A novel multinuclear composite agarose gel microsphere and its preparation method

Abstract

This invention discloses a novel multi-core composite agarose gel microsphere and its preparation method. The core of the microsphere consists of agarose microspheres with a particle size of no more than 60 μm, which are uniformly dispersed inside the microsphere. The shell of the microsphere is a neutral polysaccharide composite material. First, the core agarose microspheres are prepared, then they are cross-linked and activated. Finally, the activated and cross-linked agarose gel microspheres are used as the core, and the pre-cross-linked polysaccharide gel microspheres are used as the shell to prepare the novel multi-core composite agarose gel microsphere. This core-shell type microsphere combines the fine pore structure of the core with the macropore structure of the shell. The high-resolution chromatography medium prepared after cross-linking treatment has a rich pore structure that facilitates efficient mass transfer of biomolecules within the medium, and its mechanical strength is also greatly improved.

Description

Technical Field

[0001] This invention relates to the field of bioseparation engineering technology, and in particular to a novel multinucleated composite agarose gel microsphere and its preparation method. Background Technology

[0002] Agarose is a natural marine linear polysaccharide isolated from red algae such as Gracilaria, Porphyra, and others. It possesses high hydrophilicity, electroneutrality, abundant hydroxyl groups, and thermoreversible sol-gel properties. Gel microspheres prepared from agarose are among the most ideal polysaccharide-based chromatographic media. They are important carrier tools in the field of bioengineering separation and purification, and can be prepared into gel filtration chromatography media, ion exchange chromatography media, and hydrophobic chromatography media, widely used for the separation and purification of biomolecules such as proteins, cells, nucleic acids, antibodies, and enzymes.

[0003] Dextran is a glucose polymer produced by bacteria cultured in sucrose through a catalytic reaction. Its molecular formula is (C6H2O)2O2. 10 Based on the type of glycosidic bond, dextran can be divided into α-glucan and β-glucan, with α-glucan being more commonly used and known as dextran. Dextran possesses excellent biodegradability, gelling properties, biocompatibility, hydrophilicity, and pH and temperature stability, making it an excellent raw material for the preparation of polysaccharide microspheres. Grafting branched dextran chains at multiple sites onto the surface and pores of agarose media allows for the utilization of the three-dimensional adsorption space provided by dextran, offering advantages such as higher adsorption capacity and accelerated mass transfer rate.

[0004] The rapid development of life sciences and technologies has placed higher demands on bio-separation and purification technologies and media. In the future, this field will primarily focus on developing efficient, high-throughput, and rapid chromatography media materials. To address the problems of low separation efficiency of polysaccharide-based chromatography media for large biomolecules, significant raw material loss, structural damage leading to inactivation, and poor pressure resistance, there is an urgent need to develop a separation and purification media with high resolution, high flow rate, and high pressure resistance. Summary of the Invention

[0005] This invention aims to improve upon the shortcomings of traditional agarose microspheres as a soft gel matrix, which suffer from poor mechanical strength and a simple internal network structure. A novel multi-core composite agarose gel microsphere is developed, featuring cross-linked agarose gel microspheres as the core and pre-cross-linked polysaccharide gel microspheres as the shell. This core-shell microsphere combines the fine pore structure of the core with the macropore structure of the shell. The high-resolution chromatography medium prepared through cross-linking treatment exhibits a rich pore structure that facilitates efficient mass transfer of biomolecules within the medium, while also significantly improving mechanical strength.

[0006] To achieve the above objectives, the present invention provides a novel multi-core agarose gel microsphere, wherein the core of the microsphere is agarose microspheres with a particle size of no more than 60 μm, and the agarose microspheres are uniformly dispersed inside the microsphere; the shell of the microsphere is a neutral polysaccharide composite material.

[0007] Furthermore, its preparation method includes the following steps:

[0008] S1. Preparation of kernel agarose microspheres, including:

[0009] S1.1. Preparation of the first oil phase: The surfactant and the first oily substance that is immiscible with water are mixed;

[0010] S1.2. Preparation of the first aqueous phase: Prepare a polysaccharide solution containing 4-6% w / v agarose, heat until clear and transparent, and completely dissolved;

[0011] S1.3. Pour the prepared first aqueous phase into the prepared first oil phase that is being stirred to emulsify, then cool and solidify to solidify the agarose droplets into microspheres.

[0012] S1.4. Centrifuge to break the emulsion and separate the layers, discard the supernatant, and wash repeatedly until clean to obtain the kernel agarose microspheres; preferably, the centrifugation is performed at 3500 rpm for 5 min;

[0013] S2. Cross-linking and activation of kernel agarose microspheres, the specific steps of which include:

[0014] S2.1. Mix the obtained kernel agarose microspheres with water, concentrated alkali, 1,4-dioxane and the first crosslinking agent until homogeneous;

[0015] S2.2. React the above-obtained mixture at 150-400 rpm and 20-35℃ for 2-6 h;

[0016] S2.3. The above-obtained material is repeatedly washed with ultrapure water or ethanol to remove the NaOH solution and organic solvent, thereby obtaining the post-crosslinked core agarose microspheres.

[0017] S3. Obtaining novel multinuclear agarose gel microspheres:

[0018] S3.1 Preparation of the second oil phase: Mix the surfactant and the second oily substance that is immiscible with water;

[0019] S3.2 Preparation of the second aqueous phase: Prepare a polysaccharide solution containing 4-6% w / v agarose, heat until clear and transparent to completely dissolve it, then add the post-crosslinked core agarose microspheres and mix evenly; if there are bubbles, heat to defoam;

[0020] S3.3 Pour the prepared second aqueous phase into the prepared second oil phase that is being stirred, continue stirring to emulsify, and then solidify; so that the W / O emulsion droplets cool and solidify into agarose gel microspheres;

[0021] S3.4 Add concentrated alkali to react with the second crosslinking agent;

[0022] S3.5 Centrifugation causes the layers to separate, the supernatant is discarded, and the mixture is repeatedly washed until clean to obtain novel multinucleated agarose gel microspheres; preferably, the centrifugation is performed at 3500 rpm for 5 min.

[0023] Furthermore, in step S1.1, the surfactant is 0.75-2% w / v Span 80, and the first oily substance is liquid paraffin.

[0024] Furthermore, in step S1.2, the polysaccharide solution also contains dextran.

[0025] Further, in step S1.3, the emulsification is carried out at 800-1400 rpm for 10-30 min; the solidification is carried out at 500-600 rpm with the temperature reduced to 15-25℃; and the volume ratio of the liquid paraffin to the polysaccharide solution of agarose is 200:60.

[0026] Further, in step S2.1, the first crosslinking agent is at least one of a halogenated epoxy compound and a diglycidyl ether reagent; preferably, the halogenated epoxy compound is epichlorohydrin, and the diglycidyl ether reagent is selected from any one of ethylene glycol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.

[0027] Optionally, the ratio of the kernel agarose microspheres: concentrated alkali: crosslinking agent is 36g:14ml:14ml.

[0028] Furthermore, in step S3.1, the surfactant is 0.75-2% w / v Span 80, and the second oily substance is liquid paraffin.

[0029] Furthermore, in step S3.2, the polysaccharide solution also contains dextran; the mass ratio of the post-crosslinked core agarose microspheres to the polysaccharide solution is 1:2-5.

[0030] Furthermore, in step S3.3, the stirring speed for continued emulsification is 800-1400 rpm, and the emulsification time is 10-30 min;

[0031] Optionally, the curing process involves cooling the temperature to 15-25°C at 500-600 rpm.

[0032] Optionally, the ratio of liquid paraffin: agarose-containing polysaccharide solution: post-crosslinked core agarose microspheres is 180 ml: 36 g: 18 g.

[0033] Furthermore, in step S3.4, the second crosslinking agent is at least one of a halogenated epoxy compound and a diglycidyl ether reagent;

[0034] Preferably, the halogenated epoxy compound is epichlorohydrin, and the diglycidyl ether reagent is selected from any one of ethylene glycol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.

[0035] Optional, the volume ratio of concentrated alkali to crosslinking agent is 1:2.

[0036] Optionally, the reaction is carried out by stirring at a speed of 300 rpm; preferably, the reaction time is 30 min. Attached Figure Description

[0037] Figure 1 These are optical microscope images and particle size distribution diagrams of the composite polysaccharide microspheres (agarose:glucan = 5:1, 4:2, 3:3, 2:4) in Example 1 of this invention.

[0038] Figure 2 The optical microscope images prepared for comparative examples 1-3 are magnified 100 times.

[0039] Figure 3 These are particle size and particle size distribution diagrams of the novel multinucleated agarose gel microspheres and their core agarose gel microspheres in Examples 2-4 of this invention.

[0040] Figure 4 The images are optical microscope images magnified 100 times for the novel multinucleated agarose gel microspheres prepared in Examples 2-5 of this invention.

[0041] Figure 5 This is a diagram showing the average particle size and particle size distribution of a series of novel multi-core agarose gel microspheres with 6% agarose microspheres as the core, as described in Example 5 of this invention.

[0042] Figure 6 The images are optical microscope images magnified 100 times for the novel multinucleated agarose gel microspheres prepared in Examples 6-10 of this invention.

[0043] Figure 7 These are optical microscope images and particle size distribution diagrams of novel multinucleated agarose gel microspheres with different core microsphere sizes in Embodiment 11 of the present invention.

[0044] Figure 8These are optical microscope images and particle size distribution diagrams of the novel multinucleated agarose gel microspheres at different emulsification speeds in Example 12 of this invention.

[0045] Figure 9 These are optical microscope images and particle size distribution diagrams of novel multinuclear agarose gel microspheres with different crosslinking times in Example 13 of this invention.

[0046] Figure 10 These are infrared analysis images of the novel multinucleated agarose gel microspheres in Examples 2-4 of this invention compared with other types of microspheres.

[0047] Figure 11 This is a density diagram of the novel multinucleated agarose gel microspheres in Examples 2-4 of the present invention, their core agarose gel microspheres, and the epoxy group modification density of Comparative Example 1.

[0048] Figure 12 This is a flow rate-pressure test graph comparing the novel multinucleated agarose gel microspheres of Example 3 with those of Comparative Examples 3-5. It reflects the pressure resistance and mechanical strength of the agarose microspheres.

[0049] Figure 13 This is a flow rate-pressure test diagram of a series of novel multi-core agarose gel microspheres with 6% agarose microspheres as the core in Example 5 of the present invention.

[0050] Figure 14 This is a gel filtration chromatography performance test diagram of a series of novel multi-core agarose gel microspheres with 6% agarose microspheres as the core in Example 5 of the present invention. Detailed Implementation

[0051] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially. In the following embodiments, unless otherwise explicitly stated, "%" refers to weight percentage.

[0052] Example 1: Optimization Experiment of Multi-core Composite Agarose Gel Microsphere Shell Material

[0053] S1. Preparation of the oil phase: Heat 180ml of liquid paraffin to 65℃, add 1.5% w / v Span 80 surfactant while mechanically stirring at 1000rpm, maintain constant temperature, mix evenly, and set aside.

[0054] S2. Preparation of aqueous phase: Prepare 54g of a complex polysaccharide solution containing a certain weight ratio of agarose to dextran (5:1, 4:2, 3:3, 2:4), heat until clear and transparent, and completely dissolved;

[0055] S3. Pour the prepared aqueous phase into the prepared oil phase (800-1400 rpm) that is being stirred, stir and emulsify for 10 min, and then solidify in an ice-water bath at 600 rpm for 10 min; so that the W / O emulsion droplets cool and solidify into agarose gel microspheres.

[0056] S4. Add 1% v / v NaOH and 2% v / v ethylene glycol diglycidyl ether, and then stir at 300 rpm for 0.5 h.

[0057] S5. Centrifuge the above mixture to separate the layers, discard the supernatant, and wash repeatedly until clean. This yields novel multi-nuclear composite agarose gel microspheres.

[0058] Figure 1 These are optical microscope images and particle size distribution diagrams of the composite polysaccharide microspheres (agarose:glucan = 5:1, 4:2, 3:3, 2:4) in this embodiment. It can be seen that as the proportion of glucan in the composite polysaccharide composition increases, the average particle size of the microspheres decreases. Preferably, when the agarose to glucan ratio is 4:2, the particle size distribution is uniform.

[0059] Example 2: Preparation of novel multinucleated agarose gel microspheres

[0060] S1. Preparation of kernel agarose microspheres, including:

[0061] S1.1. Preparation of the oil phase: Heat 200ml of liquid paraffin to 80℃, add 0.75% w / v Span 80 surfactant while mechanically stirring at 900rpm, maintain constant temperature, mix evenly, and set aside.

[0062] S1.2. Preparation of the aqueous phase: Prepare 60 ml of a polysaccharide solution containing 4% w / v agarose, and heat (heat until the agarose solution boils, about 90-100℃) until clear and transparent and completely dissolved.

[0063] S1.3. Pour the prepared aqueous phase into the prepared oil phase (800-1400 rpm) that is being stirred, stir and emulsify for 10 min, then cool down to 20°C at 600 rpm to solidify the agarose droplets into microspheres.

[0064] S1.4. Centrifuge at 3500 rpm for 5 min to break the emulsion and separate the layers (to achieve the purpose of centrifugation to break the emulsion and sedimentation). Discard the supernatant (oil and surfactant), and wash repeatedly until clean to obtain the core agarose microspheres.

[0065] S2. Cross-linking and activation of kernel agarose microspheres, the specific steps of which include:

[0066] S2.1. Mix 18g of the kernel agarose microspheres obtained above with 10ml of water, 7ml of 20% NaOH solution, 7ml of 1,4-dioxane, and 7ml of epichlorohydrin until homogeneous;

[0067] S2.2. The mixture obtained above was reacted at 200 rpm and 30°C for 2 h;

[0068] S2.3. The above-obtained material is repeatedly washed with ultrapure water or ethanol to remove the NaOH solution and organic solvent, thereby obtaining the post-crosslinked core agarose microspheres.

[0069] S3. Preparation of novel multinuclear agarose gel microspheres, including the following steps:

[0070] S3.1. Preparation of the oil phase: Heat 180ml of liquid paraffin to 65℃, add 1.5% w / v Span 80 surfactant while mechanically stirring at 1000rpm, maintain constant temperature, mix evenly, and set aside.

[0071] S3.2. Preparation of the aqueous phase: Prepare 36g of a polysaccharide solution containing 4% w / v agarose, heat until clear and transparent, and completely dissolved. While hot, add 18g of the kernel agarose microspheres obtained after cross-linking treatment in S2, and mix thoroughly; if bubbles are present, heat at 80-90℃ for 5 minutes to defoam.

[0072] S3.3. Pour the prepared aqueous phase into the prepared oil phase (800-1400 rpm) that is being stirred, stir and emulsify for 10 min, and then solidify in an ice-water bath at 600 rpm for 10 min; so that the W / O emulsion droplets cool and solidify into agarose gel microspheres.

[0073] S3.4. Add 1% v / v NaOH and 2% v / v ethylene glycol diglycidyl ether, and then stir the mixture at 300 rpm for 0.5 h.

[0074] S3.5. Centrifuge the above mixture (3500 rpm for 5 min) to separate the layers, discard the supernatant, and wash repeatedly until clean. This yields novel multinuclear agarose gel microspheres.

[0075] Example 3: Preparation of novel multinuclear composite agarose gel microspheres

[0076] S1. Preparation of kernel agarose microspheres: Same as in Example 2.

[0077] S2. Kernel agarose microsphere cross-linking activation: Same as Example 2.

[0078] S3. Preparation of novel multinuclear composite agarose gel microspheres, including the following steps:

[0079] S3.1. Preparation of the oil phase: Heat 180ml of liquid paraffin to 65℃, add 1.5% w / v Span 80 surfactant while mechanically stirring at 1000rpm, maintain constant temperature, mix evenly, and set aside.

[0080] S3.2. Preparation of the aqueous phase: Prepare 36g of a complex polysaccharide solution containing 2% dextran and 4% agarose, and heat until clear and transparent, and completely dissolved. While hot, add 18g of kernel agarose microspheres and mix well; if bubbles are present, heat at 80-90℃ for 5 minutes to defoam.

[0081] S3.3. Pour the prepared aqueous phase into the prepared oil phase (800-1400 rpm) that is being stirred, stir and emulsify for 10 min, and then solidify in an ice-water bath at 600 rpm for 10 min; so that the W / O emulsion droplets cool and solidify into agarose gel microspheres.

[0082] S3.4. Add 1% v / v NaOH and 2% v / v ethylene glycol diglycidyl ether, and then stir the mixture at 300 rpm for 0.5 h.

[0083] S3.5. Centrifuge the above mixture to separate the layers, discard the supernatant, and wash repeatedly until clean. This yields novel multi-nuclear composite agarose gel microspheres.

[0084] The average particle size and particle size distribution of the novel multinuclear composite agarose gel microspheres obtained in this embodiment were measured using a Winner2000 laser particle size analyzer. The average particle size of the microspheres in water was 125 μm, and the particle size distribution coefficient R0 was [missing value]. span It is 1.38.

[0085] Example 4: Preparation of novel multinuclear composite agarose gel microspheres

[0086] S1. Preparation of kernel agarose microspheres: Same as in Example 2.

[0087] S2. Kernel agarose microsphere cross-linking activation: Same as Example 2.

[0088] S3. Preparation of novel multinuclear composite agarose gel microspheres, including the following steps:

[0089] S3.1. Preparation of the oil phase: Heat 180ml of liquid paraffin to 65℃, add 1.5% w / v Span 80 surfactant while mechanically stirring at 1000rpm, maintain constant temperature, mix evenly, and set aside.

[0090] S3.2. Preparation of the aqueous phase: Prepare 36g of a composite polysaccharide solution containing 2% dextran and 4% agarose, and heat until clear and transparent, and completely dissolved. While hot, add 18g of the cross-linked kernel agarose microspheres obtained in S2 and mix well; if there are bubbles, heat at 80-90℃ for 5 minutes to defoam.

[0091] S3.3. Pour the prepared aqueous phase into the prepared oil phase (800-1400 rpm) that is being stirred, stir and emulsify for 10 min, and then solidify in an ice-water bath at 600 rpm for 10 min; so that the W / O emulsion droplets cool and solidify into agarose gel microspheres.

[0092] S3.4. Add 1% v / v NaOH, 2% v / v 1,4-dioxane and 2% v / v epichlorohydrin, and stir at 300 rpm for 0.5 h.

[0093] S3.5. Centrifuge the above mixture to separate the layers, discard the supernatant, and wash repeatedly until clean. This yields novel multi-nuclear composite agarose gel microspheres.

[0094] The average particle size and particle size distribution of the novel multinuclear composite agarose gel microspheres obtained in this embodiment were measured using a Winner2000 laser particle size analyzer. The average particle size of the microspheres in water was 133 μm, and the particle size distribution coefficient R0 was [missing value]. span It is 1.51.

[0095] Example 5: Preparation of novel multinuclear composite agarose gel microspheres

[0096] S1. Preparation of kernel agarose microspheres, including:

[0097] S1.1. Preparation of the oil phase: Heat 200ml of liquid paraffin to 80℃, add 0.75% w / v Span 80 surfactant while mechanically stirring at 3000rpm, maintain constant temperature, mix evenly, and set aside.

[0098] S1.2. Preparation of aqueous phase: Prepare 60 ml of polysaccharide solution containing 6% w / v agarose, heat until clear and transparent, and completely dissolved.

[0099] S1.3. Pour the prepared aqueous phase into the prepared oil phase (2000-4000 rpm) that is being stirred, stir and emulsify for 20 min, then cool down to 20°C at 2800 rpm to solidify the agarose droplets into microspheres.

[0100] S1.4. Centrifuge again to separate the layers (to achieve centrifugation, demulsification, sedimentation, and separation), discard the supernatant (oil and surfactant), and wash repeatedly until clean to obtain the core agarose microspheres.

[0101] S2. Kernel agarose microsphere cross-linking activation: Same as Example 1.

[0102] S3. Preparation of novel multinuclear composite agarose gel microspheres, including the following steps:

[0103] S3.1. Preparation of the oil phase: Heat 180ml of liquid paraffin to 65℃, add 1.5% w / v Span 80 surfactant while mechanically stirring at 1000rpm, maintain constant temperature, mix evenly, and set aside.

[0104] S3.2. Preparation of the aqueous phase: Prepare 36g of three composite polysaccharide solutions: containing 5% agarose and 2.5% dextran; or 4% agarose and 2% dextran; or 3% agarose and 1.5% dextran. Heat until clear and transparent, and completely dissolved. While hot, add 18g of the kernel agarose microspheres obtained in S2 and mix thoroughly; if bubbles are present, heat at 80-90℃ for 5 minutes to defoam.

[0105] S3.3. Pour the prepared aqueous phase into the prepared oil phase (800-1400 rpm) that is being stirred, stir and emulsify for 10 min, and then solidify in an ice-water bath at 600 rpm for 10 min; so that the W / O emulsion droplets cool and solidify into agarose gel microspheres.

[0106] S3.4. Add 1% v / v NaOH, 2% v / v 1,4-dioxane and 2% v / v epichlorohydrin, and stir at 300 rpm for 0.5 h.

[0107] S3.5. Centrifuge the above mixture to separate the layers, discard the supernatant, and wash repeatedly until clean. This yields three novel multi-core composite agarose gel microspheres with a core of 6% agarose microspheres and a shell of three composite polysaccharides (5% agarose and 2.5% dextran; 4% agarose and 2% dextran; 3% agarose and 1.5% dextran).

[0108] The average particle size and particle size distribution of the novel multinuclear composite agarose gel microspheres obtained in this embodiment were measured using a Winner2000 laser particle size analyzer. The average particle size of the microspheres in water was 140 μm, and the particle size distribution coefficient R0 was [missing value]. span It is 1.63.

[0109] Table 1 shows the partition coefficients K of a series of novel multi-core agarose gel microspheres with 6% agarose microspheres as the core in Example 5 of this invention. d Value table. As can be seen from Table 1, with the increase of shell concentration, the K value of bovine serum albumin... d The value decreases progressively. For the same protein sample, smaller K values ​​result in... d The value is due to the smaller pores in the microspheres blocking the flow, thus allowing the material to flow out of the chromatography column more quickly.

[0110] Table 1

[0111]

[0112] Example 6: Preparation of novel multinuclear composite agarose gel microspheres

[0113] S1. Preparation of kernel agarose microspheres: Same as in Example 2.

[0114] S2. Kernel agarose microsphere cross-linking activation: Same as Example 2.

[0115] S3. Preparation of novel multinuclear composite agarose gel microspheres: Same as Example 3, except that the pre-crosslinking was performed using 1% v / v NaOH and 2% v / v butanediol diglycidyl ether.

[0116] The average particle size and particle size distribution of the novel multinuclear composite agarose gel microspheres obtained in this embodiment were measured using a Winner2000 laser particle size analyzer. The average particle size of the microspheres in water was 143 μm, and the particle size distribution coefficient R0 was [missing value]. span It is 1.52.

[0117] Example 7: Preparation of novel multinuclear composite agarose gel microspheres

[0118] S1. Preparation of kernel agarose microspheres: Same as in Example 2.

[0119] S2. Kernel agarose microsphere cross-linking activation: Same as Example 2.

[0120] S3. Preparation of novel multinuclear composite agarose gel microspheres: Same as Example 2, except that the pre-crosslinking was performed using 1% v / v NaOH and 2% v / v hexanediol diglycidyl ether.

[0121] The average particle size and particle size distribution of the novel multinuclear composite agarose gel microspheres obtained in this embodiment were measured using a Winner2000 laser particle size analyzer. The average particle size of the microspheres in water was 149 μm, and the particle size distribution coefficient R0 was [missing value]. span It is 1.58.

[0122] Example 8: Preparation of novel multinuclear composite agarose gel microspheres

[0123] S1. Preparation of kernel agarose microspheres: Same as in Example 2.

[0124] S2. Kernel agarose microsphere cross-linking activation: Same as Example 2.

[0125] S3. Preparation of novel multinuclear composite agarose gel microspheres:

[0126] S3.1. Preparation of the oil phase: Heat 160ml of liquid paraffin and 20ml of soybean oil to 65℃, and while mechanically stirring at 1000rpm, add 1.5% w / v Span 80 surfactant, maintain constant temperature, and mix thoroughly. Set aside.

[0127] S3.2. Preparation of the aqueous phase: Prepare 36g of a complex polysaccharide solution containing 2% dextran and 4% agarose, and heat until clear and transparent and completely dissolved. While still hot, add the kernel agarose microspheres and mix well; if bubbles are present, heat at 80-90℃ for 5 minutes to defoam.

[0128] S3.3. Pour the prepared aqueous phase into the prepared oil phase (800-1400 rpm) that is being stirred, stir and emulsify for 10 min, and then solidify in an ice-water bath at 600 rpm for 10 min; so that the W / O emulsion droplets cool and solidify into agarose gel microspheres.

[0129] S3.4. Add 1% v / v NaOH and 2% v / v ethylene glycol diglycidyl ether, and then stir the mixture at 300 rpm for 0.5 h.

[0130] S3.5. Centrifuge the above mixture to separate the layers, discard the supernatant, and wash repeatedly until clean. This yields novel multinuclear agarose gel microspheres.

[0131] The average particle size and particle size distribution of the novel multinuclear composite agarose gel microspheres obtained in this embodiment were measured using a Winner2000 laser particle size analyzer. The average particle size of the microspheres in water was 127 μm, and the particle size distribution coefficient R0 was [missing value]. span It is 1.55.

[0132] Example 9: Preparation of novel multinuclear composite agarose gel microspheres

[0133] S1. Preparation of kernel agarose microspheres: Same as in Example 2.

[0134] S2. Kernel agarose microsphere cross-linking activation: Same as Example 2.

[0135] S3. Preparation of novel multinuclear composite agarose gel microspheres:

[0136] S3.1. Preparation of the oil phase: Heat 180ml of liquid paraffin to 65℃, add 1.5% w / v Span 80 surfactant while mechanically stirring at 1000rpm, maintain constant temperature, mix evenly, and set aside.

[0137] S3.2. Preparation of the aqueous phase: Prepare 45g of a complex polysaccharide solution containing 2% dextran and 4% agarose, and heat until clear and transparent and completely dissolved. While hot, add 9g of kernel agarose microspheres and mix well; if bubbles are present, heat at 80-90℃ for 5 minutes to defoam.

[0138] S3.3. Pour the prepared aqueous phase into the prepared oil phase (800-1400 rpm) that is being stirred, stir and emulsify for 10 min, and then solidify in an ice-water bath at 600 rpm for 10 min; so that the W / O emulsion droplets cool and solidify into agarose gel microspheres.

[0139] S3.4. Add 1% v / v NaOH and 2% v / v ethylene glycol diglycidyl ether, and then stir the mixture at 300 rpm for 0.5 h.

[0140] S3.5. Centrifuge the above mixture to separate the layers, discard the supernatant, and wash repeatedly until clean. This yields novel multinuclear agarose gel microspheres.

[0141] The average particle size and particle size distribution of the novel multinuclear composite agarose gel microspheres obtained in this embodiment were measured using a Winner2000 laser particle size analyzer. The average particle size of the microspheres in water was 120 μm, and the particle size distribution coefficient R0 was [missing value]. span It is 1.74.

[0142] Example 10: Preparation of novel multinuclear composite agarose gel microspheres

[0143] S1. Preparation of kernel agarose microspheres: Same as in Example 2, except that the polysaccharide solution is a polysaccharide solution containing 5% w / v agarose.

[0144] S2. Kernel agarose microsphere cross-linking activation: Same as Example 2.

[0145] S3. Preparation of novel multinuclear composite agarose gel microspheres: Same as Example 3, except that the pre-crosslinking was performed using 1% v / v NaOH and 2% v / v ethylene glycol diglycidyl ether.

[0146] The average particle size and particle size distribution of the novel multinuclear composite agarose gel microspheres obtained in this embodiment were measured using a Winner2000 laser particle size analyzer. The average particle size of the microspheres in water was 118 μm, and the particle size distribution coefficient R0 was [missing value]. span It is 1.67.

[0147] Example 11: Preparation of novel multinuclear composite agarose gel microspheres

[0148] S1. Preparation of kernel agarose microspheres: Same as in Example 2.

[0149] S2. Kernel agarose microsphere cross-linking activation: Same as Example 2.

[0150] S3. Preparation of novel multinuclear composite agarose gel microspheres, including the following steps:

[0151] S3.1. Preparation of the oil phase: Heat 180ml of liquid paraffin to 65℃, add 1.5% w / v Span 80 surfactant while mechanically stirring at 1000rpm, maintain constant temperature, mix evenly, and set aside.

[0152] S3.2. Preparation of the aqueous phase: Prepare 36g of a composite polysaccharide solution containing 2% dextran and 4% agarose, and heat until clear and transparent and completely dissolved. While hot, add kernel agarose microspheres with a certain particle size range (10-45μm, 45-60μm, 60-75μm, 75-105μm), mix evenly with the composite polysaccharide solution, and heat to defoam for 5min.

[0153] Prepare a 36g solution of a complex polysaccharide containing 2% dextran and 4% agarose, and heat until clear and transparent and completely dissolved. While still hot, add kernel agarose microspheres and mix well; if bubbles are present, heat at 80-90℃ for 5 minutes to defoam.

[0154] S3.3. Pour the prepared aqueous phase into the prepared oil phase (800-1400 rpm) that is being stirred, stir and emulsify for 10 min, and then solidify in an ice-water bath at 600 rpm for 10 min; so that the W / O emulsion droplets cool and solidify into agarose gel microspheres.

[0155] S3.4. Add 1% v / v NaOH and 2% v / v ethylene glycol diglycidyl ether, and then stir the mixture at 300 rpm for 0.5 h.

[0156] S3.5. Centrifuge the above mixture to separate the layers, discard the supernatant, and wash repeatedly until clean. This yields novel multi-nuclear composite agarose gel microspheres.

[0157] Example 12: Preparation of novel multinuclear composite agarose gel microspheres

[0158] S1. Preparation of kernel agarose microspheres: Same as in Example 2.

[0159] S2. Kernel agarose microsphere cross-linking activation: Same as Example 2.

[0160] S3. Preparation of novel multinuclear composite agarose gel microspheres, including the following steps:

[0161] S3.1. Preparation of the oil phase: Heat 180ml of liquid paraffin to 65℃, and add 1.5% w / v Span 80 surfactant while maintaining a constant temperature and mixing evenly. Set aside for later use.

[0162] S3.2. Preparation of the aqueous phase: Prepare 36g of a complex polysaccharide solution containing 2% dextran and 4% agarose, and heat until clear and transparent and completely dissolved. While still hot, add the kernel agarose microspheres and mix well; if bubbles are present, heat at 80-90℃ for 5 minutes to defoam.

[0163] S3.3. Pour the prepared aqueous phase into the prepared oil phase (800 rpm, 1000 rpm, 1200 rpm, 1400 rpm) that is being stirred, and after stirring and emulsifying for 10 min, solidify it in an ice-water bath at 600 rpm for 10 min; so that the W / O emulsion droplets cool and solidify into agarose gel microspheres.

[0164] S3.4. Add 1% v / v NaOH and 2% v / v ethylene glycol diglycidyl ether, and then stir the mixture at 300 rpm for 0.5 h.

[0165] S3.5. Centrifuge the above mixture to separate the layers, discard the supernatant, and wash repeatedly until clean. This yields novel multi-nuclear composite agarose gel microspheres.

[0166] Example 13: Preparation of novel multinuclear composite agarose gel microspheres

[0167] S1. Preparation of kernel agarose microspheres: Same as in Example 2.

[0168] S2. Kernel agarose microsphere cross-linking activation: Same as Example 2.

[0169] S3. Preparation of novel multinuclear composite agarose gel microspheres, including the following steps:

[0170] S3.1. Preparation of the oil phase: Heat 180ml of liquid paraffin to 65℃, add 1.5% w / v Span 80 surfactant while stirring at a certain mechanical speed (800-1400rpm), keep the temperature constant, mix evenly, and set aside.

[0171] S3.2. Preparation of the aqueous phase: Prepare 36g of a complex polysaccharide solution containing 2% dextran and 4% agarose, and heat until clear and transparent and completely dissolved. While still hot, add the kernel agarose microspheres and mix well; if bubbles are present, heat at 80-90℃ for 5 minutes to defoam.

[0172] S3.3. Pour the prepared aqueous phase into the prepared oil phase (800-1400 rpm) that is being stirred, stir and emulsify for 10 min, and then solidify in an ice-water bath at 600 rpm for 10 min; so that the W / O emulsion droplets cool and solidify into agarose gel microspheres.

[0173] S3.4. Add 1% v / v NaOH and 2% v / v ethylene glycol diglycidyl ether, and then stir at 300 rpm for a certain time (5 min, 15 min, 0.5 h, 1 h, 2 h, 4 h, 6 h);

[0174] S3.5. Centrifuge the above mixture to separate the layers, discard the supernatant, and wash repeatedly until clean. This yields novel multi-nuclear composite agarose gel microspheres.

[0175] Preparation of Comparative Example 1: 4% Agarose Gel Microspheres

[0176] The procedure is the same as step S1 in Example 2. The difference is that the mechanical stirring speed is 800 rpm. 4% agarose gel microspheres are obtained.

[0177] Comparative Example 2: Preparation of 6% Agarose Gel Microspheres

[0178] The procedure is the same as step S1 in Example 2. The difference is that a 6% w / v agarose solution is prepared in aqueous phase, and the mechanical stirring speed is 800 rpm. This yields 6% agarose gel microspheres.

[0179] Comparative Example 3: Preparation of Composite Polysaccharide Gel Microspheres

[0180] The steps S1-S5 are the same as in Example 1. The difference is that a composite polysaccharide solution is prepared by using 4% w / v agarose and 2% w / v dextran in the aqueous phase, resulting in a composite polysaccharide gel microsphere containing 4% agarose and 2% dextran.

[0181] Comparative Example 4: Preparation of cross-linked 4% agarose gel microspheres

[0182] S1: Same as step 1 in Comparative Example 1.

[0183] S2: Same as step S2 in Example 2. Cross-linked 4% agarose gel microspheres are obtained.

[0184] Comparative Example 5: Preparation of cross-linked 4% agarose gel microspheres

[0185] Same as Comparative Example 4. The only difference is that the 4% w / v agarose solution was changed to a 6% w / v agarose solution.

[0186] S1: Same as Comparative Example 2, Step 2.

[0187] S2: Same as step S2 in Example 2. Cross-linked 6% agarose gel microspheres are obtained.

[0188] Test case

[0189] 1) Morphology and particle size testing:

[0190] The morphology of the microspheres prepared in the examples and comparative examples was characterized by optical microscopy: wet core-shell microspheres were dropped onto the surface of a glass slide, covered with a coverslip, and observed and photographed using an ML11-Ⅱ optical microscope.

[0191] The average particle size and particle size distribution were measured using a laser particle size analyzer: A sample was prepared by uniformly dispersing core-shell microspheres in distilled water, and an appropriate amount of wet sample was added to the measuring cup of the laser particle size analyzer. The particle size distribution is commonly measured using the particle size distribution coefficient R. span The value is used to represent the uniformity of particle size; the smaller the value, the more uniform the particle size.

[0192] Figure 1 Optical microscope images and particle size distribution diagrams at 100x magnification of the composite polysaccharide microspheres (agarose:dextran = 5:1, 4:2, 3:3, 2:4) prepared in Example 1 of this invention. As can be seen from the figures, when the agarose:dextran ratio is 5:1, 4:2, 3:3, or 2:4, the average particle size of the microspheres decreases as the proportion of dextran in the composite polysaccharide composition increases. Preferably, when the agarose:dextran ratio is 4:2, the particle size distribution is uniform and conforms to the particle size of the shell layer of the core-shell microspheres.

[0193] Figure 2 The images shown are optical microscope images magnified 100 times, obtained from Comparative Examples 1-3. As can be seen from the images, the microspheres shown in Comparative Examples 1-3 exhibit good sphericity and monodispersity.

[0194] Figure 3 These are particle size and particle size distribution diagrams of the novel multi-core agarose gel microspheres and their core agarose gel microspheres in Examples 2-4 of this invention. As can be seen from the diagrams, when the core-shell microspheres encapsulate core agarose microspheres with an average particle size of 25 μm, the average particle size increases accordingly; and when the shell layer is composited with dextran, the microsphere particle size further increases.

[0195] Figure 4 These are optical microscope images, magnified 100x, of the novel multinucleated agarose gel microspheres prepared in Examples 2-5 of this invention. As can be seen from the images, when the shell material in Example 2 contains only agarose, the microspheres are clear and transparent; the microspheres shown in Examples 2-5 exhibit good sphericity and monodispersity. In Example 5, with three different shell concentrations, it can be observed that the average particle size gradually increases with increasing shell concentration.

[0196] Figure 5 This is a diagram showing the average particle size and particle size distribution of a series of novel multi-core agarose gel microspheres with 6% agarose microspheres as the core, as described in Example 5 of this invention. When the shell concentration is 4.5-7.5% (i.e., shell concentrations of 3% agarose and 1.5% dextran, 4% agarose and 2% dextran, and 5% agarose and 2.5% dextran), it indicates that under the same experimental conditions, the average particle size gradually increases with increasing shell concentration, and the particle size distribution coefficient R... span The size is reduced, resulting in a more uniform particle size.

[0197] Figure 6 These are optical microscope images, magnified 100x, of the novel multinucleated agarose gel microspheres prepared in Examples 6-10 of this invention. As can be seen from the images, all examples 6-10 produced microspheres with good sphericity and monodispersity.

[0198] Figure 7 These are optical microscope images and particle size distribution maps of the novel multi-core agarose gel microspheres with different core microsphere sizes in Example 11 of this invention. As can be seen from the figures, the particle size analysis map has two peaks: the first peak represents the volume percentage of unencapsulated core microspheres, and the second peak represents the volume percentage of formed core-shell microspheres. When the particle size of the core microspheres is less than 60 μm (i.e., 10-45 μm, 45-60 μm), the total volume of the formed core-shell microspheres is the highest, and the core-shell microspheres show good morphology in the optical microscope images. When the particle size of the core microspheres is greater than 60 μm (i.e., 60-75 μm, 75-105 μm), the volume percentage in the particle size map is dominated by core microspheres, and it can be seen from the optical microscope that the formed core-shell microspheres are too large and easily break.

[0199] Figure 8 These are optical microscope images and particle size distribution diagrams of the novel multinucleated agarose gel microspheres at different emulsification speeds in Example 12 of this invention. As can be seen from the figures, core-shell microspheres can be formed at emulsification speeds ranging from 800 to 1400 rpm.

[0200] Figure 9These are optical microscope images and particle size distribution diagrams of the novel multinuclear agarose gel microspheres with different crosslinking times in Example 13 of this invention. Both 15 min and 30 min of crosslinking time resulted in well-formed multinuclear agarose gel microspheres. Preferably, a crosslinking time of 30 min resulted in the most uniform particle size distribution of the multinuclear agarose gel microspheres.

[0201] 2) Infrared Spectroscopy Test: After drying, pulverizing, and sieving the microspheres, they were mixed with potassium bromide, compressed into tablets, and measured using a Fourier transform infrared spectrometer. (See attached image.) Figure 10 Different types of microspheres all exhibited obvious polysaccharide absorption characteristic peaks, ranging from 3016 to 3679 cm⁻¹. -1 The decrease in the intensity of the stretching vibration band of the associated hydroxyl groups in Examples 3 and 4 indicates that a large number of hydroxyl groups in the composite polysaccharide molecule participated in the cross-linking reaction; 2824-3008 cm -1 For the -CH stretching vibration belt, 2855cm in Examples 3 and 4 -1 The appearance of the peak value is a result of the increased strength of the methylene groups. Due to the use of crosslinking agents during the preparation process, more methylene groups were introduced into the microspheres.

[0202] 3) Epoxy group modification density test: The microspheres prepared in the examples and the kernel agarose gel microspheres used were simultaneously activated with epichlorohydrin, sodium thiosulfate was added, and titrated with standard hydrochloric acid solution. The OH groups released from the activated gel microspheres were determined by titration. － The concentration is the concentration of epoxy groups, and the epoxy group modification density is calculated by conversion. The epoxy group modification density of the core agarose microspheres is 66.82 μmol / g, the epoxy group modification density of the multi-core agarose microspheres in Example 2 is 61.97 μmol / g, the epoxy group modification density of the multi-core agarose microspheres in Example 3 is 52.77 μmol / g, and the epoxy group modification density of the multi-core agarose microspheres in Example 4 is 50.53 μmol / g. (See [link to example]). Figure 11 .

[0203] 4) Flow rate-pressure test: Different types of agarose gel microspheres were packed into a 5 ml chromatography column. A certain flow rate was introduced, and after the column bed stabilized, a constant pressure was applied. The highest flow rate was measured by gradually increasing the pressure at intervals, and after stabilizing for 5 minutes, the corresponding flow rate at each pressure was recorded. The highest flow rate of the microsphere medium was determined when the flow rate stabilized within a certain pressure range. A flow rate-pressure curve was then plotted. See [link to relevant documentation]. Figure 12 This reflects the novel multi-core composite agarose gel microspheres prepared by this invention. The core agarose microspheres provide support, while the shell layer utilizes pre-crosslinked composite polysaccharides to form an interpenetrating gel network structure, resulting in significantly improved mechanical properties, exceeding those of 6% crosslinked agarose gel microspheres. See [link to related documentation]. Figure 13 This reflects that the mechanical properties of the novel multinucleated composite agarose gel microspheres prepared by this invention are effectively improved with the increase of the concentration of composite polysaccharides in the shell.

[0204] 5) Gel filtration chromatography performance test: Core-shell agarose microspheres were packed into a chromatography column (10 cm × 1.5 cm I.D.) and equilibrated with buffer (0.01 mol / L Tris-HCl, pH 6.8) until the baseline stabilized. Preparation of standard protein mixtures: Each standard protein was dissolved in buffer (1 mg / mL), and 100 μL of the solution was injected into the chromatography column. Elution was performed using the same buffer. The detection wavelength was set to 280 nm, and the chromatograms were recorded. Each chromatography operation was repeated using the AKTA protein purification system at a flow rate of 1.0 mL / min. d The value is calculated as follows:

[0205]

[0206] In the formula, Vr is the retention volume of the sample (mL); the sample in this test example is bovine serum albumin.

[0207] V0 – External water volume (mL), determined by the retention volume of blue dextran 2000;

[0208] Vt — Total mobile phase volume, including internal and external water volumes (mL), determined by the retention volume of uracil.

[0209] The difference between the total mobile phase volume and the external water volume represents the internal water volume filling the pores of the agarose microspheres, reflecting the density of the network structure and the porosity within the microspheres. See [link / reference]. Figure 14 As shown in Table 1, with the increase of the shell concentration of the multinuclear composite agarose gel microspheres, the Kc of the solute molecules in the bovine serum albumin sample... d The smaller the value, the faster the outflow and the smaller the pores formed by the microspheres.

[0210] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A method for preparing multinuclear agarose gel microspheres, characterized in that, The core of the microspheres consists of agarose microspheres with a particle size of no more than 60 μm, which are uniformly dispersed inside the microspheres; the shell of the microspheres is a neutral polysaccharide composite material. Its preparation method includes the following steps, S1. Preparation of kernel agarose microspheres, including: S1.

1. Preparation of the first oil phase: A surfactant and a first oily substance immiscible with water are mixed; the surfactant is 0.75-2% w / v Span 80, and the first oily substance is liquid paraffin; S1.

2. Preparation of the first aqueous phase: Prepare a polysaccharide solution containing 4-6% w / v agarose, heat until clear and transparent, and completely dissolved; S1.

3. Pour the prepared first aqueous phase into the prepared first oil phase that is being stirred to emulsify, then cool and solidify to solidify the agarose droplets into microspheres. S1.

4. Centrifuge to break the emulsion and separate the layers, discard the supernatant and wash repeatedly until clean to obtain the kernel agarose microspheres; S2. Cross-linking and activation of kernel agarose microspheres, the specific steps of which include: S2.

1. Mix the obtained kernel agarose microspheres with water, concentrated alkali, 1,4-dioxane and the first crosslinking agent until homogeneous; S2.

2. React the above-obtained mixture at 150-400 rpm and 20-35℃ for 2-6 h; S2.

3. The above-obtained material is repeatedly washed with ultrapure water or ethanol to remove the NaOH solution and organic solvent, thereby obtaining the post-crosslinked core agarose microspheres. S3. Obtaining multinucleated agarose gel microspheres: S3.1 Preparation of the second oil phase: A surfactant and a second oily substance immiscible with water are mixed; the surfactant is 0.75-2% w / v Span 80, and the second oily substance is liquid paraffin; S3.2 Preparation of the second aqueous phase: Prepare a polysaccharide solution containing 4.5-7.5% w / v agarose, heat until clear and transparent to completely dissolve it, then add post-crosslinked core agarose microspheres and mix evenly; if there are bubbles, heat to defoam; the polysaccharide solution also contains dextran; S3.3 Pour the prepared second aqueous phase into the prepared second oil phase which is being stirred, continue stirring to emulsify, and then solidify; so that the W / O emulsion droplets cool and solidify into agarose gel microspheres; the solidification is to cool down to 15-25℃ at 500-600 rpm; S3.4 Add concentrated alkali to react with the second crosslinking agent; S3.5 Centrifuge to separate the layers, discard the supernatant, and wash repeatedly until clean to obtain multinucleated agarose gel microspheres.

2. The method for preparing multinuclear agarose gel microspheres as described in claim 1, characterized in that, In step S1.2, the polysaccharide solution also contains dextran.

3. The method for preparing multinuclear agarose gel microspheres as described in claim 1, characterized in that, In step S1.3, the emulsification is carried out at 800-1400 rpm for 10-30 min; the solidification is carried out by cooling to 15-25℃ at 500-600 rpm; and the volume ratio of the liquid paraffin to the polysaccharide solution of agarose is 200:

60.

4. The method for preparing multinuclear agarose gel microspheres as described in claim 1, characterized in that, In steps S1.4 and S3.5, the centrifugation conditions are 3500 rpm and 5 min.

5. The method for preparing multinuclear agarose gel microspheres as described in claim 1, characterized in that, In step S2.1, the first crosslinking agent is at least one of a halogenated epoxy compound and a diglycidyl ether reagent.

6. The method for preparing multinuclear agarose gel microspheres as described in claim 5, characterized in that, The halogenated epoxy compound is epichlorohydrin, and the diglycidyl ether reagent is selected from any one of ethylene glycol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether. Optionally, in step S2.1, the ratio of the kernel agarose microspheres: concentrated alkali: first crosslinking agent is 36g:14mL:14mL.

7. The method for preparing multinuclear agarose gel microspheres as described in claim 1, characterized in that, In step S3.2, the mass ratio of the post-crosslinked kernel agarose microspheres to the polysaccharide solution is 1:2-5.

8. The method for preparing multinuclear agarose gel microspheres as described in claim 1, characterized in that, In step S3.3, the stirring speed for continued emulsification is 800-1400 rpm, and the emulsification time is 10-30 min; Optionally, the ratio of liquid paraffin: agarose-containing polysaccharide solution: post-crosslinked core agarose microspheres is 180 mL: 36 g: 18 g.

9. The method for preparing multinuclear agarose gel microspheres as described in claim 1, characterized in that, In step S3.4, the second crosslinking agent is at least one of a halogenated epoxy compound and a diglycidyl ether reagent; Optional, the volume ratio of concentrated alkali to crosslinking agent is 1:2; Optionally, the reaction is a stirred reaction at a speed of 300 rpm.

10. The method for preparing multinuclear agarose gel microspheres as described in claim 9, characterized in that, The halogenated epoxy compound is epichlorohydrin, and the diglycidyl ether reagent is selected from any one of ethylene glycol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.

11. The method for preparing multinuclear agarose gel microspheres as described in claim 9, characterized in that, In step S3.4, the reaction time is 30 min.

12. The method for preparing multinuclear agarose gel microspheres as described in claim 1, characterized in that, In step S3.5, the centrifugation is performed at 3500 rpm for 5 minutes.

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