Preparation method and application of glucan microspheres with high crosslinking degree

Abstract

The invention discloses a preparation method of high-crosslinking-degree glucan microspheres. The preparation method comprises the following steps: S1, preparing primary crosslinking glucan microspheres; and S2, carrying out secondary chemical crosslinking to obtain the high-crosslinking-degree glucan microspheres. According to the preparation method disclosed by the invention, the primary cross-linked dextran microspheres with low cross-linking degree are firstly prepared and then are subjected to secondary cross-linking, so that the cross-linking degree of the dextran microspheres is improved, the mechanical property and the stability of the dextran microspheres are improved, the cross-linking degree of the pre-cross-linked dextran microspheres and the process conditions of secondary cross-linking can be regulated and controlled according to actual requirements, and the preparation method is suitable for industrial production. According to the preparation method, non-toxic liquid paraffin is adopted as an oil phase, organic solvents which are high in toxicity and dangerous in production are not used, the difficulty of large-scale production can be greatly reduced, and the microspheres which are high in crosslinking degree, smooth and round, low in crosslinking agent residue, qualified in cytotoxicity and good in biocompatibility are obtained.

Description

Technical Field

[0001] The invention relates to A61K and the field of drug preparations, and in particular to a preparation method and application of highly cross-linked dextran microspheres. Background Art

[0002] After cross-linking, dextran has good water absorption, water insolubility and water swelling, and is widely used in the field of biomedicine. However, the existing dextran microspheres have low strength. In order to improve the cross-linking degree of dextran microspheres, the method of increasing the feed amount and raising the cross-linking temperature is usually adopted, but this will bring the problem of easy agglomeration of dextran gel microspheres. In order to reduce the agglomeration of microspheres, it is necessary to increase the continuous phase, and the viscosity of the continuous phase is large, which will cause the problem of subsequent cleaning difficulties, impurities, and increased by-product content. Therefore, it is very important to develop a method for high-cross-linked dextran gel microspheres with low impurity content and round and uniform particle size.

[0003] Chinese invention patent CN201811523471.5 discloses a method for improving the crosslinking degree of crosslinked dextran gel microspheres, which involves soaking the crosslinked dextran gel microspheres in an alkaline solution to carry a sufficient amount of alkaline substances, dispersing them in the continuous phase and encountering a crosslinking agent, and then undergoing an intramolecular crosslinking reaction again, thereby increasing the crosslinking degree; filtering out excess alkaline solution after soaking can avoid agglomeration caused by the increase in the volume of the dispersed phase, and avoid the undesirable condition of adhesion caused by secondary crosslinking. However, it involves secondary emulsification, which is a cumbersome process, and the crosslinking agent uses n-octane, which involves the problem of low safety of residual crosslinking agent. Chinese invention patent CN202010831711.9 discloses a method for preparing polymer microspheres for protein separation and analysis based on a core-shell structure and its application. Highly cross-linked polymer microspheres are prepared by surface graft copolymerization or surface-initiated active polymerization and by surface epoxy ring-opening bonding or substitution reaction of halogen-containing functional groups. The prepared core-shell structured polymer microspheres have uniform particle size, are resistant to high pressure, and are resistant to acids and alkalis. However, they use highly toxic and flammable benzene organic solvents, and their safety of use is poor. Summary of the invention

[0004] In order to develop a method for high cross-linked dextran gel microspheres with low impurity content and round and uniform particle size, the present invention first prepares primary cross-linked dextran microspheres with low cross-linking degree, and then performs secondary cross-linking on them, so that the obtained dextran microspheres have high cross-linking degree. The first aspect of the present invention provides a method for preparing high cross-linked dextran microspheres, comprising the following steps:

[0005] S1: dissolving dextran in a first alkaline solution, then adding a first cross-linking agent and mixing evenly to obtain a dispersed phase for use; emulsifying a surfactant and an oil phase to obtain a continuous phase for use; mixing the dispersed phase with the continuous phase, performing a chemical cross-linking, and washing to obtain a cross-linked dextran microsphere;

[0006] S2: adding the once cross-linked dextran microspheres into a second alkaline solution containing a second cross-linking agent, stirring to perform secondary chemical cross-linking, and washing and removing impurities to obtain highly cross-linked dextran microspheres.

[0007] As a preferred embodiment, the method for preparing the highly cross-linked dextran microspheres specifically comprises the following steps:

[0008] S1 Preparation of primary cross-linked dextran microspheres:

[0009] S1.1 Dissolve dextran in the first alkaline solution, stir until the dextran is completely dissolved, then add the first cross-linking agent, stir at 200-400 rpm for 10-30 min at room temperature to obtain a dispersed phase for use;

[0010] S1.2 Add the surfactant to the oil phase and mix to obtain a continuous phase for later use;

[0011] S1.3 Add the dispersed phase into the continuous phase for emulsification at a temperature of 25-50°C for 10-30 minutes;

[0012] S1.4 After emulsification, cross-linking is performed;

[0013] S1.5 After cross-linking, wash, cool to room temperature, stand for 5-15 minutes, remove the upper oil phase, add ethanol aqueous solution containing Tween to the remaining material, stir well for 5-15 minutes, stand for 5-15 minutes, remove the upper oil phase, add water for multiple washing, solid-liquid separation, and obtain primary cross-linked dextran microspheres;

[0014] S2 Preparation of highly cross-linked dextran microspheres:

[0015] S2.1 adding the primary cross-linked dextran microspheres into the second alkaline solution for alkaline pretreatment at a temperature of 60-80°C and 200-400rpm for 20-40min;

[0016] S2.2 After the alkali pretreatment, a second crosslinking agent is added and stirred for secondary chemical crosslinking;

[0017] S2.3 After the reaction is completed, cool to room temperature and stand for 5-15 minutes, remove the supernatant alkaline solution, rinse the dextran microspheres with running water, separate the solid and liquid, and obtain highly cross-linked dextran microspheres;

[0018] S2.4 After cleaning, add ethanol aqueous solution to the highly cross-linked dextran microspheres, completely immerse the microspheres, stir thoroughly for 20-40 minutes, separate the solid and liquid, repeat 3-5 times, then add purified water, completely immerse the microspheres, stir thoroughly for 20-40 minutes, separate the solid and liquid, repeat 3-5 times, and obtain.

[0019] As a preferred embodiment, the Tween is Tween 80, the mass concentration of Tween 80 is 0.5%, and the mass concentration of the ethanol aqueous solution is 50%.

[0020] As a preferred embodiment, the temperature of the first chemical cross-linking in step S1 is 25-70°C, and the reaction time is 10-20h; the temperature of the second chemical cross-linking in step S2 is 25-70°C, and the reaction time is 20-30h.

[0021] As a preferred embodiment, the temperature of the first chemical cross-linking in step S1 is 60-70°C, and the reaction time is 13-18h; the temperature of the second chemical cross-linking in step S2 is 60-70°C, and the reaction time is 20-25h.

[0022] As a preferred embodiment, the temperature of the first chemical cross-linking in step S1 is 70° C., and the reaction time is 15 h; the temperature of the second chemical cross-linking in step S2 is 70° C., and the reaction time is 24 h.

[0023] As a preferred embodiment, in step S2, the mass ratio of the primary cross-linked dextran microspheres to the second alkaline solution is 1:(1-5).

[0024] As a preferred embodiment, in step S2, the mass ratio of the primary cross-linked dextran microspheres to the second alkaline solution is 1:(2-4).

[0025] As a preferred embodiment, in step S2, the mass ratio of the primary cross-linked dextran microspheres to the second alkaline solution is 1:3.

[0026] As a preferred embodiment, the mass ratio of dextran to the first cross-linking agent in step S1 is 1:(0.5-1), and the mass ratio of the primary cross-linked dextran microspheres to the second cross-linking agent in step S2 is 1:(0.5-1).

[0027] As a preferred embodiment, the mass ratio of dextran to the first cross-linking agent in step S1 is 1:(0.8-1), and the mass ratio of the primary cross-linked dextran microspheres to the second cross-linking agent in step S2 is 1:(0.5-0.8).

[0028] As a preferred embodiment, the mass ratio of dextran to the first cross-linking agent in step S1 is 1:1, and the mass ratio of the primary cross-linked dextran microspheres to the second cross-linking agent in step S2 is 1:0.5.

[0029] As a preferred embodiment, the molar concentration of the first alkaline solution is 0.2-0.5M, and the molar concentration of the second alkaline solution is 0.2-0.5M.

[0030] As a preferred embodiment, the molar concentration of the first alkaline solution is 0.5M, and the molar concentration of the second alkaline solution is 0.5M.

[0031] As a preferred embodiment, the volume ratio of the dispersed phase to the continuous phase in step S1 is 1:(5-10).

[0032] As a preferred embodiment, in step S1, the volume ratio of the dispersed phase to the continuous phase is 1:5.

[0033] During the experiment, the applicant found that by using 1,4-butanediol diglycidyl ether as a cross-linking agent for dextran gel microspheres and performing secondary cross-linking on the dextran microspheres, dextran gel microspheres with a high degree of cross-linking can be obtained, the degradation performance of the gel microspheres can be reduced, and the enzyme resistance activity can be improved. The possible reason is speculated to be: in order to increase the degree of cross-linking, the existing gel microspheres need to increase the amount of raw materials fed, or increase the reaction temperature, but the increase in the amount of raw materials fed, the viscosity increases, and the subsequent difficulty of cleaning and removing impurities from the gel microspheres increases. If the reaction is not complete, the residual impurities may generate by-products or affect the safety of use. The present application uses 1,4-butanediol diglycidyl ether as a cross-linking agent to perform secondary cross-linking on the pre-cross-linked gel microspheres. On the basis of not changing the single amount of raw materials fed, the degree of cross-linking of the gel microspheres is increased, and the reaction is promoted to be complete, avoiding the generation of by-products.

[0034] The applicant further discovered that using a low-concentration alkaline solution as the dispersed phase can reduce the collision and agglomeration of the gel microspheres and avoid the formation of irregular gels. The reason is that the contact resistance of the low-concentration dispersed phase in the continuous phase increases, reducing the probability of contact between the gel microspheres, thereby forming gel microspheres with uniform particle size.

[0035] As a preferred embodiment, after the dispersed phase and the continuous phase are mixed in step S1, they are first emulsified and then cross-linked. The emulsification temperature is 25-50° C. and the emulsification time is 10-30 min.

[0036] As a preferred embodiment, after the dispersed phase and the continuous phase are mixed in step S1, they are first emulsified and then cross-linked. The emulsification temperature is 25° C. and the emulsification time is 20 min.

[0037] As a preferred embodiment, the first cross-linking agent and the second cross-linking agent are both selected from at least one of epichlorohydrin, 1,4-butanediol diglycidyl ether, and divinyl sulfone.

[0038] As a preferred embodiment, the first cross-linking agent and the second cross-linking agent are both 1,4-butanediol diglycidyl ether.

[0039] As a preferred embodiment, the first alkaline solution and the second alkaline solution are both aqueous sodium hydroxide solutions.

[0040] As a preferred embodiment, in step S1, the mass fraction of the surfactant in the oil phase is 3-6%.

[0041] As a preferred embodiment, the surfactant is Span 80, and the oil phase is liquid paraffin.

[0042] The second aspect of the present invention provides an application of a method for preparing highly cross-linked dextran microspheres, which is applied to the preparation of highly cross-linked dextran microspheres, wherein the water absorption of the highly cross-linked dextran microspheres is 1-2%, and the degradation time of the highly cross-linked dextran microspheres under the action of dextranase is greater than 25 days.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] (1) The method for preparing highly cross-linked dextran microspheres of the present invention first prepares primary cross-linked dextran microspheres with a low degree of cross-linking, and then performs secondary cross-linking on the microspheres, thereby increasing the degree of cross-linking of the dextran microspheres, improving the mechanical properties and stability of the dextran microspheres, and adjusting the degree of cross-linking of the pre-cross-linked dextran microspheres and the process conditions of the secondary cross-linking according to actual needs to obtain dextran microspheres with different degrees of cross-linking.

[0045] (2) The method for preparing highly cross-linked dextran microspheres of the present invention uses a low-concentration alkaline solution as the dispersed phase, which can reduce the collision and agglomeration of the gel microspheres and avoid the formation of irregular gels.

[0046] (3) The method for preparing highly cross-linked dextran microspheres of the present invention uses a low concentration of alkaline solution during the secondary cross-linking. Under the catalysis of the alkaline substance, the dextran microspheres will undergo intermolecular cross-linking reaction again, thereby increasing the degree of cross-linking, avoiding the re-emulsification process, simplifying the production process, and effectively reducing the residue of the cross-linking agent, thereby improving biosafety and biocompatibility.

[0047] (4) The method for preparing highly cross-linked dextran microspheres of the present invention uses non-toxic liquid paraffin as the oil phase and does not use highly toxic and dangerous organic solvents, which can greatly reduce the difficulty of large-scale production and obtain microspheres with high cross-linking degree, smooth and round shape, low cross-linking agent residue, qualified cytotoxicity and good biocompatibility.

[0048] (5) The method for preparing highly cross-linked dextran microspheres of the present invention also includes multiple washing and impurity removal, which improves the purity and quality of the dextran microspheres, maintains the integrity of the microstructure of the dextran microspheres, and makes them smooth and round. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a microscopic morphology of the dextran microspheres prepared after step S1 in Example 1;

[0050] Figure 2 This is a microscopic morphology of the dextran microspheres prepared after step S2 in Example 1;

[0051] Figure 3 This is a line graph of the in vitro degradation of the dextran microspheres prepared in Example 1;

[0052] Figure 4 This is a microscopic morphology of the dextran microspheres prepared after step S1 in Example 2;

[0053] Figure 5 This is a microscopic morphology of the dextran microspheres prepared after step S2 in Example 2;

[0054] Figure 6 This is a line graph of the in vitro degradation of the dextran microspheres prepared in Example 2;

[0055] Figure 7 This is a microscopic morphology of the dextran microspheres prepared in Comparative Example 1;

[0056] Figure 8 This is a microscopic morphology of the dextran microspheres prepared in Comparative Example 2;

[0057] Fig. 9 This is a microscope morphology of the dextran microspheres prepared in Comparative Example 3. DETAILED DESCRIPTION

[0058] Example 1

[0059] A method for preparing highly cross-linked dextran microspheres, comprising the following steps:

[0060] S1 Preparation of primary cross-linked dextran microspheres:

[0061] S1.1 Dissolve 50 g of dextran in 100 g of the first alkaline solution, stir until the dextran is completely dissolved, then add the first cross-linking agent, stir at 300 rpm for 20 min at room temperature to obtain a dispersed phase for use;

[0062] S1.2 Add 30g of surfactant into 500g of oil phase and mix to obtain a continuous phase for later use;

[0063] S1.3 Add the dispersed phase into the continuous phase for emulsification at 25°C and 300 rpm for 20 min;

[0064] S1.4 After emulsification, cross-linking was performed at a temperature of 70°C and a reaction speed of 300 rpm for 15 h;

[0065] S1.5 After cross-linking, wash, cool to room temperature, stand for 10 minutes, remove the upper oil phase, add 500mL of 50wt% ethanol aqueous solution containing 0.5wt% Tween 80 to the remaining material, stir well for 10 minutes, stand for 10 minutes, remove the upper oil phase, add water for multiple washings, separate the solid and liquid, and obtain the primary cross-linked dextran microspheres;

[0066] S2 Preparation of highly cross-linked dextran microspheres:

[0067] S2.1 Add 50g of the primary cross-linked dextran microspheres into 150g of the second alkaline solution for alkaline pretreatment at a temperature of 70°C and 300rpm for 30min;

[0068] S2.2 After alkali pretreatment, 45 g of the second crosslinking agent was added and stirred for secondary chemical crosslinking. The crosslinking temperature was 70°C and the reaction was carried out at 300 rpm for 24 h.

[0069] S2.3 After the reaction is completed, cool to room temperature and stand for 10 minutes, remove the supernatant alkaline solution, rinse the dextran microspheres with running water, separate the solid and liquid, and obtain highly cross-linked dextran microspheres;

[0070] S2.4 After cleaning, add 1L of ethanol aqueous solution to the highly cross-linked dextran microspheres, completely immerse the microspheres, stir thoroughly for 30 minutes, separate the solid and liquid, repeat 3 times, then add 1L of purified water, completely immerse the microspheres, stir thoroughly for 30 minutes, separate the solid and liquid, repeat 3 times, and you have it.

[0071] In the step S2.1, the mass ratio of the primary cross-linked dextran microspheres to the second alkaline solution is 1:3.

[0072] The mass ratio of dextran to the first cross-linking agent in the step S1.1 is 5:3, and the mass ratio of the primary cross-linked dextran microspheres to the second cross-linking agent in the step S2.2 is 1:45 g.

[0073] The molar concentration of the first alkaline solution is 0.2 M, and the molar concentration of the second alkaline solution is 0.5 M. Both the first alkaline solution and the second alkaline solution are aqueous sodium hydroxide solutions.

[0074] The volume ratio of the dispersed phase to the continuous phase in step S1.3 is 1:5.

[0075] The first cross-linking agent and the second cross-linking agent are both 1,4-butanediol diglycidyl ether.

[0076] The surfactant is Span 80, and the oil phase is liquid paraffin purchased from GENERAL-REAGENT, with a content of >99 wt%.

[0077] The dextran was purchased from Macklin, with an average molecular weight of 70,000 Da.

[0078] Example 2

[0079] A method for preparing highly cross-linked dextran microspheres, comprising the following steps:

[0080] S1 Preparation of primary cross-linked dextran microspheres:

[0081] S1.1 Dissolve 50 g of dextran in 100 g of the first alkaline solution, stir until the dextran is completely dissolved, then add the first cross-linking agent, stir at 300 rpm for 20 min at room temperature to obtain a dispersed phase for use;

[0082] S1.2 Add 60g of surfactant into 1000g of oil phase and mix to obtain a continuous phase for later use;

[0083] S1.3 Add the dispersed phase into the continuous phase for emulsification at 25°C and 300 rpm for 20 min;

[0084] S1.4 After emulsification, cross-linking was performed at a temperature of 70°C and a reaction speed of 300 rpm for 15 h;

[0085] S1.5 After cross-linking, wash, cool to room temperature, stand for 10 minutes, remove the upper oil phase, add 500mL of 50wt% ethanol aqueous solution containing 0.5wt% Tween 80 to the remaining material, stir well for 10 minutes, stand for 10 minutes, remove the upper oil phase, add water for multiple washings, separate the solid and liquid, and obtain the primary cross-linked dextran microspheres;

[0086] S2 Preparation of highly cross-linked dextran microspheres:

[0087] S2.1 Add the primary cross-linked dextran microspheres into 150 g of the second alkaline solution for alkaline pretreatment at 70°C and 300 rpm for 30 min;

[0088] S2.2 After alkali pretreatment, add 25g of the second crosslinking agent and stir to perform secondary chemical crosslinking. The crosslinking temperature is 70°C and the reaction is carried out at 300rpm for 24h.

[0089] S2.3 After the reaction is completed, cool to room temperature and stand for 10 minutes, remove the supernatant alkaline solution, rinse the dextran microspheres with running water, separate the solid and liquid, and obtain highly cross-linked dextran microspheres;

[0090] S2.4 After cleaning, add 1L of ethanol aqueous solution to the highly cross-linked dextran microspheres, completely immerse the microspheres, stir thoroughly for 30 minutes, separate the solid and liquid, repeat 3 times, then add 1L of purified water, completely immerse the microspheres, stir thoroughly for 30 minutes, separate the solid and liquid, repeat 3 times, and you have it.

[0091] In the step S2.1, the mass ratio of the primary cross-linked dextran microspheres to the second alkaline solution is 1:3.

[0092] The mass ratio of dextran to the first cross-linking agent in the step S1.1 is 1:1, and the mass ratio of the primary cross-linked dextran microspheres to the second cross-linking agent in the step S2.2 is 1:0.5.

[0093] The molar concentration of the first alkaline solution is 0.5 M, and the molar concentration of the second alkaline solution is 0.5 M. Both the first alkaline solution and the second alkaline solution are aqueous sodium hydroxide solutions.

[0094] The volume ratio of the dispersed phase to the continuous phase in step S1.3 is 1:5.

[0095] The first cross-linking agent and the second cross-linking agent are both 1,4-butanediol diglycidyl ether.

[0096] The surfactant is Span 80, and the oil phase is liquid paraffin purchased from GENERAL-REAGENT, with a content of >99 wt%.

[0097] The dextran was purchased from Macklin, with an average molecular weight of 70,000 Da.

[0098] Comparative Example 1

[0099] A method for preparing highly cross-linked dextran microspheres, the specific implementation method is the same as that of Example 1, except that the molar concentration of the second alkaline solution is 3M.

[0100] Comparative Example 2

[0101] A method for preparing highly cross-linked dextran microspheres, the specific implementation method is the same as Example 1, the difference is that in step S1.1, the mass ratio of dextran to the first cross-linking agent is 4:1, the molar concentration of the first alkaline solution is 0.5M, and step S2 is not included.

[0102] Comparative Example 3

[0103] A method for preparing highly cross-linked dextran microspheres, comprising the following steps:

[0104] S1 Preparation of primary cross-linked dextran microspheres:

[0105] S1.1 Dissolve 50 g of dextran in 100 g of the first alkaline solution, stir until the dextran is completely dissolved, and stir at 300 rpm for 20 min at room temperature to obtain a dispersed phase for use;

[0106] S1.2 Add 30g of surfactant into 500g of oil phase and mix to obtain a continuous phase for later use;

[0107] S1.3 Add the dispersed phase into the continuous phase for emulsification at 25°C and 300 rpm for 20 min;

[0108] S1.4 After emulsification, add 75g of cross-linking agent for cross-linking at a temperature of 70°C and react at 300rpm for 15h;

[0109] S1.5 was washed after cross-linking, cooled to room temperature, allowed to stand for 10 minutes, the upper oil phase was removed, 500 mL of 50 wt% ethanol aqueous solution containing 0.5 wt% Tween 80 was added to the remaining material, stirred thoroughly for 10 minutes, allowed to stand for 10 minutes, the upper oil phase was removed, water was added for multiple washings, and solid-liquid separation was performed to obtain dextran microspheres.

[0110] The molar concentration of the first alkaline solution is 0.2M;

[0111] The volume ratio of the dispersed phase to the continuous phase in step S1.3 is 1:5.

[0112] The cross-linking agent is 1,4-butanediol diglycidyl ether.

[0113] The surfactant is Span 80, and the oil phase is liquid paraffin.

[0114] Comparative Example 4

[0115] A method for preparing highly cross-linked dextran microspheres, comprising the following steps:

[0116] S1 Preparation of primary cross-linked dextran microspheres:

[0117] S1.1 Dissolve 50 g of dextran in 100 g of the first alkaline solution, stir until the dextran is completely dissolved, add 75 g of a cross-linking agent, and stir at 300 rpm for 20 min at room temperature.

[0118] The molar concentration of the first alkaline solution is 0.2M;

[0119] The cross-linking agent is 1,4-butanediol diglycidyl ether.

[0120] Result: Gel has formed in the system and subsequent emulsification and cross-linking reaction cannot occur. Excessive cross-linking agent cannot be pre-mixed into dispersed phase.

[0121] Performance Testing

[0122] 1. Light microscopy test

[0123] The morphology of the dextran microspheres prepared in Examples 1-2 and Comparative Examples 1-3 was observed using a WD300-48LT biological microscope.

[0124] like Figure 1 , Figure 2 As shown, Figure 1 This is a photo of the cross-linked dextran microspheres of Example 1 under a microscope. Figure 2 This is a photo of the highly cross-linked dextran microspheres obtained after secondary cross-linking in Example 1 under a microscope; Figure 4 This is a photo of the primary cross-linked dextran microspheres of Example 2 under a microscope. Figure 5 This is a photo of the highly cross-linked dextran microspheres obtained after secondary cross-linking in Example 2 under a microscope; Figure 7 This is a photo of the dextran microspheres prepared in Example 1 under a microscope; as shown in the figure: the once cross-linked dextran microspheres will agglomerate when undergoing secondary cross-linking in a high-concentration alkaline solution. This is because the pre-cross-linking degree of the pre-cross-linked dextran microspheres is too low, and the cross-linking reaction occurs rapidly in the high-concentration alkaline solution, and they are ultimately prone to agglomeration. Figure 8 This is a photo of the dextran microspheres prepared in comparative example 2 under a microscope; as shown in the figure: the product obtained in comparative example 2 includes agglomerates, irregular gels formed after incomplete emulsification, and microsphere particles. This is because the concentration of sodium hydroxide in the aqueous phase is high, the cross-linking reaction between the dextran microemulsions is rapid, and at the same time, there is too little continuous phase in the reaction system, and the collision probability of the dextran microemulsions is too high, resulting in unsatisfactory emulsification effect, and finally forming a variety of irregular gels. Fig. 9 This is a photo of the dextran microspheres prepared in Comparative Example 3 under a microscope, as shown in the figure: the product obtained in Comparative Example 3 is mainly a gel block with almost no microspheres. This is because the amount of cross-linking agent added is too high, the cross-linking reaction between the dextran microemulsions is rapid, and at the same time, there is too little continuous phase in the reaction system, and the collision probability of the dextran microemulsions is too high, resulting in the final formation of gel blocks.

[0125] 2. Cytotoxicity

[0126] The test was performed according to the MTT method in reference standard GB / T 16886.5-2017. Four concentrations (100%, 75%, 50%, 25%) of test sample extract, blank, 100% negative control and positive control were prepared with MEM medium (containing 10% fetal bovine serum), extracted for 24 hours, and the extraction ratio was 0.2g:1mL. Under the conditions of 37°C and 5vol% CO2, semi-confluent monolayer cells of L-929 (NIH3T3) mouse fibroblasts were cultured on 96-well plates with test sample extract, blank and the other two controls. After 24±2 hours, MTT colorimetric determination was performed, and the cell survival rate was calculated by reading on a microplate reader at 570nm and 650nm.

[0127] The cell survival rate was calculated according to the following formula: Survival rate % = R / R0×100

[0128] R: average optical density of the test group, positive control group, and negative control group;

[0129] Ro: average optical density of the blank group;

[0130] The test results were all qualified (all > 70%).

[0131] The test results of Example 1 and Example 2 are shown in Table 1.

[0132] Table 1

[0133] Sample name Example 1 Example 2 Culture conditions 120rpm for three days 120rpm for three days 100% extraction 85.88％ 93.52％ 75% extraction 97.73％ 95.88％ 50% extraction 95.40％ 97.24％ 25% extraction 104.90％ 98.39％ Positive Control 2.69％ 7.70％ Negative control 94.29％ 102.24％ Test Results qualified qualified

[0134] 3. Determination of equilibrium water content

[0135] The dextran microspheres that have reached water absorption equilibrium are evacuated with a vacuum pump to remove surface moisture, and placed in an empty watch glass weighing m0. The total mass of the wet bulb and the empty watch glass is weighed as m1. The dextran microsphere sample is dried in a 60°C oven to constant weight. Finally, the total mass of the dry bulb and the empty watch glass is weighed as m2. The equilibrium water content of the dextran microspheres is calculated as X = (m1-m2) / (m2-m0). The lower the equilibrium water content of the microspheres, the higher the degree of cross-linking of the microspheres.

[0136] The test results of Example 1 and Example 2 are shown in Table 2.

[0137] Table 2

[0138]

[0139] 4. In vitro accelerated degradation test

[0140] First, prepare a dextranase / PBS solution with a concentration of 0.05g / mL (1250-1285U / mL) and a pH of 5.0. Preparation method of dextranase solution: weigh 40g of dextranase stock solution, add 800mL of pH 5.0 PBS phosphate buffer to prepare a 0.05g / mL pH 5.0 dextranase / PBS solution. The preparation method of pH 5.0 PBS phosphate buffer is as follows: weigh 8g of sodium chloride, 0.2g of potassium chloride, 0.2g of potassium dihydrogen phosphate, 2.9g of 12-hydrated sodium dihydrogen phosphate, add 4L of purified water, and after complete dissolution, add orthophosphoric acid to adjust the pH value of the solution to 5.0.

[0141] The dextran microspheres that have reached water absorption equilibrium are vacuumed with a vacuum pump to remove surface moisture to obtain wet microspheres. 1 g of wet microsphere sample is weighed and transferred to a 15 mL plastic test tube. 5 mL of 0.05 g / mL pH=5.0 dextranase solution is added and the sample volume V0 is recorded. The microspheres are placed in a 37°C water bath shaker for degradation. The shaker speed is set to 40 rpm. The supernatant is replaced every 1 day. The degradation of the microspheres is observed and the sample volume V is recorded. n . The calculation formula of microsphere degradation degree is as follows (V n-V0) / V0.

[0142] The test results of Example 1 are shown in Figure 3 The test results of Example 2 are shown in Figure 5 In the figure, A embodiment 1 is a single cross-linked dextran microsphere, B embodiment 1 is a double cross-linked dextran microsphere, C embodiment 2 is a single cross-linked dextran microsphere, and D embodiment 2 is a double cross-linked dextran microsphere.

[0143] After testing, the equilibrium water content of the once-crosslinked dextran microspheres of Example 1 was 3.02. Based on the in vitro degradation, it was shown that the once-crosslinked dextran microspheres had a low degree of crosslinking and a loose crosslinking structure, and degraded faster in vitro under the action of dextranase.

[0144] The equilibrium water content of the secondary cross-linked dextran microspheres is 1.83, which is significantly lower than that of the single cross-linked dextran microspheres. Combined with the in vitro degradation, it is shown that the dextran microspheres obtained after secondary cross-linking have a higher degree of cross-linking, and their in vitro degradation is significantly slower, and the enzyme resistance of the secondary cross-linked dextran microspheres is greatly improved.

[0145] The equilibrium water content of the once-cross-linked dextran microspheres in Example 2 is 2.34. The comprehensive degradation in vitro indicates that the once-cross-linked dextran microspheres have a low degree of cross-linking and a loose cross-linking structure. Under the action of dextranase, they degrade faster in vitro.

[0146] The equilibrium water content of the secondary cross-linked dextran microspheres is 1.73, which is significantly lower than that of the pre-cross-linked dextran microspheres. Combined with the in vitro degradation, it is shown that the dextran microspheres obtained after secondary cross-linking have a higher degree of cross-linking, and their in vitro degradation is significantly slower, and the enzyme resistance of the secondary cross-linked dextran microspheres is greatly improved.

Claims

1. A method for preparing highly cross-linked dextran microspheres, characterized in that: The following steps are involved: S1: dissolving dextran in a first alkaline solution, then adding a first cross-linking agent and mixing evenly to obtain a dispersed phase for use; emulsifying a surfactant and an oil phase to obtain a continuous phase for use; mixing the dispersed phase with the continuous phase, performing a chemical cross-linking, and washing to obtain a cross-linked dextran microsphere; S2: adding the once cross-linked dextran microspheres into a second alkaline solution containing a second cross-linking agent, stirring to perform secondary chemical cross-linking, washing, and removing impurities to obtain highly cross-linked dextran microspheres.

2. The method for preparing highly cross-linked dextran microspheres according to claim 1, characterized in that: The temperature of the first chemical cross-linking in step S1 is 25-70° C., and the reaction time is 10-20 hours; the temperature of the second chemical cross-linking in step S2 is 25-70° C., and the reaction time is 20-30 hours.

3. The method for preparing highly cross-linked dextran microspheres according to claim 1, characterized in that: In the step S2, the mass ratio of the primary cross-linked dextran microspheres to the second alkaline solution is 1:(1-5).

4. The method for preparing highly cross-linked dextran microspheres according to claim 1, characterized in that: The mass ratio of dextran to the first cross-linking agent in step S1 is 1:(0.5-1), and the mass ratio of the primary cross-linked dextran microspheres to the second cross-linking agent in step S2 is 1:(0.5-1).

5. The method for preparing highly cross-linked dextran microspheres according to claim 1, characterized in that: The molar concentration of the first alkaline solution is 0.2-0.5M, and the molar concentration of the second alkaline solution is 0.2-0.5M.

6. The method for preparing highly cross-linked dextran microspheres according to claim 1, characterized in that: In step S1, the volume ratio of the dispersed phase to the continuous phase is 1:(5-10).

7. The method for preparing highly cross-linked dextran microspheres according to claim 1, characterized in that: In the step S1, after the dispersed phase and the continuous phase are mixed, they are first emulsified and then cross-linked. The emulsification temperature is 25-50° C. and the emulsification time is 10-30 min.

8. The method for preparing highly cross-linked dextran microspheres according to claim 1, characterized in that: The first cross-linking agent and the second cross-linking agent are both selected from at least one of epichlorohydrin, 1,4-butanediol diglycidyl ether, and divinyl sulfone.

9. The method for preparing highly cross-linked dextran microspheres according to claim 1, characterized in that: In step S1, the mass fraction of the surfactant in the oil phase is 3-6%.

10. An application of the method for preparing highly cross-linked dextran microspheres according to any one of claims 1 to 9, characterized in that: The method is used for preparing highly cross-linked dextran microspheres, wherein the equilibrium water content of the highly cross-linked dextran microspheres is 1-2, and the degradation time of the highly cross-linked dextran microspheres under the action of dextranase is greater than 25 days.

Images