CN118895002A - A kind of degradable cross-linked dextran microsphere and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of degradable microspheres, in particular to a degradable crosslinked dextran microsphere and a preparation method thereof, wherein the preparation raw materials comprise a water phase, an oil phase and a crosslinking agent; the aqueous phase comprises glucan and an alkaline aqueous solution, the oil phase comprises a surfactant and paraffin, the cross-linking agent comprises a cross-linking agent 1 and a cross-linking agent 2, and the mass ratio of the glucan to the cross-linking agent 1 is 1: (0.4-2). The invention takes glucan as raw material, 1, 4-butanediol diglycidyl ether as cross-linking agent, liquid paraffin as continuous phase, and the glucan is cross-linked by the cross-linking agent to obtain stable gel microsphere, and the stable gel microsphere is prepared by different parameters: the dextran microspheres with adjustable particle size and crosslinking degree can be obtained by changing the structural parameters of the microemulsion, adjusting the microstructure and the like, such as water phase concentration, crosslinker input, temperature and rotation speed, water-oil ratio and the like. The degradable cross-linked dextran microspheres prepared by the method have low cross-linking agent residue and good enzyme resistance, and meanwhile, the cross-linked dextran microspheres have qualified cytotoxicity and good biocompatibility.

Description

A kind of degradable cross-linked dextran microsphere and preparation method thereof

Technical Field

The invention relates to the technical field of degradable microspheres, and in particular to degradable cross-linked dextran microspheres and a preparation method thereof.

Background Art

Dextran, also known as dextran, is a polymer mainly composed of D-glucose connected by α-1→6 glycosidic bonds. Cross-linked dextran microspheres are polymer microspheres prepared by reacting dextran as a raw material with a cross-linking agent. Commonly used cross-linking agents include epichlorohydrin, 1,4-butanediol diglycidyl ether, divinyl sulfone, etc. Dextran is non-toxic and has good bioadhesion, biocompatibility, biodegradability and gel properties. Due to the unique gelling properties of the dextran raw material itself, cross-linked dextran microspheres not only have good water absorption, but also have the characteristics of water insolubility and water absorption and swelling. Since its development, it has been widely used in the field of biomedicine.

The currently commercialized cross-linked dextran gel, whose foreign trade name is Sephadex, uses epichlorohydrin as a cross-linking agent and can be used as a column chromatography gel medium for material separation and purification; it can also be used as a microcarrier in tissue engineering and cell culture.

Due to its good biocompatibility, stability and in vivo degradability, cross-linked dextran gel has been increasingly used in clinical practice as a new type of tissue filler in recent years. For example, the foreign product "Deflux" is a sterile injectable gel, the main components of which are cross-linked dextran gel and cross-linked sodium hyaluronate gel. It is mainly used clinically for the treatment of grade II-IV vesicoureteral reflux in children. After cross-linked dextran microspheres are injected into the body as implants, they can promote the production of fibroblasts and collagen between the microspheres, thereby stabilizing the volume of the implant and achieving a lasting filling effect. As a new type of tissue filling gel, Deflux has the unique advantage of being more convenient and faster than traditional antibacterial and surgical treatments. As an effective filling component, cross-linked dextran gel can function in the body for up to 1 to 3 years, ensuring the durability of the therapeutic effect.

The existing dextran microsphere gel preparation technology currently uses epichlorohydrin (ECH) as a crosslinker, and BDDE (1,4-butanediol diglycidyl ether) is rarely used to crosslink dextran. Currently, 1,4-butanediol diglycidyl ether is mostly used to crosslink sodium hyaluronate as a soft tissue filler. After reacting with hyaluronic acid, the epoxy groups of 1,4-butanediol diglycidyl ether are neutralized, and only trace amounts of unreacted 1,4-butanediol diglycidyl ether remain in the product (< 2 parts per million). When cross-linked hyaluronic acid, uncross-linked hyaluronic acid, and unreacted 1,4-butanediol diglycidyl ether degrade, they decompose into harmless byproducts or byproducts that are identical to substances already found in the skin. More than 15 years of clinical and biocompatibility data support the good clinical safety of 1,4-butanediol diglycidyl ether cross-linked hyaluronic acid and its degradation products.

In the prior art, in the preparation process of cross-linked dextran microspheres disclosed in patent CN108721206A "Composition for treating vesicoureteral reflux, stress urinary incontinence and fecal incontinence and its preparation method", toluene and other highly toxic organic solvents are used as the continuous phase, which not only affects the health of operators, but also pollutes the natural environment.

Summary of the invention

In order to solve the problems in the prior art, the present invention provides a degradable cross-linked dextran microsphere, and the preparation raw materials include an aqueous phase, an oil phase and a cross-linking agent; the aqueous phase includes dextran and an alkaline aqueous solution, the oil phase includes a surfactant and paraffin, the cross-linking agent includes a cross-linking agent 1 and a cross-linking agent 2, and the mass ratio of the dextran to the cross-linking agent 1 is 1: (0.4-2).

In one embodiment, the mass ratio of the dextran to the cross-linking agent 1 is 1:(0.4-1).

In one embodiment, the particle size of the microspheres is 100 μm to 350 μm.

In one embodiment, the alkaline aqueous solution is a sodium hydroxide aqueous solution, and the concentration of the sodium hydroxide aqueous solution is 0.2 to 1.5 mol/L.

In one embodiment, the concentration of the sodium hydroxide aqueous solution is 0.2-1 mol/L.

In one embodiment, the paraffin is liquid paraffin, and the mass ratio of the sodium hydroxide aqueous solution in the water phase to the liquid paraffin in the oil phase is 1:(5-20).

In one embodiment, the mass ratio of the sodium hydroxide aqueous solution in the water phase to the liquid paraffin in the oil phase is 1:(5-15).

In one embodiment, the concentration of the dextran in the aqueous phase is 0.3-0.6 g/mL.

In one embodiment, the concentration of the dextran in the aqueous phase is 0.4-0.5 g/mL.

In one embodiment, the surfactant in the oil phase is one or more of Span 80 and Span 85; the concentration of the surfactant is 0.02-0.1 g/g.

In one embodiment, the cross-linking agent 1 and the cross-linking agent 2 are both 1,4-butanediol diglycidyl ether.

In one embodiment, the concentration of the surfactant is 0.04-0.08 g/g.

The second aspect of the present invention provides a method for preparing degradable cross-linked dextran microspheres, comprising the following steps:

(1) dissolving dextran in a sodium hydroxide aqueous solution to obtain an aqueous phase, and premixing the crosslinking agent 1 with the aqueous phase to obtain a solution A;

(2) dissolving the surfactant in liquid paraffin to obtain an oil phase, and transferring the oil phase to a device with stirring and temperature control functions;

(3) adding the solution A obtained in step (1) to the oil phase in step (3) to carry out an emulsification reaction, wherein the temperature during the emulsification reaction is 20 to 80° C.; after the reaction is completed, adding the crosslinking agent 2 to carry out a crosslinking reaction, wherein the temperature during the crosslinking reaction is 20 to 80° C.;

(4) After the reaction in step (3) is completed, the degradable cross-linked dextran microspheres are obtained by washing with an organic solvent, passing through a sieve for solid-liquid separation, and drying;

In one embodiment, the premixing time in step (1) is 0.5 to 2 hours, the premixing temperature is 10 to 25° C., and the rotation speed is 100 to 600 rpm.

In one embodiment, the temperature of the emulsification reaction process in step (3) is 40-70°C.

In one embodiment, the rotation speed of the emulsification reaction in step (3) is 100 to 800 rpm, preferably 300 to 600 rpm.

In one embodiment, the emulsification reaction time in step (3) is 0.1 to 2 hours, preferably 10 to 60 minutes.

In one embodiment, the cross-linking process temperature in step (3) is 40-70°C.

In one embodiment, the rotation speed of the cross-linking reaction in step (3) is 100 to 800 rpm, and the cross-linking time is 4 to 50 hours.

In one embodiment, the rotation speed of the cross-linking reaction in step (3) is 300-600 rpm.

In one embodiment, in step (3), the mass ratio of the cross-linking agent 2 to the dextran is (0-3):1.

In one embodiment, in step (3), the mass ratio of the cross-linking agent 2 to the dextran is (0-1):1.

The present invention chooses to add or not add the crosslinking agent 2 to the reaction system according to the crosslinking degree requirement of the microspheres.

In one embodiment, the washing times in step (4) are 3 times.

In one embodiment, the organic solvent in step (4) is an aqueous solution containing Tween 80.

In one embodiment, the organic solvent in step (4) is a 50% ethanol aqueous solution containing 1-5% Tween 80.

In one embodiment, the sieve in step (4) is a 200-mesh sieve.

Beneficial Effects

1. The cross-linked dextran microsphere preparation process of the present invention uses liquid paraffin, ethanol, etc. with better biological safety. No dangerous organic solvents, such as toluene and n-hexane, are used in the microsphere preparation process. The process is simple and safe.

2. The present invention premixes the aqueous phase with a crosslinking agent, and can pre-crosslink the dextran solution under the same initial dextran concentration conditions, thereby increasing the viscosity of the aqueous phase and facilitating the acquisition of microspheres with larger particle sizes, with a particle size range of 100 μm to 350 μm. The cross-linked dextran microspheres prepared by the present invention are smooth and round.

3. The present invention uses dextran as a raw material, 1,4-butanediol diglycidyl ether as a cross-linking agent, and liquid paraffin as a continuous phase. Dextran is cross-linked by a cross-linking agent to obtain stable gel microspheres. By changing different parameters such as water phase concentration, cross-linking agent input, temperature and rotation speed, water-oil ratio, etc., the structural parameters of the microemulsion are changed, the microstructure is adjusted, and the agglomeration of microspheres is avoided, so that dextran microspheres with adjustable particle size and cross-linking degree can be obtained.

4. The degradable cross-linked dextran microspheres prepared by the method of the present invention have low cross-linking agent residues and good enzyme resistance. At the same time, the cross-linked dextran microspheres have qualified cytotoxicity and good biocompatibility.

5. The degradable cross-linked dextran microspheres prepared by the method of the present invention have readily available raw materials, are easy to prepare, have low cost, and are easy to industrialize.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a micrograph of the dextran microspheres prepared in Example 3.

FIG. 2 is a micrograph of the dextran microspheres prepared in Example 5.

FIG3 is a micrograph of the freeze-dried powder of the dextran microspheres prepared in Example 5.

FIG4 is a micrograph of the freeze-dried dextran microspheres prepared in Example 5 after the dry powder has swelled.

FIG5 is a micrograph of the dextran microspheres prepared in Example 8.

FIG. 6 is a micrograph of the dextran microspheres prepared in Example 9.

FIG. 7 is a micrograph of dextran microspheres prepared in Example 10

FIG8 is a micrograph of the dextran microspheres prepared in Comparative Example 1.

FIG. 9 is a micrograph of the dextran microspheres prepared in Comparative Example 2.

FIG. 10 is a micrograph of the dextran microspheres prepared in Comparative Example 3.

FIG. 11 is a micrograph of the Sephadex G25 dry powder after swelling in Comparative Example 4.

FIG. 12 is a micrograph of the comparative example 5NW Dex G-25 dextran gel.

FIG. 13 is a graph showing the in vitro degradation of the dextran microspheres prepared in Examples 3, 4, 6, 7 and Comparative Examples 3 and 4.

DETAILED DESCRIPTION

In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The experimental methods for which specific conditions are not specified in the examples are carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be purchased commercially.

Example 1

In a first aspect, the present embodiment provides a degradable cross-linked dextran microsphere, wherein the raw materials for preparation include an aqueous phase, an oil phase and a cross-linking agent; the aqueous phase includes dextran and an aqueous sodium hydroxide solution, the oil phase includes Span 80 and liquid paraffin, the cross-linking agent includes cross-linking agent 1, and the mass ratio of the dextran to the cross-linking agent 1 is 1:0.4. The cross-linking agent 1 is 1,4-butanediol diglycidyl ether.

The concentration of the sodium hydroxide aqueous solution is 0.5 mol/L.

The mass ratio of the sodium hydroxide aqueous solution in the water phase to the liquid paraffin in the oil phase is 1:10.

The second aspect of this embodiment provides a method for preparing degradable cross-linked dextran microspheres, comprising the following steps:

(1) 40 g of dextran was dissolved in 100 g of 0.5 mol/L sodium hydroxide aqueous solution to obtain an aqueous phase, and 16 g of 1,4-butanediol diglycidyl ether was premixed with the aqueous phase for 1 h at a premixing temperature of 22° C. and a rotation speed of 300 rpm to obtain a solution A;

(2) Dissolve 60 g of Span 80 in 1000 g of liquid paraffin to obtain an oil phase, and transfer the oil phase to a device with stirring and temperature control functions;

(3) adding the solution A obtained in step (1) to the oil phase in step (3) to carry out an emulsification reaction, wherein the temperature during the emulsification reaction is 40° C., the rotation speed is 300 rpm, and the emulsification time is 20 min; after the reaction is completed, no crosslinking agent is added to the reaction system, and the temperature during the crosslinking process is 40° C., the rotation speed is 300 rpm, and the crosslinking time is 15 h;

(4) After the reaction in step (3) is completed, the microspheres are washed three times with a 50% ethanol aqueous solution containing 2% Tween 80, passed through a 200-mesh sieve for solid-liquid separation, and dried to obtain degradable cross-linked dextran microspheres.

Example 2

This embodiment is basically the same as Embodiment 1, except that:

Step (1) Add 24 g of 1,4-butanediol diglycidyl ether to the aqueous phase, and the mass ratio of dextran to cross-linking agent is 1:0.6.

Example 3

This embodiment is basically the same as Embodiment 1, except that:

The concentration of the sodium hydroxide aqueous solution is 0.75 mol/L.

FIG1 is a micrograph of the dextran microspheres prepared in this example.

Example 4

This embodiment is basically the same as Embodiment 1, except that:

Step (1) adding 40 g of 1,4-butanediol diglycidyl ether to the aqueous phase, the mass ratio of dextran to the cross-linking agent being 1:1;

The emulsification temperature in step (3) is 50° C.

The cross-linking temperature in step (3) is 50°C.

Example 5

This embodiment is basically the same as Embodiment 1, except that:

The emulsification temperature in step (3) is 70° C.

The cross-linking temperature in step (3) is 70°C.

FIG. 2 is a micrograph of the dextran microspheres prepared in this example.

FIG3 is a micrograph of the freeze-dried powder of the dextran microspheres prepared in this example.

FIG4 is a microscopic photograph of the freeze-dried dextran microspheres prepared in this example after swelling of the dry powder.

Example 6

In a first aspect, the present embodiment provides a degradable cross-linked dextran microsphere, wherein the raw materials for preparation include an aqueous phase, an oil phase and a cross-linking agent; the aqueous phase includes dextran and an aqueous sodium hydroxide solution, the oil phase includes Span 80 and liquid paraffin, the cross-linking agent includes a cross-linking agent 1 and a cross-linking agent 2, and the mass ratio of the dextran to the cross-linking agent 1 is 1:1. The cross-linking agent 1 and the cross-linking agent 2 are both 1,4-butanediol diglycidyl ether.

The concentration of the sodium hydroxide aqueous solution is 0.5 mol/L.

The mass ratio of the sodium hydroxide aqueous solution in the water phase to the liquid paraffin in the oil phase is 1:10.

The second aspect of this embodiment provides a method for preparing degradable cross-linked dextran microspheres, comprising the following steps:

(1) Dissolve 40 g of dextran in 100 g of 0.5 mol/L sodium hydroxide aqueous solution to obtain an aqueous phase, premix 40 g of 1,4-butanediol diglycidyl ether with the aqueous phase for 1 h at a premixing temperature of 22° C. and a rotation speed of 300 rpm to obtain solution A;

(2) Dissolve 60 g of Span 80 in 1000 g of liquid paraffin to obtain an oil phase, and transfer the oil phase to a device with stirring and temperature control functions;

(3) adding the solution A obtained in step (1) to the oil phase in step (3) to carry out an emulsification reaction, wherein the temperature during the emulsification reaction is 70° C., the rotation speed is 300 rpm, and the emulsification time is 20 min; after the reaction is completed, 40 g of the crosslinking agent 2 is added to the reaction system, and the temperature during the crosslinking process is 70° C., the rotation speed is 300 rpm, and the crosslinking time is 20 h;

(4) After the reaction in step (3) is completed, the microspheres are washed three times with a 50% ethanol aqueous solution containing 2% Tween 80, passed through a 200-mesh sieve for solid-liquid separation, and dried to obtain degradable cross-linked dextran microspheres.

Example 7

This embodiment is basically the same as embodiment 4, except that:

The cross-linking process temperature in step (3) is 70° C. and the cross-linking time is 40 h.

Example 8

This embodiment is basically the same as Embodiment 1, except that:

Step (1) Dissolve 50 g of dextran in 100 g of 0.5 mol/L sodium hydroxide aqueous solution, stir until the dextran is completely dissolved, obtain a dextran alkaline aqueous solution, and obtain an aqueous phase.

Step (2): Add 50 g of 1,4-butanediol diglycidyl ether to the dextran alkaline aqueous solution in step (1), premix for 30 minutes, and the mass ratio of dextran to premixed 1,4-butanediol diglycidyl ether is 1:1.

The emulsification temperature in step (3) is 70° C.

The cross-linking temperature in step (3) is 70°C.

FIG5 is a micrograph of the dextran microspheres prepared in this example.

Example 9

In a first aspect, the present embodiment provides a degradable cross-linked dextran microsphere, wherein the raw materials for preparation include an aqueous phase, an oil phase and a cross-linking agent; the aqueous phase includes dextran and an aqueous sodium hydroxide solution, the oil phase includes Span 80 and liquid paraffin, the cross-linking agent includes cross-linking agent 1, and the mass ratio of the dextran to the cross-linking agent 1 is 1:0.6. The cross-linking agent is 1,4-butanediol diglycidyl ether.

The concentration of the sodium hydroxide aqueous solution is 1.5 mol/L.

The mass ratio of the sodium hydroxide aqueous solution in the water phase to the liquid paraffin in the oil phase is 1:15.

The second aspect of this embodiment provides a method for preparing degradable cross-linked dextran microspheres, comprising the following steps:

(1) Dissolve 50 g of dextran in 100 g of 1.5 mol/L sodium hydroxide aqueous solution to obtain an aqueous phase, premix 30 g of 1,4-butanediol diglycidyl ether with the aqueous phase for 30 min at a premixing temperature of 22° C. and a rotation speed of 300 rpm to obtain solution A;

(2) Dissolve 90 g of Span 80 in 1500 g of liquid paraffin to obtain an oil phase, and transfer the oil phase to a device with stirring and temperature control functions;

(3) adding the solution A obtained in step (1) to the oil phase in step (3) to carry out an emulsification reaction, wherein the temperature during the emulsification reaction is 40° C., the rotation speed is 300 rpm, and the emulsification time is 20 min; after the reaction is completed, no crosslinking agent is added to the reaction system, and the temperature during the crosslinking process is 40° C., the rotation speed is 300 rpm, and the crosslinking time is 15 h;

(4) After the reaction in step (3) is completed, the microspheres are washed three times with a 50% ethanol aqueous solution containing 2% Tween 80, passed through a 200-mesh sieve for solid-liquid separation, and dried to obtain degradable cross-linked dextran microspheres.

FIG. 6 is a microscopic photograph of the dextran microspheres prepared in this example.

Example 10

In a first aspect, the present embodiment provides a degradable cross-linked dextran microsphere, wherein the raw materials for preparation include an aqueous phase, an oil phase and a cross-linking agent; the aqueous phase includes dextran and an aqueous sodium hydroxide solution, the oil phase includes Span 80 and liquid paraffin, the cross-linking agent includes a cross-linking agent 1 and a cross-linking agent 2, and the mass ratio of the dextran to the cross-linking agent 1 is 1:1. The cross-linking agent 1 and the cross-linking agent 2 are both 1,4-butanediol diglycidyl ether.

The concentration of the sodium hydroxide aqueous solution is 0.5 mol/L.

The mass ratio of the sodium hydroxide aqueous solution in the water phase to the liquid paraffin in the oil phase is 1:10.

The second aspect of this embodiment provides a method for preparing degradable cross-linked dextran microspheres, comprising the following steps:

(1) Dissolve 40 g of dextran in 100 g of 0.5 mol/L sodium hydroxide aqueous solution to obtain an aqueous phase, premix 40 g of 1,4-butanediol diglycidyl ether with the aqueous phase for 1 h at a premixing temperature of 22° C. and a rotation speed of 300 rpm to obtain solution A;

(2) Dissolve 60 g of Span 80 in 1000 g of liquid paraffin to obtain an oil phase, and transfer the oil phase to a device with stirring and temperature control functions;

(3) adding the solution A obtained in step (1) to the oil phase in step (3) to carry out an emulsification reaction, wherein the temperature during the emulsification reaction is 70° C., the rotation speed is 300 rpm, and the emulsification time is 20 min; after the reaction is completed, 20 g of crosslinking agent 2 is added to the reaction system, and the temperature during the crosslinking process is 70° C., the rotation speed is 300 rpm, and the crosslinking time is 20 h;

(4) After the reaction in step (3) is completed, the microspheres are washed three times with a 50% ethanol aqueous solution containing 2% Tween 80, passed through a 200-mesh sieve for solid-liquid separation, and dried to obtain degradable cross-linked dextran microspheres.

FIG. 7 is a micrograph of the dextran microspheres prepared in this example.

Comparative Example 1

This embodiment is basically the same as embodiment 9, except that:

The concentration of the sodium hydroxide aqueous solution is 2 mol/L.

FIG8 is a micrograph of the dextran microspheres prepared in this example.

Comparative Example 2

This embodiment is basically the same as embodiment 3, except that:

The rotation speed during the cross-linking process of step (3) is 1000 rpm.

FIG. 9 is a micrograph of the dextran microspheres prepared in this example.

Comparative Example 3

This embodiment is basically the same as embodiment 5, except that:

The cross-linking process temperature in step (3) is 80°C.

FIG. 10 is a microscopic photograph of the dextran microspheres prepared in this example.

Comparative Example 4

Commercially available dextran gel product: McLean Sephadex G25, CAS number: 9041-35-4.

FIG. 11 is a micrograph of Sephadex G25 dry powder after swelling.

Comparative Example 5

Commercially available dextran gel products: Suzhou Nanovita NW Dex G-25 series dextran gel.

FIG. 12 is a micrograph of NW Dex G-25 dextran gel.

Performance Testing

1. Balanced moisture content test

Test objects: samples prepared in Examples 1-10.

Test method: The dextran microspheres that have reached water absorption equilibrium are vacuumed with a vacuum pump to remove surface moisture, and placed in an empty watch glass weighing m _0. The total mass of the wet ball and the empty watch glass is weighed as m ₁ . The dextran microsphere sample is dried in a 50°C oven to constant weight, and finally the total mass of the dry ball and the empty watch glass is weighed as m ₂ . The equilibrium water content of the dextran microspheres is calculated as X = (m ₁ -m ₂ )/(m ₂ -m ₀ ). The lower the equilibrium water content of the microspheres, the higher the cross-linking degree of the microspheres. The test results are shown in Table 1.

2. In vitro accelerated degradation test

Test objects: samples prepared in Examples 3, 4, 6, 7 and Comparative Examples 3 and 4.

Test method: First, prepare a dextranase/PBS solution with a concentration of 0.05 g/mL (1250-1285 U/mL) and pH = 5.0.

Preparation method of dextranase solution: weigh 40g of dextranase stock solution, add 800mL of pH=5.0 PBS phosphate buffer to prepare 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 1L of purified water, and after complete dissolution, add orthophosphoric acid to adjust the pH value of the solution to 5.0.

The dextran microspheres that have reached water absorption equilibrium were vacuumed with a vacuum pump to remove surface moisture to obtain wet microspheres. 1 g of wet microsphere sample was transferred to a 15 mL plastic test tube, 5 mL of 0.05 g/mL pH=5.0 dextranase solution was added, and the tube was placed in a 37°C water bath shaker for degradation. The shaker speed was set to 40 rpm, and the supernatant was replaced every 1 day to observe the degradation of the microspheres. The test results are shown in Figure 13.

The percentage of the ordinate in Figure 13 indicates the degradation degree of the microspheres: taking the curve of Example 3 as an example, it shows that the microspheres are completely degraded within 1 day; taking Comparative Example 4 as an example, it shows that the microspheres are degraded by 53% (i.e. -53%) within 4 days and 93% within 10 days; taking Comparative Example 3 as an example, it shows that the microspheres swell by 133% (i.e. +133%) within 4 days and degrade by 67% (i.e. -67%) within 17 days. The higher the cross-linking degree of the microspheres, the slower the in vitro degradation rate and the stronger the enzyme resistance.

3. Cytotoxicity test

Test objects: samples prepared in Examples 4 and 10.

Test method: Examples 4 and 10 were subjected to cytotoxicity tests. The specific method was tested according to the MTT method in reference standard GB/T16886.5-2017. Four concentrations (100%, 75%, 50%, 25%) of test sample extracts, blanks, 100% negative controls and positive controls were prepared with MEM culture medium (containing 10% fetal bovine serum), extracted for 24 hours, and the extraction ratio was 0.2g:1mL. Under the conditions of 37C and 5% _CO2 , semi-confluent monolayers of L-929 (NIH3T3) mouse fibroblasts were cultured on 96-well plates with test sample extracts, blanks 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. A survival rate of >70% is qualified.

The cell survival rate was calculated according to the following formula: Survival rate % = R/R ₀ × 100

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

R ₀ : average optical density of the blank group. See Table 2 for test results.

Table 1

Table 2

Sample name 100% extraction 75% extraction 50% extraction 25% extraction Positive Control Negative control Test Results Example 4 85.88% 97.73% 95.40% 104.90% 2.69% 94.29% qualified Example 10 94.64% 98.24% 100.01% 98.57% 2.66% 92.78% qualified

Claims (10)

1. A degradable cross-linked dextran microsphere, characterized in that the preparation raw materials include an aqueous phase, an oil phase and a cross-linking agent; the aqueous phase includes dextran and an alkaline aqueous solution, the oil phase includes a surfactant and paraffin, the cross-linking agent includes cross-linking agent 1 and cross-linking agent 2, and the mass ratio of the dextran to the cross-linking agent 1 is 1: (0.4-2). 2 . The degradable cross-linked dextran microspheres according to claim 1 , wherein the particle size of the microspheres is 100 μm to 350 μm. 3. The degradable cross-linked dextran microspheres according to claim 1, characterized in that the alkaline aqueous solution is a sodium hydroxide aqueous solution, and the concentration of the sodium hydroxide aqueous solution is 0.2 to 1.5 mol/L. 4. The degradable cross-linked dextran microspheres according to claim 3, characterized in that the paraffin is liquid paraffin, and the mass ratio of the sodium hydroxide aqueous solution in the water phase to the liquid paraffin in the oil phase is 1:(5-20). 5 . The degradable cross-linked dextran microspheres according to claim 1 , wherein the concentration of the dextran in the aqueous phase is 0.3 to 0.6 g/mL. 6. The degradable cross-linked dextran microspheres according to claim 1, characterized in that the surfactant in the oil phase is one or more of Span 80 and Span 85; and the concentration of the surfactant is 0.02-0.1 g/g. 7. A method for preparing the degradable cross-linked dextran microspheres according to any one of claims 1 to 6, characterized in that it comprises the following steps: (1) dissolving dextran in a sodium hydroxide aqueous solution to obtain an aqueous phase, and premixing the crosslinking agent 1 with the aqueous phase to obtain a solution A; (2) dissolving the surfactant in liquid paraffin to obtain an oil phase, and transferring the oil phase to a device with stirring and temperature control functions; (3) adding the solution A obtained in step (1) to the oil phase in step (3) to carry out an emulsification reaction, wherein the temperature during the emulsification reaction is 20 to 80° C.; after the reaction is completed, adding the crosslinking agent 2 to carry out a crosslinking reaction, wherein the temperature during the crosslinking reaction is 20 to 80° C.; (4) After the reaction in step (3) is completed, the degradable cross-linked dextran microspheres are obtained by washing with an organic solvent, passing through a sieve for solid-liquid separation, and drying. 8. The method for preparing degradable cross-linked dextran microspheres according to claim 7, characterized in that the rotation speed of the cross-linking reaction in step (3) is 100 to 800 rpm and the cross-linking time is 4 to 50 hours. 9. The method for preparing degradable cross-linked dextran microspheres according to claim 7, characterized in that the mass ratio of the cross-linking agent 2 to the dextran in the step (3) is (0-3):1. 10 . The method for preparing degradable cross-linked dextran microspheres according to claim 7 , characterized in that the organic solvent in step (4) is an aqueous solution containing Tween 80.