CN112915205B

CN112915205B - Photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization treatment and preparation method thereof

Info

Publication number: CN112915205B
Application number: CN202110151182.2A
Authority: CN
Inventors: 丁杨; 周建平; 孙晨凯; 程皓; 张华清
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-02-03
Filing date: 2021-02-03
Publication date: 2023-08-01
Anticipated expiration: 2041-02-03
Also published as: CN112915205A

Abstract

The invention discloses a photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization treatment and a preparation method thereof, belonging to the field of pharmaceutical preparations. The combined delivery system comprises polymer nanoparticles and enzyme nanocapsules, wherein the polymer nanoparticles are coated with photosensitizers, the polymer nanoparticles and the enzyme nanocapsules are connected through chemical bonds to form a nanocomposite, and the surface of the nanocomposite is further modified with a targeting biological macromolecule. The combined delivery system can realize the high-efficiency enrichment of the tumor part co-delivered by the same target area of the photosensitizer and the enzyme, and can improve the oxygen yield and the oxygen utilization rate of the focus part by flexibly adjusting the assembly proportion of the polymer nanoparticles and the nanocapsules, effectively improve the hypoxia microenvironment of tumor tissues and enhance the photodynamic treatment effect, and can be used for the application of the preparation in tumor diagnosis or treatment medicaments.

Description

Photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization treatment and preparation method thereof

Technical Field

The invention belongs to the field of pharmaceutical preparations, relates to a nano-drug combination delivery system and a preparation method thereof, and in particular relates to a photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization treatment and a preparation method thereof.

Background

For the treatment of tumors, surgical excision, chemotherapy and radiation therapy are mainly adopted. In recent years, with the rapid development of nano-technology and radiation technology, optical treatment has become a modern non-invasive novel radiation treatment means. Photodynamic therapy (Photodynamic therapy, PDT) can be divided into PDT type I and PDT type II, wherein PDT type II is the main research direction, and the action mechanism is that photosensitizers excite oxygen in tumor cells to form singlet oxygen, and the singlet oxygen can oxidize nucleic acids, proteins and biological membranes in stressed cells, destroy normal metabolism of the tumor cells and further kill the cells. Because II type photodynamic therapy is very dependent on oxygen, aiming at the characteristic that a great amount of hydrogen peroxide is contained in the tumor microenvironment, the improvement of the photodynamic therapy effect by adopting novel nanometer and radiation technology is very important.

Hydrogen peroxide degrading enzyme (CAT) is a catalytic enzyme with extremely high turnover rate, can rapidly catalyze the decomposition of hydrogen peroxide to generate oxygen, and has been used for improving tumor hypoxia condition with other therapeutic means. Since hydrogen peroxide degrading enzyme is an exogenous enzyme, it is degraded by proteases and rendered immunogenic in vivo. Therefore, it is important to encapsulate and deliver it to the target site using immobilized enzyme technology.

At present, related researches on photodynamic therapy anti-tumor nano delivery systems mainly focus on the aspects of modifying the structure of a photosensitizer, improving the drug loading rate of the photosensitizer, catalyzing hydrogen peroxide by adopting an organic or inorganic material and the like, and are difficult to solve the problems of poor oxygen dependence, targeting property, biocompatibility and the like of the photosensitizer. In addition, there are patents to modify catalase on liposomes in co-incubation, lack protection for catalase, and low encapsulation efficiency, and difficulty in achieving enzyme/photosensitizer targeted co-delivery. There are also patents to coat catalase with polymer, but exposing photosensitizer on nanoparticle surface, it is difficult to improve photosensitizer stability and achieve nano-targeting delivery.

Therefore, a co-delivery system with high-efficiency photosensitizer entrapment and improved tumor hypoxia condition at the same target area is needed to solve the above drawbacks. The construction of the nano delivery system with high drug loading capacity, biocompatibility and good targeting property, which can improve the hypoxia condition of the tumor part and promote the photodynamic therapy, is a new breakthrough in the field of photodynamic therapy.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides a photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization treatment, which has the advantages of improving the stability, tumor targeting, co-delivery and bionic characteristics of photosensitizer and hydrogen peroxide degrading enzyme, has high oxygen production efficiency, and can be used for enhancing photodynamic anti-tumor treatment.

In order to achieve the above object, the present invention adopts the following technical scheme:

the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment comprises polymer nanoparticles and enzyme nanocapsules, wherein the polymer nanoparticles and the enzyme nanocapsules are coated with the photosensitizer, the polymer nanoparticles and the enzyme nanocapsules are connected through chemical bonds to form a nanocomposite, and the surface of the nanocomposite is further modified with a targeting biological macromolecule;

the polymer nanoparticles are formed by self-assembly of polymers modified with boric acid groups and epoxy groups;

the enzyme nanocapsule is formed by in-situ free radical polymerization of ethylenated hydrogen peroxide degrading enzyme and amine, alcohol or mercaptan-containing monomers;

the targeting biological macromolecule is polysaccharide or protein, and is selected from one or more of dextran, hyaluronic acid, polylysine, heparin or protein or polypeptide with tumor targeting property and derivatives thereof.

Further, the polymer modified with boric acid groups and epoxy groups is prepared by the following method:

step 1, dispersing boric acid compound and acrylic acid derivative monomer in organic solvent, reacting at room temperature in dark condition, purifying and freeze-drying to obtain acrylic acid compound modified with boric acid group;

And 2, dispersing the acrylic acid compound modified with the boric acid group, a polymerizable monomer, a cross-linking agent and a free radical initiator in an organic solvent for free radical polymerization reaction, and obtaining the polymer modified with the boric acid group and the epoxy group after extraction, purification and spin evaporation.

Further, the boric acid compound is selected from a halogen-containing phenylboronic acid monomer, a halogen-containing pyridinylboronic acid monomer, or a halogen-containing thiopheneboronic acid monomer; the acrylic acid derivative monomer is an acrylic acid monomer containing tertiary amine groups and is selected from one or more of diethylaminoethyl methacrylate, N-vinyl pyrrolidone, dimethylaminoethyl acrylate or N, N-diethylaminoethyl acrylate and derivatives thereof; the polymerizable monomer is an acrylic monomer containing epoxy groups and is selected from one or more of glycidyl methacrylate, glycidyl acrylate, acetone glycidyl methacrylate, allyl glycidyl ether and derivatives thereof.

Preferably, the boric acid compound is selected from one or more of 4-bromomethylbenzoic acid, 3-chloro-4-pyridineboronic acid pinacol ester, 5-bromo-2-thiopheneboronic acid, 6-fluoropyridine-3-boronic acid and derivatives thereof.

Further, in the step 1, the mass ratio of the acrylic acid monomer to the boric acid monomer is 1:0.5-1, and the concentration of the acrylic acid monomer is 4 mg/mL-5 mg/mL; in the step 2, the molar ratio of the boric acid-acrylic acid compound to the polymerizable monomer to the cross-linking agent to the free radical initiator is 1:0.5-1:0.5:0.25-0.5, the concentration of the boric acid-acrylic acid compound is 4-5 mg/mL, and the free radical polymerization reaction time is 8-14 h.

Further, the photosensitizer is selected from one or more of indocyanine green, chlorin e6, methylene blue, IR780 or hematoporphyrin monomethyl ether.

Further, the vinylated hydrogen peroxide degrading enzyme is prepared by the following method: dissolving hydrogen peroxide degrading enzyme and a monomer containing succinimidyl ester in a buffer solution with the pH of 8.0-9.5, reacting at room temperature, and dialyzing the reaction solution to obtain the ethylenated hydrogen peroxide degrading enzyme;

the succinyl ester-containing monomer is N- (allyloxycarbonyl oxygen) succinimide or acrylic acid-N-succinimide ester.

Further, the mass ratio of the hydrogen peroxide degrading enzyme to the monomer containing succinimidyl ester is 1:5-8.

Further, the amine, alcohol or mercaptan-containing monomer is selected from one or more of allylamine hydrochloride, 3-methyl-butenamine, 3-butenamine, allylmercaptan and derivatives thereof.

The preparation method of the combined delivery system comprises the following steps:

step 1, dissolving a polymer modified with a boric acid group and an epoxy group and a photosensitizer in an organic solvent, dispersing the mixture in pure water according to the volume ratio of 1/10-1/50, and carrying out ultrasonic and dialysis purification under the condition of avoiding light at room temperature to obtain polymer nanoparticles coated with the photosensitizer;

Step 2, dissolving the ethylenated hydrogen peroxide degrading enzyme, a primary amine or thiol-containing monomer, a cross-linking agent and a redox initiator in a buffer solution, carrying out in-situ free radical polymerization at room temperature under the protection of nitrogen, and dialyzing the reaction solution to obtain an enzyme nanocapsule;

step 3, dissolving the polymer nanoparticles obtained in the step 1 and the enzyme nanocapsules obtained in the step 2 in a buffer solution according to a mass ratio of 1:1-1:15, and reacting at room temperature to obtain a nanocomposite;

and 4, dissolving the nano-composite obtained in the step 3 and the targeting biomacromolecule in a buffer solution for co-incubation to obtain the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment.

Further, in the step 1, the mass ratio of the polymer to the photosensitizer is 1:0.5-1, and the concentration of the polymer is 0.4 mg/mL-0.6 mg/mL.

In the step 2, the mass ratio of the styrenated hydrogen peroxide degrading enzyme to the primary amine, alcohol or mercaptan-containing monomer to the cross-linking agent to the redox agent is 1:1-10:2:5, and the in-situ free radical polymerization reaction time is 2-3 h.

The nano capsule formed on the surface of the protein can effectively protect the activity of the protein in vivo by using a chemical combination means. The structure of the hydrogen peroxide degrading enzyme contains a plurality of lysine groups, the residue end of the hydrogen peroxide degrading enzyme is amino, and the hydrogen peroxide degrading enzyme can be used as a high-activity site to be amidated with a monomer containing succinimidyl ester, so that double bonds of a polymer reaction skeleton group are introduced into the surface of the hydrogen peroxide degrading enzyme. The vinyl hydrogen peroxide degrading enzyme and the monomer containing the polymerizable group undergo in-situ polymerization reaction to form the enzyme nanocapsule, and the carrier can overcome the obstacle of the hydrogen peroxide degrading enzyme in photodynamic anti-tumor treatment.

According to the particle size, the dispersion coefficient and the stability of the co-delivery system under physiological conditions, when the mass ratio of the photosensitizer-polymer nanoparticles to the enzyme nanocapsules is 1:1-1:5, the particle size and the dispersion coefficient of the obtained compound are larger and unstable under physiological conditions; when the ratio of the two is 1:5-1:10, the particle size of the obtained nano-composite is moderate, the nano-composite is uniformly dispersed, and good physiological condition stability can be maintained; when the ratio of the two is 1:10-1:15, the surface reactive groups of the polymer nanoparticles reach saturation, and excessive enzyme nanocapsules exist to damage the uniformity of the nano delivery system. Therefore, the preferable mass ratio of the photosensitizer-polymer nanoparticle to the enzyme nanocapsule is 1:5-10.

The photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment provided by the invention has the advantages that when the photosensitizer/enzyme combination delivery system is stimulated by hydrogen peroxide, hydrogen peroxide degrading enzyme catalyzes the photosensitizer to generate a large amount of oxygen for the photosensitizer; when the photosensitizer is irradiated by laser, the photosensitizer is converted from a ground state into an excited state, and the photosensitizer is identical to triplet state oxygen in spin, so that the excited state energy is transmitted to the triplet state oxygen, and the singlet state oxygen is induced to be generated. The photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment has the advantages of protecting the stability of catalase in vivo, catalyzing oxygen generation, enhancing photodynamic treatment and having good application prospects in the aspects of preparing tumor diagnosis and treatment preparations, targeted photodynamic treatment and synergistic photodynamic antigravity treatment.

Compared with the prior art, the invention has the beneficial effects that:

1. the photosensitizer/enzyme combination delivery system can not only enhance photodynamic treatment effect, but also protect photosensitizer and catalase through two carriers respectively, and improve stability and enzyme activity of the medicine under physiological conditions;

2. the photosensitizer/enzyme combined delivery system is used as an oxygen supply system according to the requirement, and the oxygen production rate and the oxygen utilization rate of catalase are improved by adjusting the assembly proportion of photosensitizer-polymer nanoparticles and enzyme nanocapsules, so that the hypoxic microenvironment of tumor parts is improved efficiently;

3. the photosensitizer/enzyme combined delivery system provided by the invention incubates the targeting biomacromolecule, improves the tumor targeting of the delivery system, realizes that the photosensitizer and the enzyme are co-delivered to the tumor part with the target area through covalent combination of two carriers, and further enhances the curative effect of photodynamic therapy.

Drawings

FIG. 1 is a graph showing the particle size distribution of photosensitizer-polymer nanoparticles;

FIG. 2 is a graph showing particle size distribution of enzyme nanocapsules;

FIG. 3 is a graph showing particle size distribution of polymer nanoparticles combined with nanocapsules at different ratios;

FIG. 4 is a graph showing particle size distribution of polymer nanoparticles and nanocapsules (example 38) at a fixed ratio over time;

FIG. 5 is a graph showing the experimental results of investigation of the binding rate of polymer nanoparticles and nanocapsules ninhydrin at a fixed ratio;

FIG. 6 shows the results of an oxygen production experiment of a photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization;

FIG. 7 is a photosensitizer release profile for a photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy (example 40);

FIG. 8 is the results of enzyme activity studies of a photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization (example 41);

FIG. 9 is a cytotoxicity test results of a photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization;

FIG. 10 is a graph depicting the results of an investigation of the ROS production capability of a photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization;

FIG. 11 is a graph depicting the results of an examination of the oxygen generating capacity of RDPP in a photosensitizer/enzyme combination delivery system for photodynamic sensitization therapy against tumors;

FIG. 12 is a graph showing the results of a cell uptake assay for a photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization;

fig. 13 is an experimental result of apoptosis of a photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization.

Detailed Description

The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.

Example 1

Synthesis of boric acid-epoxy polymers

According to the mol ratio of diethylaminoethyl methacrylate to 4-bromomethylbenzoic acid of 1:0.5, adding trace potassium iodide, dissolving in 6 mL of N, N-dimethylformamide, and carrying out light-shielding reaction for 24 h. The prepared monomer, glycidyl methacrylate and N, N-methylenebisacrylamide are dissolved in 4 mL of N, N-dimethylformamide according to a molar ratio of 1:1:0.5 under the protection of nitrogen. According to the mol ratio of boric acid-acrylic acid compound to initiator azo-diisobutyronitrile of 1:0.5, under 65 ℃, the reaction is carried out vigorously for 12 h, then n-hexane is used for extraction and purification, and the boric acid-epoxy polymer is obtained after rotary evaporation.

Example 2

Synthesis of boric acid-epoxy polymers

Adding a trace amount of potassium iodide according to the molar ratio of N-vinyl pyrrolidone to 4-bromomethyl phenylboronic acid of 1:1, dissolving in 6 mL of N, N-dimethylformamide, and carrying out light-shielding reaction for 24 h. The prepared compound, glycidyl methacrylate and N, N-methylenebisacrylamide are dissolved in 4 mL of N, N-dimethylformamide according to a molar ratio of 1:1:0.5 under the protection of nitrogen. According to the mol ratio of boric acid-acrylic acid compound to initiator azodiisobutyronitrile of 1:0.5, the mixture is subjected to violent reaction at 65 ℃ for 12 h, and then is extracted and purified by n-hexane, and the boric acid-epoxy polymer is obtained after rotary evaporation.

Example 3

Synthesis of boric acid-epoxy polymers

According to the mol ratio of diethylaminoethyl methacrylate to 4-bromomethylbenzoic acid of 1:2, adding a trace amount of potassium iodide, dissolving in 6 mL of N, N-dimethylformamide, and carrying out light-shielding reaction for 24 h. The prepared compound, glycidyl acrylate and N, N-methylenebisacrylamide are dissolved in 4 mL of N, N-dimethylformamide according to a molar ratio of 1:1:0.5 under the protection of nitrogen. According to the mol ratio of boric acid-acrylic acid compound to initiator azodiisobutyronitrile of 1:0.5, the mixture is subjected to violent reaction at 65 ℃ for 12 h, and then is extracted and purified by n-hexane, and the boric acid-epoxy polymer is obtained after rotary evaporation.

Example 4

Synthesis of boric acid-epoxy polymers

Adding a trace amount of potassium bromide according to the mol ratio of N-vinyl pyrrolidone to 3-chloro-4-pyridine boric acid pinacol ester of 1:1, dissolving in 6 mL tetrahydrofuran, and carrying out light-shielding reaction for 24 h. The prepared compound, glycidyl acrylate and N, N-methylenebisacrylamide are dissolved in 4 mL of N, N-dimethylformamide according to a molar ratio of 1:1:0.5 under the protection of nitrogen. According to the mol ratio of boric acid-acrylic acid compound to initiator azodiisobutyronitrile of 1:0.5, the mixture is subjected to violent reaction at 65 ℃ for 12 h, and then is extracted and purified by n-hexane, and the boric acid-epoxy polymer is obtained after rotary evaporation.

Example 5

Synthesis of boric acid-epoxy polymers

Adding a trace amount of sodium iodide according to the molar ratio of dimethylaminoethyl acrylate to 5-bromo-2-thiopheneboronic acid of 1:1, dissolving in 6 mL methanol, and carrying out light-shielding reaction for 24 h. The prepared compound, allyl glycidyl ether and N, N-methylene bisacrylamide are dissolved in 4 mL of N, N-dimethylformamide according to the mol ratio of 1:1:0.5 under the protection of nitrogen. According to the mol ratio of boric acid-acrylic acid compound to initiator azodiisobutyronitrile of 1:0.5, the mixture is subjected to violent reaction at 65 ℃ for 12 h, and then is extracted and purified by n-hexane, and the boric acid-epoxy polymer is obtained after rotary evaporation.

Example 6

Synthesis of boric acid-epoxy polymers

According to the mol ratio of the N, N-diethylaminoethyl acrylate to the 6-fluoropyridine-3-boric acid of 1:1, adding a trace amount of potassium iodide, dissolving in 6 mL tetrahydrofuran, and carrying out light-shielding reaction for 24: 24 h. The prepared compound, 3, 4-epoxy-1-butene and N, N-methylenebisacrylamide are dissolved in 4 mL of N, N-dimethylformamide according to a molar ratio of 1:1:0.5 under the protection of nitrogen. According to the mol ratio of boric acid-acrylic acid compound to initiator azodiisobutyronitrile of 1:0.5, the mixture is subjected to violent reaction at 65 ℃ for 12 h, and then is extracted and purified by n-hexane, and the boric acid-epoxy polymer is obtained after rotary evaporation.

Example 7

Synthesis of boric acid-epoxy polymers

According to the mol ratio of diethylaminoethyl methacrylate to 4-bromomethyl phenylboronic acid of 1:1, adding a trace amount of potassium iodide, dissolving in 6 mL methanol, and carrying out light-shielding reaction for 24: 24 h. The prepared compound, 1, 2-epoxy-9-decene and N, N-methylenebisacrylamide were dissolved in 4 mL of N, N dimethylformamide at a molar ratio of 1:1:0.5 under nitrogen protection. According to the mol ratio of boric acid-acrylic acid compound to initiator azodiisobutyronitrile of 1:0.5, the mixture is subjected to violent reaction at 65 ℃ for 12 h, and then is extracted and purified by n-hexane, and the boric acid-epoxy polymer is obtained after rotary evaporation.

Example 8

Synthesis of boric acid-epoxy polymers

According to the mole ratio of N-vinyl pyrrolidone to 3-chloro-4-pyridine boric acid pinacol ester of 1:1, adding trace potassium iodide, dissolving in 6 mL of N, N-dimethylformamide, and carrying out light-shielding reaction for 24 h. The prepared compound, glycidyl methacrylate and N, N-methylenebisacrylamide are dissolved in 4 mL of N, N-dimethylformamide according to a molar ratio of 1:0.2:0.5 under the protection of nitrogen. According to the mol ratio of boric acid-acrylic acid compound to initiator azodiisobutyronitrile of 1:0.5, the mixture is subjected to violent reaction at 65 ℃ for 12 h, and then is extracted and purified by n-hexane, and the boric acid-epoxy polymer is obtained after rotary evaporation.

Example 9

Synthesis of boric acid-epoxy polymers

Adding a trace amount of potassium iodide according to the molar ratio of dimethylaminoethyl acrylate to 5-bromo-2-thiopheneboronic acid of 1:1, dissolving in 6 mL of N, N-dimethylformamide, and carrying out light-shielding reaction for 24 h. The prepared compound, glycidyl methacrylate and N, N-methylenebisacrylamide are dissolved in 4 mL of N, N-dimethylformamide according to a molar ratio of 1:0.5:0.5 under the protection of nitrogen. According to the mol ratio of boric acid-acrylic acid compound to initiator azodiisobutyronitrile of 1:0.5, the mixture is subjected to violent reaction at 65 ℃ for 12 h, and then is extracted and purified by n-hexane, and the boric acid-epoxy polymer is obtained after rotary evaporation.

Example 10

Synthesis of boric acid-epoxy polymers

According to the mol ratio of the N, N-diethylaminoethyl acrylate to the 6-fluoropyridine-3-boric acid of 1:1, adding a trace amount of potassium iodide, dissolving in 6 mL of N, N-dimethylformamide, and carrying out light-shielding reaction for 24 h. The prepared compound, acrylamide and N, N-methylenebisacrylamide are dissolved in 4 mL of N, N-dimethylformamide according to a molar ratio of 1:0.5:0.5 under the protection of nitrogen. According to the mol ratio of boric acid-acrylic acid compound to initiator azodiisobutyronitrile of 1:0.5, the mixture is subjected to violent reaction at 65 ℃ for 12 h, and then is extracted and purified by n-hexane, and the boric acid-epoxy polymer is obtained after rotary evaporation.

Example 11

Synthesis of boric acid-epoxy polymers

Adding a trace amount of potassium iodide according to the molar ratio of dimethylaminoethyl acrylate to 4-bromomethylphenylboronic acid of 1:1, dissolving in 6 mL of N, N-dimethylformamide, and carrying out light-shielding reaction for 24 h. The prepared compound, methyl methacrylate and N, N-methylenebisacrylamide are dissolved in 4 mL of N, N-dimethylformamide according to a molar ratio of 1:0.5:0.5 under the protection of nitrogen. According to the mol ratio of boric acid-acrylic acid compound to initiator azodiisobutyronitrile of 1:0.5, the mixture is subjected to violent reaction at 65 ℃ for 12 h, and then is extracted and purified by n-hexane, and the boric acid-epoxy polymer is obtained after rotary evaporation.

Example 12

Synthesis of boric acid-epoxy polymers

Adding trace potassium iodide according to the molar ratio of N-vinyl pyrrolidone to 5-bromo-2-thiopheneboronic acid of 1:1, dissolving in 6 mL of N, N-dimethylformamide, and carrying out light-shielding reaction for 24 h. The prepared compound, glycidyl methacrylate and N, N-methylenebisacrylamide are dissolved in 4 mL of N, N-dimethylformamide according to a molar ratio of 1:1:0.5 under the protection of nitrogen. The boric acid-epoxy polymer is obtained by carrying out a vigorous reaction at 65 ℃ for 12 h according to the mol ratio of the boric acid-acrylic acid polymer to the initiating agent toluene peroxide of 1:0.5, extracting and purifying with n-hexane and carrying out rotary evaporation.

Example 13

Preparation of indocyanine green entrapped photosensitizer-polymer nanoparticles

The boric acid-epoxy polymer (example 1) was dissolved in 40. Mu.L of methanol at a mass ratio of indocyanine green of 1:0.2, and then dispersed in 1.5. 1.5 mL pure water. The drug-loaded nanoparticles are prepared by adopting a nano precipitation method, and a probe ultrasonic dispersing instrument is used for carrying out ultrasonic dispersion on the solution with the power of 85 and W for 1 min. And (3) putting the solution into a 3500Da dialysis bag, dialyzing away from light to remove the non-encapsulated indocyanine green, and freeze-drying to obtain the photosensitizer-polymer nanoparticle encapsulated with indocyanine green. The drug loading rate of the nanoparticle is 14.6 percent and the encapsulation rate is 90.5 percent measured by an ultraviolet spectrophotometry. The nano-delivery system particle size obtained in this example was measured by a malvern particle size meter, and a in fig. 1 is the measurement result of the malvern particle size meter, and the nano-delivery system particle size was 83.5. 83.5 nm.

Example 14

The boric acid-epoxy polymer (example 2) was dissolved in 40. Mu.L of methanol at a mass ratio of indocyanine green of 1:0.3, and then dispersed in 1.5. 1.5 mL pure water. The drug-loaded nanoparticles are prepared by adopting a nano precipitation method, and a probe ultrasonic dispersing instrument is used for carrying out ultrasonic dispersion on the solution with the power of 85 and W for 1 min. And (3) putting the solution into a 3500Da dialysis bag, dialyzing away from light to remove the non-encapsulated indocyanine green, and freeze-drying to obtain the photosensitizer-polymer nanoparticle encapsulated with indocyanine green. The drug loading rate of the nanoparticle is 25.2 percent and the encapsulation rate is 91.3 percent measured by an ultraviolet spectrophotometry. The nano-delivery system particle size obtained in this example was measured by a malvern particle size meter, and b in fig. 1 is the measurement result of the malvern particle size meter, and the nano-delivery system particle size was 86.5. 86.5 nm.

Example 15

Preparation of photosensitizer-Polymer nanoparticles entrapped methylene blue

The boric acid-epoxy polymer (example 3) was dissolved in 40. Mu.L of methanol at a mass ratio of 1:0.4 to methylene blue and then dispersed in 1.5. 1.5 mL pure water. The drug-loaded nanoparticles are prepared by adopting a nano precipitation method, and a probe ultrasonic dispersing instrument is used for carrying out ultrasonic dispersion on the solution with the power of 85 and W for 1 min. And (3) putting the solution into a 3500 Da dialysis bag, dialyzing away from light to remove the non-entrapped methylene blue, and freeze-drying to obtain the photosensitizer-polymer nanoparticle entrapped methylene blue. The drug loading rate of the nanoparticle is 36.3 percent and the encapsulation rate is 88.3 percent measured by an ultraviolet spectrophotometry. The nano-delivery system particle size obtained in this example was measured by a malvern particle size meter, and g in fig. 1 is the measurement result of the malvern particle size meter, and the nano-delivery system particle size was 168.4 and nm.

Example 16

Preparation of photosensitizer-Polymer nanoparticles entrapped methylene blue

The boric acid-epoxy polymer (example 4) was dissolved in 40. Mu.L of methanol at a mass ratio of 1:0.5 to methylene blue and then dispersed in 1.5. 1.5 mL pure water. The drug-loaded nanoparticles are prepared by adopting a nano precipitation method, and a probe ultrasonic dispersing instrument is used for carrying out ultrasonic dispersion on the solution with the power of 85 and W for 1 min. And (3) putting the solution into a 3500 Da dialysis bag, dialyzing away from light to remove the non-entrapped methylene blue, and freeze-drying to obtain the photosensitizer-polymer nanoparticle entrapped methylene blue. The drug loading rate of the nanoparticle is 42.1 percent and the encapsulation rate is 90.2 percent measured by an ultraviolet spectrophotometry. The nano-delivery system particle size obtained in this example was measured by a malvern particle size meter, and j in fig. 1 is the measurement result of the malvern particle size meter, and the nano-delivery system particle size was 290.2 nm.

Example 17

Preparation of photosensitizer-polymer nanoparticles entrapped with chlorin e6

According to the mass ratio of boric acid-epoxy polymer (example 5) to chlorin e6 of 1:0.3, dissolved in 40. Mu.L of methanol and then dispersed in 1.5. 1.5 mL pure water. The drug-loaded nanoparticles are prepared by adopting a nano precipitation method, and the solution is subjected to ultrasonic dispersion by using a probe ultrasonic dispersion instrument with power of 170 and W for 1 min. And (3) putting the solution into a 3500 Da dialysis bag, dialyzing away from light to remove unencapsulated chlorin e6, and freeze-drying to obtain the photosensitizer-polymer nanoparticle encapsulating chlorin e 6. The drug loading rate of the nanoparticle is 24.4% and the encapsulation rate is 90.3% measured by an ultraviolet spectrophotometry. The nano-delivery system particle size obtained in this example was measured by a malvern particle size meter, and h in fig. 1 is the measurement result of the malvern particle size meter, and the nano-delivery system particle size was 126.5. 126.5 nm.

Example 18

Preparation of photosensitizer-polymer nanoparticles entrapped with chlorin e6

According to the mass ratio of boric acid-epoxy polymer (example 6) to chlorin e6 of 1:0.3, dissolved in 80. Mu.L of methanol and then dispersed in 1.5. 1.5 mL pure water. The drug-loaded nanoparticles are prepared by adopting a nano precipitation method, and a probe ultrasonic dispersing instrument is used for carrying out ultrasonic dispersion on the solution with the power of 85 and W for 1 min. And (3) putting the solution into a 3500 Da dialysis bag, dialyzing away from light to remove unencapsulated chlorin e6, and freeze-drying to obtain the photosensitizer-polymer nanoparticle encapsulating chlorin e 6. The drug loading rate of the nanoparticle is 21.2 percent and the encapsulation rate is 87.5 percent as measured by an ultraviolet spectrophotometry. The nano-delivery system particle size obtained in this example was measured by a malvern particle size meter, and i in fig. 1 is the measurement result of the malvern particle size meter, and the nano-delivery system particle size was 254.5 nm.

Example 19

Preparation of photosensitizer-Polymer nanoparticles entrapped with IR780 iodide

The boric acid-epoxy polymer (example 8) was dissolved in 40. Mu.L of methanol at a mass ratio of 1:0.3 to IR780 iodide, and then dispersed in 1.5. 1.5 mL pure water. The drug-loaded nanoparticles are prepared by adopting a nano precipitation method, and a probe ultrasonic dispersing instrument is used for carrying out ultrasonic dispersion on the solution with the power of 85 and W for 1 min. And (3) putting the solution into a 3500 Da dialysis bag, dialyzing away from light to remove the unencapsulated IR780 iodide, and freeze-drying to obtain the photosensitizer-polymer nanoparticle encapsulated with the IR780 iodide. The drug loading rate of the nanoparticle is 19.2 percent and the encapsulation rate is 86.5 percent measured by an ultraviolet spectrophotometry. The nano-delivery system particle size obtained in this example was measured by a malvern particle size meter, and d in fig. 1 is the measurement result of the malvern particle size meter, and the nano-delivery system particle size was 124.5. 124.5 nm.

Example 20

The boric acid-epoxy polymer (example 10) was dissolved in 40. Mu.L of methanol at a mass ratio of 1:0.3 to IR780 iodide, and then dispersed in 1.5. 1.5 mL pure water. The drug-loaded nanoparticles are prepared by adopting a nano precipitation method, and a probe ultrasonic dispersing instrument is used for carrying out ultrasonic dispersion on the solution with the power of 85 and W for 1 min. And (3) putting the solution into a 3500 Da dialysis bag, dialyzing away from light to remove the unencapsulated IR780 iodide, and freeze-drying to obtain the photosensitizer-polymer nanoparticle encapsulated with the IR780 iodide. The drug loading rate of the nanoparticle is 20.7% and the encapsulation rate is 88.2% measured by an ultraviolet spectrophotometry. The nano-delivery system particle size obtained in this example was measured by a malvern particle size meter, and e in fig. 1 is the measurement result of the malvern particle size meter, and the nano-delivery system particle size was 114.2 nm.

Example 21

Preparation of hematoporphyrin monomethyl ether-entrapped photosensitizer-polymer nanoparticles

According to the mass ratio of boric acid-epoxy polymer (example 11) to hematoporphyrin monomethyl ether of 1:0.3, the mixture was dissolved in 40. Mu.L of methanol and then dispersed in 1.5. 1.5mL pure water. The drug-loaded nanoparticles are prepared by adopting a nano precipitation method, and a probe ultrasonic dispersing instrument is used for carrying out ultrasonic dispersion on the solution with the power of 85 and W for 1 min. And (3) putting the solution into a 3500 Da dialysis bag, dialyzing away from light to remove the non-encapsulated hematoporphyrin monomethyl ether, and freeze-drying to obtain the photosensitizer-polymer nanoparticle encapsulated with hematoporphyrin monomethyl ether. The drug loading rate of the nanoparticle is 23.1% and the encapsulation rate is 89.4% as measured by an ultraviolet spectrophotometry. The nano-delivery system particle size obtained in this example was measured by a malvern particle size meter, and f in fig. 1 is the measurement result of the malvern particle size meter, and the nano-delivery system particle size was 120.5. 120.5 nm.

Example 22

According to the mass ratio of boric acid-epoxy polymer (example 12) to hematoporphyrin monomethyl ether of 1:0.3, dissolved in 40. Mu.L of methanol and then dispersed in 1.5mL of pure water. The drug-loaded nanoparticles are prepared by adopting a nano precipitation method, and a probe ultrasonic dispersing instrument is used for carrying out ultrasonic dispersion on the solution with the power of 85W for 1 min. And (3) putting the solution into a 3500 Da dialysis bag, dialyzing away from light to remove the non-encapsulated hematoporphyrin monomethyl ether, and freeze-drying to obtain the photosensitizer-polymer nanoparticle encapsulated with hematoporphyrin monomethyl ether. The drug loading rate of the nanoparticle is 21.9 percent and the encapsulation rate is 86.8 percent measured by an ultraviolet spectrophotometry. The nano-delivery system particle size obtained in this example was measured by a malvern particle size meter, and in fig. 1 c is the measurement result of the malvern particle size meter, and the nano-delivery system particle size was 113.2 nm.

Example 23

Nanometer capsule of synthetic hydrogen peroxide degrading enzyme

And respectively adding the hydrogen peroxide degrading enzyme and the acrylic acid-N-succinimidyl ester into sodium bicarbonate buffer solution with the pH value of 8.0-9.5 according to the mass ratio of 1:10. Reaction 2h was carried out at room temperature. The reaction solution was filled into a 3500 Da dialysis bag, and unreacted acrylic acid-N-succinimidyl ester was removed to obtain a styrenated hydrogen peroxide degrading enzyme. According to the mass ratio of the vinylation hydrogen peroxide degrading enzyme to the allylamine hydrochloride, the cross-linking agent N, N-methylene bisacrylamide and the initiator ammonium persulfate of 1:10:2:5, respectively adding the two into a sodium bicarbonate buffer solution. Reaction 2h was stirred slowly at room temperature under nitrogen blanket. After the reaction, the mixture is filled into a 3500 Da dialysis bag, unreacted monomers and the like are removed, and the enzyme nanocapsule is obtained after freeze drying.

The particle size of the enzyme nanocapsules obtained in this example was measured by a malvern particle size meter, and in fig. 2, a is the measurement result of the malvern particle size meter, the particle size of the enzyme nanocapsules was 36.1. 36.1 nm, and the encapsulation efficiency of hydrogen peroxide degrading enzyme was 86.5% as measured by BCA method.

Example 24

Nanometer capsule of synthetic hydrogen peroxide degrading enzyme

And respectively adding the hydrogen peroxide degrading enzyme and N- (allyloxycarbonyl oxygen) succinimide into sodium bicarbonate buffer solution with pH value of 8.0-9.5 according to the mass ratio of 1:10. The reaction was carried out at room temperature for 2h. The reaction solution was filled into a 3500 Da dialysis bag, and unreacted N- (allyloxycarbonyl-oxy) succinimide was removed to obtain a styrenated hydrogen peroxide degrading enzyme. The method comprises the steps of respectively adding the vinylation hydrogen peroxide degrading enzyme, 3-methyl-butenamine, a cross-linking agent N, N-methylene bisacrylamide and an initiator ammonium persulfate in a mass ratio of 1:10:2:5 into a sodium bicarbonate buffer solution. Reaction 2h was stirred slowly at room temperature under nitrogen blanket. After the reaction, the mixture is filled into a 3500 Da dialysis bag, unreacted monomers and the like are removed, and the enzyme nanocapsule is obtained after freeze drying.

The particle size of the enzyme nanocapsules obtained in this example was measured by a malvern particle size meter, and c in fig. 2 is the measurement result of the malvern particle size meter, the particle size of the enzyme nanocapsules was 46.3. 46.3 nm, and the encapsulation efficiency of hydrogen peroxide degrading enzyme was 66.5% as measured by BCA method.

Example 25

Nanometer capsule of synthetic hydrogen peroxide degrading enzyme

And respectively adding the hydrogen peroxide degrading enzyme and the acrylic acid-N-succinimidyl ester into sodium bicarbonate buffer solution with the pH value of 8.0-9.5 according to the mol ratio of 1:5. Reaction 2 h was carried out at room temperature. The reaction solution was filled into a 3500 Da dialysis bag, and unreacted acrylic acid-N-succinimidyl ester was removed to obtain a styrenated hydrogen peroxide degrading enzyme. And respectively adding the vinylation hydrogen peroxide degrading enzyme, 3-butenamine, a cross-linking agent N, N-methylene bisacrylamide and an initiator ammonium persulfate in a mass ratio of 1:10:2:5 into a sodium bicarbonate buffer solution. Reaction 2 h was stirred slowly at room temperature under nitrogen blanket. After the reaction, the mixture is filled into a 3500 Da dialysis bag, unreacted monomers and the like are removed, and the enzyme nanocapsule is obtained after freeze drying.

The particle size of the enzyme nanocapsules obtained in this example was measured by a malvern particle size meter, and e in fig. 2 is the measurement result of the malvern particle size meter, the particle size of the enzyme nanocapsules was 49.2. 49.2 nm, and the encapsulation efficiency of hydrogen peroxide degrading enzyme was 66.4% as measured by BCA method.

Example 26

Nanometer capsule of synthetic hydrogen peroxide degrading enzyme

And respectively adding the hydrogen peroxide degrading enzyme and the acrylic acid-N-succinimidyl ester into sodium bicarbonate buffer solution with pH value of 8.0-9.5 according to the mol ratio of 1:3. Reaction 2 h was carried out at room temperature. The reaction solution was filled into a 3500 Da dialysis bag, and unreacted acrylic acid-N-succinimidyl ester was removed to obtain a styrenated hydrogen peroxide degrading enzyme. According to the mass ratio of the vinylation hydrogen peroxide degrading enzyme to the allyl mercaptan, the cross-linking agent N, N-methylene bisacrylamide to the initiator ammonium persulfate of 1:10:2:5, respectively adding the two components into the sodium bicarbonate buffer solution. Reaction 2 h was stirred slowly at room temperature under nitrogen blanket. After the reaction, the mixture is filled into a 3500 Da dialysis bag, unreacted monomers and the like are removed, and the enzyme nanocapsule is obtained after freeze drying.

The particle size of the enzyme nanocapsules obtained in this example was measured by a malvern particle size meter, and in fig. 2 b, the particle size of the enzyme nanocapsules was 44.2. 44.2 nm, and the encapsulation efficiency of hydrogen peroxide degrading enzyme was 40.1% as measured by BCA method.

Example 27

Nanometer capsule of synthetic hydrogen peroxide degrading enzyme

And respectively adding the hydrogen peroxide degrading enzyme and the acrylic acid-N-succinimidyl ester into sodium bicarbonate buffer solution with the pH value of 8.0-9.5 according to the molar ratio of 1:10. Reaction 4 h was carried out at room temperature. The reaction solution was filled into a 3500 Da dialysis bag, and unreacted acrylic acid-N-succinimidyl ester was removed to obtain a styrenated hydrogen peroxide degrading enzyme. According to the mass ratio of the vinylation hydrogen peroxide degrading enzyme to the allylamine hydrochloride, the cross-linking agent N, N-methylene bisacrylamide and the initiator ammonium persulfate of 1:10:2:5, respectively adding the two into a sodium bicarbonate buffer solution. Under the condition of room temperature and nitrogen protection, the reaction is carried out slowly for 2 h. After the reaction, the mixture is filled into a 3500 Da dialysis bag, unreacted monomers and the like are removed, and the enzyme nanocapsule is obtained after freeze drying.

The particle size of the enzyme nanocapsules obtained in this example was measured by a malvern particle size meter, d in fig. 2 is the measurement result of the malvern particle size meter, the particle size of the enzyme nanocapsules was 62.2. 62.2 nm, and the encapsulation efficiency of hydrogen peroxide degrading enzyme was 75.6% as measured by BCA method.

Example 28

Nanometer capsule of synthetic hydrogen peroxide degrading enzyme

And respectively adding the hydrogen peroxide degrading enzyme and the acrylic acid-N-succinimidyl ester into sodium bicarbonate buffer solution with the pH value of 8.0-9.5 according to the molar ratio of 1:10. Reaction 2 h was carried out at room temperature. The reaction solution was filled into a 3500 Da dialysis bag, and unreacted acrylic acid-N-succinimidyl ester was removed to obtain a styrenated hydrogen peroxide degrading enzyme. The method comprises the steps of respectively adding the vinylation hydrogen peroxide degrading enzyme, 3-methyl-butenamine, a cross-linking agent N, N-methylene bisacrylamide and an initiator ammonium persulfate in a mass ratio of 1:15:2:5 into a sodium bicarbonate buffer solution. Reaction 2 h was stirred slowly at room temperature under nitrogen blanket. After the reaction, the mixture is filled into a 3500 Da dialysis bag, unreacted monomers and the like are removed, and the enzyme nanocapsule is obtained after freeze drying.

The particle size of the enzyme nanocapsules obtained in this example was measured by a malvern particle size meter, g in fig. 2 is the measurement result of the malvern particle size meter, the particle size of the enzyme nanocapsules was 58.3. 58.3 nm, and the encapsulation efficiency of hydrogen peroxide degrading enzyme was 77.4% as measured by BCA method.

Example 29

Nanometer capsule of synthetic hydrogen peroxide degrading enzyme

And respectively adding the hydrogen peroxide degrading enzyme and the acrylic acid-N-succinimidyl ester into sodium bicarbonate buffer solution with the pH value of 8.0-9.5 according to the molar ratio of 1:10. Reaction 2 h was carried out at room temperature. The reaction solution was filled into a 3500 Da dialysis bag, and unreacted acrylic acid-N-succinimidyl ester was removed to obtain a styrenated hydrogen peroxide degrading enzyme. According to the mass ratio of the vinylation hydrogen peroxide degrading enzyme to 3-butenamine, the cross-linking agent N, N-methylene bisacrylamide and the initiator ammonium persulfate of 1:5:2:5, respectively adding the two into the sodium bicarbonate buffer solution. Reaction 2 h was stirred slowly at room temperature under nitrogen blanket. After the reaction, the mixture is filled into a 3500 Da dialysis bag, unreacted monomers and the like are removed, and the enzyme nanocapsule is obtained after freeze drying.

The particle size of the enzyme nanocapsules obtained in this example was measured by a malvern particle size meter, and f in fig. 2 is the measurement result of the malvern particle size meter, the particle size of the enzyme nanocapsules was 48.8 nm, and the encapsulation efficiency of hydrogen peroxide degrading enzyme was 67.9% as measured by BCA method.

Example 30

Nanometer capsule of synthetic hydrogen peroxide degrading enzyme

And respectively adding the hydrogen peroxide degrading enzyme and the acrylic acid-N-succinimidyl ester into sodium bicarbonate buffer solution with the pH value of 8.0-9.5 according to the molar ratio of 1:10. Reaction 2 h was carried out at room temperature. The reaction solution was filled into a dialysis bag of 3500 Da, and unreacted acrylic acid-N-succinimidyl ester was removed to obtain a styrenated catalase. According to the mass ratio of the vinylation hydrogen peroxide degrading enzyme to the allyl mercaptan, the cross-linking agent N, N-methylene bisacrylamide to the initiator ammonium persulfate of 1:10:2:5, respectively adding the two components into the sodium bicarbonate buffer solution. Under the condition of room temperature and nitrogen protection, the reaction is carried out slowly for 2 h. After the reaction, the mixture is filled into a 3500 Da dialysis bag, unreacted monomers and the like are removed, and the enzyme nanocapsule is obtained after freeze drying.

The particle size of the enzyme nanocapsules obtained in this example was measured by a malvern particle size meter, i in fig. 2 is the measurement result of the malvern particle size meter, the particle size of the enzyme nanocapsules was 55.9. 55.9 nm, and the encapsulation efficiency of hydrogen peroxide degrading enzyme was 72.8% as measured by BCA method.

Example 31

Nanometer capsule of synthetic hydrogen peroxide degrading enzyme

And respectively adding the hydrogen peroxide degrading enzyme and the acrylic acid-N-succinimidyl ester into sodium bicarbonate buffer solution with the pH value of 8.0-9.5 according to the molar ratio of 1:10. Reaction 2 h was carried out at room temperature. The reaction solution was filled into a 3500 Da dialysis bag, and unreacted acrylic acid-N-succinimidyl ester was removed to obtain a styrenated hydrogen peroxide degrading enzyme. According to the mass ratio of the vinylation hydrogen peroxide degrading enzyme to the allylamine hydrochloride, the cross-linking agent N, N-methylene bisacrylamide and the initiator ammonium persulfate of 1:10:2:5, respectively adding the two into a sodium bicarbonate buffer solution. Under the condition of room temperature and nitrogen protection, the reaction is carried out slowly for 2 h. After the reaction, the mixture is filled into a 3500 Da dialysis bag, unreacted monomers and the like are removed, and the enzyme nanocapsule is obtained after freeze drying.

The particle size of the enzyme nanocapsules obtained in this example was measured by a markov particle size meter, j in fig. 2 is the measurement result of the markov particle size meter, the particle size of the enzyme nanocapsules was 45.7. 45.7 nm, and the encapsulation efficiency of hydrogen peroxide degrading enzyme was 72.8% as measured by BCA method.

Example 32

Preparation of BSA nanocapsules without catalytic Capacity

Respectively adding the bovine serum albumin and the acrylic acid-N-succinimidyl ester into sodium bicarbonate buffer solution with the pH value of 8.0-9.5 according to the mass ratio of 1:10. Reaction 2 h was carried out at room temperature. The reaction solution was filled into a 3500 Da dialysis bag, and unreacted acrylic acid-N-succinimidyl ester was removed to obtain styrenated BSA. The method comprises the steps of respectively adding the vinyl BSA, allylamine hydrochloride, a cross-linking agent N, N-methylene bisacrylamide and an initiator ammonium persulfate in a mass ratio of 1:10:2:5 into a sodium bicarbonate buffer solution. Reaction 2 h was stirred slowly at room temperature under nitrogen blanket. After the reaction, the mixture was filled into a dialysis bag of 3500 Da, unreacted monomers and the like were removed, and the mixture was freeze-dried to obtain BSA nanocapsules.

The particle size of the BSA nanocapsules obtained in this example was measured by a malvern particle size meter, and h in fig. 2 is the measurement result of the malvern particle size meter, and the particle size of the BSA nanocapsules was 35.7. 35.7 nm, and the BSA encapsulation efficiency was 84.8% as measured by the BCA method.

Example 33

Photosensitizer/enzyme combination delivery system for synthetic anti-tumor photodynamic sensitization therapy

Precisely weighing a certain amount of photosensitizer-polymer nanoparticles in example 13 and enzyme nanocapsule solid powder in example 30, adding the solid powder into sodium bicarbonate buffer solution according to the mass ratio of the photosensitizer-polymer nanoparticles to the enzyme nanocapsules of 1:5, and stirring for reaction under the condition of avoiding light at room temperature for 4 h. According to the mass ratio of the polymer nanoparticle to the glucan of 1:1, adding the polymer nanoparticle to the reaction solution, and reacting at room temperature for 6 h. Placing the reaction solution into a 5 mL centrifuge tube, centrifuging at 5000 rpm/min for 5 min, collecting the upper layer liquid, and lyophilizing to obtain the photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization.

The particle size of the nano delivery system of this example was measured by a malvern particle size meter, and a in fig. 3 is the measurement result of the malvern particle size meter, and the nano delivery system particle size was 181.5 and nm.

Example 34

Precisely weighing a certain amount of photosensitizer-polymer nanoparticles in example 14 and enzyme-entrapped protein nanocapsule solid powder in example 23, adding the solid powder into sodium bicarbonate buffer solution according to the mass ratio of the photosensitizer-polymer nanoparticles to the enzyme nanocapsules of 1:10, and stirring for reaction under the condition of avoiding light at room temperature for 4 h. According to the mass ratio of the polymer nanoparticle to the hyaluronic acid of 1:0.5, adding the polymer nanoparticle to the reaction solution, and reacting at room temperature of 6 h. Placing the reaction solution into a 5 mL centrifuge tube, centrifuging at 5000 rpm/min for 5 min, collecting the upper layer liquid, and lyophilizing to obtain the photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization.

The particle size of the nano delivery system of this example was measured by a malvern particle sizer, and in fig. 3 c is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 129.7 nm.

Example 35

Precisely weighing a certain amount of photosensitizer-polymer nanoparticles in example 16 and enzyme nanocapsule solid powder in example 25, adding the solid powder into sodium bicarbonate buffer solution according to the mass ratio of the photosensitizer-polymer nanoparticles to the enzyme nanocapsules of 1:15, and stirring for reaction under the condition of avoiding light at room temperature for 4 h. According to the mass ratio of the polymer nanoparticle to the heparin of 1:1, adding the polymer nanoparticle to the reaction solution, and reacting at room temperature for 6 h. Placing the reaction solution into a 5 mL centrifuge tube, centrifuging at 5000 rpm/min for 5 min, collecting the upper layer liquid, and lyophilizing to obtain the photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization.

The particle size of the nano delivery system of this example was measured by a malvern particle sizer, and b in fig. 3 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 81.5. 81.5 nm.

Example 36

Precisely weighing a certain amount of photosensitizer-polymer nanoparticles in example 17 and enzyme nanocapsule solid powder in example 26, adding the solid powder into sodium bicarbonate buffer solution according to the mass ratio of the photosensitizer-polymer nanoparticles to the enzyme nanocapsules of 1:10, and stirring for reaction under the condition of avoiding light at room temperature for 4 h. According to the mass ratio of the polymer nano particles to the polylysine of 1:1, the polymer nano particles and the polylysine are added into a reaction solution to react at room temperature of 6 h. Placing the reaction solution into a 5 mL centrifuge tube, centrifuging at 5000 rpm/min for 5 min, collecting the upper layer liquid, and lyophilizing to obtain the photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization.

The particle size of the nano delivery system of this example was measured by a malvern particle sizer, and d in fig. 3 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 181.4 nm.

Example 37

Precisely weighing a certain amount of photosensitizer-polymer nanoparticles in the example 18 and catalase nanocapsule solid powder in the example 28, adding the solid powder into sodium bicarbonate buffer solution according to the mass ratio of the photosensitizer-polymer nanoparticles to the enzyme nanocapsules of 1:10, and stirring for reaction under the condition of avoiding light at room temperature for 4 h. According to the mass ratio of the polymer nanoparticle to the Apoa-1 of 1:1, the polymer nanoparticle and the Apoa-1 are added into a reaction solution to react at room temperature 6 h. Placing the reaction solution into a 5 mL centrifuge tube, centrifuging at 5000 rpm/min for 5 min, collecting the upper layer liquid, and lyophilizing to obtain the photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization.

The particle size of the nano delivery system of this example was measured by a malvern particle sizer, and e in fig. 3 is the measurement result of the malvern particle sizer, and the nano delivery system particle size was 133.6 nm.

Example 38

Precisely weighing a certain amount of photosensitizer-polymer nanoparticles in example 20 and catalase nanocapsule solid powder in example 29, adding the solid powder into sodium bicarbonate buffer solution according to the mass ratio of the photosensitizer-polymer nanoparticles to the enzyme nanocapsules of 1:10, and stirring for reaction under the condition of avoiding light at room temperature for 4 h. According to the mass ratio of the polymer nano particles to the polylysine of 1:1, the polymer nano particles and the polylysine are added into a reaction solution to react at room temperature of 6 h. Placing the reaction solution into a 5 mL centrifuge tube, centrifuging at 5000 rpm/min for 5 min, collecting the upper layer liquid, and lyophilizing to obtain the photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization.

The particle size of the nano delivery system of this example was measured by a malvern particle sizer, and f in fig. 3 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 153.9 and nm.

Example 39

Precisely weighing a certain amount of photosensitizer-polymer nanoparticles in the example 22 and enzyme nanocapsule solid powder in the example 31, adding the solid powder into sodium bicarbonate buffer solution according to the mass ratio of the photosensitizer-polymer nanoparticles to the enzyme nanocapsules of 1:10, and stirring for reaction under the condition of avoiding light at room temperature for 4 h. According to the mass ratio of the polymer nanoparticle to the hyaluronic acid of 1:1, the polymer nanoparticle and the hyaluronic acid are added into a reaction solution to react at room temperature of 6 h. Placing the reaction solution into a 5 mL centrifuge tube, centrifuging at 5000 rpm/min for 5 min, collecting the upper layer liquid, and freeze-drying to obtain the photodynamic therapy enhanced enzyme/photosensitizer combined delivery system.

The particle size of the nano delivery system of this example was measured by a malvern particle sizer, and h in fig. 3 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 173.1 and nm.

Example 40

Synthetic BSA/photosensitizer combination delivery system

Precisely weighing a certain amount of photosensitizer-polymer nanoparticles in example 14 and BSA nanocapsule solid powder coated with BSA in example 32, adding the solid powder into sodium bicarbonate buffer solution according to the mass ratio of the photosensitizer-polymer nanoparticles to the BSA nanocapsules of 1:10, and stirring for reaction under the condition of avoiding light at room temperature for 4 h. According to the mass ratio of the polymer nano particles to the polylysine of 1:1, the polymer nano particles and the polylysine are added into a reaction solution to react at room temperature of 6 h. Placing the reaction solution into a 5 mL centrifuge tube, centrifuging at 5000 rpm/min for 5 min, collecting the upper layer liquid, and freeze-drying to obtain the photodynamic therapy enhanced enzyme/photosensitizer combined delivery system.

The particle size of the nano delivery system of this example was measured by a malvern particle sizer, and g in fig. 3 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 128.6 nm.

Example 41

Experiment of variation of particle size of photosensitizer/enzyme combination delivery system of anti-tumor photodynamic sensitization treatment along with reaction time

A certain amount of photosensitizer-polymer nanoparticles in example 14 and enzyme nanocapsule solid powder in example 23 are precisely weighed, added into sodium bicarbonate buffer solution according to the mass ratio of photosensitizer-polymer nanoparticles to enzyme nanocapsules of 1:10, reacted at room temperature, and the reaction solution is taken out to measure the particle size respectively at 0 h, 0.5 h, 1 h, 2 h and 4 h.

The particle size of the nano delivery system at different time points in the present example was measured by a malvern particle size meter, and fig. 4 shows the measurement result of the malvern particle size meter, the particle size of the combined delivery system tends to be stable with the extension of the reaction time, and when the reaction time is less than 2 h, the two carriers are not completely combined, and free enzyme nanocapsules exist; when the reaction time is longer than 4 h, the two carriers are gradually combined completely, and the particle size of the nano delivery system is uniform to be 131.2 nm.

Example 42

Light sensitizer/enzyme combination delivery system ninhydrin binding rate investigation experiment for anti-tumor photodynamic sensitization treatment

Precisely weighing a certain amount of photosensitizer-polymer nanoparticles in example 16 and enzyme nanocapsule solid powder in example 26, adding the solid powder into sodium bicarbonate buffer solution according to the mass ratio of the photosensitizer-polymer nanoparticles to the enzyme nanocapsules of 1:10, reacting at room temperature, taking out the reaction solution and indene trione solution respectively for heating reaction of 0.5 h at 0. 0 h, 0.5 h, 1 h, 2 h and 4 h.

The absorbance after the combination of the nano delivery system and ninhydrin at different time points in this example was measured by an ultraviolet-visible spectrophotometer, and fig. 5 shows the measurement result of the combination rate of the nano delivery system, and the absorbance of the nano delivery system decreases with the extension of the reaction time, indicating that the combination rate of the two nanoparticles increases. When the reaction time is less than 2 h, free amino groups exist to react with ninhydrin, so that the absorbance is high but the binding rate is low; when the reaction time is longer than 4 h, the two carriers are gradually combined completely, and the combination rate is maintained at about 80%.

Example 43

Photosensitizer/enzyme combined delivery system oxygen production amount investigation experiment for anti-tumor photodynamic sensitization treatment

Precisely weighing a certain amount of lyophilized powder of the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment prepared in the example 35, and preparing a solution (methylene blue: 150 mug/mL) by adopting a sodium bicarbonate buffer solution; 500. Mu.M hydrogen peroxide was prepared as a reaction substrate, and 3 parallel samples of the free catalase group, BSA control group (example 40) and formulation group were added to the hydrogen peroxide solution, respectively, and stirred at room temperature and dissolved oxygen was measured using an oxygen dissolving meter. The oxygen content test results of the preparation are shown in fig. 6.

The result shows that the combined delivery system can quickly catalyze hydrogen peroxide to generate oxygen, and compared with free catalase, the catalase nanocapsule does not influence the catalysis effect of the catalase, so that the combined delivery system can effectively realize immobilized enzyme technology, and the catalytic capability of the nano delivery system is provided.

Example 44

Photosensitizer release assay for photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy

Precisely weighing a certain amount of freeze-dried powder of the antitumor optical synergistic catalase/photosensitizer combined delivery system prepared in the example 38, and preparing a solution (IR 780 iodide: 150 mu g/mL) by adopting sodium bicarbonate buffer solution; 3 parts of the solution were taken, 1mL of each solution was placed in 3500 Da dialysis bags and placed in 20 mL of PBS solution at pH 6.8. Three samples were run in parallel with shaking uniformly in a shaker at 100 rpm/min at 37℃and sampling and replenishment of the response volume and pH release medium at specific time points. After the experiment is finished, the cumulative release percentage of each indocyanine green is calculated by adopting a fluorescence spectrophotometry method, and the release effect of the photosensitizer is shown in figure 7.

The result shows that the nanometer delivery system can effectively protect the photosensitizer from being released, and the cumulative release percentage of indocyanine green is only 10.1+/-1.4% along with the extension of the release time, so that the combined delivery system can effectively prevent the photosensitizer from being aggregated or degraded in an in-vivo environment and improve the killing effect of photodynamic therapy.

Example 45

Enzyme activity investigation experiment of photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization treatment

Precisely weighing a certain amount of lyophilized powder of the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment prepared in the example 34, and preparing a solution by adopting a sodium bicarbonate buffer solution; preparing 500 mu M hydrogen peroxide as a reaction substrate, respectively taking 3 parts of parallel samples of a free catalase group and a nano delivery system preparation group, adding the parallel samples into a hydrogen peroxide solution, respectively incubating 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h and 12 h at a water temperature of 37 ℃, stirring at room temperature, and measuring by using a catalase activity detection kit, wherein the experimental results are shown in figure 8.

As can be seen from the results, the immobilized enzyme technology of the combined delivery system of the invention can effectively protect catalase, and the activity of free catalase is reduced along with the extension of the reaction time, and after 4 h, the activity is no longer available; the preparation group can effectively improve the stability of the catalase, and can still have 50% of enzyme activity up to 12 h. Experimental results show that the protein nanocapsule can permeate a solution and improve the stability of catalase.

Example 46

Cytotoxicity assay of photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy

The killing effect of the anti-tumor optical synergistic catalase/photosensitizer combined delivery system on tumor cells is examined through an in-vitro cytotoxicity experiment, and 4T1 breast cancer cells are selected as a study object.

The method comprises the following specific steps: culturing 4T1 breast cancer cells in DMEM high-sugar culture medium containing 10% fetal bovine serum, digesting with pancreatin after the cells grow to logarithmic phase, centrifuging to collect cells, inoculating into 96-well plate, culturing at 37deg.C for 24 h, removing culture medium, adding 100 μl of free photosensitizer, photosensitizer-polymer nanoparticle prepared in example 14 and photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization prepared in example 36, respectively, wherein photosensitizer concentration range is 0.1-10 μg/mL, incubating at 37deg.C for 6 h, and performing near infrared wavelength light irradiation treatment (808 nm,1.8 per well with laser W/cm ² 5 min), and the medium containing the preparation was discarded and washed twice with PBS buffer. After adding 20. Mu.L of 5 mg/mL MTT phosphate solution and incubating at 37℃for 4 h, the supernatant was discarded, 200. Mu.L of DMSO was added, absorbance was measured with a microplate reader and at 570 nm wavelength, and cell viability was calculated (results are shown in FIG. 9).

IC of free photosensitizer group ₅₀ IC of photosensitizer-polymeric nanoparticles with a value of 4.106. Mu.g/mL ₅₀ IC of catalase/photosensitizer combination delivery system with value of 4.04 mug/mL and anti-tumor optical synergy ₅₀ The value was reduced to 2.566. Mu.g/mL. According to the result, the preparation has concentration dependency, and compared with a control group, the anti-tumor optical synergistic catalase/photosensitizer combined delivery system has better tumor cell killing effect, which is probably due to the active targeting effect brought by the bionic hyaluronic acid, so that accumulation of the photosensitizer in tumor cells is improved, and the killing effect on the tumor cells is promoted.

Example 47

Singlet oxygen production capability investigation of photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization treatment

The treatment effect of the anti-tumor optical synergistic catalase/photosensitizer combined delivery system in tumor cells is examined through an in-vitro singlet oxygen generation capability experiment, and 4T1 breast cancer cells are selected as a study object. The method comprises the following specific steps: culturing 4T1 cells to logarithmic phase, digesting with pancreatin, centrifuging to collect cells, inoculating into 24-well plate, and cell density of about 1×10 ⁵ Each well containing 5% CO at 37 ℃ ₂ After overnight incubation in incubator, the medium was aspirated and washed three times with PBS, 100. Mu.L of free photosensitizer, photosensitizer-polymer nanoparticles prepared in example 14, and photosensitizer/enzyme combination delivery system (photosensitizer concentration: 2. Mu.g/mL) for anti-tumor photodynamic sensitization treatment prepared in example 37 were added, respectively, at 37℃with 5% CO ₂ The incubator continues to incubate 6 h.6 h the medicated medium was discarded, 0.2 mL serum-free medium containing singlet oxygen probe R was added, and the mixture was irradiated with 808 laser for 5 min (1.5W/cm) ² ) Containing 5% CO at 37 DEG C ₂ Incubation in incubator continued to 0.5 h. After incubation, the culture medium is discarded, and after three times of washing with precooled PBS, the culture medium is placed in an inverted fluorescence microscope for observing the green fluorescence intensity

The singlet oxygen generating capacity results are shown in fig. 10, and the results show that the intracellular fluorescence of the blank group, the NIR group and the free photosensitizer group is weak, while the fluorescence of the photosensitizer-polymer nanoparticle group and the anti-tumor optically synergistic catalase/photosensitizer combined delivery system is strong, which indicates that the polymer is favorable for protecting the photosensitizer after being coated with the photosensitizer and playing a photodynamic role in tumor cells. And after the catalase and the bionic material hyaluronic acid are introduced, the tumor targeting capability and photodynamic therapy level can be better improved.

Example 48

Intracellular oxygen production capacity RDPP (RDPP) investigation of photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization treatment

The oxygen generating capacity of the anti-tumor optical synergistic catalase/photosensitizer combination delivery system in tumor cells is examined through the oxygen generating capacity in cells, and 4T1 breast cancer cells are selected as study objects. The method comprises the following specific steps: culturing 4T1 cells to logarithmic phase, inoculating into cell culture dish, and culturing at 37deg.C with 5% CO ₂ Culturing overnight in incubator, sucking off the culture medium, washing twice with PBS, adding 100 μl free photosensitizer, photosensitizer-polymer nanoparticle prepared in example 17, and photosensitizer/enzyme combination delivery system (photosensitizer concentration: 2 μg/mL) for anti-tumor photodynamic sensitization treatment prepared in example 34, respectively, performing anoxic sealing treatment on part of the culture dish, and containing 5% CO at 37deg.C ₂ Incubator culture 8 h. After incubation, the medium was discarded, washed twice with PBS, 100 μl of RDPP solution was added to each dish, the anaerobic sealing treatment was continued, and incubation was performed for 0.5. 0.5 h. After the incubation, the RDPP solution was discarded, washed three times with PBS, and placed under a fluorescence microscope to give rise to a red fluorescence intensity during the observation period.

As shown in FIG. 11, the results of examination of intracellular oxygen production ability show that RDPP is an oxygen sensitive dye, and generates stronger red fluorescence when the oxygen content is low, conversely, quenching occurs when the oxygen content is high, and the fluorescence signal is low. According to the results, the fluorescence of the normoxic control group is weak; in the cells subjected to hypoxia treatment, the group without catalase has stronger red fluorescence, and the catalase protein nanocapsule group and the catalase/photosensitizer combined delivery system with anti-tumor optical synergy have weaker fluorescence, so that the protein nanocapsule can effectively protect the catalase from being degraded by the protease and can play a role in catalyzing degradation in tumor microenvironment. Meanwhile, after the photosensitizer-carrying polymer nanoparticles and hyaluronic acid are introduced, the catalytic capability of catalase is not affected. Is beneficial to enhancing the photodynamic anti-tumor treatment effect of the photosensitizer.

Example 49

Cell uptake experiments of photosensitizer/enzyme combination delivery systems for anti-tumor photodynamic sensitization therapy

The targeting effect and uptake effect of the anti-tumor optical synergistic catalase/photosensitizer combined delivery system on tumor cells are examined through an in-vitro cell uptake experiment, and 4T1 breast cancer cells are selected as a study object.

The method comprises the following specific steps: culturing 4T1 breast cancer cells in DMEM high sugar culture medium containing 10% foetal calf serum, culturing until the cells grow to logarithmic phase, digesting with pancreatin, centrifuging to collect cells, inoculating into copolymer Jiao Min, and culturing at cell density of 1×10 ⁵ Each well containing 5% CO at 37 ℃ ₂ After overnight incubation in incubator, the medium was discarded and washed twice with PBS, and 500. Mu.L of the photosensitizer/enzyme combination delivery system (photosensitizer concentration: 2. Mu.g/mL) for anti-tumor photodynamic sensitization prepared in example 33 after serum-free dilution was added to each of 1 h, 2 h, 4 h and 8 h, and after fixing the cells with 4% paraformaldehyde solution, the cells were washed twice with PBS, and after staining the nuclei with 200. Mu.L of DAPI working solution for 8min, they were observed with a laser confocal microscope.

The results of the qualitative cell uptake experiments are shown in FIG. 12, which shows that the intracellular fluorescence intensity of the photosensitizer increases gradually with the prolonged incubation time. And the photosensitizer is distributed in the whole cell, which indicates that the preparation can effectively deliver the photosensitizer into the cell to play a role, and the generated singlet oxygen can kill tumor cells in the whole cell under the photodynamic therapy effect, so that the high-efficiency anti-tumor effect is achieved.

Example 50

Photosensitizer/enzyme combination delivery system apoptosis experiments for anti-tumor photodynamic sensitization treatment

The ability of the photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization treatment to induce tumor cell apoptosis is examined through an in-vitro apoptosis experiment, and 4T1 breast cancer cells are selected as a study object.

The method comprises the following specific steps: culturing 4T1 breast cancer cells in DMEM high sugar culture medium containing 10% foetal calf serum, culturing until the cells grow to logarithmic phase, digesting with pancreatin, centrifuging to collect cells, inoculating into 6-well plate, and culturing at cell density of 1×10 ⁵ Each well containing 5% CO at 37 ℃ ₂ After overnight incubation in incubator, the medium was discarded and washed twice with PBS, 500. Mu.L of DMEM, free photosensitizer, BSA control group (example 40) and the antitumor optically enhanced catalase/photosensitizer combination delivery system prepared in example 39 (photosensitizer concentration of 2. Mu.g/mL) were added, respectively, and after further incubation in incubator for 8 h, the incubation was continued for 5 min (1.5W/cm) with 808 laser ² ) 200. Mu.L of pancreatin was added to each well after irradiation for 5 min, and stopped with serum-containing medium, centrifuged at 3000 rpm/min for 3min, and the supernatant was discarded. The lower cell pellet was resuspended in 1 Xbinding buffer and 5. Mu.L of Annexin-FITC and PI dye were added and mixed well before detection using flow cytometry.

The apoptosis results are shown in fig. 13, and the results show that the apoptosis rate of the free photosensitizer is only 23.1%, the apoptosis rate of the BSA control group is 53.9%, and the apoptosis rate induced by the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment is 88.5%. The method shows that the PDT effect of the photosensitizer can be effectively improved by introducing the enzyme nanocapsules, the apoptosis induction rate is improved by 3 times, and the tumor cell killing effect of the photosensitizer is effectively enhanced, so that the high-efficiency anti-tumor effect is achieved.

While the invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit of the invention.

Claims

1. A photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy, characterized by: the preparation method comprises the steps of encapsulating a polymer nanoparticle of a photosensitizer and an enzyme nanocapsule, wherein the polymer nanoparticle and the enzyme nanocapsule are connected through a chemical bond to form a nanocomposite, and a targeting biological macromolecule is further modified on the surface of the nanocomposite;

The mass ratio of the polymer nanoparticles to the enzyme nanocapsules is 1:5-10;

the polymer nanoparticles are formed by self-assembly of polymers modified with boric acid groups and epoxy groups; the enzyme nanocapsule is formed by in-situ free radical polymerization of ethylenated hydrogen peroxide degrading enzyme and amine or mercaptan-containing monomers; the targeting biological macromolecules are dextran, hyaluronic acid, polylysine or heparin;

the polymer modified with the boric acid groups and the epoxy groups is prepared by the following method:

step 1, dispersing boric acid compound and monomer in organic solvent, reacting at room temperature in dark condition, purifying and freeze-drying to obtain acrylic acid compound with modified boric acid group;

the boric acid compound is 4-bromomethyl phenylboronic acid, 3-chloro-4-pyridine boric acid pinacol ester, 5-bromo-2-thiophene boric acid or 6-fluoropyridine-3-boric acid; the monomer is diethylaminoethyl methacrylate, N-vinyl pyrrolidone, dimethylaminoethyl acrylate or N, N-diethylaminoethyl acrylate;

step 2, dispersing an acrylic acid compound modified with a boric acid group, a polymerizable monomer, a cross-linking agent and a free radical initiator in an organic solvent for free radical polymerization reaction, and obtaining a polymer modified with the boric acid group and the epoxy group after extraction, purification and spin evaporation;

The polymerizable monomer is glycidyl methacrylate, glycidyl acrylate, acetone glycidyl methacrylate or allyl glycidyl ether;

the photosensitizer is one or more selected from indocyanine green, chlorin e6, methylene blue, IR780 or hematoporphyrin monomethyl ether;

the vinylated hydrogen peroxide degrading enzyme is prepared by the following method:

dissolving hydrogen peroxide degrading enzyme and a monomer containing succinimidyl ester in a buffer solution with the pH of 8.0-9.5, reacting at room temperature, and dialyzing the reaction solution to obtain the ethylenated hydrogen peroxide degrading enzyme;

the monomer containing the succinimidyl ester is N- (allyloxycarbonyl oxygen) succinimidyl or acrylic acid-N-succinimidyl ester;

the amine or mercaptan-containing monomer is allylamine hydrochloride, 3-methyl-2-butenamine, 3-butenamine or allylmercaptan.

2. A method of making a combination delivery system as defined in claim 1, wherein: the method comprises the following steps:

step 1, dissolving a polymer modified with a boric acid group and an epoxy group and a photosensitizer in an organic solvent, dispersing the mixture in pure water according to the volume ratio of 1/10-1/50, and carrying out ultrasonic and dialysis purification at room temperature under the dark condition to obtain polymer nanoparticles carrying the photosensitizer;

Step 2, dissolving the ethylenated hydrogen peroxide degrading enzyme, amine or mercaptan monomer, cross-linking agent and redox initiator in buffer solution, carrying out in-situ free radical polymerization at room temperature under the protection of nitrogen, and dialyzing the reaction solution to obtain an enzyme nanocapsule;

step 3, dissolving the polymer nanoparticles obtained in the step 1 and the enzyme nanocapsules obtained in the step 2 in a buffer solution according to a mass ratio of 1:5-10, and reacting at room temperature to obtain a nanocomposite;

3. The preparation method according to claim 2, characterized in that: in the step 1, the mass ratio of the polymer to the photosensitizer is 1:0.5-1:5.

4. The preparation method according to claim 2, characterized in that: in the step 2, the mass ratio of the vinylated hydrogen peroxide degrading enzyme to the amine-containing, alcohol-containing or mercaptan-containing monomer to the cross-linking agent to the redox agent is 1:1-10:2:5, and the in-situ free radical polymerization reaction time is 2-3 h.

5. Use of the combination delivery system of claim 1 for the preparation of a medicament for tumor diagnosis or treatment.