CN104450814A - Horseradish-peroxidase-mediated free radical initiation system and method for preparing hydrogel - Google Patents

Abstract

The invention relates to a free radical initiation system mediated by horseradish peroxidase and a method for preparing hydrogel. The initiation system is composed of N-hydroxysuccinimide, horseradish peroxidase and hydrogen peroxide. The use of N-hydroxysuccinimide as the substrate of the enzyme avoids the use of acetylacetone with high biological toxicity. The preparation method of the present invention can control the initiation of monomer polymerization to prepare hydrogel within 50s to 5 minutes at room temperature by adjusting the component concentration ratio of the ternary initiation system, and can be used to prepare high-strength nanocomposite hydrogel, which is environmentally friendly. , The advantages of rapid and controllable gelation have obvious application prospects in the fields of controlled drug release, enzyme immobilization, and tissue engineering.

Description

Horseradish peroxidase-mediated free radical initiation system and method for preparing hydrogel

technical field

The invention relates to the technical field of macromolecules, in particular to a horseradish peroxidase-mediated free radical initiation system and a method for preparing hydrogel using the system.

Background technique

Free radical polymerization is often used in the production of high molecular polymers and the manufacture of fine chemicals, and is an important technology for industrial production of high molecular products. The first step in a free radical reaction is free radical initiation. The traditional free radical initiation methods mainly include: thermal initiation; redox initiation; ultraviolet light initiation; high-energy radiation (such as laser, α-ray, β-ray, γ-ray, X-ray irradiation, etc.) initiation and so on. These polymerization initiation methods either require the use of special equipment and consume a large amount of energy, or require the use of some toxic initiators, which are harmful to organisms and may affect the natural environment, and the reaction conditions are relatively harsh.

Compared with the traditional free radical initiation method, the generation of free radicals through biological enzyme-catalyzed reactions is an environmentally friendly, mild reaction condition, high efficiency, low energy consumption, and easy to operate free radical initiation method. A superior approach to molecular materials.

Oxidoreductases can catalyze electron transfer reactions to generate free radicals, which initiate the polymerization of vinyl monomers in aqueous solution. It has been reported in the literature that horseradish peroxidase (EC1.11.1.7)/β-diketone/hydrogen peroxide ternary enzyme-mediated free radical initiation system can initiate the free radical initiation of hydrophilic vinyl monomers (such as acrylamide). base polymerization, or initiate miniemulsion polymerization of hydrophobic monomers (such as styrene) (Polym. , 3793-3818). However, there will be an irreproducible and long induction period (45-360 min) in this reaction, which is not conducive to practical application. Our research group researched and developed a series of horseradish peroxidase/acetylacetone/hydrogen peroxide ternary initiation system with optimized component ratio and applied for a Chinese patent. This type of initiator system can rapidly (within 6 min) initiate free radical polymerization at room temperature, and prepare nanocomposite hydrogels with high strength and high catalytic performance (Chem. Commun., 2013, 101, 3793-3818). In this system, acetylacetone was used as the substrate of horseradish peroxidase to generate free radicals to initiate polymerization. However, the high biological toxicity of acetylacetone is not conducive to its application in biomaterials. So far, no acetylacetone-free horseradish peroxidase-mediated radical polymerization initiation system has not been reported.

N-hydroxysuccinimide is an amide compound widely used in biology and synthetic chemistry. Its structure contains active nitrogen hydroxyl groups, which can be covalently combined with antigens, antibodies, nucleic acids, etc., and is often used in pharmaceutical intermediates Body, precursor of synthetic polypeptides, antibiotics, etc., as well as diagnostic imaging and treatment of tumors, with good biocompatibility. Our research group recently found that N-hydroxysuccinimide, horseradish peroxidase, and hydrogen peroxide are prepared in a certain concentration ratio, which can generate free radicals to initiate monomer polymerization. The free radical initiating system can be used to rapidly and controllably prepare hydrogels, which is conducive to the application of the prepared hydrogels in vivo, such as embolization agents in arterial embolization, or as injectable tissue engineering scaffold materials, etc. .

Contents of the invention

The object of the present invention is to provide a free radical polymerization initiation system mediated by horseradish peroxidase based on N-hydroxysuccinimide.

Another object of the present invention is to use the above-mentioned initiation system to initiate the polymerization of vinyl monomers in aqueous solution to prepare hydrogels. The method has mild reaction conditions, easy operation, fast gelation and controllable time, and can be used to prepare high Strong nanocomposite hydrogels.

The purpose of the present invention can be achieved through the following technical solutions:

Horseradish peroxidase mediated free radical initiation system, which is an initiation system that uses water as the reaction medium to initiate free radical polymerization of vinyl monomers at room temperature, including N-hydroxysuccinimide, horseradish peroxidation enzymes and hydrogen peroxide.

The molar concentration ratio of hydrogen peroxide and horseradish peroxidase (300units/mg) is 25-960, and the molar concentration ratio of N-hydroxysuccinimide and hydrogen peroxide is 2-29. The molar concentration of N-hydroxysuccinimide is not lower than 2.65mM, and the molar concentration of hydrogen peroxide is not lower than 0.66mM.

The preparation process of the triggering system of the present invention is simple, and can be prepared in various ways. N-hydroxysuccinimide, horseradish peroxidase, and hydrogen peroxide can be mixed and used directly; or N-hydroxyl After mixing succinimide and horseradish peroxidase for a period of time, use it with hydrogen peroxide; or mix and react N-hydroxysuccinimide and hydrogen peroxide for a period of time, and then react with horseradish Oxidases are used in conjunction.

Different from the initiation system described in the prior art in which the carbon-centered free radicals derived from acetylacetone initiate polymerization, the initiation system of the present invention has a novel free radical initiation mechanism. It was proved by electron spin resonance (ESR) that the nitroxide free radical derived from N-hydroxysuccinimide was generated in the initiating system of the present invention. In the case of no spin trapping agent, the ternary initiation system of the present invention (wherein the molar concentration ratio of N-hydroxysuccinimide and hydrogen peroxide is 29, the molar concentration of hydrogen peroxide and horseradish peroxidase The ratio is 278) and the ESR scanning signal generated is the 9-fold peak of nitroxide free radicals, indicating that nitroxide free radicals derived from N-hydroxysuccinimide are generated in the system to initiate polymerization.

The method for preparing hydrogel by using horseradish peroxidase-mediated free radical initiation system comprises uniformly mixing N-hydroxysuccinimide, horseradish peroxidase and an aqueous solution of ethylenic monomers, and then adding the hydrogel to it. Hydrogen oxidation, adjust the component concentration ratio of horseradish peroxidase/N-hydroxysuccinimide/hydrogen peroxide ternary initiation system, control the generation of free radicals within 50s~5min at room temperature to initiate monomer polymerization to form a three-dimensional polymer network, resulting in a hydrogel.

The component concentration of the horseradish peroxidase/N-hydroxysuccinimide/hydrogen peroxide ternary initiation system is adjusted to: the molar concentration ratio of hydrogen peroxide and horseradish peroxidase is 25-960, and simultaneously The molar concentration ratio of N-hydroxysuccinimide and hydrogen peroxide is 2 to 29, the molar concentration of N-hydroxysuccinimide used in the initiation system is not lower than 2.65mM, and the molar concentration of hydrogen peroxide is not low at 0.66mM.

The ethylenic monomer is selected from at least one of water-soluble acrylate derivatives, acrylamide derivatives or N-vinylpyrrolidone; the amount of the ethylenic monomer accounts for 5-15% of the total weight of the reaction raw materials , preferably 8 to 10%.

The water-soluble acrylate derivative is hydroxyethyl methacrylate (HEMA), hydroxypropyl acrylate (HPA) or polyethylene glycol methyl methacrylate (PEGMA), and the acrylamide derivative is Acrylamide (AM), N,N-dimethylacrylamide (DMAA) or N-isopropylacrylamide (NIPA).

Water-soluble compounds with two or more double bonds can also be added as cross-linking agents in the reactant, including N, N-methylenebisacrylamide (BIS), polyethylene glycol diacrylate (PEGDA) or For the vinyl-modified protein, the amount of the cross-linking agent accounts for 0.3-5% of the total weight of the reaction raw materials.

Water-dispersible inorganic nanomaterials can also be added to the reactants to prepare high-strength nanocomposite hydrogels. The inorganic nanomaterials are nano-silicon dioxide or nano-hydroxyapatite, preferably nanometers with an average particle size of 10 to 40 nm. silica. The amount of the inorganic nanometer material accounts for 5-16% of the total weight of the reaction raw materials.

Ordinary polymer hydrogels generally can only resist a compressive strength of about 20-100kPa and are easily crushed. The nanocomposite hydrogel prepared by the invention can resist the compressive strength of about 1300kPa and can return to the original shape, has excellent mechanical properties and biocompatibility, and can be applied to the fields of drug controlled release, enzyme immobilization, tissue engineering and the like.

Compared with the prior art, the outstanding advantages of the present invention are: the use of N-hydroxysuccinimide with good biocompatibility as the substrate of the enzyme provides a novel horseradish peroxidase-mediated free Based on the initiation mechanism, the use of acetylacetone with high biological toxicity is avoided, which is beneficial to its application in biomaterials. Another outstanding advantage of the present invention is: by adjusting the component concentration ratio of horseradish peroxidase/N-hydroxysuccinimide/hydrogen peroxide ternary initiation system, lower initiator concentration can be used at room temperature (For example, N-hydroxysuccinimide molar concentration is 2.65 mM, while hydrogen peroxide molar concentration is 0.66 mM) Rapid and controllable preparation of hydrogels. Therefore, the method has obvious application prospects in the preparation of injectable hydrogels for application in the field of biology.

Description of drawings

Fig. 1 is the free radical signal generated by the free radical inducing system of the present invention detected by electron spin resonance.

Detailed ways

The technical scheme of the present invention is further described below in conjunction with specific examples, and the present invention is further described below in conjunction with examples, but these examples are not used to limit the present invention.

Example 1

1) Preparation of precursor solution: Take 0.11-0.13g of N,N-dimethylacrylamide, 0.07-0.09g of cross-linking agent polyethylene glycol diacrylate (average molecular weight 250), and 1.6-1.7g of deionized water into the sample bottle, mix well with a vortex mixer.

2) Preparation of hydrogel: 0.66 mg of N-hydroxysuccinimide, 100 μL of horseradish peroxidase concentrate, and 50 μL of aqueous hydrogen peroxide (N-hydroxysuccinimide in the reaction system) were sequentially added to the above precursor solution. The molar concentration ratio of amine and hydrogen peroxide is 4.35, the molar concentration ratio of hydrogen peroxide and horseradish peroxidase is 25, and the final molar concentration of hydrogen peroxide is 0.66mM), fast mixing is timed with a stopwatch simultaneously, airtight static Place to obtain a light brown-red transparent hydrogel, and the gelation time is 3min10s.

Example 2

1) Preparing the precursor solution: the steps are the same as in Example 1.

2) Preparation of hydrogel: Add 0.61 mg of N-hydroxysuccinimide, 100 μL of horseradish peroxidase concentrate, and 50 μL of aqueous hydrogen peroxide (N-hydroxysuccinimide in the reaction system The molar concentration ratio of amine and hydrogen peroxide is 2, the molar concentration ratio of hydrogen peroxide and horseradish peroxidase is 25, and the final molar concentration of hydrogen peroxide is 1.32mM), mixes quickly, and airtightly stands still to obtain light brown Red transparent hydrogel, gelation time 2min20s.

Example 3

1) Preparing the precursor solution: the steps are the same as in Example 1.

2) Preparation of hydrogel: Add 1.77 mg of N-hydroxysuccinimide, 100 μL of horseradish peroxidase concentrate, and 50 μL of aqueous hydrogen peroxide (N-hydroxysuccinimide in the reaction system The molar concentration ratio of amine and hydrogen peroxide is 5.84, and the molar concentration ratio of hydrogen peroxide and horseradish peroxidase is 25, and the final molar concentration of hydrogen peroxide is 1.32mM), mix quickly, airtight and stand still to obtain light brown Red transparent hydrogel, gelation time 1min40s.

Example 4

1) Preparing the precursor solution: the steps are the same as in Example 1.

2) Preparation of hydrogel: 8.02 mg of N-hydroxysuccinimide, 100 μL of horseradish peroxidase concentrate, and 50 μL of aqueous hydrogen peroxide solution (N-hydroxysuccinimide in the reaction system) were sequentially added to the above precursor solution. The molar concentration ratio of amine and hydrogen peroxide is 13.23, the molar concentration ratio of hydrogen peroxide and horseradish peroxidase is 50, and the final molar concentration of hydrogen peroxide is 2.63mM), mixes quickly, and airtightly stands still to obtain light brown Red transparent hydrogel, gelation time 50s.

Example 5

1) Preparation of precursor solution: Take 0.09-0.11g of N,N-dimethylacrylamide, 0.05-0.07g of polyethylene glycol diacrylate (average molecular weight 250) as a cross-linking agent, and 1.62-1.72g of deionized water into the sample bottle, mix well with a vortex mixer.

2) Preparation of hydrogel: Add 8.5 mg of N-hydroxysuccinimide, 100 μL of horseradish peroxidase concentrate, and 50 μL of aqueous hydrogen peroxide (N-hydroxysuccinimide in the reaction system The molar concentration ratio of amine and hydrogen peroxide is 5.84, the molar concentration ratio of hydrogen peroxide and horseradish peroxidase is 480, and the final molar concentration of hydrogen peroxide is 6.32mM), mixes quickly, and airtightly stands still to obtain light yellow Transparent hydrogel, gelation time 2min30s.

Example 6

1) Preparing the precursor solution: the steps are the same as in Example 5.

2) Preparation of hydrogel: Add 8.5 mg of N-hydroxysuccinimide, 100 μL of horseradish peroxidase concentrate, and 50 μL of aqueous hydrogen peroxide (N-hydroxysuccinimide in the reaction system The molar concentration ratio of amine and hydrogen peroxide is 5.84, the molar concentration ratio of hydrogen peroxide and horseradish peroxidase is 960, and the final molar concentration of hydrogen peroxide is 6.32mM), mix quickly, airtight and stand still to obtain light yellow Transparent hydrogel, the gelation time is 3min40s.

Example 7

1) Preparing the precursor solution: the steps are the same as in Example 5.

2) Preparation of hydrogel: 5.34 mg of N-hydroxysuccinimide, 100 μL of horseradish peroxidase concentrate, and 50 μL of aqueous hydrogen peroxide solution (N-hydroxysuccinimide in the reaction system) were sequentially added to the above precursor solution. The molar concentration ratio of amine and hydrogen peroxide is 3.67, the molar concentration ratio of hydrogen peroxide and horseradish peroxidase is 480, and the final molar concentration of hydrogen peroxide is 6.32mM), mix quickly, airtight and stand still to obtain light yellow Transparent hydrogel, gelation time 5min.

Example 8

1) Preparing the precursor solution: the steps are the same as in Example 5.

2) Preparation of hydrogel: 7.04 mg of N-hydroxysuccinimide, 100 μL of horseradish peroxidase concentrate, and 50 μL of aqueous hydrogen peroxide (N-hydroxysuccinimide in the reaction system) were sequentially added to the above precursor solution. The molar concentration ratio of amine and hydrogen peroxide is 4.84, and the molar concentration ratio of hydrogen peroxide and horseradish peroxidase is 480, and the final molar concentration of hydrogen peroxide is 6.32mM), mix quickly, airtight and stand still to obtain light yellow Transparent hydrogel, gelation time 3min.

Example 9

1) Preparing the precursor solution: the steps are the same as in Example 5.

2) Preparation of hydrogel: Add 42.17 mg of N-hydroxysuccinimide, 100 μL of horseradish peroxidase concentrate, and 50 μL of aqueous hydrogen peroxide solution (N-hydroxysuccinimide in the reaction system The molar concentration ratio of amine and hydrogen peroxide is 29, the molar concentration ratio of hydrogen peroxide and horseradish peroxidase is 480, and the final molar concentration of hydrogen peroxide is 6.32mM), mix quickly, airtight and stand still to obtain light yellow Transparent hydrogel, the gelation time is 2min25s.

Example 10

1) Preparation of precursor solution: Take 0.30g of N,N-dimethylacrylamide, 1.533g of nano-silica aqueous dispersion (21wt%), and 6.77mg of polyethylene glycol diacrylate (average molecular weight 250) into the sample bottle , mix well with a vortex mixer.

2) Preparation and performance test of hydrogel: Add 10.33 mg of N-hydroxysuccinimide, 100 μL of horseradish peroxidase concentrate, and 50 μL of aqueous hydrogen peroxide (N-hydroxyl The molar concentration ratio of succinimide and hydrogen peroxide is 7.1, and the molar concentration ratio of hydrogen peroxide and horseradish peroxidase is 480), mixes quickly, airtight leaves standstill to obtain light yellow transparent hydrogel (containing 16wt % nano-silica). After the above-mentioned hydrogel is made into a cylindrical sample (15.7mm in diameter and 9.0mm in height), the compressive strength measured by an electronic universal testing machine is 1350kPa (99% compressive strain), and it can be restored to its original shape after the test.

As shown in Figure 1, in the case of no spin trapping agent, the ternary initiation system of the present invention (wherein the molar concentration ratio of N-hydroxysuccinimide and hydrogen peroxide is 29, hydrogen peroxide and horseradish over The molar concentration ratio of oxidase is 278) and the ESR scanning signal produced is the 9-fold peak of nitroxide free radicals, indicating that nitroxide free radicals derived from N-hydroxysuccinimide are produced in the system to initiate polymerization.

Example 11

A method for preparing a hydrogel using a horseradish peroxidase-mediated free radical initiation system, the method uniformly mixes N-hydroxysuccinimide, horseradish peroxidase and an aqueous solution of an ethylenic monomer, and then injects it into the hydrogel Add hydrogen peroxide to adjust the component concentration ratio of horseradish peroxidase/N-hydroxysuccinimide/hydrogen peroxide ternary initiation system, wherein the molar concentration ratio of hydrogen peroxide and horseradish peroxidase 25, the molar concentration ratio of N-hydroxysuccinimide and hydrogen peroxide is 4, the molar concentration of N-hydroxysuccinimide used in the initiation system is 2.65mM, and the molar concentration of hydrogen peroxide is 0.66mM, The ethylenic monomer used is polyethylene glycol methyl methacrylate, and the amount added accounts for 5% of the total weight of the reaction raw materials. In addition, N, N-methylenebispropylene can also be added during the preparation of the hydrogel For amides, the amount of crosslinking agent accounts for 0.3% of the total weight of the reaction raw materials.

At room temperature, free radicals are generated within 4min10s to initiate polymerization of monomers to form a three-dimensional polymer network and obtain a hydrogel.

Example 12

A method for preparing a hydrogel using a horseradish peroxidase-mediated free radical initiation system, the method uniformly mixes N-hydroxysuccinimide, horseradish peroxidase and an aqueous solution of an ethylenic monomer, and then injects it into the hydrogel Add hydrogen peroxide, adjust the component concentration ratio of horseradish peroxidase/N-hydroxysuccinimide/hydrogen peroxide ternary initiation system, the molar concentration ratio of hydrogen peroxide and horseradish peroxidase is 960 , and the molar concentration ratio of N-hydroxysuccinimide and hydrogen peroxide is 29, the molar concentration of N-hydroxysuccinimide used in the initiation system is not lower than 19.1mM, and the molar concentration of hydrogen peroxide is not lower than 0.66mM. The selected ethylenic monomer is N,N-dimethylacrylamide, and the addition amount accounts for 15% of the total weight of the reaction raw materials. In addition, during the preparation of the hydrogel, nanometer disulfide with an average particle size of 10-40nm can also be added. Silicon oxide is used to prepare high-strength nanocomposite hydrogel, and the dosage accounts for 16% of the total weight of the reaction raw materials. Free radicals were generated within 5 minutes at room temperature to initiate monomer polymerization to form a three-dimensional polymer network and obtain a hydrogel.

The present invention has been exemplarily described above, but the implementation of the present invention is not limited by the above examples. Any other modifications, replacements, combinations, and simplifications made without departing from the essence of the principles of the present invention shall be equivalent replacement methods, and shall be included within the protection scope of the present invention.

Claims (9)

1. horseradish peroxidase mediation free radical initiator system, it is characterized in that, this free radical initiator system is be at room temperature the initiator system that reaction medium causes that vinyl monomer carries out radical polymerization with water, comprise N-hydroxy-succinamide, horseradish peroxidase and hydrogen peroxide, described hydrogen peroxide and the molar concentration rate of horseradish peroxidase are 25 ~ 960, and described N-hydroxy-succinamide and the molar concentration rate of hydrogen peroxide are 2 ~ 29.

2. horseradish peroxidase mediation free radical initiator system according to claim 1, it is characterized in that, the volumetric molar concentration of described N-hydroxy-succinamide is not less than 2.65mM, and the volumetric molar concentration of hydrogen peroxide is not less than 0.66mM.

3. horseradish peroxidase mediation free radical initiator system according to claim 1, it is characterized in that, described free radical initiator system is the initiator system that N-hydroxy-succinamide, horseradish peroxidase, hydrogen peroxide are directly mixed to get radical polymerization;

Or by after N-hydroxy-succinamide and horseradish peroxidase mixing, then add hydrogen peroxide with the use of the initiator system obtaining radical polymerization;

Or by N-hydroxy-succinamide and or hydrogen peroxide mixing after, then add horseradish peroxidase with the use of the initiator system obtaining radical polymerization.

4. utilize the mediation of the horseradish peroxidase according to any one of claim 1-3 free radical initiator system to prepare the method for hydrogel, it is characterized in that, the method is by even for the aqueous solution of N-hydroxy-succinamide, horseradish peroxidase and vinyl monomer, then hydrogen peroxide is added wherein, regulate the concentration of component ratio of horseradish peroxidase/N-hydroxy-succinamide/hydrogen peroxide ternary initiator system, at room temperature control in 50s ~ 5min, produce the polymerization of free radical trigger monomer and form Space network of polymer, obtain hydrogel.

5. horseradish peroxidase mediation free radical initiator system according to claim 4 prepares the method for hydrogel, it is characterized in that, described hydrogen peroxide and the molar concentration rate of horseradish peroxidase are 25 ~ 960, and the molar concentration rate of N-hydroxy-succinamide and hydrogen peroxide is 2 ~ 29, the volumetric molar concentration of the N-hydroxy-succinamide adopted in initiator system is not less than 2.65mM, and the volumetric molar concentration of hydrogen peroxide is not less than 0.66mM.

6. horseradish peroxidase mediation free radical initiator system according to claim 4 prepares the method for hydrogel, it is characterized in that, described vinyl monomer is selected from least one in water miscible acrylate derivative, acrylamide derivative or NVP: the add-on of vinyl monomer accounts for 5 ~ 15% of reaction raw materials gross weight, and preferably 8 ~ 10%.

7. horseradish peroxidase mediation free radical initiator system according to claim 6 prepares the method for hydrogel, it is characterized in that, described water miscible acrylate derivative is hydroxyethyl methylacrylate, Propylene glycol monoacrylate or polyoxyethylene glycol methyl methacrylate; Described acrylamide derivative is acrylamide, N,N-DMAA or NIPA.

8. horseradish peroxidase mediation free radical initiator system according to claim 4 prepares the method for hydrogel, it is characterized in that, water-soluble cpds with two or more double bonds can also be added as linking agent in the preparation process of hydrogel, comprise the protein of N,N methylene bis acrylamide, polyethyleneglycol diacrylate or modified by vinyl; Described dosage of crosslinking agent accounts for 0.3 ~ 5% of reaction raw materials gross weight.

9. horseradish peroxidase mediation free radical initiator system according to claim 4 prepares the method for hydrogel, it is characterized in that, the Nanometer composite hydrogel that water dispersible inorganic nano material prepares high strength can also be added in the preparation process of hydrogel, described inorganic nano material is nano silicon or nanometer hydroxyapatite, and preferred median size is the nano silicon of 10 ~ 40nm; The consumption of described inorganic nano material accounts for 5 ~ 16% of reaction raw materials gross weight.