A multi-armed anti-CD40 antibody-mediated nano dual-drug delivery system and its preparation method and application

Abstract

The present invention provides a multi-arm Anti-CD40 antibody-mediated nanometer dual-drug delivery system and its preparation method and application, belonging to the field of biomedicine technology. In the present invention, chromium ion-doped zinc gallate nanocrystals are embedded in silica-coated gold nanorod nanoparticles GNR@MSNs to prepare composite nanoparticles cGNR@MSNs, and then multi-arm DOTV is modified on the surface of the nanoparticles, and then anti-CD40 antibodies are covalently coupled to obtain the multi-arm Anti-CD40 antibody-mediated nanometer dual-drug delivery system. The multi-arm Anti-CD40 antibody-mediated nanometer dual-drug delivery system improves diagnostic accuracy by combining multimodal imaging, promotes the development of imaging-guided combined therapy, exerts the synergistic effect of chemotherapy, immunotherapy and photothermal therapy, optimizes the tumor treatment effect, is suitable for the treatment of various malignant solid tumors, and has good practicality.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system and a preparation method and application thereof. Background Art

[0002] Cancer treatment traditionally includes three main methods: surgery, chemotherapy and radiotherapy. Although great progress has been made in these fields, tumor treatment strategies such as surgery, chemotherapy and radiotherapy are often accompanied by the disadvantages of incomplete treatment, easy recurrence, easy metastasis and easy to cause multiple complications, which often bring more serious side effects and great pain to patients. It is also not conducive to the recovery of cancer patients. Therefore, it is necessary to find more effective and safer alternative treatment methods. Modifying targeting molecules (including DNA, aptamers, proteins, etc.) on the surface of nanocarriers and releasing loaded drugs through designed chemistry and enzyme-responsive chemistry mechanisms to obtain a new environmentally sensitive stimulus-responsive drug delivery system. By designing biomacromolecule-gated mesoporous silica nanodrug delivery systems, the simultaneous delivery of multiple drugs and stimulus-responsive controlled release has become one of the current research hotspots in the field of drug delivery.

[0003] Gold nanorods (GNRs) have good and controllable surface chemical properties and unique localized surface plasmon resonance (LSPR) effect. They have strong absorption and scattering effects in the near-infrared light region (650-900nm), continuously adjustable LSPR absorption peak and high photothermal conversion efficiency (50%-100%). They have been widely used in biomedical fields such as photothermal therapy, disease diagnosis and drug delivery.

[0004] Mesoporous silica nanoparticles (MSNs) are nanomaterials composed of numerous honeycomb-shaped mesoporous channels. They not only possess properties such as large specific surface area, large pore volume, and adjustable pore size, but also possess advantages such as stable chemical properties and good biocompatibility. Therefore, mesoporous silica nanomaterials can not only encapsulate chemical anticancer drugs of different properties, but also achieve drug delivery and stimuli-responsive controlled release by modifying their surfaces with various functional groups. The tumor microenvironment has complex and variable characteristics such as acidification, hypoxia, inflammatory reactivity, and immunosuppression, and many drugs often have difficulty reaching the lesion area due to shortcomings such as instability and lack of target targeting. Therefore, achieving stimuli-responsive release of drugs is of great significance for improving the therapeutic efficacy of drugs and achieving precise treatment.

[0005] Near-infrared long-lasting luminescent material ZnGa2O4:Cr 3+(ZGC) has unique deep tissue penetration and rechargeable long afterglow luminescence properties. In addition, the material has good chemical stability, biocompatibility and cyclic recharge characteristics, and can be used in biosensing, medical imaging, surgical guidance and tumor diagnosis. Near-infrared long afterglow materials are used in the imaging field and have higher signal-to-noise ratio and resolution compared with other optical materials. The excitation light source wavelength of traditional long afterglow materials is mostly limited to the ultraviolet region. The development of long afterglow materials that emit light in the near-infrared region has significant advantages and can realize the application of long afterglow luminescent materials in a variety of new fields.

[0006] The CD40 receptor (Cluster of differentiation 40) is a transmembrane protein belonging to the tumor necrosis factor (TNF) superfamily. It is primarily expressed on the surface of malignant tumors, including breast, lung, and renal cancers. Inspired by the CD40 / CD40L (CD40 ligand) interaction that enhances immune responses, anti-CD40 antibodies have demonstrated promising activity in enhancing anti-tumor responses and have proven to be an attractive cancer treatment strategy. Furthermore, gadolinium(III)-based magnetic resonance imaging contrast agents have garnered significant attention in recent years. Nitrogen-containing heterocyclic polycarboxylic acid ligands (such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid, DOTA) are readily modified with functional groups, and their gadolinium(III) complexes exhibit greater thermodynamic stability and kinetic inertness than linear complexes. Protein-bound gadolinium-based contrast agents offer improved biocompatibility and imaging contrast, making them highly sought after in the development of new gadolinium-based contrast agents.

[0007] Therefore, it is necessary to develop a biomacromolecule-gated mesoporous silica nanodrug delivery system that can simultaneously exert the synergistic effects of chemotherapy, immunotherapy, and photothermal therapy, achieve simultaneous delivery of multiple drugs and stimulus-responsive controlled release, and thus optimize the tumor treatment effect. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the present invention provides a multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system and its preparation method and application; in the present invention, chromium ion-doped zinc gallate ZnGa2O4:Cr 3+(ZGC) nanocrystals are embedded in silica-coated gold nanorod nanoparticles GNR@MSNs to prepare composite nanoparticles cGNR@MSNs, and then 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is modified on the surface of the nanoparticles to connect divinyl pyrimidine derivatives (DOTV), and then anti-CD40 antibodies are covalently coupled as nanogates to obtain the multi-arm anti-CD40 antibody-mediated nano dual-drug delivery system; the multi-arm anti-CD40 antibody-mediated nano dual-drug delivery system improves diagnostic accuracy by combining multimodal imaging, promotes the development of imaging-guided combined therapy, exerts the synergistic effect of chemotherapy, immunotherapy and photothermal therapy, optimizes the tumor treatment effect, is suitable for the treatment of various malignant solid tumors, and has good practicality.

[0009] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0010] The present invention first provides a multi-arm anti-CD40 antibody-mediated nano dual-drug delivery system, in which multi-arm DOTV and anti-CD40 antibodies are self-assembled step by step on the surface of cGNR@MSNs nanocomposite to form a multilayer membrane structure; the cGNR@MSNs nanocomposite is a spherical nanostructure, which is formed by embedding ZnGa2O4:Cr in a core-shell structure of mesoporous silica (MSNs) coated with gold nanorods (GNRs). 3+ Nanocrystals are obtained; the anti-tumor drug is loaded in the shell mesopores of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system.

[0011] Preferably, the multi-arm DOTV is evenly distributed in the shell and mesoporous channels of the cGNR@MSNs nanocomposite, and the Anti-CD40 antibody is distributed on the surface of the cGNR@MSNs nanocomposite to form the outermost antibody layer;

[0012] The anti-tumor drug includes camptothecin.

[0013] The present invention also provides a method for preparing the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system, which specifically comprises the following steps:

[0014] S1. Synthesis of gold nanorods (GNRs) using seed-mediated growth method:

[0015] A chloroauric acid solution, a cetyltrimethylammonium bromide (CTAB) solution, and a sodium borohydride solution were mixed and stirred, and then aged at room temperature to obtain a reaction solution 1 after the reaction was completed, which was set aside;

[0016] Dissolve CTAB and sodium oleate in deionized water, mix well, add silver nitrate aqueous solution, and then carry out reaction 1 under stirring to obtain reaction solution 2;

[0017] HAuCl4 solution was added to reaction solution 2 and reaction 2 was carried out under stirring. After the reaction was completed, the pH value was adjusted, and then ascorbic acid was added. After stirring and mixing, reaction solution 1 was added, and the mixture was allowed to stand and finally centrifuged to obtain GNRs, which were dispersed in deionized water for later use.

[0018] Preferably, in step S1, the volume ratio of the chloroauric acid solution, the CTAB solution, and the sodium borohydride solution is 5:5:1, the concentration of the chloroauric acid solution is 0.3-0.6 mM, the concentration of the CTAB solution is 0.1-0.2 mM, and the sodium borohydride solution is 0.6 mL of 0.01 M sodium borohydride solution diluted to 1 mL;

[0019] The aging reaction time is 30 to 60 minutes;

[0020] The dosage ratio of CTAB, sodium oleate and deionized water is 9g:1.234g:250mL

[0021] The usage ratio of the CTAB silver nitrate solution, HAuCl4 solution, ascorbic acid and reaction solution 1 is 9 g:14.5 mL:250 mL:1.25 mL:0.8 mL; the concentration of the silver nitrate aqueous solution is 4 mM, the concentration of the HAuCl4 solution is 1 mM, and the concentration of ascorbic acid is 0.064 M;

[0022] The reaction 1 is stirred at 30°C for 15 to 30 minutes;

[0023] The reaction 2 is stirred at 30°C until the solution becomes colorless;

[0024] The pH value is adjusted to 1.30 using 2.1 mL of 12 M concentrated HCl;

[0025] The standing is standing at 30° C. for 12 hours.

[0026] S2. Preparation of core-shell silica-coated gold nanorod nanoparticles (GNR@MSNs):

[0027] The GNRs dispersion and CTAB were dissolved in deionized water, and then NaOH solution and tetraethyl silicate solution were added and reacted under stirring. After the reaction was completed, the solid precipitate was centrifuged and dried to obtain a solid powder.

[0028] The solid powder was then dispersed in a mixed solution of hydrochloric acid and methanol and refluxed for reaction. After the reaction, the mixture was centrifuged, washed, and dried to obtain GNR@MSNs.

[0029] Preferably, in step S2, the usage ratio of the GNRs dispersion, CTAB, deionized water, NaOH solution and tetraethyl silicate solution is 5 mL: 0.1 g: 0.5 mL: 450 μL;

[0030] The concentration of the GNRs dispersion is 1.5-2 mg·mL -1 , the concentration of the NaOH solution is 0.1M;

[0031] The tetraethyl silicate solution uses methanol as solvent and has a volume fraction of 20%;

[0032] The tetraethyl silicate solution was added dropwise three times, and the mixture was stirred for 6 hours after each addition.

[0033] The amount ratio of the GNRs dispersion, hydrochloric acid and methanol is 5 mL:0.2 mL:20 mL, and the concentration of the hydrochloric acid is 12 M;

[0034] The reflux reaction was carried out at room temperature for 1 h.

[0035] Preparation of S3.cGNR@MSNs Nanocomposites:

[0036] Ga(NO3)3, Zn(NO3)2 and Cr(NO3)3 were dissolved in a water / ethanol solution to obtain a precursor mixed solution containing Ga(NO3)3, Zn(NO3)2 and Cr(NO3)3;

[0037] The precursor mixed solution is then incubated with the GNR@MSNs nanomaterial, centrifuged after the incubation, and dried to obtain a solid powder; the solid powder is calcined to obtain the cGNR@MSNs nanocomposite material.

[0038] Preferably, in step S3, Ga in the precursor mixed solution 3+ 、Zn 2+ and Cr 3+ The final ion concentrations were 1 M, 0.5 M, and 0.002 M, respectively;

[0039] The dosage ratio of the GNR@MSNs and precursor mixed solution is 500 mg:300 μL;

[0040] The incubation is carried out at room temperature;

[0041] The calcination conditions are: heating to 600° C. at a rate of 5° C. / min, and calcining at 600° C. for 1 hour.

[0042] S4. Preparation of DOTA-derived divinylpyrimidine (DOTV) functionalized cGNR@MSNs (DOTV-cGNR@MSNs):

[0043] S4.1. Preparation of NH2-cGNR@MSNs:

[0044] The cGNR@MSNs nanocomposite was dispersed in tetrahydrofuran, and then 3-aminopropyltrimethoxysilane was added thereto. The mixture was refluxed under heating and stirring conditions. After the reaction, the mixture was centrifuged, washed, and dried to obtain primary amine-functionalized cGNR@MSNs (NH2-cGNR@MSNs).

[0045] S4.2. Preparation of CH3CO-cGNR@MSNs:

[0046] NH2-cGNR@MSNs, levulinic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS) and triethylamine were dissolved in tetrahydrofuran and stirred at room temperature. After the reaction, the mixture was centrifuged, washed and dried to obtain β-levulinamide derivative functionalized cGNR@MSNs (CH3CO-cGNR@MSNs).

[0047] S4.3. Preparation of NH2-N=cGNR@MSNs:

[0048] CH3CO-cGNR@MSNs were dispersed in a methanol solution, to which a methanol solution containing hydrazine dihydrochloride and NaOH was added for reflux reaction 2. After the reaction, the mixture was centrifuged, washed, and dried to obtain hydrazine-functionalized cGNR@MSNs nanoparticles (NH2-N=cGNR@MSNs).

[0049] S4.4. Preparation of DOTA-cGNR@MSNs Nanoparticles

[0050] NH2-N=cGNR@MSNs nanoparticles were dispersed in tetrahydrofuran, and EDC·HCl, NHS, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and triethylamine were added thereto. The mixture was stirred at room temperature for 2 seconds. After the reaction, the mixture was centrifuged, washed and dried to obtain DOTA-cGNR@MSNs nanoparticles.

[0051] S4.5. Preparation of DOTV-cGNR@MSNs:

[0052] DOTA-cGNR@MSNs nanoparticles were dispersed in tetrahydrofuran, and EDC·HCl, NHS, 2-amino-4,6-divinylpyrimidine (DOTV), and triethylamine were added thereto. The mixture was stirred at room temperature for 3 minutes. After the reaction, the mixture was centrifuged, washed, and dried to obtain DOTV-cGNR@MSNs.

[0053] Preferably, in step S4.1, the ratio of the amount of cGNR@MSNs nanocomposite material to 3-aminopropyltrimethoxysilane is 0.1 g: 1-2 mmol; the reflux reaction is carried out under stirring and reflux at 80° C. for 12 h;

[0054] In step S4.2, the ratio of NH2-cGNR@MSNs, levulinic acid, EDC·HCl, NHS, and triethylamine is 0.1 g: 1-2 mmol: 2 mmol: 2 mmol: 2 mmol, and the stirring reaction time is 12 h;

[0055] In step S4.3, the ratio of CH3CO-cGNR@MSNs, hydrazine dihydrochloride, and NaOH is 0.1 g: 0.5-1 mmol: 1-2 mmol, and the reflux reaction 2 is carried out at 80°C for 2 h.

[0056] In step S4.4, the ratio of NH2-N=cGNR@MSNs nanoparticles, EDC·HCl, NHS, DOTA and triethylamine is 0.1 g: 1-2 mmol: 1 mmol: 1 mmol: 1 mmol; the stirring reaction 2 is carried out at room temperature for 12 h;

[0057] In step S4.5, the amount ratio of DOTA-cGNR@MSNs nanoparticles, EDC·HCl, NHS, 2-amino-4,6-divinylpyrimidine and triethylamine is 0.2 g: 2-3 mmol: 3 mmol: 3 mmol: 3 mmol; the stirring reaction 3 is carried out at room temperature for 12 h.

[0058] S5. Preparation of multi-armed anti-CD40 antibody-mediated nanoparticle dual-drug delivery system:

[0059] Dissolving the anti-tumor drug in Tris-HCl buffer to obtain an anti-tumor drug loading solution; adding Anti-CD40 and tris(2-carboxyethyl)phosphine hydrochloride to the Tris-HCl buffer, incubating to obtain an Anti-CD40 antibody solution for later use;

[0060] DOTV-cGNR@MSNs were mixed with the anti-tumor drug loading liquid and stirred for reaction. After the reaction, Anti-CD40 antibody solution was added to the reaction liquid and stirred for reaction. After the reaction, the mixture was centrifuged and washed to obtain a multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system.

[0061] Preferably, in step S5, the anti-tumor drug includes camptothecin (CPT);

[0062] The concentration of the anti-tumor drug loading solution is 0.2 mM and the pH value is 7.4;

[0063] During the preparation of the Anti-CD40 antibody solution, the dosage ratio of Anti-CD40, tris(2-carboxyethyl)phosphine hydrochloride, and Tris-HCl buffer was 0.1 mL (3 μM): 0.2 mg: 10 mL. The incubation condition was 2 h at 0°C.

[0064] The dosage ratio of the DOTV-cGNR@MSNs, CPT loading solution and Anti-CD40 antibody solution was 10 mg: 10 mL (0.2 mM): 10 mL;

[0065] The reaction was carried out under the conditions of continuous stirring at 0°C for 6 h.

[0066] The present invention also provides the use of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system as a multimodal imaging preparation.

[0067] Preferably, the multimodal imaging includes magnetic resonance imaging, long-afterglow luminescence imaging and fluorescence imaging.

[0068] The present invention also provides the use of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system in the preparation of drugs for treating tumors.

[0069] Preferably, the tumor comprises a malignant tumor that overexpresses the CD40 receptor.

[0070] Compared with the prior art, the present invention has the following beneficial effects:

[0071] (1) In the present invention, zinc gallate ZnGa2O4:Cr doped with chromium ions is prepared. 3+ The preparation method of silica-coated gold nanorod nanoparticles (GNR@MSNs) embedded with ZGC nanocrystals yields cGNR@MSNs composite nanoparticles. The cGNR@MSNs composite nanoparticles can be used as drug carriers for targeted delivery of anti-tumor drugs. Furthermore, the ZGC nanocrystals doped in the cGNR@MSNs composite nanoparticles can serve as inorganic long-lasting probes, maintaining a persistent luminescence time of 10 minutes under LED illumination. This demonstrates a long afterglow lifetime and is suitable for medical imaging.

[0072] (2) The multi-armed Anti-CD40 antibody-mediated nano dual-drug delivery system of the present invention modifies the surface of cGNR@MSNs composite nanoparticles with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) linked to a divinyl pyrimidine derivative (DOTV), and then covalently couples the Anti-CD40 antibody as a nano-gate. The Anti-CD40 monoclonal antibody is used as an immunomodulatory drug and gating element, and is suitable for the treatment of various tumors, especially for the combined treatment of malignant tumors with overexpression of CD40 receptors. It can improve the safety of the delivery system and the therapeutic effect of chemical drugs, realize a combined treatment strategy combining immunotherapy and chemotherapy, and improve the medication experience and therapeutic effect of tumor patients.

[0073] Moreover, the multi-armed Anti-CD40 antibody on the surface of cGNR@MSNs not only promotes the encapsulation of drug molecules by the antibody nanogate, achieving high delivery efficiency of targeted drug delivery, but also enhances the binding force between the Anti-CD40 antibody and the CD40 receptor on the surface of tumor cells, thereby improving the immune regulation effect and can be applied in the field of tumor cell immunotherapy.

[0074] (3) The multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system of the present invention simultaneously realizes the combination of chemotherapy, immunotherapy and photothermal therapy, has a significant synergistic effect on inhibiting tumor cell proliferation, and develops nanomedicines for combined tumor treatment, which can be applied to the clinical treatment of various tumors such as lung cancer, liver cancer and breast cancer. Under weakly acidic pH conditions, the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system disconnects the acylhydrazone bond modified on the surface of the cGNR@MSNs composite nanomaterial, opens the Anti-CD40 antibody nanogate, and realizes the controlled release of the drug-loaded CPT; under near-infrared irradiation, the gold nanorods act as photothermal conversion devices, converting light energy into heat energy, causing the temperature of the solution around the nanoparticles to rise, thereby enhancing the dynamic vibration of the multi-arm antibody, causing the antibody nanogate to open, and realizing the controlled release of CPT.

[0075] (4) The multi-armed Anti-CD40 antibody-mediated nano dual-drug delivery system of the present invention can achieve multimodal imaging combining fluorescence imaging, near-infrared persistent luminescence imaging, and magnetic resonance imaging. The present invention develops an Anti-CD40 antibody-gated nanocomposite particle drug delivery system, which improves diagnostic accuracy by combining multimodal imaging, promotes the development of imaging-guided combined therapy, exerts the synergistic effect of chemotherapy, immunotherapy, and photothermal therapy, optimizes tumor treatment effects, and is suitable for the treatment of various malignant solid tumors.

[0076] (5) The multi-armed Anti-CD40 antibody-mediated nano dual-drug delivery system of the present invention achieves targeted delivery of the loaded drug CPT to specific tumor cells. The Anti-CD40 antibody can recognize and bind to the CD40 receptor overexpressed on the surface of tumor cells. The antibody can guide the drug delivery system to target tumor cells and improve the efficiency of drug uptake by cells. In addition, the construction of the antibody nanogate reduces the premature release of the drug, so that its toxicity only acts on tumor cells, thereby improving the toxic and side effects of the drug delivery system.

[0077] (6) The multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system of the present invention can be used as a magnetic resonance imaging (MRI) contrast agent after chelating gadolinium ions, and the antibody-mediated imaging mode can enhance the sensitivity of magnetic resonance imaging. The system can simultaneously realize magnetic resonance imaging, long-lasting luminescence imaging and fluorescence imaging. The multimodal imaging method can be applied to fields such as biomedical imaging, tumor diagnosis and surgical navigation. Compared with a single imaging mode, it can improve the resolution and accuracy of imaging diagnosis. In addition, the system can realize repeated recharging under the irradiation of LED light source, has a long tissue penetration depth, and has obvious advantages in deep tissue imaging in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Schematic diagram of the preparation method of multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system.

[0079] Figure 2 Transmission electron microscopy images of GNRs (A), cGNR@MSNs (B), DOTV-cGNR@MSNs (C) and multi-armed Anti-CD40 antibody-mediated nano dual-drug delivery system (D).

[0080] Figure 3 UV-visible full-wavelength scanning spectra of GNR, GNR@MSNs and cGNR@MSNs.

[0081] Figure 4 Long afterglow luminescence emission spectrum of cGNR@MSNs.

[0082] Figure 5 FTIR spectra of blank and surface functionalized nanocarriers: a is cGNR@MSNs, b is NH2-N=cGNR@MSNs, c is DOTA-cGNR@MSNs, d is DOTV-cGNR@MSNs, and e is a multi-armed Anti-CD40 antibody-mediated nano dual-drug delivery system.

[0083] Figure 6 This is the stimulus-responsive controlled release kinetic curve of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system.

[0084] Figure 7 Confocal laser scanning microscopy (CLSM) images of in vitro cytotoxicity experiments and cell uptake: (A) MCF cells; (B) A549 cells; (C) CLSM images of MCF-7 and A549 cells cellular uptake of the multi-armed Anti-CD40 antibody-mediated nano-drug delivery system and drug CPT release.

[0085] Figure 8 Fluorescence and long afterglow luminescence imaging of the nano dual-drug delivery system mediated by multi-arm Anti-CD40 antibody injection in mouse models under LED irradiation; A is the blank control (λ ex =400nm,λ em =450nm); B, C are fluorescence imaging of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system group without CPT loading at the time of first excitation (B) and 10 minutes later (C); D, E are fluorescence imaging of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system group loaded with CPT at the time of first excitation (D) and 10 minutes later (E); F, G are fluorescence imaging of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system group loaded with CPT at the time of second excitation (F) and 10 minutes later (G);

[0086] H is the blank control under long afterglow luminescence mode (λ em=730nm); I, J are the fluorescence imaging of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system group without CPT loading at the time of the first excitation (I) and 10 minutes later (J); K, L are the fluorescence imaging of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system group loaded with CPT at the time of the first excitation (K) and 10 minutes later (L); M, N are the fluorescence imaging of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system group loaded with CPT at the time of the second excitation (M) and 10 minutes later (N).

[0087] Figure 9 Magnetic resonance imaging (T1-weighted MR images) of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system Anti-CD40-DOTVcGNR@MSNs-Gd. DETAILED DESCRIPTION

[0088] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, unless otherwise specified, are conventional methods, equipment, and materials in the art and can be purchased from the market.

[0089] Example 1:

[0090] The preparation process of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system of the present invention is as follows: Figure 1 As shown, the specific methods include the following:

[0091] S1. Preparation of Gold Nanorods (GNRs):

[0092] Mix 5 mL of 0.5 mM chloroauric acid solution (HAuCl4) and 5 mL of 0.2 mM cetyltrimethylammonium bromide solution (CTAB) in a flask to obtain Mixture 1. Dilute 0.6 mL of freshly prepared 0.01 M sodium borohydride solution (NaBH4) to 1 mL with water to obtain a diluted sodium borohydride solution for later use. Add the diluted sodium borohydride solution to Mixture 1, stir for 2 minutes to mix thoroughly, and then age at room temperature for 30 minutes to obtain Reaction Solution 1.

[0093] Dissolve 9 g of CTAB and 1.234 g of sodium oleate in 250 mL of deionized water to obtain Mixture 2. To ensure complete dissolution, the mixture was heated to 50°C and stirred until completely dissolved, then cooled to 30°C to obtain Reaction Solution 2. Silver nitrate aqueous solution (AgNO3, 14.5 mL, 4 mM) was added to Reaction Solution 2 and stirred at 30°C for 15 minutes. After stirring, Reaction Solution 3 was obtained. 250 mL of HAuCl4 solution (1 mM) was added to Reaction Solution 3 and stirred continuously for 90 minutes to obtain a colorless solution. The pH of the colorless solution was then adjusted with 2.1 mL of concentrated HCl (12 M). After slow stirring for 15 minutes, 1.25 mL of ascorbic acid (0.064 M) was added and stirred for 30 seconds to obtain Reaction Solution 4.

[0094] 0.8 mL of reaction solution 1 was added to reaction solution 4 and stirred for 30 s. After stirring, the mixture was allowed to stand at 30 °C for 12 h. After standing, the mixture was centrifuged at 10,000 rpm for 30 min to obtain gold nanorods (GNRs). The gold nanorods (GNRs) were dispersed in 30 mL of deionized water to obtain a GNRs solution (1.6 mg mL -1 ),spare.

[0095] S2. Preparation of core-shell silica-coated gold nanorods (GNR@MSNs) nanoparticles:

[0096] 5 mL of GNRs solution and 0.1 g of CTAB were dissolved in 50 mL of deionized water at 55 °C, and then 0.5 mL of NaOH solution (0.1 M) was added. Then, 450 μL of tetraethyl silicate solution (20% TEOS / methanol) was added dropwise three times, for a total of 3 times. Each addition of tetraethyl silicate solution was separated by 30 minutes. After all the additions were made, the mixture was slowly stirred for 6 hours to coat the GNRs surface with a silica shell layer, and a solid precipitate was obtained by centrifugation.

[0097] In order to remove the template agent CTAB, the solid powder was dispersed in a mixed solution of 0.2 mL of hydrochloric acid (12 M) and 20 mL of methanol, refluxed for 1 h, and then centrifuged to obtain a solid precipitate, which was thoroughly washed with methanol and vacuum dried to obtain core-shell structured silica-coated gold nanorods (GNR@MSNs) nanoparticles.

[0098] Preparation of S3.cGNR@MSNs Nanocomposites:

[0099] The following steps were used to obtain ZGC nanocrystal-embedded cGNR@MSNs:

[0100] Ga(NO3)3, Zn(NO3)2 and Cr(NO3)3 were dissolved in a water / ethanol (1:1, v / v) mixed solution to obtain Ga 3+ 、Zn 2+ and Cr 3+ The final ion concentrations of the mixed solutions containing Ga(NO3)3, Zn(NO3)2 and Cr(NO3)3 were 1M, 0.5M and 0.002M respectively. The Ga(NO3)3, Zn(NO3)2 and Cr(NO3)3 precursor solutions were used to load metal nitrates in the mesopores of GNR@MSNs. 3+ 、Zn 2+ and Cr 3+ The stoichiometric ratio is 2 / 1 / 0.004, which is favorable for ZnGa2O4:Cr 3+ (ZGC) Generation of long-lasting luminescent nanocrystals.

[0101] Then 500 mg of GNR@MSNs nanoparticles were incubated with 300 μL of the precursor mixed solution for 2 h. The solid after centrifugation was vacuum dried for 12 h. The obtained solid powder was heated to 600 °C in a muffle furnace at a rate of 5 °C / min and calcined for 1 h to obtain ZGC embedded in GNR@MSNs nanocomposite material, namely cGNR@MSNs nanocomposite material.

[0102] S4. Preparation of DOTA-derived divinylpyrimidine (DOTV) functionalized cGNR@MSNs (DOTV-cGNR@MSNs):

[0103] S4.1. Preparation of NH2-cGNR@MSNs:

[0104] 0.1 g of cGNR@MSNs nanocomposite was dispersed in tetrahydrofuran, and then 2 mmol of 3-aminopropyltrimethoxysilane was added thereto. The mixture was refluxed at 80°C with stirring for 12 h. After the reaction, the mixture was centrifuged, washed, and dried to obtain primary amine-functionalized cGNR@MSNs (NH2-cGNR@MSNs).

[0105] S4.2. Preparation of CH3CO-cGNR@MSNs:

[0106] 0.1 g NH2-cGNR@MSNs, 2 mmol levulinic acid, 2 mmol N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl), 2 mmol N-hydroxysuccinimide (NHS) and 2 mmol triethylamine were dissolved in tetrahydrofuran and stirred at room temperature for 12 h. After the reaction, the mixture was centrifuged, washed and dried to obtain β-levulinamide derivative functionalized cGNR@MSNs (CH3CO-cGNR@MSNs).

[0107] S4.3. Preparation of NH2-N=cGNR@MSNs:

[0108] 0.1 g of CH3CO-cGNR@MSNs was dispersed in a methanol solution, and a methanol solution containing 1 mmol of hydrazine dihydrochloride and 2 mmol of NaOH was added thereto. The mixture was refluxed at 80 °C for 2 h. After the reaction, the mixture was centrifuged, washed, and dried to obtain hydrazine-functionalized cGNR@MSNs nanoparticles (NH2-N=cGNR@MSNs).

[0109] S4.4. Preparation of DOTA-cGNR@MSNs Nanoparticles

[0110] 0.1 g of NH2-N=cGNR@MSNs nanoparticles were dispersed in tetrahydrofuran, and 1 mmol of EDC·HCl, 1 mmol of NHS, 1 mmol of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1 mmol of triethylamine were added thereto. The mixture was stirred at room temperature for 2 hours. After the reaction, the mixture was centrifuged, washed, and dried to obtain DOTA-cGNR@MSNs nanoparticles.

[0111] S4.5. Preparation of DOTV-cGNR@MSNs:

[0112] 0.2 g of DOTA-cGNR@MSNs nanoparticles were dispersed in tetrahydrofuran, and 3 mmol of EDC·HCl, 3 mmol of NHS, 3 mmol of 2-amino-4,6-divinylpyrimidine (DOTV) and 3 mmol of triethylamine were added thereto. The mixture was stirred at room temperature for 3 hours. The reaction time was 12 hours. After the reaction, the mixture was centrifuged, washed and dried to obtain DOTV-cGNR@MSNs.

[0113] S5. Preparation of multi-armed anti-CD40 antibody-mediated nanoparticle dual-drug delivery system:

[0114] 3.50 mg of camptothecin (CPT) was dissolved in 50 mL of Tris-HCl buffer (10 mM, pH 7.4) to obtain a CPT loading solution (0.2 mM, pH 7.4).

[0115] Anti-CD40 (0.1 mL, 3 μM) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 1 mM) were added to 10 mL of Tris-HCl buffer (10 mM, pH 7.4) and incubated at 0°C for 2 h to complete the disulfide bond reduction of the Anti-CD40 antibody to obtain an Anti-CD40 antibody solution for later use.

[0116] 10 mg of DOTV-cGNR@MSNs was mixed with 10 mL of CPT loading liquid and stirred at 25 °C for 12 h to obtain a reaction solution. Subsequently, the above-mentioned reduced Anti-CD40 antibody solution (10 mL) was added to the reaction solution, and the reaction was continuously stirred at 0 °C for 6 h to covalently capture the antibody. After the reaction, centrifugation and washing were performed to obtain a multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system, which was recorded as Anti-CD40-DOTV-cGNR@MSNs.

[0117] The morphology and particle size of GNRs and cGNR@MSNs nanocomposites were characterized by transmission electron microscopy. Figure 2 shown. Figure 2 Transmission electron microscopy images of GNRs (A) and cGNR@MSNs (B). Figure 2 A shows that the average longitudinal diameter of gold nanorods (GNRs) is 49nm and the lateral diameter is 18nm. Figure 2 B It can be seen that cGNR@MSNs maintain uniform size and mesoporous structure, and the average diameter of the uniform ZGC nanocrystals indicated by the arrows is 2.60 nm.

[0118] GNRs, GNR@MSNs and cGNR@MSNs solutions with appropriate concentrations were prepared respectively and full wavelength scanning was performed using a UV-visible spectrophotometer. The scanning results are shown in Figure 2. Figure 3 As shown. Figure 3 As can be seen in the figure, the longitudinal surface plasmon resonance (LSPR) wavelength of GNRs is 770nm, and the LSPR wavelength of GNR@MSNs is about 785nm. The UV-vis spectrum of cGNR@MSNs shows that its absorption bands are 350-450 and 550-650nm, which can be attributed to the successful generation of ZGC nanoparticles.

[0119] The cGNR@MSNs were characterized by the phosphorescence mode of fluorescence spectroscopy. Figure 4 As shown. Figure 4 It can be seen that the long afterglow luminescence spectrum of cGNR@MSNs under 254 nm excitation shows that the phosphorescence emission peak is around 730 nm.

[0120] Figure 5 FTIR spectra of blank and surface functionalized nanocarriers: a is cGNR@MSNs, b is NH2-N=cGNR@MSNs, c is DOTA-cGNR@MSNs, d is DOTV-cGNR@MSNs, and e is anti-CD40-DOTV-cGNR@MSNs. Figure 5 It can be seen that the main infrared absorption peaks of cGNR@MSNs appear at 1210, 1060, and 800 cm-1 At 3430 and 1640 cm, the stretching vibration absorption peaks of the silica matrix are -1 The stretching and bending vibration absorption peaks of OH and adsorbed water are at 2355cm -1 A new peak appears at 1645,1560cm, which is attributed to the stretching vibration of =N-NH2. -1 The amide I band and amide II band appeared at 1720 cm -1 There is an absorption peak at 1580, 1400 cm -1 The new peaks appearing at 1720 cm-1 are attributed to the stretching vibration absorption peaks of the C=C and C=N double bonds in DVP. After antibody conjugation, enhanced absorption peaks of the C=O double bond, amide I band, and amide II band are shown, which are located at 1720 cm-1, respectively. -1 , 1645cm -1 , and 1560cm -1 and the newly emerged CH stretching vibration absorption peak is located at 2980 cm -1 FTIR spectroscopy results confirmed the successful modification of functional groups on the surface of the nanoparticles and the successful coupling of Anti-CD40 antibodies to construct a multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system.

[0121] Example 2:

[0122] In this example, the stimuli-responsive controlled release of the multi-armed Anti-CD40 antibody-mediated nano dual-drug delivery system was investigated under acidic pH triggering. The release kinetics of CPT in Anti-CD40-DOTV-cGNR@MSNs were monitored using a fluorescence spectrometer, and the fluorescence emission spectrum of CPT (λ ex =365nm,λ em =440nm), the results are as follows Figure 6 shown.

[0123] Figure 6 The stimulation-responsive controlled release kinetics curve of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system is shown in Figure 2. Figure 6As can be seen in the figure, the pre-release amount of CPT after 4 hours under the condition of physiological pH 7.4 is negligible (<5%), which indicates that the blocking performance of the antibody nanogate in encapsulating the drug in the mesopore is good. Acidic pH (5.0 and 3.0) releases the drug in tris-HCl buffer, showing the external diffusion and fluorescence recovery of CPT, and reaches the equilibrium state of maximum release after 3.5 hours. The release efficiency of CPT is 85% under the triggering of pH 5.0, indicating the disconnection of the acylhydrazone bond and the separation of the antibody. In contrast, pH 3.0 triggering can release 97% of the CPT load, because at lower pH, most of the connecting chains can be disconnected to open the antibody nanogate.

[0124] Therefore, the developed Anti-CD40 antibody-mediated nanodrug delivery system can well achieve "zero release" of drugs, and achieve "intelligent" targeted therapeutic effects after the acylhydrazone bond breaks under different acidic conditions when external conditions change, thereby reducing toxic side effects and better exerting tumor therapeutic effects.

[0125] Example 3:

[0126] In this example, the MTT method was used to determine the effects of different concentrations of cGNR@MSNs, DOTV-cGNR@MSNs, and the multi-armed Anti-CD40 antibody-mediated nano dual-drug delivery system of the present invention on the viability of MCF-7 (human breast cancer cells, purchased from Wuhan Pronose Life Science Technology Co., Ltd.) and A549 (human non-small cell lung cancer cells, purchased from Wuhan Pronose Life Science Technology Co., Ltd.) cells. The specific steps are as follows:

[0127] MCF-7 and A549 cells in the logarithmic growth phase were cultured at a rate of approximately 5 × 10 cells per well. 4 The cells were seeded into a 96-well plate at a density of 100 μL per well and cultured for 24 hours. After the cells adhered to the wall, the culture medium was aspirated and 100 μL of culture medium containing different concentrations of cGNR@MSNs, DOTV-cGNR@MSNs and the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system of the present invention (0, 12.5, 50, 100, 200, 400 μg / mL) was added. The culture was continued. After 24 hours, the original culture medium was aspirated and rinsed 3 times with sterile PBS. 100 μL (1 mg / mL) of MTT solution was added under light-proof conditions, and the cells were placed in an incubator and cultured for 4 hours. The MTT solution was removed and 100 μL of DMSO solution was added to each well. The cells were shaken for 10 minutes and the OD was measured by a microplate reader. 570 The cell survival rate was calculated according to the following formula, and 5 replicate wells were set for each concentration.

[0128] Cell viability (%) = (OD of experimental group / OD of control group) × 100%.

[0129] The results of the investigation are as follows Figure 7 (A) and Figure 7 As shown in (B), it can be seen from the figure that after incubation for 24 h, blank cGNR@MSNs and DOTV-cGNR@MSNs showed no significant difference in the DOTV content, even at high concentrations (>100 μg·mL -1 ), also showed low cytotoxicity, which indicates that the nanocomposite material has good biosafety as a drug delivery carrier. The multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system (400 μg mL -1 ) has high cytotoxicity and induces apoptosis in MCF-7 and A549 cells. The cell survival rates after 24 h are 30.5% and 13.3%, respectively. CPT loaded Anti-CD40-DOTV-cGNR@MSNs at 12.5, 25, 50, 100, 200, and 400 μg·mL -1 At all concentrations, cell viability decreased significantly with increasing dose concentration. At all concentrations, the multi-armed Anti-CD40 antibody-mediated nano-drug delivery system had a higher cytotoxicity against A549 cells than against MCF-7 cells. The antibody promoted the endocytosis of CD40 receptor-positive cells and drug toxicity.

[0130] In summary, the developed multi-arm Anti-CD40 antibody-mediated nanoparticle dual-drug delivery system exhibits high cytotoxicity and strong tumor-killing ability against tumor cells. Compared to CD40 receptor-negative MCF-7 cells, the multi-arm Anti-CD40 antibody-mediated nanoparticle dual-drug delivery system exhibits even higher tumor-killing ability against CD40 receptor-positive A549 cells, enabling better targeted tumor therapy.

[0131] Example 4:

[0132] In this example, laser scanning confocal microscopy was used to characterize the uptake of the multi-armed Anti-CD40 antibody-mediated nano dual-drug delivery system by MCF-7 and A549 cells. The specific steps are as follows:

[0133] MCF-7 and A549 cells in the logarithmic growth phase were collected and divided into 5 × 10 4The cells were seeded at a density of 100 cells per dish into a 35mm single cell culture dish covered with a coverslip, 1 mL each, and cultured for 24 hours. After the cells adhered to the wall, the culture medium was aspirated and the culture medium containing Anti-CD40-DOTV-cGNR@MSNs (1 mL, 100 μg / mL) was added. The cells were placed in a cell culture incubator and cultured for 0 hours, 1 hour, 3 hours, 6 hours and 12 hours. After washing with PBS three times, the cells were fixed with 4% paraformaldehyde for 30 minutes and washed gently with PBS three times to remove excess paraformaldehyde. The coverslip was removed and sealed with 50% glycerol. Finally, the cells were observed under a fluorescence microscope. The results are as follows: Figure 7 (C) shown.

[0134] Figure 7 (C) CLSM images of the cellular uptake and CPT release of Anti-CD40-DOTV-cGNR@MSNs by MCF-7 and A549 cells. As can be seen from the figure, the fluorescence intensity of CPT increases significantly with increasing incubation time (3h, 6h, 12h, and 24h), indicating that cellular uptake and drug release are achieved in A549 and MCF-7. Due to the acidic microenvironment of the two tumor cells, the modified acylhydrazone bond on the nanocarrier is disconnected, opening the Anti-CD40 antibody nanogate and releasing CPT to generate a fluorescent signal. A549 cells exhibit a stronger fluorescence signal than MCF-7 cells, indicating that higher cellular uptake and drug release efficiency can be achieved in CD40 receptor-positive tumor cells.

[0135] Example 5:

[0136] In vivo multimodal imaging of model mice was performed with 200 μg·g -1 The injection concentration was 200 mg / L, and the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system and PBS buffer solution were injected intravenously according to the mouse body weight to obtain a CPT-loaded group and a blank group. White light LED was used to excite deep tissue. After 5 minutes of LED irradiation, a multimodal small animal imaging system (In-VivoXtreme, Bruker) was used to immediately capture fluorescence images (FL Imaging) and long afterglow luminescence images (PL Imaging). The images were taken again after an interval of 10 minutes. The shooting results are shown in the figure. Figure 8 shown.

[0137] Figure 8 Fluorescence and long afterglow luminescence imaging of mouse models injected with Anti-CD40-DOTV-cGNR@MSNs under LED irradiation. Figure 8In the AG fluorescence mode, the CPT fluorescence signal of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system group increased with the extension of irradiation time, indicating that LED irradiation promoted the release of CPT through photothermal conversion. In addition, in the PL mode, the mice showed clear and high signal-to-noise ratio PL images ( Figure 8 HN,λ em =730nm), although the PL signal weakens over time, the long afterglow luminescence property of the nanocomposite material can be repeatedly charged to obtain a recoverable and clear PL image.

[0138] Therefore, the prepared multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system can achieve photothermal conversion through LED irradiation to promote the release of CPT, realize PL imaging, and achieve long-lasting luminescence performance for repeated recharging, and has good long-lasting luminescence imaging capabilities.

[0139] Example 6:

[0140] The multi-armed Anti-CD40 antibody-mediated nano dual-drug delivery system of the present invention prepares a magnetic resonance imaging contrast agent through the chelation of the tetraazacyclic ring in DOTV with gadolinium ions. The specific preparation process is as follows:

[0141] The multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system (10 mg) was dissolved in Tris-HCl (10 mM) buffer solution, and then gadolinium (III) chloride hexahydrate (10 mL, 1 mM) was added. The mixture was stirred at 0°C for more than 6 hours, and the magnetic resonance imaging contrast agent was obtained by centrifugation.

[0142] Figure 9 Magnetic resonance imaging (T1-weighted MR images) of the multi-armed Anti-CD40 antibody-mediated nanoparticle dual-drug delivery system, Anti-CD40-DOTVcGNR@MSNs-Gd. As shown in the figure, the multi-armed Anti-CD40 antibody-mediated nanoparticle dual-drug delivery system was prepared into solutions of varying concentrations after chelating gadolinium ions. MRI images were acquired using a 3.0T MRI system. T1 longitudinal relaxation-weighted imaging showed clear MRI images and a high signal-to-noise ratio, and the MRI signal increased with increasing sample concentration. These results demonstrate the broad application prospects of this drug delivery system in the field of MRI.

[0143] In summary, the multi-armed Anti-CD40 antibody-mediated nano-drug delivery system of the present invention can achieve multimodal imaging combining fluorescence imaging, near-infrared persistent luminescence imaging, and magnetic resonance imaging. The present invention develops an Anti-CD40 antibody-gated nanocomposite particle drug delivery system, which further improves diagnostic accuracy by combining multimodal imaging, promotes the development of imaging-guided combination therapy, and exerts the synergistic effects of chemotherapy, immunotherapy, and photothermal therapy to optimize tumor treatment effects. It is suitable for the treatment of various malignant solid tumors.

[0144] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection of the present invention.

Claims

1. A method for preparing a multi-arm anti-CD40 antibody-mediated nano dual-drug delivery system, characterized in that: include: S1. Preparation of gold nanorods (GNRs); S2. Preparation of core-shell silica-coated gold nanorod nanoparticles GNR@MSNs; Preparation of S3.cGNR@MSNs Nanocomposites: Ga(NO3)3, Zn(NO3)2 and Cr(NO3)3 were dissolved in a water / ethanol solution to obtain a precursor mixed solution containing Ga(NO3)3, Zn(NO3)2 and Cr(NO3)3; The precursor mixed solution is then incubated with the GNR@MSNs nanomaterials. After the incubation, the mixture is centrifuged and dried to obtain a solid powder, which is then calcined to obtain the cGNR@MSNs nanocomposite material. Preparation of S4.DOTV-cGNR@MSNs: S4.

1. Preparation of NH2-cGNR@MSNs: The cGNR@MSNs nanocomposite was dispersed in tetrahydrofuran, and then 3-aminopropyltrimethoxysilane was added thereto. The mixture was refluxed under heating and stirring conditions. After the reaction, the mixture was centrifuged, washed, and dried to obtain NH2-cGNR@MSNs. S4.

2. Preparation of CH3CO-cGNR@MSNs: NH2-cGNR@MSNs, levulinic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride EDC·HCl, N-hydroxysuccinimide NHS and triethylamine were dissolved in tetrahydrofuran and stirred at room temperature. After the reaction, the mixture was centrifuged, washed and dried to obtain CH3CO-cGNR@MSNs. S4.

3. Preparation of NH2-N=cGNR@MSNs: CH3CO-cGNR@MSNs were dispersed in a methanol solution, to which a methanol solution containing hydrazine dihydrochloride and NaOH was added for reflux reaction 2. After the reaction, the mixture was centrifuged, washed, and dried to obtain NH2-N=cGNR@MSNs. S4.

4. Preparation of DOTA-cGNR@MSNs Nanoparticles NH2-N=cGNR@MSNs nanoparticles were dispersed in tetrahydrofuran, and EDC·HCl, NHS, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DOTA, and triethylamine were added thereto. The mixture was stirred at room temperature for 2 minutes. After the reaction, the mixture was centrifuged, washed, and dried to obtain DOTA-cGNR@MSNs nanoparticles. S4.

5. Preparation of DOTV-cGNR@MSNs: DOTA-cGNR@MSNs nanoparticles were dispersed in tetrahydrofuran, and EDC·HCl, NHS, 2-amino-4,6-divinylpyrimidine DOTV, and triethylamine were added thereto. The mixture was stirred at room temperature for 3 minutes. After the reaction, the mixture was centrifuged, washed, and dried to obtain DOTV-cGNR@MSNs. S5. Preparation of multi-armed anti-CD40 antibody-mediated nanoparticle dual-drug delivery system: Dissolving the anti-tumor drug in Tris-HCl buffer to obtain an anti-tumor drug loading solution; adding the anti-CD40 antibody and tris(2-carboxyethyl)phosphine hydrochloride to the Tris-HCl buffer, incubating, and obtaining an anti-CD40 antibody solution for later use; DOTV-cGNR@MSNs were mixed with the anti-tumor drug loading liquid and stirred for reaction. After the reaction, Anti-CD40 antibody solution was added to the reaction liquid and stirred for reaction. After the reaction, the mixture was centrifuged and washed to obtain a multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system.

2. The method for preparing the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system according to claim 1, characterized in that: In step S3, Ga 3+ 、Zn 2+ and Cr 3+ The final ion concentrations were 1 M, 0.5 M, and 0.002 M, respectively; The dosage ratio of the GNR@MSNs and precursor mixed solution is 500 mg:300 μL; The incubation is carried out at room temperature; The calcination conditions are: heating to 600° C. at a rate of 5° C. / min, and calcining at 600° C. for 1 hour.

3. The method for preparing the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system according to claim 1, characterized in that: In step S4.1, the ratio of the cGNR@MSNs nanocomposite material to 3-aminopropyltrimethoxysilane is 0.1 g: 1-2 mmol; the reflux reaction is carried out under stirring and reflux at 80° C. for 12 h; In step S4.2, the ratio of NH2-cGNR@MSNs, levulinic acid, EDC·HCl, NHS, and triethylamine is 0.1 g: 1~2 mmol: 2 mmol: 2 mmol: 2 mmol, and the stirring reaction time is 12 h; In step S4.3, the ratio of CH3CO-cGNR@MSNs, hydrazine dihydrochloride, and NaOH is 0.1 g: 0.5~1 mmol: 1~2 mmol, and the reflux reaction 2 is carried out at 80°C for 2 h. In step S4.4, the ratio of NH2-N=cGNR@MSNs nanoparticles, EDC·HCl, NHS, DOTA, and triethylamine is 0.1 g: 1~2 mmol: 1 mmol: 1 mmol: 1 mmol; the stirring reaction 2 is carried out at room temperature for 12 h; In step S4.5, the amount ratio of DOTA-cGNR@MSNs nanoparticles, EDC·HCl, NHS, 2-amino-4,6-divinylpyrimidine and triethylamine is 0.2 g: 2~3 mmol: 3 mmol: 3 mmol: 3 mmol; the stirring reaction 3 is carried out at room temperature for 12 h.

4. The method for preparing the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system according to claim 1, characterized in that: In step S5, the anti-tumor drug includes camptothecin CPT; The concentration of the anti-tumor drug loading solution is 0.2 mM and the pH value is 7.4; During the preparation of the Anti-CD40 antibody solution, the Anti-CD40 antibody, tris(2-carboxyethyl)phosphine hydrochloride, and Tris-HCl buffer were used in a ratio of 0.1 mL:0.2 mg:10 mL. The incubation conditions were 0°C for 2 hours. The concentration of the Anti-CD40 antibody was 3 µM. The dosage ratio of the DOTV-cGNR@MSNs, CPT loading solution, and Anti-CD40 antibody solution is 10 mg: 10 mL: 10 mL; the concentration of the CPT loading solution is 0.2 mM; The reaction was carried out under the conditions of continuous stirring at 0°C for 6 h.

5. The multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system prepared by the method according to any one of claims 1 to 4, characterized in that: In the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system, multi-arm DOTV and Anti-CD40 antibodies are self-assembled step by step on the surface of the cGNR@MSNs nanocomposite material to form a multilayer film structure; The cGNR@MSNs nanocomposite material is a spherical core-shell nanostructure, and ZnGa2O4:Cr is embedded in the core-shell nanostructure. 3+ Nanocrystal; the anti-tumor drug is loaded in the shell mesopores of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system.

6. The multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system according to claim 5, characterized in that: The multi-arm DOTV is evenly distributed in the shell and mesoporous channels of the cGNR@MSNs nanocomposite material, and the anti-CD40 antibody is distributed on the surface of the cGNR@MSNs nanocomposite material to form the outermost antibody layer; and the anti-tumor drug includes camptothecin.

7. Use of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system prepared by the method according to any one of claims 1 to 4, or the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system according to claim 5 or 6 in the preparation of a multimodal imaging preparation.

8. The use according to claim 7, characterized in that The multimodal imaging includes magnetic resonance imaging, long-afterglow luminescence imaging and fluorescence imaging.

9. Use of the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system prepared by the method according to any one of claims 1 to 4, or the multi-arm Anti-CD40 antibody-mediated nano dual-drug delivery system according to claim 5 or 6 in the preparation of drugs for treating tumors.

10. The use according to claim 9, characterized in that The tumor includes a malignant tumor that overexpresses the CD40 receptor.

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