Porous sodium hyaluronate microspheres as well as preparation method and application thereof

Abstract

The invention relates to a porous sodium hyaluronate microsphere as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) preparing a water phase: dissolving sodium hyaluronate in an alkaline solution containing a chemical foaming agent to obtain an alkaline aqueous solution containing the sodium hyaluronate and the chemical foaming agent, and then mixing the alkaline aqueous solution with a cross-linking agent to obtain the water phase; preparing an oil phase: dissolving an emulsifier in the oil phase matrix to obtain the oil phase; (2) adding the water phase obtained in the step (1) into the oil phase, carrying out a cross-linking reaction, and then carrying out a foaming reaction by adopting a heating method or an acid titration method; and after the reaction is completed, cleaning and removing an oil phase by a gradient centrifugation method to obtain the porous sodium hyaluronate microspheres. According to the method, the occupation effect of a foaming agent in a cross-linking agent is utilized, the foaming agent generates gas through a heating method or an acid titration method, holes are reserved in the cross-linking gel and on the surface of the cross-linking gel in the gas escaping process, and therefore the porous sodium hyaluronate microspheres are prepared.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of high polymer materials and biomedical materials, and particularly relates to a porous sodium hyaluronate microsphere as well as a preparation method and application thereof. BACKGROUND

[0002] As a natural linear polysaccharide, sodium hyaluronate has become a key material in the biomedical field due to its excellent biocompatibility, degradability and non-immunogenicity, and is widely used in the fields of tissue engineering scaffolds such as cartilage repair, drug release carriers such as antitumor drug delivery, injection fillers such as soft tissue repair and filling, and postoperative anti-adhesion.

[0003] At present, the application of sodium hyaluronate is mainly in the form of cross-linked sodium hyaluronate gel and non-cross-linked sodium hyaluronate solution, and the preparation of sodium hyaluronate microspheres is mainly by emulsion cross-linking method or composite microsphere method.

[0004] CN111686664A discloses a preparation method of emulsion cross-linked sodium hyaluronate gel microspheres for injection. First, a sodium hyaluronate microsphere particle emulsion is prepared; then a cross-linking agent is added to the emulsion to obtain a microsphere emulsion, which is washed with n-hexane, then with ethyl acetate, and then with anhydrous ethanol under stirring.

[0005] CN115414528A discloses a composite sodium hyaluronate microsphere gel, which is obtained by adding polycaprolactone microspheres to cross-linked sodium hyaluronate gel.

[0006] CN114369264A discloses a cross-linked sodium hyaluronate microsphere and a preparation method thereof. Sodium hyaluronate is dissolved in an alkali solution, dried, and then ground in liquid nitrogen to prepare hydroxyl-activated sodium hyaluronate microspheres, which are then cross-linked to obtain cross-linked sodium hyaluronate microspheres.

[0007] However, the above-mentioned preparation methods of dense sodium hyaluronate microspheres still have certain limitations, including poor controllability of microsphere pore size, low drug loading efficiency, limited cell infiltration, and unbalanced metabolic rate, etc.

[0008] Therefore, it has become one of the technical problems to be solved to develop a preparation method of porous sodium hyaluronate microspheres with controllable process and high cost-effectiveness. SUMMARY

[0009] In view of the deficiencies of the prior art, the purpose of the present application is to provide a porous sodium hyaluronate microsphere as well as a preparation method and application thereof.

[0010] To achieve the purpose of the present application, the following technical solutions are adopted:

[0011] In a first aspect, the present application provides a method for preparing porous sodium hyaluronate microspheres, comprising the following steps:

[0012] (1) Water phase preparation: dissolve sodium hyaluronate in an alkaline solution containing a chemical foaming agent to obtain an alkaline aqueous solution containing sodium hyaluronate and a chemical foaming agent, and then mix with a crosslinking agent to obtain a water phase; oil phase preparation: dissolve an emulsifier in an oil phase matrix to obtain an oil phase;

[0013] (2) Add the water phase obtained in step (1) to the oil phase, and perform a crosslinking reaction, and then perform a foaming reaction by using a temperature rising method or an acid titration method; after the reaction is completed, the oil phase is removed by gradient centrifugation to obtain porous sodium hyaluronate microspheres.

[0014] The present application utilizes the space occupation of the foaming agent in the crosslinking agent, and the gas generated by the foaming agent through the temperature rising method (by high temperature decomposition) or the acid titration method (by acid-base neutralization), and the process of gas escaping will leave holes in the inside and surface of the crosslinked gel, thereby preparing porous sodium hyaluronate microspheres.

[0015] Compared with traditional solid sodium hyaluronate microsphere materials, the porous sodium hyaluronate microspheres have better active ingredient transport function, and can slowly release after reaching the implanted position to prolong the action maintenance time; at the same time, the pore size of the porous sodium hyaluronate microspheres can be controlled and adjusted according to the concentration of the foaming agent, the parameters of the foaming reaction, etc.; and no other toxic organic reagents are added in the whole experimental process, the operation process is simple, the properties are stable, and it is suitable for storage and use.

[0016] Preferably, the chemical foaming agent in step (1) includes any one or a combination of at least two of sodium carbonate, sodium bicarbonate or ammonium bicarbonate.

[0017] Preferably, in the alkaline solution containing a chemical foaming agent in step (1), the concentration of the chemical foaming agent is 0.1-3 mol / L, and the concentration of the base is 0.02-0.25 mol / L.

[0018] Specific point values in 0.1-3 mol / L can be selected as 0.1 mol / L, 0.5 mol / L, 1 mol / L, 1.5 mol / L, 2 mol / L, 2.5 mol / L, 3 mol / L, etc., and specific point values in 0.02-0.25 mol / L can be selected as 0.02 mol / L, 0.05 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, etc.

[0019] Preferably, the alkaline solution containing a chemical foaming agent includes a sodium hydroxide solution containing a chemical foaming agent.

[0020] Preferably, the mass percentage of sodium hyaluronate in the basic solution containing sodium hyaluronate and chemical foaming agent in step (1) is 1-15%, for example, it can be 1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%, etc.

[0021] Preferably, the molecular weight of the sodium hyaluronate is 20-250 wDa, for example, it can be 20 wDa, 50 wDa, 100 wDa, 150 wDa, 200 wDa, 250 wDa, etc.

[0022] Preferably, the crosslinking agent in step (1) includes any one or a combination of at least two of 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), or polyethylene glycol diglycidyl ether (PEGDE).

[0023] Preferably, the mass percentage of the crosslinking agent in the aqueous phase is 0.5-5%, for example, it can be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.

[0024] Preferably, the emulsifier in step (1) includes Tween and / or Span.

[0025] Preferably, the oil phase matrix in step (1) includes any one or a combination of at least two of liquid paraffin, vegetable oil, or silicone oil.

[0026] Preferably, the process of adding the aqueous phase to the oil phase in step (2) further includes stirring, and the stirring speed is 100-700 rpm, for example, it can be 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, etc.

[0027] Preferably, the crosslinking reaction time is 12-72 h, and the crosslinking reaction temperature is 15-40℃.

[0028] Specific point values in 12-72 h can be selected as 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, etc., and specific point values in 15-40℃ can be selected as 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc.

[0029] Preferably, the foaming reaction time is 1-8 h, for example, it can be 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h.

[0030] Preferably, the foaming reaction in step (2) using the temperature rising method is to rise the temperature to 40-100℃ and react for 1-8 h.

[0031] The specific point value in the range of 40-100℃ can be 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, 100℃, etc., and the specific point value in the range of 1-8 h can be 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, etc.

[0032] Preferably, the concentration of the acid solution in the process of the foaming reaction by acid titration in step (2) is 0.2-5 mol / L, and the dropping speed is 1×10 -5 -1×10 -3 mol / min.

[0033] The specific point value in the range of 0.2-5 mol / L can be 0.2 mol / L, 0.5 mol / L, 1 mol / L, 1.5 mol / L, 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L, 4.5 mol / L, 5 mol / L, etc., and the specific point value in the range of 1×10 -5 -1×10 -3 mol / min can be 1×10 -5 mol / min, 5×10 -5 mol / min, 1×10 -4 mol / min, 5×10 -4 mol / min, 1×10 -3 mol / min, etc.

[0034] Preferably, the acid solution comprises an aqueous solution of hydrochloric acid and / or acetic acid.

[0035] Preferably, the temperature of the foaming reaction by acid titration is 15-40℃, and the time is 1-8 h.

[0036] The specific point value in the range of 15-40℃ can be 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the specific point value in the range of 1-8 h can be 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, etc.

[0037] Preferably, the cleaning agent used in the gradient centrifugation method for removing the oil phase in step (2) is an aqueous solution of water and / or ethanol.

[0038] Preferably, the gradient centrifugation method for removing the oil phase in step (2) comprises at least four gradients, and the cleaning agents are water, an aqueous solution of ethanol with a volume percentage of 20-50%, an aqueous solution of ethanol with a volume percentage of 50-70%, and an aqueous solution of ethanol with a volume percentage of 70-90%, in sequence.

[0039] Among them, the specific point value in 20-50% can be selected as 20%, 25%, 30%, 35%, 40%, 45%, 50%, etc., the specific point value in 50-70% can be selected as 50%, 55%, 60%, 65%, 70%, etc., and the specific point value in 70-90% can be selected as 70%, 75%, 80%, 85%, 90%, etc.

[0040] In a second aspect, the application provides a porous sodium hyaluronate microsphere prepared by the method for preparing the porous sodium hyaluronate microsphere according to the first aspect.

[0041] In a third aspect, the application provides an application of the porous sodium hyaluronate microsphere according to the second aspect in preparing a tissue engineering scaffold, a drug sustained-release carrier or an injection filler.

[0042] Compared with the prior art, the application has the following beneficial effects:

[0043] The application utilizes the space-occupying effect of the foaming agent in the crosslinking agent, and the foaming agent generates gas through the heating method or the acid titration method. The process of gas escaping leaves holes in the inside and surface of the crosslinked gel, thereby preparing the porous sodium hyaluronate microsphere. Compared with the traditional solid sodium hyaluronate microsphere material, the porous sodium hyaluronate microsphere has a better active ingredient transport function, and can be slowly released after reaching the implantation position, so as to prolong the action maintenance time. At the same time, the size of the holes of the porous sodium hyaluronate microsphere can be controlled and adjusted according to the concentration of the foaming agent and the parameters of the foaming reaction. In addition, no other toxic organic reagent is added in the whole experimental process, the operation process is simple, the property is stable, and the porous sodium hyaluronate microsphere is suitable for storage and use. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 Figure 1 is an SEM characterization diagram of the porous sodium hyaluronate microsphere obtained in Example 1.

[0045] Figure 2 Figure 4 is a characterization diagram of the porous sodium hyaluronate microsphere obtained in Example 4 under a microscope. DETAILED DESCRIPTION

[0046] In order to further illustrate the technical means adopted by the application and its effects, the technical solutions of the application will be further described below in combination with the preferred embodiments of the application, but the application is not limited in the scope of the embodiments.

[0047] Example 1

[0048] The embodiment provides a preparation method of a porous sodium hyaluronate microsphere, which comprises the following steps:

[0049] (1) Preparation of the water phase:

[0050] Take 2.34 g NH4CO3 and 0.3 g NaOH dissolved in 30 mL water, and prepare a basic aqueous solution containing 1 mol / L NH4CO3 and 0.25 mol / L NaOH; add 0.8 g of hyaluronic acid sodium powder with a molecular weight of 163 wDa to the above basic aqueous solution, and mix and stir until homogeneous to obtain a basic aqueous solution containing hyaluronic acid sodium and a chemical foaming agent; add 1.0 mL of BDDE to the basic aqueous solution, and continue to stir and mix uniformly, which is the water phase.

[0051] (2) Oil phase preparation:

[0052] Take 2 g Span-80 and dissolve it in 198 g liquid paraffin. After stirring uniformly, an oil phase with a Span-80 mass percentage of 1% is obtained.

[0053] (3) Microsphere preparation:

[0054] Under a stirring speed of 300 rpm, the water phase obtained in step (1) is added dropwise to the oil phase obtained in step (2) by using a syringe, and a crosslinking reaction is carried out at 25°C for 60 h. In an acid burette, 20 mL of hydrochloric acid with a concentration of 1 mol / L is added, and the dropping speed is controlled at 2 min / drop (about 2.5×10 -5 mol / min) to drop the hydrochloric acid into the crosslinked mixed solution. After the dropping is completed, the reaction is continued for 6 h. After the reaction is completed, gradient ethanol aqueous solutions with volume percentages of 0% (water), 30%, 50%, 70%, and 90% are used for washing and centrifugation in sequence to remove the oil phase in the reaction solution, and porous hyaluronic acid sodium microspheres are obtained, which are freeze-dried for preservation.

[0055] Example 2

[0056] The present embodiment provides a method for preparing porous hyaluronic acid sodium microspheres, which comprises the following steps:

[0057] (1) Water phase preparation:

[0058] Take 0.252 g NaHCO3 and 0.024 g NaOH dissolved in 30 mL water, and prepare a basic aqueous solution containing 0.1 mol / L NaHCO3 and 0.02 mol / L NaOH; add 3 g of hyaluronic acid sodium powder with a molecular weight of 20 wDa to the above basic aqueous solution, and mix and stir until homogeneous to obtain a basic aqueous solution containing hyaluronic acid sodium and a chemical foaming agent; add 0.2 mL of BDDE to the basic aqueous solution, and continue to stir and mix uniformly, which is the water phase.

[0059] (2) Oil phase preparation:

[0060] Take 1 g Span-80 dissolved in 199 g liquid paraffin, stirring evenly after the Span-80 mass percentage content of 0.5% oil phase.

[0061] (3) Microsphere preparation:

[0062] The water phase obtained in step (1) is added dropwise into the oil phase obtained in step (2) at a stirring speed of 700 rpm, and the crosslinking reaction is carried out at 15°C for 72 h. 5 mL of hydrochloric acid with a concentration of 0.5 mol / L is added in an acid burette, and the dropping speed is controlled to be 2 min / drop (about 1.25 x 10 -5 mol / min). The hydrochloric acid is added dropwise into the crosslinked mixed solution, and after the addition is completed, the reaction is continued at 15°C for 2 h. After the reaction is completed, the oil phase in the reaction solution is removed by washing and centrifuging with gradient ethanol aqueous solution with a volume percentage of 0% (water), 40%, 60%, and 80% successively, to obtain porous sodium hyaluronate microspheres, which are freeze-dried for preservation.

[0063] Example 3

[0064] The present embodiment provides a method for preparing porous sodium hyaluronate microspheres, comprising the following steps:

[0065] (1) Preparation of water phase:

[0066] 7.56 g of NaHCO3 and 0.12 g of NaOH are dissolved in 30 mL of water to prepare an alkaline aqueous solution containing 3 mol / L of NaHCO3 and 0.1 mol / L of NaOH; 0.3 g of sodium hyaluronate powder with a molecular weight of 250 wDa is added to the above-mentioned alkaline aqueous solution, and mixed and stirred until homogeneous to obtain an alkaline aqueous solution containing sodium hyaluronate and a chemical foaming agent; 1.5 mL of BDDE is added to the alkaline aqueous solution, and the stirring and mixing are continued until uniform, to obtain the water phase.

[0067] (2) Preparation of oil phase:

[0068] 4 g of Span-80 is dissolved in 196 g of liquid paraffin, and after stirring evenly, the Span-80 mass percentage content of 2% oil phase is obtained.

[0069] (3) Microsphere preparation:

[0070] The water phase obtained in step (1) is added dropwise into the oil phase obtained in step (2) at a stirring speed of 100 rpm, and the crosslinking reaction is carried out at 40°C for 30 h. 10 mL of hydrochloric acid with a concentration of 0.5 mol / L is added in an acid burette, and the dropping speed is controlled to be 30 seconds / drop (about 5 x 10 -4mol / min) to the crosslinked mixed solution, and after the dropping was completed, the reaction was continued at 40°C for 8 h. After the reaction was completed, the porous sodium hyaluronate microspheres were obtained by sequentially using gradient ethanol aqueous solutions with volume percentage contents of 0% (water), 20%, 40%, 60%, and 80% to centrifugally wash and remove the oil phase in the reaction solution, and were freeze-dried for preservation.

[0071] Example 4

[0072] The present example provides a method for preparing porous sodium hyaluronate microspheres, comprising the following steps:

[0073] (1) Preparation of the water phase:

[0074] 2.34 g of NH4CO3 and 0.15 g of NaOH were dissolved in 30 mL of water to prepare an alkaline aqueous solution containing 1 mol / L of NH4CO3 and 0.125 mol / L of NaOH; 1.2 g of sodium hyaluronate powder with a molecular weight of 163 wDa was added to the above alkaline aqueous solution, and the mixture was stirred until homogeneous to obtain an alkaline aqueous solution containing sodium hyaluronate and a chemical foaming agent; 0.75 mL of BDDE was added to the alkaline aqueous solution, and the mixture was continuously stirred until homogeneous, thereby obtaining the water phase.

[0075] (2) Preparation of the oil phase:

[0076] 2 g of Span-80 was dissolved in 198 g of liquid paraffin, and after the mixture was stirred until homogeneous, an oil phase with a Span-80 concentration of 1% was obtained.

[0077] (3) Preparation of the microspheres:

[0078] The water phase obtained in step (1) was added dropwise to the oil phase obtained in step (2) using a syringe under stirring at a speed of 320 rpm, and the crosslinking reaction was performed at 25°C for 24 h. The crosslinked mixed solution was heated, and the temperature was controlled at 60°C, and the reaction was continued for 6 h. After the reaction was completed, the porous sodium hyaluronate microspheres were obtained by sequentially using gradient ethanol aqueous solutions with volume percentage contents of 0% (water), 30%, 50%, 70%, and 90% to centrifugally wash and remove the oil phase in the reaction solution, and were freeze-dried for preservation.

[0079] Example 5

[0080] The present example provides a method for preparing porous sodium hyaluronate microspheres, comprising the following steps:

[0081] (1) Preparation of the water phase:

[0082] Take 0.252 g NaHCO3 and 0.024 g NaOH dissolved in 30 mL water, and prepare a basic aqueous solution containing 1 mol / L NaHCO3 and 0.02 mol / L NaOH; add 3 g of hyaluronic acid sodium powder with a molecular weight of 20 wDa to the above basic aqueous solution, and mix and stir until homogeneous to obtain a basic aqueous solution containing hyaluronic acid sodium and a chemical foaming agent; add 0.2 mL of BDDE to the basic aqueous solution, and continue to stir and mix uniformly, which is the water phase.

[0083] (2) Oil phase preparation:

[0084] Take 1 g Span-80 and dissolve it in 199 g liquid paraffin, and stir uniformly to obtain an oil phase with a Span-80 mass percentage of 0.5%.

[0085] (3) Microsphere preparation:

[0086] Under a stirring speed of 700 rpm, the water phase obtained in step (1) is added dropwise to the oil phase obtained in step (2) by using a syringe, and the crosslinking reaction is carried out at 15°C for 60 h. The crosslinked mixed solution is heated, and the temperature is controlled at 40°C, and the reaction is continued for 8 h. After the reaction is completed, gradient ethanol aqueous solutions with volume percentages of 0% (water), 40%, 60%, and 80% are used for washing and centrifugation in sequence to remove the oil phase in the reaction solution, and porous hyaluronic acid sodium microspheres are obtained, which are freeze-dried and stored.

[0087] Example 6

[0088] The present embodiment provides a method for preparing porous hyaluronic acid sodium microspheres, comprising the following steps:

[0089] (1) Water phase preparation:

[0090] Take 7.56 g NaHCO3 and 0.12 g NaOH dissolved in 30 mL water, and prepare a basic aqueous solution containing 3 mol / L NaHCO3 and 0.1 mol / L NaOH; add 0.5 g of hyaluronic acid sodium powder with a molecular weight of 250 wDa to the above basic aqueous solution, and mix and stir until homogeneous to obtain a basic aqueous solution containing hyaluronic acid sodium and a chemical foaming agent; add 1.5 mL of crosslinking agent BDDE to the basic aqueous solution, and continue to stir and mix uniformly, which is the water phase.

[0091] (2) Oil phase preparation:

[0092] Take 4 g Span-80 and dissolve it in 196 g liquid paraffin, and stir uniformly to obtain an oil phase with a Span-80 mass percentage of 2%.

[0093] (3) Microsphere preparation:

[0094] The water phase obtained in step (1) was added drop by drop into the oil phase obtained in step (2) using a syringe at a stirring speed of 100 rpm, and a cross-linking reaction was carried out at 40°C for 30 h. The cross-linked mixed solution was heated, and the temperature was controlled at 85°C, and the reaction was continued for 2 h. After the reaction was completed, the oil phase in the reaction solution was removed by gradient ethanol aqueous solution with a volume percentage of 0% (water), 20%, 40%, 60%, and 80% for centrifugal cleaning, and porous sodium hyaluronate microspheres were obtained, which were freeze-dried and stored.

[0095] Example 7

[0096] This example provides a method for preparing porous sodium hyaluronate microspheres, which is different from example 1 only in that the cross-linking reaction time in step (3) is adjusted to 30 h, and the other parameters remain the same as in example 1.

[0097] Example 8

[0098] This example provides a method for preparing porous sodium hyaluronate microspheres, which is different from example 1 only in that the cross-linking reaction time in step (3) is adjusted to 24 h, and the other parameters remain the same as in example 1.

[0099] Example 9

[0100] This example provides a method for preparing porous sodium hyaluronate microspheres, which is different from example 1 only in that the cross-linking reaction time in step (3) is adjusted to 10 h, and the other parameters remain the same as in example 1.

[0101] Example 10

[0102] This example provides a method for preparing porous sodium hyaluronate microspheres, which is different from example 1 only in that the dropping speed of hydrochloric acid in step (3) is adjusted to 6 min / drop (about 8.3×10 -6 mol / min), and the other parameters remain the same as in example 1.

[0103] Example 11

[0104] This example provides a method for preparing porous sodium hyaluronate microspheres, which is different from example 1 only in that the dropping speed of hydrochloric acid in step (3) is adjusted to 2 s / drop (about 1.5×10 -3 mol / min), and the other parameters remain the same as in example 1.

[0105] Example 12

[0106] The embodiment provides a preparation method of porous sodium hyaluronate microspheres, which is only different from the embodiment 4 in that the temperature of heating the mixed solution after crosslinking in step (3) is adjusted to 35 DEG C, and the rest parameters are consistent with those of the embodiment 4.

[0107] Example 13

[0108] The embodiment provides a preparation method of porous sodium hyaluronate microspheres, which is only different from the embodiment 4 in that the temperature of heating the mixed solution after crosslinking in step (3) is adjusted to 110 DEG C, and the rest parameters are consistent with those of the embodiment 4.

[0109] Test Example 1

[0110] (1) SEM characterization:

[0111] The structure of the porous sodium hyaluronate microspheres prepared by the embodiment 1 and the embodiment 4 is characterized in the form of morphology, and the results are shown in Figure 1 , Figure 2 respectively. It can be seen that the porous sodium hyaluronate microspheres can be prepared by using the temperature raising method or the acid titration method, the microsphere surface leaves relatively dense and uniform pore structure, and a plurality of visible bubbles exist in the microsphere.

[0112] (2) Pore size characterization:

[0113] The size and pore size of the porous sodium hyaluronate microspheres prepared by the embodiment 1 to the embodiment 13 are characterized, and the results are shown in Table 1. It can be seen that when the crosslinking reaction time is controlled to be 12-72 h, the uniform porous pore size can be formed. When the porous structure of the sodium hyaluronate microspheres is prepared by the acid titration method, the acid dropping speed is controlled to be 1x10 -5 -1x10 -3 mol / min, the porous microspheres with more pores, smaller and uniform porous pore size can be obtained; when the porous structure of the sodium hyaluronate microspheres is formed by the temperature raising method, the temperature is controlled to be 40-100 DEG C, the obtained microspheres can present full spherical shape and have more pore structures and smaller porous pore size.

[0114] Table 1

[0115]

[0116] The applicant declares that the technical scheme of the present application is illustrated by the above embodiments, but the present application is not limited to the above embodiments, that is, it does not mean that the present application must rely on the above embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes and the like all fall within the protection scope and disclosure scope of the present application.

[0117] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details of the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0118] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not describe various possible combinations again.

Claims

1. A method for preparing porous sodium hyaluronate microspheres, characterized in that, The preparation method includes the following steps: (1) Aqueous phase preparation: Sodium hyaluronate is dissolved in an alkaline solution containing a chemical foaming agent to obtain an alkaline aqueous solution containing sodium hyaluronate and a chemical foaming agent, which is then mixed with a crosslinking agent to obtain an aqueous phase; Oil phase preparation: Emulsifier is dissolved in an oil phase matrix to obtain an oil phase; (2) Add the aqueous phase obtained in step (1) to the oil phase to carry out cross-linking reaction, and then carry out foaming reaction by heating method or acid titration method; after the reaction is completed, wash and remove the oil phase by gradient centrifugation method to obtain porous sodium hyaluronate microspheres.

2. The preparation method according to claim 1, characterized in that, The chemical foaming agent in step (1) includes any one or a combination of at least two of sodium carbonate, sodium bicarbonate, or ammonium bicarbonate; Preferably, in the alkaline solution containing the chemical foaming agent in step (1), the concentration of the chemical foaming agent is 0.1-3 mol / L, and the concentration of the alkali is 0.02-0.25 mol / L; Preferably, the alkaline solution containing a chemical foaming agent includes a sodium hydroxide solution containing a chemical foaming agent.

3. The preparation method according to claim 1 or 2, characterized in that, In step (1), the alkaline solution containing sodium hyaluronate and a chemical foaming agent has a sodium hyaluronate content of 1-15% by mass. Preferably, the molecular weight of the sodium hyaluronate is 20-250 wDa.

4. The preparation method according to any one of claims 1-3, characterized in that, The crosslinking agent in step (1) includes any one or a combination of at least two of 1,4-butanediol glycidyl ether, divinyl sulfone, or polyethylene glycol diglycidyl ether; Preferably, the mass percentage of the crosslinking agent in the aqueous phase is 0.5-5%; Preferably, the emulsifier in step (1) includes Tween and / or Span; Preferably, the oil phase matrix in step (1) includes any one or a combination of at least two of liquid paraffin, vegetable oil or silicone oil.

5. The preparation method according to any one of claims 1-4, characterized in that, Step (2) in which the aqueous phase is added to the oil phase also includes stirring, wherein the stirring speed is 100-700 rpm; Preferably, the crosslinking reaction is carried out at a temperature of 15-40°C for 12-72 hours. Preferably, the foaming reaction takes 1-8 hours.

6. The preparation method according to any one of claims 1-5, characterized in that, Step (2) involves heating the foaming reaction to 40-100℃ for 1-8 hours.

7. The preparation method according to any one of claims 1-5, characterized in that, In step (2), the concentration of the acidic solution during the foaming reaction using acid titration is 0.2-5 mol / L, and the dropping rate is 1×10⁻⁶. -5 -1×10 -3 mol / min; Preferably, the acidic solution comprises an aqueous solution of hydrochloric acid and / or acetic acid; Preferably, the temperature for the foaming reaction using acid titration is 15-40℃, and the time is 1-8 h.

8. The preparation method according to any one of claims 1-7, characterized in that, Step (2) The cleaning agent used to remove the oil phase by gradient centrifugation is an aqueous solution of water and / or ethanol; Preferably, step (2) gradient centrifugation cleaning to remove the oil phase includes at least four gradients, with the cleaning agents being water, an aqueous solution of ethanol with a volume percentage of 20-50%, an aqueous solution of ethanol with a volume percentage of 50-70%, and an aqueous solution of ethanol with a volume percentage of 70-90%.

9. Porous sodium hyaluronate microspheres prepared by the method described in any one of claims 1-8.

10. The use of the porous sodium hyaluronate microspheres according to claim 9 in the preparation of tissue engineering scaffolds, drug sustained-release carriers or injectable fillers.

Images