CN112915205A

CN112915205A - Photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy and preparation method thereof

Info

Publication number: CN112915205A
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: 2021-06-08
Anticipated expiration: 2041-02-03
Also published as: CN112915205B

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 and the enzyme nanocapsules are coated with a photosensitizer, the polymer nanoparticles and the enzyme nanocapsules are connected through chemical bonds to form a nano compound, and targeting biological macromolecules are modified on the surface of the nano compound. The combined delivery system can realize the high-efficiency enrichment of the tumor part co-delivered in the same target area of the photosensitizer and the enzyme, improves the oxygen production amount and the oxygen utilization rate of the focus part by flexibly adjusting the assembly proportion of the polymer nanoparticles and the nanocapsules, effectively improves the hypoxic microenvironment of tumor tissues, enhances the photodynamic treatment effect, and can be applied to the application of preparations in tumor diagnosis or treatment medicines.

Description

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

Technical Field

The invention belongs to the field of medicinal preparations, relates to a nano-medicament combined delivery system and a preparation method thereof, and particularly relates to a photosensitizer/enzyme combined delivery system for anti-tumor photodynamic sensitization treatment and a preparation method thereof.

Background

The main approaches to tumor treatment are surgical resection, chemotherapy and radiotherapy. In recent years, with the rapid development of nano-radiation technology, optical therapy has become a modern non-invasive new radiation therapy approach. Photodynamic therapy (PDT) can be divided into type i PDT and type ii PDT, wherein type ii PDT is the main research direction, and the action mechanism is that a photosensitizer excites oxygen in tumor cells into singlet oxygen, and the singlet oxygen can oxidize nucleic acid, protein and biomembrane in the stress cells, destroy normal metabolism of the tumor cells and further kill the cells. Because type II photodynamic therapy is very dependent on oxygen, aiming at the characteristic that the tumor microenvironment contains a large amount of hydrogen peroxide, the adoption of a novel nano and radiation technology to improve the photodynamic therapy effect on the tumor is very important.

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

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

Therefore, it is necessary to construct a co-delivery system with a high efficiency photosensitizer entrapment and improved tumor hypoxia for the same target region to solve the above disadvantages. The construction of the nano delivery system which has high drug-loading rate, biocompatibility and good targeting property and can improve the hypoxic 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 in the prior art, the invention provides a photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy, the nanometer delivery system has the characteristics of improving the stability, tumor targeting property, same target area, co-delivery and bionic property of the photosensitizer and hydrogen peroxide degrading enzyme, has high-efficiency oxygen production efficiency and can be used for enhancing photodynamic anti-tumor therapy.

In order to achieve the purpose, the invention adopts the following technical scheme:

a photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy comprises a photosensitizer-coated polymer nanoparticle and an enzyme nanocapsule, wherein the polymer nanoparticle and the enzyme nanocapsule are connected through a chemical bond to form a nano-composite, and a targeting biological macromolecule is modified on the surface of the nano-composite;

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 ethylene hydrogen peroxide degrading enzyme and a monomer containing amines, alcohols or thiols;

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

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

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

and 2, dispersing the boric acid group-modified acrylic acid compound, the polymerizable monomer, the cross-linking agent and the free radical initiator in an organic solvent, carrying out free radical polymerization reaction, and carrying out extraction, purification and rotary evaporation to obtain the boric acid group-and epoxy group-modified polymer.

Further, the boric acid compound is selected from a halogen-containing phenyl boronic acid monomer, a halogen-containing pyridine boronic acid monomer or a halogen-containing thiophene boronic acid monomer; the acrylic acid derivative monomer is an acrylic acid monomer containing a tertiary amine group, 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 boronic acid compound is selected from one or more of 4-bromomethylbenzeneboronic acid, 3-chloro-4-pyridineboronic acid pinacol ester, 5-bromo-2-thiopheneboronic acid boronic 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 succinimide ester in a buffer solution with the pH value of 8.0-9.5, reacting at room temperature, and dialyzing the reaction solution to obtain vinylated hydrogen peroxide degrading enzyme;

the monomer containing succinyl ester is N- (allyloxycarbonyloxy) succinimide or acrylic acid-N-succinimidyl ester.

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

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

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

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

step 2, dissolving vinylated hydrogen peroxide degrading enzyme, primary amine-containing or thiol-containing 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 enzyme nanocapsules;

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 nano compound;

and 4, dissolving the nano compound obtained in the step 3 and the targeting biological macromolecule in a buffer solution for 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.

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

By utilizing a chemical combination means, the nanocapsule is formed on the surface of the protein, so that the activity of the protein in vivo can be effectively protected. The structure of the hydrogen peroxide degrading enzyme contains a plurality of lysine groups, the residual 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 succinimide ester-containing monomer, so that a polymer reaction skeleton group double bond is introduced to the surface of the hydrogen peroxide degrading enzyme. The ethylene hydrogen peroxide degrading enzyme and the monomer containing the polymerizable group have 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 invention, 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 large, and the compound is unstable under physiological conditions; when the ratio of the two is 1: 5-1: 10, the obtained nano-composite has moderate particle size, is uniformly dispersed, and can keep good stability under physiological conditions; when the ratio of the two is 1: 10-1: 15, the reactive groups on the surfaces of the polymer nanoparticles are saturated, and excessive enzyme nanocapsules exist, so that the uniformity of a nano delivery system is damaged. Therefore, the optimal mass ratio of the photosensitizer-polymer nanoparticles to the enzyme nanocapsules is 1: 5-10.

According to the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization therapy, 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 to be supplied to the photosensitizer; when the photosensitizer is irradiated by laser, the photosensitizer is converted into an excited state from a ground state, and the photosensitizer and triplet oxygen have the same spin, so that excited state energy is favorably transferred to triplet oxygen, and singlet oxygen is induced to be generated. The photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy has the advantages of protecting the internal stability of catalase, catalyzing the generation of oxygen, enhancing photodynamic therapy, and having good application prospects in the aspects of preparing tumor diagnosis and treatment preparations, targeted photodynamic therapy and synergistic photodynamic antigravity therapy.

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

1. the photosensitizer/enzyme combination delivery system can enhance the photodynamic therapy effect, more importantly, the photosensitizer and catalase are respectively protected by two carriers, and the stability and the enzyme activity of the drug under physiological conditions are improved;

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

3. the photosensitizer/enzyme combined delivery system provided by the invention incubates targeting biomacromolecules, improves the tumor targeting of the delivery system, and realizes co-delivery of the photosensitizer and the enzyme to a tumor part through covalent combination of two carriers, thereby further enhancing 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 particle size distribution diagram of an enzyme nanocapsule;

FIG. 3 is a graph showing the distribution of particle sizes of polymer nanoparticles and nanocapsules in different proportions;

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

FIG. 5 shows the results of the experiment for examining the binding rate of polymer nanoparticles and nanocapsules ninhydrin at a fixed ratio;

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

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

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

FIG. 9 shows the results of cytotoxicity investigation of photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy;

FIG. 10 shows the results of the examination of ROS production capacity of the photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy;

FIG. 11 shows the results of the RDPP oxygen production capacity investigation of the photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy;

FIG. 12 shows the results of cell uptake experiments for the photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy;

FIG. 13 shows the results of apoptosis experiments on the photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy.

Detailed Description

The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.

Example 1

Synthesis of boronic acid-epoxy based polymers

Adding a trace amount of potassium iodide according to the molar ratio of the diethylaminoethyl methacrylate to the 4-bromomethylbenzeneboronic acid of 1:0.5, dissolving in 6 mL of N, N-dimethylformamide, and reacting for 24 hours in a dark place. The prepared monomer, glycidyl methacrylate and N, N-methylene-bisacrylamide 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 molar ratio of 1:0.5 of boric acid-acrylic acid compound to azodiisobutyronitrile as an initiator, after violent reaction for 12 hours at 65 ℃, extracting and purifying by using normal hexane, and performing rotary evaporation to obtain the boric acid-epoxy group polymer.

Example 2

Synthesis of boronic acid-epoxy based polymers

Adding a trace amount of potassium iodide according to the molar ratio of the N-vinyl pyrrolidone to the 4-bromomethylbenzeneboronic acid of 1:1, dissolving in 6 mL of N, N-dimethylformamide, and reacting for 24 hours in a dark place. The prepared compound, glycidyl methacrylate and N, N-methylene-bis-acrylamide 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 molar ratio of 1:0.5 of boric acid-acrylic acid compound to azodiisobutyronitrile as an initiator, after violent reaction for 12 hours at 65 ℃, normal hexane is used for extraction and purification, and rotary evaporation is carried out to obtain the boric acid-epoxy group polymer.

Example 3

Synthesis of boronic acid-epoxy based polymers

Adding a trace amount of potassium iodide according to a molar ratio of the diethylaminoethyl methacrylate to the 4-bromomethylbenzeneboronic acid of 1:2, dissolving in 6 mL of N, N-dimethylformamide, and reacting for 24 hours in a dark place. The prepared compound, glycidyl acrylate 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 molar ratio of 1:0.5 of boric acid-acrylic acid compound to azodiisobutyronitrile as an initiator, after violent reaction for 12 hours at 65 ℃, normal hexane is used for extraction and purification, and rotary evaporation is carried out to obtain the boric acid-epoxy group polymer.

Example 4

Synthesis of boronic acid-epoxy based polymers

Adding a trace amount of potassium bromide according to the molar ratio of 1:1 between N-vinyl pyrrolidone and 3-chloro-4-pyridineboronic acid pinacol ester, dissolving in 6 mL of tetrahydrofuran, and reacting for 24 hours in a dark place. The prepared compound, glycidyl acrylate 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 molar ratio of 1:0.5 of boric acid-acrylic acid compound to azodiisobutyronitrile as an initiator, after violent reaction for 12 hours at 65 ℃, normal hexane is used for extraction and purification, and rotary evaporation is carried out to obtain the boric acid-epoxy group polymer.

Example 5

Synthesis of boronic acid-epoxy based polymers

Adding a trace amount of sodium iodide according to the molar ratio of the dimethylaminoethyl acrylate to the 5-bromo-2-thiopheneboronic acid of 1:1, dissolving in 6 mL of methanol, and reacting for 24 hours in a dark place. The prepared compound, allyl glycidyl ether and N, N-methylene bisacrylamide were dissolved in 4 mL of N, N dimethylformamide at a molar ratio of 1:1:0.5 under nitrogen protection. According to the molar ratio of 1:0.5 of boric acid-acrylic acid compound to azodiisobutyronitrile as an initiator, after violent reaction for 12 hours at 65 ℃, normal hexane is used for extraction and purification, and rotary evaporation is carried out to obtain the boric acid-epoxy group polymer.

Example 6

Synthesis of boronic acid-epoxy based polymers

Adding a trace amount of potassium iodide according to the molar ratio of the N, N-diethylaminoethyl acrylate to the 6-fluoropyridine-3-boric acid of 1:1, dissolving in 6 mL of tetrahydrofuran, and reacting for 24 hours in a dark place. The prepared compound, 3, 4-epoxy-1-butene and N, N-methylene bisacrylamide 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 molar ratio of 1:0.5 of boric acid-acrylic acid compound to azodiisobutyronitrile as an initiator, after violent reaction for 12 hours at 65 ℃, normal hexane is used for extraction and purification, and rotary evaporation is carried out to obtain the boric acid-epoxy group polymer.

Example 7

Synthesis of boronic acid-epoxy based polymers

Adding a trace amount of potassium iodide according to the molar ratio of the diethylaminoethyl methacrylate to the 4-bromomethylbenzeneboronic acid of 1:1, dissolving in 6 mL of methanol, and reacting for 24 hours in a dark place. The prepared compound, 1, 2-epoxy-9-decene and N, N-methylene bisacrylamide 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 molar ratio of 1:0.5 of boric acid-acrylic acid compound to azodiisobutyronitrile as an initiator, after violent reaction for 12 hours at 65 ℃, normal hexane is used for extraction and purification, and rotary evaporation is carried out to obtain the boric acid-epoxy group polymer.

Example 8

Synthesis of boronic acid-epoxy based polymers

Adding a trace amount of potassium iodide according to the molar ratio of the N-vinyl pyrrolidone to the 3-chloro-4-pyridineboronic acid pinacol ester of 1:1, dissolving in 6 mL of N, N-dimethylformamide, and reacting for 24 hours in a dark place. The prepared compound, glycidyl methacrylate and N, N-methylene-bis-acrylamide 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 molar ratio of 1:0.5 of boric acid-acrylic acid compound to azodiisobutyronitrile as an initiator, after violent reaction for 12 hours at 65 ℃, normal hexane is used for extraction and purification, and rotary evaporation is carried out to obtain the boric acid-epoxy group polymer.

Example 9

Synthesis of boronic acid-epoxy based polymers

Adding a trace amount of potassium iodide according to the molar ratio of 1:1 between dimethylaminoethyl acrylate and 5-bromo-2-thiopheneboronic acid, dissolving in 6 mL of N, N-dimethylformamide, and reacting for 24 hours in a dark place. The prepared compound, glycidyl methacrylate and N, N-methylene-bis-acrylamide 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 molar ratio of 1:0.5 of boric acid-acrylic acid compound to azodiisobutyronitrile as an initiator, after violent reaction for 12 hours at 65 ℃, normal hexane is used for extraction and purification, and rotary evaporation is carried out to obtain the boric acid-epoxy group polymer.

Example 10

Synthesis of boronic acid-epoxy based polymers

Adding a trace amount of potassium iodide according to the molar ratio of the N, N-diethylaminoethyl acrylate to the 6-fluoropyridine-3-boric acid of 1:1, dissolving in 6 mL of N, N-dimethylformamide, and reacting for 24 hours in a dark place. The prepared compound, acrylamide and N, N-methylene-bisacrylamide were dissolved in 4 mL of N, N-dimethylformamide at a molar ratio of 1:0.5:0.5 under nitrogen protection. According to the molar ratio of 1:0.5 of boric acid-acrylic acid compound to azodiisobutyronitrile as an initiator, after violent reaction for 12 hours at 65 ℃, normal hexane is used for extraction and purification, and rotary evaporation is carried out to obtain the boric acid-epoxy group polymer.

Example 11

Synthesis of boronic acid-epoxy based polymers

Adding a trace amount of potassium iodide according to the mol ratio of the dimethylaminoethyl acrylate to the 4-bromomethylbenzeneboronic acid of 1:1, dissolving in 6 mL of N, N-dimethylformamide, and reacting for 24 hours in a dark place. The prepared compound, methyl methacrylate and N, N-methylenebisacrylamide were dissolved in 4 mL of N, N-dimethylformamide at a molar ratio of 1:0.5:0.5 under nitrogen protection. According to the molar ratio of 1:0.5 of boric acid-acrylic acid compound to azodiisobutyronitrile as an initiator, after violent reaction for 12 hours at 65 ℃, normal hexane is used for extraction and purification, and rotary evaporation is carried out to obtain the boric acid-epoxy group polymer.

Example 12

Synthesis of boronic acid-epoxy based polymers

Adding a trace amount of potassium iodide according to the molar ratio of the N-vinyl pyrrolidone to the 5-bromo-2-thiopheneboronic acid of 1:1, dissolving in 6 mL of N, N-dimethylformamide, and reacting for 24 hours in a dark place. The prepared compound, glycidyl methacrylate and N, N-methylene-bis-acrylamide 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 molar ratio of 1:0.5 of boric acid-acrylic acid polymer to initiator toluene peroxide, after violent reaction for 12 hours at 65 ℃, normal hexane is used for extraction and purification, and rotary evaporation is carried out to obtain the boric acid-epoxy group polymer.

Example 13

Preparation of photosensitizer-polymer nanoparticles encapsulating indocyanine green

The boronic acid-epoxy group polymer (example 1) to indocyanine green mass ratio of 1:0.2 was dissolved in 40 μ L of methanol and redispersed in 1.5mL of pure water. The drug-loaded nanoparticles are prepared by a nano precipitation method, and a probe ultrasonic disperser is used for carrying out ultrasonic dispersion on the solution at the power of 85W for 1 min. And filling the solution into a 3500Da dialysis bag, dialyzing away from light to remove the indocyanine green which is not encapsulated, and freeze-drying to obtain the indocyanine green-encapsulated photosensitizer-polymer nanoparticles. The drug loading of the nanoparticles was 14.6% and the encapsulation efficiency was 90.5% as determined by uv spectrophotometry. The particle size of the nano delivery system obtained in this example was measured by a malvern particle sizer, and a in fig. 1 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 83.5 nm.

Example 14

The boronic acid-epoxy group polymer (example 2) to indocyanine green mass ratio of 1:0.3 was dissolved in 40 μ L of methanol and redispersed in 1.5mL of pure water. The drug-loaded nanoparticles are prepared by a nano precipitation method, and a probe ultrasonic disperser is used for carrying out ultrasonic dispersion on the solution at the power of 85W for 1 min. And filling the solution into a 3500Da dialysis bag, dialyzing away from light to remove the indocyanine green which is not encapsulated, and freeze-drying to obtain the indocyanine green-encapsulated photosensitizer-polymer nanoparticles. The drug loading of the nanoparticles is 25.2% and the encapsulation rate is 91.3% as measured by an ultraviolet spectrophotometry. The particle size of the nano delivery system obtained in this example was measured by a malvern particle sizer, and b in fig. 1 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 86.5 nm.

Example 15

Preparation of photosensitizer-polymer nanoparticles carrying methylene blue

The boric acid-epoxy based polymer (example 3) was dissolved in 40. mu.L of methanol in a mass ratio of 1:0.4 to methylene blue, and redispersed in 1.5mL of pure water. The drug-loaded nanoparticles are prepared by a nano precipitation method, and a probe ultrasonic disperser is used for carrying out ultrasonic dispersion on the solution at the power of 85W for 1 min. And filling the solution into a 3500Da dialysis bag, dialyzing in a dark place to remove the methylene blue which is not coated, and freeze-drying to obtain the photosensitizer-polymer nanoparticles coated with the methylene blue. The drug loading of the nanoparticles was 36.3% and the encapsulation efficiency was 88.3% as measured by uv spectrophotometry. The particle size of the nano delivery system obtained in this example was measured by a malvern particle sizer, g in fig. 1 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 168.4 nm.

Example 16

Preparation of photosensitizer-polymer nanoparticles carrying methylene blue

The boric acid-epoxy based polymer (example 4) was dissolved in 40. mu.L of methanol in a mass ratio of 1:0.5 to methylene blue, and redispersed in 1.5mL of pure water. The drug-loaded nanoparticles are prepared by a nano precipitation method, and a probe ultrasonic disperser is used for carrying out ultrasonic dispersion on the solution at the power of 85W for 1 min. And filling the solution into a 3500Da dialysis bag, dialyzing in a dark place to remove the methylene blue which is not coated, and freeze-drying to obtain the photosensitizer-polymer nanoparticles coated with the methylene blue. The drug loading of the nanoparticles was 42.1% and the encapsulation efficiency was 90.2% as determined by uv spectrophotometry. The particle size of the nano delivery system obtained in this example was measured by a malvern particle sizer, and j in fig. 1 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 290.2 nm.

Example 17

Preparation of photosensitizer-polymer nanoparticles entrapping chlorin e6

According to the mass ratio of the boric acid-epoxy group polymer (example 5) to chlorin e6 of 1:0.3, the mixture is dissolved in 40. mu.L of methanol and then dispersed in 1.5mL of pure water. The drug-loaded nanoparticles are prepared by a nano precipitation method, and a probe ultrasonic disperser is used for carrying out ultrasonic dispersion on the solution at the power of 170W for 1 min. And filling the solution into a 3500Da dialysis bag, dialyzing away from light to remove the chlorin e6 which is not entrapped, and freeze-drying to obtain the photosensitizer-polymer nanoparticles entrapped with the chlorin e 6. The drug loading of the nanoparticles is 24.4% and the encapsulation efficiency is 90.3% as measured by an ultraviolet spectrophotometry. The particle size of the nano delivery system obtained in this example was measured by a malvern particle sizer, h in fig. 1 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 126.5 nm.

Example 18

Preparation of photosensitizer-polymer nanoparticles entrapping chlorin e6

According to the mass ratio of the boric acid-epoxy group polymer (example 6) to the chlorin e6 of 1:0.3, the mixture is dissolved in 80 μ L of methanol and then dispersed in 1.5mL of pure water. The drug-loaded nanoparticles are prepared by a nano precipitation method, and a probe ultrasonic disperser is used for carrying out ultrasonic dispersion on the solution at the power of 85W for 1 min. And filling the solution into a 3500Da dialysis bag, dialyzing away from light to remove the chlorin e6 which is not entrapped, and freeze-drying to obtain the photosensitizer-polymer nanoparticles entrapped with the chlorin e 6. The drug loading of the nanoparticles was 21.2% and the encapsulation efficiency was 87.5% as determined by uv spectrophotometry. The particle size of the nano delivery system obtained in this example was measured by a malvern particle sizer, and i in fig. 1 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 254.5 nm.

Example 19

Preparation of photosensitizer-polymer nanoparticles carrying IR780 iodide

The boric acid-epoxy based polymer (example 8) was dissolved in 40. mu.L of methanol at a mass ratio of 1:0.3 to IR780 iodide and redispersed in 1.5mL of purified water. The drug-loaded nanoparticles are prepared by a nano precipitation method, and a probe ultrasonic disperser is used for carrying out ultrasonic dispersion on the solution at the power of 85W for 1 min. And (3) filling the solution into a 3500Da dialysis bag, carrying out dark dialysis to remove the IR780 iodide which is not carried, and carrying out freeze drying to obtain the photosensitizer-polymer nanoparticles carrying the IR780 iodide. The drug loading of the nanoparticles was 19.2% and the encapsulation efficiency was 86.5% as determined by uv spectrophotometry. The particle size of the nano delivery system obtained in this example is measured by a malvern particle sizer, and d in fig. 1 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system is 124.5 nm.

Example 20

Preparation of photosensitizer-polymer nanoparticles carrying IR780 iodide

The boric acid-epoxy based polymer (example 10) was dissolved in 40. mu.L of methanol at a mass ratio of 1:0.3 to IR780 iodide and redispersed in 1.5mL of pure water. The drug-loaded nanoparticles are prepared by a nano precipitation method, and a probe ultrasonic disperser is used for carrying out ultrasonic dispersion on the solution at the power of 85W for 1 min. And (3) filling the solution into a 3500Da dialysis bag, carrying out dark dialysis to remove the IR780 iodide which is not carried, and carrying out freeze drying to obtain the photosensitizer-polymer nanoparticles carrying the IR780 iodide. The drug loading of the nanoparticles was 20.7% and the encapsulation efficiency was 88.2% as determined by uv spectrophotometry. The particle size of the nano delivery system obtained in this example was measured by a malvern particle sizer, and e in fig. 1 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 114.2 nm.

Example 21

Preparation of photosensitizer-polymer nanoparticles entrapping hematoporphyrin monomethyl ether

According to the mass ratio of the boric acid-epoxy group polymer (example 11) to hematoporphyrin monomethyl ether of 1:0.3, the mixture was dissolved in 40. mu.L of methanol and redispersed in 1.5mL of pure water. The drug-loaded nanoparticles are prepared by a nano precipitation method, and a probe ultrasonic disperser is used for carrying out ultrasonic dispersion on the solution at the power of 85W for 1 min. And filling the solution into a 3500Da dialysis bag, dialyzing away from light to remove the unencapsulated hematoporphyrin monomethyl ether, and freeze-drying to obtain the photosensitizer-polymer nanoparticles encapsulated with hematoporphyrin monomethyl ether. The drug loading of the nanoparticles was 23.1% and the encapsulation efficiency was 89.4% as determined by uv spectrophotometry. The particle size of the nano delivery system obtained in this example was measured by a malvern particle sizer, and f in fig. 1 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 120.5 nm.

Example 22

According to the mass ratio of the boric acid-epoxy group polymer (example 12) to hematoporphyrin monomethyl ether of 1:0.3, the mixture was dissolved in 40. mu.L of methanol and redispersed in 1.5mL of pure water. The drug-loaded nanoparticles are prepared by a nano precipitation method, and a probe ultrasonic disperser is used for carrying out ultrasonic dispersion on the solution at the power of 85W for 1 min. And filling the solution into a 3500Da dialysis bag, dialyzing away from light to remove the unencapsulated hematoporphyrin monomethyl ether, and freeze-drying to obtain the photosensitizer-polymer nanoparticles encapsulated with hematoporphyrin monomethyl ether. The drug loading of the nanoparticles was 21.9% and the encapsulation efficiency was 86.8% as determined by uv spectrophotometry. The particle size of the nano delivery system obtained in this example was measured by a malvern particle sizer, and c in fig. 1 is the measurement result of the malvern particle sizer, and the particle size of the nano delivery system was 113.2 nm.

Example 23

Synthetic hydrogen peroxide degrading enzyme nanocapsules

Adding hydrogen peroxide degrading enzyme and acrylic acid-N-succinimide ester into sodium bicarbonate buffer solution with the pH value of 8.0-9.5 respectively according to the mass ratio of 1: 10. The reaction was carried out at room temperature for 2 h. The reaction solution was filled in a 3500Da dialysis bag, and unreacted N-succinimidyl acrylate was removed to obtain an vinylated hydrogen peroxide-degrading enzyme. According to the mass ratio of the ethylene 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 ethylene hydrogen peroxide degrading enzyme to the sodium bicarbonate buffer solution. The mixture is slowly stirred and reacted for 2 hours at room temperature under the protection of nitrogen. After the reaction, the reaction mixture was put into a 3500Da dialysis bag, and unreacted monomers were removed therefrom, followed by lyophilization to obtain enzyme nanocapsules.

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

Example 24

Synthetic hydrogen peroxide degrading enzyme nanocapsules

Adding hydrogen peroxide degrading enzyme and N- (allyloxycarbonyloxy) succinimide into sodium bicarbonate buffer solution with pH of 8.0-9.5 according to the mass ratio of 1: 10. The reaction was carried out at room temperature for 2 h. The reaction solution was packed in a 3500Da dialysis bag, and unreacted N- (allyloxycarbonyloxy) succinimide was removed to obtain an ethenylated hydrogen peroxide-degrading enzyme. According to the mass ratio of the ethylene hydrogen peroxide degrading enzyme to the 3-methyl-butenamine to the cross-linking agent N, N-methylene bisacrylamide to the initiator ammonium persulfate of 1:10:2:5, respectively adding the ethylene hydrogen peroxide degrading enzyme to the sodium bicarbonate buffer solution. The mixture is slowly stirred and reacted for 2 hours at room temperature under the protection of nitrogen. After the reaction, the reaction mixture was put into a 3500Da dialysis bag, and unreacted monomers were removed therefrom, followed by lyophilization to obtain enzyme nanocapsules.

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

Example 25

Synthetic hydrogen peroxide degrading enzyme nanocapsules

Adding hydrogen peroxide degrading enzyme and acrylic acid-N-succinimide ester into sodium bicarbonate buffer solution with pH of 8.0-9.5 according to the molar ratio of 1: 5. The reaction was carried out at room temperature for 2 h. The reaction solution was filled in a 3500Da dialysis bag, and unreacted N-succinimidyl acrylate was removed to obtain an vinylated hydrogen peroxide-degrading enzyme. According to the mass ratio of the ethylene hydrogen peroxide degrading enzyme to the 3-butenamine to the cross-linking agent N, N-methylene-bisacrylamide to the initiating agent ammonium persulfate of 1:10:2:5, respectively adding the ethylene hydrogen peroxide degrading enzyme to the sodium bicarbonate buffer solution. The mixture is slowly stirred and reacted for 2 hours at room temperature under the protection of nitrogen. After the reaction, the reaction mixture was put into a 3500Da dialysis bag, and unreacted monomers were removed therefrom, followed by lyophilization to obtain enzyme nanocapsules.

The particle size of the enzyme nanocapsule obtained in this example was measured by a malvern particle sizer, and e in fig. 2 is a measurement result of the malvern particle sizer, and the enzyme nanocapsule particle size was 49.2 nm, and the encapsulation efficiency of the hydrogen peroxide-degrading enzyme was determined to be 66.4% by the BCA method.

Example 26

Synthetic hydrogen peroxide degrading enzyme nanocapsules

Adding hydrogen peroxide degrading enzyme and acrylic acid-N-succinimide ester into sodium bicarbonate buffer solution with pH of 8.0-9.5 according to the molar ratio of 1: 3. The reaction was carried out at room temperature for 2 h. The reaction solution was filled in a 3500Da dialysis bag, and unreacted N-succinimidyl acrylate was removed to obtain an vinylated hydrogen peroxide-degrading enzyme. According to the mass ratio of the ethylene hydroperoxide 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 ethylene hydroperoxide degrading enzyme to the sodium bicarbonate buffer solution. The mixture is slowly stirred and reacted for 2 hours at room temperature under the protection of nitrogen. After the reaction, the reaction mixture was put into a 3500Da dialysis bag, and unreacted monomers were removed therefrom, followed by lyophilization to obtain enzyme nanocapsules.

The particle size of the enzyme nanocapsule obtained in this example was measured by a malvern particle sizer, and b in fig. 2 is a measurement result of the malvern particle sizer, and the particle size of the enzyme nanocapsule was 44.2 nm, and the encapsulation efficiency of the hydrogen peroxide-degrading enzyme was 40.1% by the BCA method.

Example 27

Synthetic hydrogen peroxide degrading enzyme nanocapsules

Adding hydrogen peroxide degrading enzyme and acrylic acid-N-succinimide ester into sodium bicarbonate buffer solution with pH of 8.0-9.5 according to a molar ratio of 1: 10. The reaction was carried out at room temperature for 4 h. The reaction solution was filled in a 3500Da dialysis bag, and unreacted N-succinimidyl acrylate was removed to obtain an vinylated hydrogen peroxide-degrading enzyme. According to the mass ratio of the ethylene 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 ethylene hydrogen peroxide degrading enzyme to the sodium bicarbonate buffer solution. Slowly reacting for 2h at room temperature under the protection of nitrogen. After the reaction, the reaction mixture was put into a 3500Da dialysis bag, and unreacted monomers were removed therefrom, followed by lyophilization to obtain enzyme nanocapsules.

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

Example 28

Synthetic hydrogen peroxide degrading enzyme nanocapsules

Adding hydrogen peroxide degrading enzyme and acrylic acid-N-succinimide ester into sodium bicarbonate buffer solution with pH of 8.0-9.5 according to a molar ratio of 1: 10. The reaction was carried out at room temperature for 2 h. The reaction solution was filled in a 3500Da dialysis bag, and unreacted N-succinimidyl acrylate was removed to obtain an vinylated hydrogen peroxide-degrading enzyme. According to the mass ratio of the ethylene hydrogen peroxide degrading enzyme to the 3-methyl-butenamine to the cross-linking agent N, N-methylene bisacrylamide to the initiator ammonium persulfate of 1:15:2:5, respectively adding the ethylene hydrogen peroxide degrading enzyme to the sodium bicarbonate buffer solution. The mixture is slowly stirred and reacted for 2 hours at room temperature under the protection of nitrogen. After the reaction, the reaction mixture was put into a 3500Da dialysis bag, and unreacted monomers were removed therefrom, followed by lyophilization to obtain enzyme nanocapsules.

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

Example 29

Synthetic hydrogen peroxide degrading enzyme nanocapsules

Adding hydrogen peroxide degrading enzyme and acrylic acid-N-succinimide ester into sodium bicarbonate buffer solution with pH of 8.0-9.5 according to a molar ratio of 1: 10. The reaction was carried out at room temperature for 2 h. The reaction solution was filled in a 3500Da dialysis bag, and unreacted N-succinimidyl acrylate was removed to obtain an vinylated hydrogen peroxide-degrading enzyme. According to the mass ratio of the vinylated hydrogen peroxide degrading enzyme to the 3-butenamine, the cross-linking agent N, N-methylene-bisacrylamide and the initiator ammonium persulfate of 1:5:2:5, respectively adding the vinylated hydrogen peroxide degrading enzyme and the 3-butenamine into the sodium bicarbonate buffer solution. The mixture is slowly stirred and reacted for 2 hours at room temperature under the protection of nitrogen. After the reaction, the reaction mixture was put into a 3500Da dialysis bag, and unreacted monomers were removed therefrom, followed by lyophilization to obtain enzyme nanocapsules.

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

Example 30

Synthetic hydrogen peroxide degrading enzyme nanocapsules

Adding hydrogen peroxide degrading enzyme and acrylic acid-N-succinimide ester into sodium bicarbonate buffer solution with pH of 8.0-9.5 according to a molar ratio of 1: 10. The reaction was carried out at room temperature for 2 h. The reaction solution was packed in a 3500Da dialysis bag, and unreacted N-succinimidyl acrylate was removed to obtain an vinylated catalase. According to the mass ratio of the ethylene hydroperoxide 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 ethylene hydroperoxide degrading enzyme to the sodium bicarbonate buffer solution. Slowly reacting for 2h at room temperature under the protection of nitrogen. After the reaction, the reaction mixture was put into a 3500Da dialysis bag, and unreacted monomers were removed therefrom, followed by lyophilization to obtain enzyme nanocapsules.

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

Example 31

Synthetic hydrogen peroxide degrading enzyme nanocapsules

Adding hydrogen peroxide degrading enzyme and acrylic acid-N-succinimide ester into sodium bicarbonate buffer solution with pH of 8.0-9.5 according to a molar ratio of 1: 10. The reaction was carried out at room temperature for 2 h. The reaction solution was filled in a 3500Da dialysis bag, and unreacted N-succinimidyl acrylate was removed to obtain an vinylated hydrogen peroxide-degrading enzyme. According to the mass ratio of the ethylene 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 ethylene hydrogen peroxide degrading enzyme to the sodium bicarbonate buffer solution. Slowly reacting for 2h at room temperature under the protection of nitrogen. After the reaction, the reaction mixture was put into a 3500Da dialysis bag, and unreacted monomers were removed therefrom, followed by lyophilization to obtain enzyme nanocapsules.

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

Example 32

Preparation of BSA nanocapsules without catalytic Capacity

Respectively adding the bovine serum albumin and the acrylic acid-N-succinimide ester into sodium bicarbonate buffer solution with the pH value of 8.0-9.5 according to the mass ratio of 1: 10. The reaction was carried out at room temperature for 2 h. The reaction solution was packed in a 3500Da dialysis bag, and unreacted N-succinimidyl acrylate was removed to obtain vinylated BSA. According to the mass ratio of 1:10:2:5, vinylated BSA, allylamine hydrochloride, a cross-linking agent N, N-methylene-bisacrylamide and an initiator ammonium persulfate are respectively added into a sodium bicarbonate buffer solution. The mixture is slowly stirred and reacted for 2 hours at room temperature under the protection of nitrogen. After the reaction, the mixture was put into a 3500Da dialysis bag, and unreacted monomers were removed therefrom, followed by lyophilization to obtain BSA nanocapsules.

The particle size of the BSA nanocapsules obtained in this example was measured by a malvern particle sizer, h in fig. 2 was the measurement result of the malvern particle sizer, the particle size of the BSA nanocapsules was 35.7 nm, and the encapsulation rate of BSA was determined to be 84.8% by the BCA method.

Example 33

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

Accurately weighing a certain amount of photosensitizer-polymer nanoparticles in example 13 and enzyme nanocapsule solid powder in example 30, adding the photosensitizer-polymer nanoparticles and the enzyme nanocapsule solid powder into sodium bicarbonate buffer solution according to the mass ratio of 1:5, and stirring and reacting for 4 hours at room temperature in a dark place. Adding the polymer nanoparticles and the glucan into the reaction solution according to the mass ratio of 1:1, and reacting for 6 hours at room temperature. And placing the reaction solution in a 5mL centrifuge tube, centrifuging for 5 min at 5000 rpm/min, taking the upper layer liquid, and freeze-drying to obtain the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment.

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

Example 34

Accurately weighing a certain amount of photosensitizer-polymer nanoparticles in example 14 and protein nanocapsule solid powder carrying enzyme in example 23, adding the photosensitizer-polymer nanoparticles and the enzyme nanocapsule solid powder into sodium bicarbonate buffer solution according to the mass ratio of 1:10, and stirring and reacting for 4 hours at room temperature in a dark place. Adding the polymer nanoparticles and hyaluronic acid into the reaction solution according to the mass ratio of 1:0.5, and reacting for 6 hours at room temperature. And placing the reaction solution in a 5mL centrifuge tube, centrifuging for 5 min at 5000 rpm/min, taking the upper layer liquid, and freeze-drying to obtain the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment.

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

Example 35

Accurately weighing a certain amount of photosensitizer-polymer nanoparticles in example 16 and enzyme nanocapsule solid powder in example 25, adding the photosensitizer-polymer nanoparticles and the enzyme nanocapsule solid powder into sodium bicarbonate buffer solution according to the mass ratio of 1:15, and stirring and reacting for 4 hours at room temperature in a dark place. Adding the polymer nanoparticles and heparin into the reaction solution according to the mass ratio of 1:1, and reacting for 6 h at room temperature. And placing the reaction solution in a 5mL centrifuge tube, centrifuging for 5 min at 5000 rpm/min, taking the upper layer liquid, and freeze-drying to obtain the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment.

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 nm.

Example 36

Accurately weighing a certain amount of photosensitizer-polymer nanoparticles in example 17 and enzyme nanocapsule solid powder in example 26, adding the photosensitizer-polymer nanoparticles and the enzyme nanocapsule solid powder into sodium bicarbonate buffer solution according to the mass ratio of 1:10, and stirring and reacting for 4 hours at room temperature in a dark place. Adding the polymer nanoparticles and the polylysine into the reaction solution according to the mass ratio of 1:1, and reacting for 6 h at room temperature. And placing the reaction solution in a 5mL centrifuge tube, centrifuging for 5 min at 5000 rpm/min, taking the upper layer liquid, and freeze-drying to obtain the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment.

The particle size of the nano delivery system of the present embodiment 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 is 181.4 nm.

Example 37

Accurately weighing a certain amount of photosensitizer-polymer nanoparticles in example 18 and catalase nanocapsule solid powder in example 28, adding the photosensitizer-polymer nanoparticles and the catalase nanocapsule solid powder into sodium bicarbonate buffer solution according to the mass ratio of 1:10, and stirring and reacting for 4 hours at room temperature in a dark place. Adding the polymer nanoparticles and the Apoa-1 into the reaction solution according to the mass ratio of 1:1, and reacting at room temperature for 6 h. And placing the reaction solution in a 5mL centrifuge tube, centrifuging for 5 min at 5000 rpm/min, taking the upper layer liquid, and freeze-drying to obtain the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment.

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 of the malvern particle sizer, which is 133.6 nm.

Example 38

Accurately weighing a certain amount of photosensitizer-polymer nanoparticles in example 20 and catalase nanocapsule solid powder in example 29, adding the photosensitizer-polymer nanoparticles and the catalase nanocapsule solid powder into sodium bicarbonate buffer solution according to the mass ratio of 1:10, and stirring and reacting for 4 hours at room temperature in a dark condition. Adding the polymer nanoparticles and the polylysine into the reaction solution according to the mass ratio of 1:1, and reacting for 6 h at room temperature. And placing the reaction solution in a 5mL centrifuge tube, centrifuging for 5 min at 5000 rpm/min, taking the upper layer liquid, and freeze-drying to obtain the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment.

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 of the malvern particle sizer, which is 153.9 nm.

Example 39

Accurately weighing a certain amount of photosensitizer-polymer nanoparticles in example 22 and enzyme nanocapsule solid powder in example 31, adding the photosensitizer-polymer nanoparticles and the enzyme nanocapsule solid powder into sodium bicarbonate buffer solution according to the mass ratio of 1:10, and stirring and reacting for 4 hours at room temperature in a dark place. Adding the polymer nanoparticles and hyaluronic acid into the reaction solution according to the mass ratio of 1:1, and reacting for 6 hours at room temperature. And placing the reaction solution in a 5mL centrifuge tube, centrifuging for 5 min at 5000 rpm/min, taking 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 of the malvern particle sizer, which is 173.1 nm.

Example 40

Synthetic BSA/photosensitizer combination delivery system

Accurately weighing a certain amount of the photosensitizer-polymer nanoparticles in the example 14 and BSA nanocapsule solid powder loaded with BSA in the example 32, adding the photosensitizer-polymer nanoparticles and the BSA nanocapsule solid powder into sodium bicarbonate buffer solution according to the mass ratio of 1:10, and stirring and reacting for 4 hours at room temperature in a dark place. Adding the polymer nanoparticles and the polylysine into the reaction solution according to the mass ratio of 1:1, and reacting for 6 h at room temperature. And placing the reaction solution in a 5mL centrifuge tube, centrifuging for 5 min at 5000 rpm/min, taking 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 of the malvern particle sizer, which is 128.6 nm.

EXAMPLE 41

Experiment of particle size of photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy along with change of reaction time

Accurately weighing a certain amount of photosensitizer-polymer nanoparticles in example 14 and enzyme nanocapsule solid powder in example 23, adding the photosensitizer-polymer nanoparticles and the enzyme nanocapsule solid powder into a sodium bicarbonate buffer solution according to the mass ratio of 1:10, reacting at room temperature, and taking out reaction solutions at 0 h, 0.5 h, 1 h, 2h and 4 h respectively to measure the particle size.

The particle sizes of the nano delivery system at different time points in the embodiment are measured by a malvern particle size analyzer, fig. 4 is a measurement result of the malvern particle size analyzer, the particle size of the combined delivery system tends to be stable along with the extension of the reaction time, when the reaction time is less than 2h, 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 combined completely and the particle size of the nano delivery system is 131.2 nm.

Example 42

Experiment for investigating ninhydrin combination rate of photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy

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

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

Example 43

Oxygen production amount investigation experiment of photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization treatment

Precisely weighing a certain amount of the photosensitizer/enzyme combination delivery system lyophilized powder for anti-tumor photodynamic sensitization therapy prepared in example 35, and preparing the lyophilized powder into a solution (methylene blue: 150. mu.g/mL) by adopting a sodium bicarbonate buffer solution; 500 μ M hydrogen peroxide was prepared as a reaction substrate, and 3 parts of each of the free catalase group, BSA control group (example 40) and preparation group were added to the hydrogen peroxide solution, stirred at room temperature and subjected to dissolved oxygen measurement using an oxygen dissolution apparatus. The results of the formulation oxygen content experiments are shown in figure 6.

The results show that the combined delivery system can rapidly catalyze hydrogen peroxide to generate oxygen, and compared with free catalase, the catalase nanocapsule does not influence the catalytic effect of catalase, so that the combined delivery system can effectively realize an immobilized enzyme technology, and the catalytic capability of the nano delivery system is endowed.

Example 44

Photosensitizer release experiments for photosensitizer/enzyme combination delivery systems for anti-tumor photodynamic sensitization therapy

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

The results show that the nano delivery system can effectively protect the photosensitizer from being released, and the cumulative release percentage of the indocyanine green is only 10.1 +/-1.4% along with the prolonging of the release time, which indicates that the combined delivery system can effectively prevent the photosensitizer from aggregating or degrading 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 therapy

Precisely weighing a certain amount of the photosensitizer/enzyme combination delivery system lyophilized powder for anti-tumor photodynamic sensitization therapy prepared in example 34, and preparing a solution by using 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 samples into a hydrogen peroxide solution, respectively incubating for 0 h, 0.5 h, 1 h, 2h, 4 h, 6 h, 8 h and 12 h at 37 ℃ and stirring at room temperature, and measuring by using a catalase activity detection kit, wherein the experimental result is shown in figure 8.

The results show that the immobilized enzyme technology of the combined delivery system can effectively protect catalase, and the activity of free catalase is reduced along with the prolonging of the reaction time, and the catalase does not have activity after 4 hours; the preparation group can effectively improve the stability of catalase, and the enzyme activity can still be 50% up to 12 h. The experimental result shows that the protein nano capsule can play a role in permeating solution and improving the stability of catalase.

Example 46

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

The killing effect of the catalase/photosensitizer combined delivery system with the function of anti-tumor optical synergism on tumor cells is examined through an in vitro cytotoxicity experiment, and 4T1 breast cancer cells are selected as research objects.

The method comprises the following specific steps: culturing 4T1 breast cancer cells in a DMEM high-glucose medium containing 10% fetal calf serum, after the cells grow to a logarithmic phase, digesting by pancreatin, centrifugally collecting the cells, inoculating the cells into a 96-well plate, culturing the complete culture solution at 37 ℃ for 24 h, removing the culture medium, respectively adding 100 mu L of free photosensitizer, the photosensitizer-polymer nanoparticles prepared in the example 14 and the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization treatment prepared in the example 36, wherein the concentration range of the photosensitizer is 0.1-10 mu g/mL, and after incubating at 37 ℃ for 6 h, performing near-infrared wavelength light irradiation treatment (808 nm, 1.8W/cm) on each well by using a laser (1.8W/cm )²5 min), then the medium containing the preparation was discarded and washed twice with PBS buffer. mu.L of 5 mg/mL MTT phosphate solution was added, the mixture was incubated at 37 ℃ for 4 hours, the supernatant was discarded, 200. mu.L DMSO was added, and the cell viability was calculated by measuring the absorbance at a wavelength of 570 nm using a microplate reader (results are shown in FIG. 9).

IC of free photosensitizer group₅₀Photosensitizer-polymeric nanoparticle IC with value of 4.106 μ g/mL₅₀IC of catalase/photosensitizer combination delivery system with 4.04 μ g/mL, antitumor optical synergy₅₀The value dropped to 2.566. mu.g/mL. According to results, the preparation has concentration dependence, and compared with a control group, the catalase/photosensitizer combined delivery system with the antitumor optical synergistic effect has better tumor cell killing effect, which is probably due to the active targeting effect brought by the bionic hyaluronic acid, so that the accumulation of the photosensitizer in the tumor cells is improved, and the killing effect on the tumor cells is promoted.

Example 47

Singlet oxygen Generation capability examination of photosensitizer/enzyme combination delivery System for anti-tumor photodynamic sensitization therapy

The therapeutic effect of the catalase/photosensitizer combined delivery system with the antitumor optical synergistic effect in tumor cells is examined through an in vitro singlet oxygen generation capacity experiment, and 4T1 breast cancer cells are selected as a research object. The method comprises the following specific steps: when the 4T1 cells are cultured to logarithmic phase, pancreatin is used for digestion, the cells are collected by centrifugation and inoculated in a 24-well plate, and the cell density is highAbout 1 × 10⁵One/well, 5% CO at 37 ℃₂After overnight incubation in an incubator, after aspirating the medium and adding PBS to wash three times, 100. mu.L of free photosensitizer, the photosensitizer-polymer nanoparticles prepared in example 14, and the photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy prepared in example 37 (photosensitizer concentration: 2. mu.g/mL) were added, respectively, at 37 ℃ in 5% CO₂The incubation was continued in the incubator for 6 h. After 6 h, the drug-containing medium was discarded, 0.2 mL of serum-free medium containing singlet oxygen probe R was added, and laser irradiation was performed for 5 min (1.5W/cm) using 808²) 5% CO at 37 ℃₂The incubation was continued in the incubator for 0.5 h. After incubation, the culture medium is discarded, washed with precooled PBS for three times, and placed in an inverted fluorescence microscope for observing the green fluorescence intensity

The singlet oxygen generation capacity results are shown in fig. 10, and the results show that the blank control group, the NIR group and the free photosensitizer group have weak intracellular fluorescence, while the photosensitizer-polymer nanoparticle group and the anti-tumor optically synergistic catalase/photosensitizer combined delivery system have strong fluorescence, which indicates that the photosensitizer is protected from exerting photodynamic action in tumor cells after being wrapped by the polymer. After catalase and bionic material hyaluronic acid are introduced, the tumor targeting capability and the photodynamic therapy level can be better improved.

Example 48

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

Examination of oxygen production capacity of the anti-tumor optically synergistic catalase/photosensitizer combination delivery system in tumor cells by intracellular oxygen production capacity, 4T1 breast cancer cells were selected as the study object. The method comprises the following specific steps: after 4T1 cells were cultured to logarithmic phase, they were seeded into cell culture dishes containing 5% CO at 37 deg.C₂After overnight incubation in an incubator, the medium was aspirated and washed twice with PBS, 100. mu.L of free photosensitizer, the photosensitizer-polymer nanoparticles prepared in example 17, and the photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy (photosensitizer concentration: 2. mu.g/mL) prepared in example 34 were added, respectively, to the incubatorPerforming anoxic sealing treatment in culture dish at 37 deg.C with 5% CO₂And culturing in an incubator for 8 h. After incubation, the medium was discarded, washed twice with PBS, 100 μ L RDPP solution was added to each dish, the anaerobic sealing treatment was continued, and incubation was continued for 0.5 h. After incubation, the RDPP solution was discarded, washed three times with PBS, and placed under a microscope to induce weak red fluorescence during observation.

The results of the examination of intracellular oxygen production capacity are shown in fig. 11, RDPP is an oxygen sensitive dye, and generates strong red fluorescence when the oxygen content is low, and conversely, quenching occurs when the oxygen content is high, and the fluorescence signal is low. According to the results, the control group treated by the normal oxygen has weak fluorescence; in the cells subjected to the anoxic treatment, the group without catalase has strong red fluorescence, and the catalase protein nanocapsule group and the catalase/photosensitizer combined delivery system with the antitumor optical synergistic effect have weak fluorescence, so that the protein nanocapsule can effectively protect catalase from being degraded by protease and can play a role in catalytic degradation in a tumor microenvironment. Meanwhile, after the photosensitizer-carrying polymer nanoparticles and hyaluronic acid are introduced, the catalytic capability of catalase is not influenced. Is beneficial to enhancing the photosensitizer and playing the role of photodynamic anti-tumor therapy.

Example 49

Cell uptake assay for photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy

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

The method comprises the following specific steps: culturing 4T1 breast cancer cells in DMEM high-sugar medium containing 10% fetal calf serum, collecting cells by trypsinization and centrifugation after the cells grow to logarithmic phase, inoculating in a confocal dish with cell density of 1 × 10⁵One/well, 5% CO at 37 ℃₂After overnight incubation in an incubator, the medium was discarded and washed twice with PBS, and 500. mu.L of the photosensitizer for anti-tumor photodynamic sensitization therapy prepared in example 33 after serum-free dilution was added at 1 h, 2h, 4 h and 8 hThe cells were fixed with 4% paraformaldehyde solution, washed twice with PBS, stained with 200. mu.L of DAPI working solution for 8min, and observed with a confocal laser microscope.

The results of the qualitative cell uptake experiments are shown in FIG. 12, and show that the intracellular fluorescence intensity of the photosensitizer gradually increases with the increase of the incubation time. And the photosensitizer can be observed to be distributed in the whole cell, which shows 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 action of photodynamic therapy, so that the high-efficiency anti-tumor effect is achieved.

Example 50

Apoptosis test of photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy

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

The method comprises the following specific steps: culturing 4T1 breast cancer cells in DMEM high-sugar medium containing 10% fetal calf serum, collecting cells by trypsinization and centrifugation after the cells grow to logarithmic phase, inoculating in 6-well plate with cell density of 1 × 10⁵One/well, 5% CO at 37 ℃₂After overnight incubation in an incubator, the medium was discarded and washed twice with PBS, 500. mu.L of DMEM, free photosensitizer, BSA control (example 40) and the catalase/photosensitizer combination delivery system with antitumor optical potentiation prepared in example 39 (photosensitizer concentration: 2. mu.g/mL) were added, and after further incubation in an incubator for 8 hours, the cells were irradiated with 808 laser light for 5 min (1.5W/cm)²) After irradiation, 200. mu.L of trypsin was added to each well for digestion for 5 min and terminated with serum-containing medium, centrifuged at 3000 rpm/min for 3min, and the supernatant was discarded. The lower layer cell sediment is re-suspended by 1 × Binding buffer, 5 μ L Annexin-FITC and PI staining solution are added, and detection is carried out by adopting a flow cytometer after uniform mixing.

The apoptosis results are shown in fig. 13, and 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 of the photosensitizer/enzyme combination delivery system for the anti-tumor photodynamic sensitization therapy is 88.5%. The introduction of the enzyme nanocapsule can effectively improve the PDT effect of the photosensitizer, increase the apoptosis rate by 3 times, and effectively enhance the killing effect of the photosensitizer on tumor cells, thereby achieving the effect of efficiently resisting tumors.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A photosensitizer/enzyme combination delivery system for anti-tumor photodynamic sensitization therapy, characterized in that: the nano-composite is formed by connecting the polymer nanoparticles and the enzyme nanocapsules through chemical bonds, and targeting biological macromolecules are modified on the surface of the nano-composite;

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

2. The combination delivery system of claim 1, wherein:

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

step 1, dispersing a boric acid compound and an acrylic acid derivative monomer in an organic solvent, reacting at room temperature in a dark place, and purifying and freeze-drying to obtain an acrylic acid compound for modifying a boric acid group;

3. The combination delivery system of claim 2, wherein:

the boric acid compound is selected from halogen-containing phenyl boric acid monomer, halogen-containing pyridine boric acid monomer or halogen-containing thiophene boric acid monomer; the acrylic acid derivative monomer is an amine-containing acrylic acid derivative monomer 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.

4. The combination delivery system of claim 1, wherein: the photosensitizer is selected from one or more of indocyanine green, chlorin e6, methylene blue, IR780 or hematoporphyrin monomethyl ether.

5. The combination delivery system of claim 1, wherein:

the ethylene-based hydrogen peroxide degrading enzyme is prepared by the following method:

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

6. The combination delivery system of claim 1, wherein: the amine, alcohol or thiol monomer is one or more selected from allylamine hydrochloride, 3-methyl-butenamine, 3-butenamine, allylthiol and its derivatives.

7. A method of preparing the combination delivery system of claim 1, characterized in that: the method comprises the following steps:

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

step 2, dissolving the vinylated hydrogen peroxide degrading enzyme, amine, alcohol or thiol 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 enzyme nanocapsules;

8. The method of claim 7, wherein: in the step 1, the mass ratio of the polymer to the photosensitizer is 1: 0.5-1: 5.

9. The method of claim 7, wherein: in the step 2, the mass ratio of the vinylated hydrogen peroxide degrading enzyme to the amine, alcohol or thiol monomer, 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.

10. Use of the combination delivery system of claim 1 in a medicament for the diagnosis or treatment of tumors.