Preparation method of agarose microspheres by reverse phase emulsification

Abstract

This invention belongs to the field of microsphere preparation and discloses a method for preparing agarose microspheres using a reverse emulsification method. The preparation method includes the following steps: preparing an agarose aqueous solution of a certain concentration as the aqueous phase; pouring the prepared oil phase into a self-made reaction vessel; and preheating the reaction vessel by turning on a circulating constant-temperature water bath. Preheating the oil phase to the required temperature and adjusting the rotation speed to ensure sufficient preheating. Slowly pouring the heated aqueous phase into the oil phase, adjusting the rotation speed, and emulsifying for a certain time. Stopping heating in the water bath, replacing the hot water with cold water to slowly cool the emulsion, and controlling the time to solidify it into microspheres.

Description

Technical Field

[0001] This invention belongs to the field of microsphere preparation, specifically designing a method for preparing agarose microspheres by reverse emulsification. Background Technology

[0002] Agarose gel is a non-toxic, biocompatible, and non-irritating biodegradable natural high-molecular-weight polymer. It possesses a series of advantages, such as being rich in hydroxyl groups, having good hydrophilicity, strong modifying ability, low non-specific adsorption, and good stability. Simultaneously, its matrix has an excellent porous structure, which facilitates macromolecular diffusion, making it ideal for preparing chromatographic media. Agarose-based chromatographic media are among the most commonly used chromatographic media to date in gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and atomic number chromatography, and are important separation chromatographic media for the purification of biomolecules such as proteins, nucleic acids, and peptides.

[0003] The main methods for preparing agarose microspheres include suspension stirring, membrane emulsification, spraying, and microfluidics. Membrane emulsification is slow and the membrane module is prone to contamination; spraying is complex and difficult to scale up for production. Chinese Patent Publication No. CN118022641A discloses a microfluidic method for preparing microspheres, but this method has low yield per batch, is carried out under high temperature and pressure, and requires sophisticated equipment. In contrast, reverse emulsification is simple to operate, has low equipment requirements, and is easily scalable for mass production.

[0004] Currently, the reverse emulsification method for preparing agarose microspheres has been widely used due to its advantages such as simple process, low equipment requirements, and ease of scale-up production. However, existing reverse emulsification processes still have the following shortcomings: During emulsification, droplets are affected by shear forces, collisions, and aggregation, easily generating droplets with large differences in particle size, resulting in a wide particle size distribution and poor uniformity of the final prepared agarose microspheres. Furthermore, the microsphere particle size is greatly affected by fluctuations in emulsification conditions, making size control difficult and failing to meet the requirements for particle size consistency and batch stability in chromatographic media and other fields.

[0005] Therefore, we need to develop a method for preparing agarose microspheres that can improve the particle size distribution and controllability of the microspheres. Summary of the Invention

[0006] This invention provides a method for preparing agarose microspheres by reverse emulsification, which is simple to operate, requires low equipment standards, and is easy to scale up for production.

[0007] This invention provides a method for preparing agarose microspheres by reverse emulsification, comprising the following steps: Step S1, Aqueous phase preparation: Prepare an agarose solution with a concentration of [missing information] and dissolve it by heating; Step S2, oil phase preparation: Add a certain amount of oil-soluble emulsifier to the base material in the constructed reaction vessel, and stir continuously for a period of time under a water bath at a suitable temperature; Step S3, Emulsification: While maintaining the temperature of the oil phase, the aqueous agarose solution is slowly added dropwise to the oil phase in the reactor while it is still hot. Stirring is maintained during the dropwise addition to form a water-in-oil primary emulsion. After the dropwise addition is completed, the stirring speed is increased and emulsification is continued for a period of time to form a stable water-in-oil emulsion. Step S4, Curing: Stop heating the water bath and replace the hot water with cold water to slowly cool the emulsion, controlling the time to solidify it into microspheres; keep stirring during the cooling process, and stop stirring after gelation is complete, and let it stand to allow the microspheres to settle. Step S5, Washing: After standing for a period of time, pour off the upper oil phase and wash with water and anhydrous ethanol alternately to finally obtain clean agarose microspheres.

[0008] Preferably, the concentration of the aqueous agarose solution prepared in step S1 is 2%-8%, the heating temperature is 90℃-100℃, and the heating time is 70min-120min.

[0009] As a preferred option The base material for step S2 includes one or more of cyclohexane, liquid paraffin, petroleum ether, olive oil, soybean oil, and sunflower oil.

[0010] Preferably, the emulsifier in step S2 is PO-5S emulsifier with a concentration of 6% and an HLB value of 4.7; the stirring speed in step S2 is 300 rpm. In step S3, while the aqueous agarose solution is being slowly poured into the oil phase while still hot, the mixture is stirred at 500 rpm. During the continuous emulsification process in step S3, the heating and emulsification temperature is 50℃~80℃, the emulsification time is 10min~50min, and the emulsification speed is 600rpm. The volume ratio of the aqueous phase to the oil phase is 1:2 to 1:3.

[0011] Preferably, step S2 uses a 70°C water bath.

[0012] As a preferred option Oil-soluble emulsifiers include one or more of PO-5S, Span 40, Span 60, Span 80, Span 85, Tween 20, Tween 40, Tween 60 and Tween 80, used to adjust the HLB value.

[0013] Preferably, when replacing hot water with cold water to slowly cool the emulsion, the cooling rate is adjusted to 2-15°C / min or direct ice-water bath cooling is used to cool the emulsion to below 10-20°C.

[0014] Preferably, the stirring speed during the curing process in step S4 is 400 rpm.

[0015] Preferably, step S5 requires the plant to stand for 1 hour.

[0016] The present invention also provides agarose microspheres, which are prepared by the method for preparing agarose microspheres by the reverse emulsification method described above.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention first prepares the aqueous phase: an agarose solution of a certain concentration is prepared and heated to dissolve; then, an oil phase is prepared: a certain amount of oil-soluble emulsifier is added to the base material in the constructed reaction vessel, and the mixture is continuously stirred for a period of time under a water bath at a suitable temperature; while maintaining the temperature of the oil phase, the aqueous agarose solution is slowly added dropwise to the oil phase in the reaction vessel while hot, and stirring is maintained during the dropwise addition to form a water-in-oil primary emulsion. After the dropwise addition is completed, the stirring speed is increased, and emulsification is continued for a period of time to form a stable water-in-oil emulsion. In the emulsification stage, the heated and dissolved agarose aqueous phase is added to a pre-prepared oil phase system while maintaining fluidity. The stirring conditions during emulsification are controlled to stabilize droplet formation. Unlike traditional reverse emulsification processes that rely solely on mechanical shearing to form droplets, this invention achieves a stable particle size distribution by controlling the emulsifier system and stirring conditions during emulsification. The preferred scheme, determined through experiments, is an emulsifier concentration of 6%, an emulsifier HLB value of 4.7 (PO-5S), and an emulsification speed of 600 rpm. This ensures good interfacial stability during droplet formation, reducing droplet aggregation. Furthermore, since the distribution of microspheres in ratios of 1:2 and 1:3 is similar, this method maintains system stability even under low oil phase conditions.

[0018] This invention employs a process of emulsification followed by solidification. After emulsification, the water bath is stopped, and cold water is used instead of hot water to slowly cool the emulsion, allowing it to solidify into microspheres within a controlled time. Stirring is maintained throughout the cooling process. Once gelation is complete, stirring is stopped, and the microspheres are allowed to settle. After a period of settling, the upper oil phase is poured off, and the microspheres are washed alternately with water and anhydrous ethanol to obtain clean agarose microspheres. This application utilizes a process route of emulsification followed by cooling and solidification. By controlling the cooling process of the emulsion system, the formed droplets are gradually solidified, avoiding particle size fluctuations caused by rapid solidification or droplet instability, thereby improving the consistency of the microsphere particle size distribution. Experimental verification has shown that this method achieves superior preparation results. Attached Figure Description

[0019] Figure 1 Microscopic images and particle size distribution of agarose microspheres with an emulsifier concentration of 3%; Figure 2 Microscopic images and particle size distribution of agarose microspheres at an emulsifier concentration of 5%; Figure 3 Microscopic images and particle size distribution of agarose microspheres at an emulsifier concentration of 6%; Figure 4 Microscopic images and particle size distribution of agarose microspheres at an emulsifier concentration of 8%; Figure 5 Microscopic images and particle size distribution of agarose microspheres with an HLB value of 4.3; Figure 6 Microscopic images and particle size distribution of agarose microspheres with an HLB value of 4.7; Figure 7 Microscopic images and particle size distribution of agarose microspheres at an HLB value of 5; Figure 8 Microscopic images and particle size distribution of agarose microspheres at an HLB value of 6; Figure 9 Microscopic images and particle size distribution of agarose microspheres at an emulsification speed of 400 rpm; Figure 10 Microscopic images and particle size distribution of agarose microspheres at an emulsification speed of 600 rpm; Figure 11 Microscopic images and particle size distribution of agarose microspheres at an emulsification speed of 800 rpm; Figure 12 Microscopic images and particle size distribution of agarose microspheres at an emulsification speed of 1000 rpm. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0021] The core of this invention lies in the control of the emulsification and solidification processes. During the emulsification stage, the heated and dissolved agarose aqueous phase is added to a pre-prepared oil phase system while maintaining fluidity. The stirring conditions during emulsification are controlled to stabilize the droplet formation process. Unlike traditional reverse emulsification processes that rely solely on mechanical shearing to form droplets, this application controls the emulsifier system and emulsification conditions to maintain good interfacial stability during droplet formation, thereby reducing droplet aggregation. This application employs a process route of emulsification followed by cooling and solidification. By controlling the cooling process of the emulsion system, the formed droplets are gradually solidified, avoiding particle size fluctuations caused by rapid solidification or droplet instability, thus improving the consistency of the microsphere particle size distribution.

[0022] Unless otherwise specified, all raw materials and equipment used in the following examples were commercially available.

[0023] Example 1 Reference Figure 1 This embodiment exemplifies a method for preparing agarose microspheres using a reverse emulsification method, the specific steps of which are as follows: (1) Prepare 100 mL of 6% agarose solution and heat it for 120 min at a temperature of 95℃; (2) Prepare 200 mL of oil phase cyclohexane and add 15 mL of PO-5S emulsifier into the prepared 1L reactor. Stir for about 15 to 20 minutes at 300 rpm under 70℃ water bath conditions to make it fully mixed.

[0024] (3) While the aqueous agarose solution is hot, slowly add it dropwise to the oil phase at a constant rate. During the dropwise addition process, stir at 500 rpm. Under the stirring of the stirrer, disperse it into uniformly sized droplets to form a water-in-oil primary emulsion. After the dropwise addition is completed, adjust the speed to 600 rpm and continue strong emulsification for 30 minutes to form a stable water-in-oil emulsion. The temperature of the aqueous agarose solution when slowly adding it dropwise to the oil phase should be above 90℃. (4) Stop heating the water bath and replace the hot water with cold water to slowly cool the emulsion. Control the temperature for 30 minutes to solidify it into microspheres. Keep the temperature of the cold water below 20°C. Continue stirring at 400 rpm during the cooling process. After gelation is complete, stop stirring and let the microspheres settle. This speed is the speed during solidification, not the speed during emulsification. (5) After standing for 1 hour, pour off the upper oil phase and wash with petroleum ether, water and anhydrous ethanol respectively; Comparative Experiment 1 Combination Figures 1-4 The difference from Example 1 is that all other experimental conditions are the same, with emulsifier concentrations of 3%, 5%, 6%, and 8%, respectively. The emulsifier selected in this experiment is PO-5S. By observing the microscopic images and particle size distribution of agarose microspheres at different concentrations, it was found that at an emulsifier concentration of 6%, the proportion of microspheres with a particle size of 90-110 μm was the largest; therefore, an emulsifier concentration of 6% is appropriate.

[0025] Comparative Experiment 2 Combination Figures 5-8 The difference from Experiment 1 is that, with other experimental conditions remaining the same, and emulsifier HLB values ​​of 4.3, 4.7, 5, and 6, a suitable HLB value was achieved by combining Span80 (HLB=4.3) and Tween40 (HLB=15.6). Microscopic images and particle size distributions of agarose microspheres at different HLB values ​​were observed. At an HLB value of 4.7, the proportion of microspheres with a size of 90-110 μm was the largest. Since PO-5S has an HLB value of 4.7, it is more convenient to use; therefore, PO-5S emulsifier was selected.

[0026] Comparative Experiment 3 Combination Figures 9-12 The difference from Experiment 1 is that the emulsification speeds were 400 rpm, 600 rpm, 800 rpm, and 1000 rpm. Observing the microscopic images and particle size distribution of agarose microspheres at different speeds, the proportion of microspheres with a size of 90-110 μm was the largest when the emulsification speed was 600 rpm. Therefore, an emulsification speed of 600 rpm is more suitable.

[0027] Comparative Experiment 4 The difference from Experiment 1 is that all other experimental conditions are the same, with the water-to-oil ratio being 1:2, 1:4, 1:6, and 1:8. Microscopic images and particle size distributions of agarose microspheres at different water-to-oil ratios were observed. The microsphere distributions were similar at ratios of 1:2 and 1:3. Therefore, this experiment can maintain system stability even under low oil phase conditions.

[0028] Comparative Experiment 5 Synergistic effect experiment of HLB value and emulsifier concentration The difference from Experiment 1 is that, with other experimental conditions unchanged, the relationship between HLB value and emulsifier concentration was studied. Microscopic images and particle size distribution maps of agarose microspheres were observed. The optimal distribution was found when the HLB value was 4.7 and the emulsifier concentration was 6, indicating a synergistic relationship between HLB value and emulsifier concentration. A specific concentration of emulsifier is required to match the HLB value.

[0029] Comparative Experiment 6 HLB value and emulsification speed synergistic experiment The difference from Experiment 1 is that, with other experimental conditions unchanged, the relationship between HLB value and emulsification speed was studied, and the microscopic images and particle size distribution of agarose microspheres were observed. The optimal distribution was found when the HLB value was 4.7 and the emulsification speed was 600 rpm. Therefore, simply increasing the speed cannot improve the particle size distribution; it needs to be matched with a specific HLB value.

[0030] In summary, this invention provides an innovative and simple method for preparing agarose. Agarose microspheres of 90-110 μm in size are prepared by a reverse emulsification method.

Claims

1. A method for preparing agarose microspheres using a reverse emulsification method, characterized in that, Includes the following steps: Step S1, Aqueous phase preparation: Prepare an agarose solution with a concentration of [missing information] and dissolve it by heating; Step S2, oil phase preparation: Add a certain amount of oil-soluble emulsifier to the base material in the constructed reaction vessel, and stir continuously for a period of time under a water bath at a suitable temperature; Step S3, Emulsification: While maintaining the temperature of the oil phase, the aqueous agarose solution is slowly added dropwise to the oil phase in the reactor while it is still hot. Stirring is maintained during the dropwise addition to form a water-in-oil primary emulsion. After the dropwise addition is completed, the stirring speed is increased and emulsification is continued for a period of time to form a stable water-in-oil emulsion. Step S4, Curing: Stop heating the water bath and replace the hot water with cold water to slowly cool the emulsion, controlling the time to solidify it into microspheres; keep stirring during the cooling process, and stop stirring after gelation is complete, and let it stand to allow the microspheres to settle. Step S5, Washing: After standing for a period of time, pour off the upper oil phase and wash with water and anhydrous ethanol alternately to finally obtain clean agarose microspheres.

2. The method for preparing microspheres by reverse emulsification according to claim 1, characterized in that: In step S1, the concentration of the aqueous agarose solution is 2%-8%, the heating temperature is 90℃-100℃, and the heating time is 70min-120min.

3. The method for preparing microspheres by reverse emulsification according to claim 1, characterized in that: The base material for step S2 includes one or more of cyclohexane, liquid paraffin, petroleum ether, olive oil, soybean oil, and sunflower oil.

4. The method for preparing microspheres by reverse emulsification according to claim 1, characterized in that: The emulsifier in step S2 is PO-5S emulsifier with a concentration of 6% and an HLB value of 4.7; the stirring speed in step S2 is 300 rpm. In step S3, while the aqueous agarose solution is being slowly poured into the oil phase while still hot, the mixture is stirred at 500 rpm. During the continuous emulsification process in step S3, the heating and emulsification temperature is 50℃~80℃, the emulsification time is 10min~50min, and the emulsification speed is 600rpm. The volume ratio of the aqueous phase to the oil phase is 1:2 to 1:

3.

5. The method for preparing microspheres by reverse emulsification according to claim 1, characterized in that: Step S2 uses a 70℃ water bath.

6. The method for preparing agarose microspheres by reverse emulsification according to claim 1, characterized in that: Oil-soluble emulsifiers include one or more of PO-5S, Span 40, Span 60, Span 80, Span 85, Tween 20, Tween 40, Tween 60 and Tween 80, used to adjust the HLB value.

7. The method for preparing agarose microspheres by reverse emulsification according to claim 1, characterized in that: When replacing hot water with cold water to slowly cool the emulsion, the cooling rate is adjusted to 2-15°C / min or direct ice-water bath cooling is used to cool it down to below 10-20°C.

8. The method for preparing agarose microspheres by reverse emulsification according to claim 1, characterized in that: The stirring speed during the curing process in step S4 is 400 rpm.

9. The method for preparing agarose microspheres by reverse emulsification according to claim 1, characterized in that: Step S5 requires standing for 1 hour.

10. An agarose microsphere, characterized in that, The agarose microspheres were prepared by the reverse emulsification method according to any one of claims 1-8.

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