Antibody gold nanocage drug delivery system, preparation method, and use

Abstract

An antibody gold nanocage drug delivery system, a preparation method, and a use. LA-DOX-mPEG and a gold nanocage are first prepared independently; reaction between LA-DOX-mPEG and the gold nanocage is enabled to obtain DOX@GNC; then HS-PEG-COOH and an antibody are added for reaction to obtain the antibody gold nanocage drug delivery system. In addition to the advantages of nano drug delivery systems in the prior art, the gold nanocage drug delivery system further has the active targeting capability for tumor cells and can reduce the toxic and side effects of chemotherapeutic drugs on normal cells; pH responsive DOX drug release in an acidic tumor microenvironment can improve drug accumulation in tumor tissue. In addition, under the irradiation of near-infrared light, the gold nanocage converts light energy into heat energy, and the pH responsive drug release combined with high temperature enables rapid pulsed release of DOX carried by the gold nanocage, thereby achieving synergistic treatment of photothermal therapy and chemotherapy.

Description

Antibody gold nanocage drug delivery system, preparation method and use Technical Field

[0001] The present invention belongs to the technical field of nanomedicines and relates to an antibody gold nanocage drug delivery system, a preparation method and uses. Background Art

[0002] Malignant tumors pose a serious threat to human health. Due to the high heterogeneity of tumors and the unique characteristics of the tumor microenvironment, anti-tumor drugs are difficult to accumulate in tumor lesions, unable to diffuse deeply and penetrate into the tumor parenchyma to exert their effects, thus limiting their clinical efficacy. Utilizing the characteristics of the tumor microenvironment to construct intelligent anti-tumor nanomedicines with strong targeting, minimal side effects, and the ability to combine multiple therapies is of great clinical significance. B7-H3 is specifically and highly expressed in tumors and their associated blood vessels, making it an excellent target for tumor treatment. Gold nanocages (GNCs) have good biocompatibility, surface modifiability, and high photothermal conversion efficiency. Their hollow structure makes them excellent drug carriers. Nanomedicines precisely target tumor sites. Photothermal therapy can not only kill tumor cells that are difficult to remove surgically, but synergistic photothermal / chemotherapy can also accelerate the cellular internalization of drugs and enhance anti-tumor efficacy.

[0003] Photothermal therapy exposes tumor tissue to a high temperature environment (41-47°C). Tumor cells cannot tolerate high temperatures, causing their proteins to denature and their cell membranes to be destroyed, leading to cell death through apoptosis. In addition, many studies have shown that photothermal therapy not only has the effect of directly killing tumor cells, but can also inhibit tumor metastasis. Local photothermal therapy of tumors also induces immunogenic death of tumor cells. This process is regulated by tumor antigens and heat shock proteins released by dead cells, which are then captured by antigen-presenting cells, activating the body's immune system to identify and attack distant metastatic tumors. For example, a study found that when nude mice with melanoma tumor models that received intravenous injections of gold nanoshells were given photothermal therapy, tumor growth was significantly inhibited or even shrunk, and the subcutaneous tumor implants that had not received care also had CD4 + Helper T cells and CD8 +Cytotoxic T cell infiltration. However, single photothermal therapy cannot effectively eradicate tumors. This is because photothermal therapy may upregulate heat shock proteins (HSPs) in tumor cells, increase the heat stress tolerance of cancer cells, and result in insufficient heat to kill tumor cells. Photothermal therapy is often combined with traditional chemotherapy to construct a multimodal combined treatment system to achieve the purpose of combined synergy in anti-cancer effects. It can also prevent problems such as cancer recurrence and drug resistance after the use of chemotherapy drugs alone. The local high temperature of the tumor generated by photothermal therapy can inhibit the activity of DNA repair enzymes in tumor cells after chemotherapy, disrupt DNA synthesis and repair, and enhance the cytotoxicity of chemotherapy drugs. In addition, local high temperature inhibits ATP production by inducing mitochondrial dysfunction, inhibits the expression of multidrug resistance genes and multidrug resistance-related proteins, and avoids the development of tumor resistance. In addition, it can also increase the solubility of drugs in the tumor microenvironment and accelerate thermal motion, promote the cross-linking reaction between drugs and cell DNA, and enhance the effect of chemotherapy.

[0004] Furthermore, the localized hyperthermia generated by photothermal therapy can improve the physiological conditions of the tumor microenvironment, positively impacting the penetration of nanomedicines into tumor tissue. When tumors are heated to 43°C (typically at the periphery of the tumor tissue), blood flow in tumor vessels can increase approximately twofold, improving ineffective blood flow in newly formed vessels and causing increased vascular pressure. Furthermore, heat-induced cytoskeletal damage causes endothelial cell damage, further widening the pores between vascular spaces. This change facilitates the penetration of nanomedicines from blood vessels into tumor tissue. Enhanced blood perfusion also increases the permeability of newly formed tumor vessels and cell membranes, promoting tumor cell uptake of nanomedicines and enabling greater penetration of nanomedicines into the tumor. A study found that when gold nanorods were intravenously injected into a mouse sarcoma (S-180) tumor model, the amount of Evans blue dye entering the tumor site after photothermal therapy was compared at different temperatures. It was observed that when the tumor temperature reached 43°C or above, the dye content in the tumor site increased (up to 1.8-fold). These results demonstrate the feasibility of enhancing tumor drug penetration through photothermal therapy.

[0005] The gold nanocomposite carrier closest to the present invention in the prior art can overcome the defects of sudden release of chemotherapy drugs and poor single chemotherapy effect, but lacks active targeting to tumor cells.

[0006] Summary of the Invention

[0007] The present invention aims to address the shortcomings of the prior art by providing a gold nanocage drug delivery system that can precisely target tumor tissue and release DOX chemotherapy drugs in response to the pH of the tumor microenvironment. The gold nanocage surface is modified with a B7-H3 antibody fragment and an intelligent responsive material. The B7-H3 antibody fragment modified on the gold nanocage surface imparts active targeting properties to tumor tissue. By modifying the B7-H3 antibody fragment on its surface, specific tumor tissue targeting is further achieved. When the nanodrug is locally enriched in the tumor, under near-infrared light irradiation, the gold nanocage converts light energy into heat energy due to the surface plasmon resonance effect, causing a sharp increase in local temperature (high temperature can effectively kill tumor cells) and accelerating the pulsed release of the DOX drug at the hyperthermia temperature of the tumor microenvironment (the weakly acidic tumor microenvironment cleaves the hydrazone bond of the modified DOX, and the high temperature of the gold nanocage accelerates the release of the chemotherapy drug, resulting in a pulsed release of the drug). This achieves a combined therapeutic effect of photothermal therapy and chemotherapy.

[0008] To achieve the above object, the present invention provides a method for preparing an antibody-gold nanoparticle drug delivery system, which is characterized by comprising the following steps:

[0009] S1. Synthesizing LA-DOX-mPEG, wherein the molecular weight of polyethylene glycol in the functional LA-DOX-mPEG is 1 kDa to 5 kDa;

[0010] S2. Preparation of gold nanocages: Gold nanocages with a particle size of 30 to 200 nm were prepared by electrochemical replacement.

[0011] S3. The LA-DOX-mPEG obtained in S1 reacts with the gold nanocage solution obtained in S2 to obtain DOX@GNC:

[0012] Modification of S4.B7-H3 antibody: The DOX@GNC obtained in S3 was reacted with HS-PEG-COOH and antibody to obtain a monoclonal antibody gold nanoparticle drug delivery system.

[0013] Further, the S1 is,

[0014] First, ethyl lipoate is prepared, and then the ethyl lipoate is dissolved in an anhydrous organic solvent and reacted with hydrated hydrazine and ethyl acetate solution, followed by extraction, drying, centrifugation, concentration, and separation to obtain lipoic acid hydrazine LA-NHNH2; then the hydrazine and doxorubicin hydrochloride are dissolved in an anhydrous organic solvent and trifluoroacetic acid is added dropwise to react to obtain lipoic acid hydrazine doxorubicin;

[0015] Polyethylene glycol monomethyl ether and triethylamine are dissolved in an anhydrous organic solvent, and a dichloromethane solution containing 4-nitrophenyl chloroformate is added dropwise to react, washed, and separated to obtain mPEG-NPC;

[0016] The obtained lipoyl doxorubicin and mPEG-NPC were dissolved in anhydrous dimethylformamide, TEA was added for reaction, and then purified to obtain LA-DOX-mPEG.

[0017] Further, the S2 is,

[0018] After heating ethylene glycol, sodium sulfide, ethylene glycol solution of polyvinyl pyrrolidone, and ethylene glycol solution of silver nitrate are added in sequence for reaction. After the reaction is completed, silver cubes are obtained by washing;

[0019] Polyvinyl pyrrolidone is added to the silver cube solution obtained above, and then chloroauric acid solution is added to react under heating and stirring. After the reaction is completed, the reaction is centrifuged, washed, and resuspended to obtain 30-200 nm gold nanocages, namely GNCs.

[0020] Further, said S3 is,

[0021] The gold nanocage obtained in step S2 reacts with mPEG-SH to obtain solution A;

[0022] The LA-DOX-mPEG obtained in S1 reacts with NaBH4 to obtain solution B;

[0023] Solution B was added to solution A for reaction, centrifuged, and resuspended to obtain DOX@GNC;

[0024] Furthermore, the molar ratio of LA-DOX-mPEG to gold nanocage is 1:20 to 1:80.

[0025] Further, the S4 is,

[0026] HS-PEG-COOH was added to the DOX@GNC obtained in S3 and stirred. N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were then added and stirred. The mixture was centrifuged and resuspended. The antibody was then added to the resuspended solution and continued to be stirred. The monoclonal antibody gold nanoparticle drug delivery system was obtained by centrifugation.

[0027] Furthermore, the molar ratio of the HS-PEG-COOH to the gold nanocage is 1:300 to 1:400, and the molar ratio of the antibody to the HS-PEG-COOH is 1:2 to 1:20.

[0028] Furthermore, the antibody is a monoclonal antibody, a single-chain antibody, or an antibody Fab or Fc antibody fragment.

[0029] The present invention also provides an antibody-gold nanoparticle drug delivery system prepared by a method for preparing the antibody-gold nanoparticle drug delivery system.

[0030] The present invention also protects the use of the antibody-gold nanoparticle drug delivery system for preparing cancer therapeutic drugs; preferably, the cancer is not limited to blood cancer, bone cancer, lymphoma, intestinal cancer, liver cancer, gastric cancer, pelvic cancer, lung cancer, brain cancer, nerve cancer, breast cancer, esophageal cancer, and kidney cancer.

[0031] The LA-DOX-mPEG described in the present invention is also the linker described in the text.

[0032] The gold nano drug delivery system that is closest to the present invention in the prior art overcomes the defects of sudden release of chemotherapy drugs and poor effect of single chemotherapy to a certain extent, but its targeting to tumors and related cells (tumor lesions) is insufficient. Therefore, the present invention is further developed on this basis, and the composite material that is closest to the present invention in the prior art is cleverly modified with the help of B7-H3 antibodies and smart response DOX. It has the ability to actively target tumor tissue. The controlled release of drugs in the acidic microenvironment of the tumor can be achieved through pH-sensitive hydrazone bonds, providing a new method for preparing a gold nanocage drug delivery system for combined photothermal and chemotherapy treatment, realizing photothermal / chemotherapy synergistic treatment of tumors, and providing important scientific basis and reference for the design of smart response carriers and multi-modal combined treatment of tumors. There is currently no report on a gold nanocage drug delivery system that simultaneously carries a B7-H3 antibody fragment and DOX.

[0033] In addition to the advantages of existing nano-drug delivery systems, the gold nanocage drug delivery system of this invention also demonstrates that B7-H3 antibody modification can achieve active targeting of tumor cells, reducing the toxic side effects of chemotherapy drugs on normal cells. It also demonstrates that pH-responsive DOX drug release in the acidic tumor microenvironment can enhance drug accumulation in tumor tissue. Furthermore, under near-infrared light irradiation, the gold nanocage converts light energy into heat energy. The pH-responsive drug release and high temperature result in rapid pulsed release of DOX carried by the gold nanocage, achieving synergistic photothermal and chemotherapy treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Fig. 1 is a LA-DOX-mPEG synthesis flow chart;

[0035] Figure 2 shows the FTIR and 1 H-NMR spectrum;

[0036] FIG3 is a TEM image of the prepared silver cube, gold nanocage, and gold nanocage drug-loaded system;

[0037] Figure 4 shows the in vitro cumulative drug release curves of Anti-B7-H3-scFv@GNC@DOX under different pH conditions;

[0038] FIG5 is a diagram showing the uptake behavior of tumor cells of different materials observed using FACS and CLSM in Example 2;

[0039] Figure 6 is a bar graph of cell viability at different concentrations of DOX, mPEG@GNC, Anti-B7-H3-scFv@GNC, DOX@GNC, and Anti-B7-H3-scFv@GNC@DOX in Example 3 with or without near-infrared light irradiation;

[0040] Figure 7 shows the flow cytometry results of cell cycle distribution obtained by treating cells with DOX, mPEG@GNC, Anti-B7-H3-scFv@GNC, DOX@GNC, and Anti-B7-H3-scFv@GNC@DOX (1 μg / mL) for 24 hours with or without near-infrared light treatment in Example 4;

[0041] Figure 8 is a graph showing the flow cytometry results of reactive oxygen species production in cells treated with mPEG@GNC, Anti-B7-H3-scFv@GNC, DOX, DOX@GNC or Anti-B7-H3-scFv@GNC@DOX (1 μg / mL) for 24 hours with or without near-infrared light treatment in Example 5.

[0042] Figure 9 is a graph showing the plasma drug concentration-time curves of DOX, DOX@GNC, and Anti-B7-H3-scFv@GNC (DOX dose is 3 mg / kg) in rats and the antitumor efficacy results in mice in Example 6.

[0043] Figure 10 shows the results of pathological damage and tumor neovascularization in five major tissues of the heart, liver, spleen, lung and kidney of mice treated with DOX, DOX@GNC and Anti-B7-H3-scFv@GNC (DOX dose of 3 mg / kg) in Example 6.

[0044] FIG11 is a graph showing the binding capacity of mouse B7-H3 antibody measured by Octet instrument. DETAILED DESCRIPTION

[0045] Below in detail embodiments of the present invention, the example of described embodiment is shown in the accompanying drawings, wherein identical or similar reference numerals represent identical or similar elements or elements with identical or similar functions throughout. The embodiment described below by reference to the accompanying drawings is exemplary, is intended to be used for explaining the present invention, and is not to be construed as limiting the present invention. In the embodiment, those not indicating specific techniques or conditions are carried out according to the technology or conditions described in the document in this area or according to the product specification. Reagents used or instruments not indicating manufacturers are conventional products that can be obtained commercially.

[0046] The present invention provides a novel targeted controlled-release gold nanocage drug delivery system for combined photothermal and chemotherapy treatment, which comprises a gold nanocage with photothermal conversion efficiency, a pH-smart responsive DOX, and a B7-H3 antibody modification.

[0047] Example 1 Preparation of Anti-B7-H3-scFv@GNC@DOX Gold Nanocage Drug Delivery System

[0048] The reaction process is shown in Figure 1.

[0049] (1) Synthesis of pH-responsive LA-DOX-mPEG (i.e., linker)

[0050] 1) Synthesis and Purification of LAOEt: Under nitrogen and in the dark, lipoic acid (LA), ethanol (EtOH), and p-dimethylaminopyridine (DAMP) were dissolved in dichloromethane (DCM) to obtain solution A. N,N'-dicyclohexylcarbodiimide (DCC) was dissolved in dichloromethane to obtain solution B. Solution B was added dropwise to solution A at -10-20°C. After stirring for 1-3 hours, the temperature was raised to 25-45°C and the reaction was continued for 12-48 hours. The mixture was filtered, and the filtrate was concentrated. The yellow oily substance, lipoic acid ethyl ester (LAOEt), was separated by column chromatography.

[0051] 2) Synthesis and purification of LA-NHNH2: Under nitrogen and in the dark, LAOEt was dissolved in anhydrous methanol. Hydrated hydrazide was added dropwise with stirring at 30-60°C. After reaction for 8-24 hours, ethyl acetate was added. The mixture was extracted with saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and centrifuged. The supernatant was concentrated and separated by column chromatography to obtain LA-NHNH2 as a pale yellow solid.

[0052] 3) Synthesis and purification of LA-DOX: Under nitrogen and in the dark, LA-NHNH2 and doxorubicin hydrochloride (DOX) were dissolved in anhydrous methanol. Trifluoroacetic acid was then added dropwise. The mixture was stirred at 0-35°C for 4-12 h. The mixture was concentrated, allowed to stand, and then centrifuged. The mixture was washed with acetonitrile and dried to obtain lipoyl-doxorubicin LA-DOX as a dark red powder.

[0053] 4) Synthesis and Purification of mPEG-NPC: Under nitrogen, polyethylene glycol monomethyl ether and triethylamine were dissolved in anhydrous dichloromethane, stirred at -10-15°C, and a dichloromethane solution containing 4-nitrophenyl chloroformate was added dropwise. The reaction was carried out at -10-15°C for 0.5-3 hours, followed by 12-48 hours at room temperature. The reaction solution was diluted with 1-5 times the volume equivalent of dichloromethane and washed three times with saturated brine. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and the excess solvent was removed by rotary evaporation. The mixture was then washed with petroleum ether and impurities were separated using a Sephadex LH-20 column. The product, mPEG-NPC, was obtained by rotary evaporation as a buttery product.

[0054] 5) Synthesis and purification of LA-DOX-mPEG: The LA-DOX and mPEG-NPC obtained above were dissolved in anhydrous dimethylformamide under nitrogen and light protection at room temperature. TEA was added to the solution and stirred for 8-48 hours. The solvent was removed by rotary evaporation at 25-55°C. The solution was purified by column chromatography and freeze-dried to obtain a red solid LA-DOX-mPEG. Figure 2 shows the FTIR and 1 H-NMR spectra, where (A) is the FTIR spectrum of pH-responsive LA-DOX-mPEG, and (B) is the FTIR spectrum of pH-responsive LA-DOX-mPEG. 1 H-NMR spectrum. As can be seen from Figure 2, the infrared functional group characteristic peaks and the number of hydrogen spectra and their characteristic peaks of the synthesized target compound are consistent with expectations.

[0055] (2) Preparation of gold nanocages

[0056] 1) Ethylene glycol was heated to 120-180° C. under magnetic stirring, and 0.5-0.8 mg / mL sodium sulfide was added. After 5-25 minutes, a 10-30 mg / mL ethylene glycol solution of polyvinyl pyrrolidone was added. Then, a 30-50 mg / mL ethylene glycol solution of silver nitrate was gradually added while stirring. The reaction was stopped for 5-30 minutes, cooled to room temperature, washed with acetone, and the product was collected by centrifugation and washed with deionized water to obtain silver cubes. The TEM image of the silver cubes is shown in FIG3 (A).

[0057] 2) Polyvinylpyrrolidone was added to the silver cube solution obtained above, and then chloroauric acid solution was added dropwise with stirring at 70-130°C. The reaction was stopped and cooled to room temperature. The supernatant was removed by centrifugation, and the solution was washed with saturated sodium chloride solution and then with deionized water to obtain gold nanocages (i.e., GNCs). The solution was resuspended in ultrapure water by ultrasonication to obtain a gold nanocage solution (i.e., GNCs solution, sometimes also collectively referred to as GNCs), a TEM image of which is shown in Figure 3 (B).

[0058] 3) Add mPEG-SH to the 30 μg / mL GNC solution to a final concentration of 100 nmol / mL. Stir at room temperature for 4-6 hours. Centrifuge (10,000 rpm, 10 minutes) to remove unbound matter, and resuspend in an equal volume of deionized water to synthesize mPEG@GNC. (To ensure that the experimental and control groups were compared using a single variable, the mPEG@GNC prepared in this step served as the experimental group GNC.)

[0059] 4) Add mPEG-SH to the 30 μg / mL GNC solution to a final concentration of 0.5 nmol / mL, and stir at room temperature for 4-6 hours to obtain solution A;

[0060] Then, add an appropriate amount of LA-DOX-mPEG and 1.5-2.5 times the mass equivalent of NaBH4, and stir at 0°C for 1-3 hours to obtain solution B;

[0061] Solution B was added to solution A and stirred at room temperature for 4 to 8 h. The unbound portion was removed by centrifugation (10,000 rpm, 10 min). The precipitate was resuspended in an equal volume of deionized water to prepare DOX@GNC.

[0062] (3) Modification of B7-H3 Antibody

[0063] 0.5-0.8 nmol / mL of HS-PEG-COOH was added to the 30-50 μg / mL gold nanocage solution (i.e., GNC solution) prepared above and stirred at room temperature for 2-8 hours. 20-30 nmol / mL of N-hydroxysuccinimide (NHS) and 20-30 nmol / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were added and stirred at 0-30°C for 2-8 hours. After centrifugation, the precipitate was resuspended in an equal volume of deionized water. Anti-B7-H3-scFv single-chain antibody was then added to the resuspended solution, and stirring was continued at 0-30°C for 2-8 hours. Unbound antibody was removed by centrifugation to obtain the Anti-B7-H3-scFv@GNC@DOX gold nanocage drug delivery system. The TEM image of the Anti-B7-H3-scFv@GNC@DOX gold nanocage drug delivery system is shown in Figure 3 (C).

[0064] Preparation process of Anti-B7-H3-scFv single-chain antibody:

[0065] 1. Production of anti-B7-H3 monoclonal antibody (mouse origin)

[0066] 1.1 Immunization of mice

[0067] Eight-week-old female Balb / c mice were immunized subcutaneously at multiple sites with a mixture of human B7-H3 recombinant antigen protein (purchased from Shanghai YEASEN, product number 93111ES60, 100 μg, RMB 3185, Uniport No. Q5ZPR3.1) and complete Freund's adjuvant (CFA, Sigma) emulsion, at a dose of 80-100 μg protein per mouse. Three weeks later, booster immunizations were performed with an emulsion of protein and incomplete adjuvant (IFA, Sigma) for a total of three immunizations, at a dose of 80-100 μg protein per mouse. Sera were collected approximately two weeks after each immunization for antibody titer determination using ELISA on a SpectraMax i3 microplate reader. When serum titers reached a sufficiently high level, a booster immunization was performed intraperitoneally with 100 μg of human B7-H3 recombinant antigen protein dissolved in PBS. Three days later, spleens were removed and fused.

[0068] 1.2 Preparation and screening of hybridomas

[0069] Hybridoma cells were generated by fusing spleen cells from immunized mice with a mouse myeloma cell line (SP2 / 0-Ag14) using polyethylene glycol (PEG1450, SIGMA) as a fusion agent. The fused cells were suspended in HAT selection medium (Sigma Aldrich) and cultured in 96-well plates for approximately 11 days. The culture supernatant was then identified using ELISA on a SpectraMax i3. Positive clones were then subcloned using limiting dilution methods three or more times to ensure they were single-origin positive clones.

[0070] 1.3 Purification of anti-B7-H3 monoclonal antibody (mouse origin)

[0071] The subclones described in 1.2 were cultured with conventional hybridoma cells, and the culture supernatant was captured using HiTrap Protein G HP (purchased from GE Healthcare, Catalog No. 29048581). The supernatant was eluted with 0.1 M glycine (pH 3.5), and the eluted peak was collected and desalted using a HiTrap Desalting column (purchased from GE Healthcare, Catalog No. 29048684) with 1× PBS (pH 7.4) as the replacement buffer. The supernatant was stored at -20°C until use.

[0072] II. Characteristics of Anti-B7-H3 Monoclonal Antibody (Mouse) (Specific Binding Ability)

[0073] SPC-A1, A549, and CA-9-22 (human oral epithelial cancer cell lines) that highly express B7-H3 were used, and B7-H3 / Fc of human IgG1 (sequence derived from the Fc segment of human IgG1, sequence number: AAB2181.1, as shown in SEQ ID As shown in NO:12, a total of 456 amino acids were cloned into the vector according to standard methods, and then transferred to 293T cells to express B7-H3 / IgG1-Fc. The expressed B7-H3 / IgG1-Fc was purified using a protein A column and thoroughly mixed with the anti-B7-H3 monoclonal antibody (mouse source) purified according to 1.3 above. The mixture was incubated at 4°C for 20 to 30 minutes, washed, and detected on a FACSVerse machine. The results are shown in Figure 11. Figure 11 shows the binding capacity of the mouse B7-H3 antibody measured by the Octet instrument (molecular interaction instrument detection method, using bio-layer interferometry technology). The horizontal axis represents the time course (in seconds) of antibody binding to the B7-H3 antigen on cells. The first half of the rising curve represents the time required for the antibody to bind to the antigen (Association), while the second half of the flat curve represents the dissociation of the antibody after binding (Dissociation). A shorter binding time (a steeper curve) indicates stronger antibody binding, while a flat curve (no dip) after binding indicates minimal dissociation of the antibody after binding. This experiment demonstrates that the single-chain antibody FD3-34-scFv binds well to the B7-H3 antigen. See Figure 11 and Table 1 for detailed data.

[0074] Table 1 Binding parameters of FD3-34-scfv single chain antibody to B7-H3 antigen

[0075] Note: KD represents the binding affinity of the antibody; Kon represents the binding constant between the antibody and the antigen; Kdis represents the separation constant between the antibody and the antigen.

[0076] 3. Sequence Determination of Anti-B7-H3 Antibody (Mouse)

[0077] According to the hybridoma preparation technique (Kohler and Milstein, 1975) and the sections on cDNA library preparation, gene identification, and DNA sequencing in Molecular Cloning (3rd edition, by J. Sambrook and D.W. Russell, translated by Huang Peitang et al.), total RNA was extracted from UM1-51, UM1-56, and UM1-69 hybridoma cells, and cDNA was synthesized and sequenced.

[0078] First, total RNA was extracted and purified from hybridoma cells using RNA purification kits (RAeasy Mini Kit, Qiagen 74104, QIAshredder, Qiagen 79654). The RNA was then reverse transcribed into first-strand cDNA using the SMARter RACE cDNA Amplification Kit (Clontech) according to the company's reagent manual. PCR amplification was performed using the 5' RACE technique, using the universal primer UPM (provided by the kit) as the upstream primer and the gene-specific primer GSP (designed based on the mouse IgG1 heavy chain variable region and light chain kappa chain gene sequences) as the downstream primer, using the SMARter first-strand cDNA as the template. The PCR products were then ligated into T-vectors (Zero Blunt TOPO PCR Cloning Kit, Invitrogen K2875-J10). Select clones for sequencing and analysis to obtain the light and heavy chain variable region sequences of the antibody, respectively. Primers were then designed based on the heavy chain variable region sequence and the mouse IgG1 sequence. The full-length heavy chain sequence was obtained by PCR. The sequence was analyzed using the IGBLAST analysis tool (KABAT) to determine the light and heavy chains and their CDR regions, as shown below.

[0079] The light chain sequence is as follows:

[0080] Among them, the single underline "____" represents the CDR1 (GASENIYGALN, SEQ ID NO: 1) sequence; the double underline "____" represents the CDR1 (GASENIYGALN, SEQ ID NO: 1) sequence; Represents CDR2( SEQ ID NO: 2) sequence; lower wavy line Represents CDR3( SEQ ID NO:3) sequence.

[0081] The heavy chain sequence is as follows:

[0082] Among them, the single underline "____" represents the CDR1 (GFNIKDT, SEQ ID NO: 4) sequence; the double underline "____" represents the CDR1 (GFNIKDT, SEQ ID NO: 4) sequence; Represents CDR2( SEQ ID NO: 5) sequence; lower wavy line Represents CDR3( SEQ ID NO:6) sequence.

[0083] IV. Humanization of anti-B7-H3 antibody (mouse origin)

[0084] In order to make antibodies better applicable to clinical patient treatment, it is necessary to carry out humanization transformation to the antibody sequence so that it retains high affinity to antigen and other favorable biological properties (such as reducing its immunogenicity in the human body). To achieve this goal, according to a preferred method, humanized antibodies are prepared by analyzing the parent sequence and various conceptual humanized products using a three-dimensional model of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Almost all mouse antibodies can be humanized by CDR grafting, thereby maintaining antigen binding. For related technical literature, reference may be made to Sims et al., 1987, J. Immunol., 151: 2296; Chothia et al., 1987, J. Mol. Biol., 196: 901; Presta et al., 1993, J. Immnol., 151: 2623; Carter et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 4285; Lo Benny KC et al., in Antibody Engineering: Methods and Protocols, Vol. 248, Humana Press, New Jersey, 2004.

[0085] Based on the above work, FD3-34-scFv is preferably humanized. First, the antibody heavy chain constant region is replaced with the human IgG4 constant region sequence, and the antibody light chain constant region is replaced with the human κ chain constant region to obtain a human-mouse chimeric parent antibody (Chimeric Ab). After confirming the functional activity of the parent antibody, the heavy chain variable region and light chain variable region sequences are humanized, and the key amino acids in the FR region are reversely mutated on the basis of CDR transplantation. For amino acids between FRs that are not sure whether they affect the binding of antigen and antibody, merger synthesis is performed to construct a series of IgG mutants. After screening based on antibody affinity and expression level, one heavy chain and one light chain sequence were screened and cloned into the pcDNA3.1 expression vector using conventional molecular cloning technology. The 5' end of the light chain or heavy chain sequence carries a Kozak sequence and a human IL-2 signal peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 11), and its corresponding nucleotide sequence is:

[0086] The light chain sequence is as follows:

[0087] Among them, the single underline "____" represents the CDR1 (GASENIYGALN, SEQ ID NO: 1) sequence; the double underline "____" represents the CDR1 (GASENIYGALN, SEQ ID NO: 1) sequence; Represents CDR2( SEQ ID NO: 2) sequence; lower wavy line Represents CDR3( SEQ ID NO:3) sequence.

[0088] The heavy chain sequence is as follows:

[0089] Among them, the single underline "____" represents the CDR1 (GFNIKDT, SEQ ID NO: 4) sequence; the double underline "____" represents the CDR1 (GFNIKDT, SEQ ID NO: 4) sequence; Represents CDR2( SEQ ID NO: 5) sequence; lower wavy line Represents CDR3( SEQ ID NO:6) sequence.

[0090] Transient transfection of the above light and heavy chain expression vectors into ExpiCHO-S (Invitrogen) cells yielded a humanized antibody (FD3-34-scFv). The purified antibody was screened by ELISA and then biochemically analyzed by biochemical interaction assay (BLI), identifying the humanized antibody with the best binding affinity ( Figure 11 ).

[0091] As can be seen from Figure 3, a halo appears around the gold nanocage, indicating that it is successfully modified with the pH-responsive conjugate and actively targets the B7-H3 antibody fragment.

[0092] The constructed Anti-B7-H3-scFv@GNC@DOX gold nanocage drug delivery system has a size of about 70 nm (see Figure 3) and a potential of about -23 mV.

[0093] 1 mL of the Anti-B7-H3-scFv@GNC@DOX gold nanocage drug delivery system was precisely measured and placed in a dialysis bag (molecular weight cut-off = 3500 Da). Dialysis was then used to investigate the in vitro release behavior of the drug from EP tubes containing release media at different pH values ​​(pH 7.4, 6.5, and 5.5). Figure 4 shows the in vitro cumulative drug release curves of the Anti-B7-H3-scFv@GNC@DOX gold nanocage drug delivery system under different pH conditions. It can be seen that the release rates of DOX vary significantly under different pH media. Under normal physiological conditions of pH 7.4, DOX in the multifunctional gold nanocarrier is released slowly and the final cumulative release amount is only about 30%, indicating that before the multifunctional gold nanocarrier reaches the tumor site, it will not release the drug rapidly under normal physiological conditions, reducing the damage of the drug to normal tissues; when the drug delivery system accumulates in the weakly acidic tumor microenvironment, at pH 6.5, the cumulative release amount is about 50%; at pH 5.5, the release of DOX is very rapid, with a cumulative release of more than 50% within 24 hours, and the final cumulative release amount exceeds 90%.

[0094] The targeted controlled-release gold nanocage drug delivery system of the present invention uses the B7-H3 antibody fragment to specifically recognize tumor cells, tumor vascular endothelial cells and stromal fibroblasts, thereby achieving precise delivery of drugs to tumor tissues; the intelligent responsive DOX realizes the sustained and controlled release of DOX drugs in the tumor microenvironment.

[0095] The gold nanocage drug-carrying system for combined photothermal and chemotherapy treatment of the present invention can be applied to near-infrared photothermal therapy. The gold nanocage converts light energy into heat energy under near-infrared light irradiation. The pH-responsive drug release and high temperature enable rapid pulse release of DOX carried by the gold nanocage, thereby achieving synergistic photothermal and chemotherapy treatment.

[0096] Example 2

[0097] NCI-H1299 human non-small cell lung cancer cells (available from ATCC) in the logarithmic growth phase were cultured and flow cytometry was used to examine the differences in cellular uptake of free DOX (DOX is used clinically as a control, i.e., DOX dissolved in PBS), DOX@GNC, and Anti-B7-H3-scFv@GNC@DOX nanoparticles. Flow cytometry was also used to examine the differences in tumor cell uptake of Anti-B7-H3-scFv@GNC@DOX at different times, including 1, 2, 4, 12, and 24 hours. Laser confocal microscopy was used to evaluate the tumor cell uptake of DOX, Anti-B7-H3-scFv@GNC, DOX@GNC, and Anti-B7-H3-scFv@GNC@DOX. The results are shown in Figure 5, which shows the uptake behavior of tumor cells for different materials observed by FACS and CLSM, where the PBS solvent control is used. Among them, (A) is the intracellular chemotherapy drug DOX content after incubation with different drug delivery systems for 12 hours. In cells treated with Anti-B7-H3-scFv@GNC@DOX, the DOX fluorescence intensity was significantly higher than that of free DOX (2.14 times; p<0.001) or DOX@GNC (1.72 times; p<0.01); (B) is the uptake capacity of tumor cells for the constructed Anti-B7-H3-scFv@GNC@DOX at different times, and the maximum uptake was achieved after 12 hours; (C) is the intracellular localization and drug release of the multifunctional gold nanocarrier detected by confocal fluorescence microscopy. DOX is red fluorescence, and Anti-B7-H3-scFv single-chain antibody is The treatment shows green fluorescence, and DAPI shows blue fluorescence, which is used to mark the location of the cell nucleus. It can be seen that the cells treated with Anti-B7-H3-scFv@GNC@DOX show that the red fluorescence of DOX is diffused throughout the cells, and the fluorescence is stronger than that of cells treated with DOX and DOX@GNC, indicating that under antibody-mediated endocytosis, the cells are able to take up more multifunctional gold nanocarriers.

[0098] Example 3

[0099] Using logarithmic growth phase NCI-H1299 human non-small cell lung cancer cells, MTT was used to investigate the inhibitory effects of free DOX, mPEG@GNC, Anti-B7-H3-scFv@GNC, DOX@GNC, and Anti-B7-H3-scFv@GNC@DOX on tumor cell proliferation when combined with photothermal therapy at the same DOX concentration (0, 0.25, 0.5, 1, 2, and 4 μg / mL). GNCs demonstrated no significant cytotoxicity and demonstrated good biocompatibility when treated with the same near-infrared light. The photothermal effect was positively correlated with the concentration of GNCs (consistent with the mPEG@GNC and GNCs concentrations in all experimental groups). The results are shown in Figure 6.

[0100] Figure 6 is a bar graph of cell survival rates at different concentrations of DOX, mPEG@GNC, Anti-B7-H3-scFv@GNC, DOX@GNC, and Anti-B7-H3-scFv@GNC@DOX in Example 3, with or without near-infrared light irradiation. It can be seen that when DOX@GNC is linked to Anti-B7-H3-scFv, the uptake of DOX is significantly enhanced under B7-H3 receptor-mediated endocytosis, demonstrating a stronger cell-killing effect. When cells were treated with NIR laser, their cell survival rate changed significantly compared to the no-light group. The higher the GNC content in the culture medium, the stronger the cell-killing effect. The Anti-B7-H3-scFv@GNC@DOX plus NIR laser irradiation group had a significant inhibitory effect on cell proliferation.

[0101] Example 4

[0102] NCI-H1299 human non-small cell lung cancer cells in the logarithmic growth phase were used to investigate the fluorescence produced by the double-stranded DNA fluorescent dye propidium iodide (PI) binding to DNS. The fluorescence intensity was proportional to the double-stranded DNA content. DOX, mPEG@GNC, Anti-B7-H3-scFv@GNC, DOX@GNC, and Anti-B7-H3-scFv@GNC@DOX (1 μg / mL DOX equivalent) were examined. One group was illuminated (808 nm laser, 3 minutes). The other group was not illuminated and continued to culture for 24 hours. The cells were then digested with 0.25% trypsin (EDTA-free) and digested with complete culture medium. After dilution and counting, the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was carefully aspirated, rinsed twice with pre-chilled PBS, and centrifuged again at 1000 rpm twice, and the supernatant was carefully discarded. Add 1 mL of pre-chilled 70% ethanol solution and fix at 4°C for 12 hours. Wash away the ethanol, add 100 μL of RNase A solution to the cell pellet, resuspend the cells, incubate at 37°C in a water bath for 30 minutes, add 400 μL of PI solution, incubate at 4°C in the dark for 30 minutes, and then perform flow cytometry analysis. This study examined the effects of combined photothermal and chemotherapy treatment on the tumor cell cycle.

[0103] Figure 7 shows flow cytometry results of cell cycle distribution obtained after cells were treated with DOX, mPEG@GNC, Anti-B7-H3-scFv@GNC, DOX@GNC, and Anti-B7-H3-scFv@GNC@DOX (1 μg / mL) for 24 hours with or without near-infrared light irradiation in Example 4; the control is PBS. It can be seen that GNCs alone have no effect on cell cycle and proliferation. However, when cells are treated with Anti-B7-H3-scFv@GNC@DOX and irradiated with NIR, a significant shift in the S and G2 / M phases is observed, with a stronger arrest effect than that in the DOX-treated group (the G0 / G1 ratio decreased by 10.33%, p < 0.05). This suggests that the mechanism of cell cycle arrest (S and G2 / M phases) induced by photothermal therapy is different from that induced by DOX (G2 / M phase), enhancing the sensitivity of cells to the chemotherapeutic drug DOX and significantly arresting the cell cycle in the S and G2 / M phases.

[0104] Example 5

[0105] The level of intracellular reactive oxygen species is evaluated by utilizing the characteristic of 2',7-dichlorofluorescein diacetate (DCFH-DA), which is non-fluorescent and can freely pass through the cell membrane. Once inside the cell, it can be hydrolyzed by intracellular esterases to produce DCFH. The reactive oxygen species in the cell can oxidize the non-fluorescent DCFH to produce fluorescent DCF.

[0106] NCI-H1299 human non-small cell lung cancer cells (from ATCC) in the logarithmic growth phase were obtained, trypsinized and counted using a cell counting plate, diluted with culture medium containing 20 ng / mL IFN-γ to a cell concentration of 5×105 / mL, and 500 μL / well were evenly inoculated into a 24-well plate and incubated in an incubator for 10 hours. The old culture medium was aspirated and replaced with culture medium containing DOX, mPEG@GNC, Anti-B7-H3-scFv@GNC, DOX@GNC and Anti-B7-H3-scFv@GNC@DOX (1 μg / mL DOX equivalent). The culture was continued for 12 hours. One group was irradiated with light (808 nm laser, 3 minutes), and the other group was not irradiated with light and cultured for 24 hours. The cells were digested with 0.25% trypsin and complete culture medium was added to terminate the digestion. After dilution and counting, the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was carefully aspirated and the cells were treated with 10 μM freshly prepared DCFH-DA solution for 15 minutes, and then detected by flow cytometry. The results are shown in Figure 8, which shows the flow cytometry results of reactive oxygen species production in cells treated with mPEG@GNC, Anti-B7-H3-scFv@GNC, DOX, DOX@GNC, or Anti-B7-H3-scFv@GNC@DOX (1 μg / mL) for 24 hours with or without near-infrared light treatment as in Example 5; the control is PBS. As can be seen, without light treatment, cells treated with GNCs and Anti-B7-H3-scFv@GNC produced comparable reactive oxygen species equivalents to those in the negative control group. Treatment of cells with near-infrared light and mPEG@GNC or Anti-B7-H3-scFv@GNC increased reactive oxygen species production. When cells were treated with near-infrared light and DOX together, the combined effects of photothermal therapy and chemotherapy greatly increased the ROS level. The intracellular ROS levels in the DOX@GNC and Anti-B7-H3-scFv@GNC@DOX plus near-infrared light treatment groups were 2.15 times (p < 0.001) and 2.43 times (p < 0.001) that of the DOX group.

[0107] Example 6

[0108] To reveal the improved pharmacokinetic and pharmacodynamic properties of Anti-B7-H3-scFv@GNC@DOX, the plasma DOX concentration in rats was determined by LC-ESI-MS / MS. The drug activity of DOX (i.e., Dox·HCl in the table), DOX@GNC (i.e., DOX@GNC in the table) and DOX@GNC in the table were evaluated in SD rats. S) and Anti-B7-H3-scFv@GNC@DOX (B7-H3 / Dox@GNCs in the table) in vivo pharmacokinetics. An NCI-H1299 xenograft mouse model was established, revealing improved tumor lesion accumulation and anti-tumor activity of Anti-B7-H3-scFv@GNC@DOX compared to clinically used DOX and non-targeted DOX@GNC.

[0109] By comparing the PK parameters of Anti-B7-H3-scFv@GNC@DOX, DOX@GNC and clinical DOX (Table 1), the inventors found that the AUC of Anti-B7-H3-scFv@GNC@DOX 0-∞ The MRT of DOX in the Anti-B7-H3-scFv@GNC@DOX group was 2.23 times that of DOX@GNC and 15.58 times that of DOX, indicating that the DOX absorption of the constructed active targeted drug delivery system was significantly increased. 0- ∞ The results showed that the in vivo retention time of the constructed drug delivery system was significantly enhanced and the in vivo clearance rate was significantly reduced.

[0110] Table 1 Pharmacokinetic parameters of Dox·HCl, Dox@GNCs, and B7-H3 / Dox@GNCs after single intravenous injection (Mean±SD, n=6)

[0111] The tissue distribution and antitumor activity of different drug groups were evaluated in the NCI-H1299 xenograft tumor mouse model (when the tumor volume reached an average of 80-100 mm 3After the successful construction of the anti-tumor drug (dose of 3 mg / kg), group treatment was carried out. Figure 9 shows the plasma drug concentration-time curves of DOX, DOX@GNC and Anti-B7-H3-scFv@GNC (DOX dose is 3 mg / kg) in rats in Example 6, and the anti-tumor efficacy results in mice. As shown in Figure 9A, the blood drug concentrations of Anti-B7-H3-scFv@GNC@DOX and DOX@GNC were significantly increased compared to DOX in mice, and the half-life and retention time of the drug in the body were significantly prolonged. Among them, the area under the drug-time curve (AUC) of Anti-B7-H3-scFv@GNC@DOX was the highest, followed by DOX@GNC. DOX is easily and quickly cleared in the body. Furthermore, as shown in Figure 9B, the accumulation of Anti-B7-H3-scFv@GNC@DOX in tumor tissues was significantly increased compared to DOX@GNC and DOX, while drug accumulation in other normal organs was reduced, reducing drug toxicity to non-target organs in mice and maintaining normal body weight (Figure 9C). GNCs, on the other hand, were similar to the control group, with no significant differences in body weight or tumor volume compared to the other drug groups, demonstrating the good biocompatibility of GNCs as drug carriers. Therefore, under the action of near-infrared light (NIR), the photothermal / chemotherapy synergistic treatment effect of the active targeted accumulation drug delivery system for tumor lesions was further enhanced, and the in vivo antitumor activity of Anti-B7-H3-scFv@GNC@DOX was significantly higher than that of the other drug groups (Figure 9D and E).

[0112] Figure 10 shows the pathological damage and tumor angiogenesis in five major tissues (heart, liver, spleen, lung, and kidney) of mice treated with DOX, DOX@GNC, and Anti-B7-H3-scFv@GNC (DOX dose 3 mg / kg) in Example 6. The damage to the heart, liver, spleen, lung, and kidney in the treated mice is evident. The DOX group showed disorganized myocardial fibers and significant myocardial damage. Anti-B7-H3-scFv@GNC@DOX actively targeted tumor lesions and accumulated in them, mitigating drug damage to normal tissues. Major tissues and organs remained normal with no significant damage. In contrast, the GNCs group showed consistent results in the heart, liver, spleen, lung, and kidney, as in the control group, with no parenchymal organ damage observed, and no change in tumor tissue neovascularization density. Anti-B7-H3-scFv@GNC@DOX actively targeted tumors and their neovascular cells, significantly reducing the number of tumor neovascularization under chemo / photothermal therapy.

[0113] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing an antibody gold nanoparticle drug delivery system, characterized in that: The following steps are included: S1. Synthesizing LA-DOX-mPEG, wherein the molecular weight of polyethylene glycol in the functional LA-DOX-mPEG is 1 kDa to 5 kDa; S2. Preparation of gold nanocages: Gold nanocages with a particle size of 30 to 200 nm were prepared by electrochemical replacement method; S3. The LA-DOX-mPEG obtained in S1 reacts with the gold nanocages obtained in S2 to obtain DOX@GNC: Modification of S4.B7-H3 antibody: The DOX@GNC obtained in S3 was reacted with HS-PEG-COOH and antibody to obtain a monoclonal antibody gold nanoparticle drug delivery system.

2. The method for preparing the antibody-gold nanoparticle drug delivery system according to claim 1, characterized in that: The S1 is, First, ethyl lipoate is prepared, and then the ethyl lipoate is dissolved in an anhydrous organic solvent and reacted with hydrated hydrazine and ethyl acetate solution, and then extracted, dried, centrifuged, concentrated and separated to obtain lipoic acid acylhydrazine LA-NHNH2; then the lipoic acid acylhydrazine and doxorubicin hydrochloride are dissolved in an anhydrous organic solvent and trifluoroacetic acid is added dropwise to react to obtain lipoic acid acyl doxorubicin; Polyethylene glycol monomethyl ether and triethylamine are dissolved in anhydrous organic solvents respectively, and a dichloromethane solution containing 4-nitrophenyl chloroformate is added dropwise to react, washed, and separated to obtain mPEG-NPC; The obtained lipoic acid acyl doxorubicin and mPEG-NPC were dissolved in anhydrous dimethylformamide, TEA was added for reaction, and then purified to obtain LA-DOX-mPEG.

3. The method for preparing the antibody-gold nanoparticle drug delivery system according to claim 1, characterized in that: The S2 is, After the ethylene glycol is heated, sodium sulfide, ethylene glycol solution of polyvinyl pyrrolidone, and ethylene glycol solution of silver nitrate are added in sequence for reaction, and after the reaction is completed, silver cubes are obtained by washing; Polyvinyl pyrrolidone is added to the silver cube solution obtained above, and then chloroauric acid solution is added to react under heating and stirring. After the reaction is completed, the solution is centrifuged, washed, and resuspended to obtain 30-200 nm gold nanocages, namely GNCs.

4. The method for preparing the antibody-gold nanoparticle drug delivery system according to claim 1, characterized in that: The S3 is, The gold nanocage obtained in step S2 reacts with mPEG-SH to obtain solution A; The LA-DOX-mPEG obtained in S1 reacts with NaBH4 to obtain solution B; Solution B was added to solution A for reaction, centrifuged, and resuspended to obtain DOX@GNC.

5. The method for preparing the antibody-gold nanoparticle drug delivery system according to claim 4, characterized in that: The molar ratio of LA-DOX-mPEG to gold nanocage is 1:20 to 1:

80.

6. The method for preparing the antibody-gold nanoparticle drug delivery system according to claim 1, characterized in that: The S4 is, HS-PEG-COOH was added to the DOX@GNC obtained in S3 and stirred, and then N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added and stirred, centrifuged, resuspended, and then the antibody was added to the resuspended solution and continued to stir and centrifuged to obtain the monoclonal antibody gold nanoparticle drug delivery system.

7. The method for preparing the antibody-gold nanoparticle drug delivery system according to claim 6, characterized in that: The molar ratio of the HS-PEG-COOH to the gold nanocage is 1:300-1:400, and the molar ratio of the antibody to the HS-PEG-COOH is 1:2-1:

20.

8. The method for preparing the antibody-gold nanoparticle drug delivery system according to claim 1, characterized in that: The antibody is a monoclonal antibody, a single-chain antibody, or an antibody Fab or Fc antibody fragment.

9. The antibody-gold nanoparticle drug delivery system prepared by the method for preparing the antibody-gold nanoparticle drug delivery system according to any one of claims 1 to 8.

10. Use of the antibody-gold nanoparticle drug delivery system according to claim 9 for preparing cancer therapeutic drugs.

11. The use according to claim 10, characterized in that The cancers include but are not limited to blood cancer, bone cancer, lymphoma, intestinal cancer, liver cancer, stomach cancer, pelvic cancer, lung cancer, brain cancer, nerve cancer, breast cancer, esophageal cancer, and kidney cancer.

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