A method for preparing biocompatible rice aggregate particles based on a microreactor system

Abstract

The present invention discloses a method for preparing biocompatible rice aggregate particles based on a microreactor system. The present invention adopts excellent biocompatible materials such as shellac and polylactic acid, dissolves the shellac and polylactic acid together in tetrahydrofuran, and then quickly performs solvent exchange in a microfluidic chip. The advantages of the microreactor's long residence time, controllable temperature and two-phase flow rate are used to dynamically regulate the nano-coprecipitation and phase separation processes, thereby accurately controlling the particle size, composition, and morphology of the nano-aggregates. The biocompatible nano-aggregate particles prepared by this method have a biomimetic structure of the new coronavirus and a similar size, and can be used for the development of biomimetic new coronavirus vaccines.

Description

Technical Field

[0001] The invention relates to the fields of granular materials and microchemical industry, and relates to a method for preparing biocompatible rice aggregate particles based on a microreactor system. Background Art

[0002] Nanoparticles that are non-toxic, safe, and have excellent biocompatibility have broad market applications and development prospects in the field of biomedical technology, such as targeted drug delivery, cell separation and detection, tumor hyperthermia, and biosensors. As functional particles, aggregate particles can have their micromorphology regulated and used as biomimetic COVID-19 particles for the development of biomimetic COVID-19 vaccines. However, existing methods for preparing aggregate particles cannot precisely and dynamically control preparation conditions such as residence time and temperature during the preparation process, making it impossible to obtain nanoaggregate particles with precise size, composition, and morphology.

[0003] Nanocoprecipitation under rapid mixing is an effective method for obtaining biocompatible nanoparticles, and different particle carriers can be obtained by controlling phase separation through interfacial tension. This method uses biocompatible materials, combines interfacial tension and the dynamic regulation of the nanocoprecipitation process and phase separation process by the microreactor system, and prepares shellac-polylactic acid aggregate particles with adjustable properties. The new coronavirus is spherical as a whole, with a thorn structure on the surface, and its diameter generally ranges from 60nm to 140nm. The nanoaggregate particles with good biocompatibility prepared by this method have a biomimetic structure similar to that of the new coronavirus, and by regulating parameters such as the two-phase flow rate and concentration, aggregate particles with a diameter of about 100nm can be obtained. Such particles have great potential in the development of biomimetic new coronavirus vaccines. Summary of the Invention

[0004] In view of the shortcomings of the existing agglomerate preparation methods, the present invention provides a method for preparing biocompatible shellac-polylactic acid nanoaggregate particles based on a microreactor system. The preparation method uses materials such as shellac and polylactic acid. Such materials have excellent biocompatibility and can automatically degrade in the human body. They are safe and non-toxic and can be used in the field of biomedicine. The preparation method has a simple process and can control the size, composition, and morphology of the nanoaggregate particles by precisely regulating parameters such as the two-phase flow rate, the two-phase concentration, the residence time, and the temperature. The prepared biocompatible nanoaggregate particles have a bionic structure of the new coronavirus and a similar size, and can be used for the development of bionic new coronavirus vaccines.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for continuously preparing nanoaggregate particles by using a microreactor system through a nano-coprecipitation method combined with controllable phase separation, comprising the following steps:

[0007] (1) Shellac is dissolved in tetrahydrofuran (THF) and stirred until completely dissolved. Polylactic acid (PLA) is then added to the shellac solution to prepare a mixed solution of shellac and PLA in THF. Tween 80 is added to deionized water and ultrasonically shaken to accelerate the dissolution to obtain a Tween 80 aqueous solution.

[0008] (2) pumping the shellac-polylactic acid solution obtained in step (1) into microreactor A and allowing it to remain there for a period of time; pumping the Tween 80 solution into microreactor B and allowing it to remain there for a period of time; heating the two solutions in the coil of the microreactor until the solution temperature rises to the preheating temperature and remains constant;

[0009] (3) The preheated tetrahydrofuran mixed solution of shellac and polylactic acid in step (2) and the Tween 80 solution are introduced into the microfluidic chip and uniformly mixed. Since the solubility of shellac and polylactic acid in Tween 80 is greatly reduced, the two polymers undergo a nano-coprecipitation process and precipitate together. At the same time, a thermodynamic phase separation process begins. The phase separation process refers to the process in which the solubility of the polymer decreases, the polymer condenses from the solution, and precipitates to form a particle structure. This process is dominated by kinetic equilibrium under rapid cooling conditions and takes a period of time of 10-30 minutes. In the microfluidic chip, due to the exchange of good solvent and poor solvent, the polymers in the mixed solution only begin the phase separation process, and an intermediate mixed solution that is not completely phase-separated is obtained;

[0010] (4) The incomplete phase-separated intermediate mixed solution obtained in step (3) is passed into the microreactor C for cooling. During the cooling process, the intermediate mixed solution continues to undergo a phase separation process dominated by kinetic equilibrium, thereby obtaining a uniform dispersion of biocompatible shellac-polylactic acid nanoaggregate particles in Tween 80 solution. The uniform dispersion of the aggregate particles in Tween 80 solution is then passed into a collection tank for storage.

[0011] According to a preferred embodiment of the present invention, in step (1),

[0012] In the tetrahydrofuran mixed solution of shellac and polylactic acid, the concentration of shellac ranges from 0.1 mg / mL to 4 mg / mL;

[0013] In the tetrahydrofuran mixed solution of shellac and polylactic acid, the concentration of polylactic acid ranges from 0.1 mg / mL to 4 mg / mL;

[0014] The molecular weight of the polylactic acid is in the range of 2000-5000;

[0015] The volume fraction of the Tween 80 aqueous solution is in the range of 0.5% to 5%.

[0016] According to a preferred embodiment of the present invention, in step (2),

[0017] The introduction rate of the tetrahydrofuran mixed solution of shellac and polylactic acid is 0.1 mL / h-1 mL / h;

[0018] The introduction rate of the Tween 80 aqueous solution is 4 mL / h-40 mL / h;

[0019] The ratio of the introduction rate of the tetrahydrofuran mixed solution of shellac and polylactic acid to the Tween 80 aqueous solution is 1:20-1:40;

[0020] The volume ratio of the mixed solution to the Tween 80 aqueous solution is in the range of 1:15-1:60.

[0021] In the preparation process, the mixed solution and Tween 80 aqueous solution need to stay in the coil for a certain time to be preheated. The generated particles can be stable for a long time at room temperature. The preheating temperature range is 65°C-90°C; the preheating residence time range is 10min-30min;

[0022] The diameter of the microreactor coil is in the range of 0.3 mm to 3 mm.

[0023] According to a preferred embodiment of the present invention, in step (3),

[0024] The microfluidic chip is a flow-focusing microfluidic chip, consisting of a series of connected glass capillaries. The chip includes a square capillary tube and two cylindrical glass capillaries nested within the square capillary tube. The two cylindrical glass capillaries are coaxially located, with their adjacent ends positioned within the square capillary tube. Furthermore, the square capillary tube has an outer diameter of 1.4 mm and a wall thickness of 0.2 mm, while the cylindrical glass capillaries have an outer diameter of 0.96 mm and a wall thickness of 0.2 mm.

[0025] According to a preferred embodiment of the present invention, in step (4),

[0026] In the preparation process, the intermediate mixed liquid that is not completely phase-separated needs to be passed into the coil of the microreactor C and stay for a period of time for rapid cooling, which is conducive to the formation and stabilization of the agglomerate structure. The cooling temperature range is 0°C-20°C; the cooling residence time range is 10min-30min;

[0027] The diameter of the coil of the microreactor C ranges from 0.5 mm to 2 mm.

[0028] According to a preferred embodiment of the present invention, in step (2),

[0029] The shellac-polylactic acid solution is pumped into microreactor A by a horizontal flow pump, and the Tween 80 solution is pumped into microreactor B by a horizontal flow pump;

[0030] The microreactor system includes heat exchange oil. The temperature and flow rate of the heat exchange oil in the microreactor system are adjusted to raise the temperature of the shellac-polylactic acid solution and the Tween 80 solution to the preheating temperature and keep it constant.

[0031] According to a preferred embodiment of the present invention, in step (4),

[0032] The intermediate mixed liquid that is not completely phase-separated is quickly introduced into the microreactor C for heat exchange with the heat exchange oil for rapid cooling.

[0033] The beneficial effects of the present invention are:

[0034] (1) Select materials with excellent biocompatibility, which can be automatically degraded in the human body, are safe and non-toxic, and can be used in the biomedical field.

[0035] (2) The microreactor system can precisely control the residence time of the raw material solution preheating process by regulating the flow rate and the length of the connected coil tube, and adjust the temperature flow rate of the cooling oil in the microreactor system so that the temperature of the raw material solution rises rapidly to the specified temperature and can be kept constant. At the same time, the solution after the two-phase mixture and the generated particles are allowed to cool quickly. The nano-coprecipitation and phase separation process changes from being dominated by thermodynamic equilibrium to being dominated by kinetic equilibrium, thereby ensuring that the size, composition, and morphology of the generated agglomerate particles are adjustable.

[0036] (3) The shellac-polylactic acid aggregate particles prepared by the present invention have a biomimetic structure similar to that of the new coronavirus and a similar size, and have great potential in the development and research of biomimetic vaccines for the new coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic diagram of the process of preparing biocompatible nanoaggregate particles of the present invention. After the two-phase solution is mixed, it undergoes a rapid cooling process through a microreactor, and the nano-coprecipitation and phase separation processes are dominated by kinetic equilibrium.

[0038] Figure 2 This is a schematic diagram of the process for preparing biocompatible nano dimer particles of the present invention. Unlike the process for preparing agglomerate particles, after the two-phase solution is mixed, it undergoes a heat preservation and natural cooling process, and the nano coprecipitation and phase separation process is dominated by thermodynamic equilibrium.

[0039] Figure 3 These are model diagrams and actual diagrams of the nano-aggregate particles with shellac as the core prepared by the present invention.

[0040] Figure 4 These are the model diagram and actual diagram of the nano-aggregate particles with polylactic acid as the core prepared by the present invention.

[0041] Figure 5These are the model diagram and actual diagram of the nano dimer particles prepared by the present invention.

[0042] Figure 6 This is a diagram of a microfluidic chip for preparing biocompatible nanoaggregate particles according to the present invention. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to the accompanying drawings and examples.

[0044] Example 1: Preparation of biocompatible nanoaggregate particles with shellac as the core and an average particle size of about 100 nm.

[0045] Refer to the attached Figure 1 The method of the present invention is used to prepare biocompatible aggregate particles, and the specific steps are as follows:

[0046] (1) Add 9 mg of shellac to 10 mL of tetrahydrofuran, place a magnetic rotor and magnetically stir until the shellac is completely dissolved to obtain a shellac solution. Then, add 1 mg of polylactic acid to the shellac solution and ultrasonically disperse it in an ultrasonic cleaning apparatus until it is completely dissolved. Finally, a mixed solution with a shellac concentration of 0.9 mg / mL and a polylactic acid concentration of 0.1 mg / mL is obtained. Add 0.5 g of Tween 80 to 50 mL of deionized water, ultrasonically disperse it in an ultrasonic cleaning apparatus, and filter it using a disposable syringe filter (specification: Φ25 mm*0.45 μm) to obtain a Tween 80 aqueous solution.

[0047] (2) The mixed solution of lac polylactic acid and the Tween 80 aqueous solution obtained in step (1) were pumped to the coils in microreactor A and microreactor B respectively by a horizontal flow pump for preheating, and the heat exchange oil temperature of the microreactor was adjusted to 70°C, the heat exchange oil flow rate was 100 mL / h, and the temperature of the mixed solution of lac polylactic acid and the Tween 80 aqueous solution was kept constant at 70°C. The residence time of the raw material solution was 25 min.

[0048] (3) The mixed solution of shellac polylactic acid and Tween 80 aqueous solution preheated in step (2) were introduced into the Figure 6 The mixture is uniformly mixed in the flow focusing microfluidic chip shown. Due to the low solubility of shellac and polylactic acid in Tween 80 solution, a nano-coprecipitation process occurs and precipitates, and a thermodynamic phase separation process begins to occur, resulting in an intermediate mixed liquid that is not completely phase-separated.

[0049] (4) The intermediate mixed liquid that is not completely phase-separated in step (3) is quickly passed into the coil in the microreactor C for rapid cooling, and the heat exchange oil temperature of the microreactor is adjusted to 0°C, and the heat exchange oil flow rate is 150 mL / h, so that the uniform dispersion of the shellac-polylactic acid nanoaggregate particles in the Tween 80 solution is cooled to a temperature of about 0°C in the coil, and the cooling residence time is 25 min. The intermediate mixed liquid continues to undergo a phase separation process dominated by kinetic equilibrium during the rapid cooling process, and a uniform dispersion of the shellac-polylactic acid nanoaggregate particles in the Tween 80 solution with biocompatibility is obtained as shown in the attached figure. Figure 3 As shown in (a), the aggregate particles are finally stored in a collection tank. The scanning electron microscope image of the aggregate particles is shown in the attached figure. Figure 3 As shown in (b), the average particle size is about 100 nm.

[0050] Example 2: Preparation of biocompatible nanoaggregate particles with a polylactic acid core and an average particle size of about 100 nm.

[0051] Refer to the attached Figure 1 The method of the present invention is used to prepare biocompatible aggregate particles, and the specific steps are as follows:

[0052] (1) Add 1 mg of shellac to 10 mL of tetrahydrofuran, place a magnetic rotor, and stir magnetically until the shellac is completely dissolved to obtain a shellac solution. Then, add 9 mg of polylactic acid to the shellac solution and ultrasonically disperse it in an ultrasonic cleaning apparatus until it is completely dissolved. Finally, a mixed solution with a shellac concentration of 0.1 mg / mL and a polylactic acid concentration of 0.9 mg / mL is obtained. Add 0.5 g of Tween 80 to 50 mL of deionized water, ultrasonically disperse it in an ultrasonic cleaning apparatus, and filter it using a disposable syringe filter (specification: Φ25 mm*0.45 μm) to obtain a Tween 80 aqueous solution.

[0053] (2) The mixed solution of lac polylactic acid and the Tween 80 aqueous solution obtained in step (1) were pumped to the coils in microreactor A and microreactor B respectively by a horizontal flow pump for preheating, and the heat exchange oil temperature of the microreactor was adjusted to 70°C, the heat exchange oil flow rate was 100 mL / h, and the temperature of the mixed solution of lac polylactic acid and the Tween 80 aqueous solution was kept constant at 70°C. The residence time of the raw material solution was 25 min.

[0054] (3) The mixed solution of shellac and polylactic acid and the Tween 80 aqueous solution preheated in step (2) are introduced into the flow focusing microfluidic chip at a rate of 0.5 mL / h and 20 mL / h, respectively, and are uniformly mixed. Since shellac and polylactic acid have low solubility in the Tween 80 solution, a nano-coprecipitation process occurs and they precipitate, and a thermodynamic phase separation process begins to occur, thereby obtaining an intermediate mixed solution that is not completely phase-separated.

[0055] (4) The intermediate mixed liquid that is not completely phase-separated in step (3) is quickly passed into the coil in the microreactor C for rapid cooling, and the heat exchange oil temperature of the microreactor is adjusted to 0°C, and the heat exchange oil flow rate is 150 mL / h, so that the uniform dispersion of the shellac-polylactic acid nanoaggregate particles in the Tween 80 solution is cooled to 0°C in the coil, and the cooling residence time is 25 min. The intermediate mixed liquid continues to undergo a phase separation process dominated by kinetic equilibrium during the rapid cooling process, and a uniform dispersion of the shellac-polylactic acid nanoaggregate particles in the Tween 80 solution with biocompatibility is obtained as shown in the attached figure. Figure 4 As shown in (a), the aggregate particles are finally stored in a collection tank. The scanning electron microscope image of the aggregate particles is shown in the attached figure. Figure 4 As shown in (b), the average particle size is about 100 nm.

[0056] Comparative Example 3: Preparation of biocompatible nano-dimer particles with an average particle size of about 400 nm.

[0057] Refer to the attached Figure 2 The method of the present invention is used to prepare biocompatible aggregate particles, and the specific steps are as follows:

[0058] (1) Add 120 mg of shellac to 120 mL of tetrahydrofuran, place a magnetic rotor and perform magnetic stirring until the shellac is completely dissolved to obtain a shellac solution. Then, add 120 mg of polylactic acid to the shellac solution and ultrasonically disperse it in an ultrasonic cleaning apparatus until it is completely dissolved, finally obtaining a mixed solution with a shellac concentration of 24 mg / mL and a polylactic acid concentration of 24 mg / mL. Add 1 mg of Tween 80 to 100 mL of deionized water, ultrasonically disperse it in an ultrasonic cleaning apparatus, and then filter it using a disposable syringe filter (specification: Φ25 mm*0.45 μm) to obtain a Tween 80 aqueous solution.

[0059] (2) The mixed solution of lac polylactic acid and the Tween 80 aqueous solution obtained in step (1) were pumped to the coils in microreactor A and microreactor B respectively by a horizontal flow pump for preheating, and the heat exchange oil temperature of the microreactor was adjusted to 70°C, the heat exchange oil flow rate was 100 mL / h, and the temperature of the mixed solution of lac polylactic acid and the Tween 80 aqueous solution was kept constant at 70°C. The residence time of the raw material solution was 25 min.

[0060] (3) The mixed solution of shellac and polylactic acid and the Tween 80 aqueous solution preheated in step (2) are introduced into the flow focusing microfluidic chip at a rate of 0.5 mL / h and 20 mL / h, respectively, and are uniformly mixed. Since shellac and polylactic acid have low solubility in the Tween 80 solution, a nano-coprecipitation process occurs and they precipitate, and a thermodynamic phase separation process begins to occur, thereby obtaining an intermediate mixed solution that is not completely phase-separated.

[0061] (4) The intermediate mixed liquid that is not completely phase-separated in step (3) is directly introduced into a collection tank through a PTFE pipe and kept at 70°C for ten minutes, and then cooled naturally. At this time, the phase separation process is dominated by thermodynamic equilibrium, and there is sufficient time for the particle morphology to tend to the lowest energy state, and a uniform dispersion of shellac-polylactic acid dimer particles in Tween 80 solution is obtained (such as the attached Figure 5 (a)), the scanning electron micrograph of the dimer particles is shown in the attached Figure 5 As shown in (b), the average particle size is about 400 nm.

[0062] Comparison between Example 1 and Example 2 illustrates that by swapping the concentrations of shellac and PLA, agglomerate particles with different cores can be prepared in this method. Experiments have shown that the concentrations of shellac and PLA and the flow rate do not affect whether the final particle morphology is agglomerates or dimers, and can only microscopically adjust the morphology, such as the size of the hemispheres of the dimer particles. Whether the cooling process is rapid cooling or natural cooling after heat preservation affects whether the particles ultimately have an agglomerate structure or a dimer structure. Comparison between Example 1 or Example 2 and Example 3 illustrates that in Example 1 or Example 2, after the microfluidic chip begins the phase separation process, the mixed solution is passed into microreactor C for rapid cooling. Due to the rapid drop in temperature, the phase separation process is dominated by kinetic equilibrium, and agglomerate particles can be formed. In contrast, in Example 3, after the microfluidic chip begins the phase separation process, the mixed solution is kept warm for ten minutes and then naturally cooled. The phase separation process is dominated by thermodynamic equilibrium, and therefore only dimer particles can be formed.

Claims

1. A method for preparing biocompatible rice aggregate particles based on a microreactor system, characterized in that: The steps include: (1) Shellac is dissolved in tetrahydrofuran (THF) and stirred until completely dissolved. Polylactic acid (PLA) is then added to the shellac solution to prepare a mixed solution of shellac and PLA in THF. Tween 80 is added to deionized water and ultrasonically shaken to accelerate the dissolution to obtain a Tween 80 aqueous solution. (2) pumping the shellac-polylactic acid solution obtained in step (1) into microreactor A and allowing it to remain there for a period of time; The Tween 80 solution is pumped into microreactor B and allowed to remain there for a period of time. The two solutions are heated in the coil of the microreactor until the solution temperature rises to the preheating temperature and remains constant. The microreactor system includes heat exchange oil, and the temperature and flow rate of the heat exchange oil in the microreactor system are adjusted so that the temperature of the shellac-polylactic acid solution and the Tween 80 solution rises to the preheating temperature and remains constant; (3) The preheated tetrahydrofuran mixed solution of shellac and polylactic acid in step (2) and the Tween 80 solution are introduced into the microfluidic chip and uniformly mixed. Since the solubility of shellac and polylactic acid in Tween 80 is greatly reduced, the two polymers undergo a nano-coprecipitation process and precipitate together. At the same time, a thermodynamic phase separation process begins. In the microfluidic chip, the polymers in the mixed solution have just begun the phase separation process, and an intermediate mixed solution that is not completely phase separated is obtained. (4) the intermediate mixed solution obtained in step (3) that is not completely phase-separated is passed into the microreactor C for cooling, and the intermediate mixed solution continues to undergo a phase separation process dominated by kinetic equilibrium during the cooling process, thereby obtaining a uniform dispersion of biocompatible shellac-polylactic acid nanoaggregate particles in Tween 80 solution, and then the uniform dispersion of the aggregate particles in Tween 80 solution is passed into a collection tank for storage; In step (4), the intermediate mixed liquid that is not completely phase-separated needs to be passed into the coil of the microreactor C and stay for a period of time for rapid cooling, which is conducive to the formation and stabilization of the agglomerate structure. The cooling temperature range is 0°C-20°C; the cooling residence time range is 10min-30min; The intermediate mixed liquid that is not completely phase-separated is quickly introduced into the microreactor C for heat exchange with the heat exchange oil for rapid cooling.

2. The method for preparing biocompatible aggregate particles based on a microreactor system according to claim 1, characterized in that: In step (1), In the tetrahydrofuran mixed solution of shellac and polylactic acid, the concentration of shellac ranges from 0.1 mg / mL to 4 mg / mL; In the tetrahydrofuran mixed solution of shellac and polylactic acid, the concentration of polylactic acid ranges from 0.1 mg / mL to 4 mg / mL.

3. The method for preparing biocompatible aggregate particles based on a microreactor system according to claim 1, characterized in that: In step (1), The molecular weight of the polylactic acid is in the range of 2000-5000; The volume fraction of the Tween 80 aqueous solution is in the range of 0.5% to 5%.

4. The method for preparing biocompatible aggregate particles based on a microreactor system according to claim 1, characterized in that: In step (2), The introduction rate of the tetrahydrofuran mixed solution of shellac and polylactic acid is 0.1 mL / h-1 mL / h; The introduction rate of the Tween 80 aqueous solution is 4 mL / h-40 mL / h; The ratio of the introduction rate of the tetrahydrofuran mixed solution of shellac and polylactic acid to the Tween 80 aqueous solution is 1:20-1:40; The volume ratio of the mixed solution to the Tween 80 aqueous solution is in the range of 1:15-1:

60.

5. The method for preparing biocompatible aggregate particles based on a microreactor system according to claim 1, characterized in that: In step (2), The mixed solution and Tween 80 aqueous solution need to stay in the coil for a certain period of time for preheating. The generated particles can be stable for a long time at room temperature. The preheating temperature range is 65℃-90℃; the preheating residence time range is 10min-30min; The diameter of the microreactor coil is in the range of 0.3 mm to 3 mm.

6. The method for preparing biocompatible aggregate particles based on a microreactor system according to claim 1, characterized in that: In step (3), The microfluidic chip is a flow-focusing microfluidic chip, which is composed of capillary glass tubes, including a square capillary tube and two cylindrical glass capillaries arranged inside the square capillary tube. The two cylindrical glass capillaries are located on the same axis and the adjacent ends of the two cylindrical glass capillaries are both located inside the square capillary tube.

7. The method for preparing biocompatible aggregate particles based on a microreactor system according to claim 6, characterized in that: In step (3), The dimensions of the square capillary glass tube are 1.4 mm in outer side length and 0.2 mm in wall thickness, and the dimensions of the cylindrical glass capillary tube are 0.96 mm in outer diameter and 0.2 mm in wall thickness.

8. The method for preparing biocompatible aggregate particles based on a microreactor system according to claim 1, characterized in that: In step (4), The diameter of the coil of the microreactor C ranges from 0.5 mm to 2 mm.

9. The method for preparing biocompatible aggregate particles based on a microreactor system according to claim 1, characterized in that: In step (2), The shellac-polylactic acid solution is pumped into microreactor A through a horizontal flow pump, and the Tween 80 solution is pumped into microreactor B through a horizontal flow pump.

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