Anti-PD-L1 and Anti-HER2 double-antibody guide nano-drug system as well as preparation method and application of anti-PD-L1 and Anti-HER2 double-antibody guide nano-drug system

Abstract

The invention provides an Anti-PD-L1 and Anti-HER2 double-antibody guide nano-drug system as well as a preparation method and application thereof, and belongs to the technical field of composite material preparation and biological medicine. Perovskite quantum dots with a wide photoluminescence spectrum are combined to the surface of accurately designed and prepared Gd-MOF to obtain a nano composite material, and then the nano composite material is used as a carrier to be covalently coupled with Anti-PD-L1 and Anti-HER2 antibodies and loaded with an anti-cancer drug to obtain a double-antibody guided nano drug system. The double-antibody guided nano-drug system provides a multi-mode imaging strategy for tumor diagnosis, exerts the treatment effect of combining multiple treatment methods in tumor combined treatment, and realizes the synergistic effect of chemical treatment, immunotherapy and photodynamic therapy; the invention can be applied to the development and clinical practice of tumor diagnosis technology and tumor treatment drugs.

Description

Technical Field

[0001] The present invention belongs to the field of composite material preparation and biomedicine technology, and specifically relates to an Anti-PD-L1 and Anti-HER2 dual antibody-guided nanomedicine system and a preparation method and application thereof. Background Art

[0002] In recent years, the incidence of malignant tumors has continued to rise, posing a major threat to public health. Traditional anticancer drugs have serious defects such as high toxicity and poor tumor region specificity. Therefore, it is necessary to find more effective and safer alternative treatments. Designing a drug delivery system that can accurately release drugs in cancer cells and delay the release of drugs in normal cells is considered to be a new strategy to solve these problems.

[0003] Metal-organic frameworks (MOFs) consist of metal ions linked by organic ligands and have properties such as highly ordered structures, tunable porosity, and large surface areas, making them suitable for a variety of applications. MOFs can serve as multifunctional platforms for catalysis, energy storage, and drug delivery. The three-dimensional lattice structure of MOFs facilitates the encapsulation of a variety of cargoes, from small molecules to DNA sequences and biomacromolecules. Due to their biosafety in biomedical applications, MOFs offer considerable promise for advancing disease diagnosis and treatment.

[0004] Coupling specific targeting molecules (including DNA, aptamers, proteins, etc.) to the surface of materials helps to deliver drugs to designated cells. In addition, the surface of materials is modified with nano-gates with stimulus-responsive mechanisms, which can control release using a series of triggering factors, including pH, light, temperature, and redox agents. Due to their unique design and easy functionalization, nano drug delivery systems have attracted widespread attention for their ability to protect drug molecules from degradation, thereby improving delivery efficiency. The prospect of clinical benefits has promoted the development of therapeutic platforms and image-guided combination therapies, highlighting their value in achieving efficient and synergistic effects.

[0005] Multimodal imaging plays a vital role in precision medicine, providing higher accuracy and resolution for disease diagnosis and evaluation of treatment effects. Various imaging modalities can work together to achieve the common goal of accurate disease diagnosis, including fluorescence, radionuclide, and magnetic resonance imaging (MRI). Immunotherapy stimulates the body's immune response to recognize and eliminate diseased cells, using immune agonist antibodies or immune checkpoint inhibitors for immune regulation. PD-1 (programmed death-1) and PD-L1 (programmed death ligand-1) are immune checkpoint molecules that play an important role in regulating the immune system. PD-1 is a membrane receptor protein that is mainly present on the surface of activated T cells, B cells, and monocytes, while PD-L1 is the ligand of PD-1. When PD-L1 binds to PD-1, it can inhibit the activation and proliferation of T cells, thereby reducing the attack on tumor cells. Therefore, the PD-1 and PD-L1 pathways have become important targets for tumor immunotherapy. Inhibiting the PD-1 or PD-L1 pathway can restore the ability of immune cells to attack tumor cells, and this treatment method is widely used in the treatment of various malignancies. Human epidermal growth factor receptor-2 HER2 is a member of the EGFR receptor tyrosine kinase family. Heterodimerization of this receptor with other members of the EGFR family is usually due to HER2 overexpression, leading to autophosphorylation of tyrosine residues within the cytoplasmic domain of the heterodimer and initiating various signaling pathways, leading to cell proliferation and tumorigenesis.

[0006] Therefore, it is necessary to develop a biomacromolecule-gated metal-organic framework nanodrug system and a multimodal imaging platform to simultaneously exert the synergistic effects of chemotherapy, immunotherapy, and photodynamic therapy (PDT), achieve targeted drug delivery and stimulus-responsive controlled release, and improve the effectiveness of precise diagnosis and tumor treatment. Summary of the invention

[0007] In view of some deficiencies in the prior art, the present invention provides an Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system and a preparation method and application thereof; the present invention combines perovskite quantum dots with a wide photoluminescence spectrum to the surface of a precisely designed and prepared Gd-MOF metal organic framework material to obtain a nanocomposite material, and then uses the nanocomposite material as a carrier to covalently couple Anti-PD-L1 and Anti-HER2 antibodies to the nanocomposite material through divinyl pyrimidine to obtain a dual antibody-guided nanodrug system; the antibody coupled to the surface of the dual antibody-guided nanodrug system acts as a nanogate to regulate the delivery of camptothecin chemotherapy drugs, and as a specific antibody to bind to target antigens overexpressed on the surface of tumor cells, thereby achieving "precision guidance" of the nanodrug system to tumor tissues, improving drug utilization efficiency and achieving optimal tumor treatment effects; the dual antibody-guided nanodrug system provides a multimodal imaging strategy for tumor diagnosis, exerts the therapeutic efficacy of combining multiple treatment methods in combined tumor treatment, achieves the synergistic effect of chemotherapy, immunotherapy and photodynamic therapy, and can be applied to the development and clinical practice of tumor diagnostic technology and tumor treatment drugs.

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0009] The present invention first provides a nanocomposite material, wherein the perovskite quantum dots are uniformly distributed on the surface of a Gd-MOF metal organic framework material; the Gd-MOF metal organic framework material is a three-dimensional cubic material with a diameter between 60 and 200 nm, an average size of about 120 nm, a porous structure, an average pore diameter of 1.8 nm, and a corresponding pore volume of 0.17 cm 3 ·g -1 .

[0010] The present invention also provides a method for preparing the above-mentioned nanocomposite material, the preparation method comprising:

[0011] (1) Preparation of Gd-MOF:

[0012] Gadolinium chloride hexahydrate and 1,2,4,5-tetrakis(4-carboxyphenyl)benzene were dissolved in N,N-dimethylformamide, and then acetic acid was added. The solution was ultrasonically treated and transferred to a reactor for hydrothermal reaction. The solution was then allowed to stand and cooled, and then centrifuged. The precipitate was washed with deionized water and ethanol, and then dried in a vacuum oven to obtain a MOF white solid.

[0013] (2) Preparation of PQDs:

[0014] PbX2 and CsX are added to a DMF solution and heated and stirred for a first reaction, and then 5-bromovaleric acid and oleylamine solution are added and heated and stirred for a second reaction to obtain a precursor solution; the precursor solution is added to deionized water at a certain temperature to obtain PQDs; X in the PbX2 and CsX is a mixture of one or more of Cl, Br and I;

[0015] (3) Preparation of nanocomposite materials: Gd-MOF was dispersed in tetrahydrofuran, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added, and the mixture was stirred to modify the surface of Gd-MOF;

[0016] Adding dithiodivinylpyrimidine (DVP) and PQDs to the modified Gd-MOF, reacting under stirring, and centrifuging after the reaction to obtain the nanocomposite material, which is recorded as DVP-MOF-PQDs;

[0017] The structural formula of the dithiodivinylpyrimidine is:

[0018]

[0019] Steps (1) and (2) are performed in no particular order.

[0020] Preferably, in step (1), the molar ratio of 1,2,4,5-tetrakis(4-carboxyphenyl)benzene to gadolinium chloride hexahydrate is 1:1-1:3;

[0021] The dosage ratio of N,N-dimethylformamide and acetic acid is 15-30mL:0.1-1.5mL;

[0022] The hydrothermal reaction conditions are 100-150° C. for 6-15 hours.

[0023] Preferably, in step (2), the molar ratio of PbX2, CsX, 5-bromovaleric acid and oleylamine is 1:1:5:5;

[0024] The conditions for the first heating and stirring reaction are: stirring and reacting at 60-80° C. for 40-60 min; the conditions for the second heating and stirring reaction are: stirring and reacting at 60-80° C. for 15 min;

[0025] The PQDs are inorganic perovskite quantum dots CsPbX3, where X is a mixture of one or more of Cl, Br and I.

[0026] Preferably, the PQDs include PQDs1 and PQDs2, wherein PQDs1 is CsPbCl3, λ em =504nm, the PQDs2 is CsPbBr0.75 I 2.25 ,λ em =653nm.

[0027] Preferably, in step (3), the usage ratio of Gd-MOF, EDC and NHS is 0.1-0.2 mg: 1 mol: 1 mol;

[0028] The MOF surface modification time is 4-8h;

[0029] The modified MOF, dithiodivinylpyrimidine (DVP) and PQDs are used in a ratio of 0.1-0.2 mg: 0.05 mmol: 0.05 mmol;

[0030] The reaction time under stirring is 4-8h.

[0031] Preferably, in step (3), the method for preparing dithiodivinylpyrimidine (DVP) comprises:

[0032] S1. Dissolve 3-chloropropylamine hydrochloride and sodium thiosulfate pentahydrate in a methanol aqueous solution, heat and stir to react, then add a methanol solution of iodine, continue the reaction in the dark, and then remove the solvent to obtain a yellow oily 3,3'-dithiodipropylamine;

[0033] S2. adding 3,3'-dithiobispropylamine to a methanol solvent, and then adding ethyl trifluoroacetate, reacting at low temperature, and then removing the methanol under vacuum to obtain 3,3'-dithiobispropylamine with NH2 at one end protected by trifluoroacetyl;

[0034] S3. Add 3,3'-dithiobispropylamine with trifluoroacetyl protection at one end of the product obtained in S2 and di-tert-butyl dicarbonate to tetrahydrofuran, stir to react, and obtain an intermediate product. Then remove the solvent, add a mixed solvent of ammonia water and methanol, stir and remove the solvent, and purify the residue by silica gel chromatography to obtain 3,3'-dithiobispropylamine with BOC protection at one end;

[0035] S4. 4,6-dichloropyrimidine-2-amine and oxalyl chloride were added to dichloromethane, heated and stirred to react 1, and after the reaction was completed, the solvent was removed under high vacuum to obtain 4,6-dichloro-2-isocyanate pyrimidine;

[0036] The single-end BOC-protected 3,3'-dithiobispropylamine and 4,6-dichloro-2-isocyanatopyrimidine obtained in S3 are dissolved in dichloromethane, heated and stirred to react 2, and after the reaction is completed, the solvent is removed and purified by silica gel chromatography to obtain product a;

[0037] S5. The product obtained in S4 is dissolved in tetrahydrofuran solvent with Pd(dppf)Cl2·CH2Cl2, potassium vinyl trifluoroborate and triethylamine, and the mixture is heated and stirred for reaction. After the reaction is completed, the solvent is removed and purified by chromatography to obtain product b;

[0038] The product b is dissolved and stirred in a solvent of trifluoroacetic acid and dichloromethane, and then the solvent is removed under vacuum, and purified by chromatography to obtain the disulfide divinyl pyrimidine linker molecule (DVP) responsive to glutathione (GSH).

[0039] Preferably, in step S1, the volume ratio of water to methanol in the methanol aqueous solution is 1:1-1:1.5; the molar ratio of iodine, 3-chloropropylamine hydrochloride and sodium thiosulfate pentahydrate in the iodine methanol solution is 1:2:2-1:2:2.5; the heating and stirring reaction conditions are: stirring at 80-90°C for 8-10h; the conditions for continuing the reaction in the dark are: stirring and reacting at 75-95°C for 6-8h;

[0040] In step S2, the molar ratio of 3,3'-dithiobispropylamine to ethyl trifluoroacetate is 1:1-1:1.5; the reaction conditions are 0°C for 2-3h;

[0041] In step S3, the molar ratio of di-tert-butyl dicarbonate to the product in S2 is 1:1-1:1.5; the reaction conditions are 20-30°C for 8-14h; the ammonia and methanol mixed solvent is 5mL of ammonia solution (30%wt) and 5mL of methanol; the silica gel chromatography purification mobile phase is petroleum ether and ethyl acetate, wherein petroleum ether:ethyl acetate=1:1;

[0042] In step S4, the molar ratio of 4,6-dichloropyrimidine-2-amine to oxalyl chloride is 1:4-1:5, and the conditions for heating and stirring reaction 1 are stirring and reacting at 35-45°C for 8-12h; the molar ratio of the product obtained in S3 to 4,6-dichloro-2-isocyanate pyrimidine is 1:1-1:1.5; the conditions for heating and stirring reaction 2 are stirring and reacting at 35-45°C for 8-12h; the mobile phase for silica gel chromatography purification is petroleum ether and ethyl acetate, wherein petroleum ether:ethyl acetate=3:2;

[0043] In step S5, the molar ratio of the S5 product to Pd(dppf)Cl2·CH2Cl2, potassium vinyl trifluoroborate, and triethylamine is 30:1:100:10-40:1:100:10; the heating and stirring reaction conditions are stirring the reaction at 80-100°C for 10-15h; and in the trifluoroacetic acid and dichloromethane solvent, the volume ratio of trifluoroacetic acid to dichloromethane is 1:1-1:1.5.

[0044] The present invention also provides the use of the nanocomposite material in preparing a double antibody-guided nanomedicine system.

[0045] The present invention also provides an Anti-PD-L1 and Anti-HER2 dual antibody-directed nanodrug system, wherein the dual antibody-directed nanodrug system is a mixture of an Anti-PD-L1 antibody-directed nanodrug and an Anti-HER2 antibody-directed nanodrug, and the molar ratio of the Anti-PD-L1 antibody-directed nanodrug to the Anti-HER2 antibody-directed nanodrug is 1:1;

[0046] In the Anti-PD-L1 antibody-directed nanomedicine, the nanocomposite material is a carrier, and the anti-tumor drug is loaded in the porous structure of the nanocomposite material; the Anti-PD-L1 antibody is covalently coupled to the nanocomposite material;

[0047] In the Anti-HER2 antibody-guided nanomedicine, the anti-tumor drug is loaded in the porous structure of the nanocomposite material; and the Anti-HER2 antibody is covalently coupled to the nanocomposite material.

[0048] Preferably, the anti-tumor drug is camptothecin.

[0049] The present invention also provides a method for preparing the above-mentioned Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system, the preparation method comprising:

[0050] The nanocomposite material DVP-MOF-PQDs is dispersed in a CPT loading solution, stirred for reaction, and centrifuged and washed after the reaction to obtain a CPT-loaded nanocomposite material;

[0051] Pre-treating the Anti-HER2 antibody and the Anti-PD-L1 antibody with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to obtain an Anti-HER2 antibody solution and an Anti-PD-L1 antibody solution, respectively;

[0052] The anti-HER2 antibody solution and the anti-PD-L1 antibody solution are incubated with the CPT-loaded nanocomposite material to obtain the anti-PD-L1 antibody-directed nanomedicine and the anti-HER2 antibody-directed nanomedicine;

[0053] The Anti-PD-L1 antibody-directed nanodrug and the Anti-HER2 antibody-directed nanodrug are mixed in equal molar ratios to obtain the Anti-PD-L1 and Anti-HER2 dual antibody-directed nanodrug system.

[0054] Preferably, the mass ratio of the nanocomposite material DVP-MOF-PQDs to the CPT loading solution is 1000:7; the stirring reaction is carried out at 25° C. for 12 hours;

[0055] The mass ratio of the Anti-HER2 antibody to tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is 2:1; the mass ratio of the Anti-PD-L1 antibody to tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is 2:1;

[0056] The pretreatment conditions were all stirring reaction at 0° C. for 4 hours, and after the reaction was completed, washing with a filter membrane (MWCO 30 kDa) was used to remove excess TCEP.

[0057] Preferably, the mass ratio of the Anti-HER2 antibody solution, the Anti-PD-L1 antibody solution, and the CPT-loaded nanocomposite material is 1:1000;

[0058] The incubation condition is stirring at 0°C for 12 hours.

[0059] The present invention also provides the use of the above-mentioned dual antibody-guided nanodrug system as a multimodal imaging agent; preferably, the multimodal imaging includes magnetic resonance imaging and fluorescence imaging.

[0060] The present invention also provides the use of the above-mentioned dual antibody-guided nanodrug system in the preparation of drugs for treating tumors; preferably, the tumors include but are not limited to malignant tumors that overexpress PD-L1 and HER2 receptors, such as liver and gastric tumors.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] (1) The dual-antibody-guided nanodrug system of Anti-PD-L1 and Anti-HER2 described in the present invention can achieve multimodal imaging combining fluorescence imaging and magnetic resonance imaging. This multimodal imaging method can be applied to the fields of biomedical imaging, tumor diagnosis, and surgical navigation. Compared with a single imaging mode, it can improve the resolution and accuracy of imaging diagnosis, improve the diagnostic accuracy of nanocomposites, and facilitate the use of image-guided combined therapy. In addition, the treatment system exerts the synergistic effect of chemotherapy, immunotherapy, and photodynamic therapy to improve the performance of tumor diagnosis and imaging-guided combined therapy.

[0063] (2) The dual antibody-guided nanodrug system of the present invention comprises MOF, PQDs, disulfide divinyl pyrimidine, loaded anti-tumor drugs and surface-modified antibodies, wherein Gd-MOF combines CsPbX3 PQDs for fluorescence imaging and Gd ions for MRI to achieve multimodal imaging; MOF is functionalized with disulfide divinyl pyrimidine to capture antibodies, which act as nanogates to block the anti-tumor drugs in the nanopores of the nanocomposite material and the antibodies can play an immunotherapeutic role. Meanwhile, DVP can act as a gating molecule responsive to GSH stimulation. The Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system, in the presence of GSH, disconnects the disulfide bonds modified on the surface of the composite nanomaterial, opens the Anti-PD-L1 or Anti-HER2 antibody nanogate, and achieves the controlled release of the CPT loaded with drugs.

[0064] The dual antibody-guided nanodrug system of the present invention combines chemotherapy, immunotherapy and photodynamic therapy to achieve a synergistic effect in tumor treatment, and provides a new multimodal imaging method for accurate diagnosis of tumors, thereby improving the effect of tumor treatment.

[0065] (3) The Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system of the present invention realizes the targeted delivery of the loaded drug CPT to specific tumor cells. The Anti-PD-L1 and Anti-HER2 antibodies can recognize and bind to the PD-L1 and HER2 receptors overexpressed on the surface of tumor cells. The antibodies can guide the drug delivery system to target tumor cells and improve the efficiency of drug uptake by cells. 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.

[0066] (4) The Anti-PD-L1 and Anti-HER2 antibodies not only promote the encapsulation effect of the antibody nanogate on the drug molecules, achieving a high delivery efficiency of targeted drug delivery, but also improve the binding force between the antibody and the surface receptors of tumor cells. They are suitable for the treatment of various tumors, especially for the combined treatment of malignant tumors with overexpression of PD-L1 or HER2 receptors. They can improve the safety of the delivery system and the therapeutic effect of chemical drugs, realize the combined treatment strategy of immunotherapy and chemotherapy, and improve the medication experience and treatment effect of tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Schematic diagram of the preparation method of the dual antibody-guided nanodrug system and its application in tumor diagnosis and treatment drugs.

[0068] Figure 2Scanning and transmission electron microscopy images; in the figure, (A) SEM image of MOF; (B) TEM image of MOF; (C) TEM image of nanocomposite material (DVP-MOF-PQDs); (D) TEM image of Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system.

[0069] Figure 3 Nitrogen adsorption-desorption isotherms of Gd-MOF and nanocomposites (DVP-MOF-PQDs).

[0070] Figure 4 Pore ​​volume and pore size distribution of Gd-MOF and nanocomposites (DVP-MOF-PQDs).

[0071] Figure 5 FTIR spectra of Anti-PD-L1 and Anti-HER2 dual antibody-directed nanodrug system: (a) 1,2,4,5-tetrakis(4-carboxyphenyl)benzene; (b) MOF; (c) nanocomposite material (DVP-MOF-PQDs); (d) antibody-directed nanodrug system.

[0072] Figure 6 UV-visible absorption spectra of Gd-MOF, divinylpyrimidine disulfide (DVP), and nanocomposites (DVP-MOF-PQDs).

[0073] Figure 7 Fluorescence emission spectra of Gd-MOF and nanocomposites (DVP-MOF-PQDs).

[0074] Figure 8 Stimulus response and CPT release behavior of Anti-HER2 and Anti-PD-L1 antibody-directed nanodrug systems: Kinetic curves of GSH reduction-responsive controlled release of anti-tumor drugs in nanodrug systems of (A) Anti-HER2 and (B) Anti-PD-L1 antibodies.

[0075] Fig. 9 Optical imaging of tumor-bearing mice for in vivo tumor diagnosis of antibody-guided nanodrug system; (A) shows the in vivo optical imaging and metabolic distribution of Anti-HER2-guided nanodrug system, Anti-PD-L1-guided nanodrug system or 1:1 mixture of dual antibody-guided nanodrug system 2 hours after administration; (B) shows the biological distribution of antibody-guided nanodrug system as a tumor diagnostic probe in tumors and important organs.

[0076] Fig.10Figure 2 shows the magnetic resonance imaging and in vivo tumor diagnosis of the antibody-directed nanodrug system; in the figure, (A) is the longitudinal relaxation T1 of the antibody-directed nanodrug system; (B) is the transverse relaxation T2 relaxation rate curve changing with the concentration of the antibody-directed nanodrug system; (C) is the in vitro T1 and T2 magnetic resonance imaging of antibody-directed nanodrug systems of different concentrations; (D) is the in vivo diagnostic effect of the antibody-directed nanodrug system on AGS tumor-bearing mice and the T1-weighted MR image.

[0077] Fig.11 Cell survival rate of HepG2 cells (A) and AGS cells (B) after incubation with MOF, nanocomposites, and dual antibody-guided nanodrug system for 24 hours.

[0078] Fig.12 The in vivo tumor therapeutic effect of the antibody-guided nanodrug system on tumor-bearing mice and the combined therapeutic effect of chemotherapy, immunotherapy and photodynamic therapy; (A) is the tumor growth curve of AGS tumor-bearing mice after 20 days of continuous drug administration; (B) is the tumor weight of AGS tumor-bearing mice in different treatment groups after 20 days of continuous drug administration; (C) is the H&E staining result of tumor tissue of mice in different treatment groups at the end of treatment. DETAILED DESCRIPTION

[0079] The present invention is 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. In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. The sources, trade names, and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are not specifically stated, and are the same as the sources indicated for the first time; the reagents, materials, etc. involved are not specifically stated, and are all obtained for commercial purposes.

[0080] In the following examples, BALB / c mice were purchased from Cavens Laboratory Animal Co., Ltd., all of which were female and 2-3 weeks old. HepG2 tumor cells were purchased from Wuhan Pronocell Life Science Co., Ltd. (CL-0103); AGS tumor cells were purchased from Wuhan Pronocell Life Science Co., Ltd. (CL-0022).

[0082] Example 1: Preparation of nanocomposite materials

[0083] (1) Preparation of Gd-MOF:

[0084] 0.7 mmol of gadolinium chloride hexahydrate and 0.5 mmol of 1,2,4,5-tetrakis(4-carboxyphenyl)benzene were dissolved in 15 mL of N,N-dimethylformamide, and then 1 mL of acetic acid was added thereto, and ultrasonication was performed until the solution was completely dissolved. Then, the mixture was transferred to a reactor and heated at 120° C. for 12 hours. After the reaction was completed, the mixture was cooled, and then the product was centrifuged at a speed of 10,000 rpm, washed with ethanol and deionized water, and then dried in a vacuum drying oven at 60° C. for 12 hours to obtain the Gd-MOF.

[0085] The morphology and chemical composition of the synthesized Gd-MOF were comprehensively analyzed using field emission scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 2 As shown in the SEM image of A, the morphology analysis shows that the nanoparticles are three-dimensional cubic materials; Figure 2 B is a transmission electron microscope image of Gd-MOF. It can be seen from the figure that the diameter of Gd-MOF is between 60-200nm, and it is in a uniform cubic shape with an average size of about 120nm. Figure 3 The nitrogen adsorption-desorption isotherm analysis showed that Gd-MOF has a microporous structure, and the calculated BET surface area is 391 m 2 ·g -1 . Figure 4 The pore size distribution analysis shows that the Gd-MOF has a narrow pore size range of 1 to 5 nm, with an average pore size of 1.8 nm and a corresponding pore volume of 0.17 cm 3 ·g -1 .

[0086] (2) Preparation of disulfide divinyl pyrimidine linker molecules (DVP) with glutathione response:

[0087] S1. Add 21.50mmol of 3-chloropropylamine hydrochloride and 21.72mmol of sodium thiosulfate pentahydrate into a double-necked flask containing 80mL of methanol aqueous solution (50%wt), heat to 80°C, and stir magnetically for 9 hours to generate a sulfate intermediate; then add 50mL of methanol solution containing 10.54mmol of iodine and continue stirring for 15 hours. After stirring, remove the methanol solvent under high vacuum, then add 20mL of sodium hydroxide solution (6M) to the remaining solution, extract with chloroform three times, 20mL each time, separate the organic layer with a separatory funnel, and then concentrate under reduced pressure to obtain a light yellow liquid, i.e., 3,3'-dithiobispropylamine.

[0088] S2. Dissolve 11 mmol of 3,3'-dithiobispropylamine in 10 mL of methanol, slowly add 11 mmol of ethyl trifluoroacetate at 0°C, maintain magnetic stirring at 0°C for 2 hours, and then naturally heat the reaction mixture to room temperature. After TLC analysis shows that the reaction is complete, remove the methanol solvent under high vacuum to obtain a light yellow oily liquid, i.e., 3,3'-dithiobispropylamine with NH2 protected at one end by trifluoroacetyl.

[0089] S3. Add 10 mmol of the product of S2 to 10 mL of tetrahydrofuran, then add 8 mL of tetrahydrofuran containing 11 mmol of di-tert-butyl decarbonate to the above solution, reflux and stir at 25°C for 12 hours, TLC analysis shows that the reaction is complete, remove the organic solvent under high vacuum to obtain an intermediate product. Then add 4.3 mmol of the intermediate product to 5 mL of methanol, add 5 mL of 30% ammonia water, reflux and stir at 25°C for 20 hours to remove the trifluoroacetyl group, and obtain a light yellow liquid, i.e., 3,3'-dithiodipropylamine with single-end NH2 protected by BOC.

[0090] S4. Add 12.5 mmol of 4,6-dichloropyrimidine-2-amine to 15 mL of dichloromethane and mix well. Then add 58.0 mmol of dichloro oxalic acid. Reflux at 40° C. and stir magnetically for 10 hours. Remove the organic solvent under high vacuum to obtain a yellow solid, i.e., 4,6-dichloro-2-isocyanatopyrimidine.

[0091] 3.75 mmol of 4,6-dichloro-2-isocyanatopyrimidine and 3.57 mmol of the product of S3 were dissolved in 20 mL of dichloromethane, heated to 40°C and stirred for 7 hours to obtain a crude product, which was purified by silica gel column chromatography to obtain the product as a yellow solid.

[0092] S5. 2.02 mmol of the product of S4, 0.06 mmol of Pd(dppf)Cl2·CH2Cl2, 6.06 mmol of potassium vinyl trifluoroborate and 0.45 mmol of triethylamine were dissolved in 30 mL of tetrahydrofuran solution, mixed evenly, stirred and refluxed at 90°C for 12 hours, and then the solvent was removed under high vacuum. The residue was purified by silica gel column chromatography to obtain an intermediate product, and then the intermediate product was dissolved in 5 ml of a TFA / CH2Cl2 mixed solution with a TFA / CH2Cl2 volume ratio of 1:1. The mixture was stirred at room temperature for 6 hours, and then the solvent was removed under high vacuum to obtain a disulfide divinyl pyrimidine linker molecule with glutathione response.

[0093] The structural formula of the dithiodivinylpyrimidine is:

[0094]

[0095] (3) Preparation of PQDs:

[0096] In this step, the composition of PQDs is adjusted by changing the ratio of halogen ions X, and PQDs1 and PQDs2 are prepared respectively. When X is ..., PQDs1 is prepared, and the PQDs1 is CsPbCl3, λ em =504nm; when X is a mixture of Br and I, wherein the molar ratio of Br to I is 1:3, PQDs2 is prepared, wherein PQDs2 is CsPbBr 0.75 I 2.25 ,λ em =653nm, the specific preparation method is as follows. Preparation of PQDs1: Add 0.4mmol PbCl2 and 0.4mmol CsCl to a flask containing 10mL DMF solution, heat to 70°C, and stir magnetically for 50min, then continuously and rapidly inject 2mmol 5-bromovaleric acid and 2mmol oleylamine solution into the above solution. Continue heating and stirring for 15min to obtain a precursor solution. Then, at 70°C, quickly add 2mL of the precursor solution into 25mL of deionized water to obtain PQDs1 (CsPbCl3, λ em =504nm).

[0097] Preparation of PQDs2: 0.1mmol PbBr2, 0.1mmol CsBr, 0.3mmol PbI2, and 0.3mmol CsI were added to a flask containing 10mL DMF solution, and heated and stirred for 50min at 70℃ for the first time. Then, 2mmol 5-bromovaleric acid and 2mmol oleylamine solution were continuously and rapidly injected into the above solution. The second heating and stirring was performed at 70℃ for 15min to obtain a precursor solution. Then, 2mL of the precursor solution was quickly added to 25mL of deionized water at 70℃ to obtain PQDs2 (CsPbBr 0.75 I 2.25 ,λ em =653nm).

[0098] (4) Preparation of nanocomposites:

[0099] 0.1g of Gd-MOF was dispersed in 10ml of tetrahydrofuran, 0.1mmol of NHS and 0.1mmol of EDC were added, and the mixture was stirred at 25°C for 6 hours to modify the MOF surface; 0.05mmol of DVP linker molecules and 0.05mmol of PQDs were added to the modified Gd-MOF and stirred for 6 hours, and the mixture was centrifuged at 10000rpm after stirring, and washed to obtain a nanocomposite material, which was recorded as DVP-MOF-PQDs. When PQDs was PQDs1, the nanocomposite material was recorded as DVP-MOF-PQDs1; when PQDs was PQDs2, the nanocomposite material was recorded as DVP-MOF-PQDs2. Figure 2 A and 2B show that MOF is a three-dimensional cubic structure with uniform morphology, indicating that MOF was successfully synthesized. After the PQDs were modified, the Gd-MOF was covalently coupled with the carboxyl groups on the surface of MOF due to the presence of oleylamine ligands in the preparation of PQDs. Figure 2 From the TEM image of C, it can be seen that PQDs are evenly distributed on the surface of MOF.

[0100] Figure 3 and Figure 4 It shows that DVP-MOF-PQDs2 maintains the porous structure of MOF, presents a type I isotherm, and has a BET surface area of ​​316 m2 g -1 , pore volume is 0.17cm3·g -1 The average pore size is 1.7 nm, which indicates that it is a promising carrier for drug delivery.

[0101] from Figure 6 It can be seen that DVP-MOF-PQDs1 and DVP-MOF-PQDs2 show UV-visible absorption peaks corresponding to disulfide divinyl pyrimidine, PQDs1 and PQDs2 at 240nm, 503nm and 613nm, respectively, indicating that disulfide divinyl pyrimidine and PQDs were successfully modified on the surface of MOF material.

[0102] from Figure 7 It can be seen that Gd-MOF exhibits an apparent fluorescence signal from the aggregation-induced emission characteristics of the H4TCPB ligand, with an emission peak at 405nm under 323nm excitation, and the nanocomposite of DVP-MOF-PQDs (1 or 2) retains the fluorescence emission peaks corresponding to Gd-MOF and PQDs 1 or 2 at 400nm, 504nm and 653nm, respectively. This shows that PQDs have been successfully modified on the MOF material, proving the successful preparation of the nanocomposite.

[0103] Example 2: Preparation of dual antibody-directed nanomedicine system

[0104] S1. Disperse 10 mg of the nanocomposite in 10 mL of CPT loading solution (0.2 mM) to obtain the CPT-loaded nanocomposite (DVP-MOF-PQDs).

[0105] S2. 25 μg of Anti-PD-L1 antibody and Anti-HER2 antibody were respectively added to 10 mL of Tris-HCl buffer, and then 10 μL of 0.3 mmol / L tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the mixture was stirred at 0°C for 4 hours. After the reaction, the mixture was washed with a filter membrane (MWCO 30 kDa) to remove excess TCEP to obtain pretreated Anti-PD-L1 antibody solution and Anti-HER2 antibody solution.

[0106] S3. Add 10 mL of the pretreated Anti-PD-L1 antibody solution and 10 mL of the Anti-HER2 antibody solution into 5 mg of the CPT-loaded nanocomposite material, respectively, stir at 0°C for 12 hours, centrifuge after stirring, and wash the solid precipitate thoroughly with Tris-HCl buffer to obtain Anti-PD-L1-guided nanodrugs and Anti-HER2-guided nanodrugs, respectively.

[0107] S4. Mix the Anti-PD-L1 directed nanodrug and the Anti-HER2 directed nanodrug in equal proportions to obtain the Anti-PD-L1 and Anti-HER2 dual antibody directed nanodrug system.

[0108] Figure 2 D is a TEM image of the Anti-PD-L1 antibody-guided nanodrug system. It can be seen from the figure that a layer of Anti-PD-L1 antibody is coated around DVP-MOF-PQDs. The thickness of the Anti-PD-L1 antibody layer on the surface of the nanocomposite is about 12nm, which is consistent with the size predicted by dynamic simulation. Since MOF is a porous structure, CPT small molecules can be loaded in the pores of MOF, and then the antibody is coupled to the MOF surface, which can prevent the premature release of CPT in the nanocomposite and construct a drug delivery system for combined therapy.

[0109] Figure 5 The FTIR spectrum of the nanocomposite material confirmed that the Anti-PD-L1 antibody was constructed on the surface of the nanocomposite material. As can be seen from the figure, the FTIR spectrum of the 1,2,4,5-tetrakis(4-carboxyphenyl)benzene ligand is similar to that of Gd-MOF at 3020 cm -1 、1602cm -1 and 1405cm -1There are similar absorption peaks at 2930 cm-1, corresponding to the CH stretching vibration of the aromatic ring, the C=O stretching vibration of the carboxyl group, and the CC stretching vibration of the aromatic ring. After functionalization with DVP and PQDs, the FTIR spectrum of DVP-MOF-PQDs shows several obvious features, including the alkyl chain in the linker at 2930 cm-1. -1 CH stretching vibration at 1668 and 1545 cm -1 The amide I and amide II bands appeared at 1420 cm -1 The stretching vibration of C=C and C=N caused by the nearby pyrimidine ring. After coupling with the antibody, the FTIR spectrum showed that the amide I and amide II bands were enhanced, which proved that the antibody nanogate was constructed on the surface of the nanocomposite material and the Anti-PD-L1 antibody-guided nanodrug system was successfully obtained.

[0110] Embodiment 3:

[0111] In this example, a fluorescence spectrometer was used to monitor the release kinetics of CPT in the Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system. The fluorescence emission spectrum of CPT at λex = 365 nm, λem = 440 nm was recorded to investigate the stimulation response and controllable release ability of the Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system under GSH triggering.

[0112] like Figure 8 A and Figure 8 B is shown in the release curve of CPT over time. Under the condition of physiological pH of 7.4, the pre-release after 6 hours is negligible (<6%). "Zero pre-release" will improve the efficiency of targeted delivery and reduce side effects on healthy tissues. After adding GSH (5mM), the release rate of CPT is very fast and reaches equilibrium after 5.5 hours. It shows that the drug-carrying system directed by Anti-HER2 and Anti-PD-L1 can release chemotherapy drugs in a controlled manner, which proves that the DVP connector of the dual antibody-directed nanodrug system mixed in equal proportions of the two can also be selectively disconnected, the antibody is detached, and the drug is released. In the presence of GSH, the co-delivery and stimulus-responsive controlled release of antibodies and anti-tumor drugs simulates the tumor environment with elevated GSH levels, allowing chemotherapy and immunotherapy to be combined for combined treatment.

[0113] Embodiment 4:

[0114] In this example, in vivo fluorescence imaging was performed on model mice to investigate the in vivo biomedical imaging capability of the antibody-guided nanodrug system in tumor diagnosis. The specific steps are as follows:

[0115] (1) Construction of model mice:

[0116] Construction of AGS tumor-bearing mice: BALB / c mice were subcutaneously injected with AGS tumor cells, with an injection volume of approximately 2×10 6 Tumor cells were cultured for 15 days until the tumor volume reached 50-100mm 3 , and AGS tumor-bearing mice were obtained.

[0117] Construction of HepG2 tumor-bearing mice: BALB / c mice were subcutaneously injected with HepG2 tumor cells, with an injection volume of approximately 2×10 6 Tumor cells were cultured for 15 days until the tumor volume reached 50-100mm 3 , and obtained HepG2 tumor-bearing mouse model.

[0118] (2) Experimental groups:

[0119] The mice were divided into six groups according to the experimental requirements. The specific groups are as follows:

[0120] Blank group: 200 μl PBS was injected into the tail vein of nude mice without tumor;

[0121] Anti-PD-L1 directed nanodrug system + nude mouse group: Anti-PD-L1 directed nanodrug system was injected into the tail vein of nude mice without tumors, and the drug was administered according to the weight of mice (10 mg kg -1 ), if the weight of the mouse is 20 g, the mass of the contained Anti-PD-L1 directed nanodrug system is 200 μg;

[0122] Anti-HER2 directed nanodrug system + HepG2 tumor-bearing mice group: Anti-HER2 directed nanodrug system was injected into the tail vein of HepG2 tumor-bearing mice, and the drug was administered according to the weight of mice (10 mg kg -1 );

[0123] Double antibody-guided nanodrug system + HepG2 tumor-bearing mice group: The double antibody-guided nanodrug system was injected into the tail vein of the HepG2 tumor-bearing mice group, and the drug was administered according to the weight of the mice (10 mg kg -1 );

[0124] Anti-PD-L1 directed nanodrug system + AGS tumor-bearing mice group: Anti-PD-L1 directed nanodrug system was injected into the tail vein of AGS tumor-bearing mice, and the drug was administered according to the weight of the mice (10 mg kg -1 );

[0125] Double antibody-guided nanodrug system + AGS tumor-bearing mice group: Double antibody-guided nanodrug system was injected into the tail vein of AGS tumor-bearing mice, and the drug was administered according to the weight of mice (10 mg kg -1 ).

[0126] (3) In vivo fluorescence imaging:

[0127] Two hours after administration, a microscope with two optical windows (λ ex1 =410nm,λ ex2 The mice obtained from each group in step (2) were scanned using a multimodal small animal imaging system (in vivo Xtreme, Bruker) with a wavelength of 535 nm. The in vivo imaging results are shown in Figure 2. Fig. 9 As shown in A.

[0128] from Fig. 9 A It can be seen that the nude mice injected with the Anti-PD-L1-guided nanodrug system showed brighter imaging under two optical windows compared with the nude mice injected with only the PBS group. The Anti-PD-L1-guided nanodrug system is evenly distributed in the nude mice without tissue enrichment. In the HepG2 tumor-bearing mouse group, the Anti-HER2-guided nanodrug system was injected into the tail vein, and the fluorescence window 1 showed a strong fluorescence. The fluorescence signal was the overlap of the fluorescence of CPT and PQD1 in the Anti-HER2-guided nanodrug system and showed an enhanced fluorescence signal, indicating that the Anti-HER2-guided nanodrug system can be targeted and enriched around the HepG2 tumor. In addition, the AGS tumor-bearing mice injected with the Anti-PD-L1-guided nanodrug system showed an increase in the fluorescence signal at the tumor tissue under two optical windows, of which optical window 1 showed the fluorescence of CPT, and optical window 2 showed the fluorescence of PQD2 of the Anti-PD-L1-guided nanodrug system, indicating that the Anti-PD-L1-guided nanodrug system can be targeted and enriched in AGS tumor cells. Fluorescence imaging of HepG2 tumor-bearing mice and AGS tumor-bearing mice injected with the dual antibody-guided nanodrug system also demonstrated that the antibody-guided nanodrug system can be targeted to the tumor site.

[0129] In this step, the mice obtained from each group were euthanized to analyze the distribution of the antibody-guided nanodrug system in tumor tissues and major organs (heart, liver, spleen, lungs, and kidneys). The analysis results are as follows: Fig. 9 As shown in B.

[0130] from Fig. 9 B It can be seen that the tumor tissues of AGS tumor-bearing mice and HepG2 tumor-bearing mice have different degrees of fluorescence enhancement signals, which is consistent with the results described above. After the non-tumor-bearing nude mice were injected with the Anti-PD-L1-guided nanodrug system, they had weaker fluorescence mainly in the liver and kidneys, which was mainly related to the metabolism of the drug in the body.

[0131] In summary, the combination of antibodies and nanocomposites can identify tumor markers and promote early detection of tumors, thereby supporting accurate tumor diagnosis and treatment applications. The Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system of the present invention improves the quality of tumor imaging compared to the single Anti-PD-L1-guided nanodrug system and Anti-HER2-guided nanosystem, and has good reliability and versatility. Therefore, it can be used as a PL probe for tumor diagnosis.

[0132] Embodiment 5:

[0133] In this example, the MRI contrast agent effect of the Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system was evaluated by in vitro and in vivo experiments, and T1 and T2 weighted MRI analysis was performed using a 7.0T Bruker BioSpec instrument. The specific steps are as follows:

[0134] (1) In vitro experiments:

[0135] Different concentrations of antibody-guided nano-drug systems were set, and the antibody-guided nano-drug systems of different concentrations were placed on a nuclear magnetic resonance instrument for T1 and T2 scanning to obtain magnetic resonance images. The repetition time (TR) and echo time (TE) were set to TR = 3000.0 ms and TE = 11.0 ms, respectively.

[0136] The results are as follows Fig.10 A and Fig.10 As shown in B, by plotting the relationship between 1 / T1 and 1 / T2 values ​​and the concentration of the antibody-directed nanocomposite, the longitudinal relaxation rate r1 and the transverse relaxation rate r2 were calculated to be 0.02722 μg, respectively. -1 mL s -1 and 0.03994 μg -1 mL s -1 This indicates that there is a strong linear relationship between the T1 or T2 relaxation rate and the concentration of the antibody-directed nanodrug system. Fig.10 As shown in C, MRI imaging shows that as the particle concentration increases, the signal intensity on the T1-weighted image increases, producing a brighter image, and the signal intensity on the T2-weighted image decreases, producing a darker image. This also shows that there is a strong relationship between the concentration of the antibody-guided nanodrug system and the magnetic resonance signal, indicating that the antibody-guided nanodrug system has the potential to be used as a magnetic resonance imaging probe.

[0137] (2) In vivo experiments:

[0138] The antibody-directed nanodrug system was injected into the tail vein of AGS tumor-bearing mice, and the drug was administered according to body weight (100 mg kg -1), and MRI scan was performed 2 hours later. Spin echo sequence was used to obtain in vivo MRI images, TR = 437.7ms, TE = 8.5ms.

[0139] The results are as follows Fig.10 As shown in D, it can be seen from the figure that in the T1-weighted magnetic resonance imaging of AGS tumor-bearing mice injected with the antibody-directed nanodrug system, the magnetic resonance signal of the tumor area is brighter than that of the uninjected control group mice. This indicates that the antibody-directed nanodrug system is enriched in the tumor area.

[0140] In summary, the prepared antibody-guided nanodrug system can be used as a magnetic resonance contrast agent for in vivo magnetic resonance imaging, which is beneficial for precision medical diagnosis.

[0141] Embodiment 6:

[0142] In this example, the MTT method was used to evaluate the in vitro cytotoxicity of Gd-MOF, DVP-MOF-PQDs, Anti-HER2 directed nanodrug system, Anti-PD-L1 directed nanodrug system, and the Anti-PD-L1 and Anti-HER2 dual antibody directed nanodrug system of the present invention on cancer cells (HepG2 and AGS cells). The specific steps are as follows:

[0143] HepG2 and AGS cells in the logarithmic growth phase were taken and approximately 5×10 4 The cells were inoculated 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 then 100 μL of culture medium containing different concentrations of Gd-MOF, DVP-MOF-PQDs, Anti-HER2 guided nanodrug system, Anti-PD-L1 guided nanodrug system and Anti-PD-L1 and Anti-HER2 dual antibody guided nanodrug system of the present invention (0, 12.5, 25, 50, 100, 200, 300 μg / mL) was added respectively. After culturing for 24 hours, the original culture medium was aspirated and rinsed with sterile PBS for 3 times.

[0144] Add 100 μL (1 mg / mL) MTT solution under light-proof conditions and continue culturing in an incubator for 4 hours. After the incubation, remove the MTT solution and add 100 μL dimethyl sulfoxide (DMSO) solution to each well. Oscillate for 10 minutes. Measure OD570 with an enzyme reader and calculate the cell survival rate. The settlement results are as follows: Fig.11 shown.

[0145] from Fig.11It can be seen that Gd-MOF and DVP-MOF-PQDs have high biosafety against HepG2 and AGS cells in the range of 12.5 to 300 μg mL -1 Within the concentration range of 300 μg·mL, the cell survival rate remained above 90%. After treatment with the Anti-HER2-guided nanodrug system, the survival rate of HepG2 and AGS cells decreased significantly with the increase of the concentration of the Anti-HER2-guided nanodrug system. -1 The viability of HepG2 and AGS cells was 8.9% and 16.9% at 300 μg mL -1 Under the Anti-PD-L1 guided nanodrug system, the survival rates of HepG2 and AGS cells were 45.6% and 8.2%, respectively. In contrast, after treatment with the Anti-PD-L1 and Anti-HER2 dual antibody guided nanodrug system described in the present invention, the survival rates of HepG2 and AGS cells were only 6.15% and 5.57%. This shows that the antibody-guided nanodrug delivery system uses Anti-HER2 and Anti-PD-L1 as targeting ligands to promote cell uptake and drug toxicity of the corresponding antigen-positive cells.

[0146] Embodiment 7:

[0147] This example measures the in vitro anti-tumor effect of the Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system of the present invention, and the specific steps are as follows:

[0148] BALB / c mice were injected subcutaneously with AGS tumor cells. When the tumor volume of the tumor-bearing mice (AGS) reached about 100 mm 3 The mice were randomly divided into 5 groups, including PBS, DVP-MOF-PQDs combined with PDT, Anti-PD-L1 guided nanodrug system without CPT combined with PDT, Anti-PD-L1 guided nanodrug system combined with PDT and Anti-PD-L1 and Anti-HER2 dual antibody guided nanodrug system group, with 3 mice in each group.

[0149] PBS group: AGS tumor-bearing mice were injected with 200 μl PBS via the tail vein twice a day;

[0150] DVP-MOF-PQDs combined with PDT group: AGS tumor-bearing mice were injected with antibody-free DVP-MOF-PQDs nanocomposites twice a day through the tail vein. The dose was based on body weight (5 mg kg -1 ), and further irradiated the tumor site with full-spectrum LED (10 W) for 10 min 2 h after tail vein injection, twice a day.

[0151] Anti-PD-L1 directed nanodrug system without CPT combined with PDT group: Anti-PD-L1 directed nanodrug system without CPT was a system that omitted the step of loading CPT in the CPT loading solution before coupling the DVP-MOF-PQDs nanocomposite with the Anti-PD-L1 antibody. The anti-PD-L1 directed nanodrug system without CPT was injected into the tail vein of AGS tumor-bearing mice twice a day, and the drug was administered according to body weight (5 mg kg -1 ), and further irradiated the tumor site with full-spectrum LED (10 W) for 10 min 2 h after tail vein injection, twice a day.

[0152] Anti-PD-L1 directed nanodrug system combined with PDT group: AGS tumor-bearing mice were injected with the anti-PD-L1 directed nanodrug system twice a day through the tail vein, and the drug was administered according to body weight (5 mg kg -1 ), and further irradiated the tumor site with full-spectrum LED (10 W) for 10 min 2 h after tail vein injection, twice a day.

[0153] Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system group: AGS tumor-bearing mice were injected with the Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system twice a day through the tail vein, and the drug was administered according to body weight (5 mg kg -1 ).

[0154] The above combined PDT groups were treated with 10W full-spectrum LED (10 minutes) 2 hours after each intravenous injection. Tumor growth was monitored every 2 days, and tumor volume was calculated using the formula V = L × W 2 / 2, where L and W are the longest and shortest dimensions respectively. The result is as follows Fig.12 shown.

[0155] from Fig.12 A shows that in the AGS tumor-bearing group, compared with PBS (tumor volume = 2261 mm 3 ), nanocomposite material DVP-MOF-PQDs+PDT (tumor volume = 1865 mm 3 ) and the Anti-PD-L1 directed nanodrug system without CPT combined with PDT treatment (tumor volume = 926mm 3 Compared with the mice treated with anti-PD-L1 nanomedicine system combined with PDT (tumor volume = 100 mm 3 ) and the Anti-PD-L1 and Anti-HER2 dual antibody guided nanodrug system of the present invention, tumor volume = 94mm 3) showed a smaller tumor volume after 20 days of treatment. This shows that the Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system significantly enhanced the efficacy of gastric cancer inhibition and achieved a synergistic effect through the integration of multiple treatment methods. In addition, the Anti-PD-L1-guided nanodrug system without CPT and the PDT co-administration group also showed effective tumor inhibition, providing a safer treatment option and avoiding the use of chemical drugs in sensitive cases.

[0156] from Fig.12 As can be seen in B, after 20 days of treatment, the tumor weight after dissection confirmed the enhanced anti-tumor effect of the antibody-directed combination therapy.

[0157] from Fig.12 C As can be seen, hematoxylin and eosin (H&E) staining of the main organs of AGS tumor-bearing mice (including heart, lung, spleen, liver and kidney) showed clear cell morphology and no obvious pathological abnormalities compared with the control group. After antibody-directed combined treatment, H&E staining images showed that both single treatment and combined treatment induced large areas of tumor cell necrosis and apoptosis, highlighting the significant tumor damage in the treatment group. The results of tissue section analysis after combined treatment were consistent with the tumor treatment results, confirming the effectiveness of combined treatment in pathophysiology.

[0158] In summary, the present invention combines perovskite quantum dots with a wide photoluminescence spectrum to the surface of a precisely designed and prepared Gd-MOF metal organic framework material to obtain a nanocomposite material, and then uses the nanocomposite material as a carrier to covalently couple Anti-PD-L1 and Anti-HER2 antibodies to the nanocomposite material through divinyl pyrimidine to obtain a dual-antibody-guided nanodrug system; the antibody coupled to the surface of the dual-antibody-guided nanodrug system acts as a nanogate to regulate the delivery of camptothecin chemotherapy drugs, and as a specific antibody to bind to target antigens overexpressed on the surface of tumor cells, thereby achieving "precision guidance" of the nanodrug system to tumor tissues, improving drug utilization efficiency and achieving optimal tumor treatment effects; the dual-antibody-guided nanodrug system provides a multimodal imaging strategy for tumor diagnosis, exerts the therapeutic efficacy of combining multiple treatment methods in combined tumor treatment, achieves the synergistic effect of chemotherapy, immunotherapy and photodynamic therapy, and can be applied to the development and clinical practice of tumor diagnostic technology and tumor treatment drugs.

[0159] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A nanocomposite material, characterized in that: The perovskite quantum dots in the nanocomposite material are uniformly distributed on the surface of the Gd-MOF metal organic framework material; the Gd-MOF metal organic framework material is a three-dimensional cubic material with a diameter between 60-200nm, an average size of about 120nm, a porous structure, an average pore diameter of 1.8nm, and a corresponding pore volume of 0.17cm 3 ·g -1 .

2. The method for preparing the nanocomposite material according to claim 1, characterized in that: The preparation method comprises: (1) Preparation of Gd-MOF: Gadolinium chloride hexahydrate and 1,2,4,5-tetrakis(4-carboxyphenyl)benzene were dissolved in N,N-dimethylformamide, and then acetic acid was added. The solution was ultrasonically treated and transferred to a reactor for hydrothermal reaction. The solution was then allowed to stand and cooled, and then centrifuged. The precipitate was washed with deionized water and ethanol, and then dried in a vacuum oven to obtain a MOF white solid. (2) Preparation of PQDs: PbX2 and CsX are added to a DMF solution and heated and stirred for a first reaction, and then 5-bromovaleric acid and oleylamine solution are added and heated and stirred for a second reaction to obtain a precursor solution; the precursor solution is added to deionized water at a certain temperature to obtain PQDs; X in the PbX2 and CsX is a mixture of one or more of Cl, Br and I; (3) Preparation of nanocomposite materials: Gd-MOF was dispersed in tetrahydrofuran, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added, and the mixture was stirred to modify the surface of Gd-MOF; Adding dithiodivinylpyrimidine (DVP) and PQDs to the modified Gd-MOF, reacting under stirring, and centrifuging after the reaction to obtain the nanocomposite material, which is recorded as DVP-MOF-PQDs; The structural formula of the dithiodivinylpyrimidine is: Steps (1) and (2) are performed in no particular order.

3. The method for preparing the nanocomposite material according to claim 2, characterized in that: In step (1), the molar ratio of 1,2,4,5-tetrakis(4-carboxyphenyl)benzene to gadolinium chloride hexahydrate is 1:1-1:3; The dosage ratio of N,N-dimethylformamide and acetic acid is 15-30mL:0.1-1.5mL; The hydrothermal reaction conditions are 100-150° C. for 6-15 hours; In step (2), the molar ratio of PbX2, CsX, 5-bromovaleric acid and oleylamine is 1:1:5:5; The conditions for the first heating and stirring reaction are: stirring and reacting at 60-80° C. for 40-60 min; the conditions for the second heating and stirring reaction are: stirring and reacting at 60-80° C. for 15 min; The PQDs are inorganic perovskite quantum dots CsPbX3, where X is a mixture of one or more of Cl, Br and I; In step (3), the usage ratio of Gd-MOF, EDC and NHS is 0.1-0.2 mg: 1 mol: 1 mol; The MOF surface modification time is 4-8h; The modified MOF, dithiodivinylpyrimidine (DVP) and PQDs are used in a ratio of 0.1-0.2 mg: 0.05 mmol: 0.05 mmol; The reaction time under stirring is 4-8h.

4. Use of the nanocomposite material according to claim 1 in the preparation of a dual antibody-guided nanodrug system.

5. An Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system, characterized in that: The dual antibody-directed nanodrug system is a mixture of an Anti-PD-L1 antibody-directed nanodrug and an Anti-HER2 antibody-directed nanodrug, and the molar ratio of the Anti-PD-L1 antibody-directed nanodrug to the Anti-HER2 antibody-directed nanodrug is 1:1; In the Anti-PD-L1 antibody-directed nanomedicine, the nanocomposite material is a carrier, and the anti-tumor drug is loaded in the porous structure of the nanocomposite material; the Anti-PD-L1 antibody is covalently coupled to the nanocomposite material; In the Anti-HER2 antibody-guided nanomedicine, the anti-tumor drug is loaded in the porous structure of the nanocomposite material; and the Anti-HER2 antibody is covalently coupled to the nanocomposite material.

6. The method for preparing the Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system according to claim 5, characterized in that: The preparation method comprises: The nanocomposite material DVP-MOF-PQDs is dispersed in a CPT loading solution, stirred for reaction, and centrifuged and washed after the reaction to obtain a CPT-loaded nanocomposite material; Pre-treating the Anti-HER2 antibody and the Anti-PD-L1 antibody with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to obtain an Anti-HER2 antibody solution and an Anti-PD-L1 antibody solution, respectively; The anti-HER2 antibody solution and the anti-PD-L1 antibody solution are incubated with the CPT-loaded nanocomposite material to obtain the anti-PD-L1 antibody-directed nanomedicine and the anti-HER2 antibody-directed nanomedicine; The Anti-PD-L1 antibody-directed nanodrug and the Anti-HER2 antibody-directed nanodrug are mixed in equal molar ratios to obtain the Anti-PD-L1 and Anti-HER2 dual antibody-directed nanodrug system.

7. The method for preparing the Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system according to claim 6, characterized in that: The mass ratio of the nanocomposite material DVP-MOF-PQDs and the CPT loading solution is 1000:7; the stirring reaction condition is stirring at 25° C. for 12 hours; The mass ratio of the Anti-HER2 antibody to tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is 2:1; the mass ratio of the Anti-PD-L1 antibody to tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is 2:1; The pretreatment conditions were all stirring reaction at 0° C. for 4 hours, and after the reaction was completed, washing with a filter membrane (MWCO 30 kDa) was used to remove excess TCEP.

8. The method for preparing the Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system according to claim 7, characterized in that: The mass ratio of Anti-HER2 antibody solution, Anti-PD-L1 antibody solution, and CPT-loaded nanocomposite was 1:1000; The incubation condition is stirring at 0°C for 12 hours.

9. Use of the Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system of claim 5 as a multimodal imaging agent.

10. Use of the Anti-PD-L1 and Anti-HER2 dual antibody-guided nanodrug system according to claim 5 in the preparation of drugs for treating tumors.

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