Antibody-modified co-loaded drug nanomicelles, and preparation method and application thereof

Abstract

The application discloses an antibody-modified co-loaded drug nanomicelle and a preparation method and application thereof, utilizes a reduction-responsive disulfide bond to construct a polymer-doxorubicin prodrug, and is co-assembled with a photosensitizer-loaded polymer main chain to form a mixed nanomicelle, wherein the mixed nanomicelle has suitable particle size and good physiological stability, can specifically release drugs under a tumor microenvironment, and realizes enrichment at a tumor site by modifying a monoclonal antibody on the surface of the mixed nanomicelle, the monoclonal antibody being combined with a human epidermal growth factor receptor highly expressed on the surface of tumor cells, so that the mixed nanomicelle is actively targeted to the tumor cells; the mixed nanomicelle breaks the reduction-sensitive disulfide bond under the microenvironment of a high glutathione concentration of the tumor, stimulates the responsive release of doxorubicin, and kills tumor cells; meanwhile, under the condition of laser irradiation, the monoxide oxygen generated by the photosensitizer causes damage to the tumor cells; the two cooperate to inhibit the proliferation of tumor cells, enhance the anti-tumor effect, and realize the combined treatment of chemotherapy and photodynamic therapy for cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biomedical polymer materials, and particularly relates to a preparation method of a single antibody modified combined chemotherapy and photodynamic therapy mixed micelle with reduction responsiveness and active targeting and application thereof in cancer treatment. BACKGROUND

[0002] Nanomedicine has great prospects in the diagnosis and treatment of various diseases including cancer in recent years. Block copolymer micelles are common drug carriers in the treatment of many diseases such as cancer. Amphiphilic block copolymers can self-assemble into "core-shell structure" nanomicelles as drug carriers in water. The hydrophobic core of the micelles can be used to encapsulate hydrophobic small molecule drugs to increase the solubility of the drugs. The hydrophilic shell forms a protective barrier through hydration, thereby reducing the protein adsorbent reticuloendothelial system clearance of the drugs during blood circulation and effectively prolonging the half-life of the drugs.

[0003] As a broad-spectrum anticancer chemotherapy drug, doxorubicin has good antitumor effect and achieves therapeutic effect on various tumor diseases. However, single doxorubicin chemotherapy can cause serious cardiotoxicity and other toxic side effects, so its clinical application is limited. Photodynamic therapy (PDT) can assist chemotherapy. Photosensitizer is a light-activated chemical substance that generates cytotoxic singlet oxygen 1 O2 under laser irradiation, leading to cell apoptosis and tissue damage. PDT can also mediate better drug enrichment in tumor sites by changing vascular permeability. At the same time, chemotherapy can also assist PDT to a certain extent, further removing residual cancer cells and inhibiting the regeneration of damaged blood vessels, and the two achieve combined therapy, which not only enhances the anti-tumor treatment effect and reduces the systemic toxic side effects.

[0004] Previous studies have used doxorubicin (DOX) and chlorin e6 (Ce6) for chemotherapy-photodynamic combined therapy, but due to the encapsulation method, this drug delivery system may cause premature leakage of therapeutic drugs, causing certain toxic side effects to normal cells; in addition, it is difficult to achieve a large amount of drug enrichment in tumor sites by simply enhancing the enhanced permeability and retention (EPR) effect, and the lack of targeting of the nanodrug carrier also causes a decrease in the bioavailability of the drug. SUMMARY

[0005] The application aims to provide a preparation method and application of a mixed nanomicelle of antibody-modified combined chemotherapy and photodynamic therapy, in particular, a preparation method of a mixed nanomicelle of monoclonal antibody-modified co-loaded anti-tumor drugs and phototherapy drugs. Direct treatment of relevant tumors by using small molecule drugs can cause strong toxic side effects, and construction of prodrug nanoparticles by using polymers as drug carriers can achieve anti-tumor effects while reducing damage of drugs to normal cells. The mixed micelle has biocompatibility, reduction sensitivity and active targeting, can significantly inhibit proliferation of tumor cells, has excellent anti-tumor effect, and realizes chemical-photodynamic combined treatment.

[0006] The application adopts the following technical scheme:

[0007] The application discloses a preparation method of a co-loaded drug nanomicelle, which comprises the following steps: self-assembling a chemotherapy drug prodrug and a phototherapy drug prodrug to obtain a co-loaded drug nanomicelle.

[0008] The application discloses a preparation method of a co-loaded drug nanomicelle, which comprises the following steps: self-assembling a chemotherapy drug prodrug and a phototherapy drug prodrug to obtain a co-loaded drug nanomicelle.

[0009] In the application, the monoclonal antibody comprises nimotuzumab, pertuzumab, cetuximab, CD147 monoclonal antibody or CD163 monoclonal antibody; the chemotherapy drug comprises doxorubicin, camptothecin or gemcitabine; and the phototherapy drug comprises chlorin e6 or tetracarboxy zinc phthalocyanine.

[0010] In the application, the chemotherapy drug prodrug contains a disulfide bond; a block copolymer is reacted with the chemotherapy drug prodrug modified by the disulfide bond to obtain the chemotherapy drug prodrug; preferably, a hydrophilic segment of the block copolymer is polyethylene glycol, and a hydrophobic segment contains an epoxy group; for example, the block copolymer is polyethylene glycol-glycidyl methacrylate. b - polyglycidyl methacrylate.

[0011] In the application, a polymer is reacted with a phototherapy drug prodrug to obtain the phototherapy drug prodrug, and preferably, a hydrophilic polymer is reacted with the phototherapy drug prodrug to obtain the phototherapy drug prodrug; the hydrophilic polymer is polyethylene glycol; and preferably, the phototherapy drug prodrug has a group that can react with the monoclonal antibody.

[0012] In the application, the mass ratio of the chemotherapy drug prodrug to the phototherapy drug prodrug is 1: (0.5-2), and preferably 1: (1-1.5).

[0013] The application discloses the antibody modified co-loaded drug nanomicelles prepared by the preparation method or the co-loaded drug nanomicelles and application thereof in preparation of nanodrugs.

[0014] The application utilizes the reduction sensitive disulfide bond to construct micelles, uses polyethylene glycol as a hydrophilic shell to load chemotherapeutic drugs and phototherapeutic drugs to form mixed nanomicelles, and modifies the surface of the mixed micelles with antibodies to form mixed nanomicelles with tumor cell targeting effect.

[0015] Taking doxorubicin and a photosensitizer as an example, the preparation method of the antibody modified co-loaded drug nanomicelles disclosed by the application comprises the following steps.

[0016] (1) using a block copolymer with an epoxy group in a side chain and a doxorubicin derivative modified by a disulfide bond as raw materials, reduction responsive polymer-doxorubicin prodrug nanoparticles are prepared;

[0017] (2) using maleimide polyethylene glycol and a photosensitizer as raw materials, polyethylene glycol-photosensitizer is prepared;

[0018] (3) the reduction responsive polymer-doxorubicin prodrug nanoparticles and the polyethylene glycol-photosensitizer are self-assembled to form mixed nanomicelles co-loading doxorubicin and the photosensitizer;

[0019] (4) a monoclonal antibody is modified on the mixed nanomicelles co-loading doxorubicin and the photosensitizer to obtain the antibody modified co-loaded drug nanomicelles.

[0020] The application discloses a preparation method of mixed nanomicelles co-loading doxorubicin and a photosensitizer, which comprises the following steps.

[0021] (1) using a block copolymer with an epoxy group in a side chain and a doxorubicin derivative modified by a disulfide bond as raw materials, reduction responsive polymer-doxorubicin prodrug nanoparticles are prepared;

[0022] (2) using maleimide polyethylene glycol and a photosensitizer as raw materials, polyethylene glycol-photosensitizer is prepared;

[0023] (3) the reduction responsive polymer-doxorubicin prodrug nanoparticles and the polyethylene glycol-photosensitizer are self-assembled to form mixed nanomicelles co-loading doxorubicin and the photosensitizer.

[0024] In the application, the antibody modified co-loaded drug nanomicelles are mixed nanomicelles co-loading an antibody modified anti-tumor drug and a phototherapeutic drug.

[0025] In the present application, the reduction-responsive polymer-doxorubicin prodrug nanoparticles are prepared by ring-opening reaction of the block copolymer containing epoxy group in the side chain and the doxorubicin derivative modified by disulfide bond in a solvent in the presence of a catalyst; preferably, the reduction-responsive polymer-doxorubicin prodrug nanoparticles are prepared by ring-opening reaction of the block copolymer containing epoxy group in the side chain and the doxorubicin derivative modified by disulfide bond in anhydrous N,N-dimethylformamide as a solvent in the presence of a catalyst.

[0026] In the present application, the photosensitizer is preferably chlorin e6; the polyethylene glycol modified by maleimide group at one end and amino group at the other end and the photosensitizer chlorin e6 are used as raw materials to prepare polyethylene glycol-chlorin e6 by amidation reaction in a solvent in the presence of a catalyst; preferably, the polyethylene glycol modified by maleimide group at one end and amino group at the other end and the photosensitizer chlorin e6 are used as raw materials to prepare polyethylene glycol-chlorin e6 by amidation reaction in anhydrous N,N-dimethylformamide as a solvent in the presence of a catalyst.

[0027] In the present application, the reduction-responsive polymer-doxorubicin prodrug nanoparticles and the polyethylene glycol-chlorin e6 are dispersed in a good solvent, and the mixed nanomicelles co-loading doxorubicin and chlorin e6 are formed by self-assembly in water.

[0028] In the present application, the mixed nanomicelles co-loading doxorubicin and chlorin e6 and the monoclonal antibody are used as raw materials to realize antibody-modified nanoparticles by Michael addition reaction of the maleimide group on the surface of the mixed nanomicelles with the thiol group of the monoclonal antibody in an acidic buffer solution, so as to obtain the mixed nanomicelles co-loading doxorubicin and chlorin e6 modified by the monoclonal antibody.

[0029] There are studies on the chemical-photodynamic combination therapy using doxorubicin (DOX) and chlorin e6 (Ce6), but most of them adopt the drug loading mode, which may cause the premature leakage of the therapeutic drugs and cause certain toxic side effects to normal cells; in addition, it is difficult to realize the large enrichment of the drugs at the tumor site only by enhancing the permeability retention (EPR) effect, and the lack of targeting of the nanodrug carrier also causes the decrease of the bioavailability of the drugs. The present application improves the above problems: the reduction-sensitive disulfide bond is used to realize the coupling of DOX and the polymer, the generated polymer prodrug stably exists under normal physiological conditions and blood circulation, and the disulfide bond is broken under the action of high-concentration glutathione in the tumor microenvironment and DOX is released, so as to improve the stability and safety of the drug-loaded micelles; in addition, the monoclonal antibody is modified on the surface of the mixed micelles, specifically binds to the EGFR receptor highly expressed on the surface of tumor cells, targets the surface of tumor cells, and increases the drug enrichment amount at the tumor site, so as to realize the targeted anti-tumor treatment and significantly improve the treatment effect.

[0030] The application discloses a preparation method of an antibody modified co-loaded drug nanomicelle, which comprises the following steps:

[0031] (1) obtaining a block copolymer polyethylene glycol-glycidyl methacrylate from bromine-terminated methoxypolyethylene glycol and glycidyl methacrylate as raw materials; preferably, under an inert atmosphere, taking bromine-terminated methoxypolyethylene glycol as a macroinitiator, glycidyl methacrylate as a monomer, anhydrous tetrahydrofuran as a solvent, and in the presence of a catalyst and a ligand, the block copolymer polyethylene glycol-glycidyl methacrylate is obtained through atom transfer radical polymerization; b b (2) preparing an adriamycin derivative from dithiodipropionic anhydride and hydrochloric acid adriamycin; preferably, under an inert atmosphere, taking dithiodipropionic anhydride and hydrochloric acid adriamycin as raw materials, and anhydrous N,N-dimethylformamide as a solvent, the adriamycin derivative modified by a disulfide bond is prepared through the reaction between an anhydride and an amino group;

[0032] (3) under an inert atmosphere, taking the block copolymer containing an epoxy group in a side chain and the adriamycin derivative modified by a disulfide bond as raw materials, and anhydrous N,N-dimethylformamide as a solvent, the reductive-responsive polymer-adriamycin prodrug nanoparticles are prepared through a ring-opening reaction in the presence of a catalyst;

[0033] (4) preparing polyethylene glycol-dihydrophenophor e6 from polyethylene glycol and photosensitizer dihydrophenophor e6; preferably, taking polyethylene glycol modified by a maleimide group at one end and an amino group at the other end (maleimide-polyethylene glycol-amino) and the photosensitizer dihydrophenophor e6 as raw materials, and anhydrous N,N-dimethylformamide as a solvent, the polyethylene glycol-dihydrophenophor e6 is prepared through amidation in the presence of a catalyst;

[0034] (5) mixing the polymer-adriamycin prodrug and the polyethylene glycol-dihydrophenophor e6 in a certain proportion to form the mixed nanomicelle co-loaded with adriamycin and dihydrophenophor e6 (abbreviated as DCMMs) through self-assembly; preferably, the reductive-responsive polymer-adriamycin prodrug and the polyethylene glycol-dihydrophenophor e6 are dispersed in a good solvent in a certain proportion, and the mixed nanomicelle co-loaded with adriamycin and dihydrophenophor e6 is formed through self-assembly in water or a buffer (preferably a neutral buffer);

[0035] (5) mixing the polymer-adriamycin prodrug and the polyethylene glycol-dihydrophenophor e6 in a certain proportion to form the mixed nanomicelle co-loaded with adriamycin and dihydrophenophor e6 (abbreviated as DCMMs) through self-assembly; preferably, the reductive-responsive polymer-adriamycin prodrug and the polyethylene glycol-dihydrophenophor e6 are dispersed in a good solvent in a certain proportion, and the mixed nanomicelle co-loaded with adriamycin and dihydrophenophor e6 is formed through self-assembly in water or a buffer (preferably a neutral buffer);

[0036] ​(6) Modify the surface of the mixed nanomicelles with monoclonal antibodies to obtain a mixed nanomicelles (abbreviated as NTZ-DCMMs) for combined chemotherapy and photodynamic therapy with antibody modification; preferably, using mixed nanomicelles and monoclonal antibodies as raw materials, under acidic buffer conditions such as 2-morpholine ethanesulfonic acid buffer (MES buffer), the maleimide groups on the surface of the mixed nanomicelles undergo a Michael addition reaction with the thiol groups of the monoclonal antibodies to achieve antibody modification of nanoparticles, thereby obtaining mixed nanomicelles modified with monoclonal antibodies and co-loaded with doxorubicin and dihydroporphyrin e6.

[0037] In the above technical solution:

[0038] In step (1), the inert atmosphere is nitrogen; the catalyst is cuprous bromide; the ligand is pentamethyldiethylenetriamine; the molar ratio of bromine-terminated methoxy polyethylene glycol, glycidyl methacrylate, cuprous bromide, and pentamethyldiethylenetriamine is 1:60 to 80:1:2; the atom transfer radical polymerization reaction temperature is 60 to 65 °C; and the reaction time is 1 to 3 h.

[0039] In step (2), the molar ratio of doxorubicin hydrochloride to dithiodipropionic anhydride is 1:1 to 2; the inert atmosphere is nitrogen; the acylation reaction temperature is room temperature to 40 °C; and the reaction time is 24 h to 48 h.

[0040] In step (3), the inert atmosphere is nitrogen; the catalyst is tetrabutylammonium hydroxide; polyethylene glycol- b The molar ratio of poly(glycidyl methacrylate), doxorubicin derivative, and tetrabutylammonium hydroxide is 1:1.2 to 1.8:1; the ring-opening reaction temperature is 60 ℃ to 80 ℃; and the reaction time is 12 h to 24 h.

[0041] In step (4), the molar ratio of maleimide-polyethylene glycol-amino to dihydroporphyrin E6 is 1:1.2-1.6; the catalyst is N-hydroxysuccinimide or 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the amidation reaction temperature is 20℃-30℃; and the reaction time is 12 h-48 h.

[0042] In step (5), the good solvent is dimethyl sulfoxide, and the stirring time during self-assembly is 2 to 4 hours.

[0043] In step (6), the mass ratio of monoclonal antibody to mixed micelle nanoparticles is 1:500 to 1500; the Michael addition reaction is carried out at room temperature for 3 to 10 hours.

[0044] The application provides a drug delivery system with safety and stability, avoids early release of drugs in blood circulation in vivo, reduces damage to normal cells, and improves bioavailability of the drugs; meanwhile, the surface of mixed nanomicelles is combined with a monoclonal antibody, so that the targeting and specificity can be obviously enhanced, and good inhibiting effect on tumor cells with high expression of EGFR can be achieved. The introduction of photodynamic therapy avoids serious toxic side effects caused by single traditional chemotherapy, and Ce6 and DOX have good synergistic effect, so that the chemical-photodynamic combined therapy significantly improves the anti-tumor effect.

[0045] In the application, the block copolymer polyethylene glycol- b The chemical structure of the block copolymer polyethylene glycol-glycidyl methacrylate is as follows:

[0046] ;

[0047] In the formula, x is 45-113, and y is 17-38.

[0048] The chemical structure of the disulfide bond modified doxorubicin derivative is as follows:

[0049] ;

[0050] The chemical structure of the reduction-responsive polymer-doxorubicin prodrug is as follows:

[0051] ;

[0052] In the formula, DOX represents doxorubicin, x is 45-113, y is 17-38, and z is 5-12.

[0053] The chemical structure of the polyethylene glycol-chlorin e6 is as follows:

[0054] ;

[0055] In the formula, m is 45-113.

[0056] This invention assembles a polymer backbone containing a reduction-responsive polymer—doxorubicin prodrug—and a photosensitizer, dihydroporphyrin E6, into hybrid nanomicelles. These micelles not only release the chemotherapeutic drug doxorubicin in response to the tumor microenvironment but also induce singlet oxygen production in the photosensitizer dihydroporphyrin E6 under 660 nm laser irradiation, achieving combined chemotherapy and photodynamic therapy. Furthermore, the monoclonal antibody modified on the surface of the hybrid nanomicelles enables targeted drug delivery, reduces toxic side effects on normal cells, enhances drug accumulation at the tumor site, significantly inhibits tumor cell proliferation, and further improves the anti-tumor therapeutic effect. Therefore, this invention discloses the application of the aforementioned monoclonal antibody-modified co-loaded drug nanomicelles in the preparation of nanomedicines, such as anti-tumor drugs, specifically in the preparation of targeted reduction-responsive anti-tumor polymeric prodrugs.

[0057] Due to the implementation of the above method, the present invention has the following advantages compared with the prior art:

[0058] This invention is the first to synthesize doxorubicin derivatives modified with disulfide bonds and carboxyl groups, and to synthesize them with block copolymers (polyethylene glycol- b The epoxy groups on the side group of poly(glycidyl methacrylate) undergo a bonding reaction to form a reduction-responsive polymer-doxorubicin prodrug, enabling reduction-responsive drug release in the microenvironment of high glutathione concentration in tumor cells;

[0059] This invention proposes to co-assemble a reduction-responsive polymer—doxorubicin prodrug—with polyethylene glycol—dihydroporphyrin E6 in an aqueous phase to form mixed nanomicelles. These micelles exhibit good stability and biocompatibility, enabling combined chemotherapy-photodynamic therapy for antitumor treatment.

[0060] This invention utilizes the Michael addition reaction between the maleimide groups on the surface of mixed micelle nanoparticles and the thiol groups of antibodies to achieve antibody-micelle nanoparticle coupling. The mixed nanomicelles modified with monoclonal antibodies have good active targeting properties and exhibit superior anti-tumor therapeutic effects.

[0061] The monoclonal antibody-modified mixed nanomicelle structure co-loaded with doxorubicin and dihydroporphyrin E6 provided by this invention has a reasonable structure, high drug loading capacity, simple synthesis, readily available raw materials, and simple purification. Attached Figure Description

[0062] Figure 1 The methoxylated polyethylene glycol (PEG) with bromine-terminated initiator in Example 1 2000 The NMR spectrum of 1H NMR (-Br) was obtained using deuterated chloroform as the solvent.

[0063] Figure 2 The block copolymer polyethylene glycol in Example 1 b - Polyglycidyl methacrylate (PEG-b

[0064] Figure 3 2000

[0065] Figure 4 ss ss

[0066] Figure 5

[0067] Figure 6 -1

[0068] Figure 7

[0069] Figure 8

[0070] Figure 9

[0071] Figure 10

[0072] Figure 11 b ​​​​​​​​​​​​​​- Cell survival rate after co-incubating PGMA with human umbilical vein endothelial cells (HUVEC cells) and mouse liver cancer cells (H22) for 48 h.

[0073] Figure 12 The original drug doxorubicin (Free DOX) and polymer-doxorubicin prodrug (PEG-) in Example 9 ss Cytotoxicity of mouse hepatocellular carcinoma cells H22 by DOX, photosensitizer prodrug (PEG-Ce6), mixed nanomicelles (DCMMs), and monoclonal antibody-modified mixed nanomicelles (NTZ-DCMMs).

[0074] Figure 13 The images show the fluorescence uptake by H22 cells after incubation with PBS, Free DOX, Free Ce6, DCMMs, and NTZ-DCMMs for 3 h and 6 h, respectively, in Example 10.

[0075] Figure 14 The intracellular ROS levels of H22 cells after treatment with PBS, Free Ce6, DCMMs, and NTZ-DCMs under light or no light conditions in Example 11 are shown.

[0076] Figure 15 Fluorescent images of major internal organs and tumors in mice 4 h and 24 h after tail vein injection of the drug in Example 12.

[0077] Figure 16 The images show the changes in mouse body weight, tumor volume, and solid tumors removed after 14 days of drug treatment, as well as the tumor weight, recorded during the treatment period in Example 13. (In the one-way ANOVA, *, *, and **** represent P<0.05, P<0.01, P<0.001, and P<0.0001, respectively.)

[0078] Figure 17 The images show the hematoxylin-eosin staining, polynucleotide chain break detection, and immunohistochemical images of mouse tumors after treatment in Example 13.

[0079] Figure 18 This is a schematic diagram of the synthetic route and micelle structure of monoclonal antibody-modified mixed nanomicelles co-loaded with doxorubicin and dihydroporphyrin E6. Detailed Implementation

[0080] The micelles formed by physically entrapping drugs can cause premature drug leakage and other problems during in vivo circulation, and the strategy of polymer prodrug can effectively improve the stability of drugs in blood circulation by using chemical bonds sensitive to tumor microenvironment to connect drugs and micelles. The present application utilizes the characteristics of high concentration glutathione (GSH) microenvironment of tumor cells, introduces reduction-sensitive disulfide bond in the prodrug, and realizes the release of drugs in tumor cells. A mixed nanomicelle co-loading chemotherapeutic drug doxorubicin and phototherapeutic drug chlorin e6 is constructed, and the mixed nanomicelle is modified with monoclonal antibody, so that the mixed nanomicelle modified by the antibody can actively target the receptors highly expressed on the surface of cancer cells. On the one hand, the disulfide bond is broken to release the original drug under the high concentration GSH environment of tumor, so as to realize the enrichment of drugs in tumor sites and kill tumor cells; on the other hand, under the condition of laser irradiation, the active oxygen generated by the photosensitizer causes damage to tumor cells, and the combination of chemotherapy and photodynamic therapy realizes the combined anti-tumor treatment.

[0081] The application discloses a preparation method of an antibody-modified co-loaded drug nanomicelle, which comprises the following steps:

[0082] (1) Under the condition of inert atmosphere, methoxypolyethylene glycol terminated by bromine is used as a macromolecular initiator, glycidyl methacrylate is used as a monomer, anhydrous tetrahydrofuran is used as a solvent, and a block copolymer polyethylene glycol-g-polyglycidyl methacrylate is obtained by atom transfer radical polymerization in the presence of a catalyst and a ligand, wherein the block copolymer is a block copolymer with an epoxy group in a side chain; b

[0083] (2) Under the condition of inert atmosphere, dithiodipropionic anhydride and doxorubicin hydrochloride are used as raw materials, and anhydrous N,N-dimethylformamide is used as a solvent, and a doxorubicin derivative modified by a disulfide bond is prepared by the reaction of an acid anhydride and an amino group;

[0084] (3) Under the condition of inert atmosphere, the block copolymer with an epoxy group in a side chain and the doxorubicin derivative modified by the disulfide bond are used as raw materials, anhydrous N,N-dimethylformamide is used as a solvent, and a reduction-responsive polymer-doxorubicin prodrug nanoparticle is prepared by ring-opening reaction in the presence of a catalyst;

[0085] (4) Polyethylene glycol modified with a maleimide group at one end and an amino group at the other end and a photosensitizer chlorin e6 are used as raw materials, anhydrous N,N-dimethylformamide is used as a solvent, and polyethylene glycol-chlorin e6 is prepared by amidation reaction in the presence of a catalyst;

[0086] (5) The reduction-responsive polymer-doxorubicin prodrug and the polyethylene glycol-chlorin e6 are dispersed in a good solvent according to a certain proportion, and a mixed nanomicelle co-loading doxorubicin and chlorin e6 is formed by self-assembly in water; ​

[0087] (6) Using mixed nanomicelles and monoclonal antibodies as raw materials, a Michael addition reaction is carried out between the maleimide groups on the surface of the mixed nanomicelles and the sulfhydryl groups of the monoclonal antibodies in an acidic buffer solution, so as to realize antibody-modified nanoparticles, and obtain mixed nanomicelles co-loaded with doxorubicin and chlorin e6.

[0088] The raw materials involved in the present application are all existing products, and the specific preparation operation and performance test are conventional technologies. The animal experiments meet the relevant requirements of Suzhou University, and part of them are statistically analyzed. Example 1

[0089] Synthesis of amphiphilic block polymer main chain PEG b -PGMA

[0090] Synthesis of amphiphilic block polymer main chain PEG b -PGMA is prepared by atom transfer radical polymerization.

[0091] Preparation of macromolecular initiator PEG 2000 -Br by substitution reaction, and the specific synthesis method is as follows: 50 mL single-necked flask is added mPEG 2000 (2.5 mmol, 5 g), TEA (5 mmol, 0.5 g), anhydrous toluene (20 mL), and stirred and dissolved at room temperature; then 2-bromoisobutyryl bromide (5 mmol, 1.15 g) is dispersed in 20 mL of anhydrous toluene, and is added dropwise into the single-necked flask at a rate of 1 drop / 2 seconds using a dry 100 mL constant-pressure dropping funnel in an ice-water bath, and after the dropwise addition is completed, the reaction is continued at room temperature for 24 h, and then transferred to a 35°C oil bath (600 r / min) for continuous reaction for 24 h. After the reaction is completed, the reaction residue is hot-filtered to remove the generated triethylamine salt, and most of the solvent is removed by a rotary evaporator, and the concentrated residue is precipitated in ice n-hexane for 2 times, and finally dried in a vacuum drying box for 12 h to obtain white powder product PEG 2000 -Br (yield: 3.7 mg, yield: 68.3%). The proton nuclear magnetic resonance spectrum of the product is as shown in Figure 1 .

[0092] Preparation of amphiphilic block copolymer polyethylene glycol-polyglycidyl methacrylate PEG b -PGMA by atom transfer radical polymerization, and the specific synthesis method is as follows: monomer glycidyl methacrylate GMA (262.5 mg), PEG 2000-Br (200 mg), pentamethyldiethylenetriamine (PMDETA, 48 mg) was dissolved in 20 mL tetrahydrofuran (THF), the system was deoxygenated by two freeze-thaw cycles, CuBr (13.3 mg) was added, and another freeze-thaw cycle was performed, and finally filled with nitrogen, then reacted at 60°C for 2 h. After the reaction was completed, the atmosphere was passed and the reaction was terminated by cooling. The reaction retention liquid was passed through an alkaline aluminum oxide column to remove most of the copper ions, and then loaded into a dialysis bag with a molecular weight cut-off of 3.5 kDa, dialyzed in THF for 24 h, and then dialyzed in deionized water for 48 h (the dialysis water was replaced every 6 h), 20 g of Seachem was added to the dialysis water to remove the remaining small amount of copper ions, and the solution in the dialysis bag changed from light green to colorless. The solution was freeze-dried to obtain a white flocculent solid, which was polyethylene glycol- b - glycidyl methacrylate (PEG- b -PGMA, yield: 221.3 mg; yield: 72.6%). The product was characterized by nuclear magnetic resonance hydrogen spectrum ( Figure 2 ) and gel permeation chromatogram ( Figure 3 ). Example Two

[0093] Synthesis of amphiphilic reduction-responsive polymer-doxorubicin prodrug PEG- ss -DOX

[0094] Reduction-responsive polymer prodrug PEG- ss -DOX is prepared by ring-opening reaction between doxorubicin derivative DOX- ss -COOH and the epoxy group of GMA in block copolymer PEG- b -PGMA.

[0095] Synthesis of disulfide linker 3,3'-dithiodipropionic anhydride (DTDPA). The synthesis method is as follows: 3,3'-dithiodipropionic acid (3.0 g, 48 mmol) was dissolved in acetyl chloride (30 mL), and refluxed in a coiled condenser tube at 70°C for 12 h. After the reaction was completed, the temperature was lowered to room temperature, and the solvent was removed by a rotary evaporator. The residue was precipitated with ethyl ether and washed three times, and finally dried under vacuum to obtain a white solid, which was 3,3'-dithiodipropionic anhydride (DTDPA, yield: 1.4 mg; yield: 51.1%).

[0096] Synthesis of disulfide bond modified doxorubicin derivative DOX- ss-COOH. The specific synthesis method is as follows: Doxorubicin hydrochloride (DOX·HCl) (100 mg) and triethylamine (TEA) (55 mg) were dissolved in 10 mL of DMF. The mixture was stirred in the dark for 4 h to remove hydrochloric acid. Then, DTDPA (40 mg) dissolved in 10 mL of DMF was added. The mixture was reacted in an oil bath at 35 °C in the dark for 24 h. After the reaction was completed, the mixture was cooled to room temperature, and most of the DMF was removed by rotary evaporation. The mixture was precipitated three times in ice-cold ether, and finally dried under vacuum to obtain a red solid powder, namely DOX- ss -COOH (Yield: 120 mg; Yield: 79%).

[0097] DOX- ss -COOH carboxyl group and PEG- b The epoxy groups of PGMA undergo a ring-opening reaction, and DOX is grafted onto the side groups of the block copolymer to synthesize the reduction-responsive polymer - doxorubicin prodrug PEG-. ss -DOX. The specific synthesis method is as follows: Under nitrogen purging, PEG-DOX is added sequentially to a 50 mL side-burning flask. b -PGMA (99.2 mg), DOX- ss -COOH (300 mg), tetrabutylammonium hydroxide (TBAH) (56.44 mg), and 20 mL DMF were reacted in an oil bath at 70 °C in the dark for 12 h. After the reaction was completed and cooled to room temperature, the reaction residue was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed. First, it was dialyzed in DMF for 24 h, then transferred to deionized water for another 48 h. The solution in the dialysis bag was freeze-dried, finally yielding a red flocculent solid, namely PEG- ss -DOX (yield: 143 mg; yield: 41%). The product contains DTDPA and DOX- ss -COOH, PEG- ss -DOX's 1H NMR spectrum is as follows Figure 4 As shown. Example 3

[0098] Synthesis of polyethylene glycol-dihydroporphyrin e6 (PEG-Ce6)

[0099] The photosensitizer backbone PEG-Ce6 was generated by an amidation reaction mediated by N-hydroxysuccinimide (NHS) and carbodiimide (EDC). The specific synthesis method is as follows: Ce6 (59.7 mg) was first dissolved in 5 mL of dimethyl sulfoxide (DMSO), followed by the sequential addition of NHS (14.4 mg) and EDC (24.0 mg). The mixture was stirred at room temperature for 6 h to activate the carboxyl groups. Then, Mal-PEG-NH2 (216.7 mg) dissolved in 10 mL of DMSO was added... The product was added dropwise to the Ce6 solution (dropwise time: 1 minute), and stirring continued at room temperature for 24 h. The reaction mixture was dialyzed using a dialysis bag with a molecular weight cutoff of 3.5 kDa to purify the product. Dialysis was first performed in DMSO for 24 h, then transferred to deionized water for another 48 h. Finally, the solution in the dialysis bag was freeze-dried to obtain a black solid powder, namely polyethylene glycol-dihydroporphyrin e6 (PEG-Ce6, yield: 167 mg, 60.4%). The 1H NMR spectra of the raw material Mal-PEG-NH2 and the product PEG-Ce6 are shown below. Figure 5 As shown. Example 4

[0100] Preparation of mixed micelle DCMMs and nimotuzumab-modified prodrug nanoparticles NTZ-DCMMs

[0101] Mixed nanomicelles co-loaded with doxorubicin and dihydroporphyrin E6 were prepared by dialysis. The specific method is as follows: Weigh 50 mg of PEG- ss -DOX and 60 mg of PEG-Ce6 were placed in a single-necked round-bottom flask, and 2 mL of dimethyl sulfoxide was added to dissolve them completely. A micro-injection pump (WZS-50F) equipped with PBS was used to inject the solution at a rate of 2 mL / h. -1 The sample was added dropwise to the flask at a controlled dropping rate under light-protected stirring conditions. After complete injection, stirring was continued for 6 hours in the dark. The mixed micelle solution was then transferred to a dialysis bag with a molecular weight cutoff of 5 kDa and dialyzed in deionized water for 24 hours (with the dialysate changed every 6 hours). Finally, the mixed micelle solution obtained after dialyzing was brought to a final volume of 25 mL with deionized water to obtain mixed nanomicelles (DCMMs) co-loaded with doxorubicin and dihydroporphyrin E6 at a concentration of 0.5 mg / mL. -1 The particle size distribution histogram and corresponding transmission electron microscopy image of the mixed nanomicelles are shown below. Figure 6 As shown. Dynamic light scattering instrumentation measured the size of the mixed nanomicelles to be approximately 185 nm, with a narrow particle size distribution and a dispersion index of 0.054; TEM observation revealed that the nanoparticles were uniformly dispersed spherical with a particle size of approximately 180 nm.

[0102] Nimotuzumab (purchased from Shanghai Saimei Biotechnology) modified prodrug nanoparticles NTZ-DCMMs were prepared via a Michael addition reaction between maleimide on the surface of mixed micelle nanoparticles and free thiol groups contained in nimotuzumab. The specific procedure is as follows: First, a 0.5 M 2-morpholinoethanesulfonic acid buffer solution was prepared, and the pH was adjusted to 6 by titration with 1 M NaOH aqueous solution. Then, 100 μL of the monoclonal antibody and the mixed micelle nanoparticles were reacted in MES buffer solution at room temperature for 12 h, yielding the monoclonal antibody-modified mixed micelle nanoparticles. Example 5

[0103] Particle size stability of mixed nanomicelles DCMMs under different conditions

[0104] The average particle size and particle size distribution of nanoparticles are important parameters for evaluating whether they can pass through the tumor vascular barrier. The hydrophobic core of mixed nanomicelles co-loaded with doxorubicin and chlorin e6 can improve the solubility of small molecule drugs and prolong the circulation half-life, while the polyethylene glycol hydrophilic chain isolates the hydrophobic core from the outside world, reducing nonspecific protein adsorption, thereby ensuring the stable existence of nanoparticles under normal physiological conditions. The specific experimental steps are as follows: First, prepare mixed nanomicelles using different media (PB 7.4 solution, 10% FBS PB 7.4 solution, and PB 7.4 solution containing 10 mM GSH): dissolve 50 mg of PEG-DOX and 60 mg of PEG-Ce6 in 2 mL of DMSO solution, use a micro-injection pump to add PB 7.4 solution, 10% FBS PB 7.4 solution, or PB 7.4 solution containing 10 mM GSH, stir for 4 h, then dialyze in deionized water for 24 h, and finally dilute to 220 mL, with a uniform concentration of 0.5 mg mL ss -1 .

[0105] Stir at room temperature, test the particle size and particle size change at intervals, and the test results are shown in Figure 7 (Left). In PB7.4 medium, the particle size and particle size distribution of mixed nanomicelles hardly change within 48 h, verifying the stability of mixed micelles under normal physiological conditions; in 10% FBS PB 7.4 solution, the particle size and distribution hardly change with time, indicating that mixed micelles can remain stable in blood circulation and avoid premature drug leakage; PB 7.4 solution containing 10 mM GSH is used to simulate the tumor microenvironment. Under this condition, the particle size and particle size distribution change significantly with time, and the particle size tends to increase, which is due to the breaking of the disulfide bond of mixed micelles DCMMs under reducing conditions, the destruction of the structure of the nanoparticles, the dissociation of the hydrophilic segment and the hydrophobic segment, the dissolution or agglomeration of the hydrophobic segment, and the broadening of the particle size distribution, resulting in multiple peaks, as shown in Figure 7 (right). Example Six

[0106] Drug release behavior of mixed nanomicelles DCMMs in vitro

[0107] ​The mixed nanomicelles DCMMs can break the disulfide bond and release the doxorubicin under the reducing condition of GSH. The in vitro drug release behavior of DCMMs was studied by dialysis method. The specific method is as follows: first, two different buffer solutions are configured: (1) pH 7.4 phosphate buffer solution containing 0.5 w t% Tween 80 PB 7.4 phosphate buffer solution; (2) 10 mM GSH + pH 7.4 phosphate buffer solution + 0.5 w t% Tween 80. Take 5 mL of mixed micelles DCMMs with a concentration of 0.5 mg mL -1 , and place them in a dialysis bag with a molecular weight cut-off of 10 kDa. Add 40 mL of different buffer solutions to the dialysis bag in a large centrifuge tube, and oscillate in a constant temperature shaking incubator at 37°C at a speed of 160 rpm. At the set time point, take out 5 mL of dialysate, and supplement with an equal volume of buffer solution. Each group is performed in triplicate, and the average value is calculated and the error is calculated. The DOX concentration in the taken buffer solution is determined by fluorescence spectrophotometry, the excitation wavelength is set to 488 nm, the scanning range is 520-620 nm, and the fluorescence intensity at 596 nm is read. The DOX concentration corresponding to the absorbance value is calculated according to the DOX aqueous standard curve. The cumulative drug release amount is calculated according to formula (1):

[0108] (1);

[0109]

[0110] V e : the volume of each release liquid taken out;

[0111] V 0: the initial release liquid volume;

[0112] C i : the drug concentration in the release liquid taken out for the ith time (mg mL -1 );

[0113] C n : the drug concentration in the release liquid taken out for the nth time (mg mL -1 );

[0114] m drug : the initial mass of drug contained in the nanoparticles (mg);

[0115] n: the number of sampling times.

[0116] The results are as follows: Figure 8 ​As shown, the cumulative release of DCMMs in PB 7.4 medium was less than 10% in 100 h, indicating that DCMMs could exist stably under normal physiological conditions and avoid drug leakage. However, under the reducing condition of 10 mM GSH, the cumulative release of DCMMs was more than 60%, which embodied its reduction responsiveness and could realize drug enrichment at the tumor site. Example Seven

[0117] Blood compatibility of mixed nanomicelles DCMMs

[0118] Hemolysis reaction affects the degree of red blood cell lysis and the release of hemoglobin caused by the material. Good blood compatibility determines whether the drug can be widely used in the body circulation. The degree of red blood cell membrane damage after incubation of the drug with red blood cells was determined by spectrophotometry. The specific experimental steps are as follows: collect mouse orbital blood with a heparin sodium pretreated blood collection tube, add 5 mL of PBS and blow evenly, and centrifuge at 3000 rpm to make the intact red blood cells precipitate; remove the supernatant, continue to add 10 mL of PBS to the red blood cell suspension and blow evenly, and remove the supernatant, repeat the same steps three times until the supernatant becomes colorless; finally, add 10 mL of PBS and mix well, and store in the refrigerator for standby. Prepare 21 centrifuge tubes, each tube contains 0.5 mL of evenly dispersed red blood cell suspension, use ultrapure water as positive control and PBS as negative control, divide into three groups for experiment, the first group is DOX PBS solution, the concentration range is 3.13-50 mg L -1 ; the second group is Ce6 PBS solution, the concentration range is 2.5-40 mg L -1 ; the third group is mixed nanomicelles DCMMs co-loaded with doxorubicin and chlorin e6. The doxorubicin content of the third group is the same as the first group, and the Ce6 content of the third group is the same as the second group. Place all centrifuge tubes in a constant temperature shaking incubator (37°C, 160 rpm) and shake for 4 h, then use a high-speed centrifuge (4°C, 10000 rpm, 5 min) to centrifuge the red blood cells, and take 300 μL of supernatant from each sample and add it to a 96-well plate, all samples are tested in triplicate. Measure the absorbance at 540 nm using a microplate reader, and calculate the hemolysis percentage using formula (2):

[0119] (2);

[0120] In the formula, OD sample is the absorbance of the sample well, OD (-)control is the absorbance of the negative control group, and OD (+)control is the absorbance of the positive control group. The test results are as follows: Figure 9The hemolysis rate of DCMMs group was significantly lower than that of free DOX group and free Ce6 group, and was less than 6%, indicating that DCMMs had good blood compatibility and did not cause significant damage to red blood cells. Example Eight

[0121] In vitro ROS detection of mixed nanomicelles DCMMs

[0122] 1,3-diphenyl isobenzofuran (DPBF) was used as a singlet oxygen 1 O2probe to evaluate the photodynamic effect of DCMMs in vitro. DPBF is a highly efficient singlet oxygen trapping agent, which has a strong ultraviolet absorption peak at 410 nm when it is in a reduced state. Once it 1 O2binds, DPBF is irreversibly oxidized, and the ultraviolet absorption intensity at 410 nm rapidly decreases. Based on this, DPBF was used for in vitro ROS detection experiments.

[0123] The specific operation is as follows: first, prepare a DMSO solution with a concentration of 30 μM DPBF. Under dark conditions, add 10 μL of the DPBF solution to free Ce6, PEG-Ce6 and DCMMs respectively (the concentration of Ce6 in all samples is 1 μg mL -1 ), and irradiate the samples with a 660 nm red light laser (power density: 0.2 W cm -2 ). At 20, 60, 120, 180 and 300 s, respectively, measure the ultraviolet absorption peak of the samples at 408 nm using an ultraviolet spectrophotometer, with DCMMs without light as a blank control group. From Figure 10 it can be seen that the absorbance of the blank control group without light at 408 nm hardly changes; while under red light irradiation, the absorbance gradually decreases with the extension of the irradiation time, indicating that the ultraviolet absorption intensity of DPBF decreases, and PEG-Ce6 and DCMMs do not destroy the ability of the photosensitizer Ce6 to generate 1 O2under light irradiation. Example Nine

[0124] Cell compatibility and cytotoxicity experiment

[0125] As drug-loaded micellar nanoparticles, they should respond to release drugs in the tumor microenvironment, and not cause cytotoxicity under normal physiological conditions. Therefore, the conventional tetramethyl azo salt micro-enzyme reaction colorimetric method (MTT method) was used to detect the cytotoxicity of the polymer backbone PEG- b -PGMA to verify its biocompatibility with human umbilical vein endothelial cells (HUVEC cells). The CCK-8 method was used to verify the cytotoxicity of the polymer PEG- b- The biocompatibility of PGMA with mouse hepatocellular carcinoma cells (H22 cells) was evaluated, along with free DOX and PEG- ss - The toxicity of DOX, PEG-Ce6, DCMMs and NTZ-DCMMs to H22 cells.

[0126] Detection of polymer PEG- using the MTT assay b -PGMA is for biocompatibility. The specific steps are as follows: The culture medium consists of DMEM containing 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin, and streptomycin. First, cryopreserved human umbilical vein endothelial cells (HUVECs) are thawed and passaged. Cells in good logarithmic growth phase are selected to prepare a cell suspension. The cells are then seeded into 96-well plates (100 μL is added to each well except the outermost well, and the seeding density is 5 × 10⁶ cells / well). 4 Cells were then placed in a cell culture incubator (37 °C, 5% CO2) for 12 h to allow for full cell adhesion. Different concentrations of polymer PEG- were then prepared. b Add 25 μL of sample solution and 100 μL of culture medium (sample diluted 5-fold) to each well of the PGMA PBS solution. Set up four parallel experiments with PBS as a blank control group. After sample addition, continue incubation in a cell culture incubator for 48 h. Prepare MTT PBS solution (concentration 5 mg / mL). -1 Add 25 μL of MTT solution to each well and incubate in a cell culture incubator for 4 h to reduce the MTT in the living cells to blue-purple formazan crystals and deposit them in the cells. Then, remove the supernatant from each well and add 150 μL of DMSO to dissolve the formed formazan crystals. Finally, use a microplate reader to detect the absorbance (optical density, OD) of each well at 570 nm. The cell viability is calculated using formula (3). Figure 11 ).

[0127] (3);

[0128] In the formula, OD treated The absorbance of the sample being measured is OD. control The absorbance is for the PBS blank control group.

[0129] The toxicity of mixed nanomicelles to H22 cells was detected using the CCK-8 assay. The specific procedures were as follows: RPMI-1640 culture medium containing 10% high-quality fetal bovine serum and 1% penicillin / streptomycin was used. Frozen H22 cells were revived and passaged. Cells in the logarithmic growth phase were collected, and cell counts were performed using a cell counting chamber. The cell suspension concentration was adjusted, and 100 μL of mouse hepatocellular carcinoma cell (H22 cell) suspension was added to each well of a 96-well plate, resulting in a final seeding density of 5 × 10⁶ cells / well. 4 Each well contains [number] samples. The plate is incubated in a cell culture incubator for 12 hours. Samples of different concentrations (mixed micelles based on DOX concentration) are prepared: free DOX, PEG- [concentration not specified]. ss -DOX, PEG-Ce6, DCMMs and NTZ-DCMMs, wherein the concentration of DMSO added to the well plate is guaranteed to be no more than 0.1%. The sample addition operation is the same as the MTT method. After the sample addition is completed, the 96-well plate of PEG-Ce6, DCMMs and NTZ-DCMMs groups is incubated in a cell culture incubator for 4 h, and then irradiated with a 660 nm red laser (200 mW) for 5 min, and then placed in a cell culture incubator for 44 h. Then, 10 μL of CCK-8 solution is added to each well, and the cell culture incubator is incubated for 4 h. Finally, the absorbance of each well is measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability is calculated by formula (3). Figure 12 Among them, free DOX and PEG- ss The half-maximal inhibitory concentrations (IC50) of DOX, PEG-Ce6, DCMMs, and NTZ-DCMMs groups were as follows: 50 The DOX concentrations (μg / mL) were 0.200, 1.125, 3.025, 0.842, and 0.699, respectively. Example 10

[0130] Cell uptake experiment

[0131] The process of nanoparticle uptake by cells was observed using laser confocal microscopy. The uptake of H22 cells by mixed nanomicelles (DCMMs) co-loaded with doxorubicin and dihydroporphyrin e6, and nimotuzumab-modified nanoparticles (NTZ-DCMMs) and the release of drugs under high GHS conditions within tumor cells were investigated. The specific steps are as follows: First, DCMMs and NTZ-DCMMs (containing 50 mg / L of DOX) were prepared. -1 The Ce6 content is 40 mg / L. -1 After resuscitation, H22 cells were cultured until they multiplied, counted, diluted several times, and seeded into 35 mm glass-bottomed culture dishes (NEST, China) at a seeding density of 2 × 10⁻⁶. 5mL, 3 mL of cell suspension was added to each dish, a total of 10 dishes, and placed in the cell incubator for 12 h. The sample was prepared with PBS: free DOX, free Ce6, DCMMs and NTZ-DCMMs, diluted ten times with DMEM, and added to the dish in two groups of 3 h and 6 h, the DOX content in the sample after dilution was 5 mg / L -1 , and the Ce6 content was 4 mg / L -1 . PBS was used as a blank control group. Then the medium was aspirated and rinsed with PBS for 3 times, and then 1 mL of Hoechst 33342 staining agent (10 μg / mL -1 ) was added to each dish, wrapped with tin foil and placed in the incubator for 20 min, and then the staining agent was aspirated and rinsed with PBS for 3 times to ensure complete removal of the dye. Finally, it was observed and photographed using a Zeiss laser confocal microscope, and the fluorescence images were taken in the DAPI channel (blue), PE channel (red), and APC channel (green), respectively. The test results are shown in Figure 13 After 6 h of incubation of H22 cells with DCMMs and NTZ-DCMMs, the red and green fluorescence were higher than that of the 3 h group, and the amount of nanoparticles taken up by the cells increased with time. In addition, the antibody-modified mixed nanomicelles NTZ-DCMMs showed more obvious red and green fluorescence than the mixed nanomicelles DCMMs without antibody modification, which proved that NTZ-DCMMs could actively target and accumulate in H22 cells. Then the Free DOX group without nanoparticle modification and the Free Ce6 group showed the weakest fluorescence intensity, because small molecule drugs are mainly endocytosed and excreted by the difference between intracellular and extracellular concentrations, and are difficult to accumulate in tumor cells. The results prove that the antibody-modified mixed nanomicelles NTZ-DCMMs can be better taken up by cells and have a certain targeting function for liver cancer cells. Example Eleven

[0132] Measurement of intracellular ROS

[0133] DCFH-DA was used to detect the level of intracellular ROS. DCFH-DA itself does not produce fluorescence, can freely pass through the cell membrane and be hydrolyzed by intracellular esterase to DCFH, and active oxygen can convert non-fluorescent DCFH to fluorescent DCF through oxidation, so that the level of active oxygen can be detected by detecting the fluorescence of DCF.

[0134] The specific experimental steps are as follows: prepare PBS solutions of free Ce6, DCMMs and NTZ-DCMMs, and the Ce6 concentration of all samples is uniformly 20 mg / L -1 . H22 cells were recovered and subcultured to exponential growth, diluted and added to 35 mm glass bottom culture dishes after cell counting, with a seeding density of 2×105 The sample was diluted 5 times with culture medium and added to the dish. After 4 h of incubation, the sample was washed 3 times with PBS, and 20 μM DCFH-DA was added to the sample and incubated with the H22 cells for 20 min. The sample was washed 2 times with PBS to remove the un-loaded active oxygen probe. The sample was irradiated with a 660 nm red laser (0.2 W cm -2 ) for 3 min, and then observed in a live cell workstation. Figure 14 The results of the assay are shown. It can be observed that the green fluorescence of the sample is significantly enhanced after laser irradiation, indicating that the photosensitizer Ce6 generates 1 O2 under laser irradiation. In addition, since the small molecule free Ce6 is endocytosed and exocytosed only by the concentration difference inside and outside the cell, the efficiency of cell uptake is low, and only a weak fluorescence signal can be observed. The NTZ-DCMMs group after light irradiation showed the strongest fluorescence, indicating that the mixed nanomicelles modified by antibodies can specifically recognize the tumor cell surface overexpressed epidermal growth factor receptor EGFR, and break the reduction-sensitive disulfide bond to release the drug under the high concentration GSH environment in the tumor site. Example Twelve

[0135] In vivo drug distribution experiment

[0136] To observe the penetration behavior of the mixed nanomicelles co-loaded with doxorubicin and chlorin e6 in tumor cells, the fluorescence images of the main organs and tumors of the mice after tail vein injection were observed by in vivo imaging. The specific experimental operation is as follows: first, the subcutaneous model of H22 hepatoma cell line in mice was constructed, and the H22 cells were passaged to a sufficient number and resuspended in PBS. The female BALB / c mice (6 weeks old) were anesthetized with 60 μL of sodium pentobarbital (10 mg mL -1 ) and injected subcutaneously between the skin and mucosa of the right thigh. Each mouse was inoculated with 2.5×10 6 cells. When the tumor volume reached 100 mm 3 , the mice were divided into two groups for the experiment. The prepared PBS solutions of free DOX, PEG- ss -DOX, DCMMs and NTZ-DCMMs were injected into the H22 tumor-bearing mice through the tail vein. The concentration of DOX in all samples was 0.4 mg mL -1 , and the concentration of Ce6 was 0.3 mg mL -1 . After 4 h and 24 h, the mice were sacrificed and the heart, liver, spleen, lung, kidney and tumor tissues were removed. The blood on the surface of the internal organs was washed with PBS and then placed on a glass slide. The 4 h group and the 24 h group were photographed using a small animal in vivo imaging instrument (IVIS Lumina II). The experimental results are shown inFigure 15 As shown, free DOX, as a small molecule drug, mainly accumulates in the liver and is rapidly metabolized, making it difficult to achieve long-term circulation in mice. After forming mixed nanomicelles using polymers as carriers, the fluorescence signal of the drug at the tumor site is significantly increased, with the NTZ-DCMMs group showing the strongest tumor fluorescence intensity and the largest drug accumulation. In the 4-h group, the liver, kidneys, and tumors all showed strong fluorescence signals. As time progressed, due to the large-scale metabolism of the drug by mice, the fluorescence in the liver and kidneys almost disappeared after 24 hours, while weak fluorescence could still be detected in the tumor site. The NTZ-DCMMs group showed the strongest fluorescence signal in the tumor tissue, demonstrating that the EPR effect and the active targeting of the antibody enable NTZ-DCMMs to achieve specific drug delivery to the tumor site in vivo, resulting in higher tumor accumulation, and thus potentially exerting a better anti-tumor therapeutic effect. Example 13

[0137] In vivo anti-tumor efficacy experiment

[0138] To investigate the antitumor efficacy of in vivo chemotherapy-photodynamic therapy combined with mixed nanomicelles carrying doxorubicin and dihydroporphyrin e6, tumor volume was recorded and observed in real time during a 14-day, 7-times administration. The specific procedures were as follows: H22 tumor-bearing mice (same as in Example XII) were established, with 6 mice per group divided into 5 groups for the experiment. Each group was administered 100 μL of the following sample: PBS, free DOX, PEG- ss -DOX, DCMMs and NTZ-DCMMs, wherein the concentration of uniform DOX is 0.4 mg / mL -1 The concentration of Ce6 was 0.3 mg / mL. -1 Two hours after each administration, DCMMs and NTZ-DCMMs were subjected to a 660 nm (0.2 W cm⁻¹) treatment. -2 Mouse tumor tissue was irradiated with laser for 5 minutes per mouse, administered every two days for a total of 7 doses. Before each dose, mice were weighed and tumor volume was measured. After treatment, all mice were sacrificed, and their heart, liver, spleen, lungs, kidneys, and tumor tissues were dissected. All extracted solid tumors were photographed and weighed. The heart, liver, spleen, lungs, and kidneys were fixed with 4% polymethyl methacrylate for further immunohistochemical analysis. Mouse weight records are as follows. Figure 16 As shown in (A), the weight of mice in the free DOX group decreased significantly, and the original drug doxorubicin produced extremely high toxic side effects, leading to emaciation in the mice; while the weight of mice in the PBS group and the prodrug group remained stable, indicating that DCMMs and NTZ-DCMMs have good biocompatibility and can significantly enhance anti-tumor efficacy without producing large toxic side effects. Figure 16(B) shows the changes in tumor volume during treatment. The PBS group showed the fastest tumor volume growth. All treatment groups demonstrated good anti-tumor efficacy, with the NTZ-DCMMs group showing the best treatment effect, reflecting the good tumor microenvironment responsiveness of NTZ-DCMMs in drug release and active targeted anti-tumor therapy. Solid tumor photograph. Figure 16 (C) and tumor tissue weight Figure 16 (D) It was also confirmed that DCMMs and NTZ-DCMMs penetrated tumor tissue and released drugs in a responsive manner, significantly inhibiting tumor growth.

[0139] The pathological changes in tumor tissues were further investigated using hematoxylin and eosin (H&E) staining, Ki 67 staining, and TdT-mediated dUTP nick-end labeling (TUNEL). Results are as follows: Figure 17 As shown, NTZ-DCMMs exhibited the highest number of necrotic and apoptotic tumor cells. This antibody-modified mixed nanomicelles co-loaded with DOX and Ce6 achieved the enrichment of DOX at the tumor tissue, directly killing tumor cells. Under laser irradiation, the singlet oxygen generated by the photosensitizer Ce6 simultaneously induced apoptosis and necrosis of cancer cells. NTZ-DCMMs showed the best inhibitory effect on tumor cell proliferation, confirming the optimal therapeutic effect of chemotherapy-photodynamic therapy.

[0140] This invention relates to the synthetic route and schematic diagram of the mixed nanomicelles modified with monoclonal antibodies and co-loaded with doxorubicin and dihydroporphyrin E6, as shown in the figure. Figure 18 As shown. The polymer-doxorubicin prodrug is abbreviated as PEG- ss -DOX; polyethylene glycol-dihydroporphyrin e6 is abbreviated as PEG-Ce6; mixed nanomicelles co-loaded with doxorubicin and dihydroporphyrin e6 are abbreviated as DCMMs; mixed nanomicelles co-loaded with monoclonal antibody modified with doxorubicin and dihydroporphyrin e6 are abbreviated as NTZ-DCMMs. This invention utilizes reduction-responsive disulfide bonds to construct a polymer-doxorubicin prodrug, which is then co-assembled with a photosensitizer-loaded polymer backbone to form mixed nanomicelles. These nanomicelles have suitable particle size and good physiological stability, enabling them to specifically release the drug in the tumor microenvironment. By modifying the surface of the mixed nanomicelles with monoclonal antibodies, the monoclonal antibodies bind to the human epidermal growth factor receptor highly expressed on the surface of tumor cells, allowing the mixed nanomicelles to actively target tumor cells and accumulate at the tumor site. In the high glutathione concentration microenvironment of the tumor, the mixed nanomicelles break and reduce-sensitive disulfide bonds, stimulating the responsive release of doxorubicin, which kills tumor cells. Simultaneously, under laser irradiation, the singlet oxygen generated by the photosensitizer damages tumor cells. Both synergistically inhibit tumor cell proliferation, enhance anti-tumor efficacy, and achieve combined chemotherapy and photodynamic therapy for cancer.

Claims

1. A method for preparing antibody-modified co-loaded drug nanomicelles, characterized in that, The process includes the following steps: self-assembling chemotherapy drug prodrugs and phototherapy drug prodrugs to obtain co-loaded drug nanomicelles; then modifying monoclonal antibodies onto the co-loaded drug nanomicelles to obtain antibody-modified co-loaded drug nanomicelles. The following are prodrugs used in chemotherapy: ； DOX stands for doxorubicin; x ranges from 45 to 113; y ranges from 17 to 38; z ranges from 5 to 12. The following are the pre-medications for phototherapy: ； The range of m is 45 to 113.

2. The preparation method according to claim 1, characterized in that, The monoclonal antibodies include nimotuzumab, pertuzumab, cetuximab, CD147 monoclonal antibody, or CD163 monoclonal antibody.

3. A method for preparing co-loaded drug nanomicelles, characterized in that, The process includes the following steps: self-assembling chemotherapy drug prodrugs and phototherapy drug prodrugs to obtain co-loaded drug nanomicelles; The following are prodrugs used in chemotherapy: ； DOX stands for doxorubicin; x ranges from 45 to 113; y ranges from 17 to 38; z ranges from 5 to 12. The following are the pre-medications for phototherapy: ； The range of m is 45 to 113.

4. Antibody-modified co-loaded drug nanomicelles or co-loaded drug nanomicelles prepared by the preparation method according to claim 1 or 3.

5. The use of the antibody-modified co-loaded drug nanomicelles as described in claim 4, or the use of co-loaded drug nanomicelles in the preparation of nanomedicines.

6. The application according to claim 5, characterized in that, Nanomedicines are combination therapies.

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