Co-assembled nano-drug carrying monoclonal antibody and chemotherapeutic drug as well as preparation method and application of co-assembled nano-drug

Abstract

The invention relates to the technical field of biomedical engineering, and provides a co-assembled nano-drug carrying a monoclonal antibody and a chemotherapeutic drug as well as a preparation method and application of the co-assembled nano-drug. The co-assembled nano-drug provided by the invention is formed by co-assembling a chemotherapy drug-polypeptide compound and a monoclonal antibody through enzyme catalysis, the nano-drug can target an epidermal growth factor receptor on the surface of oral squamous carcinoma cells, so that the release of a chemotherapeutic drug at a tumor site is realized; moreover, co-assembly is carried out by adopting an enzymatic method, so that the stability of a nano-drug system can be improved, and the curative effect of the drug is improved. In conclusion, the co-assembled nano-drug provided by the invention is clear in chemical structure, strong in stability, good in biocompatibility and easy to store, can effectively inhibit growth of oral squamous carcinoma cells, realizes step-by-step delivery of anti-cancer drugs from cell membranes to cell nucleuses, has a good anti-tumor effect, and has a wide application prospect in the aspects of cancer treatment and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and in particular to a co-assembled nano drug carrying a monoclonal antibody and a chemotherapeutic drug, and a preparation method and application thereof. Background Art

[0002] Oral squamous cell carcinoma (HNSCC) is the most common malignant tumor in the oral and maxillofacial region. It is highly malignant and has a poor prognosis, with a 5-year survival rate of only 50% to 60%. At present, traditional treatments for oral squamous cell carcinoma include surgery, anticancer drug therapy, and radiotherapy. Chemotherapy is the administration of chemical drugs into the body through oral or intravenous injection to selectively kill rapidly proliferating tumor cells. It is usually used as an adjuvant treatment before and after surgery; radiotherapy is the use of high-dose radiation to irradiate the target area, which can kill tumor cells and relieve symptoms. It is particularly suitable for locally advanced squamous cell carcinoma.

[0003] Molecular targeted therapy is a new type of tumor treatment method. It targets specific tumor-related molecules and uses target molecule-specific preparations or drugs for precise treatment. With the rapid development of treatment technology, molecular targeted therapy is increasingly recognized and valued by clinicians. Molecular targeted technology is divided into active targeting and passive targeting. Active targeting technology refers to the combination of some targeting molecules for certain tumor-overexpressed receptors on carriers containing anticancer drugs, so that nano drug carriers can target tumor cells through the specific recognition effect between receptors and ligands, and nano drugs accumulate on the surface of target cells in large quantities to achieve cell internalization. Different from the mode of small molecule drugs freely diffusing into cells, active targeting technology allows more anticancer drugs to enter cells and better play an anti-cancer role.

[0004] Peptide compounds are an important class of bioactive molecules, which are easy to design and synthesize, easy to metabolize in the human body, and have few toxic side effects. However, since they cannot maintain a stable structural form in aqueous solution, have a short half-life, and low bioavailability, their application in drug carriers is limited, especially the use of peptide carriers to combine molecular targeted drugs with traditional anticancer drugs is rarely reported. Summary of the invention

[0005] In view of this, the present invention provides a co-assembled nano drug carrying a monoclonal antibody and a chemotherapeutic drug, and a preparation method and application thereof. The present invention uses a polypeptide as a carrier and realizes effective co-loading of a monoclonal antibody and a chemotherapeutic drug through enzymatic co-assembly. The obtained nano drug has good stability, high bioavailability, targeting, good drug efficacy, low toxicity and side effects, and can exert a better anti-cancer effect.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A co-assembled nano-drug carrying a monoclonal antibody and a chemotherapeutic drug is formed by enzymatic co-assembly of a chemotherapeutic drug-peptide complex and a monoclonal antibody; the monoclonal antibody is cetuximab; the structure of the chemotherapeutic drug-peptide complex is shown in Formula I:

[0008]

[0009] The present invention also provides a method for preparing the co-assembled nanomedicine carrying a monoclonal antibody and a chemotherapeutic drug as described in the above scheme, comprising the following steps:

[0010] The chemotherapeutic drug-polypeptide complex, monoclonal antibody, enzyme and solvent are mixed and co-assembled to obtain the co-assembled nano drug carrying the monoclonal antibody and the chemotherapeutic drug.

[0011] Preferably, the preparation method of the chemotherapy drug-polypeptide complex comprises: mixing 10-hydroxycamptothecin, glutaric anhydride and an organic solvent for esterification reaction to obtain a 10-hydroxycamptothecin-glutaric acid derivative, wherein the structural formula of the 10-hydroxycamptothecin-glutaric acid derivative is shown in Formula II;

[0012]

[0013] The solid phase peptide synthesis method is used to sequentially connect amino acids to synthesize a peptide chain on a resin, and the N-terminus of the peptide chain is capped with the 10-hydroxycamptothecin-glutaric acid derivative, and then the peptide chain is cut off from the resin to obtain the chemotherapy drug-polypeptide complex.

[0014] Preferably, the molar ratio of 10-hydroxycamptothecin to glutaric anhydride is 1:(3-5); and the esterification reaction time is 48-72 hours.

[0015] Preferably, the end-capping comprises: mixing the compound having the structure shown in Formula II, a condensing agent, an activating agent and a solvent, and adding the obtained mixture into a solid phase tube used in a solid phase peptide synthesis method for end-capping.

[0016] Preferably, the enzyme is alkaline phosphatase.

[0017] Preferably, the dosage ratio of the chemotherapy drug-polypeptide complex, monoclonal antibody and enzyme is 1 mg: 1-5 μg: 8-10 U.

[0018] Preferably, the co-assembly temperature is 3-5°C, the time is 24-72 hours, and the pH value is 6-7.

[0019] The present invention also provides the use of the co-assembled nano drug carrying a monoclonal antibody and a chemotherapeutic drug described in the above scheme or the co-assembled nano drug carrying a monoclonal antibody and a chemotherapeutic drug prepared by the preparation method described in the above scheme in the preparation of anti-tumor drugs.

[0020] Preferably, the tumor is oral squamous cell carcinoma.

[0021] The present invention provides a co-assembled nano-drug carrying a monoclonal antibody and a chemotherapeutic drug (denoted as HP@antibody), which is formed by enzymatic co-assembly of a chemotherapeutic drug-polypeptide complex and a monoclonal antibody; the monoclonal antibody is cetuximab; and the structure of the chemotherapeutic drug-polypeptide complex is shown in Formula I (see above). The present invention uses a hydrophilic short peptide as a carrier, uses cetuximab as a tumor targeting factor, and connects 10-hydroxycamptothecin (HCPT) to the polypeptide through an ester bond, thereby achieving effective co-loading of molecular targeted drugs (cetuximab) and traditional chemotherapy drugs (10-hydroxycamptothecin), which can achieve anti-tumor synergistic effects, significantly improve anti-tumor efficacy, and has good targeting and small toxic and side effects; and, epidermal growth factor receptor (EGFR) is a cell membrane receptor with tyrosine protein kinase activity, which is related to the degree of cell malignancy. Oral squamous cell carcinoma can highly express EGFR. The nano drug of the present invention can target the EGFR receptor on the surface of oral squamous cell carcinoma cells. The nano drug can efficiently deliver the drug to the tumor cell through active targeting. After entering the cell, the esterase in the lysosome in the cell can hydrolyze the ester bond connecting the drug in the nano drug system, and realize the release of the chemotherapy drug at the tumor site, thereby exerting an anti-cancer effect. In summary, the nano drug provided by the present invention has good stability, high bioavailability, targeting, good drug efficacy, small toxicity and side effects, and good anti-cancer effect.

[0022] The present invention also provides a method for preparing the co-assembled nano-drug carrying a monoclonal antibody and a chemotherapeutic drug described in the above scheme. The present invention uses glutaric anhydride to connect 10-hydroxycamptothecin and a polypeptide to obtain a chemotherapeutic drug-polypeptide complex, and then co-assembles the chemotherapeutic drug-polypeptide complex and the monoclonal antibody in an enzyme-catalyzed manner to obtain the co-assembled nano-drug carrying the monoclonal antibody and the chemotherapeutic drug. The present invention uses an enzymatic method to perform co-assembly, which can optimize the assembly path of the polypeptide molecule, greatly improve its biological activity through the controllable folding of the polypeptide, thereby exerting a better functional effect; it can improve the stability of the nano-drug system, increase the efficacy of the drug, reduce the toxic and side effects of the drug, and enable the anti-cancer drug to exert a better anti-cancer effect. In addition, the preparation method provided by the present invention has a simple process, low cost, and high yield.

[0023] The present invention also provides the use of the co-assembled nano drug carrying a monoclonal antibody and a chemotherapeutic drug described in the above scheme or the co-assembled nano drug carrying a monoclonal antibody and a chemotherapeutic drug prepared by the preparation method described in the above scheme in the preparation of an anti-tumor drug. The co-assembled nano drug provided by the present invention has a clear chemical structure, strong stability, good biocompatibility, and is easy to preserve. It can effectively inhibit the growth of oral squamous cell carcinoma cells, and realizes the step-by-step delivery of anti-cancer drugs from the cell membrane to the cell nucleus, has a good anti-tumor effect, and has broad application prospects in cancer treatment and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the high-resolution mass spectrum of HCPT-GA;

[0025] Figure 2 is the H NMR spectrum of HCPT-GA;

[0026] Figure 3 HCPT-G D F D F D High-resolution mass spectrum of pYERGD;

[0027] Figure 4 HCPT-G D F D F D H NMR spectrum of pYERGD;

[0028] Figure 5 This is the test result of polyacrylamide gel electrophoresis experiment;

[0029] Figure 6 This is a transmission electron microscopy image of the co-assembled nanodrug;

[0030] Figure 7 Fluorescence test results for cell uptake experiments;

[0031] Figure 8 The drug uptake test results for the cell uptake experiment;

[0032] Fig. 9 The results of the drug release performance investigation of co-assembled nanomedicines;

[0033] Fig.10 The results are for in vitro cytotoxicity tests;

[0034] Fig.11 These are photos of tumors in mice after treatment with different drugs;

[0035] Fig.12 The tumor weights of mice after treatment with different drugs. DETAILED DESCRIPTION

[0036] The present invention provides a co-assembled nano-drug carrying a monoclonal antibody and a chemotherapeutic drug, which is formed by enzymatic co-assembly of a chemotherapeutic drug-polypeptide complex and a monoclonal antibody; the monoclonal antibody is cetuximab; and the structure of the chemotherapeutic drug-polypeptide complex is shown in Formula I:

[0037]

[0038] In the present invention, the chemotherapeutic drug-polypeptide complex is specifically composed of a polypeptide and a chemotherapeutic drug connected to the N-terminus of the polypeptide via an ester bond, the chemotherapeutic drug is 10-hydroxycamptothecin (HCPT), and the sequence of the polypeptide is G D F D F D pYERGD (glycine, D-phenylalanine, D-phenylalanine, D-phosphorylated tyrosine, glutamic acid, arginine, glycine, aspartic acid, as shown in SEQ ID No: 1). In the present invention, the chemotherapeutic drug-polypeptide complex is denoted as HCPT-G D F D F D pYERGD, referred to as HP.

[0039] The present invention also provides a method for preparing the co-assembled nanomedicine carrying a monoclonal antibody and a chemotherapeutic drug as described in the above scheme, comprising the following steps:

[0040] The chemotherapeutic drug-polypeptide complex, monoclonal antibody, enzyme and solvent are mixed and co-assembled to obtain the co-assembled nano drug carrying the monoclonal antibody and the chemotherapeutic drug.

[0041] First, the preparation method of the chemotherapeutic drug-polypeptide complex is described.

[0042] In the present invention, the method for preparing the chemotherapeutic drug-polypeptide complex preferably comprises:

[0043] 10-hydroxycamptothecin, glutaric anhydride and an organic solvent are mixed for esterification reaction to obtain a 10-hydroxycamptothecin-glutaric acid derivative, wherein the structural formula of the 10-hydroxycamptothecin-glutaric acid derivative is shown in Formula II;

[0044]

[0045] The solid phase peptide synthesis method is used to sequentially connect amino acids to synthesize a peptide chain on a resin, and the N-terminus of the peptide chain is capped with the 10-hydroxycamptothecin-glutaric acid derivative, and then the peptide chain is cut off from the resin to obtain the chemotherapy drug-polypeptide complex.

[0046] The present invention mixes 10-hydroxycamptothecin, glutaric anhydride and an organic solvent for esterification reaction to obtain a 10-hydroxycamptothecin-glutaric acid derivative (referred to as HCPT-GA). In the present invention, the molar ratio of the 10-hydroxycamptothecin to glutaric anhydride is preferably 1:(3-5), more preferably 1:(3.5-4.5); the organic solvent is preferably pyridine; the esterification reaction time is preferably 48-72 hours; the esterification reaction temperature is preferably room temperature.

[0047] After the esterification reaction is completed, the present invention preferably further comprises: evaporating the obtained reaction mixture to dryness to obtain a solid product; filtering the solid product and hydrochloric acid after mixing, washing and drying the filter cake to obtain the HCPT-GA; the evaporation is preferably carried out in a vacuum; the washing reagent is preferably hydrochloric acid; the drying is preferably vacuum drying; the concentration of the hydrochloric acid used for mixing the solid product and the hydrochloric acid used for washing are preferably 1 mol / L.

[0048] After obtaining HCPT-GA, the present invention uses a solid phase peptide synthesis method to sequentially connect amino acids to synthesize a peptide chain on a resin, and uses the HCPT-GA to cap the N-terminus of the peptide chain, and then cuts the peptide chain from the resin to obtain the chemotherapy drug-polypeptide complex. In the present invention, the resin is preferably dichlororesin; the raw material used for synthesizing the peptide chain is preferably an amino acid with a side chain protecting group and an amino protecting group, and the amino protecting group is preferably Fmoc; the amino acid is preferably prepared according to DGRE D pY D F D FG are connected in sequence. With the participation of a condensing agent, the amino acids are connected in sequence on the dichlororesin to form a peptide chain. When the last amino acid G is connected, the protecting group on its amino group is cut off to expose the amino group, and then the end is capped.

[0049] In a specific embodiment of the present invention, the method for synthesizing a peptide chain preferably comprises:

[0050] (1) Add dichlororesin into a solid phase tube and swell it with dichloromethane, then remove excess dichloromethane;

[0051] (2) mixing the first amino acid, the activating agent and dichloromethane, adding the resulting mixture into a solid phase tube for reaction, then blocking with a blocking solution, and then removing the amino protecting group with a cutting solution;

[0052] (3) mixing a second amino acid, a condensing agent, an activating agent and N,N-dimethylformamide, adding the resulting mixture to a solid phase tube for reaction, and then removing the amino protecting group with a cutting solution;

[0053] (4) According to the order of amino acids in the peptide chain, the connection of each amino acid is completed in sequence according to the method of step (3) until the last amino acid G is connected.

[0054] In the present invention, the swelling time is preferably 10 minutes; the blocking solution is preferably obtained by mixing dichloromethane (DCM), methanol (MeOH) and N,N-diisopropylethylamine (DIEA), and the volume ratio of dichloromethane, methanol and N,N-diisopropylethylamine is preferably 17:2:1; the blocking time is preferably 15 minutes; after the blocking is completed, it is preferably washed with dichloromethane and N,N-dimethylformamide in sequence.

[0055] In the present invention, the cutting liquid used in step (2) and step (3) is preferably a piperidine solution, the volume fraction of piperidine in the piperidine solution is preferably 20%, and the solvent of the piperidine solution is preferably N,N-dimethylformamide (DMF); the excision time is preferably 30 minutes; after the excision is completed, DMF is preferably used for washing.

[0056] In the present invention, the activating agent used in step (2) and step (3) is preferably N,N-diisopropylethylamine (DIEA); the condensing agent is preferably benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU); and the reaction time in step (2) and step (3) is preferably 2h.

[0057] After the peptide chain is synthesized, the present invention uses the HCPT-GA to cap the N-terminus of the peptide chain, and then cuts the peptide chain from the resin to obtain the chemotherapeutic drug-polypeptide complex. In the present invention, the capping preferably includes: mixing the HCPT-GA, condensing agent, activating agent and solvent, and adding the resulting mixture to the solid phase tube used in the solid phase peptide synthesis method for capping. In the present invention, the condensing agent is preferably HBTU, and the activating agent is preferably DIEA; the dosage ratio of the HCPT-GA, HBTU and DIEA is preferably 450-500 mg: 350-380 mg: 320-340 μL, more preferably 478 mg: 379 mg: 330 μL; the capping time is preferably 2 hours; after the capping is completed, it is preferably washed with DMF and DCM in sequence.

[0058] In the present invention, the cutting liquid used for cutting the peptide chain is preferably obtained by mixing trifluoroacetic acid (TFA), water and triisopropylsilane (TIS), the volume fraction of TFA in the cutting liquid is preferably 95%, the volume fraction of water is preferably 2.5%, and the volume fraction of TIS is preferably 2.5%; the cutting time is preferably 45 minutes; the presence of TFA in the cutting process can also simultaneously remove the side chain protecting groups on the amino acids. The present invention preferably collects the cleavage product liquid obtained by cutting, and post-treats the cleavage product liquid, and the post-treating preferably includes: adding DCM after rotary evaporation of the cleavage product liquid to dryness, rotary evaporating to dryness again, repeating the operation of adding DCM and rotary evaporating to dryness 4 times to obtain an oily substance; mixing the oily substance with anhydrous ether and filtering, washing the filter residue to obtain a crude product; subjecting the crude product to HPLC separation, and collecting the eluent corresponding to the target product peak; evaporating and freeze-drying the eluent in turn to obtain the chemotherapeutic drug-polypeptide complex; the eluent used for HPLC separation is preferably a trifluoroacetic acid-methanol solution, and the mass fraction of trifluoroacetic acid in the trifluoroacetic acid-methanol solution is 0.5%.

[0059] After obtaining the chemotherapeutic drug-polypeptide complex, the present invention mixes the chemotherapeutic drug-polypeptide complex, monoclonal antibody, enzyme and solvent for co-assembly to obtain the co-assembled nano drug carrying the monoclonal antibody and the chemotherapeutic drug.

[0060] In the present invention, the enzyme is preferably alkaline phosphatase (ALP); the dosage ratio of the chemotherapeutic drug-polypeptide complex, the monoclonal antibody and the enzyme is preferably 1 mg: 1-5 μg: 8-10 U, more preferably 1 mg: 2-5 μg: 10 U; the solvent is preferably PBS; the co-assembly temperature is preferably 3-5°C, more preferably 4°C, the time is preferably 24-72h, more preferably 24-48h, and the pH value is preferably 6-7, more preferably 6-6.5. In a specific embodiment of the present invention, it is preferred to first add the chemotherapeutic drug-polypeptide complex to PBS, adjust the pH value to 6-7 with sodium carbonate after mediation and dissolve, then add the monoclonal antibody, add alkaline phosphatase after mediation, and place at 4°C for co-assembly after mediation again.

[0061] The present invention also provides the use of the co-assembled nano drug carrying a monoclonal antibody and a chemotherapeutic drug described in the above scheme or the co-assembled nano drug carrying a monoclonal antibody and a chemotherapeutic drug prepared by the preparation method described in the above scheme in the preparation of anti-tumor drugs; the tumor is oral squamous cell carcinoma.

[0062] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] Example 1

[0064] 1. Synthesis of HCPT-GA

[0065] 0.3 g of 10-hydroxycamptothecin (HCPT) and 0.282 g of glutaric anhydride solution were weighed in 30 mL of pyridine and stirred at room temperature for 48 h. The resulting reaction mixture was then evaporated to dryness in a vacuum, and 20 mL of 1 mol / L hydrochloric acid was added to the evaporated dry product to precipitate a yellow-green precipitate, which was then filtered using a Buchner funnel, and the filter cake was washed again with 1 mol / L hydrochloric acid, and the filter cake was collected and vacuum dried to obtain the target product HCPT-GA.

[0066] Figure 1 This is the high-resolution mass spectrum of HCPT-GA; Figure 2 is the H NMR spectrum of HCPT-GA; Figure 1 It can be seen that the obtained product has the target structure.

[0067] 2. HCPT-G D F D F D Synthesis of pYERGD (abbreviated as HP)

[0068] 1) Weigh dichlororesin (0.516 g) and add it to a solid phase tube, then swell it with DCM for 10 min, and then remove the DCM with an ear bulb.

[0069] 2) According to DGRE D pY D F D In the order of FG, the first amino acid (1 mol) and DIEA (330 μL) were dissolved in dichloromethane (15 mL) and added to the solid phase tube for reaction for 2 h. Wash with DCM 5 times and block with blocking solution (DCM: MeOH: DIEA = 17 mL: 2 mL: 1 mL) for 15 min. After blocking, wash with DCM 5 times and then with DMF 5 times, each time for 1 min. Then, use 20% piperidine (piperidine: DMF = 1:4) solution to cut the Fmoc protecting group, and the cutting time is 30 min. After cutting, wash with DMF 5 times, each time for 1 min.

[0070] 3) Weigh the next amino acid (1 mol) and HBTU (379 mg) and dissolve them in DMF. Then add 330 μL of DIEA and add the mixture into the solid phase tube. React for 2 h, wash 5 times with DMF, then cut with 20% piperidine solution for 30 min and wash 5 times with DMF.

[0071] 4) According to DGRE D pY D F D In the order of FG, step (3) is repeated to complete the connection of each amino acid in sequence until the last amino acid G is connected.

[0072] 5) Weigh HCPT-GA (478 mg) and HBTU (379 mg) and dissolve them in DMF. Then add 330 μL of DIEA into the solid phase tube and react for 2 h. Wash with DMF 5 times and DCM 5 times.

[0073] 6) Prepare cutting solution: 95% TFA (19 mL) + 2.5% H2O (500 μL) + 2.5% TIS (500 μL). Add the cutting solution to the solid phase tube and cut for 45 minutes.

[0074] 7) The cutting solution was collected and TFA was evaporated on a rotary evaporator, and then 20 mL of DCM was added and evaporated on a rotary evaporator. This was repeated 4 times to obtain an oily substance.

[0075] 8) Anhydrous ether was added to the oily substance, and solid precipitated. The residue was filtered using a Buchner funnel. The residue was washed with ether three times, and the solid was collected to obtain a crude product.

[0076] 9) Separate the crude product by HPLC, using a trifluoroacetic acid-methanol solution as the eluent, wherein the mass fraction of trifluoroacetic acid is 0.5%, collect the eluate corresponding to the peak of the target product, evaporate the methanol in the eluate under vacuum, and then freeze-dry to obtain HP.

[0077] Figure 3 HCPT-G D F D F D High-resolution mass spectrum of pYERGD; Figure 4 HCPT-G D F D F D H NMR spectrum of pYERGD; Figure 3-4 It can be seen that the obtained product has the target structure.

[0078] 3. Preparation of co-assembled nanomedicines carrying monoclonal antibodies and chemotherapeutic drugs

[0079] 3 mg HP was added to the EP tube, followed by 480 μL PBS, swirled to completely dissolve the HP, and the pH was adjusted to 6 with sodium carbonate. 15 μL cetuximab (5 mg / mL) was added after another swirling, and 10 U ALP was added after another swirling. The mixture was swirled and reacted at 4°C for 24 h to obtain the co-assembled nanodrug.

[0080] 4. Polyacrylamide gel electrophoresis experiment

[0081] Polyacrylamide gel electrophoresis was used to test whether cetuximab could co-assemble with the polypeptide, as follows:

[0082] 3 mg HP was dissolved in 500 μL PBS, the pH was adjusted to 6 with sodium carbonate, 5 μg and 10 μg of cetuximab were added respectively, and 10 U ALP was added after mediation. After mediation, the system was assembled at 4 ° C for 24 h, and then the obtained nanodrug system was centrifuged (12000 / min, 15 min), and 20 μL of the supernatant was taken for SDS gel electrophoresis experiment.

[0083] Figure 5 is the test result of polyacrylamide gel electrophoresis experiment; according to Figure 5 It can be seen that HP can be completely co-assembled with 5 μg of monoclonal antibody. When 10 μg of monoclonal antibody is added, complete assembly cannot be achieved. Therefore, the present invention selects the co-assembled nanomedicine (HP@antibody) formed by adding 5 μg of monoclonal antibody and polypeptide for subsequent experiments.

[0084] Example 2 Microscopic morphology of co-assembled nanomedicine under transmission electron microscope

[0085] 20 μL of co-assembled nanodrug was dropped onto the carbon-coated copper mesh. After 1 minute, excess liquid was absorbed with filter paper, washed with ultrapure water, and then dried with filter paper. Then 10 μL of uranyl acetate was added to the copper mesh for staining. After 1 minute, the sample was dried and placed in a desiccator to dry overnight. The microscopic morphology of the sample was observed using a transmission electron microscope (Tecnai G2 F20 system, operating voltage 200 kV). The results are shown in Figure 2. Figure 6 As shown. Figure 6 It can be seen that the co-assembled nanomedicine of the present invention presents a uniform and dense nanofiber structure under an electron microscope.

[0086] In addition, testing has shown that the co-assembled nanodrug has good water solubility and good stability in distilled water and culture medium containing 10% serum.

[0087] Example 3 Detection of the ability of oral squamous cell carcinoma HN6 cells to take up co-assembled nanomedicine

[0088] Cell culture must be strictly sterile. In order to verify the cell's ability to absorb the assembly, this example uses oral squamous cell carcinoma cell HN6 to test cell uptake, and the culture medium is DMEM (containing 10% FBS and 1% double antibody). The cells are first revived and cultured, and then plated and tested. The specific steps are as follows:

[0089] 1) Add HN6 cell suspension to a 24-well plate at a density of 50,000 cells / well, set up 3 parallel wells for each group and each time point, and incubate in a 37°C incubator for 24 h.

[0090] 2) After removing the upper culture medium, fresh culture medium containing 100 μM co-assembled nanodrug was added to each well and incubated in an incubator for 1, 2, and 4 h, respectively.

[0091] 3) Aspirate the culture medium and gently rinse with PBS solution three times.

[0092] 4) Add cell lysis solution containing 10% DMSO to each well, mix well, transfer to a 1.5 mL centrifuge tube, and centrifuge at 15000 r / min for 10 min.

[0093] 5) After centrifugation, the supernatant was collected and transferred into a black 96-well plate. The fluorescence intensity was detected by an ELISA instrument and compared with the standard curve to determine the drug uptake in each group.

[0094] The same experiment was performed using HCPT and HP.

[0095] The results are as follows Figure 7-Figure 8 As shown, Figure 7 For the fluorescence test results, Figure 8 This is the result of the drug intake test. Figure 7-Figure 8 The results of the cell uptake experiment in the present invention show that the co-assembled nanomedicine prepared by the present invention can be taken up by oral squamous cell carcinoma cells with high expression of EGFR, and the uptake amount is significantly higher than that of HCPT and HP.

[0096] Example 4 Investigation of drug release properties of co-assembled nanomedicine

[0097] First, the co-assembled nanodrug was diluted with lysosome simulation liquid to a concentration of 1 mg / mL, and 5 mL of the diluted sample was placed in an Eppendorf tube and placed on a shaker at 37°C. 200 μL of sample was taken from the tube at fixed time points, and the release of HCPT was detected by LC-MS. The standard used was HCPT, and the sampling time points were 0, 4, 8, 12, 16, 20, and 24 hours.

[0098] The same experiment was performed by replacing the co-assembled nanodrug with HP.

[0099] Fig. 9 The results of the drug release performance investigation of co-assembled nanomedicines are shown in Figure 2. Fig. 9 It can be seen that HCPT can be continuously released from the co-assembled nanodrug.

[0100] Example 5 Toxic effect of co-assembled nanomedicine on HN6 cells (in vitro cytotoxicity test)

[0101] 1) After digestion, HN6 cells were seeded in a 96-well plate at a density of 6,000 to 8,000 cells / well. 100 μL of cell suspension was added to each well and the plates were incubated overnight in an incubator.

[0102] 2) After the cells have attached to the wall, the upper culture medium is aspirated and fresh culture medium containing drugs (HCPT, HP or HP@antibody) is added again. Five wells of each concentration are set as parallels, and a total of 10 concentration gradients are set up and cultured in the incubator for 48 hours.

[0103] 3) Discard the supernatant and wash twice with new culture medium to eliminate the influence of drugs. Add 100 μL of diluted CCK-8 solution to each well, then place in a cell culture incubator for 4 hours, and measure the absorbance at 450 nm with an enzyme marker.

[0104] Fig.10 The results of in vitro cytotoxicity tests. Fig.10 It can be seen that the co-assembled nanomedicine of the present invention exhibits stronger cytotoxicity than free HCPT chemotherapy drugs.

[0105] Example 6 In vivo antitumor activity test

[0106] Healthy male BALB / cA-nu mice (5 weeks old) were randomly divided into four groups and injected subcutaneously with 100 μL of 5×10 6 When the tumor volume was about 50 mm 3 When the drug was injected into the tail vein (HCPT, HP or HP@antibody), the drug concentration was shown in Table 1. The drug was administered once a week, the weight of the mice and the size of the tumor were measured every three days, and the tumor volume was calculated by the following formula: V = length × width 2 / 2. After two doses, the mice were killed, venous blood was collected, and the tumor and internal organs were completely removed for HE staining.

[0107] Table 1 Drug concentration

[0108]

[0109] Fig.11 These are photos of tumors in mice after treatment with different drugs; Fig.12 The tumor weights of mice after different groups of drug treatment. Figure 11-12 It can be seen that the co-assembled nanomedicine of the present invention can significantly inhibit the growth of tumors in mice, and the therapeutic effect is significantly better than that of HCPT and HP.

[0110] In summary, the co-assembled nanomedicine constructed by enzyme catalysis in the present invention has a clear chemical structure, a simple preparation process, low cost, high yield, and is easy to preserve; the nanofibers formed by the co-assembled nanomedicine have strong stability and good biocompatibility, can effectively inhibit the growth of oral squamous cell carcinoma cells, and realize the step-by-step delivery of anticancer drugs from the cell membrane to the cell nucleus, have good anti-tumor effects, and have broad application prospects in cancer treatment and other aspects.

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A co-assembled nanodrug carrying a monoclonal antibody and a chemotherapeutic drug, characterized in that: The chemotherapeutic drug-polypeptide complex and the monoclonal antibody are co-assembled by enzymatic means; the monoclonal antibody is cetuximab; the structure of the chemotherapeutic drug-polypeptide complex is shown in Formula I:

2. The method for preparing the co-assembled nanodrug carrying a monoclonal antibody and a chemotherapeutic drug according to claim 1, characterized in that: The following steps are involved: The chemotherapeutic drug-polypeptide complex, monoclonal antibody, enzyme and solvent are mixed and co-assembled to obtain the co-assembled nano drug carrying the monoclonal antibody and the chemotherapeutic drug.

3. The preparation method according to claim 2, characterized in that: The preparation method of the chemotherapy drug-polypeptide complex comprises: mixing 10-hydroxycamptothecin, glutaric anhydride and an organic solvent for esterification reaction to obtain a 10-hydroxycamptothecin-glutaric acid derivative, wherein the structural formula of the 10-hydroxycamptothecin-glutaric acid derivative is shown in Formula II; The solid phase peptide synthesis method is used to sequentially connect amino acids to synthesize a peptide chain on a resin, and the N-terminus of the peptide chain is capped with the 10-hydroxycamptothecin-glutaric acid derivative, and then the peptide chain is cut off from the resin to obtain the chemotherapy drug-polypeptide complex.

4. The preparation method according to claim 3, characterized in that: The molar ratio of the 10-hydroxycamptothecin to glutaric anhydride is 1:(3-5); and the esterification reaction time is 48-72 hours.

5. The preparation method according to claim 3, characterized in that: The end-capping method comprises: mixing the compound with the structure shown in formula II, a condensing agent, an activating agent and a solvent, and adding the obtained mixed solution into a solid phase tube used in a solid phase polypeptide synthesis method for end-capping.

6. The preparation method according to claim 2, characterized in that: The enzyme is alkaline phosphatase.

7. The preparation method according to claim 2, characterized in that: The dosage ratio of the chemotherapy drug-polypeptide complex, monoclonal antibody and enzyme is 1 mg: 1-5 μg: 8-10 U.

8. The preparation method according to claim 2, characterized in that: The co-assembly temperature is 3-5° C., the time is 24-72 hours, and the pH value is 6-7.

9. Use of the co-assembled nanodrug carrying a monoclonal antibody and a chemotherapeutic drug according to claim 1 or the co-assembled nanodrug carrying a monoclonal antibody and a chemotherapeutic drug prepared by the preparation method according to any one of claims 2 to 8 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumor is oral squamous cell carcinoma.

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