A method for preparing agarose-based gel microspheres
By using an esterifying agent to modify and perform alkali treatment during the preparation of agarose-based gel microspheres, the problem of insufficient mechanical strength of agarose-based gel microspheres was solved, and good flow performance at high flow rates and a simplified preparation process were achieved.
Patent Information
- Application Number
- CN202411404468.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-09
- Publication Date
- 2025-10-03
- Estimated Expiration
- 2044-10-09
AI Technical Summary
Agarose-based gel microspheres have low mechanical strength and are difficult to work at high flow rates. Existing cross-linking methods are cumbersome and use a lot of organic reagents.
During the preparation of agarose-based gel microspheres, their pressure resistance was improved by modification with an esterifying agent and alkali treatment at different stages.
The flow properties of agarose-based gel microspheres are improved, enabling their application at higher flow rates, simplifying the preparation process, and maintaining the skeleton structure and biocompatibility of the polysaccharide.
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Figure CN119034706B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of agarose, and in particular to a method for preparing agarose-based gel microspheres. Background Art
[0002] Agarose is a natural polysaccharide, refined and processed from agar extracted from marine red algae. It has a wide range of applications in biology, chemical engineering, medicine, and the environment. Microspherized agarose gel exhibits excellent biocompatibility and pH stability, allowing it to gently permeate biomacromolecules without denaturation. The presence of large internal pores also increases the separation range for biomacromolecules.
[0003] Agarose microspheres are hydrogels containing double helices arranged into bundles through hydrogen bonds. The molecular chains form a network structure, ultimately forming pores. The presence of pores facilitates the transport of protein molecules through the gel network; varying pore sizes also result in varying protein transit times. However, the agarose-based gel microsphere matrix is relatively soft and mechanically weak, making it difficult to operate at high flow rates. Therefore, cross-linking and activation methods are commonly used to enhance the mechanical strength of agarose-based gel microspheres. However, this method requires a large number of organic reagents and is complex. Summary of the Invention
[0004] The purpose of the present invention is to solve the problem of poor pressure resistance of agarose-based gel microspheres. By performing alkali treatment at different stages of the preparation of agarose-based gel microspheres and performing alkali treatment on the agarose-based gel microspheres after cross-linking and activation to improve their pressure resistance, a method for preparing agarose-based gel microspheres is proposed.
[0005] The inventors unexpectedly discovered in experiments that alkali treatment increases the gel strength of modified agarose. They subsequently applied this method to the preparation of agarose-based gel microspheres. Agarose is first modified with an esterifying agent, and then used to prepare agarose-based gel microspheres. After alkali treatment of the agarose solution or agarose-based gel microspheres, the flow properties of the agarose-based gel microspheres are significantly improved, making them suitable for chromatography experiments at higher flow rates.
[0006] To achieve the above object, the present invention provides a method for preparing agarose-based gel microspheres, which is characterized by comprising the following steps:
[0007] S1. Fully mixing agarose and an ethanol solution to obtain a mixed solution, adding an esterifying agent to the mixed solution to modify the agarose, filtering and washing, and drying and crushing the obtained solid to obtain modified agarose;
[0008] S2, by modified agarose, or, a mixture of modified agarose and dextran or konjac gum, is added to water and dissolved at 70-120 ℃ to obtain an agarose aqueous solution with a mass fraction of 2-8%, or a mixed solution of 2-8% agarose and dextran or konjac gum;
[0009] S3, preparation of first agarose-based gel microspheres: preparing first agarose-based gel microspheres from the agarose aqueous solution or mixed solution obtained in S2;
[0010] S4, adding an alkaline solution to the agarose aqueous solution obtained in S2, mixing, and then preparing agarose-based gel microspheres, and washing them; or
[0011] Soaking the first agarose-based gel microspheres obtained in S3 in an alkaline solution and washing them clean; or
[0012] Cross-linking and activating the first agarose-based gel microspheres obtained in S3 with a cross-linking agent in an alkaline environment, washing them clean, and then immersing the cross-linked agarose-based gel microspheres in an alkaline solution and washing them clean;
[0013] Agarose-based gel microspheres were obtained.
[0014] Furthermore, in step S1, the esterification agent is any one of an organic acid, an acid anhydride, and an acid chloride. Preferably, the organic acid is valeric acid; the acid anhydride is any one of succinic anhydride, glutaric anhydride, and maleic anhydride; and the acid chloride is acetyl chloride.
[0015] The esterifying agent is dissolved in an organic solvent and added dropwise during the reaction or directly added in batches during the reaction.
[0016] Furthermore, in step S2, the mass ratio of the modified agarose to dextran or konjac gum is 1:0-0.5.
[0017] Furthermore, in step S4, the cross-linking agents are epichlorohydrin and 1,4-dioxane, and the amount of cross-linking agent added is 4% of the mass of the first agarose-based gel microspheres; preferably, the cross-linking agents include epichlorohydrin and 1,4-dioxane in approximately equal amounts.
[0018] Furthermore, in step S4, the base is at least one of sodium hydroxide, potassium hydroxide, and sodium carbonate.
[0019] Furthermore, in step S4, the concentration of the alkaline solution is 0.1M-0.2M.
[0020] The advantages of the present invention are:
[0021] The present invention directly adds an alkaline solution to the modified agarose and then performs an emulsification reaction, thereby directly obtaining agarose-based gel microspheres with good flow performance. The preparation process is simple and does not require a multi-step or repeated cross-linking process.
[0022] The present invention soaks the formed agarose-based gel microspheres in an alkaline solution, or soaks the cross-linked agarose-based gel microspheres in an alkaline solution, thereby improving the flow performance of the agarose-based gel microspheres without changing the skeleton structure of the natural polysaccharide and the good hydrophilicity and biocompatibility of the microspheres.
[0023] The invention provides a method for preparing agarose-based gel microspheres. The method utilizes different alkalis to treat different stages of agarose-based gel microspheres to obtain agarose-based gel microspheres with different flow properties, thereby realizing the wide application of agarose-based gel microspheres. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 It is an infrared analysis diagram of the microspheres without alkali treatment in Comparative Examples 3 and 6 of the present invention and the microspheres after alkali treatment in Examples 1-3.
[0025] Figure 2 These are optical microscope images and particle size distribution diagrams of agarose microspheres with different modified agarose concentrations (2%, 4%, and 8%) prepared by the emulsification solidification method in Comparative Example 3 of the present invention.
[0026] Figure 3 It is an optical microscope image and particle size distribution diagram of the composite polysaccharide microspheres (1:0.5) prepared by the emulsification solidification method in Comparative Example 4 of the present invention.
[0027] Figure 4 It is an optical microscope image and particle size distribution diagram of the composite polysaccharide microspheres (1:0.5) prepared by the spray cooling method in Comparative Example 5 of the present invention.
[0028] Figure 5 These are optical microscope images and particle size distribution diagrams of the cross-linked activated modified agarose microspheres before and after alkali treatment in Comparative Example 6 and Example 3 of the present invention.
[0029] Figure 6 1 is an optical microscope image and a particle size distribution diagram of agarose-based microspheres prepared by adding an alkaline solution to the modified agarose solution in Example 1 of the present invention.
[0030] Figure 7 1 and 2 are optical microscope images and particle size distribution diagrams of the modified agarose microspheres before and after being immersed in different types of alkali solutions in Example 2 of the present invention.
[0031] Figure 8 It is a flow rate pressure test graph of the microspheres without alkali treatment in Comparative Examples 3 and 6 of the present invention and the microspheres after alkali treatment in Examples 1-3. DETAILED DESCRIPTION
[0032] Below in detail embodiments of the present invention, the example of described embodiment is shown in the accompanying drawings, wherein identical or similar reference numerals represent identical or similar elements or elements with identical or similar functions throughout. The embodiment described below by reference to the accompanying drawings is exemplary, is intended to be used for explaining the present invention, and is not to be construed as limiting the present invention. In the embodiment, those not indicating specific techniques or conditions are carried out according to the technology or conditions described in the document in this area or according to the product specification. Reagents used or instruments not indicating manufacturers are conventional products that can be obtained commercially.
[0033] 1. Experimental methods:
[0034] 1. Determination of Agarose Gel Strength
[0035] Prepare 1.5 wt% glutaric anhydride modified agarose solution, heat it in a boiling water bath until it is completely dissolved, pour the glue solution into the plate until it is just full, cool it to room temperature, solidify it, and let it stand overnight. Place the plate on the left tray of the tray balance, and 2 Secure the plunger just enough to touch the gel. Place a beaker on the tray to the right of the balance and slowly and evenly pour distilled water into the beaker. Stop pouring water as soon as the surface of the gel breaks. Record the weight of the beaker and distilled water at this point. This value represents the gel strength of the glutaric anhydride-modified agarose. Use unmodified agarose as a control.
[0036] 2. Determination of degree of substitution
[0037] Take 1.00g modified agarose sample in beaker, add the hydrochloric acid-ethanol solution of appropriate 2.5mol / L, be placed on magnetic stirring apparatus and stir 30min, then use deionized water and 60%vol ethanol alternating washing, filtrate AgNO solution check until finish washing (chloride ion check: appropriate sample filtrate is poured in test tube, adds 0.01mol / L AgNO solution, with 60%vol ethanol and deionized water in contrast, compares three colors, if all do not have white turbidity to produce then do not have chloride ion), then 100mL deionized water is added in sample and carries out microwave heating and dissolves, finally drip phenolphthalein, be titrated to light pink and 1min with 0.01mol / LNaOH solution and do not fade.Now record the NaOH solution volume having consumed, take former agar as blank, operate as stated above, after the NaOH solution volume that record consumes, according to formula calculation.
[0038]
[0039] A=CV / M.
[0040] Where:
[0041] 0.306: molar mass of agarobiose molecule, (g / mmol);
[0042] 0.098: molar mass of maleic anhydride, (g / mmol);
[0043] C: concentration of NaOH solution, (mol / L);
[0044] V: volume of NaOH solution consumed, (mL);
[0045] M: mass of agarose sample, (g).
[0046] 3. Use conventional testing methods to test the physical and chemical properties of glutaric anhydride-modified agarose, including transparency, solidification temperature, and melting temperature. Use unmodified agarose as a control.
[0047] 4. Morphological appearance and particle size test
[0048] The morphology of the prepared microspheres was characterized by optical microscopy: wet agarose-based gel microspheres were dropped onto the surface of a glass slide, covered with a cover glass, and observed and photographed using an ML11_II optical microscope.
[0049] The average particle size and particle size distribution are measured using a laser particle size analyzer: a sample is prepared by uniformly dispersing agarose-based gel microspheres in distilled water, and an appropriate amount of the wet sample is added dropwise to the measuring cup of the laser particle size analyzer.
[0050] 5. Flow rate-pressure test:
[0051] Different types of agarose gel microspheres were sieved through a standard 80-325 mesh sieve and loaded into a 5ml chromatography column. A constant flow rate was introduced. After the column bed stabilized, the maximum flow rate was measured using a constant pressure method. The pressure was gradually increased in intervals. After stabilization for 5 minutes, the corresponding flow rate at the corresponding pressure was recorded. When the flow rate stabilized within a certain pressure range, the maximum flow rate of the microsphere medium was obtained, and a flow rate-pressure curve was plotted.
[0052] 6. Infrared spectroscopy
[0053] The agarose sample and potassium bromide powder were dried and placed in an agate mortar in a certain proportion, mixed and ground thoroughly, and then transferred to a grinding tool for tableting. The sample was measured using a Fourier transform infrared spectrometer.
[0054] 7. Preparation of agarose-based gel microspheres (or first agarose-based gel microspheres):
[0055] The agarose-based gel microspheres are prepared by conventional methods, for example, the obtained agarose aqueous solution or mixed solution is added as the aqueous phase to the oil phase containing the emulsifier, stirred and emulsified at 90° C. for 10 minutes, and then cooled to below 25° C. for solidification for 5-15 minutes to obtain agarose-based gel microspheres; or the agarose aqueous solution or mixed solution is sprayed into mist droplets through an atomizer, and then naturally cooled and settled into a collection container at a certain height to form agarose-based gel microspheres.
[0056] 2. Experimental steps
[0057] 1 (i.e., step S1) Agarose modification part: The comparative examples are all original agarose, and the original agarose is directly tested for properties according to the experimental method
[0058] Comparative Examples - 1, 2 Esterification modification of agarose
[0059] (1) Preparation of agarose modified with organic acid, acyl chloride, and different anhydrides - Comparative Example 1
[0060] Disperse 15 g of agarose in 200 mL of 80% vol ethanol to prepare an agarose suspension with a mass concentration of 7.5% (W / V); set the stirring temperature to 30° C. (not limited to this temperature, as long as it dissolves), and dissolve the agarose suspension by stirring to form a stable agarose solution;
[0061] While stirring continuously, add 5% succinic anhydride-ethanol solution, 5% glutaric anhydride-ethanol solution, 5% maleic anhydride-ethanol solution, 5% acetyl chloride-ethanol solution, and 5% valeric acid-ethanol solution to the agarose solution, and simultaneously add 3M NaOH solution to adjust the pH of the system to 8-8.5;
[0062] After the succinic anhydride-ethanol solution, glutaric anhydride-ethanol solution, maleic anhydride-ethanol solution, acetyl chloride-ethanol solution, and valeric acid-ethanol solution are added dropwise within 1 hour, stirring is continued for 1 hour and the reaction is completed;
[0063] After the reaction is completed, the residue is washed and filtered several times with anhydrous ethanol, 80% vol ethanol and a vacuum filter to obtain a filter residue;
[0064] The filter residue was placed in a 55° C. forced air drying oven and dried for 12 h, then pulverized and passed through an 80-mesh sieve to obtain modified agarose powder.
[0065] (2) Preparation of Glutaric Anhydride Modified Agarose with Different Concentrations - Comparative Example 2
[0066] Disperse 15 g of agarose in 200 mL of 80% vol ethanol to prepare an agarose suspension with a mass concentration of 7.5% (W / V); set the stirring temperature to 30°C and dissolve the agarose suspension by stirring to form a stable agarose solution;
[0067] While stirring continuously, add 1%, 3%, 5%, and 7% glutaric anhydride-ethanol solutions to the agarose solution, and simultaneously add 3M NaOH solution to adjust the pH of the system to 8-8.5;
[0068] After the glutaric anhydride-ethanol solution was added dropwise within 1 hour, stirring was continued for 1 hour and the reaction was completed;
[0069] After the reaction is completed, the residue is washed and filtered several times with anhydrous ethanol, 80% vol ethanol and a vacuum filter to obtain a filter residue;
[0070] The filter residue was placed in a 55° C. forced air drying oven and dried for 12 h, then pulverized and passed through an 80-mesh sieve to obtain modified agarose powder.
[0071] 2. (i.e., steps S2 and S3): Preparation of the first agarose-based gel microspheres (two methods were used: the emulsification solidification method in Comparative Examples 3 and 4; and the spray cooling method in Comparative Example 5)
[0072] The modified agarose used below is the agarose modified with the 5% glutaric anhydride-ethanol solution in Comparative Example 1.
[0073] Comparative Example 3- Figure 2 (2%, 4%, 8% modified agarose microspheres by emulsion solidification method);
[0074] Comparative Example 4- Figure 3 (mixed modified agarose microspheres);
[0075] Preparation of the first agarose-based gel microspheres by emulsification solidification method (the modified agarose concentration is 2%, 4%, 8%)
[0076] At 90° C., 1.8, 3.6, or 7.2 g of modified agarose, or modified agarose (1.2, 2.4, or 4.8 g) and dextran or konjac gum (0.6, 1.2, or 2.4 g) were dissolved in 90 mL of water to obtain aqueous agarose solutions or mixed solutions with mass fractions of 2%, 4%, or 8% as the aqueous phase;
[0077] Liquid paraffin (containing 2% Span80) was heated to 80°C as the oil phase, and all the aqueous phase was added to the oil phase. The mixture was stirred at 80°C and 1000 rpm for 10 minutes, and then cooled to below 20°C within 10 minutes. After washing, agarose-based gel microspheres were obtained.
[0078] Comparative Example 5- Figure 4 .
[0079] Preparation of the first agarose-based gel microspheres by spray cooling method (with agarose concentration of 4%) - This method was only used to prepare agarose-based gel microspheres without any alkali treatment experiments.
[0080] 4 g of modified agarose, or 2.66 g of modified agarose and 1.33 g of dextran or konjac gum, were dissolved in 100 mL of water at 90° C. to obtain a 4% agarose aqueous solution or mixed solution as the aqueous phase;
[0081] The agarose aqueous solution or mixed solution is sprayed into mist droplets through an atomizer, and the spray pressure is adjusted to 0.25 MPa;
[0082] The distance between the atomizing nozzle and the surface of the collected liquid was 160 cm. The mist droplets settled in the air at this height and naturally cooled to form agarose-based gel microspheres. The air temperature was 25°C.
[0083] Comparative Example 6 - Results Figure 5 .
[0084] 3. (i.e., step S4) cross-linking activation of agarose gel microspheres
[0085] 20 g of the 4% first agarose-based gel microspheres obtained in the emulsification solidification step were weighed and added to a system containing 12 ml of water, 8 ml of epichlorohydrin, 8 ml of 5 M sodium hydroxide solution and 8 ml of 1,4-dioxane. The mixture was stirred at 30°C and 250 rpm for 2 h to obtain cross-linked activated agarose-based gel microspheres.
[0086] 4. Example 1 ( Figure 6 )、Example 2( Figure 7 )、Example 3( Figure 5 , same figure as Comparative Example 6) - Alkali treatment method of agarose-based gel microspheres (mainly 4% modified agarose microspheres prepared by emulsification solidification method)
[0087] (1) Preparation of agarose-based gel microspheres by alkali treatment of modified agarose solution - Example 1
[0088] S1. 15 g of agarose and 200 ml of 80% vol ethanol solution were thoroughly mixed to obtain a mixture. 5% glutaric anhydride ethanol solution was added to the mixture to modify the agarose. The mixture was filtered and washed. The resulting solid was dried and crushed to obtain modified agarose.
[0089] S2. Add 3.6 g of modified agarose to 90 ml of water and dissolve at 120°C to obtain a 4% agarose aqueous solution;
[0090] S4. Add 9 ml of 0.2 M sodium carbonate solution to the agarose aqueous solution obtained in S2, mix well to prepare the first agarose-based gel microspheres, and wash them clean.
[0091] (2) Alkali treatment of modified agarose gel microspheres - Example 2
[0092] S1. 15 g of agarose and 200 ml of 80% vol ethanol solution were thoroughly mixed to obtain a mixture. 5% glutaric anhydride ethanol solution was added to the mixture to modify the agarose. The mixture was filtered and washed. The resulting solid was dried and crushed to obtain modified agarose.
[0093] S2. Add 3.6 g of modified agarose to 90 ml of water and dissolve at 120°C to obtain a 4% agarose aqueous solution;
[0094] S3, preparing the agarose aqueous solution obtained in S2 into first agarose-based gel microspheres;
[0095] S4. Soak the first agarose-based gel microspheres obtained in S3 in 0.2 M sodium carbonate solution, 0.2 M sodium hydroxide solution, and 0.2 M potassium hydroxide solution for 24 hours, respectively, to obtain agarose-based gel microspheres treated with different alkalis.
[0096] (3) Alkali treatment of cross-linked activated microspheres prepared from modified agarose - Example 3
[0097] S1. 15 g of agarose and 200 ml of 80% vol ethanol solution were thoroughly mixed to obtain a mixture. 5% glutaric anhydride ethanol solution was added to the mixture to modify the agarose. The mixture was filtered and washed. The resulting solid was dried and crushed to obtain modified agarose.
[0098] S2. Add 3.6 g of modified agarose to 90 ml of water and dissolve at 120°C to obtain a 4% agarose aqueous solution;
[0099] S3, preparing the agarose aqueous solution obtained in S2 into first agarose-based gel microspheres;
[0100] S4. The first agarose-based gel microspheres obtained in S3 are cross-linked and activated according to the steps of Comparative Example 6. After washing, the cross-linked agarose-based gel microspheres are immersed in a 0.2 M sodium carbonate solution for 24 hours. After washing, the cross-linked activated agarose-based gel microspheres are obtained after alkali treatment.
[0101] 3. Experimental Results
[0102] 1. Esterified modified agarose and its property characterization
[0103] (1) Characterization of properties of agarose modified with organic acids, acyl chlorides and different anhydrides - Comparative Example 1 data table
[0104] Table 1 Comparative Example 1 Data Table
[0105]
[0106] The results showed that the degree of substitution of agarose modified with valeric acid, acetyl chloride, and various anhydrides varied. This was primarily due to an esterification reaction, in which the free hydroxyl groups in the agarose were replaced by ester groups. Different anhydrides, valeric acid, and acetyl chloride reacted with different degrees of esterification in an alkaline environment, resulting in varying degrees of ester substitution for hydroxyl groups.
[0107] (2) Characterization of properties of agarose modified with different glutaric anhydride concentrations - Comparative Example 2 data table
[0108] Table 2 Comparative Example 2 Data Table
[0109]
[0110] The results showed that the gel strength of agarose modified with different concentrations of glutaric anhydride decreased significantly. This is because the ester groups in the anhydride replaced the free hydroxyl groups in the agarose, loosening the molecular structure and changing the order to disorder. Consequently, the gel strength of the modified agarose decreased. Accordingly, different concentrations of glutaric anhydride contain different numbers of ester groups, and the degree of substitution with hydroxyl groups also varies. When the glutaric anhydride concentration was 7%, the degree of substitution was the highest, and the gel strength of the modified agarose was the lowest.
[0111] In addition, the transparency is also significantly improved due to the introduction of hydrophilic groups, which enhances the dispersion of agarose molecules in water and increases the permeability of light to it.
[0112] In addition, the solidification temperature and melting temperature decreased, which was due to the introduction of ester groups weakening the binding force between agarose molecules.
[0113] (3) Infrared spectroscopy analysis (original agarose, modified agarose, modified agarose microspheres before alkali treatment, modified agarose microspheres after alkali treatment (cross-linking activation)) Figure 1
[0114] The glutaric anhydride modified agarose prepared in Comparative Example 2, the cross-linked activated modified agarose microspheres prepared in Comparative Example 6, the modified agarose solution prepared in Example 1 was added with 0.2M sodium carbonate to prepare microspheres, the modified agarose microspheres prepared in Example 2 were soaked in different alkaline solutions: the modified agarose microspheres were soaked in 0.2M sodium carbonate, the modified agarose microspheres were soaked in 0.2M sodium hydroxide and the modified agarose microspheres were soaked in 0.2M potassium hydroxide, and the cross-linked activated modified agarose microspheres prepared in Example 3 were soaked in 0.2M sodium carbonate. Infrared spectroscopy analysis was performed, and the analysis results are shown in FIG. Figure 1 The results showed that when the esterification reaction occurred, 1720cm -1 and 1570cm -1 New absorption peaks are generated at 1720cm -1 The stretching vibration absorption peak of C=O is 1570cm-1 The antisymmetric stretching vibration of the free -COO- is shown at 1720 cm. This indicates that esterification occurs after agarose is modified with glutaric anhydride. The infrared spectrum shows that the ester group can be hydrolyzed by adding alkali to the modified agarose solution, treating the modified agarose microspheres with alkali, or performing cross-linking activation (also in the presence of high concentrations of alkali). -1 The absorption peak of -C=O at the position is weakened or disappears. Therefore, the process of alkali treatment of modified agarose microspheres or cross-linked modified agarose microspheres is essentially a process of using the hydroxyl groups contained in the alkali solution to hydrolyze the ester groups contained in the modified agarose, thereby rearranging the gel network structure. After alkali treatment, the loose network structure of the modified agarose becomes ordered and aggregated, thereby improving the pressure flow rate (circulation) performance of the modified agarose microspheres.
[0115] (4) Microscope image and particle size distribution diagram
[0116] Comparative Example 3— Figure 2
[0117] As can be seen from the figure, with the increase of agarose concentration, the particle size distribution of agarose gel microspheres gradually moves towards the direction of increasing particle size. The average particle size of 2% modified agarose gel microspheres is 24 μm, and the average particle size of 8% modified agarose gel microspheres increases to 103 μm. This is because the agarose concentration is positively correlated with the viscosity of the liquid. The viscosity of the liquid will hinder the shear force of stirring, and the degree of hindrance increases with the increase of liquid viscosity.
[0118] Comparative Example 4- Figure 3
[0119] Depend on Figure 3 It can be seen that the sphericity of the two mixed microspheres is good and the monodispersity is good. The average particle size of the microspheres prepared by mixing modified agarose and dextran is smaller than that of the microspheres prepared by mixing modified agarose and konjac gum, which is proportional to the viscosity of the mixed solution of dextran, konjac gum and agarose. Under the same preparation conditions, the higher the solution viscosity, the larger the particle size of the obtained microspheres.
[0120] Comparative Example 5- Figure 4
[0121] Depend on Figure 4 It can be seen that modified agarose and modified agarose compounded with dextran or konjac gum can successfully prepare agarose-based gel microspheres under spray conditions. The particle size distribution of the composite microspheres is wider than that of the 4% modified agarose gel microspheres, that is, the particle size is more uneven. This is because the viscosity of the composite solution is greater than that of the 4% modified agarose solution, the degree of obstruction during atomization is increased, and the atomization effect is worse.
[0122] Comparative Example 6 and Example 3- Figure 5
[0123] like Figure 5 As shown, there is no significant change in the morphology and particle size distribution of the cross-linked activated agarose gel microspheres before and after alkali treatment. Example 1 (Preparation of agarose gel microspheres (4%) by adding sodium carbonate to the modified agarose solution) Figure 6 , Figure 6 These are the microscope images and particle size images of the microspheres prepared in Example 1. Observation under a microscope shows that the microspheres have good morphology, good dispersed phase, and relatively concentrated particle size distribution.
[0124] Example 2 - 4% modified agarose gel microspheres before and after different alkali treatment (0.2M sodium hydroxide, 0.2M sodium carbonate, 0.2M potassium hydroxide) - Figure 7 , Figure 7 This shows that the morphology of 4% modified agarose gel microspheres remains smooth after treatment with different alkalis, and the particle size distribution does not change significantly.
[0125] (5) Pressure and flow rate Figure 8
[0126] Emulsion solidification method: modified agarose microspheres, microspheres prepared from alkali-treated modified agarose solution, modified agarose microspheres treated with different alkalis, cross-linked activated modified agarose microspheres, and alkali-treated cross-linked activated modified agarose microspheres.
[0127] Depend on Figure 8 It can be seen that the mechanical properties of 4% modified agarose gel microspheres after different alkali treatments are significantly improved compared with those of 4% modified agarose gel microspheres. Among them, the pressure-flow rate of the modified agarose gel microspheres after 0.2M immersion is 211.11% higher than that of the 4% modified agarose gel microspheres. This is because the ester groups contained in the modified agarose are hydrolyzed after alkali treatment, and the gel strength of the modified agarose is improved. Accordingly, the modified agarose gel microspheres after alkali treatment improve the pressure-flow rate (circulation) performance of the modified agarose microspheres without changing the skeleton structure of the natural polysaccharide and the good hydrophilicity and biocompatibility of the microspheres, which is consistent with the results of the infrared spectrum. The pressure flow rate of the modified agarose microspheres after alkali treatment was similar to that of the modified agarose microspheres activated by direct cross-linking. This was because the alkaline solution was also present during cross-linking activation, the ester groups were hydrolyzed by the hydroxyl groups, and a cross-linking reaction occurred. The mechanical properties of the cross-linked and activated modified agarose microspheres were better after alkali treatment. In addition to the effect of cross-linking activation, the gel strength of the modified agarose was further improved because the ester groups contained in the modified agarose continued to fall off during the cross-linking activation and alkali treatment process.
[0128] Table 3 Determination of degree of substitution of agarose gel microspheres treated with different alkalis in Example 1-3
[0129]
[0130] As shown in the table above, the degree of substitution of microspheres prepared from modified agarose solutions treated with alkali, or after different alkali treatments, is significantly reduced, approaching zero. This is because the ester groups in the modified agarose microspheres are almost completely hydrolyzed by hydroxyl groups, resulting in an ordered gel network structure and improved pressure flow rate (flow) performance. The degree of substitution of modified agarose microspheres treated with direct alkali treatment is significantly lower than that of modified agarose microspheres treated with direct crosslinking. This is because hydroxyl groups can directly hydrolyze ester groups during direct alkali treatment. Although the crosslinking activation process occurs in an alkaline environment, the degree of hydrolysis is affected by other factors, such as temperature and crosslinking agent.
[0131] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.
Claims
1. A method for preparing agarose-based gel microspheres, characterized in that: The steps are as follows: S1. Fully mixing agarose and an ethanol solution to obtain a mixed solution, adding an esterifying agent to the mixed solution to modify the agarose, filtering and washing, and drying and crushing the obtained solid to obtain modified agarose; S2, by modified agarose, or, a mixture of modified agarose and dextran or konjac gum, is added to water and dissolved at 70-120 ℃ to obtain an agarose aqueous solution with a mass fraction of 2-8%, or a mixed solution of 2-8% agarose and dextran or konjac gum; S3, preparing the agarose aqueous solution or mixed solution obtained in S2 into first agarose-based gel microspheres; S4, adding an alkaline solution to the agarose aqueous solution obtained in S2, mixing, and then preparing agarose-based gel microspheres, and washing them; or Soaking the first agarose-based gel microspheres obtained in S3 in an alkaline solution and washing them clean; or Cross-linking and activating the first agarose-based gel microspheres obtained in S3 with a cross-linking agent in an alkaline environment, washing them clean, and then immersing the cross-linked agarose-based gel microspheres in an alkaline solution and washing them clean; Agarose-based gel microspheres were obtained.
2. The preparation method according to claim 1, characterized in that In step S1, the esterification agent is any one of organic acid, acid anhydride, and acid chloride. The esterifying agent is dissolved in an organic solvent and added dropwise during the reaction or directly added in batches during the reaction.
3. The preparation method according to claim 2, characterized in that: In step S1, the organic acid is valeric acid; the acid anhydride is any one of succinic anhydride, glutaric anhydride, and maleic anhydride; and the acyl chloride is acetyl chloride.
4. The preparation method according to claim 1, characterized in that In step S2, the mass ratio of the modified agarose to dextran or konjac gum is 1:0-0.
5.
5. The preparation method according to claim 1, characterized in that: In step S4, the cross-linking agents are epichlorohydrin and 1,4-dioxane, and the amount of the cross-linking agents added is 4% of the mass of the first agarose-based gel microspheres.
6. The preparation method according to claim 5, characterized in that: In step S4, the cross-linking agent contains approximately equal amounts of epichlorohydrin and 1,4-dioxane.
7. The preparation method according to claim 1, characterized in that: In step S4, the base is at least one of sodium hydroxide, potassium hydroxide, and sodium carbonate.
8. The preparation method according to claim 1, characterized in that: In step S4, the concentration of the alkaline solution is 0.1M-0.2M.
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