Method for preparing high-viscosity sodium hyaluronate microspheres

Abstract

A specific preparation method of the high-viscosity sodium hyaluronate microspheres comprises the following steps: by taking a water phase containing high-viscosity sodium hyaluronate as an outer phase and an oil phase containing a cross-linking agent as an inner phase, preparing an oil-in-water emulsion with uniform particle size in two-phase flow through a microfluidic technology, and then introducing the oil-in-water emulsion into an oleophylic environment to obtain the high-viscosity sodium hyaluronate microspheres. The preparation method comprises the following steps: adding sodium hyaluronate into an oil-in-water emulsion, carrying out phase transfer on the oil-in-water emulsion, changing an oil phase into an external phase, changing a water phase containing high-viscosity hyaluronic acid into an internal phase, thus forming a water-in-oil emulsion with a uniform size, and absorbing a cross-linking agent in the oil phase by the high-viscosity sodium hyaluronate to carry out cross-linking in the water-in-oil emulsion to obtain the microspheres. The high-viscosity sodium hyaluronate microspheres with uniform particle size can be prepared by utilizing transfer of an oil phase and a water phase; the cross-linking agent can be prevented from being directly added into the high-viscosity hyaluronic acid, and the situation that the internal phase of the hyaluronic acid mixed with the cross-linking agent changes along with time, the viscosity of the internal phase of the hyaluronic acid changes along with time, and the size of the prepared microspheres changes along with the change of the internal phase of the hyaluronic acid is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, and particularly to a method for preparing high-viscosity sodium hyaluronate microspheres. Background Art

[0002] Hyaluronic acid is a natural polymer linear polysaccharide composed of D-glucuronic acid and N-acetylglucosamine as disaccharide units repeatingly linked, and is widely present in animal tissues and body fluids, providing a spatial scaffold for collagen fibers and elastin. Due to its good biocompatibility and moisturizing effect, hyaluronic acid is widely used as a filler to achieve effects such as correcting mild wrinkles and improving skin texture. However, hyaluronic acid will be degraded in the human body due to the presence of hyaluronidase in the body, ultimately resulting in the loss of its scaffold function. Increasing the cross-linking degree can extend the retention time of hyaluronic acid in the body and improve the viscoelasticity of the hydrogel, making it better meet the role of the filler, but the subsequent chemical residue of the cross-linking agent will cause biocompatibility problems in the human body and even severe rejection reactions. High-molecular-weight hyaluronic acid can naturally extend the time of hyaluronic acid in the body due to the advantage of its own chain length. At the same time, the long chains of high polymers are also more conducive to product processing, enabling the hyaluronic acid chains to form physical entanglements, improving the mechanical properties of the hydrogel, and further reducing the demand for chemical cross-linking agents. However, the viscosity of hyaluronic acid with the same concentration increases significantly with the increase in the molecular weight of hyaluronic acid. In addition, because the necessary condition for hyaluronic acid cross-linking is a relatively high concentration of hyaluronic acid and the cross-linking agent, a high-viscosity hyaluronic acid solution is often a necessary condition for preparing a high-performance hyaluronic acid gel.

[0003] Due to the too high viscosity of the high-viscosity sodium hyaluronate solution, it is difficult to shear the internal phase into spheres through a droplet microfluidic device. After adding the cross-linking agent to the high-viscosity sodium hyaluronate, it will cross-link rapidly, and it may solidify before entering the microfluidic chip. This not only makes it more difficult for the high-viscosity sodium hyaluronate to be sheared into spheres by the droplet microfluidic device, but also may block the microfluidic chip. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing microspheres from high-viscosity sodium hyaluronate based on microfluidic technology.

[0005] Another purpose of the present invention is to provide high-viscosity sodium hyaluronate microspheres prepared by the method for preparing high-viscosity sodium hyaluronate microspheres described above.

[0006] The present invention is realized through the following technical solutions: A method for preparing high-viscosity sodium hyaluronate microspheres uses an aqueous phase containing high-viscosity sodium hyaluronate as the outer phase and an oil phase containing a cross-linking agent as the inner phase. By means of microfluidic technology, a water-in-oil emulsion with uniform particle size is prepared in the two-phase flow. Then, the water-in-oil emulsion is introduced into a lipophilic environment, causing the phase transfer of the water-in-oil emulsion, making the oil phase become the outer phase and the aqueous phase containing high-viscosity hyaluronic acid become the inner phase, thus forming a water-in-oil emulsion with uniform size. At the same time, the high-viscosity sodium hyaluronate absorbs the cross-linking agent in the oil phase and undergoes cross-linking in the water-in-oil emulsion to obtain microspheres.

[0007] Since the viscosity of sodium hyaluronate as the dispersed phase is relatively high, it is difficult to shear it into an emulsion in a microfluidic device. This technical solution adopts the phase inversion method. Since the generation of low-viscosity droplets is much easier than that of high-viscosity droplets, the high-viscosity sodium hyaluronate is used to shear the oil phase to form a water-in-oil emulsion. Then, the water-in-oil emulsion is placed in a lipophilic environment, and the low-viscosity oil phase and the high-viscosity aqueous phase undergo phase transfer to form a water-in-oil emulsion. Another advantage of this phase inversion method is that when high-viscosity hyaluronic acid is added with a cross-linking agent, it is extremely easy to cross-link, which is more disadvantageous for preparing hyaluronic acid microspheres using microfluidic technology. Therefore, this technical solution uses the phase inversion method to dissolve the cross-linking agent in the oil phase. In this way, no cross-linking agent needs to be added to sodium hyaluronate. When the two come into contact to form an emulsion and undergo phase transfer, the cross-linking agent dissolved in the oil phase will dissolve into the sodium hyaluronate in the aqueous phase, causing the sodium hyaluronate that becomes the inner phase to start cross-linking. Although the cross-linking agent is slightly soluble in the oil phase, a saturated solution of the cross-linking agent can be made in the oil phase. In addition, due to the phase exchange process, the oil phase becomes the outer phase, so the cross-linking agent can be continuously provided for the sodium hyaluronate in the inner phase, ensuring that the sodium hyaluronate in the inner phase has enough cross-linking agent to complete the cross-linking process.

[0008] To better implement the method of the present invention, further, its specific preparation process includes the following steps:

[0009] (1) Prepare an aqueous phase solution: Add high-viscosity hyaluronic acid or sodium hyaluronate to physiological saline and add an alkali, then stir evenly;

[0010] (2) Prepare an oil phase solution: Add a cross-linking agent to the oil phase solution and stir evenly to completely dissolve the cross-linking agent in the oil phase solution;

[0011] (3) Using the aqueous phase solution as the outer phase and the oil phase solution as the inner phase, prepare a water-in-oil emulsion through microfluidic technology;

[0012] (4) Directly place the prepared water-in-oil emulsion in a lipophilic environment and make it oscillate, so that the aqueous phase and the oil phase undergo phase transfer to obtain a water-in-oil emulsion with the outer phase being the oil phase and the inner phase being the aqueous phase;

[0013] (5) The prepared water-in-oil emulsion is placed in an oven at 10-55 °C for crosslinking for 4-72 h. After the crosslinking of sodium hyaluronate in the emulsion is completed, sodium hyaluronate microspheres are obtained, washed three times with petroleum ether, washed three times with pure water, and stored in PBS aqueous solution.

[0014] In order to better implement the method of the present invention, further, in the step (1), the component of high-viscosity hyaluronic acid or hyaluronate is 0.1-3.5 MDa.

[0015] In order to better implement the method of the present invention, further, in the aqueous solution prepared in the step (1), the content of high-viscosity hyaluronic acid or hyaluronate is 1-20 wt%, the content of alkali is 1-3 wt%, the content of sodium chloride is 0.01-1 wt%, the water is deionized water, and the alkali is at least one of sodium hydroxide and potassium hydroxide.

[0016] In order to better implement the method of the present invention, further, in the step (2), the oil used is at least one of liquid paraffin and mineral oil.

[0017] In order to better implement the method of the present invention, further, in the oil phase solution prepared in the step (2), the crosslinking agent used is at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, divinyl sulfone, 1,4-butanediol diglycidyl ether, and sodium trimetaphosphate, and the content of the crosslinking agent in the oil phase is 0.1-10 wt%.

[0018] In order to better implement the method of the present invention, further, in the step (3), the specific process of preparing the water-in-oil emulsion by microfluidic technology is as follows: One end of the inner phase outflow tube is inserted into the collection tube and fixed on a glass slide. The injection head of the aqueous phase syringe is fixed at the inlet end of the collection tube and fixed and sealed with resin glue. First, the aqueous phase of the aqueous phase syringe is injected into the collection tube through the injection head by an injection pump, and then the oil phase is injected into the inner phase outflow tube. The aqueous phase and the oil phase meet at the outlet of the inner phase outflow tube, and water-in-oil microspheres with uniform size are formed in the collection tube.

[0019] In order to better implement the method of the present invention, further, in the step (3), an oil-in-water emulsion is prepared by a microfluidic device, and the microfluidic device includes an inner-phase outflow pipe, and a collection pipe is hermetically embedded outside the outflow end of the inner-phase outflow pipe; the inner-phase outflow pipe is a circular glass pipe or a circular steel pipe with an inner diameter of 10 μm to 1000 μm and an outer diameter of 50 μm to 2000 μm; the collection pipe is a square or circular glass pipe or steel pipe with an inner diameter of 50 μm to 2000 μm and an outer diameter of 100 μm to 3000 μm; the water-phase flow rate is 1 to 50 μL / min, the oil-phase flow rate is 20 to 500 μL / min, and the diameter of the prepared oil-in-water emulsion is 30 μm to 2000 μm.

[0020] In order to better implement the method of the present invention, further, in the step (4), the method of directly placing the prepared oil-in-water emulsion in a lipophilic environment includes any one of the following:

[0021] a. A lipophilic pipeline is hermetically connected to the outlet end of the collection pipe. The oil-in-water emulsion flows into the lipophilic pipeline along with the outer phase. The original inner-phase oil-phase droplets contact the lipophilic pipeline wall, and phase transfer occurs between the water phase and the oil phase, obtaining a water-in-oil emulsion with an outer phase of oil phase and an inner phase of water phase;

[0022] b. A container containing an oil-phase solution is arranged at the outlet of the collection pipe, and a stirring mechanism is arranged in the container. The oil-in-water emulsion flowing out of the collection pipeline enters the container containing the oil-phase solution. After stirring, phase transfer occurs between the water phase and the oil phase, obtaining a water-in-oil emulsion with an outer phase of oil phase and an inner phase of water phase.

[0023] In order to better implement the method of the present invention, further, the material of the lipophilic pipeline is at least one of PE, PET, PP, PVC, and silica gel, and the inner diameter of the lipophilic pipeline is 100 to 3000 μm, and the outer diameter is 200 to 5000 μm.

[0024] A high-viscosity sodium hyaluronate microsphere is prepared by the above preparation method. The diameter of the high-viscosity sodium hyaluronate microsphere is 30 μm to 2000 μm.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] (1) By utilizing the transfer of the oil phase and the water phase, the present invention overcomes the problem of preparing high-viscosity sodium hyaluronate microspheres by microfluidics. Due to the adoption of microfluidic technology, it can prepare high-viscosity sodium hyaluronate microspheres with uniform particle size.

[0027] (2) The method provided by the present invention can avoid directly adding a crosslinking agent to high-viscosity hyaluronic acid. In this way, the problem that the viscosity of the hyaluronic acid internal phase mixed with the crosslinking agent changes over time, and the size of the prepared microspheres changes accordingly can be solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Other features, objects, and advantages of the present invention will become more apparent by reading the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0029] Figure 1 Schematic structural diagram of the device for preparing high-viscosity sodium hyaluronate of the present invention;

[0030] Figure 2 Optical image of the high-viscosity sodium hyaluronate microspheres prepared by the present invention in PBS aqueous solution;

[0031] Figure 3 Optical image of the high-viscosity sodium hyaluronate microspheres prepared in Example 6 of the present invention in PBS aqueous solution. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] To make the purpose, process conditions, and advantages of the present invention more clear, the present invention will be further described in detail below in combination with the following embodiments. However, the embodiments of the present invention are not limited thereto. Without departing from the above technical idea of the present invention, various substitutions and changes made according to common general knowledge and conventional means in the art should be included within the scope of the present invention. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] The present invention provides a high-viscosity sodium hyaluronate microsphere, which uses sodium hyaluronate with a molecular weight of 0.1 - 3.5 MDa, and the content of the high-viscosity hyaluronic acid or hyaluronate used is 1 - 20 wt%.

[0034] The specific preparation method of the high-viscosity sodium hyaluronate microsphere uses an aqueous phase containing high-viscosity sodium hyaluronate as the outer phase and an oil phase containing a crosslinking agent as the inner phase. Through microfluidic technology, a water-in-oil emulsion with uniform particle size is prepared in the two-phase flow. Then, the water-in-oil emulsion is introduced into a lipophilic environment, causing the water-in-oil emulsion to undergo a phase transfer, so that the oil phase becomes the outer phase and the aqueous phase containing high-viscosity hyaluronic acid becomes the inner phase, thereby forming a water-in-oil emulsion with uniform size. At the same time, the high-viscosity sodium hyaluronate absorbs the crosslinking agent in the oil phase and undergoes crosslinking in the water-in-oil emulsion to obtain microspheres.

[0035] Example 1:

[0036] This embodiment provides a high-viscosity sodium hyaluronate microsphere, which uses sodium hyaluronate with a molecular weight of 1.5 MDa and a high-viscosity hyaluronic acid or hyaluronate salt content of 10%. The specific preparation process is as follows:

[0037] (1) preparing an aqueous solution: pour 0.3 g of 1.5 MDa sodium hyaluronate, 0.03 g of sodium hydroxide, and 3 mL of a mixture of normal saline and pure water, wherein the volume ratio of normal saline to pure water is 2:3, into a 5 mL tin bottle in sequence and stir evenly;

[0038] (2) Preparation of oil phase solution: To 89 g of liquid paraffin, add 9 g of T1541 and 2 g of 4-butanediol diglycidyl ether (BDDE) in sequence, and stir evenly to make BDDE completely dissolved in the oil phase solution;

[0039] (3) With the aqueous solution as the external phase and the oily solution as the internal phase, an oil-in-water emulsion is prepared by microfluidic technology, and a silicone tube is installed in the outlet pipe of the microfluidic control, such as Figure 1 As shown, the water phase and the oil phase of the water-in-oil emulsion undergo phase transfer, and an oil-in-water emulsion with an outer phase of an oil phase and an inner phase of a water phase is obtained;

[0040] (4) The prepared water-in-oil emulsion was placed in an oven at 37° C. for 48 hours for cross-linking. After the cross-linking of sodium hyaluronate in the emulsion was completed, sodium hyaluronate microspheres were obtained, which were washed three times with petroleum ether and three times with pure water, and stored in a PBS aqueous solution.

[0041] Embodiment 2:

[0042] This embodiment provides a high-viscosity sodium hyaluronate microsphere, which uses sodium hyaluronate with a molecular weight of 2 MDa, and the content of high-viscosity hyaluronic acid or hyaluronate used is 7%. The specific preparation process is as follows:

[0043] (1) Prepare an aqueous solution: pour 0.21 g of 2MDa sodium hyaluronate, 0.03 g of sodium hydroxide, and 3 mL of a mixture of normal saline and pure water, wherein the volume ratio of normal saline to pure water is 2:3, into a 5 mL tin bottle in sequence and stir evenly;

[0044] (2) preparing an oil phase solution: adding 9 g of T1541 and 2 g of 4-butanediol diglycidyl ether (BDDE) to 89 g of liquid paraffin, stirring evenly to completely dissolve BDDE in the oil phase solution;

[0045] (3) With the aqueous solution as the external phase and the oily solution as the internal phase, an oil-in-water emulsion is prepared by microfluidic technology, and a silicone tube is installed in the outlet pipe of the microfluidic control, such as Figure 1As shown, the water phase and the oil phase of the water-in-oil emulsion undergo phase transfer, and an oil-in-water emulsion with an outer phase of an oil phase and an inner phase of a water phase is obtained;

[0046] (4) The prepared water-in-oil emulsion was placed in an oven at 37° C. for 48 hours for cross-linking. After the cross-linking of sodium hyaluronate in the emulsion was completed, sodium hyaluronate microspheres were obtained, which were washed three times with petroleum ether and three times with pure water, and stored in a PBS aqueous solution.

[0047] High-viscosity sodium hyaluronate microspheres with uniform size can be prepared, and remain stable in PBS aqueous solution after washing.

[0048] Embodiment 3:

[0049] This embodiment provides a high-viscosity sodium hyaluronate microsphere, which uses sodium hyaluronate with a molecular weight of 1.2 MDa and a high-viscosity hyaluronic acid or hyaluronate salt content of 15%. The specific preparation process is as follows:

[0050] (1) preparing an aqueous solution: pour 0.45 g of 1.2 MDa sodium hyaluronate, 0.03 g of sodium hydroxide, and 3 mL of a mixture of normal saline and pure water, wherein the volume ratio of normal saline to pure water is 2:3, into a 5 mL tin bottle in sequence and stir evenly;

[0051] (2) Preparation of oil phase solution: To 89 g of liquid paraffin, add 9 g of T1541 and 2 g of 4-butanediol diglycidyl ether (BDDE) in sequence, and stir evenly to make BDDE completely dissolved in the oil phase solution;

[0052] (3) With the aqueous solution as the external phase and the oily solution as the internal phase, an oil-in-water emulsion is prepared by microfluidic technology, and a silicone tube is installed in the outlet pipe of the microfluidic control, such as Figure 1 As shown, the water phase and the oil phase of the water-in-oil emulsion undergo phase transfer, and an oil-in-water emulsion with an outer phase of an oil phase and an inner phase of a water phase is obtained;

[0053] (4) The prepared water-in-oil emulsion was placed in an oven at 37° C. for 48 hours for cross-linking. After the cross-linking of sodium hyaluronate in the emulsion was completed, sodium hyaluronate microspheres were obtained, which were washed three times with petroleum ether and three times with pure water, and stored in a PBS aqueous solution.

[0054] Because the hyaluronic acid content is too high, it is impossible to prepare an internal phase solution and high-viscosity hyaluronic acid microspheres.

[0055] Embodiment 4:

[0056] This embodiment provides a high-viscosity sodium hyaluronate microsphere, which uses sodium hyaluronate with a molecular weight of 1.2 MDa and a high-viscosity hyaluronic acid or hyaluronate content of 5%. The specific preparation process is as follows:

[0057] (1) preparing an aqueous solution: pour 0.15 g of 1.2 MDa sodium hyaluronate, 0.03 g of sodium hydroxide, and 3 mL of a mixture of normal saline and pure water, wherein the volume ratio of normal saline to pure water is 2:3, into a 5 mL tin bottle in sequence and stir evenly;

[0058] (2) Preparation of oil phase solution: To 89 g of liquid paraffin, add 9 g of T1541 and 2 g of 4-butanediol diglycidyl ether (BDDE) in sequence, and stir evenly to make BDDE completely dissolved in the oil phase solution;

[0059] (3) With the aqueous solution as the external phase and the oily solution as the internal phase, an oil-in-water emulsion is prepared by microfluidic technology, and a silicone tube is installed in the outlet pipe of the microfluidic control, such as Figure 1 As shown, the water phase and the oil phase of the water-in-oil emulsion undergo phase transfer, and an oil-in-water emulsion with an outer phase of an oil phase and an inner phase of a water phase is obtained;

[0060] (4) The prepared water-in-oil emulsion was placed in an oven at 37° C. for 48 hours for cross-linking. After the cross-linking of sodium hyaluronate in the emulsion was completed, sodium hyaluronate microspheres were obtained, which were washed three times with petroleum ether and three times with pure water, and stored in a PBS aqueous solution.

[0061] High-viscosity sodium hyaluronate microspheres with uniform size can be prepared, and remain stable in PBS aqueous solution after washing.

[0062] Embodiment 5:

[0063] This embodiment provides a high-viscosity sodium hyaluronate microsphere, which uses sodium hyaluronate with a molecular weight of 1.2 MDa and a high-viscosity hyaluronic acid or hyaluronate salt content of 4%. The specific preparation process is as follows:

[0064] (1) preparing an aqueous solution: pour 0.12 g of 1.2 MDa sodium hyaluronate, 0.03 g of sodium hydroxide, and 3 mL of a mixture of normal saline and pure water, wherein the volume ratio of normal saline to pure water is 2:3, into a 5 mL tin bottle in sequence and stir evenly;

[0065] (2) Preparation of oil phase solution: To 89 g of liquid paraffin, add 9 g of T1541 and 2 g of 4-butanediol diglycidyl ether (BDDE) in sequence, and stir evenly to make BDDE completely dissolved in the oil phase solution;

[0066] (3) With the aqueous solution as the external phase and the oily solution as the internal phase, an oil-in-water emulsion is prepared by microfluidic technology, and a silicone tube is installed in the outlet pipe of the microfluidic control, such as Figure 1As shown, the water phase and the oil phase of the water-in-oil emulsion undergo phase transfer to obtain an oil-in-water emulsion with an outer phase of the oil phase and an inner phase of the water phase;

[0067] (4) The prepared water-in-oil emulsion was placed in an oven at 37° C. for 48 hours for cross-linking. After the cross-linking of sodium hyaluronate in the emulsion was completed, sodium hyaluronate microspheres were obtained, which were washed three times with petroleum ether and three times with pure water, and stored in a PBS aqueous solution.

[0068] High-viscosity sodium hyaluronate microspheres with uniform size can be prepared, and remain stable in PBS aqueous solution after washing.

[0069] Embodiment 6:

[0070] In order to verify that high-viscosity hyaluronic acid microspheres cannot be directly prepared using microfluidic technology, this example uses an aqueous phase containing high-viscosity sodium hyaluronate and BDDE as the inner phase and an oil phase as the outer phase to prepare an oil-in-water emulsion with uniform particle size in a two-phase flow using microfluidic technology, and at the same time, the sodium hyaluronate in the inner phase is cross-linked with a cross-linking agent in the oil-in-water emulsion.

[0071] The specific preparation process is as follows:

[0072] (1) Prepare an aqueous solution: pour 0.12 g of 1.2 MDa sodium hyaluronate, 0.03 g of sodium hydroxide, 0.03 g of BDDE, and 3 mL of a mixture of normal saline and pure water, wherein the volume ratio of normal saline to pure water is 2:3, into a 5 mL tin bottle in sequence and stir evenly;

[0073] (2) Preparation of oil phase solution: Add 9 g of T154 to 91 g of liquid paraffin and stir evenly to make BDDE completely dissolved in the oil phase solution;

[0074] (3) using an aqueous solution as the external phase and an oily solution as the internal phase to prepare an oil-in-water emulsion by microfluidic technology;

[0075] (4) The prepared water-in-oil emulsion was placed in an oven at 37° C. for 48 hours for cross-linking. After the cross-linking of sodium hyaluronate in the microspheres was completed, sodium hyaluronate microspheres were obtained, which were washed three times with petroleum ether and three times with pure water, and stored in a PBS aqueous solution.

[0076] The microspheres prepared Figure 3 As shown, it can be seen that high-viscosity sodium hyaluronate microspheres can be prepared by microfluidic technology using an aqueous phase containing high-viscosity sodium hyaluronate and BDDE as the inner phase and an oil phase as the outer phase, but the sizes are not uniform.

[0077] Embodiment 7:

[0078] In order to verify that high-viscosity hyaluronic acid microspheres cannot be directly prepared using microfluidic technology, this example uses an aqueous phase containing high-viscosity sodium hyaluronate and BDDE as the inner phase and an oil phase as the outer phase to prepare an oil-in-water emulsion with uniform particle size in a two-phase flow using microfluidic technology, and at the same time, the sodium hyaluronate in the inner phase is cross-linked with a cross-linking agent in the oil-in-water emulsion.

[0079] The specific preparation process is as follows:

[0080] (1) Prepare the aqueous phase solution: pour 0.21 g of 2MDa sodium hyaluronate, 0.03 g of sodium hydroxide, 0.03 g of BDDE, and 3 mL of a mixture of normal saline and pure water, wherein the volume ratio of normal saline to pure water is 2:3, into a 5 mL tin bottle in sequence and stir evenly;

[0081] (2) Preparation of oil phase solution: Add 9 g of T154 to 91 g of liquid paraffin and stir evenly to make BDDE completely dissolved in the oil phase solution;

[0082] (3) using an aqueous solution as the external phase and an oily solution as the internal phase to prepare an oil-in-water emulsion by microfluidic technology;

[0083] (4) The prepared water-in-oil emulsion was placed in an oven at 37° C. for 48 hours for cross-linking. After the cross-linking of sodium hyaluronate in the emulsion was completed, sodium hyaluronate microspheres were obtained, which were washed three times with petroleum ether and three times with pure water, and stored in a PBS aqueous solution.

[0084] The solidification occurs about half an hour after the internal phase solution is prepared. Before the stable microspheres are prepared, the internal phase has already gelled and the microspheres cannot be prepared.

[0085] Embodiment 8:

[0086] In order to verify that high-viscosity hyaluronic acid microspheres cannot be directly prepared using microfluidic technology, this example uses an aqueous phase containing high-viscosity sodium hyaluronate and BDDE as the inner phase and an oil phase as the outer phase to prepare an oil-in-water emulsion with uniform particle size in a two-phase flow using microfluidic technology, and at the same time, the sodium hyaluronate in the inner phase is cross-linked with a cross-linking agent in the oil-in-water emulsion.

[0087] The specific preparation process is as follows:

[0088] (1) Prepare the aqueous phase solution: pour 0.3 g of 1.2 MDa sodium hyaluronate, 0.03 g of sodium hydroxide, 0.03 g of BDDE, and 3 mL of a mixture of normal saline and pure water, wherein the volume ratio of normal saline to pure water is 2:3, into a 5 mL tin bottle in sequence and stir evenly;

[0089] (2) Preparation of the oil-phase solution: Add 9 g of T154 to 91 g of liquid paraffin and stir evenly to completely dissolve BDDE in the oil-phase solution;

[0090] (3) Using the aqueous-phase solution as the continuous phase and the oil-phase solution as the dispersed phase, prepare a water-in-oil emulsion through microfluidic technology;

[0091] (4) Place the prepared water-in-oil emulsion in an oven at 37 °C for 48 hours for crosslinking. After the crosslinking of sodium hyaluronate in the emulsion is completed, sodium hyaluronate microspheres are obtained. Wash them three times with petroleum ether and three times with pure water, and store them in PBS aqueous solution.

[0092] Solidification occurred about half an hour after the inner-phase solution was prepared. The inner phase had gelled before stable microspheres could be prepared, and microspheres could not be prepared.

[0093] Compare the preparation processes and results of the above examples as shown in Table 1.

[0094] Table 1 Comparison of the preparation processes and results of each example

[0095]

[0096] According to the content of Table 1, it can be seen that in Examples 1, 2, 4, and 5, highly viscous sodium hyaluronate microspheres with uniform sizes and stable existence in PBS aqueous solution after washing can be prepared. In Example 3, due to the too high HA content, the inner-phase solution could not be formulated. In Example 6, microspheres can be prepared, but the sizes are not uniform. In Examples 7 and 8, solidification occurred about half an hour after the inner-phase solution was prepared. The inner phase had gelled before stable microspheres could be prepared, and microspheres could not be prepared.

[0097] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for preparing high-viscosity sodium hyaluronate microspheres, characterized in that, Using an aqueous phase containing sodium hyaluronate with high viscosity as the outer phase and an oil phase containing a crosslinking agent as the inner phase, a water-in-oil emulsion with uniform particle size is prepared in a two-phase flow through microfluidic technology. Then, the water-in-oil emulsion is introduced into a lipophilic environment, causing the phase transfer of the water-in-oil emulsion, so that the oil phase becomes the outer phase and the aqueous phase containing high-viscosity hyaluronic acid becomes the inner phase, thereby forming a water-in-oil emulsion with uniform size. At the same time, the high-viscosity sodium hyaluronate absorbs the crosslinking agent in the oil phase and crosslinks within the water-in-oil emulsion to obtain microspheres.

2. The method for preparing high-viscosity sodium hyaluronate microspheres according to claim 1, wherein The specific preparation process includes the following steps: (1) Prepare an aqueous phase solution: Add high-viscosity hyaluronic acid or hyaluronate to physiological saline, and add an alkali, and stir evenly; (2) Prepare an oil phase solution: Add a crosslinking agent to the oil phase solution and stir evenly to completely dissolve the crosslinking agent in the oil phase solution; (3) Using the aqueous phase solution as the outer phase and the oil phase solution as the inner phase, prepare a water-in-oil emulsion through microfluidic technology; (4) Directly place the prepared water-in-oil emulsion in a lipophilic environment and make it oscillate, so that the phase transfer of the aqueous phase and the oil phase occurs, and a water-in-oil emulsion with the outer phase being the oil phase and the inner phase being the aqueous phase is obtained; (5) Place the prepared water-in-oil emulsion in an oven at 10 - 55 °C for crosslinking for 4 - 72 h. After the crosslinking of sodium hyaluronate in the emulsion is completed, sodium hyaluronate microspheres are obtained. Wash them three times with petroleum ether and three times with pure water, and obtain the microspheres and store them in a PBS aqueous solution.

3. A method for preparing high-viscosity sodium hyaluronate microspheres according to claim 2, characterized in that, In the step (1), the content of high-viscosity hyaluronic acid or hyaluronate is 0.1 - 3.5 MDa.

4. A method for preparing high-viscosity sodium hyaluronate microspheres according to claim 2 or 3, characterized in that, In the aqueous phase solution prepared in the step (1), the content of high-viscosity hyaluronic acid or hyaluronate is 1 - 20 wt%, the content of the alkali is 1 - 3 wt%, the content of sodium chloride is 0.01 - 1 wt%, the water is deionized water, and the alkali is at least one of sodium hydroxide and potassium hydroxide.

5. A method for preparing high-viscosity sodium hyaluronate microspheres according to claim 2 or 3, characterized in that, In the step (2), the oil used is at least one of liquid paraffin and mineral oil.

6. A method for preparing high-viscosity sodium hyaluronate microspheres according to claim 2 or 3, characterized in that, In the oil phase solution prepared in the step (2), the crosslinking agent used is at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, divinyl sulfone, 1,4-butanediol diglycidyl ether, and sodium trimetaphosphate, and the content of the crosslinking agent in the oil phase is 0.1 - 10 wt%.

7. A method for preparing high-viscosity sodium hyaluronate microspheres according to claim 2 or 3, characterized in that, In the step (3), a water-in-oil emulsion is prepared through a microfluidic device. The microfluidic device includes an inner phase outflow tube, and a collection tube is hermetically embedded outside the outflow end of the inner phase outflow tube; the inner phase outflow tube is a circular glass tube or a circular steel tube with an inner diameter of 10 μm - 1000 μm and an outer diameter of 50 μm - 2000 μm; the collection tube is a square or circular glass tube or steel tube with an inner diameter of 50 μm - 2000 μm and an outer diameter of 100 μm - 3000 μm; the flow rate of the aqueous phase is 1 - 50 μL / min, and the flow rate of the oil phase is 20 - 500 μL / min.

8. A method for preparing high-viscosity sodium hyaluronate microspheres according to claim 7, characterized in that, In the step (4), the method of directly placing the prepared water-in-oil emulsion in a lipophilic environment includes any one of the following: a. A lipophilic pipeline is hermetically connected to the outlet end of the collecting pipe. The oil-in-water emulsion flows into the lipophilic pipeline along with the outer phase. The oil-phase droplets of the original inner phase come into contact with the lipophilic pipe wall, and phase transfer occurs between the water phase and the oil phase, obtaining a water-in-oil emulsion with an outer phase of oil phase and an inner phase of water phase. b. A container for containing an oil-phase solution is arranged at the outlet of the collecting pipe, and a stirring mechanism is arranged in the container. The oil-in-water emulsion flowing out of the collecting pipe enters the container for containing the oil-phase solution. After stirring, phase transfer occurs between the water phase and the oil phase, obtaining a water-in-oil emulsion with an outer phase of oil phase and an inner phase of water phase.

9. The method for preparing high-viscosity sodium hyaluronate microspheres according to claim 8, wherein The material of the lipophilic pipeline is at least one of PE, PET, PP, PVC, and silica gel. The inner diameter of the lipophilic pipeline is 100 - 3000 μm, and the outer diameter is 200 - 5000 μm.

10. A high-viscosity sodium hyaluronate microsphere, characterized in that, The high-viscosity sodium hyaluronate microspheres prepared by the preparation method according to any one of claims 1 to 9, wherein the diameter of the high-viscosity sodium hyaluronate microspheres is 30 μm - 2000 μm.

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