Microgels based on emulsion method and method for their production

Abstract

The application discloses a kind of microgel based on emulsion method and its preparation method, comprising: dissolving polymer material containing aldehyde group active group, then adjusting pH value, then mixed with the solution of polymer material grafted with hydrazine group as dispersed phase, petroleum ether and emulsifier are mixed uniformly, and add acetic acid as continuous phase;After vortexing to form microdroplets after adding dispersed phase into oil phase, vortexing is placed in ice to form microgel after centrifugal tube.The microgel prepared by the application has particle size below 200 microns, narrow particle size distribution, adjustable degradation rate, and can be applied to drug loading and controlled release.

Description

Technical Field

[0001] This invention relates to the field of microgel preparation, and more specifically to a method for preparing microgels based on emulsion technology. Background Technology

[0002] The regulation of the complex in vivo microenvironment is crucial in tissue repair. The in vivo microenvironment, composed of the intercellular matrix and its fluid components, is the living space for cells. Maintaining its stability is essential for normal cellular proliferation, differentiation, metabolism, and other physiological activities. Disruption of the microenvironment can lead to cell carcinogenesis and other complications. Drugs play a significant role in regulating the in vivo microenvironment; however, drug release is a critical issue. Release that is too rapid or too slow can negatively impact tissue regeneration and repair. Therefore, researching intelligent controlled-release of drugs to effectively match the tissue regeneration and repair process is of great importance.

[0003] Compared to bulk hydrogels, microgels are polymer hydrogels ranging from tens to hundreds of micrometers in size, possessing a high specific surface area. This characteristic makes them more conducive to mass transport and cell-to-cell and cell-to-material interactions. Methods for preparing microgels mainly include mechanical fragmentation, electrospraying, and droplet microfluidics. However, microgels prepared using these methods suffer from drawbacks such as irregular structures and complex fabrication processes. Microgels prepared using emulsion methods, on the other hand, have a simpler preparation process, can be mass-produced, and are inexpensive.

[0004] Conventionally prepared microgels lack responsiveness to the pathological microenvironment, making controlled drug release difficult. However, microgels formed based on dynamic cross-linking can respond to changes in the pathological microenvironment, altering the degradation rate and thus achieving intelligent controlled drug release. The Schiff base reaction is a unique dynamic cross-linking reaction often used to prepare biodegradable hydrogels; however, due to its rapid reaction, the prepolymer solution is difficult to emulsify into spheres, making it challenging to prepare microgels using emulsion methods.

[0005] To overcome the shortcomings of existing technologies, this invention provides a method for preparing dynamic drug-loaded microgels based on an emulsion method. This method solves the problem of rapid gelation and inability to emulsify in Schiff base reactions, and successfully prepares dynamic cross-linked microgels with microenvironment responsiveness using the emulsion method, enabling intelligent controlled release of drugs and thus aiding in tissue regeneration and repair. Summary of the Invention

[0006] One objective of this application is to provide a method for preparing microgels based on an emulsion method, comprising the following steps:

[0007] (1) Dissolve material A and material B in PBS buffer solution respectively, mix and stir evenly to obtain aqueous phase A and aqueous phase B. Adjust the pH value of aqueous phase A with alkaline solution so that aqueous phase A and aqueous phase B cannot form a gel when mixed; material A is a polymer material containing aldehyde active groups, and material B is a polymer material grafted with hydrazide groups.

[0008] (2) Dilute the emulsifier with petroleum ether, add acetic acid and mix well to obtain a continuous phase;

[0009] (3) Mix and stir the pH-adjusted aqueous phase A and aqueous phase B evenly to form the dispersed phase;

[0010] (4) Add the dispersed phase to the continuous phase, vortex to form microdroplets, and place the centrifuge tube in ice after vortexing until the microdroplets crosslink to form microgels.

[0011] (5) After the microgel in step (2) has completely gelled, add petroleum ether without emulsifier to break the emulsion. After the petroleum ether evaporates naturally, wash with PBS solution and centrifuge to obtain the microgel.

[0012] In some embodiments, material A is an oxidically modified polymer material; material B is an adipic acid-hydrazide modified polymer material.

[0013] Material A and Material B are selected from biodegradable biomedical polymers; in one embodiment, at least one of the polymer materials in Material A and Material B is hyaluronic acid or gelatin.

[0014] In one embodiment, the concentrations of the oxidized modified hyaluronic acid polymer material and the adipic acid-adiazine modified polymer material in the dispersed phase are 2-4 wt% and 1.5-3.5 wt%, respectively.

[0015] In one embodiment, the preparation of the oxidized modified hyaluronic acid polymer material includes the following steps:

[0016] The polymer material is dissolved in deionized water at a concentration of 0.15-0.5 wt%, and an equal mass of sodium periodate is added. The mixture is stirred for four hours, followed by the addition of ethylene glycol to terminate the reaction for one hour. The reaction solution is dialyzed and then freeze-dried to obtain the oxidized polymer material. In some embodiments, the concentration of the polymer material is 0.2 wt%, 0.25 wt%, 0.3 wt%, 0.35 wt%, 0.4 wt%, or 0.45 wt%.

[0017] In another embodiment, the preparation of the adipic acid-adipylhydrazine modified polymer material includes the following steps:

[0018] The polymer material to be modified and adipic acid hydrazide were dissolved in deionized water. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added in two portions as a catalyst. During the reaction, the pH of the reaction solution was adjusted to acidity with 0.1 mol / L hydrochloric acid solution. After stirring at room temperature for three hours, the reaction was dialyzed and then freeze-dried to obtain the polymer material modified with adipic acid hydrazide.

[0019] In one embodiment, the pH value of aqueous phase A is adjusted with sodium hydroxide solution in step (1); preferably, the concentration of the sodium hydroxide solution is 0.5-1.5 mol / L. In one embodiment, the volume ratio of the added sodium hydroxide solution to the volume of aqueous phase A is 1:15-1:20.

[0020] The emulsifier is selected from one or more of the Tween series emulsifiers and the Span series emulsifiers; preferably, the emulsifier is selected from Tween 60 or Span 80.

[0021] In one embodiment, the concentration of the emulsifier in petroleum ether in step (2) is 4-10 wt%, and the volume concentration of the acetic acid in the continuous phase is 0.02%-0.04%.

[0022] In one embodiment, the dispersed phase in step (3) is obtained by mixing aqueous phase A and aqueous phase B in a 1:1 ratio; the ratio of the mobile phase to the dispersed phase is 1:10–1:12; and the vortex time is 0.5–5 minutes.

[0023] In another embodiment, the ratio of the mobile phase to the dispersed phase in step (4) is 1:10; the vortexing time is 0.5-1 minute, and after vortexing, the container is placed in an insulated foam box containing ice and left to stand for 1-5 hours until microgels are formed.

[0024] Another object of this application is to provide a microgel prepared by the preparation method described above.

[0025] Another object of this application is to provide an application of the microgel described above in the preparation of dynamically drug-loaded microgels.

[0026] Compared with the prior art, the present invention has the following characteristics:

[0027] This invention utilizes the dynamic cross-linking reaction of Schiff bases to successfully prepare microgels with intelligent controlled-release drugs, which can be used for tissue regeneration and repair. Simultaneously, this invention overcomes the challenge of preparing microspheres due to the rapid gelation effect of the Schiff base reaction by controlling pH and temperature. The emulsion method allows for the simple and rapid preparation of Schiff base cross-linked microgels, which can slowly degrade under normal physiological conditions. Attached Figure Description

[0028] Figure 1 Experiments on the gelation reaction of oxidatively modified hyaluronic acid and adipic acid-adipylhydrazine modified gelatin.

[0029] Figure 2 A schematic diagram of the crosslinking mechanism of oxidatively modified hyaluronic acid and adipic acid-adipylhydrazine-modified gelatin.

[0030] Figure 3 This is an observation diagram of the gelation results in Example 3.

[0031] Figure 4 Optical image of the microgel prepared in Example 4.

[0032] Figure 5 The particle size distribution diagram is shown for the microgel prepared in Example 4.

[0033] Figure 6 Optical image of the microgel prepared in Example 5.

[0034] Figure 7 Optical image of the microgel prepared in Example 6.

[0035] Figure 8 The drug release curve is shown for the microgel prepared in Example 7.

[0036] Figure 9 Degradation rates of microgels prepared from gelatin modified with different proportions of oxidized hyaluronic acid and adipic acid adihydrazide. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. The reagents used in the embodiments are all commercially available products.

[0038] Example 1

[0039] 1) Preparation of oxidized modified hyaluronic acid, including the following steps:

[0040] Weigh 1g of sodium hyaluronate with a molecular weight of 100kDa and dissolve it in 400ml of deionized water. After dissolution, add 1g of sodium periodate and stir for 4 hours. Then add 2ml of ethylene glycol and continue stirring for 1 hour. Dialyze the solution with deionized water using a 3500kDa dialysis bag for 3 days. After lyophilization, obtain oxidized modified hyaluronic acid.

[0041] 2) Preparation of adipic acid-adipylhydrazide-modified gelatin, including the following steps:

[0042] Weigh 1g of gelatin and dissolve it in 100ml of deionized water. After dissolution, add 0.696g of adipic acid hydrazide and adjust the pH of the solution to 4.75 with 1 mol / L hydrochloric acid solution. Then add 0.19g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, maintaining the pH of the solution at 4.75 during the reaction. After stirring for 1 hour, add another 0.19g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, maintaining the pH of the solution at 4.75 during the reaction, and continue stirring for 2 hours. Dialyze the solution to deionized water at pH 3.15 using a dialysis bag with a molecular weight of 8000-12000 for three days, and then freeze-dry to obtain adipic acid hydrazide-modified gelatin.

[0043] 50 mg of the oxidized hyaluronic acid and 50 mg of adipic acid-modified gelatin were dissolved separately in 1 ml of PBS solution, heated to 50 °C and stirred to dissolve, yielding oxidized hyaluronic acid solution and adipic acid-modified gelatin solution. After mixing the two polymer solutions, gelation was observed in the mixed solution, indicating that microgels could not be directly prepared using the emulsion method.

[0044] Example 2

[0045] (1) 60 mg of oxidized hyaluronic acid and 50 mg of adipic acid-modified gelatin were added to 1 ml of PBS solution and heated to 50 °C and stirred to dissolve.

[0046] (2) Add 50 μL of sodium hydroxide solution with a concentration of 1 mol / L to the dissolved oxidized hyaluronic acid solution to adjust the pH value.

[0047] (3) After mixing the pH-adjusting oxidized hyaluronic acid and the adipic acid-adipylhydrazine-modified gelatin solution, press a 5ml vial onto the solution surface and observe the liquid level.

[0048] (4) After removing the vial, add 0.02% acetic acid to the solution, stir, and then press the vial back onto the solution surface to observe the liquid level.

[0049] The observation results of this embodiment are attached. Figure 1 As shown in the figure, no gelation was observed after adjusting the pH of the polymer mixture solution with alkali. This indicates that, in this example, adjusting the alkalinity of the oxidized modified hyaluronic acid can delay the rapid gelation reaction of the Schiff base, making it suitable for emulsion preparation. Subsequent adjustment of the pH of the polymer mixture solution by adding acid solution can induce gelation again. The mechanism of gelation by the Schiff base reaction is shown in the attached figure. Figure 2 As shown.

[0050] This experiment also explored adjusting the pH of the oxidized hyaluronic acid solution using other alkaline solutions, such as sodium carbonate solution. It was found that the Schiff base reaction was effectively suppressed when the pH of the solution was adjusted to 9-10.

[0051] Example 3

[0052] (1) 60 mg of oxidized hyaluronic acid and 50 mg of adipic acid-modified gelatin were added to 1 ml of PBS solution and heated to 50 °C with stirring to dissolve. 60 μL of sodium hydroxide solution with a concentration of 1 mol / L was added to the dissolved oxidized hyaluronic acid solution so that it could not form a gel when mixed with the adipic acid-modified gelatin.

[0053] (2) Dissolve Span 80 in petroleum ether at a concentration of 5 wt%, and add acetic acid (the volume concentration of acetic acid in petroleum ether in which Span 80 is dissolved is 0.02%) to the petroleum ether in which Span 80 is dissolved, mix them evenly and use them as the oil phase continuous phase.

[0054] (3) The oxidized hyaluronic acid after pH adjustment and the gelatin modified with adipic acid and adipamide were mixed evenly in a ratio of 1:1 to obtain an aqueous dispersion phase.

[0055] (4) Mix the aqueous dispersed phase and the oil continuous phase in a centrifuge tube at a ratio of 1:10. After vortexing for 1 minute, water-in-oil microdroplets are formed. Place the centrifuge tube after vortexing in a beaker containing ice and a beaker without ice, respectively, and observe the gelation.

[0056] The observation results of this embodiment are as follows: Figure 3 As shown. The ability to form stable microgels at low temperatures is mainly due to the presence of gelatin in the raw materials. The thermosensitive nature of gelatin allows it to form a colloidal state at low temperatures, preventing the coalescence of microdroplets.

[0057] Example 4

[0058] (1) 60 mg of oxidized hyaluronic acid and 50 mg of adipic acid-modified gelatin were added to 1 ml of PBS solution and heated to 50 °C with stirring to dissolve. 60 μL of sodium hydroxide solution with a concentration of 1 mol / L was added to the dissolved oxidized hyaluronic acid solution so that it could not form a gel when mixed with the adipic acid-modified gelatin.

[0059] (2) Dissolve Span 80 in petroleum ether at a concentration of 5 wt%, and add acetic acid (the volume concentration of acetic acid in petroleum ether in which Span 80 is dissolved is 0.02%) to the petroleum ether in which Span 80 is dissolved, mix them evenly and use them as the oil phase continuous phase.

[0060] (3) The oxidized hyaluronic acid after pH adjustment and the gelatin modified with adipic acid and adipamide were mixed evenly in a ratio of 1:1 to obtain an aqueous dispersion phase.

[0061] (4) Mix the aqueous dispersed phase and the oil continuous phase in a centrifuge tube at a ratio of 1:10. After vortexing for 1 minute, form water-in-oil microdroplets. Place the centrifuge tube after vortexing in a foam box containing ice and let it stand for 2 hours to allow the microdroplets to cross-link and form microgels.

[0062] (5) The cross-linked microgel was washed three times with petroleum ether without Span 80, then fresh petroleum ether was added to break the emulsion. After the petroleum ether evaporated naturally, it was washed with PBS and centrifuged at 2000 r / min for 5 minutes to obtain the microgel (OHA3 / GelADH2.5).

[0063] The optical image of the microgel prepared in this example is shown below. Figure 4 As shown; the particle size distribution of the microgel prepared in this example is as follows. Figure 5 As shown, the polymer microgels have a narrow particle size distribution, with an average particle size of 80 micrometers.

[0064] Example 5

[0065] (1) 40 mg of oxidized hyaluronic acid and 30 mg of adipic acid-modified gelatin were added to 1 ml of PBS solution and heated to 50 °C with stirring to dissolve. 40 μL of sodium hydroxide solution with a concentration of 1 mol / L was added to the dissolved oxidized hyaluronic acid solution so that it could not form a gel when mixed with the adipic acid-modified gelatin.

[0066] (2) Dissolve Span 80 in petroleum ether at a concentration of 5 wt%, and add acetic acid (the volume concentration of acetic acid in petroleum ether in which Span 80 is dissolved is 0.013%) to the petroleum ether in which Span 80 is dissolved, mix well and use it as the oil phase continuous phase.

[0067] (3) The oxidized hyaluronic acid after pH adjustment and the gelatin modified with adipic acid and adipamide were mixed evenly in a ratio of 1:1 to obtain an aqueous dispersion phase.

[0068] (4) Mix the aqueous dispersed phase and the oil continuous phase in a centrifuge tube at a ratio of 1:10. After vortexing for 1 minute, form water-in-oil microdroplets. Place the centrifuge tube after vortexing in a foam box containing ice and let it stand for 2 hours to allow the microdroplets to cross-link and form microgels.

[0069] (5) The cross-linked microgel was washed three times with petroleum ether without Span 80, then fresh petroleum ether was added to break the emulsion. After the petroleum ether evaporated naturally, it was washed with PBS and centrifuged at 2000 r / min for 5 minutes to obtain the microgel (OHA2 / GelADH1.5).

[0070] The optical image of the microgel prepared in this example is shown below. Figure 6 As shown.

[0071] Example 6

[0072] (1) 80 mg of oxidized hyaluronic acid and 70 mg of adipic acid-modified gelatin were added to 1 ml of PBS solution and heated to 50 °C with stirring to dissolve. 80 μL of sodium hydroxide solution with a concentration of 1 mol / L was added to the dissolved oxidized hyaluronic acid solution so that it could not form a gel when mixed with the adipic acid-modified gelatin.

[0073] (2) Dissolve Span 80 in petroleum ether at a concentration of 5 wt%, and add acetic acid (the volume concentration of acetic acid in petroleum ether in which Span 80 is dissolved is 0.027%) to the petroleum ether in which Span 80 is dissolved, mix well and use it as the oil phase continuous phase.

[0074] (3) The oxidized hyaluronic acid after pH adjustment and the gelatin modified with adipic acid and adipamide were mixed evenly in a ratio of 1:1 to obtain an aqueous dispersion phase.

[0075] (4) Mix the aqueous dispersed phase and the oil continuous phase in a centrifuge tube at a ratio of 1:10. After vortexing for 1 minute, form water-in-oil microdroplets. Place the centrifuge tube after vortexing in a foam box containing ice and let it stand for 2 hours to allow the microdroplets to cross-link and form microgels.

[0076] (5) The cross-linked microgel was washed three times with petroleum ether without Span 80, then fresh petroleum ether was added to break the emulsion. After the petroleum ether evaporated naturally, it was washed with PBS and centrifuged at 2000 r / min for 5 minutes to obtain the microgel (OHA4 / GelADH3.5).

[0077] The optical image of the microgel prepared in this example is shown below. Figure 7 As shown.

[0078] Example 7

[0079] (1) 60 mg of oxidized hyaluronic acid and 50 mg of adipic acid-modified gelatin were added to 1 ml of PBS solution, and 7 mg of lysozyme was added to the oxidized hyaluronic acid solution. The mixture was heated to 40 °C and stirred to dissolve. 60 μL of 1 mol / L sodium hydroxide solution was added to the dissolved oxidized hyaluronic acid solution to prevent it from forming a gel when mixed with the adipic acid-modified gelatin.

[0080] (2) Dissolve Span 80 in petroleum ether at a concentration of 5 wt%, and add acetic acid (the volume concentration of acetic acid in petroleum ether in which Span 80 is dissolved is 0.02%) to the petroleum ether in which Span 80 is dissolved, mix well and use it as the oil phase continuous phase.

[0081] (3) The oxidized hyaluronic acid after pH adjustment and the gelatin modified with adipic acid and adipamide were mixed evenly in a ratio of 1:1 to obtain an aqueous dispersion phase.

[0082] (4) Mix the aqueous dispersed phase and the oil continuous phase in a centrifuge tube at a ratio of 1:10. After vortexing for 1 minute, form water-in-oil microdroplets. Place the centrifuge tube after vortexing in a foam box containing ice and let it stand for 2 hours to allow the microdroplets to cross-link and form microgels.

[0083] (5) Wash the cross-linked microgel three times with petroleum ether without Span 80, then add fresh petroleum ether to demulsify, wait for the petroleum ether to evaporate naturally, wash with PBS, centrifuge at 2000 r / min for 5 minutes to obtain microgel loaded with lysozyme.

[0084] (6) Divide the collected lysozyme-loaded microgels into 8 equal portions, place each portion in 2 ml of PBS solution, and place in a shaker at 37°C and 100 rpm. At regular intervals, remove 1 ml of solution and add 1 ml of fresh PBS solution to the test tube. Measure the released protein content of the collected solutions according to the detection method of the BCA kit.

[0085] The lysozyme release curve of the lysozyme-loaded microgel obtained in this example is shown below. Figure 8 As shown.

[0086] Example 8

[0087] This example investigates the degradation of microgels prepared from gelatin modified with different proportions of oxidized hyaluronic acid and adipic acid-adipylhydrazine. The specific preparation methods for the microgels are shown in Examples 4-5.

[0088] Microgels (OHA3 / GelADH2.5, OHA2 / GelADH1.5, and OHA4 / GelADH3.5) prepared by modifying gelatin with different proportions of oxidized hyaluronic acid and adipic acid hydrazide were prepared in 100 μL each and immersed in serum-free DMEM. Their remaining weight was measured at regular intervals, and the degradation rate of different microgels was evaluated by calculating their swelling ratio. The calculation formula is: ESR = Ws / Wd (Ws is the weight of the hydrogel after swelling equilibrium; Wd is the dry weight of the gel).

[0089] The result of this example is as follows: Figure 9 As shown, the degradation rate gradually decreased with increasing ratio of oxidized polymer and adipic acid-modified polymer, indicating that the degradation rate of the microgel can be controlled by adjusting the ratio of aldehyde and amino groups.

[0090] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing microgels based on an emulsion method, comprising the following steps: (1) Dissolve material A and material B in PBS buffer solution respectively, mix and stir evenly to obtain aqueous phase A and aqueous phase B respectively, add alkaline solution to adjust the pH value of aqueous phase A so that aqueous phase A and aqueous phase B cannot be gelled when mixed. Material A is a polymer material containing aldehyde active group, and material B is a polymer material grafted with hydrazide group. (2) Add the emulsifier to petroleum ether and then add acetic acid and mix thoroughly to obtain a continuous phase; (3) Mix the pH-adjusted aqueous phase A and aqueous phase B thoroughly to form the dispersed phase; (4) Add the dispersed phase to the continuous phase, vortex to form microdroplets, and place the centrifuge tube in ice after vortexing until the microdroplets crosslink to form a gel. (5) Take the gel from step (4), add petroleum ether to break the emulsion, wait for the petroleum ether to evaporate naturally, wash with PBS solution, and centrifuge to obtain microgels; Among them, the polymer material in material A and material B is at least one of hyaluronic acid and gelatin, material A is an oxidically modified polymer material; material B is an adipic acid-hydrazide modified polymer material; After adding alkali solution in step (1), the pH value of aqueous phase A is 9-10; The dispersed phase in step (3) is obtained by mixing aqueous phase A and aqueous phase B in a 1:1 ratio.

2. According to the preparation method of claim 1, the concentrations of the oxidized polymer material and the adipic acid-adipylhydrazine-modified polymer material in the dispersed phase are 2-4 wt% and 1.5-3.5 wt%, respectively.

3. The preparation method according to claim 2, characterized in that, The preparation method of the oxidatively modified polymer material is as follows: The polymer material was dissolved in deionized water at a concentration of 0.15-0.5 wt%, and sodium periodate of equal mass to the polymer material was added and stirred to react. Then, ethylene glycol was added to terminate the reaction. The reaction solution was dialyzed and then freeze-dried to obtain the oxidized polymer material. The preparation of the adipic acid-adipylhydrazine modified polymer material includes the following steps: The polymer material and adipic acid hydrazide were dissolved in deionized water. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added in two portions as a catalyst. During the reaction, the pH of the reaction solution was adjusted to acidity with hydrochloric acid solution. After stirring at room temperature for three hours, the reaction was dialyzed and then freeze-dried to obtain the polymer material modified with adipic acid hydrazide.

4. The preparation method according to any one of claims 1-3, characterized in that, The emulsifier in step (2) is a Tween series or Span series emulsifier; the concentration of the emulsifier in petroleum ether is 4-10 wt%, and the volume concentration of acetic acid in the continuous phase is 0.02%-0.04%.

5. The preparation method according to claim 1, characterized in that, In step (4), the ratio of the mobile phase to the dispersed phase is 1:10, and the vortex time is 0.5-1 minute.

6. The preparation method according to claim 5, characterized in that, In step (4), the gel obtained in step (3) is placed in an insulated foam box containing ice and left to stand for 1-5 hours to allow the microgel to cross-link internally.

7. The microgel prepared by the preparation method according to any one of claims 1-6.

8. The application of the microgel prepared according to claim 7 in drug loading and controlled release.

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