Monodisperse cross-linked hyaluronic acid microspheres and preparation method thereof

Abstract

The invention provides a preparation method of monodisperse cross-linked hyaluronic acid microspheres. The preparation method comprises the following steps: (1) preparing an internal phase fluid which contains hyaluronic acid or zinc hyaluronate and a cross-linking agent and has a pH value of 4-6, preparing an external phase fluid containing an oil-soluble surfactant, and preparing a collecting solution containing the oil-soluble surfactant and an activating agent; and (2) preparing a monodisperse water-in-oil emulsion through a microfluidic technology, collecting the water-in-oil emulsion by adopting a container containing a collecting solution, activating carboxyl of hyaluronic acid or zinc hyaluronate in the water-in-oil emulsion by using an activating agent in the collecting solution, and further carrying out a cross-linking reaction with a cross-linking agent to convert the water-in-oil emulsion into microspheres, and washing and drying the obtained microspheres. The particle size variation coefficient of the cross-linked hyaluronic acid microspheres prepared by the method does not exceed 6%, and the cross-linked hyaluronic acid microspheres prepared by the method have good monodispersity.

Description

Technical Field

[0001] The invention belongs to the technical field of medical biomaterials and relates to monodisperse cross-linked hyaluronic acid microspheres and a preparation method thereof. Background Art

[0002] Hyaluronic acid (HA) is a naturally occurring non-sulfated polysaccharide with a molecular structure composed of alternating D-glucuronic acid linked by β-4 glycosidic bonds and N-acetyl-D-glucosamine linked by β-3 glycosidic bonds. In the body, it is widely distributed in the extracellular matrix and is an important component for maintaining the structure and function of the matrix. It is also present in large quantities in the vitreous humor of the eye, providing the necessary support and light-transmitting environment for the eyeball. In addition, the synovial fluid of vertebrate joints is also rich in hyaluronic acid, which plays a key role in lubrication and cushioning of the joints. With its excellent biocompatibility - it can be well compatible with human tissue without causing rejection reactions, its near-zero immunogenicity - it does not trigger the body's immune response, and its unique viscoelasticity and lubrication properties, hyaluronic acid has now become the irreplaceable material of choice in the field of soft tissue filling, playing an important role in medical aesthetics, repair and other scenarios. However, hyaluronic acid in its free state has obvious shortcomings: its mechanical properties are weak and it is difficult to withstand high pressure or tension; and it will be rapidly degraded by related enzymes in the human body, and eventually decomposed into water and carbon dioxide, resulting in an extremely short half-life in the body of only 1 to 2 days. This greatly limits its application effect and duration. Therefore, in order to improve the mechanical strength of hyaluronic acid so that it can better adapt to the mechanical requirements in practical applications, while prolonging its retention time in the body and enhancing the durability of its effect, the industry usually uses cross-linking agents to chemically modify hyaluronic acid, and construct a more stable molecular structure through cross-linking reactions, thereby improving its various properties.

[0003] Currently, the commonly used crosslinkers for hyaluronic acid crosslinking include divinyl sulfone, 1,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, and aldehydes. However, these crosslinkers have the risk of causing side effects such as inflammation and edema in the human body. First, the biocompatibility of the crosslinkers used is limited, and residues can easily cause side effects after implantation. Second, crosslinking under alkaline conditions can easily lead to hyaluronic acid degradation, shortening the product's duration of action in the body. Third, the emulsification method used can easily cause microspheres to have uneven size and poor monodispersity. Small-particle microspheres are more sensitive to enzymes due to their large specific surface area, and degrade faster than large-particle microspheres. This results in different degradation times in the body, making it difficult to accurately assess the effect. At the same time, large-particle microspheres require greater injection force, which can increase the patient's pain. Summary of the Invention

[0004] In response to the problems of poor monodispersity, high toxicity of cross-linking agents, complex preparation process and long preparation cycle of existing cross-linked hyaluronic acid microspheres, the present invention provides monodisperse cross-linked hyaluronic acid microspheres and a preparation method thereof, so as to improve the monodispersity of cross-linked hyaluronic acid microspheres, realize the one-step preparation of monodisperse cross-linked hyaluronic acid microspheres, and simplify the preparation process.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical solutions:

[0006] A method for preparing monodisperse cross-linked hyaluronic acid microspheres comprises the following steps:

[0007] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0008] Preparation of an internal phase fluid: dissolving hyaluronic acid or zinc hyaluronate and a cross-linking agent in water and adjusting the pH of the resulting solution to 4-6 to obtain an internal phase fluid; the concentration of hyaluronic acid or zinc hyaluronate in the internal phase fluid is 0.01-0.1 g / mL, and the mass ratio of the cross-linking agent to the hyaluronic acid or zinc hyaluronate is (0.01-5):1; the water-soluble cross-linking agent is at least one of diacid dihydrazide, chitosan, gelatin, and carboxymethyl chitosan;

[0009] Prepare the external phase fluid: dissolve the oil-soluble surfactant in the oily solvent to obtain the external phase fluid; the concentration of the oil-soluble surfactant in the external phase fluid is 0.01 to 0.1 g / mL;

[0010] Preparation of a collection solution: dissolving an oil-soluble surfactant and an activator in an oily solvent to obtain a collection solution; in the collection solution, the concentration of the oil-soluble surfactant is 0.01 to 0.1 g / mL, and the concentration of the activator is 0.01 to 0.1 g / mL; the activator is at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysulfosuccinimide, and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride;

[0011] (2) Preparation of monodisperse cross-linked hyaluronic acid microspheres

[0012] The inner phase fluid is injected into the injection tube of the primary microfluidic device through a syringe pump, and the outer phase fluid is injected into the receiving tube of the primary microfluidic device to form a monodisperse oil-in-water emulsion in the receiving tube. The oil-in-water emulsion is collected in a container containing a collecting liquid and allowed to stand for a long time. During the standing period, the activator in the collecting liquid activates the carboxyl groups of the hyaluronic acid or zinc hyaluronate in the oil-in-water emulsion, causing the hyaluronic acid or zinc hyaluronate to undergo a cross-linking reaction with the cross-linking agent to convert the oil-in-water emulsion into microspheres. The obtained microspheres are washed to obtain monodisperse cross-linked hyaluronic acid microspheres.

[0013] In step (1) of the technical solution of the above preparation method, when preparing the internal phase fluid, hyaluronic acid or zinc hyaluronate and a cross-linking agent are dissolved in water, and then the pH value of the resulting solution is adjusted to 4-6 with an acid to obtain the internal phase fluid; the acid includes at least one of hydrochloric acid, phosphoric acid, and acetic acid. In the technical solution of the above preparation method, the diacid dihydrazide includes at least one of oxalic acid dihydrazide, adipic acid dihydrazide, sebacic acid dihydrazide, etc.

[0014] In the technical solution of the above preparation method, preferably, the oily solvent includes at least one of liquid paraffin, vegetable oil, mineral oil, silicone oil, synthetic oil, n-octanol, and isobutanol.

[0015] In the technical solution of the above preparation method, preferably, the oil-soluble surfactant includes at least one of Span 60, Span 80, polyisobutylene succinimide, and polyglycerol ricinoleate.

[0016] In step (2) of the technical solution of the above preparation method, the ratio of the flow rate of the external phase fluid to the flow rate of the internal phase fluid is preferably controlled to be 2 to 30. Furthermore, in step (2), the flow rate of the internal phase fluid is controlled to be 20 to 4000 μL / h, and the flow rate of the external phase fluid is controlled to be 40 to 12000 μL / h.

[0017] In step (2) of the technical solution of the above-mentioned preparation method, the primary microfluidic device used includes an injection tube, a connecting tube and a collecting tube, which are used in conjunction with a syringe pump. The injection tube is made of a cylindrical glass capillary tube, and its tail is pulled into a conical shape; the collecting tube is a cylindrical glass capillary tube; the connecting tube is a square glass tube, and a square through hole is provided in the center thereof; the tail of the injection tube is inserted into the head of the collecting tube and is connected through the connecting tube; the injection tube, the connecting tube and the collecting tube are coaxially arranged.

[0018] In step (2) of the technical solution of the above preparation method, in the primary microfluidic device used, the outlet of the injection tube is tapered, the inner diameter of the tapered mouth of the injection tube is preferably 10 to 700 μm, and the inner diameter of the receiving tube is preferably 100 to 1000 μm.

[0019] In step (2) of the technical solution of the above-mentioned preparation method, the standing time is determined according to the degree of cross-linking of the cross-linked hyaluronic acid microspheres in actual application. Generally, the standing time can be controlled to be 10 to 30 hours to allow the hyaluronic acid or zinc hyaluronate to undergo a cross-linking reaction with the cross-linking agent to convert the oil-in-water emulsion into microspheres.

[0020] In the technical solution of the above preparation method, in step (2), when washing the obtained microspheres, the oily solvent, activator, and unreacted crosslinker are first removed with an organic solvent, and then the organic solvent is removed by washing with water. Preferably, the organic solvent can be one or more of methanol, ethyl acetate, ethanol, isopropanol, and n-hexane.

[0021] In the technical solution of the above preparation method, the molecular weight of the hyaluronic acid or zinc hyaluronate is determined according to actual application requirements, and the preferred molecular weight is 5 to 1800 kDa.

[0022] In the technical solution of the above preparation method, the maximum concentration of the solution formed after the concentration of hyaluronic acid or zinc hyaluronate is dissolved in water depends on the molecular weight of the hyaluronic acid or zinc hyaluronate. The smaller the molecular weight of the hyaluronic acid or zinc hyaluronate, the greater the maximum concentration of the solution formed after dissolving in water. The larger the molecular weight of the hyaluronic acid or zinc hyaluronate, the lower the maximum concentration of the solution formed after dissolving in water. That is, the upper limit of the concentration of hyaluronic acid or zinc hyaluronate in the internal phase fluid of step (1) mainly depends on the molecular weight of the hyaluronic acid or zinc hyaluronate.

[0023] In the technical solution of the above-mentioned preparation method, the amount of the cross-linking agent in the internal phase fluid of step (1) will affect the cross-linking density of the cross-linked hyaluronic acid microspheres, and thus affect the mechanical properties, degradation properties and elastic properties of the cross-linked hyaluronic acid microspheres. In practical applications, the mass ratio of the cross-linking agent hyaluronic acid or hyaluronic acid zinc in the internal phase fluid can be determined according to specific application requirements. For example, the mass ratio of the cross-linking agent to hyaluronic acid or hyaluronic acid zinc can be (0.01~5):1, and the mass ratio of the cross-linking agent to hyaluronic acid or hyaluronic acid zinc can also be (0.1~1):1.

[0024] In the technical solution of the above preparation method, in the external phase fluid of step (1), the concentration of the oil-soluble surfactant is preferably 0.04 to 0.1 g / mL; in the collected liquid, the concentration of the oil-soluble surfactant is preferably 0.04 to 0.1 g / mL, and the concentration of the activator is 0.01 to 0.1 g / mL.

[0025] The present invention also provides monodisperse cross-linked hyaluronic acid microspheres with controllable size and excellent biocompatibility, prepared by the above method. The cross-linked hyaluronic acid microspheres are spherical, with a particle size of 50 to 400 μm, and a coefficient of variation of the particle size of the cross-linked hyaluronic acid microspheres of no more than 5%. The cross-linked hyaluronic acid microspheres can be used as medical materials, cosmetic fillers, drug control materials, cell delivery materials, and can also be used in cosmetics, tissue repair and other fields.

[0026] The technical solution of the present invention utilizes an activator in the collected liquid to activate the carboxyl groups of hyaluronic acid or sodium hyaluronate in the water-in-oil emulsion under acidic conditions, allowing the carboxyl groups of the activated hyaluronic acid or sodium hyaluronate to undergo a cross-linking reaction with the amino groups of the cross-linking agent, ultimately causing the water-in-oil emulsion to solidify and transform into cross-linked hyaluronic acid microspheres. On the one hand, conducting the cross-linking reaction under acidic conditions can effectively avoid the problem of hyaluronic acid or sodium hyaluronate being easily degraded under alkaline conditions, thereby extending the in vivo efficacy of the microsphere product. On the other hand, allowing the carboxyl groups of hyaluronic acid or sodium hyaluronate to undergo a cross-linking reaction with the cross-linking agent blocks some of the carboxyl groups of hyaluronic acid or sodium hyaluronate. The carboxyl group is one of the action sites of enzymes during the degradation of hyaluronic acid in the body. Compared with the prior art in which the hydroxyl groups of hyaluronic acid are cross-linked with the cross-linking agent, the technical solution of the present invention blocks some of the carboxyl groups of hyaluronic acid or zinc hyaluronate through a cross-linking reaction, objectively reducing the action sites of enzymes that degrade hyaluronic acid. This also helps to delay the degradation of the cross-linked hyaluronic acid microspheres in the body and extend the in vivo efficacy of the microsphere product. Based on the above points, the technical solution of the present invention is combined with microfluidic technology to achieve cross-linked hyaluronic acid microspheres with excellent monodispersity. This can solve the problem that cross-linked hyaluronic acid microsphere products cause pain to patients or have different degradation times in the body due to their wide size distribution, thereby improving the safety of microsphere products. In addition, the uniform particle size of the cross-linked hyaluronic acid microspheres is also conducive to accurate evaluation of the effect.

[0027] Compared with the prior art, the technical solution of the present invention produces the following beneficial technical effects:

[0028] The present invention provides a method for preparing monodisperse cross-linked hyaluronic acid microspheres. The method uses a cross-linking agent containing an amino group and having good biocompatibility. Based on the activation of the hyaluronic acid by an activator, the method achieves cross-linking of an oil-in-water emulsion containing hyaluronic acid and the cross-linking agent. At the same time, combined with microfluidic technology, cross-linked hyaluronic acid microspheres with uniform morphology, excellent sphericity, and excellent monodispersity are prepared. Compared with the cross-linking agents such as divinyl sulfone, 1,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, and aldehydes used in the cross-linking process of hyaluronic acid microspheres in the prior art, which have certain biological toxic side effects, the cross-linked hyaluronic acid prepared by the method of the present invention has better biocompatibility and can avoid the cross-linking agents used in the prior art from remaining in the microspheres and causing side effects such as inflammation and edema in the body. At the same time, compared with the prior art in which the hydroxyl groups of hyaluronic acid are cross-linked with the cross-linking agent under alkaline conditions, the present invention performs a cross-linking reaction between the carboxyl groups of hyaluronic acid and the cross-linking agent under acidic conditions, which can not only effectively avoid the problem of easy degradation of hyaluronic acid under alkaline conditions, but also block some of the carboxyl groups of hyaluronic acid. Both aspects are beneficial to delaying the degradation of cross-linked hyaluronic acid microspheres in the body and prolonging the effectiveness of the microsphere product in the body. DETAILED DESCRIPTION

[0029] The following examples further illustrate the present invention's monodisperse cross-linked hyaluronic acid microspheres and their preparation methods. It should be noted that the following examples are intended only to further illustrate the present invention and are not to be construed as limiting the scope of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the above-described disclosure and implemented in accordance with the present invention remain within the scope of the present invention.

[0030] In the following embodiments, the microfluidic device used is a primary microfluidic device, comprising an injection tube 1, a connecting tube 2, and a collecting tube 3, used in conjunction with a syringe pump. The injection tube 1 is made of a cylindrical glass capillary tube. The tail of the cylindrical glass capillary tube is pulled into a conical shape using a needle puller and then rolled on sandpaper to a flat end with an inner diameter of approximately 120 to 260 μm. The outer diameter of the circular tube section is 960 μm and the inner diameter is 550 μm. The collecting tube 3 is made of a cylindrical glass capillary tube. The ends of the cylindrical glass capillary tube are polished flat. The collecting tube has an outer diameter of 960 μm and an inner diameter of 550 to 700 μm. The connecting tube 2 is a square glass tube. The ends of the square glass tube are polished smooth and flat. The center portion of the square glass tube is provided with a square through-hole with a size of 1.0 × 1.0 mm. After production, the injection tube 1, connecting tube 2, and collecting tube 3 need to be ultrasonically cleaned in anhydrous ethanol and blown dry. The tail end of the injection tube 1 is inserted into the head of the collection tube 3 and connected via the connecting tube 2. The injection tube 1, connecting tube 2, and collection tube 3 are coaxially arranged and secured to a glass slide using AB glue. A steel tube can be placed over the injection tube 1, with the non-inlet end secured using AB glue to facilitate connection to a syringe pump. A flat-ended needle is secured to the inlet end of the connecting tube 2 using AB glue, and the non-inlet end of the connecting tube 2 is sealed with AB glue. Each flat-ended needle is connected to a syringe pump via a fitting.

[0031] Example 1

[0032] In this embodiment, a method for preparing monodisperse cross-linked hyaluronic acid (HA) microspheres is provided, and the steps are as follows:

[0033] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0034] Preparation of the internal phase fluid: Add hyaluronic acid (HA) with a relative molecular mass of 200,000 to 400,000 and a cross-linking agent adipic acid dihydrazide (ADH) to deionized water, stir until HA and ADH are completely dissolved, and use hydrochloric acid to adjust the pH value of the resulting solution to 5 to obtain the internal phase fluid; in the internal phase fluid, the concentration of HA is 0.01 g / mL, and the mass ratio of ADH to HA is 0.1:1.

[0035] Prepare the external phase fluid: Dissolve the surfactant Span 80 in n-octanol to obtain an external phase fluid; the concentration of Span 80 in the external phase fluid is 0.05 g / mL. Prepare the collecting solution: Dissolve the surfactant Span 80 and the activator 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in n-octanol to obtain a collecting solution; the concentration of Span 80 in the collecting solution is 0.05 g / mL and the concentration of EDC is 0.01 g / mL.

[0036] (2) Preparation of monodisperse cross-linked HA microspheres

[0037] Preparation is performed using a primary microfluidic device. An internal phase fluid is injected into the injection tube 1 of the microfluidic device via a syringe pump, and an external phase fluid is injected into the receiving tube 3 of the microfluidic device. A monodisperse water-in-oil (W / O) emulsion is formed in the receiving tube. The monodisperse W / O emulsion is collected in a container containing a collection liquid and allowed to stand for 24 hours. During this standing period, an activator in the collection liquid activates the carboxyl groups of the hyaluronic acid in the W / O emulsion, causing a cross-linking reaction between the hyaluronic acid and the cross-linking agent to convert the W / O emulsion into microspheres. The resulting microspheres are washed 3-5 times with isopropyl alcohol, then 3-5 times with deionized water, and air-dried to obtain monodisperse cross-linked HA microspheres.

[0038] A total of five groups of experiments were conducted in this step. In the first to fifth groups of experiments, the inner diameter of the cone mouth of the injection tube of the fixed microfluidic device was 120 μm, and the inner diameter of the receiving tube of the fixed microfluidic device was 550 μm. In the first to fifth groups of experiments, the flow rate of the internal phase fluid was controlled to be 200 μL / h, and the ratio of the external phase fluid flow rate to the internal phase fluid flow rate was controlled to be 5, 10, 15, 20, and 30, respectively.

[0039] Example 2

[0040] In this embodiment, a method for preparing monodisperse cross-linked HA microspheres is provided, and the steps are as follows:

[0041] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0042] Preparation of the internal phase fluid: Add HA with a relative molecular mass of 200,000 to 400,000 and the cross-linking agent ADH to deionized water, stir until HA and ADH are completely dissolved, and use hydrochloric acid to adjust the pH value of the resulting solution to 5 to obtain the internal phase fluid; in the internal phase fluid, the concentration of HA is 0.01 g / mL, and the mass ratio of ADH to HA is 0.1:1.

[0043] Prepare the external phase fluid: Dissolve the surfactant Span 80 in n-octanol to obtain an external phase fluid; the concentration of Span 80 in the external phase fluid is 0.05 g / mL. Prepare the collection solution: Dissolve the surfactant Span 80 and the activator EDC in n-octanol to obtain a collection solution; the concentration of Span 80 in the collection solution is 0.05 g / mL and the concentration of EDC is 0.01 g / mL.

[0044] (2) Preparation of monodisperse cross-linked HA microspheres

[0045] Preparation is performed using a single-stage microfluidic device. An internal phase fluid is injected into the injection tube 1 of the microfluidic device via a syringe pump, and an external phase fluid is injected into the receiving tube 3 of the microfluidic device. A monodisperse W / O emulsion is formed in the receiving tube. The monodisperse W / O emulsion is collected in a container containing a collection liquid and allowed to stand for 24 hours. During this standing period, an activator in the collection liquid activates the carboxyl groups of the hyaluronic acid in the W / O emulsion, causing a cross-linking reaction between the hyaluronic acid and the cross-linking agent, converting the W / O emulsion into microspheres. The resulting microspheres are washed 3-5 times with isopropyl alcohol, then 3-5 times with deionized water, and air-dried to obtain monodisperse cross-linked HA microspheres.

[0046] A total of five groups of experiments were conducted in this step. In the first to fifth groups of experiments, the inner diameter of the cone mouth of the injection tube of the fixed microfluidic device was 260 μm, and the inner diameter of the receiving tube of the fixed microfluidic device was 700 μm. In the first to fifth groups of experiments, the flow rate of the internal phase fluid was controlled to be 200 μL / h, and the ratio of the external phase fluid flow rate to the internal phase fluid flow rate was controlled to be 2, 5, 10, 20, and 30, respectively.

[0047] Example 3

[0048] In this embodiment, a method for preparing monodisperse cross-linked HA microspheres is provided, and the steps are as follows:

[0049] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0050] Preparation of the internal phase fluid: Add HA with a relative molecular mass of 200,000 to 400,000 and the cross-linking agent ADH to deionized water, stir until HA and ADH are completely dissolved, and use hydrochloric acid to adjust the pH value of the resulting solution to 5 to obtain the internal phase fluid; in the internal phase fluid, the concentration of HA is 0.01 g / mL, and the mass ratio of ADH to HA is 0.2:1.

[0051] Prepare the external phase fluid: Dissolve the surfactant Span 80 in n-octanol to obtain an external phase fluid; the concentration of Span 80 in the external phase fluid is 0.05 g / mL. Prepare the collection solution: Dissolve the surfactant Span 80 and the activator EDC in n-octanol to obtain a collection solution; the concentration of Span 80 in the collection solution is 0.05 g / mL and the concentration of EDC is 0.01 g / mL.

[0052] (2) Preparation of monodisperse cross-linked HA microspheres

[0053] Preparation is performed using a single-stage microfluidic device. An internal phase fluid is injected into the injection tube 1 of the microfluidic device via a syringe pump, and an external phase fluid is injected into the receiving tube 3 of the microfluidic device. A monodisperse W / O emulsion is formed in the receiving tube. The monodisperse W / O emulsion is collected in a container containing a collection liquid and allowed to stand for 24 hours. During this standing period, an activator in the collection liquid activates the carboxyl groups of the hyaluronic acid in the W / O emulsion, causing a cross-linking reaction between the hyaluronic acid and the cross-linking agent, converting the W / O emulsion into microspheres. The resulting microspheres are washed 3-5 times with isopropyl alcohol, then 3-5 times with deionized water, and air-dried to obtain monodisperse cross-linked HA microspheres.

[0054] In this step, the inner diameter of the cone of the injection tube of the fixed microfluidic device is 120 μm, and the inner diameter of the receiving tube of the fixed microfluidic device is 550 μm; the flow rate of the inner phase fluid is controlled to be 1.2 mL / h, and the flow rate of the outer phase fluid is 6 mL / h.

[0055] Example 4

[0056] In this embodiment, a method for preparing monodisperse cross-linked HA microspheres is provided, and the steps are as follows:

[0057] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0058] Preparation of the internal phase fluid: HA with a relative molecular mass of 1.7 to 1.9 million and the cross-linking agent ADH were added to deionized water and stirred until HA and ADH were completely dissolved. The pH value of the resulting solution was adjusted to 5 with hydrochloric acid to obtain the internal phase fluid; in the internal phase fluid, the concentration of HA was 0.01 g / mL, and the mass ratio of ADH to HA was 0.1:1.

[0059] Prepare the external phase fluid: Dissolve the surfactant Span 80 in n-octanol to obtain an external phase fluid; the concentration of Span 80 in the external phase fluid is 0.05 g / mL. Prepare the collection solution: Dissolve the surfactant Span 80 and the activator EDC in n-octanol to obtain a collection solution; the concentration of Span 80 in the collection solution is 0.05 g / mL and the concentration of EDC is 0.01 g / mL.

[0060] (2) Preparation of monodisperse cross-linked HA microspheres

[0061] Preparation is performed using a single-stage microfluidic device. An internal phase fluid is injected into the injection tube 1 of the microfluidic device via a syringe pump, and an external phase fluid is injected into the receiving tube 3 of the microfluidic device. A monodisperse W / O emulsion is formed in the receiving tube. The monodisperse W / O emulsion is collected in a container containing a collection liquid and allowed to stand for 24 hours. During this standing period, an activator in the collection liquid activates the carboxyl groups of the hyaluronic acid in the W / O emulsion, causing a cross-linking reaction between the hyaluronic acid and the cross-linking agent, converting the W / O emulsion into microspheres. The resulting microspheres are washed 3-5 times with isopropyl alcohol, then 3-5 times with deionized water, and air-dried to obtain monodisperse cross-linked HA microspheres.

[0062] In this step, the inner diameter of the cone of the injection tube of the fixed microfluidic device is 120 μm, and the inner diameter of the receiving tube of the fixed microfluidic device is 550 μm; the flow rate of the inner phase fluid is controlled to be 1.2 mL / h, and the flow rate of the outer phase fluid is 6 mL / h.

[0063] Example 5

[0064] In this embodiment, a method for preparing monodisperse cross-linked HA microspheres is provided, and the steps are as follows:

[0065] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0066] Preparation of the internal phase fluid: HA with a relative molecular mass of 200,000 to 400,000 and a cross-linking agent, oxalic acid dihydrazide, were added to deionized water and stirred until the HA and oxalic acid dihydrazide were completely dissolved. The pH of the resulting solution was adjusted to 5 with hydrochloric acid to obtain the internal phase fluid; the concentration of HA in the internal phase fluid was 0.02 g / mL, and the mass ratio of oxalic acid dihydrazide to HA was 0.1:1.

[0067] Prepare the external phase fluid: Dissolve the surfactant Span 80 in n-octanol to obtain an external phase fluid; the concentration of Span 80 in the external phase fluid is 0.05 g / mL. Prepare the collection solution: Dissolve the surfactant Span 80 and the activator EDC in n-octanol to obtain a collection solution; the concentration of Span 80 in the collection solution is 0.05 g / mL and the concentration of EDC is 0.01 g / mL.

[0068] (2) Preparation of monodisperse cross-linked HA microspheres

[0069] Preparation is performed using a single-stage microfluidic device. An internal phase fluid is injected into the injection tube 1 of the microfluidic device via a syringe pump, and an external phase fluid is injected into the receiving tube 3 of the microfluidic device. A monodisperse W / O emulsion is formed in the receiving tube. The monodisperse W / O emulsion is collected in a container containing a collection liquid and allowed to stand for 24 hours. During this standing period, an activator in the collection liquid activates the carboxyl groups of the hyaluronic acid in the W / O emulsion, causing a cross-linking reaction between the hyaluronic acid and the cross-linking agent, converting the W / O emulsion into microspheres. The resulting microspheres are washed 3-5 times with isopropyl alcohol, then 3-5 times with deionized water, and air-dried to obtain monodisperse cross-linked HA microspheres.

[0070] In this step, the inner diameter of the cone of the injection tube of the fixed microfluidic device is 120 μm, and the inner diameter of the receiving tube of the fixed microfluidic device is 550 μm; the flow rate of the inner phase fluid is controlled to be 1.2 mL / h, and the flow rate of the outer phase fluid is 6 mL / h.

[0071] Example 6

[0072] In this embodiment, a method for preparing monodisperse cross-linked HA microspheres is provided, and the steps are as follows:

[0073] (1) Preparation of internal phase fluid, external phase fluid and collection fluid

[0074] Preparation of the internal phase fluid: Add HA with a relative molecular mass of 200,000 to 400,000 and the cross-linking agent ADH to deionized water, stir until HA and ADH are completely dissolved, and use hydrochloric acid to adjust the pH value of the resulting solution to 5 to obtain the internal phase fluid; in the internal phase fluid, the concentration of HA is 0.01 g / mL, and the mass ratio of ADH to HA is 0.1:1.

[0075] Prepare the external phase fluid: Dissolve the surfactant Span 80 in n-octanol to obtain an external phase fluid; the concentration of Span 80 in the external phase fluid is 0.05 g / mL. Prepare the collection solution: Dissolve the surfactant Span 80 and the activator EDC in n-octanol to obtain a collection solution; the concentration of Span 80 in the collection solution is 0.05 g / mL and 0.005 g / mL.

[0076] (2) Preparation of monodisperse cross-linked HA microspheres

[0077] Preparation is performed using a single-stage microfluidic device. An internal phase fluid is injected into the injection tube 1 of the microfluidic device via a syringe pump, and an external phase fluid is injected into the receiving tube 3 of the microfluidic device. A monodisperse W / O emulsion is formed in the receiving tube. The monodisperse W / O emulsion is collected in a container containing a collection liquid and allowed to stand for 24 hours. During this standing period, an activator in the collection liquid activates the carboxyl groups of the hyaluronic acid in the W / O emulsion, causing a cross-linking reaction between the hyaluronic acid and the cross-linking agent, converting the W / O emulsion into microspheres. The resulting microspheres are washed 3-5 times with isopropyl alcohol, then 3-5 times with deionized water, and air-dried to obtain monodisperse cross-linked HA microspheres.

[0078] In this step, the inner diameter of the cone of the injection tube of the fixed microfluidic device is 120 μm, and the inner diameter of the receiving tube of the fixed microfluidic device is 550 μm; the flow rate of the inner phase fluid is controlled to be 1.2 mL / h, and the flow rate of the outer phase fluid is 6 mL / h.

[0079] The cross-linked HA microspheres prepared in Example 3 and Example 6 were respectively added to glass containers filled with deionized water, and then the cross-linked HA microspheres were compressed at a certain speed by a probe installed on a force sensor, and the force during compression was recorded by the force sensor and a computer.

Claims

1. A monodisperse cross-linked hyaluronic acid microsphere and a preparation method thereof, characterized in that: Improve the monodispersity of cross-linked hyaluronic acid microspheres and achieve one-step preparation of monodisperse cross-linked hyaluronic acid microspheres, simplify the preparation process, prepare the inner phase fluid, outer phase fluid and collection fluid, and prepare monodisperse cross-linked hyaluronic acid microspheres.