HNPs yields pre-targeting particles (HAQ@HNPs), thereby achieving active targeting of tumor tissues, while simultaneously utilizing cisplatin (Pt) IV The reduction reaction of α reduces the level of glutathione (GSH) in the tumor, creating a favorable environment for the subsequent release of alpha nuclides.

[0073] This invention uses hyaluronic acid (HAQ) as a surface-targeting modifier, which can also be replaced with RGD peptides (for angiogenesis), antibody fragments (such as anti-EGFR, PD-L1 monoclonal antibody fragments), folic acid, or small molecule targeting ligands (such as glycosyl ligands) to achieve active targeted enrichment of different tumor types.

[0074] 1.2 Structure and preparation method of active targeting system The active targeted delivery system in this embodiment is mainly composed of active targeted particles, which are based on hydrogel nanoparticles (HNPs) as the core carrier. They are loaded with alpha nuclides and connected to self-cleaving molecules through amide bonds to finally obtain active targeted particles.

[0075] Therefore, the targeted delivery system consists of two main modules: a self-immolating molecule and nanoparticles loaded with alpha nuclides, which communicate to form the active targeting nanoparticles. Thus, the core of the drug delivery system in this application lies in triggering the self-immolation of the self-immolating molecule through the inverse electron demand Diels-Alder reaction (IEEDA reaction), thereby achieving the precise release of the alpha nuclide.

[0076] The specific preparation method of the active targeting particles is as follows: 1) Preparation of hydrogel nanoparticles This step is the same as the method for preparing hydrogel nanoparticles in 1.1, and will not be repeated here.

[0077] 2) Preparation of active targeting particle precursors Due to the special properties of radioactive elements, barium is used to simulate alpha nuclides in the preparation process; in this embodiment, Ba is used. 2+ Ion simulation 223 Ra 2+ To reduce the risk of radiation, Sr²⁺ (strontium ions) and Ca²⁺ (calcium ions) can also be used as alternative simulated ions, as they belong to the alkaline earth metal group and have similar physicochemical properties, allowing for partial experimental substitution and thus reducing the radiation risk in the early stages of development.

[0078] In the preparation process, Ba²⁺ and hydrogel nanoparticles were co-incubated in the dark for 4 hours to prepare active targeting particle precursors (Ba-HNPs). 3) Preparation of self-cleaving molecules (SF) See Figure 2 The self-cleavage molecule is synthesized from 2,6-dihydroxymethyl-p-cresol in 7 organic steps. Figure 2 It is prepared by the following method: Synthesis of compound 2: Imidazole and tert-butylchlorodimethylsilane (TBSCl) were dissolved in anhydrous dichloromethane and added to anhydrous dimethylformamide (DMF) solution containing 2,6-dihydroxymethyl-p-cresol. The reaction was carried out at room temperature for 4 hours. After the reaction was completed, the reaction solution was washed with water and saturated sodium chloride solution in sequence, dried and purified by column chromatography to obtain an oily compound 2 with a yield of more than 90%.

[0079] 1 H NMR (400 MHz, CDCl3) δ: 8.05 (s, 1H), 6.93 (s, 2H), 4.85 (s, 4H), 2.28 (s, 3H), 0.97 (s, 18H), 0.15 (s, 12H); 13 C NMR (400 MHz, CDCl3) δ: 151.06, 128.42, 126.33, 125.92, 63.17, 26.04, 20.82, 18.46, -5.26; ESI-MS: m / z [MH]⁻ Calculated value is C 21 H 39 O3Si2: 395.2438, measured value is 395.2442.

[0080] Synthesis of Compound 3: Compound 2 and DIPEA were dissolved in anhydrous dichloromethane, and a solution of p-nitrophenyl chloride carbonate in dichloromethane was added dropwise at 0°C. The reaction mixture was heated to room temperature and maintained for 4 hours, then washed successively with water and saturated sodium chloride solution, dried, and purified by column chromatography to obtain a white solid compound 3 (yield >90%).

[0081] 1 H NMR (400 MHz, CDCl3) δ: 8.31 (d, J=9.2 Hz, 2H), 7.48 (d, J=9.1 Hz, 2H), 7.22 (s, 2H), 4.72 (s, 4H), 2.38 (s, 3H), 0.93 (s, 18H), 0.09 (s, 12H); 13C NMR δ: 155.73, 150.26, 145.63, 143.48, 136.80, 132.86, 128.11, 125.52, 121.57, 60.73, 31.66, 30.36, 29.84, 26.07, 21.34, 18.59; ESI-MS: m / z [M+H]⁺ Calculated value is C 28 H 44 NO7Si2: 562.2656, measured value is 562.2641.

[0082] Synthesis of compound 4: BCN and TEA were slowly injected into an anhydrous DMF solution of compound 3, and the reaction was carried out at room temperature for 12 hours. The reaction solution was diluted with ethyl acetate, washed successively with water and saturated sodium chloride solution, dried, and purified by column chromatography (CH2Cl2 / CH3OH = 20:1) to obtain compound 4.

[0083] 1 H NMR (400 MHz, CDCl3) δ: 8.59 (d, J=8.1 Hz, 2H), 7.56 (d, J=8.1 Hz, 2H), 7.21 (s, 2H), 5.52 (t, 1H), 4.66 (s, 4H), 4.57 (d, J=6.3 Hz, 2H), 3.11 (s, 3H), 2.35 (s, 3H), 0.93 (s, 18H), 0.08 (s, 12H); 13 C NMR δ: 167.46, 163.97, 162.15, 154.42, 143.21, 135.84, 131.36, 128.52, 12 7.15, 115.71, 77.37 (CDCl3), 60.25, 45.15, 26.09, 25.78, 21.44, 18.58; ESI-MS: m / z [M+H]⁺ Calculated value is C 32 H 50 N5O4Si2: 624.3401, measured value is 624.3398.

[0084] Synthesis of compound 5: Compound 4 was co-dissolved with p-toluenesulfonic acid in methanol and reacted at room temperature for 4 hours. After the solvent was removed by rotary evaporation, the mixture was diluted with ethyl acetate, washed successively with water and sodium chloride solution, dried, and purified by column chromatography (CH2Cl2 / CH3OH = 20:1) to obtain compound 5.

[0085] 1H NMR (400 MHz, DMSO-d6) δ: 8.47 (d, J=8.0 Hz, 2H), 7.60 (d, J=8.0 Hz, 2H), 7.17 (s, 2H), 5.12 (d, J=5.6 Hz, 1H), 4.41 (t, J=5.5 Hz, 8H), 3.00 (s, 3H), 2.31 (s,3H); 13 C NMR δ: 167.06, 163.17, 154.47, 144.24, 142.53, 134.61, 130.54, 127.74, 126.10, 57.72, 54.86, 48.58, 43.76, 20.81; ESI-MS: m / z [M-H2O+H]⁺ Calculated value is C 20 H 20 N5O3: 378.1566, measured value is 378.1562.

[0086] Synthesis of compound 6: Compound 5 was dissolved in pyridine, DMAP and succinic anhydride were added, and the reaction was carried out overnight under argon protection. After the reaction, the mixture was diluted with ethyl acetate and washed three times with saturated sodium chloride solution. The organic layer was dried over anhydrous Na2SO4 and purified by column chromatography (CH2Cl2 / CH3OH = 15:1) to give compound 6 as a red powder (yield >80%).

[0087] 1 H NMR (400 MHz, DMSO-d6) δ: 8.46 (d, J=8.0 Hz, 2H), 7.60 (d, J=8.0 Hz, 2H), 7.28 (d, J=2.3 Hz, 1H), 7.11 (d, J=2.3 Hz, 1H), 5.20 (m, 1H), 4.99 (s, 2H), 4.24–4.57 (m, 5H), 3.00 (s, 3H), 2.41–2.49 (m, 4H), 2.31 (s, 3H); 13 C NMR δ: 172.00, 167.07, 163.17, 154.34, 144.08, 135.45, 134.53, 130.57, 128.05, 127.87, 60.99, 57.61, 43.79, 28.89, 20.81; ESI-MS: m / z [M+H]⁺ Calculated value is C 24 H 26 N5O7: 496.1832, measured value is 496.1810.

[0088] Synthesis of Compound 7: Compound 6 and DIPEA were dissolved in anhydrous dichloromethane, and a solution of p-nitrophenyl chloride carbonate was added dropwise at 0 °C. The mixture was heated to room temperature and the reaction was continued for 4 hours. The product was washed, dried, and purified by column chromatography to give a red solid compound 7 (yield >90%), which is the self-cleaving molecule.

[0089] 1 H NMR (400 MHz, CDCl3) δ: 8.54, 8.16, 7.50, 7.16–7.30, 6.01, 5.13, 4.54, 4.49, 3.09, 2.89, 2.71, 2.33; 13 C NMR δ: 171.75, 167.47, 163.93, 155.56, 145.50, 145.35, 142.74, 136.58, 134.23, 131.74, 128.46, 125.27, 62.64, 45.14, 29.82, 21.28; ESI-MS: m / z [M–CO2+H]⁺ Calculated value is C 30 H 29 N6O9: 617.1996, measured value is 617.1994.

[0090] Synthesis of the self-immolating spacer: Biotin-PEG-NH2 (average molecular weight 1000) and TEA were slowly injected into anhydrous DMF solution of compound 7 and reacted at room temperature for 12 hours. After the solvent was removed by freeze-drying, the solution was diluted with ethyl acetate and collected by column chromatography (CH2Cl2 / CH3OH, gradient from 100:1 to 10:1).

[0091] 4) Preparation of active targeting particles The active targeting particle precursor was linked to a self-cleaving molecule via an amide bond to ultimately prepare complete active targeting particles (Ba@HNPs). See [link to specific structure] for details. Figure 3 ae.

[0092] See Figure 3 d. The TEM image clearly distinguishes between loaded and unloaded Ba. 2+ Nanoparticle morphology.

[0093] See Figure 3 e. Element Mapping also verifies the uniform distribution of Ba elements.

[0094] See Figure 3f. By using UV-Vis spectroscopy, Ba@HNPs showed a blue shift at 260 nm, corresponding to the characteristic absorption of SF molecules, confirming the successful surface modification.

[0095] See Figure 3 i. Particle size stability tests show that Ba@HNPs maintain good stability in different systems for 7 days.

[0096] See Figure 3 In the IEDDA reaction verification experiment, the self-cleaving molecule reacted rapidly with BCN, and the solution color changed from red to colorless within seconds, while the UV absorption peak disappeared. (See [link to relevant documentation]). Figure 3 h, ESI-MS further confirmed the pyrolysis products.

[0097] These data collectively indicate that 223 The key triggering process of Ra release mechanism (IEDDA reaction + autolysis) can be carried out efficiently in vivo.

[0098] Finally, it was introduced through a similar method. 223 Ra, prepared 223 Ra@HNPs were used, achieving a labeling rate of 81.44±1.36% and a purity >95%. Radioactivity stability tests showed that... 223 Ra@HNPs maintained high stability in PBS and 10% FBS for 24 hours. (See also...) Figure 3 j; ensured its activity during treatment. Example 2

[0099] This embodiment evaluates the in vitro cytotoxicity of HAQ / ²²³Ra@HNPs.

[0100] To evaluate the cytotoxic effects of HAQ / ²²³Ra@HNPs, we used a CCK-8 assay to detect the cytotoxicity of a nanoplatform containing HAQ@HNPs (pre-targeted delivery system) and ²²³Ra@HNPs (active targeted delivery system) in melanoma B16 / F10 cells and compared the Pt values. IV Changes in cell viability after 24 hours of dose gradient treatment with different treatment methods, including free Ra, HAQ@HNPs, Ra@HNPs, and HAQ / Ra@HNPs. Figure 4 As shown in Figure a, with a Ra loading of 0.025 µCi / mL and Pt... IV At a concentration of 0.6 µg / mL, after 24 hours of treatment with HAQ / ²²³Ra@HNPs, the survival rate of B16 / F10 cells was approximately 50%, demonstrating a significant therapeutic effect. Based on the dose-response relationship, this concentration was selected for subsequent in vitro mechanism experiments.

[0101] Subsequently, flow cytometry (FCM) was used to analyze the cell cycle distribution in each group. Figure 4 b to Figure 4 d). In the control group, cells were mainly distributed in the G0 / G1 phase (46.4%). However, in cells treated with free ²²³Ra, HAQ@HNPs, or ²²³Ra@HNPs, the proportion in the G0 / G1 phase decreased, while the proportion in the G2 / M phase increased. After treatment with HAQ / ²²³Ra@HNPs, the G0 / G1 phase significantly decreased to 20.6%, and the G2 / M phase significantly increased to 42.1%, suggesting that it can induce cell cycle arrest at the G2 / M checkpoint, thereby inhibiting cell proliferation.

[0102] To assess whether the treatment induced apoptosis, Annexin V / PI double staining combined with FCM analysis was further performed. Figure 4 e, Figure 4 f). The results showed that the proportion of apoptotic cells in the HAQ / ²²³Ra@HNPs group was significantly higher than that in the other treatment groups, confirming that it has the effect of inducing apoptosis.

[0103] To further elucidate its underlying mechanism, intracellular reactive oxygen species (ROS) levels were detected by DCFH-DA fluorescent probe staining combined with FCM. Figure 4 g, Figure 4 h). The results showed that HAQ / ²²³Ra@HNPs treatment significantly promoted ROS generation, suggesting that oxidative stress plays an important role in its tumor-killing effect. Considering that glutathione (GSH) is an important regulator of cellular redox homeostasis, we measured the intracellular GSH levels in cells from different treatment groups. Figure 4 As shown in i, HAQ / ²²³Ra@HNPs significantly depleted intracellular GSH levels, further supporting the involvement of ROS-mediated oxidative stress in its cytotoxic effects and potentially inducing endoplasmic reticulum (ER) stress responses.

[0104] To investigate whether ER stress occurred, transmission electron microscopy (TEM) was used to observe changes in cell ultrastructure. The results are as follows: Figure 4 As shown in Figure j, compared with the structurally intact normal ER in the control group (yellow arrow), the cells in the HAQ / ²²³Ra@HNPs treatment group showed obvious typical stress phenotypes such as ER swelling, expansion, vacuolation and degranulation (red arrow), suggesting that it can induce a significant ER stress response.

[0105] In summary, HAQ / ²²³Ra@HNPs exhibit significant in vitro cytotoxicity against B16 / F10 cells. Through a synergistic effect of multiple mechanisms, including inducing cell cycle arrest, promoting apoptosis, oxidative stress, and endoplasmic reticulum stress, they demonstrate promising potential for synergistic treatment with TAT (targeted alpha therapy) and chemotherapy. Example 3

[0106] This embodiment evaluates the pre-targeting study and anti-tumor effects of HAQ / ²²³Ra@HNPs.

[0107] To systematically evaluate the pre-targeting potential and in vivo therapeutic effects of HAQ / ²²³Ra@HNPs, we conducted a series of animal experiments in a B16 / F10 tumor-bearing mouse model.

[0108] First, the distribution of radioactivity in tumors and major organs at different time points (2 hours and 24 hours after injection) was analyzed using a gamma counter to evaluate the in vivo biodistribution of the radionuclide. Figure 5 a, Figure 5 (b) The results showed that free ²²³Ra was rapidly cleared in vivo, mainly accumulating in bone tissue in the early stages, consistent with its osteotropic biological characteristics. In contrast, HAQ / ²²³Ra@HNPs showed a significantly prolonged retention time in tumor tissue and extremely low distribution in normal organs.

[0109] In addition, to further track the in vivo behavior of nanomedicines, we used the fluorescently labeled analogue HAQ / Cy7-²²³Ra@HNPs for intravenous injection and monitored its in vivo fluorescence distribution. Figure 5 c). Its enhanced tumor enrichment capacity can be attributed to a carefully designed dual-locking mechanism, sequentially achieving primary targeting based on BR ligand recognition and secondary targeting through synergistic covalent linkage. After HAQ@HNPs are first injected into tumor tissue, the systemically infused ²²³Ra@HNPs react specifically with the tumor through bioorthogonal click chemistry, thereby inducing the dissociation of self-destructive linker groups and precisely releasing ²²³Ra and Pt. Ⅳ This mechanism enables synergistic treatment with drugs. It ensures the spatially localized release of therapeutic drugs within the tumor, thereby enhancing the combined effects of targeted alpha therapy (TAT) and chemotherapy while minimizing systemic toxicity.

[0110] Based on the above advantages, we further evaluated the in vivo antitumor therapeutic effect of HAQ / ²²³Ra@HNPs. Tumor-bearing mice were randomly divided into seven groups, receiving PBS (control group), Pt, and other treatments respectively. Ⅳ Different treatments were administered, including free ²²³Ra, HAQ@HNPs, ²²³Ra@HNPs, and HAQ / ²²³Ra@HNPs. The treatment cycle was 10 days, after which mice were sacrificed and tumor tissue was dissected and weighed for analysis. Figure 5 d, Figure 5 e). Compared with the control group, the tumor weight of the HAQ / ²²³Ra@HNPs treatment group was significantly reduced, showing a superior tumor-suppressing effect compared with other groups. The tumor growth curve recording results of each group also showed that tumor growth in the HAQ / ²²³Ra@HNPs treatment group was significantly inhibited ( Figure 5 f to Figure 5 (k), which is highly related to its long-term retention and sustained release characteristics at the tumor site.

[0111] The body weight of mice in all treatment groups did not fluctuate significantly throughout the experiment, suggesting that HAQ / ²²³Ra@HNPs have low systemic toxicity. Figure 5 To explore the potential mechanism of its tumor-suppressing effect, histopathological analysis of tumor tissue was performed, including H&E staining and TUNEL apoptosis detection (l). Figure 5 H&E staining results showed that the tumor cells in the control group and other treatment groups had relatively intact structures, while the tumor tissue in the HAQ / ²²³Ra@HNPs treatment group showed obvious necrotic areas, accompanied by morphological changes such as nuclear condensation and cell shrinkage. TUNEL staining further confirmed that the apoptosis level in the tumor tissue of the HAQ / ²²³Ra@HNPs treatment group was significantly increased, suggesting that it exerts its anti-tumor effect by inducing apoptosis and / or necrosis.

[0112] In summary, HAQ / ²²³Ra@HNPs not only exhibit excellent tumor targeting ability and prolonged tumor retention time, but also effectively inhibit tumor growth through apoptosis-mediated cytotoxicity mechanism, showing promising potential for synergistic treatment with TAT and chemotherapy. Example 4

[0113] This embodiment studies the endoplasmic reticulum stress response and immune activation induced in vivo by HAQ / ²²³Ra@HNPs.

[0114] To further explore the molecular mechanism behind the antitumor effect mediated by HAQ / ²²³Ra@HNPs, mice were sacrificed 7 days after treatment, and RNA sequencing (RNA-Seq) analysis was performed on their tumor tissues. Transcriptome analysis of the tumor tissues after treatment revealed the gene expression changes induced by HAQ / ²²³Ra@HNPs. Differentially expressed genes (DEGs) between the control group and the HAQ / ²²³Ra@HNPs-treated group were visualized using hierarchical clustering. Figure 6 (a) The results showed significant differences in gene expression between the two groups, suggesting that this treatment strategy could trigger profound changes in transcriptional regulation.

[0115] Subsequently, a Gene Ontology (GO) enrichment analysis was performed to elucidate the biological pathways affected by the treatment. Figure 6 b). The analysis results showed that pathways related to calcium-dependent protein binding, apoptosis regulation, inflammatory response regulation, and immune activation were significantly enriched, indicating that HAQ / ²²³Ra@HNPs exert their therapeutic effect through inducing cellular stress and immune regulation mechanisms.

[0116] To further explore its immune activation potential, gene set enrichment analysis (GSEA) was used to assess immune-related pathways. The results showed that, compared with the control group, the HAQ / ²²³Ra@HNPs treatment group had significant enrichment in gene sets related to "innate immune escape and cell-specific immune response" (…). Figure 6 (c) This further verifies that the nanoplatform can effectively enhance the tumor immune response.

[0117] Given the crucial role of endoplasmic reticulum (ER) stress in immunogenic cell death (ICD), we performed immunofluorescence staining analysis on key ER stress biomarkers. Figure 6 d). Under ER stress, the molecular chaperone BIP (Binding Immunoglobulin Protein) binds to unfolded proteins and simultaneously induces upregulation of the C / EBP homolog CHOP, serving as a classic marker of ER stress. During this process, calreticulin (CALR) everts to the cell membrane surface, acting as an "eat me" signal to the immune system. Significant green fluorescence signals were observed in all treatment groups, indicating that HAQ / ²²³Ra@HNPs successfully induced CALR exposure on the tumor cell membrane surface. Furthermore, the expression level of high-mobility group box 1 (HMGB1) was significantly increased in the HAQ / ²²³Ra@HNPs treatment group, further demonstrating that treatment induced ER stress and ICD, thereby activating tumor-associated immune responses.

[0118] To verify its ability to promote dendritic cell (DC) maturation and tumor-infiltrating lymphocyte accumulation in vivo, we performed flow cytometry analysis. Figure 6 The results showed that HAQ / ²²³Ra@HNPs treatment significantly promoted spleen (el). Figure 6 e, i) and tumor tissue ( Figure 6 The increased proportion of mature dendritic cells (DCs) in f and j indicates enhanced antigen-presenting capacity. Simultaneously, the presence of CD4⁺ T cells (CD4⁺ T cells) in tumor tissue... Figure 6 g, k) and CD8⁺ T cells ( Figure 6 The significant increase in h and l numbers suggests enhanced adaptive immune responses at the tumor site. These results collectively indicate that HAQ / ²²³Ra@HNPs can not only induce a strong endoplasmic reticulum stress response but also activate the adaptive immune system, thereby further enhancing its anti-tumor therapeutic effect. Example 5

[0119] This embodiment is an anti-tumor evaluation of a HAQ / ²²³Ra@HNPs combined with anti-PD-L1 therapy in a metastatic tumor model.

[0120] The aforementioned research results have confirmed that HAQ / ²²³Ra@HNPs can effectively enhance tumor immunogenicity. To further evaluate its potential to stimulate systemic anti-tumor immunity and its effect in controlling tumor metastasis when combined with immune checkpoint inhibitor (ICB) therapy, a mouse model of distant metastatic tumors was established. Figure 7 a). After different treatment regimens were administered, the tumor was dissected and weighed on day 16. Figure 7 b, Figure 7 c). The results showed that the "HAQ / ²²³Ra@HNPs + anti-PD-L1 antibody" combination therapy group exhibited significant inhibitory effects on both primary tumors and distant metastases, with therapeutic efficacy superior to any single-agent therapy, clearly demonstrating the synergistic advantage of the combination strategy in controlling tumor metastasis.

[0121] Tumor growth curves in each treatment group were also monitored. Figure 7 d to Figure 7 The combination therapy group demonstrated sustained and potent tumor suppression, while the single therapy group showed only moderate tumor suppression. Weight monitoring results showed no significant changes in body weight among the groups. Figure 7 The m) indicates that the combination therapy has good biocompatibility and tolerability and did not cause significant systemic toxicity.

[0122] To further verify whether this treatment strategy activated a systemic anti-tumor immune response, flow cytometry was used to analyze changes in the immune cell population. The results showed that the number of effector memory T cells (TEM, CD44⁺CD62L⁻) in the spleen significantly increased after combination therapy. Figure 7 The results suggest that treatment induced a durable immune memory response. Furthermore, the infiltration level of CD4⁺ T cells in the metastatic tumor microenvironment was significantly enhanced (n). Figure 7 o), while the number of CD8⁺ T cells significantly increased in the “HAQ / ²²³Ra@HNPs + anti-PD-L1” treatment group ( Figure 7 p), indicating that it triggered a strong cytotoxic T-cell immune response.

[0123] In summary, HAQ / ²²³Ra@HNPs combined with anti-PD-L1 immune checkpoint inhibitory therapy can significantly inhibit the growth of primary and metastatic tumors, enhance systemic immune responses, and promote tumor-specific T cell activation. This strategy demonstrates great potential in the treatment of metastatic tumors, providing a new pathway to overcome tumor immune escape and improve treatment efficacy. Example 6

[0124] In practical applications, the pre-targeted delivery system HAQ@HNPs is first injected, utilizing HAQ-mediated active targeting and Pt. ⅣThe tumor site is pretreated by reducing GSH levels in depleted tumor tissue, thus lowering the reducing microenvironment. Subsequently, an active targeted delivery system containing self-cleaving molecules is injected. 223 Ra@HNPs utilize the IEDDA reaction between self-cleaving molecules and existing BCN groups in tumor tissue to rapidly trigger self-cleavage and release naked HNPs. 223 Ra2+ achieves alpha particle radiation killing locally. This sequential drug delivery strategy greatly improves the accumulation and radiation efficacy of 223Ra at the tumor site, while reducing damage to normal tissues.

[0125] in conclusion: This invention revolves around alpha nuclides 223 Ra's precise delivery system is designed with multiple protection and activation mechanisms to achieve efficient release in specific tumor microenvironments.

[0126] 1) This application constructs a dual-system delivery platform based on hydrogel nanoparticles (HNPs). Nanogels were formed by crosslinking PEGDA with ultraviolet light, and then loaded with prodrugs (cisplatin) and alpha-isotopes (...). 223 (Ra) enables pre-targeted and active targeted delivery. The nanohydrogel carrier possesses softness, deformability, high biocompatibility, and particle size stability, which facilitates the penetration and retention of tumor tissue.

[0127] 2) Construction and functional design of the pre-targeting system (HAQ@HNPs) Modified HAQ was linked to Pt via ester bond modification. Ⅳ Loading hydrogel surfaces enhances active targeting of tumor cells. Pt-loaded surfaces... Ⅳ The prodrug hydrogel can be delivered via Pt in the tumor microenvironment. Ⅳ Reduction to PtII effectively lowers intracellular glutathione (GSH) levels, preparing the cell for subsequent... 223 Ra releases the preparatory conditions.

[0128] 3) Design and application of self-immolating molecules A self-cleaving molecule was designed, based on an ortho-benzyl-substituted cresol derivative, achieving rapid bi-terminal cleavage via 1,4-electron elimination. The self-cleaving molecule is cleaved via an IEDDA reaction (with the BCN group as the reactive opponent), thereby removing… 223 A protective layer on the surface of the Ra nanocarrier enables controlled release. The self-cleaving molecules possess the ability to be specifically triggered within the tumor microenvironment, enhancing... 223 Ra's release accuracy and safety.

[0129] 4) Establishment and verification of the simulation system (Ba@HNPs) With Ba2+ Alternative 223 Ra 2+ The system was prepared and its physicochemical properties were verified to ensure process safety and to demonstrate the carrier's high stability in encapsulating ions and its leak-proof characteristics.

[0130] 5) High efficiency and stability 223 Ra load and release system ( 223 Ra @HNPs) Loading within the hydrogel 223 Ra, combined with self-cleaving molecules, constructs a dual-protection system, achieving high loading rates (RCY 81.44±1.36%, RCP>95%) and good biostability (remaining stable within 24 hours). Through specifically triggered release, it minimizes radiation damage to normal tissues and improves tumor killing efficiency.

Claims

1. A dual-lock target-controlled alpha nuclide self-immolation molecular cage, characterized in that: It includes a pre-targeted delivery system and an active targeted delivery system; the active targeted delivery system includes an alpha nuclide; the pre-targeted delivery system and the active targeted system release the alpha nuclide through a pyrolysis reaction.

2. The dual-lock target-controlled alpha nuclide self-immolation molecular cage according to claim 1, characterized in that: The cleavage reaction is one of the following: reverse electron demand Diels-Alder reaction, enzyme-responsive cleavage reaction, pH-responsive autolysis reaction, or ROS-responsive autolysis reaction.

3. The dual-lock target-controlled alpha nuclide self-immolation molecular cage according to claim 1, characterized in that: The pre-targeted delivery system includes a carrier, a prodrug, a modifying group, and a cleavage group; the prodrug is loaded on the carrier; the cleavage group is connected to the surface of the carrier through the modifying group.

4. The dual-lock target-controlled alpha nuclide self-immolation molecular cage according to claim 1, characterized in that: The active targeted delivery system includes a second carrier, a self-cleaving molecule, and an alpha nuclide; the alpha nuclide is loaded on the second carrier; and the self-cleaving molecule is connected to the surface of the second carrier.

5. The dual-lock target-controlled alpha nuclide self-immolation molecular cage according to claim 3 or 4, characterized in that: Both carrier one and carrier two are selected from hydrogel nanoparticles, polypeptide hydrogels, polylactic acid-glycolic acid copolymer (PLGA), silk fibroin nanoparticles, graphene oxide functionalized nanomaterials, and liposomes.

6. The dual-lock target-controlled alpha nuclide self-immolation molecular cage according to claim 3, characterized in that: The prodrug is a glutathione depleting agent, which is one of oxaliplatin, cisplatin, a small molecule compound modified with a disulfide bridging group, or a ROS generating agent.

7. The dual-lock target-controlled alpha nuclide self-immolation molecular cage according to claim 3, characterized in that: The modifying group is one of hyaluronic acid, RGD peptide, EGFR antibody fragment, PD-L1 monoclonal antibody fragment, folic acid, and small molecule targeting ligand.

8. The dual-lock target-controlled alpha nuclide self-immolation molecular cage according to claim 1, characterized in that: The alpha nuclide is 223 Ra, 225 Ac, 211 One of the At types.

9. The method for preparing the dual-locked target-controlled alpha nuclide self-ignition molecular cage according to any one of claims 1 to 8, characterized in that: The preparation method of the pre-targeted delivery system is as follows: Step 1.1: Preparation of carrier one; Step 1.2: Load the prodrug onto the carrier 1 prepared in step 1.1 to prepare a pre-targeted nanosystem precursor; Step 1.3: Couple the modified group with the cleavage group to the surface of the pre-targeted nanosystem precursor prepared in Step 1.2 to prepare the pre-targeted delivery system; The preparation method of the active targeted delivery system is as follows: Step 2.1: Preparation of carrier two; Step 2.2: Load the alpha nuclide onto the carrier two prepared in step 2.1 to prepare the precursor of the active targeting nanosystem; Step 2.3: Preparation of self-cleaving molecules; Step 2.4: The self-cleaving molecule prepared in step 2.3 is reacted and linked with the active targeting nanosystem precursor prepared in step 2.2 to prepare an active targeting delivery system.

10. The use of the dual-lock target-controlled alpha nuclide self-ignition molecular cage according to any one of claims 1 to 8 in the preparation of tumor radiotargeting drugs.

Images