CN105860098B - A kind of preparation method and applications of the porous semi-interpenetrating network aqueous gel of temperature sensitive type - Google Patents

Abstract

The invention discloses a kind of preparation method and applications of the porous semi-interpenetrating network aqueous gel of temperature sensitive type.It is made by following five steps：First, the synthesis of linear poly hydroxy ethyl acrylate (Hydroxyethyl methacrylate, PHEMA)；Second is that the preparation of semi-interpenetrating network aqueous gel HEMA co DEAAm；Third, acrylic modifies hydrogel；Fourth, fixed papain.Mutual perforative porous structure is contained by temperature-sensitive hydrogel made from this method, there is big specific surface area, be a kind of carrier of more satisfactory immobilised enzymes to temperature change energy quick response.Enzyme immobilizatio is carried out using the click-reaction of sulfydryl on the methyl methacrylate double bond structure on carrier and enzyme molecule.So reaction selectivity is high, the enzyme containing sulfydryl is only fixed；Reaction condition is mild, and reaction temperature is at 37 DEG C or so；Activity of the immobilized enzyme obtained by the reaction is 5.5 times of conventional method glutaraldehyde immobilised enzymes, and stability is preferable, recyclable recycling.

Description

Preparation method and application of a temperature-sensitive porous semi-interpenetrating network hydrogel

technical field

The invention relates to a preparation method of a novel temperature-sensitive semi-interpenetrating network hydrogel, in particular to a preparation method and application of a temperature-sensitive porous semi-interpenetrating network hydrogel.

Background technique

The immobilization of enzymes (Immobilization of enzymes) is the use of chemical (covalent bonding method, cross-linking method) or physical (adsorption method, embedding method) and other methods to combine water-soluble enzymes with water-insoluble carriers. Preparation of new functional materials that not only maintain enzyme activity, but also reuse enzymes. While maintaining the high selectivity, high efficiency and mild conditions of the enzyme itself, the immobilized enzyme also presents many advantages, such as high storage stability, easy separation and recovery, and can be reused many times. In recent years, immobilized enzymes have been widely used in the fields of chemistry, medicine, food, environmental engineering and industrial production, and have become a current research hotspot. However, the structure and performance of the immobilized enzyme carrier material directly affect the performance of the immobilized enzyme. Therefore, it is very important to prepare support materials with excellent structure.

At present, according to the literature, the carrier materials for immobilized enzymes include inorganic materials, polymer materials, and biomass materials. Recently, new achievements in materials science have been gradually applied in the field of enzyme-immobilized materials. Among them, smart materials are the frontier and hot spot. Intelligent immobilized enzyme materials are divided into types, including pH-responsive, temperature-responsive, magnetic-responsive, etc., and in terms of material form, there are gel solids, carrier resins, etc. Among them, thermosensitive hydrogels have better advantages in immobilizing enzymes. The characteristic of thermosensitive hydrogel is that its structure contains a certain proportion of hydrophilic groups and hydrophobic groups. When the external temperature changes, the hydrophilicity and hydrophobicity of these groups and the hydrogen bond interaction will change accordingly. The hydrophilicity of most thermosensitive hydrogels will increase with the increase of temperature, but the hydrophilicity of hydrogels with lower critical solution temperature (LCST) will decrease with the increase of temperature, When the hydrogel is at a temperature lower than the LCST, the hydrophilic groups and water molecules are combined through hydrogen bonds to enhance the solubility of the gel in water; when the temperature rises, the interaction between the hydrophobic groups is strengthened, and the oxygen bonds become stronger. Weak, gel shrinks. The LCST of thermosensitive hydrogels can be changed by adjusting the ratio of hydrophilic and hydrophobic groups. Generally, the higher the content of hydrophilic groups, the higher the LCST of hydrogels, such as poly N,N-diethyl-2-acrylamide ( PDEAAM), block copolymers composed of polyethylene oxide (PEO) and polypropylene oxide (PPO), all have temperature sensitivity, and the LCST is close to human body temperature. Therefore, the progress of the enzyme-catalyzed reaction can be controlled by controlling the reaction temperature. At the same time, LCST is mostly close to room temperature, and the reaction conditions are simple and easy to control. Therefore, it is meaningful to carry out this work.

In recent years, the design and synthesis of new carriers, the development of new immobilization methods and the organic combination of the two are the research directions that many scholars have devoted themselves to in recent years. The new immobilization methods developed must follow the following principles: The immobilization is as gentle as possible to minimize or avoid the loss of enzyme activity, and thermosensitive hydrogel as a new way of immobilizing enzymes has attracted widespread attention from researchers. Gotoh et al. used NIPAm and N,N-diethylacrylamide (DEAAm) as monomers to prepare temperature-sensitive porous PNIPAm and PDEAAm hydrogels by free radical polymerization at a temperature higher than LCST. Zhang et al. used PEG as a pore-forming agent to prepare PNIPAm macroporous thermosensitive hydrogel, and the structure of the hydrogel could be adjusted by changing the amount of PEG. Alves et al. prepared pH/temperature-sensitive Semi-IPN hydrogels with isopropylpropionamide and calcium alginate. Guo et al prepared the Semi-IPN hydrogel polymer with carboxymethyl chitosan and poly(N-isopropylacrylamide), and studied the drug release properties of the polymer. Cheng et al. prepared PNIPAm macroporous hydrogels using pore-forming agents, and conducted research on the controlled release of bovine serum albumin. The controlled release rate and controlled release rate of the drug were higher. Hu Lin et al. grafted PNIPAm onto polyvinylidene fluoride membrane (PVDF) membrane pores using plasma pore-filling graft polymerization technology, and then combined with cross-linked PNIPAm hydrogel to make a temperature-sensing controlled release membrane system. The system has good temperature responsiveness. Lee et al. used PNIPAm to polymerize with several different ionic monomers, prepared a series of hydrogels with PEG of different molecular weights as pore-forming agents, and studied the release mechanism of caffeine in hydrogels. It can be seen from the given known literature that no one has completed the research on the preparation of porous semi-interpenetrating network hydrogels using DEAAm and linear hydroxyethyl methacrylate as raw materials and modifying them with the double bond structure of methyl methacrylate. The innovativeness of the research work is of great significance.

The invention prepares a porous semi-interpenetrating network hydrogel based on DEAAm as a raw material and linear hydroxyethyl methacrylate, uses the novel matrix material to react with a sulfhydryl group to immobilize a thiol enzyme, and has extremely strong selectivity and excellent temperature responsiveness. From the results obtained from the immobilized papain, it can be known that the immobilized papain can be reused, the activity recovery rate is 6.5%, the temperature response range is 35-39°C, the thermal stability is 85%, and it has excellent properties.

Contents of the invention

The technical problem to be solved by the present invention is to provide a preparation method of a temperature-sensitive porous semi-interpenetrating network hydrogel, and the application of immobilizing papain with this novel gel.

The technical solution adopted to solve the above technical problems is: a method for preparing a temperature-sensitive porous semi-interpenetrating network hydrogel, which is carried out as follows:

(1) Synthesis of PHEMA: 5 mg of initiator azobisisobutyronitrile (2,2'-Azobis (2-methylpropionitrile), AIBN), 2 mL of monomer hydroxyethyl methacrylate (Hydroxyethylmethacrylate, HEMA) were added to Into 4 mL of the solvent dimethyl sulfoxide (Dimethyl sulfoxide, DMSO), reflux in a water bath at 60° C. for 8 h under a nitrogen atmosphere to obtain PHEMA as a light yellow liquid. Precipitate with ether, filter and dry to obtain PHEMA powder.

(2) Preparation of network semi-interpenetrating hydrogel: 0.1 g of pore forming agent polyethylene glycol PEG20000, 1.8 mL of N,N-diethyl-acrylamide (N,N-diethyl-Acrylamide, DEAAm), 0.04 g of cross-linking agent N,N-methylene-bis-acrylamide (Bis) and 0.075 g of PHEMA powder were dissolved in 6 mL of DMSO, and refluxed in a water bath for 6 h under a nitrogen atmosphere to obtain a gel.

(3) Acrylate-modified gel: Soak the gel in distilled water for 14 days, weigh 1 g of the soaked gel, dissolve it in 10 mL of N,N-dimethylformamide (N,N-Dimethylformamide, DMF ), add 0.01-0.05g of sodium methoxide, dropwise add 0.11g of aminoethanol under stirring condition, stir and reflux at about 150°C for 4h. After the reaction, it was cooled to 80° C., 0.005 g of hydroquinone was added, 0.18 g of methyl methacrylate was added dropwise, and the water bath was refluxed for 1 h to obtain a transparent gel-like solid. After the reaction, the solid is washed with water for several times to obtain a temperature-sensitive porous semi-interpenetrating network hydrogel.

An application of a temperature-sensitive porous semi-interpenetrating network hydrogel, the application being: using the temperature-sensitive porous semi-interpenetrating network hydrogel to fix sulfhydryl-containing enzymes.

The beneficial effect of the invention is that the temperature-sensitive hydrogel prepared by the method contains interpenetrating porous structures, has a large specific surface area, can respond quickly to temperature changes, and is an ideal carrier for immobilized enzymes. The enzyme is immobilized by using the click reaction between the double bond structure of methyl methacrylate on the carrier and the sulfhydryl group on the enzyme molecule, which has the advantages of high reaction selectivity, mild reaction conditions, and easy operation of the method. The activity of the immobilized enzyme obtained by the reaction is The traditional method is 5.5 times faster than the glutaraldehyde-immobilized enzyme, and its stability is good, and it can be recycled.

Description of drawings

Accompanying drawing 1 is the roadmap of preparation technology of novel temperature-sensitive semi-interpenetrating network hydrogel of the present invention, in the figure, ① initiator AIBN ② solvent DMSO ③ crosslinking agent Bis ④ pore forming agent PEG20000 ⑤ DEAAm monomer ⑥ CH ₃ ONa ⑦ NH ₂ C ₂ H ₄ OH ⑧ Hydroquinone⑨methyl methacrylate

Accompanying drawing 2 is the infrared spectrogram of the novel temperature-sensitive semi-IPN hydrogel of the present invention.

Accompanying drawing 3 is the ¹³ C NMR spectrum of the novel temperature-sensitive semi-IPN hydrogel of the present invention.

Accompanying drawing 4 is the ¹ H NMR spectrum of the novel temperature-sensitive semi-IPN hydrogel of the present invention.

Accompanying drawing 5 is the scanning electron microscope picture of the novel temperature-sensitive semi-IPN hydrogel of the present invention.

Detailed ways

The present invention is further elaborated below in conjunction with embodiment and with reference to accompanying drawing:

First purchase the following raw materials: hydroxyethyl methacrylate (HEMA), azobisisobutyronitrile (AIBN), dimethylsulfoxide (DMSO), polyethylene glycol (PEG20000), N,N-diethyl Acrylamide (DEAAm), N,N-methylenebisacrylamide (Bis), methyl methacrylate, aminoethanol, dimethylformamide (DMF), sodium methoxide, hydroquinone. In this embodiment, g is used as the unit of weight, and mL is used as the unit of volume.

Example 1

1. The preparation method of porous semi-interpenetrating thermosensitive hydrogel is characterized in that it is carried out as follows:

(1) Synthesis of PHEMA: Add 5mg of initiator AIBN and 2mL of monomer HEMA into 4mL of solvent DMSO, blow nitrogen, and reflux in a water bath at 60°C for 8h. After the reaction, precipitate with ether, filter, and dry the PHEMA powder. The synthesis reaction principle is as follows:

(2): Preparation of porous network semi-interpenetrating hydrogel: Dissolve 0.1g of pore-forming agent PEG20000, 1.8mL of DEAAm, 0.04g of cross-linking agent Bis and 0.075g of PHEMA in 6mL of DMSO, and blow nitrogen , the water bath was refluxed for 6h. After the reaction, a gel was obtained. The synthesis reaction principle is as follows:

(3) Acrylate-modified gel: Soak the gel in distilled water for 14 days, weigh 1g of the soaked gel, dissolve it in 10mL of DMF, add 0.01-0.05g of sodium methoxide, stir and add 0.11g dropwise Amino ethanol was stirred and refluxed at about 150°C for 4h. After the reaction, cool to 80°C, add 0.005g of hydroquinone, slowly dropwise add 0.18g of methyl methacrylate, and reflux in the water bath for 1h. After the reaction is finished, it is washed with water for several times to obtain a temperature-sensitive porous semi-interpenetrating network hydrogel.

Infrared, ¹³ C nuclear magnetic resonance, ¹ H nuclear magnetic resonance, and scanning electron microscopy were performed on the temperature-sensitive porous semi-interpenetrating network hydrogel. The results are as follows: the infrared spectrum is shown in Figure 2, in which, 3080-2870cm ^-1 is the stretching vibration absorption peak of methyl methylene CH at the carbon-carbon double bond of the gel, and 1469-1380cm ^-1 is the bending vibration absorption peak of methyl CH Peak, 895cm ^-1 is the bending vibration absorption peak of methylene CH, and 1650cm ^-1 is the stretching vibration absorption peak of C=O. In the gel, there is no characteristic peak at 3600cm ^-1 , indicating that the methyl methacrylate modified hydrogel was successful, that is, the new temperature-sensitive porous semi-interpenetrating hydrogel was synthesized successfully. ^{The 13} C nuclear magnetic spectrum (NMR) is shown in Figure 3, and the ¹ H nuclear magnetic spectrum (NMR) is shown in Figure 4, which shows that the temperature-sensitive porous semi-interpenetrating hydrogel was successfully synthesized. The scanning electron microscope image is shown in Figure 5. It can be seen that the hydrogel is an interpenetrating porous structure, and the size and distribution of the pores are relatively uniform.

Example 2

The temperature-sensitive porous semi-interpenetrating network hydrogel prepared in Example 1 was used to immobilize sulfhydryl-containing enzymes, as follows:

Sulfhydryl click reaction immobilization of papain: Weigh 0.1 g of the prepared temperature-sensitive porous semi-IPN hydrogel, add it to the enzyme solution, heat to 30°C, the gel begins to absorb the enzyme solution and swell, and the enzyme molecules A cross-linking reaction occurs with the gel inside the gel to obtain an immobilized enzyme. The prepared immobilized papain was stored in 0.1 mol/L phosphate buffer (pH=7) at 4°C.

Through experimental comparison, it is concluded that the enzyme activity of the prepared immobilized enzyme is about 5.5 times that of the immobilized enzyme obtained by cross-linking with glutaraldehyde.

Example 3

A kind of preparation method of thermosensitive type porous semi-interpenetrating network hydrogel is carried out as follows:

(1) Synthesis of PHEMA: Add 5 mg of initiator AIBN and 2 mL of monomer HEMA to 4 mL of solvent DMSO, blow nitrogen, and reflux in a water bath at 60° C. for 8 h. After the reaction, precipitate with ether, filter, and dry to obtain PHEMA powder.

(2) Preparation of porous network semi-interpenetrating hydrogel: Dissolve 0.1g of pore-forming agent PEG20000, 1.8mL of DEAAm, 0.04g of cross-linking agent Bis and 0.075g of PHEMA in 6mL of DMSO, blow nitrogen, The water bath was refluxed for 6h. After the reaction, a gel was obtained.

(3) Acrylate-modified gel: Soak the gel in distilled water for 14 days, weigh 1g of the soaked gel, dissolve it in 10mL of DMF, add 0.01-0.05g of sodium methoxide, stir and add 0.11 g of aminoethanol, stirred and refluxed at a temperature of 1 for 4h. After the reaction, cool to temperature 2, add 0.005 g of hydroquinone, dropwise add 0.18 g of methyl methacrylate, and reflux in the water bath for 1 h. After the reaction is finished, wash with water for several times to obtain temperature-sensitive porous semi-interpenetrating network hydrogels a to f.

Table 1 Effect of temperature on gel properties

It can be seen from Table 1 that when temperature 1 is 150°C and temperature 2 is 80°C, the obtained hydrogel is optimal. When the temperature is too high, a large amount of by-products will be produced so that the yield is low, and when the temperature is low, a large amount of reactants will remain. Therefore, whether the temperature is too high or too low will make the hydrogel have too many impurities and reduce the effectiveness of the hydrogel.

Claims (2)

1. a kind of preparation method of the porous semi-interpenetrating network aqueous gel of temperature sensitive type, it is characterized in that, it carries out as follows：

（1）The synthesis of PHEMA：By the initiator azodiisobutyronitrile of 5mg（2,2'-Azobis(2- Methylpropionitrile), AIBN）, 2mL monomers hydroxy ethyl ester（Hydroxyethyl Methacrylate, HEMA）It is added to the solvent dimethyl sulfoxide (DMSO) of 4mL（Dimethyl sulfoxide, DMSO）In, in nitrogen Flow back 8h in the lower 60 DEG C of water-baths of environment, obtains PHEMA weak yellow liquids, is precipitated with ether, and filtering, drying obtains PHEMA powder；

（2）The preparation of the Semi-IPN hydrogel of network：By the N of perforating agent polyethylene glycol PEG20000,1.8mL of 0.1g, N- diethyls The crosslinking agent N,N methylene bis acrylamide of base acrylamide (N, N-diethyl-Acrylamide, DEAAm), 0.04g （Methylene-Bis-Acrylamide,Bis）It is dissolved in the DMSO of 6mL with the PHEMA powder of 0.075g, in a nitrogen environment Water-bath reflux 6h, obtains gel；

（3）Acrylate modifies gel：Gel is placed in distilled water and is impregnated 14 days, the gel after 1g impregnates is weighed, is dissolved in The N,N-dimethylformamide of 10mL（N, N-Dimethylformamide, DMF）In, the sodium methoxide of 0.01 ~ 0.05g is added in, is stirred The ethylaminoethanol of 0.11g is added dropwise under the conditions of mixing, 4h is stirred at reflux at 150 DEG C；After reaction, 80 DEG C are cooled to, is added in The hydroquinone of 0.005g, is added dropwise the methyl methacrylate of 0.18g, and water-bath reflux 1h obtains transparent gel-form solid；Reaction After, solid several times are washed with water, obtain the porous semi-interpenetrating network aqueous gel of temperature sensitive type.

2. the application of the porous semi-interpenetrating network aqueous gel of temperature sensitive type prepared by method as described in claim 1, which is characterized in that institute It states to apply and is：The porous semi-interpenetrating network aqueous gel of temperature sensitive type is used for the fixed enzyme containing sulfydryl.