Apparatus and method for preparing agarose microspheres by inverse emulsion method

Abstract

The application belongs to the field of microsphere preparation, and discloses a device and method for preparing agarose microspheres by adopting a reverse phase emulsification method. The application aims at the liquid drop stability problem existing in the emulsification process of the agarose hot melting and cold condensation system, and constructs a continuous control system of "preheating-emulsification-gel solidification". Different stage flow field switching is realized through a combined stirring structure, seamless connection of the emulsification and gel processes is realized through temperature control of the jacket program, and liquid drop coalescence in the emulsion transfer process is avoided through in-situ gelation, so as to improve the uniformity of the microsphere particle size and the batch repeatability. The application establishes a suitable process control mechanism according to the agarose hot melting and cold condensation characteristics, so that the emulsion drop size can be quickly fixed after formation, and the problems of wide particle size distribution and poor repeatability in the traditional process are solved.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and microsphere preparation technology, specifically relating to a special device for preparing agarose microspheres with uniform size and good sphericity, and a preparation method using the device. Background Technology

[0002] Reverse emulsification is a classic process for preparing agarose microspheres. Traditional laboratory methods typically involve multiple independent steps and containers: first, the aqueous phase of agarose is dissolved and heated in a beaker; then, the oil phase is prepared and preheated in another container; subsequently, the aqueous phase is poured into the oil phase with stirring for emulsification; finally, the entire emulsion is transferred to a third container for cooling and gelation. This method is cumbersome, lacks precise heat transfer and temperature control, and is difficult to standardize, resulting in a wide microsphere size distribution, poor batch reproducibility, and an opaque process that hinders observation of the emulsification and gelation processes.

[0003] Chinese patent CN119819169A discloses a microfluidic device and method for reverse emulsifying liposomes. Microfluidic methods have limited commercial applications, and the technology currently suffers from low throughput, preventing large-scale production. Double-walled glass reactors are commonly used in chemical synthesis, employing jackets for heating and cooling, and their transparency facilitates observation. However, there is currently no systematic improvement and dedicated solution for the reverse emulsification of agarose microspheres. Existing technologies lack an integrated device capable of sequentially completing the entire process—oil phase preheating, controlled emulsification, and in-situ programmed cooling gelation—within a sealed, visible container. Summary of the Invention

[0004] To address the problems of dispersion, poor controllability, and difficulty in observation in existing reverse emulsification processes, this invention provides a modularly improved device based on a standard double-layered glass reactor. The purpose of this invention is to realize the key steps of agarose microsphere preparation within a single main reaction vessel, thereby improving the consistency, controllability, and repeatability of the process.

[0005] This invention provides an apparatus for preparing agarose microspheres by a reverse emulsification method, comprising: Overall framework A double-layered glass reactor includes a reactor body mounted on an integral frame. The reactor body includes a circulating liquid jacket. The reactor body cover is provided with an oil phase feed port and an aqueous phase feed port that communicate with the interior of the reactor body. The aqueous phase feed port is used to add an agarose solution, which is the aqueous phase, to the oil phase inside the reactor at a constant rate after the oil phase is added to the reactor through the oil phase feed port. The motor's output shaft is connected via a stirring seal to a combined stirring paddle located inside the vessel and coaxially mounted with the vessel body. The motor is communicatively connected to a speed control panel for controlling the stirring speed. The speed control panel is used to control the motor to perform low-speed stirring and preheating after the oil phase is added to the vessel, medium-speed stirring and mixing during the dropwise addition of the aqueous phase, high-speed emulsification stirring after the agarose solution is added, and medium-low speed stirring during the cooling and gelation process after emulsification. A circulating water bath system is connected to the circulating liquid jacket to control the temperature inside the vessel; the circulating water bath system is communicatively connected to a programmable temperature control unit; the circulating water bath system is used for water bath heating during the low-speed stirring and preheating of the oil phase, the dripping of the water phase, and the high-speed emulsification stirring process; the circulating water bath system is also used for cooling during the gelation process after the high-speed emulsification stirring is completed. The discharge port is located at the bottom of the vessel. The discharge port is used to transfer the oil phase mixture containing microspheres in the vessel to an external beaker for washing after gelation, so as to obtain clean agarose microspheres and store them in an ethanol solution.

[0006] Preferably, the vessel lid is mounted on the vessel body using a vessel lid clamp, and the vessel lid is also provided with a condenser pipe for condensation reflux.

[0007] Preferably, the output shaft of the motor is connected to the stirring rod of the combined stirring paddle via a universal joint.

[0008] Preferably, the circulating water bath system includes an inlet located on the lower side of the vessel body and an outlet located on the upper side of the vessel body.

[0009] Preferably, the combined stirring paddle includes crescent-shaped blades and a dispersion disc coaxially mounted on the stirring rod.

[0010] Preferably, the inner wall of the vessel is equipped with baffles to increase the turbulence of the fluid, so that the material, gas or solid particles are more evenly distributed in the vessel, thereby improving the reaction rate and mixing efficiency.

[0011] Preferably, the overall frame is made of aluminum alloy and stainless steel; the parts of the double-layer glass reactor that come into contact with the materials are all made of high borosilicate glass.

[0012] Preferably, the agarose solution is pre-dissolved by heating in a three-necked flask and stored in a reservoir connected to an injection pump. The reservoir of the injection pump is used to connect to the aqueous phase feed port for dripping the agarose solution.

[0013] Preferably, the lid of the vessel is also provided with a thermometer sleeve for detecting the temperature inside the vessel.

[0014] This invention also provides a method for preparing agarose microspheres by reverse emulsification, which utilizes the aforementioned apparatus, and the method includes: Step S1: Close the discharge port at the bottom of the reactor body, open the oil phase feed port to add the oil phase containing surfactant into the reactor, control the circulating water bath system to perform external circulating water bath, heat the oil phase and keep the temperature constant at 60-70℃, and control the speed control panel to keep the motor speed at 300rpm to make the oil phase in the reactor preheat evenly. Step S2: While maintaining the oil phase temperature at 60-70℃ through the circulating water bath system, open the water phase feed port and add the pre-prepared and heated dissolved agarose solution dropwise into the oil phase in the reactor at a constant rate. Under the stirring of the agitator, the solution is dispersed into uniformly sized droplets, forming a water-in-oil primary emulsion. During the dropwise addition process, the speed control panel controls the motor to maintain a speed of 500 rpm. Step S3: After the dripping is completed, the speed control panel controls the motor to increase the speed to 600 rpm for strong emulsification, forming a stable water-in-oil emulsion; the circulating water bath system controls the heating and emulsification temperature to be maintained at 50℃~80℃, and the emulsification time is 10min~50min; Step S4: After emulsification is completed, the circulating water bath system stops heating. The circulating water bath system replaces the circulating liquid jacket with cold water to slowly cool the emulsion to below 20°C. During the cooling process, the speed control panel controls the motor to keep stirring at 400 rpm. After gelation is completed, stop stirring and let stand to allow the microspheres to settle. Step S5: After standing for 1 hour, open the discharge port at the bottom of the reactor and transfer the oil phase mixture containing microspheres in the reactor to an external beaker. Pour off the upper oil phase and wash with water and anhydrous ethanol alternately to finally obtain clean agarose microspheres, which are then stored in an ethanol solution.

[0015] Compared with the prior art, the present invention has the following outstanding advantages: 1. High Integration and Simple Operation: The three core steps of preheating, emulsification, and gelation are integrated into a single reaction vessel, avoiding losses, contamination, and temperature fluctuations caused by material transfer, and simplifying the operation process. This application addresses the droplet stability problem in the emulsification process of the agarose thermal dissolution and condensation system by constructing a continuous control system of "preheating—emulsification—gelation and solidification." A combined stirring structure enables switching of the flow field at different stages; jacketed programmed temperature control achieves seamless connection between emulsification and gelation processes; and in-situ gelation prevents droplet aggregation during emulsion transfer, thereby improving the uniformity of microsphere size and batch repeatability. This invention establishes a suitable process control mechanism for the thermal dissolution and condensation characteristics of agarose, enabling rapid fixation of droplet size after formation, solving the problems of wide particle size distribution and poor repeatability in traditional processes.

[0016] 2. High process visibility and controllability: The transparent glass vessel allows for real-time observation of the emulsion state and gelation process, facilitating process debugging and monitoring. Independently controlled feeding rate, stirring speed, and programmed cooling improve the uniformity of microsphere size.

[0017] 3. Excellent repeatability and consistency: The modular and parameterized operation mode replaces manual operation that relies on personal experience, significantly improving the repeatability and consistency between different batches of products.

[0018] 4. Low cost and easy to promote: The core of the device is based on the modification of a conventional double-layer glass reactor, which is low in cost and easy to build and promote in laboratories and small and medium-sized pilot production workshops.

[0019] 5. Good sealing and safety: The closed system reduces the leakage of volatiles and can be protected by inert gas, making it suitable for preparation processes that are sensitive to oxygen or require a more stable environment. Attached Figure Description

[0020] Figure 1 This is a diagram of the experimental setup for the apparatus used in the reverse emulsification method for preparing agarose microspheres provided by the present invention.

[0021] Figure 2 Microscopic image of agarose microspheres prepared by the reverse emulsification method according to an embodiment of the present invention.

[0022] Figure 1 The structural names of each part are as follows: 1. Speed ​​control panel; 2. Overall frame; 3. Aqueous phase feed port; 4. Oil phase feed port; 5. Reactor lid; 6. Reactor lid clamp; 7. Reactor body; 8. Circulating liquid jacket; 9. Baffle; 10. Crescent-shaped impeller; 11. Water inlet; 12. Discharge port; 13. Water outlet; 14. Stirring seal; 15. Condenser; 16. Universal joint; 17. Motor. Detailed Implementation

[0023] 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.

[0024] Unless otherwise specified, all raw materials and equipment used in the following examples were commercially available.

[0025] Combination Figure 1This embodiment provides an apparatus for preparing agarose microspheres using a reverse emulsification method, comprising a main reaction unit, a programmed temperature control unit, and an auxiliary interface unit. The main reaction unit is a jacketed double-layered glass reactor with multiple standard ground glass joints on its top. The reactor is equipped with a combined stirring paddle driven by a speed-regulating motor. This paddle preferably includes a crescent-shaped blade for medium-to-low speed mixing and a dispersion disc for high-speed emulsification. The auxiliary interface unit includes a thermometer sleeve and a reflux condenser pipe mounted on the top of the reactor.

[0026] Specifically, the double-layered glass reactor includes a reactor body 7 mounted on an integral frame 2. The reactor body 7 includes a circulating liquid jacket 8. The reactor body cover 5 is provided with an oil phase feed port 4 and an aqueous phase feed port 3 that communicate with the interior of the reactor body 7. The aqueous phase feed port 3 is used to add an agarose solution, which is the aqueous phase, to the oil phase in the reactor at a constant rate after the oil phase is added to the reactor through the oil phase feed port 4. The agarose solution (e.g., 4-10% w / v, 95°C) is preheated and dissolved in a three-necked flask and stored in a reservoir connected to an injection pump. The reservoir of the injection pump is used to connect to the aqueous phase feed port 3 to add the agarose solution.

[0027] The vessel lid 5 is mounted on the vessel body 7 via a vessel lid clamp 6. The vessel lid 5 is also equipped with a condenser pipe 15 for condensation and reflux. In addition, a baffle 9 is installed on the inner wall of the vessel body 7 to increase the turbulence of the fluid, so that the material, gas or solid particles are more evenly distributed in the vessel, thereby improving the reaction rate and mixing efficiency.

[0028] The output shaft of motor 17 is connected via a stirring seal to a combined stirring paddle located inside and coaxially with the vessel body 7. The output shaft of motor 17 is connected to the stirring rod of the combined stirring paddle via a universal joint 16. The combined stirring paddle includes crescent-shaped blades and a dispersion disc coaxially mounted on the stirring rod. The stirring rod is made of 304 stainless steel and encased in a polytetrafluoroethylene (PTFE) tube. The crescent-shaped blades are mainly used for medium- and low-speed mixing, while the dispersion disc is mainly used for high-speed emulsification. Motor 17 is communicatively connected to a speed control panel 1 for controlling the stirring speed of the combined stirring paddle. The speed control panel controls motor 17 to perform low-speed stirring and preheating (300 rpm) after the oil phase is added to the vessel, medium-speed stirring and mixing (500 rpm) during the droplet addition of the aqueous phase, high-speed emulsification stirring (600 rpm) after the agarose solution is added, and medium- and low-speed stirring (400 rpm) during the cooling and gelation process after emulsification.

[0029] The programmable temperature control unit employs an external circulating water bath system connected to the circulating liquid jacket 8 of the reactor. This system circulates cooling medium into the jacket to cool the reaction system within the reactor. The circulating water bath system is connected to the circulating liquid jacket 8 to control the internal temperature of the reactor body 7. It is also communicatively connected to the programmable temperature control unit. The circulating water bath system is used for water bath heating during low-speed stirring preheating of the oil phase, dropwise addition of the aqueous phase, and high-speed emulsification stirring. It is also used for cooling during the gelation process after high-speed emulsification stirring. The circulating water bath system includes an inlet 11 located on the lower side of the reactor body 7 and an outlet 13 located on the upper side of the reactor body 7.

[0030] The bottom of the vessel body 7 is provided with a discharge port 12, which is used to transfer the oil phase mixture containing microspheres inside the vessel body 7 to an external beaker for washing after gelation. The output shaft of the motor 17 is connected to the stirring rod of the combined stirring paddle through a universal joint 16.

[0031] This invention addresses the droplet stability issue in the emulsification process of agarose thermal dissolution and condensation systems by constructing a continuous control system of "preheating—emulsification—gel solidification." A combined stirring structure enables switching of the flow field at different stages; jacketed programmed temperature control achieves seamless integration of emulsification and gelation processes; and in-situ gelation prevents droplet aggregation during emulsion transfer, thereby improving microsphere particle size uniformity and batch repeatability. This invention establishes a process control mechanism adapted to the thermal dissolution and condensation characteristics of agarose, enabling rapid fixation of droplet size after formation, solving the problems of wide particle size distribution and poor repeatability in traditional processes. All key steps are sequentially completed within the same double-layered glass reactor.

[0032] The method for preparing agarose microspheres by reverse emulsification in this embodiment includes: Step S1: Close the discharge port at the bottom of the reactor body 7, and open the oil phase feed port 4 to add the oil phase containing surfactant (a certain amount of oil-soluble emulsifier needs to be added to the base material, which includes one or more of cyclohexane, liquid paraffin, petroleum ether, olive oil, soybean oil and sunflower oil, and the emulsifier is preferably PO-5S emulsifier with a concentration of 6% and an HLB value of 4.7) into the reactor. Control the circulating water bath system to perform external circulating water bath, heat the oil phase and keep it at a constant temperature of 60-70℃. Control the speed control panel 1 to control the motor 17 to maintain a speed of 300 rpm, so that the oil phase in the reactor is preheated evenly. Step S2: While maintaining the oil phase temperature at 60-70℃ through the circulating water bath system, open the aqueous phase feed port 3 and add the pre-prepared and heated dissolved agarose solution dropwise into the oil phase in the reactor at a constant rate. Under the stirring of the agitator, the solution is dispersed into uniformly sized droplets, forming a water-in-oil primary emulsion. During the dropwise addition process, the speed control panel 1 controls the motor 17 to maintain a speed of 500 rpm, and the stirring time is about 10 minutes. Step S3: After the dripping is completed, the speed control panel 1 controls the motor 17 to increase the speed to 600 rpm for strong emulsification, forming a stable water-in-oil emulsion; the circulating water bath system controls the heating and emulsification temperature to be maintained at 50℃~80℃, and the emulsification time is 10min~50min; Step S4: After emulsification is completed, the circulating water bath system stops heating. The circulating water bath system replaces the cold water in the circulating liquid jacket 8 to slowly cool the emulsion to below 20°C. During the cooling process, the speed control panel 1 controls the motor 17 to keep stirring at 400 rpm. After gelation is completed, stop stirring and let it stand to allow the microspheres to settle. Step S5: After standing for 1 hour, open the discharge port at the bottom of the reactor body 7, transfer the oil phase mixture containing microspheres inside the reactor body 7 to an external beaker, pour out the upper oil phase, and wash with water and anhydrous ethanol alternately to finally obtain clean agarose microspheres, which are then stored in an ethanol solution.

[0033] The following is a specific preparation method: First, prepare 100 mL of a 6% agarose solution and heat it for 120 min at 95°C as the aqueous phase. Prepare 200 mL of cyclohexane as the oil phase and add 15 mL of PO-5S emulsifier. Pour this mixture into a pre-built 1L reactor and stir at 300 rpm for 15-20 min under a 70°C water bath to ensure thorough mixing. While still hot, slowly add the agarose solution dropwise to the oil phase at a constant rate, stirring at 500 rpm for approximately 10 min. Under stirring, the mixture disperses into uniform droplets, forming a water-in-oil emulsion. After the addition is complete, adjust the stirring speed to 600 rpm and continue vigorous emulsification for 30 min to form a stable water-in-oil emulsion. The temperature of the agarose solution should be above 90°C when slowly adding it to the oil phase. After emulsification, stop heating in the circulating water bath system and replace the cold water in the circulating liquid jacket 8 to maintain emulsification. The liquid was slowly cooled, and the mixture was allowed to solidify into microspheres within 30 minutes. The temperature of the cooling water was maintained below 20°C. During the cooling process, the motor 17 was controlled by control panel 1 to maintain stirring at 400 rpm. After gelation was complete, stirring was stopped, and the mixture was allowed to settle. After settling for 1 hour, the discharge port at the bottom of the reactor 7 was opened, and the oil phase mixture containing microspheres was transferred from reactor 7 to an external beaker. The upper oil phase was poured off, and the mixture was washed alternately with water and anhydrous ethanol to obtain clean agarose microspheres. These microspheres were then stored in a 20% ethanol solution. The microscopic image of the prepared microspheres is shown below. Figure 2 As shown.

[0034] Unlike traditional reverse emulsification processes that rely solely on mechanical shearing to form droplets, this invention achieves a stable particle size distribution that meets expectations by controlling the emulsifier system and the stirring conditions during emulsification. Experiments determined the preferred scheme to be PO-5S emulsifier at a concentration of 6%, an HLB value of 4.7, and an emulsification speed of 600 rpm. This ensures good interfacial stability during droplet formation, thereby reducing droplet aggregation. Furthermore, when the distribution of microspheres in 1:2 and 1:3 ratios is similar, this method maintains system stability even under low oil phase conditions.

[0035] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An apparatus for preparing agarose microspheres by reverse emulsification, characterized in that, include: Overall framework (2), A double-layered glass reactor includes a reactor body (7) mounted on an integral frame (2). The reactor body (7) includes a circulating liquid jacket (8). The reactor body cover (5) is provided with an oil phase feed port (4) and an aqueous phase feed port (3) communicating with the interior of the reactor body (7). The aqueous phase feed port (3) is used to add an agarose solution, which is an aqueous phase, to the oil phase inside the reactor at a constant rate after the oil phase is added to the reactor through the oil phase feed port (4). The motor (17) has its output shaft connected to a combined stirring paddle located inside the vessel body (7) and coaxially arranged with the vessel body (7) via a stirring seal. The motor (17) is communicatively connected to a speed control panel (1) for controlling the stirring speed. The speed control panel is used to control the motor (17) to perform low-speed stirring and preheating after the oil phase is added to the vessel, to perform medium-speed stirring and mixing during the droplet addition of the aqueous phase, to perform high-speed emulsification stirring after the agarose solution is added, and to perform medium-low speed stirring during the cooling and gelation process after emulsification. A circulating water bath system is connected to the circulating liquid jacket (8) to control the temperature inside the vessel body (7); the circulating water bath system is communicatively connected to a programmable temperature control unit; the circulating water bath system is used for water bath heating during the process of low-speed stirring and preheating of the oil phase, dropwise addition of the water phase and high-speed emulsification stirring; the circulating water bath system is also used for cooling during the gelation process after the high-speed emulsification stirring is completed. The discharge port (12) is located at the bottom of the vessel body (7). The discharge port (12) is used to transfer the oil phase mixture containing microspheres in the vessel body (7) to an external beaker for washing after gelation, so as to obtain clean agarose microspheres and store them in an ethanol solution.

2. The apparatus for preparing agarose microspheres by reverse emulsification according to claim 1, characterized in that, The lid (5) is mounted on the body (7) by a lid clamp (6), and the lid (5) is also provided with a condenser pipe (15) for condensation reflux.

3. The apparatus for preparing agarose microspheres by reverse emulsification according to claim 1, characterized in that, The output shaft of the motor (17) is connected to the stirring rod of the combined stirring paddle via a universal joint (16).

4. The apparatus for preparing agarose microspheres by reverse emulsification according to claim 1, characterized in that, The circulating water bath system includes an inlet (11) located on the lower side of the vessel body (7) and an outlet (13) located on the upper side of the vessel body (7).

5. The apparatus for preparing agarose microspheres by reverse emulsification according to claim 1, characterized in that, The combined agitator includes crescent-shaped blades (10) and a dispersion disc coaxially mounted on the agitator rod.

6. The apparatus for preparing agarose microspheres by reverse emulsification according to claim 1, characterized in that, The inner wall of the vessel body (7) is equipped with baffles (9) to increase the turbulence of the fluid, so that the material, gas or solid particles are more evenly distributed in the vessel, thereby improving the reaction rate and mixing efficiency.

7. The apparatus for preparing agarose microspheres by reverse emulsification according to claim 1, characterized in that, The overall frame (2) is made of aluminum alloy and stainless steel; the part of the double-layer glass reactor that comes into contact with the material is made of high borosilicate glass.

8. The apparatus for preparing agarose microspheres by reverse emulsification according to claim 1, characterized in that, The agarose solution is preheated and dissolved in a three-necked flask and stored in a reservoir connected to an injection pump. The reservoir of the injection pump is used to connect to the aqueous phase feed port (3) to add the agarose solution dropwise.

9. The apparatus for preparing agarose microspheres by reverse emulsification according to claim 1, characterized in that, The kettle lid (5) is also equipped with a thermometer sleeve for detecting the temperature inside the kettle.

10. A method for preparing agarose microspheres by reverse emulsification, characterized in that, The method comprises: using the apparatus according to any one of claims 1-9 Step S1: Close the discharge port at the bottom of the reactor body (7), open the oil phase feed port (4) to add the oil phase containing surfactant into the reactor, control the circulating water bath system to perform external circulating water bath, heat the oil phase and keep the temperature at 60-70℃, control the speed control panel (1) to control the motor (17) to maintain a speed of 300rpm, so that the oil phase in the reactor is preheated evenly. Step S2: Under the premise of maintaining the oil phase temperature at 60-70℃ through the circulating water bath system, open the water phase feed port (3) and add the pre-prepared and heated dissolved agarose solution to the oil phase in the reactor at a constant rate. Under the stirring of the stirring paddle, it is dispersed into uniformly sized droplets to form a water-in-oil primary emulsion. During the droplet addition process, the speed control panel (1) controls the motor (17) to maintain a speed of 500 rpm. Step S3: After the dripping is completed, the speed control panel (1) controls the motor (17) to increase the speed to 600 rpm for strong emulsification to form a stable water-in-oil emulsion; the circulating water bath system controls the heating and emulsification temperature to be maintained at 50℃~80℃, and the emulsification time is 10min~50min; Step S4: After emulsification is completed, the circulating water bath system stops heating. The circulating water bath system replaces the cold water in the circulating liquid jacket (8) to slowly cool the emulsion to below 20°C. During the cooling process, the speed control panel (1) controls the motor (17) to keep stirring at 400 rpm. After gelation is completed, stop stirring and let it stand to allow the microspheres to settle. Step S5: After standing for 1 hour, open the discharge port at the bottom of the reactor (7), transfer the oil phase mixture containing microspheres in the reactor (7) to an external beaker, pour out the upper oil phase, and wash with water and anhydrous ethanol in turn to finally obtain clean agarose microspheres and store them in an ethanol solution.

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