Preparation method of aperture-adjustable porous flexible glue microspheres and porous flexible glue microspheres

Abstract

The invention discloses a preparation method of aperture-adjustable porous flexible glue microspheres and the porous flexible glue microspheres. Comprising the following steps: (1) adding biological polysaccharide into water, pressurizing and heating a system to hydrolyze the biological polysaccharide, and standing to obtain a dissolved and clarified water phase; (2) adding a surfactant into the solvent, heating, preserving heat, and standing to obtain a dissolved and clarified oil phase; (3) adding the water phase into the oil phase, stirring, preserving heat and cooling to obtain a porous soft rubber bare ball; (4) putting the porous soft rubber bare spheres into water, and reacting with a cross-linking agent in an alkaline environment to obtain a porous microsphere intermediate; and (5) hydrolyzing the porous microsphere intermediate under an alkaline condition, cleaning, and screening out the porous flexible glue microsphere with a certain particle size. According to the preparation method of the pore-diameter-adjustable porous soft rubber microspheres, the effects that the microspheres have different pore diameters, dependence on polysaccharide selection is small, and pressure resistance is good can be achieved.

Description

Technical Field

[0001] The invention relates to porous materials, in particular to a preparation method of porous soft gel microspheres with adjustable pore sizes and the porous soft gel microspheres. Background Art

[0002] Porous microspheres, with their unique pore structure and high specific surface area, have shown wide application potential in many fields such as catalysis, adsorption, separation, drug carriers and biosensors. This special structure gives porous microspheres significant advantages in material transport, reactivity and biocompatibility. Therefore, the preparation method of porous microspheres has always been a hot research topic in the field of materials science.

[0003] Porous soft-gel microspheres are polymer spherical particles with a porous structure prepared by polymer synthesis. This type of microsphere has a variety of preparation methods, is easy to modify, and has adjustable pore size, so it plays an important role in the field of materials science. However, compared with conventional hard-gel porous polymer microspheres, porous soft-gel microspheres have certain challenges in preparation. Their synthetic matrix is ​​usually biopolysaccharides, which have poor controllability, resulting in limited selectivity in the preparation of porous soft-gel microspheres. Compared with porous hard-gel microspheres, their surface is softer and can provide better wettability and adsorption. When used as filler carriers, they help interact with target substances. In addition, soft-gel microspheres can undergo slight deformations and can adapt to pressure changes in fluids. Low toxicity and excellent biocompatibility make them have broad application value in the field of biomedicine.

[0004] The emulsion suspension method is a common method for preparing porous soft gel microspheres. After dissolving, the polysaccharide aqueous solution is transferred to the oil phase and dispersed into oil-in-water droplets. As the aging time of the dissolved sugar increases, the hydrogen bonds between the polysaccharide chains and even the molecular chains will break. Therefore, most preparation methods try to minimize the aging time of the dissolved sugar. However, there are not many reports on whether the hydrolysis and cleavage of the polysaccharide chain segments have a negative impact on the synthesis of microspheres. In the selection process of biopolysaccharides, there are also some challenges. First, biopolysaccharides are easy to gel at low temperatures. This feature makes it particularly difficult to efficiently test their molecular weight. Therefore, it is particularly difficult to find biopolysaccharide substitutes with similar performance. Secondly, there are differences in the molecular chains of biopolysaccharides between batches. This factor increases the difficulty of preparing soft gel microspheres with similar performance through the same material ratio and process, which further makes it more difficult to control the pore size. At present, the pore size of porous soft gel microspheres can be effectively adjusted by adjusting the concentration of polysaccharides, adjusting the pH and the cross-linking process of microspheres, but these methods have a great influence on the pressure resistance of microspheres.

[0005] Affinity chromatography is a chromatographic separation technology based on the reversible and specific interaction between bioactive substances and other molecules. In this technology, porous soft gel microspheres are often used as carriers for affinity chromatography. These microspheres bind to biomacromolecules through spacer arms, so there are special requirements for the specific binding sites on the microspheres. Studies have revealed that within a certain range, as the pore size of the microspheres increases, the amount of coupling of biomacromolecules on the microspheres will also increase accordingly, resulting in better separation effects. However, the unique skeleton structure of porous soft gel microspheres makes it impossible to directly detect their pore size by methods such as BET or mercury intrusion. In order to indirectly evaluate the pore size of the microspheres, the microspheres are usually loaded into a gel column bed and gel filtration chromatography is used. Among them, the important parameter Kav can reflect the effect of screening specific biomacromolecules based on the pore size of the microspheres. Within a certain range, the larger the Kav value, the larger the pore size of the microspheres. In practical applications, in order to meet different environmental requirements and balance the pressure resistance and pore size of the microspheres, it is necessary to select porous microspheres with different Kav values ​​for large-scale production to ensure the effectiveness and reliability of affinity chromatography technology in different application scenarios. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a method for preparing porous soft gel microspheres with adjustable pore sizes, which have low dependence on the selection of polysaccharide molecular weight and good pressure resistance.

[0007] Another object of the present invention is to provide a porous soft gel microsphere with different pore sizes, less dependence on the selection of polysaccharide molecular weight, good pressure resistance and adjustable pore size.

[0008] Technical solution: The method for preparing porous soft gel microspheres with adjustable pore size of the present invention comprises the following steps:

[0009] (1) adding biopolysaccharide to water, pressurizing and heating the system to hydrolyze the biopolysaccharide, and obtaining a clear aqueous phase after standing;

[0010] (2) adding a surfactant to the solvent, heating and keeping the temperature, and allowing the solvent to stand to obtain a clear oil phase;

[0011] (3) adding the aqueous phase obtained in step (1) to the oil phase obtained in step (2), stirring, keeping warm and then cooling to obtain a porous soft gel bare ball;

[0012] (4) placing the porous soft gel bare ball obtained in step (3) in water and reacting with a crosslinking agent in an alkaline environment to obtain a porous microsphere intermediate;

[0013] (5) hydrolyzing the porous microsphere intermediate obtained in step (4) under alkaline conditions, and screening out porous soft gel microspheres of a certain particle size after washing.

[0014] Preferably, in step (1), the temperature increase is achieved by pressurizing the system; the biopolysaccharide is selected from agarose, water-soluble chitosan, or a mixture of more thereof; the system pressure is 0.05-0.25 MPa; the hydrolysis time is 1.5-5 h, more preferably 1.5-3.5 h; the average molecular weight of the polysaccharide is 100-400 kDa; the concentration of the polysaccharide solution is 5wt%-8wt%; and the standing time is 15-30 min.

[0015] Preferably, in step (2), the solvent is one or more of liquid paraffin, cyclohexane, and xylene; the surfactant is one or more of Span 80, polyethylene glycol, and sodium dodecyl sulfate; and the insulation temperature is 70-85°C.

[0016] Preferably, in step (3), the stirring linear speed is 2.5 m / s to 3.6 m / s; the stirring time is 1 to 3 h; the insulation temperature is 75 to 90° C.; and the cooling time is 0.5 to 1.2 h.

[0017] Preferably, in step (4), the cross-linking agent is one or more of 2,3-dibromopropanol diepoxide, 1,4-butanediol diglycidyl ether, and epichlorohydrin; the alkaline environment is a mixture of one or more of 10-40wt% sodium hydroxide solution and 10-40wt% potassium hydroxide solution; the cross-linking reaction temperature is 40-60°C; and the reaction time is 8-16h.

[0018] Preferably, the particle size described in step (5) is 50 to 80 μm.

[0019] Principle of the invention: When biological polysaccharides are dissolved at a relatively low temperature, the hydrogen bonds between the double helix chains break, and the chains gradually stretch to form a uniform aqueous solution. Generally, as the dissolution time increases, the chain segments are likely to be hydrolyzed, but when the degree of hydrolysis is small, it also takes a long time, up to several hours or even dozens of hours. By applying pressure to the system, the temperature of the aqueous solution is raised to above 100°C, thereby increasing the dissolution temperature of the polysaccharide. In addition to the breaking of hydrogen bonds, the glycosidic bonds between the chain segments will also break and hydrolyze during the dissolution process of the polysaccharide, forming shorter sugar chain fragments, and the molecular weight will also decrease accordingly. Since low-molecular-weight polysaccharide chains are shorter and the interaction between chains is weaker, it is easy to form a looser structure, which leads to a larger pore size. Therefore, by controlling the molecular weight of the sugar chain before the aqueous phase transfer during the synthesis process, the synthetic method provided by the above invention can be used to prepare porous soft gel microspheres with different pore sizes.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) This method increases the pressure of the system during the dissolution of the biopolysaccharide, thereby increasing the hydrolysis rate of the biopolysaccharide and controlling the hydrolysis time. When the polysaccharide chain segments are hydrolyzed to a specified average molecular weight, aqueous phase transfer is performed. The above-mentioned operation method can be used to prepare porous soft-gel microspheres with different pore sizes, thereby achieving the effect of adjustable pore size of the porous soft-gel microspheres; (2) This method uses an emulsion suspension method, and by adjusting the hydrolysis rate of the biopolysaccharide, it can be performed under conditions of high polysaccharide concentrations, thereby ensuring the pressure resistance of the porous microspheres. Even if the molecular weight of the raw polysaccharide is different, porous microspheres with similar performance can be prepared by controlling their hydrolysis degree. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a curve diagram showing the change of the second molecular weight of agarose after hydrolysis at 105° C. with the hydrolysis time;

[0022] Figure 2 This is a micrograph of the porous soft gel microspheres prepared in Example 8. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below in conjunction with embodiments.

[0024] Example 1

[0025] The method for preparing porous soft gel microspheres with adjustable pore size of the present invention comprises the following steps:

[0026] (1) 99.3 g of agarose powder with an average molecular weight of 155 kDa (referred to as the first molecular weight) was added to 1.5 kg of water, and nitrogen was introduced into the container for 3 to 5 minutes to exhaust the air therein. Subsequently, the agarose was pressurized to 0.10 MPa with the kettle temperature raised to 105° C., and heated to dissolve and stirred for 1.5 hours to hydrolyze the agarose. After the hydrolysis was completed, the agarose was allowed to stand for 30 minutes to obtain a clear aqueous phase. 100 ml of the sample was taken and kept at 80° C. for 15 minutes. The sample was subjected to a 4-minute dynamic viscosity test using a rotational viscometer (the dynamic viscosity data are shown in Table 1). Then, the average molecular weight of the sample (referred to as the second molecular weight) was estimated according to the Mark-Houwink deformation equation.

[0027] (2) Add 380 g of sodium dodecyl sulfate to 4.5 L of liquid paraffin, heat to 85°C, keep warm and stir for more than 1 hour, and let stand for 30 minutes to obtain a clear oil phase.

[0028] (3) Add the water phase system obtained in step (1) to the oil phase system obtained in step (2), start the agitator, and control the agitator linear speed to 2.72 m / s, continue stirring for 1 hour, turn on the refrigerant to control the temperature in the kettle to drop from 85°C to 35°C for 50 to 70 minutes, and thoroughly clean the solidified material to obtain a porous soft rubber bare ball.

[0029] (4) Take 1L of the porous soft gel bare ball prepared in step (3) and add it to 500g of pure water, then add 20ml of 40wt% sodium hydroxide solution and 162ml of 1,4-butanediol diglycidyl ether in sequence, react at 50°C for 14h, and wash; this process is repeated once to further improve the pressure resistance of the porous soft gel bare ball to obtain a porous microsphere intermediate.

[0030] (5) 1 L of the porous microsphere intermediate obtained in step (4) was added to 1.4 kg of 0.7 M potassium hydroxide solution to fully hydrolyze it. After washing, the microspheres were sieved using a sieving machine to obtain porous soft gel microspheres with a particle size distribution D50 of 50 to 80 μm.

[0031] The Kav value of the porous soft gel microsphere packed chromatographic column was tested. The chromatographic conditions and calculation method are as follows:

[0032] Wavelength: 218nm; Flow rate: 1mL / min; Flow rate during quantitative loop injection: 0.02mL / min; Column temperature: room temperature; Injection: 20μL; Standard sample: blue dextran (11.0mg / mL), thyroglobulin (7.0mg / ml). Kav value calculation: Kav = (Tt-T0) / (V-T0), Tt is the retention time of standard thyroglobulin, T0 is the retention time of standard blue dextran, and V is the volume of the chromatography column.

[0033] The pressure resistance test of the chromatographic column filled with porous soft gel microspheres was carried out. The test conditions and methods are as follows:

[0034] The mobile phase uses 0.1M NaCl solution, and the flow rate is increased at a linear velocity gradient of 50cm / h (when the maximum flow rate of the column is greater than 20ml / min, the flow rate is increased at a linear velocity gradient of 100cm / h), and the test is stopped when the linear velocity reaches the maximum flow rate of the column. When the pressure difference between the upper and lower columns reaches 3.0Bar, the corresponding linear velocity is recorded. In general, the greater the linear velocity of the sample, the better its pressure resistance.

[0035] Examples 2-8 and Comparative Examples 1-2

[0036] According to the preparation method and reaction conditions disclosed in Example 1, only some of the reaction conditions were changed to construct Examples 2-8 and Comparative Examples 1-2, as shown in Table 1.

[0037] Comparative Example 3

[0038] The same parts as those in Example 4 are not described in detail in this comparative example. The difference lies in that the concentration of the biopolysaccharide in step (1) is changed, that is, 60.0 g of agarose powder with an average molecular weight of 250 kDa (recorded as the first molecular weight) is added to 1.5 kg of water.

[0039] Table 1 Reaction conditions and experimental results of each experiment

[0040]

[0041] Note: (1) "Hydrolysis temperature" in the table refers to the hydrolysis temperature of agarose in step (1);

[0042] (2) *Due to the different agarose concentrations, the above specific modified equation is not suitable for estimating the second molecular weight.

[0043] As can be seen from Table 1 above, Examples 1 to 3 and 4 to 8 respectively use agarose with an average molecular weight of 155 kDa and 250 kDa. Under a certain pressure, the hydrolysis temperature and degree of hydrolysis of the polysaccharide are improved. While keeping the average molecular weight of the polysaccharide close after hydrolysis, porous soft-gel microspheres with adjustable pore size can be prepared, and the porous soft-gel microspheres also have good pressure resistance. However, it is not applicable to all polysaccharides with molecular weight distribution. When the molecular weight is large, the degree of hydrolysis will also be limited. That is, when the molecular weight of the biological polysaccharide is within a certain range, the molecular weight of the polysaccharide before the water phase transfer can be adjusted by controlling the hydrolysis time of the aqueous phase to achieve the purpose of adjustable pore size of the porous soft-gel microspheres, which are suitable for carriers of different separation and purification fillers. In addition, the present invention also reduces the dependence on the selection of biological polysaccharides, and achieves the stability of large-scale preparation of porous soft-gel microspheres by controlling the production process. Figure 1 As shown in the figure, after agarose is hydrolyzed, the molecular weight of the polysaccharide before water phase transfer tends to decrease with the extension of hydrolysis time, and the rate of decrease slows down, which further shows that the second molecular weight of agarose can be controlled by adjusting the hydrolysis time, thereby regulating the pore size of the porous soft gel microspheres.

[0044] By comparing Comparative Example 1 with Example 4, and Comparative Example 2 with Example 7, it can be seen that if the pressure of the system during the hydrolysis process is not in the range of 0.05 to 0.25 MPa, especially below this range, the purpose of controlling the average molecular weight of the polysaccharide before the aqueous phase transfer cannot be achieved, and thus the pore size of the porous soft gel microspheres cannot be regulated.

[0045] In Comparative Example 3, although the pore size of the porous soft-gel microspheres can be adjusted by adjusting the polysaccharide concentration, the pressure resistance of the sample is greatly reduced, indicating that the polysaccharide concentration has a great influence on the pressure resistance of the microspheres.

[0046] like Figure 2As shown, the present invention also provides a porous soft gel microsphere, which is obtained by the preparation method. The porous soft gel microsphere has a particle size of 50-80 μm; and the porous agarose microsphere has a porous structure.

Claims

1. A method for preparing porous soft gel microspheres with adjustable pore size, characterized in that: The following steps are involved: (1) adding biopolysaccharide to water, pressurizing and heating the system to hydrolyze the biopolysaccharide, and obtaining a clear aqueous phase after standing; (2) adding a surfactant to the solvent, heating and keeping the temperature, and allowing the solvent to stand to obtain a clear oil phase; (3) adding the aqueous phase obtained in step (1) to the oil phase obtained in step (2), stirring, keeping warm and then cooling to obtain a porous soft gel bare ball; (4) placing the porous soft gel bare ball obtained in step (3) in water and reacting with a crosslinking agent in an alkaline environment to obtain a porous microsphere intermediate; (5) hydrolyzing the porous microsphere intermediate obtained in step (4) under alkaline conditions, and screening out porous soft gel microspheres of a certain particle size after washing.

2. The method for preparing porous soft gel microspheres with adjustable pore size according to claim 1, characterized in that: In step (1), the system pressure is 0.05-0.25 MPa; and the hydrolysis time is 1.5-5 h.

3. The method for preparing porous soft gel microspheres with adjustable pore size according to claim 1, characterized in that: In step (1), the average molecular weight of the biopolysaccharide is 100 to 400 kDa.

4. The method for preparing porous soft gel microspheres with adjustable pore size according to claim 1, characterized in that: In the step (1), the biopolysaccharide is one or more of agarose and water-soluble chitosan; the concentration of the biopolysaccharide solution is 5wt% to 8wt%; and the standing time is 15 to 30 minutes.

5. The method for preparing porous soft gel microspheres with adjustable pore size according to claim 1, characterized in that: In step (2), the solvent is one or more of liquid paraffin, cyclohexane, and xylene; the surfactant is one or more of Span 80, polyethylene glycol, and sodium dodecyl sulfate; and the insulation temperature is 70-85°C.

6. The method for preparing porous soft gel microspheres with adjustable pore size according to claim 1, characterized in that: In step (3), the stirring linear speed is 2.5 m / s to 3.6 m / s; the stirring time is 1 to 3 h; the insulation temperature is 75 to 90° C.; and the cooling time is 0.5 to 1.2 h.

7. The method for preparing porous soft gel microspheres with adjustable pore size according to claim 1, characterized in that: In step (4), the cross-linking agent is one or more of 2,3-dibromopropanol diepoxide, 1,4-butanediol diglycidyl ether, and epichlorohydrin; the cross-linking reaction temperature is 40 to 60° C.; and the reaction time is 8 to 16 hours.

8. The method for preparing porous soft gel microspheres with adjustable pore size according to claim 1, characterized in that: In step (4), the alkaline environment is one or more of 10-40wt% sodium hydroxide solution and 10-40wt% potassium hydroxide solution.

9. The method for preparing porous soft gel microspheres with adjustable pore size according to claim 1, characterized in that: The particle size described in step (5) is 50 to 80 μm.

10. A porous soft gel microsphere, characterized in that: The porous soft gel microspheres are obtained by the preparation method according to any one of claims 1 to 9.

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