CN105348548B - A kind of hydrogel microsphere based on glucan and preparation method thereof - Google Patents

Abstract

The invention relates to a dextran-based hydrogel microsphere and a preparation method thereof, belonging to the technical field of biomedical polymer materials. First, dissolve the dextran in water at room temperature, add sodium periodate to obtain a mixed solution, dialyze after stirring, and freeze-dry to obtain a part of the aldylated glucan solid; Add the cyclohexane solution dissolved in the emulsifier in deionized water, add the amine cross-linking agent and stir, and finally collect the precipitate by centrifugation, and obtain the chemical cross-linked dextran hydrogel microspheres after washing; Sodium borohydride was added to the chemically cross-linked dextran hydrogel microspheres in water, stirred at room temperature for 24 hours, and finally the precipitate was collected by centrifugation, washed with deionized water to obtain reduced dextran hydrogel microspheres. The invention proposes to use the "water-in-oil" inverse microemulsion system as a reaction medium to prepare dextran-based hydrogel microspheres with controllable particle size and good stability.

Description

A kind of hydrogel microsphere based on dextran and preparation method thereof

technical field

The invention relates to a dextran-based hydrogel microsphere and a preparation method thereof, belonging to the technical field of biomedical polymer materials.

Background technique

Hydrogel is a three-dimensional network structure formed by physical or chemical cross-linking of polymers, which can swell in aqueous solution and retain a large amount of water without dissolving, and hydrophilic small molecules can diffuse in the hydrogel. Hydrogel microspheres have both properties of hydrogel and micro/nanoparticles. Due to its small size, large specific surface area, etc., as well as the dimensional stability and high solution absorption performance of hydrogels, hydrogel micro/nanoparticles are used as biomedical materials in tissue engineering, drug delivery, etc. and other fields have great application potential and broad application prospects (The development of microgels/nanogels for drug delivery applications, Progress in Polymer Science, 2008. 33: 448-477; Microgels in Drug Delivery, in Microgel Suspensions, 2011, Wiley-VCH Verlag GmbH & Co. KGaA. 375-405).

Hydrogels based on natural polymer materials have excellent biological safety and have attracted much attention as biomaterials. Polysaccharides are a class of natural polymers with good biocompatibility. Hydrogels constructed of natural polysaccharide polymer materials have good biocompatibility and biodegradability. As biomedical polymer materials, they are widely used in tissue engineering, Gene carrier and drug carrier, the current research on polysaccharide gel microspheres mainly revolves around chitosan and alginic acid. Dextran, also known as dextran, is a bacterial polysaccharide with a large number of free hydroxyl groups in its molecular structure, which has good water solubility and biological safety. Since the 1940s, dextran has been widely used clinically as a temporary plasma substitute, so dextran-based hydrogel microspheres have broad clinical application prospects as drug carrier materials.

At present, the preparation methods of hydrogel microspheres based on polymer materials are generally divided into four categories (Nanogelsas Pharmaceutical Carriers: Finite Networks of Infinite Capabilities. Angewandte Chemie International Edition , 2009. 48: 5418-5429): (1 )Using physical forces such as hydrogen bonds between polymers, hydrophilic and hydrophobic forces, and electrostatic forces to "self-assemble" to form hydrogel microspheres; The process can be carried out in a homogeneous system or in a heterogeneous environment with a nanometer size; (3) Through chemical cross-linking, the polymers are connected by covalent bonds to form gel microspheres; (4) using "plate Nanomaterial processing methods such as "printing" or "microfluidics" are used to prepare hydrogel microspheres. Among them, the microemulsion polymerization method is usually to add the polymerized monomer to the water phase (oil phase) containing an emulsifier without an initiator, add it to the oil phase (water phase) and stir vigorously to form small droplets, and then add the initiator Or initiate polymerization of monomers in the water phase (oil phase) by high-energy radiation to form nanoparticles. The "water-in-oil" inverse microemulsion method has the advantages of mild reaction conditions and easy control of the particle size of gel microspheres in the preparation of hydrogel nanoparticles, so it is a common method for preparing micro/nanoparticle materials ( The Generation of Nanoparticles in Miniemulsions. Advanced Materials, 2001. 13: 765-768; Miniemulsion Polymerization and the Structure of Polymer and Hybrid Nanoparticles. Angewandte Chemie International Edition, 2009. 48: 4488-4507), but currently there is no information about this method for the preparation of grape There are few literature reports on polysaccharide-based hydrogels, and the applicable reaction conditions and surfactant systems have not been proposed yet.

Contents of the invention

Aiming at the problems and deficiencies in the above-mentioned prior art, the present invention provides a dextran-based hydrogel microsphere and a preparation method thereof. The present invention proposes to use the "water-in-oil" inverse microemulsion system as the reaction medium, and by adjusting factors such as the surfactant type, content, surfactant compounding ratio and emulsification method that constitute the inverse microemulsion, the particle size can be prepared Controlled and stable dextran-based hydrogel microspheres, the present invention is achieved through the following technical solutions.

A kind of hydrogel microsphere based on dextran, its structural formula can be expressed as follows:

.

A kind of preparation method of above-mentioned dextran-based hydrogel microspheres, its concrete steps comprise as follows:

(1) Preparation of aldylated dextran

Dissolve dextran in water at room temperature according to the solid-to-liquid ratio of 10:100g/ml, then place it in an ice-water bath and add sodium periodate according to the sugar unit/IO ₄ ^-molar ratio of 2:1~1:1 to obtain Mixed solution, the mixed solution was stirred and reacted at room temperature and protected from light for 2-6 hours, and the obtained solution was dialyzed in deionized water with a regenerated cellulose dialysis bag for 2-3 days, and the deionized water was changed every 3-5 hours during the dialysis process , freeze-dried after dialysis to obtain partially aldylated dextran solids with a degree of aldylation of 40-60% (that is, 40-60 of every 100 sugar units were oxidized, and the degree of aldylation was titrated by hydroxylamine hydrochloride method for determination);

(2) Preparation of dextran hydrogel microspheres

Dissolve the partially formylated dextran solid obtained in step (1) in deionized water to obtain an aqueous solution of formylated dextran with a concentration of 100-200 mg/mL, and add the aqueous solution of formylated dextran to the In the cyclohexane solution in which the emulsifier is dissolved, emulsify to form a reverse microemulsion, then add an amine cross-linking agent and stir for 10-12 hours, and finally collect the precipitate by centrifugation, and wash the precipitate with ethanol to obtain a chemically cross-linked glucan Sugar hydrogel microspheres, dispersed in deionized water for preservation;

(3) Reduction of dextran hydrogel microspheres

Add sodium borohydride to the chemically cross-linked dextran hydrogel microspheres dispersed in deionized water obtained in step (2), stir at room temperature for 24 hours, and finally collect the precipitate by centrifugation, and wash the precipitate with deionized water The reduced dextran hydrogel microspheres are obtained and dispersed in deionized water for storage.

The dextran in the step (1) is any one of dextran with a molecular weight of 10000g/mol~100000g/mol.

In the step (2), the volume ratio of the aqueous solution of aldylated dextran to cyclohexane is 1:5-1:10.

The emulsifier in the step (2) is polyoxyethylene (5) nonylphenyl ether (CO-520), sorbitan oleate (Span80) or Span80/sorbitan monooleate Any one of the compounding agent of polyoxyethylene ether (Tween80) (Tween80 in the compounding agent is 5%~20% of the mass of Span80), the amount of emulsifier added is 10~50% of the mass of cyclohexane.

The emulsification in the step (2) is mechanical stirring emulsification or ultrasonic emulsification.

The amine crosslinking agent in the step (2) is any compound with two amino groups among ethylenediamine, lysine, and arginine, and the amount added is according to the The molar ratio of the aldehyde group to the amino group in the amine crosslinking agent is 1:1~2:1.

The amine crosslinking agent in the step (2) is any compound with two or more amino groups in the dendritic polyethyleneimine (PEI) with a molecular weight of 600g/mol~1800g/mol, and the addition amount is Add according to the molar ratio of the aldehyde group in the partially formylated dextran to the amino group in the amine crosslinking agent is 2:1~1:5.

In the step (3), the molar ratio of the sodium borohydride to the C=N double bonds in the chemically cross-linked dextran hydrogel microspheres is 2:1-10:1.

The schematic diagram of the preparation process of the dextran-based hydrogel microspheres of the present invention (taking ethylenediamine as a cross-linking agent as an example) is as follows:

The beneficial effects of the present invention are:

1. The dextran-based hydrogel microspheres prepared in the present invention are hydrogel microspheres based on natural polymer materials, and have good biological safety, stability and clinical application prospects.

2. The dextran-based hydrogel microspheres involved in the present invention are obtained by the reverse microemulsion cross-linking method, and their morphology and particle size can be adjusted by regulating the surfactant type, composition and emulsification method of the reverse microemulsion. regulation.

Description of drawings

Fig. 1 is the infrared spectrogram before and after reduction of Example 1 of the present invention, wherein a is the infrared spectrogram of gel microspheres before reduction, and b is the infrared spectrogram of dextran hydrogel microspheres after reduction;

Fig. 2 is the scanning electron micrograph of the gel microsphere before the reduction of Example 1 of the present invention;

3 is a scanning electron micrograph of the gel microspheres before reduction in Example 2 of the present invention;

Fig. 4 is the scanning electron micrograph of the gel microsphere before the reduction of Example 4 of the present invention;

Fig. 5 is a scanning electron micrograph of gel microspheres before reduction in Example 7 of the present invention.

Detailed ways

The present invention will be further described below in combination with the accompanying drawings and specific embodiments.

Example 1

The structural formula of the dextran-based hydrogel microspheres can be expressed as follows:

.

The preparation method of the above-mentioned dextran-based hydrogel microspheres, its specific steps include as follows:

(1) Preparation of aldylated dextran

Dissolve 5.0g dextran (dextran T10, molecular weight 10000g/mol) in water at room temperature according to the solid-to-liquid ratio of 10:100g/ml, and then place it in an ice-water bath according to the molar ratio of sugar unit/ ^IO ₄ Add sodium periodate at a ratio of 1:1 to obtain a mixed solution, stir the mixed solution at room temperature and under light-proof conditions for 4 hours, and dialyze the obtained solution in deionized water for 2 to 3 hours with a regenerated cellulose dialysis bag (molecular weight cut-off: 5kD). During the dialysis process, the deionized water was changed every 3~5h. After the dialysis was completed, freeze-dried to obtain a partially formylated dextran solid with a degree of formylation of 60% (that is, 60 out of every 100 sugar units were oxidized). );

(2) Preparation of dextran hydrogel microspheres

Dissolve 0.15 g of the partially formylated dextran solid obtained in step (1) in 1.0 mL of deionized water to obtain an aqueous solution of formylated dextran with a concentration of 150 mg/mL. Add the aqueous solution into the cyclohexane solution in which the emulsifier is dissolved, stir vigorously for 15 minutes to emulsify to form a reverse microemulsion, then add an amine cross-linking agent and stir for 12 hours, and finally collect the precipitate by centrifugation (1500rpm, 5min), and wash the precipitate with ethanol After 5-6 times, the chemically cross-linked dextran hydrogel microspheres were obtained, which were dispersed in 2.0 mL deionized water for storage. The chemically cross-linked dextran hydrogel microspheres were dried in a vacuum to obtain a light yellow solid powder. The spectrum is shown in Figure 1(a); the morphology of the obtained gel microspheres was observed with a scanning electron microscope, and the results are shown in Figure 2: the dextran gel microspheres obtained under this condition have regular Spherical structure, smooth microsphere surface, particle size distribution between 30-90μm; the amount of cyclohexane is 8.0mL, the volume ratio of aldylated dextran aqueous solution to cyclohexane is 1:8, and the emulsifier is Span80. The addition amount is 2.0g (the addition amount of the emulsifier is 32% of the mass of the cyclohexane solution), and the stirring is mechanical stirring; the amine cross-linking agent is ethylenediamine, and the addition amount is according to the partial formylated dextran The molar ratio of the aldehyde group to the amino group in the amine cross-linking agent is 2:1 and added (about 18 μL);

(3) Reduction of dextran hydrogel microspheres

Add 50 mg of sodium borohydride to the chemically cross-linked dextran hydrogel microspheres dispersed in deionized water obtained in step (2) (the molar ratio of sodium borohydride to the C=N double bond in the dextran hydrogel microspheres About 2.5:1), stirred at room temperature for 24 hours, and finally collected the precipitate by centrifugation (1500rpm, 5min). Store in 2.0 mL deionized water. The reduced dextran hydrogel microspheres obtained a white solid powder after vacuum drying, and its infrared spectrum is shown in Figure 1(b).

Example 2

The structural formula of the dextran-based hydrogel microspheres can be expressed as follows:

.

The preparation method of the above-mentioned dextran-based hydrogel microspheres, its specific steps include as follows:

(1) Preparation of aldylated dextran

Dissolve 5.0g of dextran (dextran T40, molecular weight 40000g/mol) in water at room temperature according to the solid-to-liquid ratio of 10:100g/ml, and then place it in an ice-water bath according to the molar ratio of sugar unit/ ^IO ₄ Add sodium periodate at a ratio of 1:1 to obtain a mixed solution, stir the mixed solution at room temperature and in the dark for 4 hours, and dialyze the obtained solution in deionized water for 2 to 3 hours with a regenerated cellulose dialysis bag (molecular weight cut-off: 7kD). During the dialysis process, the deionized water was changed every 3~5h. After the dialysis was completed, freeze-dried to obtain a partially formylated dextran solid with a degree of formylation of 60% (that is, 60 out of every 100 sugar units were oxidized). );

(2) Preparation of dextran hydrogel microspheres

Dissolve 0.15 g of the partially formylated dextran solid obtained in step (1) in 1.0 mL of deionized water to obtain an aqueous solution of formylated dextran with a concentration of 150 mg/mL. Add the aqueous solution into the cyclohexane solution dissolved in the emulsifier, stir vigorously for 15 minutes to emulsify to form a reverse microemulsion, then add an amine cross-linking agent and stir for 12 hours, and finally collect the precipitate by centrifugation (3000rpm, 5min), and wash the precipitate with ethanol After 5 to 6 times, the chemically cross-linked dextran hydrogel microspheres were obtained, which were dispersed in 2.0ml deionized water and stored. The morphology of the obtained gel microspheres was observed by scanning electron microscopy, and the results are shown in Figure 3 It shows that the dextran gel microspheres obtained under this condition have a regular spherical structure, the surface of the microspheres is smooth, and the particle size distribution is between 10 and 50 μm; the amount of cyclohexane is 8.0 mL, and the formaldehyde dextran The volume ratio of aqueous solution to cyclohexane is 1:8, the emulsifier is a compound of Span80/Tween80, the amount of Span80 added is 2.0g, and the amount of Tween80 is 0.2g (the amount of emulsifier added is 35% of the mass of cyclohexane), Stirring is mechanical stirring; the amine cross-linking agent is ethylenediamine, and the amount added is 2:1 according to the molar ratio of the aldehyde group in the partially formylated dextran to the amino group in the amine cross-linking agent (about 18 μL) ;

(3) Reduction of dextran hydrogel microspheres

Add 50 mg of sodium borohydride to the chemically cross-linked dextran hydrogel microspheres dispersed in deionized water obtained in step (2) (C=N double in sodium borohydride and chemically cross-linked dextran hydrogel microspheres The molar ratio of bond is 2.5:1), stirred at room temperature for 24h, and finally collected the precipitate by centrifugation (3000rpm, 5min). Balls, dispersed in 2.0mL deionized water for storage. The reduced dextran hydrogel microspheres were dispersed in a series of disodium hydrogen phosphate-citric acid buffer solutions with different pHs, and their stability was observed. The results are shown in Table 1: under the environmental conditions of pH>4.0 , the dextran gel microspheres have good stability in aqueous solution, and when the solution pH<4.0, the structure of the dextran gel microspheres is destroyed and the stability is poor.

Table 1

Example 3

The structural formula of the dextran-based hydrogel microspheres can be expressed as follows:

.

The preparation method of the above-mentioned dextran-based hydrogel microspheres, its specific steps include as follows:

(1) Preparation of aldylated dextran

Dissolve 5.0g of dextran (dextran T70, molecular weight 70000g/mol) in water at room temperature according to the solid-to-liquid ratio of 10:100g/ml, and then place it in an ice-water bath according to the molar ratio of sugar unit/ ^IO ₄ Add sodium periodate at a ratio of 1:1 to obtain a mixed solution, stir the mixed solution at room temperature and under light-proof conditions for 4 hours, and dialyze the stirred solution in deionized water for 2 For ~3 days, the deionized water was changed every 3~5h during the dialysis process. After the dialysis was completed, freeze-dried to obtain a partially aldylated dextran solid with a degree of aldylation of 60% (that is, there were 60 sugar units per 100 Oxidized);

(2) Preparation of dextran hydrogel microspheres

Dissolve 0.1 g of the partially formylated dextran solid obtained in step (1) in 1.0 mL of deionized water to obtain an aqueous solution of formylated dextran with a concentration of 100 mg/mL. Add the aqueous solution into the cyclohexane solution dissolved in the emulsifier, stir vigorously for 15 minutes to emulsify to form a reverse microemulsion, then add an amine cross-linking agent and stir for 12 hours, and finally collect the precipitate by centrifugation (3000rpm, 5min), and wash the precipitate with ethanol After 5 to 6 times, the chemically cross-linked dextran hydrogel microspheres were obtained, which were dispersed in 2.0ml deionized water for storage; the amount of cyclohexane was 8.0mL, and the volume ratio of aldylated dextran aqueous solution to cyclohexane was 1:8, the emulsifier is a compound of Span80/Tween80, the amount of Span80 added is 2.0g, and the amount of Tween80 is 0.4g (the amount of emulsifier added is 38% of the mass of the cyclohexane solution), and the stirring is mechanical; the amine The cross-linking agent is ethylenediamine, and the amount added is 2:1 according to the molar ratio of the aldehyde group in the partially formylated dextran to the amino group in the amine cross-linking agent (about 12 μL);

(3) Reduction of dextran hydrogel microspheres

Add 50 mg of sodium borohydride to the chemically cross-linked dextran hydrogel microspheres dispersed in deionized water obtained in step (2) (C=N double in sodium borohydride and chemically cross-linked dextran hydrogel microspheres The molar ratio of the bond is about 4:1), stirred at room temperature for 24 hours, and finally collected the precipitate by centrifugation (3000rpm, 5min). The precipitate was washed with deionized water for 5-6 times to obtain the reduced dextran hydrogel Microspheres, dispersed in 2.0mL deionized water for storage.

Example 4

The structural formula of the dextran-based hydrogel microspheres can be expressed as follows:

.

The preparation method of the above-mentioned dextran-based hydrogel microspheres, its specific steps include as follows:

(1) Preparation of aldylated dextran

Dissolve 5.0g of dextran (dextran T40, molecular weight 40000g/mol) in water at room temperature according to the solid-to-liquid ratio of 10:100g/ml, and then place it in an ice-water bath according to the molar ratio of sugar unit/ ^IO ₄ Add sodium periodate at a ratio of 1:1 to obtain a mixed solution, stir the mixed solution at room temperature and in the dark for 4 hours, and dialyze the obtained solution in deionized water for 2 to 3 hours with a regenerated cellulose dialysis bag (molecular weight cut-off: 7kD). During the dialysis process, the deionized water was changed every 3~5h. After the dialysis was completed, freeze-dried to obtain a partially formylated dextran solid with a degree of formylation of 60% (that is, 60 out of every 100 sugar units were oxidized). );

(2) Preparation of dextran hydrogel microspheres

Dissolve 0.1 g of the partially formylated dextran solid obtained in step (1) in 1.0 mL of deionized water to obtain an aqueous solution of formylated dextran with a concentration of 100 mg/mL. Add the aqueous solution into the cyclohexane solution dissolved in the emulsifier, stir vigorously for 30 minutes to emulsify to form a reverse microemulsion, then add an amine cross-linking agent and stir for 12 hours, and finally collect the precipitate by centrifugation (4000rpm, 5min), and wash the precipitate with ethanol After 5 to 6 times, the chemically cross-linked dextran hydrogel microspheres were obtained, which were dispersed in 2.0ml deionized water and stored. The morphology of the obtained gel microspheres was observed by scanning electron microscopy, and the results are shown in Figure 4 The results show that the dextran gel microspheres obtained under this condition have a regular spherical structure, the surface of the microspheres is smooth, and the particle size distribution is between 2 and 6 μm; the amount of cyclohexane is 8.0 mL, and the formaldehyde dextran The volume ratio of aqueous solution to cyclohexane is 1:8, the emulsifier is CO-520, the amount added is 3.0g (the amount of emulsifier added is 48% of the mass of cyclohexane), and the stirring is mechanical stirring; the amine crosslinking The agent is ethylenediamine, and the amount added is 2:1 according to the molar ratio of the aldehyde group in the partially formylated dextran to the amino group in the amine crosslinking agent (about 12 μL);

(3) Reduction of dextran hydrogel microspheres

Add 40 mg of sodium borohydride to the chemically cross-linked dextran hydrogel microspheres dispersed in deionized water obtained in step (2) (C=N double in sodium borohydride and chemically cross-linked dextran hydrogel microspheres The molar ratio of the bond is about 3:1), stirred at room temperature for 24 hours, and finally collected the precipitate by centrifugation (4000rpm, 5min). The precipitate was washed with deionized water for 5-6 times to obtain the reduced dextran hydrogel Microspheres, dispersed in 2.0mL deionized water for storage.

Example 5

The structural formula of the dextran-based hydrogel microspheres can be expressed as follows:

.

The preparation method of the above-mentioned dextran-based hydrogel microspheres, its specific steps include as follows:

(1) Preparation of aldylated dextran

Dissolve 5.0g of dextran (dextran T40, molecular weight 40000g/mol) in water at room temperature according to the solid-to-liquid ratio of 10:100g/ml, and then place it in an ice-water bath according to the molar ratio of sugar unit/ ^IO ₄ Add sodium periodate at a ratio of 2:1 to obtain a mixed solution, stir the mixed solution at room temperature and in the dark for 6 hours, and dialyze the obtained solution in deionized water for 2-3 hours with a regenerated cellulose dialysis bag (molecular weight cut-off: 7kD During the dialysis process, the deionized water was changed every 3~5h. After the dialysis was completed, freeze-dried to obtain a partially formylated dextran solid with a degree of formylation of 40% (that is, 40 out of every 100 sugar units were oxidized). );

(2) Preparation of dextran hydrogel microspheres

Dissolve 0.1 g of the partially formylated dextran solid obtained in step (1) in 1.0 mL of deionized water to obtain an aqueous solution of formylated dextran with a concentration of 100 mg/mL. Add the aqueous solution into the cyclohexane solution dissolved in the emulsifier, stir vigorously for 30 minutes to emulsify to form a reverse microemulsion, then add an amine cross-linking agent and stir for 12 hours, and finally collect the precipitate by centrifugation (3000rpm, 5min), and wash the precipitate with ethanol After 5 to 6 times, the chemically cross-linked dextran hydrogel microspheres were obtained, which were dispersed in 2.0ml deionized water and stored; the amount of cyclohexane was 8.0mL, and the volume ratio of the aldylated dextran aqueous solution to cyclohexane The emulsifier is 1:8, the emulsifier is Span80/Tween80 compound, the addition amount is 2.0g for Span80, and 0.2g for Tween80 (the addition amount of emulsifier is 35% of the mass of cyclohexane), and the stirring is mechanical stirring; The cross-linking agent is PEI (the molecular weight of PEI is 600g/mol, and its solution concentration is 0.25g/mL), and the amount added is according to the molar ratio of the aldehyde group in the partially formylated dextran to the amino group in the amine cross-linking agent. 1:1 addition (0.2mL);

(3) Reduction of chemically cross-linked dextran hydrogel microspheres

Add 40 mg of sodium borohydride to the chemically cross-linked dextran hydrogel microspheres dispersed in deionized water obtained in step (2) (C=N double in sodium borohydride and chemically cross-linked dextran hydrogel microspheres The molar ratio of bond is 2:1), stirred at room temperature for 24h, and finally collected the precipitate by centrifugation (3000rpm, 5min). Balls, dispersed in 2.0mL deionized water for storage.

Example 6

The structural formula of the dextran-based hydrogel microspheres can be expressed as follows:

.

The preparation method of the above-mentioned dextran-based hydrogel microspheres, its specific steps include as follows:

(1) Preparation of aldylated dextran

Dissolve 5.0g of dextran (dextran T70, molecular weight 70000g/mol) in water at room temperature according to the solid-to-liquid ratio of 10:100g/ml, and then place it in an ice-water bath according to the molar ratio of sugar unit/ ^IO ₄ Add sodium periodate at a ratio of 2:1 to obtain a mixed solution, stir the mixed solution at room temperature and in the dark for 6 hours, and dialyze the obtained solution in deionized water for 2-3 hours with a regenerated cellulose dialysis bag (molecular weight cut-off: 7kD During the dialysis process, the deionized water was changed every 3-5 hours. After the dialysis was completed, freeze-dried to obtain a partially aldylated dextran solid with a degree of aldylation of about 40% (that is, 40 sugar units per 100 sugar units were oxidation);

(2) Preparation of dextran hydrogel microspheres

Dissolve 0.2 g of the partially formylated dextran solid obtained in step (1) in 1.0 mL of deionized water to obtain an aqueous solution of formylated dextran with a concentration of 200 mg/mL. Add the aqueous solution into the cyclohexane solution dissolved in the emulsifier, stir vigorously for 30 minutes to emulsify to form a reverse microemulsion, then add an amine cross-linking agent and stir for 10 hours, and finally collect the precipitate by centrifugation (1500rpm, 5min), and wash the precipitate with ethanol After 5 to 6 times, the chemically cross-linked dextran hydrogel microspheres were obtained, which were dispersed in 2.0ml deionized water for storage; the amount of cyclohexane was 10.0mL, and the volume ratio of aldylated dextran aqueous solution to cyclohexane was 1:10, the emulsifier is Span80, the amount of emulsifier added is 10% of the mass of cyclohexane, and the stirring is mechanical stirring; the amine crosslinking agent is PEI (the molecular weight of PEI is 1800g/mol, and its solution concentration is 0.5g /mL), the amount added is 2:1 according to the molar ratio of the aldehyde group in the partially formylated dextran to the amino group in the amine crosslinking agent (0.2mL);

(3) Reduction of dextran hydrogel microspheres

Add sodium borohydride (sodium borohydride and chemically cross-linked dextran hydrogel microspheres C=N double bond to the chemically cross-linked dextran hydrogel microspheres dispersed in deionized water obtained in step (2) The molar ratio is 2:1), stirred at room temperature for 24h, and finally collected the precipitate by centrifugation (1500rpm, 5min). The precipitate was washed with deionized water for 5-6 times to obtain the reduced dextran hydrogel microspheres. Disperse in 2.0mL deionized water and save.

Example 7

The structural formula of the dextran-based hydrogel microspheres can be expressed as follows:

.

The preparation method of the above-mentioned dextran-based hydrogel microspheres, its specific steps include as follows:

(1) Preparation of aldylated dextran

Dissolve 5.0g of dextran (dextran T40, molecular weight 40000g/mol) in water at room temperature according to the solid-to-liquid ratio of 10:100g/ml, and then place it in an ice-water bath according to the molar ratio of sugar unit/ ^IO ₄ Add sodium periodate at a ratio of 1:1 to obtain a mixed solution, stir the mixed solution for 4 hours at room temperature and in the dark, and dialyze the obtained solution in deionized water for 2-3 During the dialysis process, the deionized water was changed every 3~5h. After the dialysis was completed, freeze-dried to obtain a partially formylated dextran solid with a degree of formylation of 60% (that is, 60 out of 100 sugar units were oxidized). );

(2) Preparation of dextran hydrogel microspheres

Dissolve 0.1 g of the partially formylated dextran solid obtained in step (1) in 1.0 mL of deionized water to obtain an aqueous solution of formylated dextran with a concentration of 100 mg/mL. The aqueous solution is added to the cyclohexane solution in which the emulsifier is dissolved, ultrasonically emulsified to form an inverse microemulsion, then an amine crosslinking agent is added and stirred for 12 hours, and finally the precipitate is collected by centrifugation (5000rpm, 5min), and the precipitate is washed with ethanol for 5~ After 6 times, the chemically cross-linked dextran hydrogel microspheres were obtained, which were dispersed in 2.0ml deionized water and preserved; the morphology of the obtained gel microspheres was observed by scanning electron microscopy, and the results are shown in Figure 5: The dextran gel microspheres obtained under this condition have a regular spherical structure, and the particle size distribution is between 200 and 500nm; the amount of cyclohexane is 8.0mL, and the volume ratio of aldylated dextran aqueous solution to cyclohexane The ratio is 1:8, the emulsifier is Span80/Tween80 compound, the amount added is 2.0g for Span80, and 0.2g for Tween80 (the amount of emulsifier added is 35% of the mass of the cyclohexane solution), and the ultrasonic emulsification condition is: power 130W, amplitude 75%; the amine cross-linking agent is ethylenediamine, the amount added is 2:1 according to the molar ratio of the aldehyde group in the partially formylated dextran to the amino group in the amine cross-linking agent (about 12 μL) ;

(3) Reduction of dextran hydrogel microspheres

Add 50 mg of sodium borohydride to the chemically cross-linked dextran hydrogel microspheres dispersed in deionized water obtained in step (2) (C=N double in sodium borohydride and chemically cross-linked dextran hydrogel microspheres The molar ratio of the bond is about 4:1), stirred at room temperature for 24h, and finally collected the precipitate by centrifugation (5000rpm, 5min). The precipitate was washed with deionized water for 5-6 times to obtain the reduced dextran hydrogel Microspheres, dispersed in 2.0mL deionized water for storage.

Example 8

The structural formula of the dextran-based hydrogel microspheres can be expressed as follows:

.

The preparation method of the above-mentioned dextran-based hydrogel microspheres, its specific steps include as follows:

(1) Preparation of aldylated dextran

Dissolve 5.0g of dextran (dextran T40, molecular weight 40000g/mol) in water at room temperature according to the solid-to-liquid ratio of 10:100g/ml, and then place it in an ice-water bath according to the molar ratio of sugar unit/ ^IO ₄ Add sodium periodate at a ratio of 1:1 to obtain a mixed solution, stir the mixed solution at room temperature and in the dark for 4 hours, and dialyze the obtained solution in deionized water for 2 to 3 hours with a regenerated cellulose dialysis bag (molecular weight cut-off: 7kD). During the dialysis process, the deionized water was changed every 3~5h. After the dialysis was completed, freeze-dried to obtain a partially formylated dextran solid with a degree of formylation of 60% (that is, 60 out of every 100 sugar units were oxidized). );

(2) Preparation of dextran hydrogel microspheres

Dissolve 0.1 g of the partially formylated dextran solid obtained in step (1) in 1.0 mL of deionized water to obtain an aqueous solution of formylated dextran with a concentration of 100 mg/mL. The aqueous solution is added to the cyclohexane solution in which the emulsifier is dissolved, ultrasonically emulsified to form an inverse microemulsion, then an amine crosslinking agent is added and stirred for 12 hours, and finally the precipitate is collected by centrifugation (5000rpm, 5min), and the precipitate is washed with ethanol for 5~ After 6 times, the chemically cross-linked dextran hydrogel microspheres were obtained, which were dispersed in 2.0ml deionized water and preserved; the morphology of the obtained gel microspheres was observed by scanning electron microscopy, and the results are shown in Figure 5: The dextran gel microspheres obtained under this condition have a regular spherical structure, and the particle size distribution is between 200 and 500nm; the amount of cyclohexane is 8.0mL, and the volume ratio of aldylated dextran aqueous solution to cyclohexane The ratio is 1:8, the emulsifier is Span80/Tween80 compound, the amount added is 2.0g for Span80, and 0.2g for Tween80 (the amount of emulsifier added is 35% of the mass of the cyclohexane solution), and the ultrasonic emulsification condition is: power 130W, amplitude 75%; the amine cross-linking agent is PEI (the molecular weight of PEI is 600g/mol, and its solution concentration is 0.5g/mL), and the amount added is based on the cross-linking of aldehyde groups and amines in partially formylated dextran. The molar ratio of the amino group in the joint agent is added at 1:5;

(3) Reduction of dextran hydrogel microspheres

Add 50 mg of sodium borohydride to the chemically cross-linked dextran hydrogel microspheres dispersed in deionized water obtained in step (2) (C=N double in sodium borohydride and chemically cross-linked dextran hydrogel microspheres The molar ratio of the bond is about 10:1), stirred at room temperature for 24h, and finally collected the precipitate by centrifugation (5000rpm, 5min). The precipitate was washed with deionized water for 5-6 times to obtain the reduced dextran hydrogel Microspheres, dispersed in 2.0mL deionized water for storage.

The specific embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Variations.

Claims (7)

1. a preparation method of dextran-based hydrogel microspheres, the dextran-based hydrogel microspheres, the structural formula can be expressed as follows: , characterized in that the specific steps include the following: (1) Preparation of aldylated dextran Dissolve dextran in water at room temperature according to the solid-to-liquid ratio of 10:100g/ml, then place it in an ice-water bath and add sodium periodate according to the sugar unit/IO ₄ ^-molar ratio of 2:1~1:1 to obtain Mixed solution, the mixed solution was stirred and reacted at room temperature and protected from light for 2-6 hours, and the obtained solution was dialyzed in deionized water with a regenerated cellulose dialysis bag for 2-3 days, and the deionized water was changed every 3-5 hours during the dialysis process , freeze-dried after dialysis to obtain a partially aldylated dextran solid with an aldylation degree of 40-60%; (2) Preparation of dextran hydrogel microspheres Dissolve the partially formylated dextran solid obtained in step (1) in deionized water to obtain an aqueous solution of formylated dextran with a concentration of 100-200 mg/mL, and add the aqueous solution of formylated dextran to the In the cyclohexane solution in which the emulsifier is dissolved, emulsify to form a reverse microemulsion, then add an amine cross-linking agent and stir for 10-12 hours, and finally collect the precipitate by centrifugation, and wash the precipitate with ethanol to obtain a chemically cross-linked glucan Sugar hydrogel microspheres, dispersed in deionized water for storage; (3) Reduction of dextran hydrogel microspheres Add sodium borohydride to the chemically cross-linked dextran hydrogel microspheres dispersed in deionized water obtained in step (2), stir at room temperature for 24 hours, and finally collect the precipitate by centrifugation, and wash the precipitate with deionized water Obtain the reduced dextran hydrogel microspheres, disperse them in deionized water and store them; The amine cross-linking agent in the step (2) is any compound with two amino groups among ethylenediamine, lysine, and arginine, and the amount added is based on the partial formylated dextran solid The molar ratio of the aldehyde group to the amino group in the amine crosslinking agent is 1:1~2:1. 2. The preparation method of dextran-based hydrogel microspheres according to claim 1, characterized in that: the dextran in the step (1) is a dextran with a molecular weight of 10000g/mol~100000g/mol any kind of sugar. 3. The preparation method of dextran-based hydrogel microspheres according to claim 1, characterized in that: in the step (2), the volume ratio of the aqueous solution of aldylated dextran to cyclohexane is 1 :5~1:10. 4. The preparation method of dextran-based hydrogel microspheres according to claim 1, characterized in that: the emulsifier in the step (2) is polyoxyethylene pentanonylphenyl ether, lost Sorbitan oleate or Span80/Sorbitan monooleate polyoxyethylene ether compound, the amount of emulsifier added is 10-50% of the mass of cyclohexane. 5. The method for preparing dextran-based hydrogel microspheres according to claim 1, characterized in that: the emulsification in the step (2) is mechanical stirring emulsification or ultrasonic emulsification. 6. The preparation method of dextran-based hydrogel microspheres according to claim 1, characterized in that: the amine crosslinking agent in the step (2) has a molecular weight of 600g/mol~1800g/mol Any compound with two or more amino groups in the dendritic polyethylenimine, the addition is 2 according to the mol ratio of the aldehyde group in the partially formylated dextran solid and the amino group in the amine cross-linking agent: 1~1:5 added. 7. The preparation method of dextran-based hydrogel microspheres according to claim 1, characterized in that: in the step (3) sodium borohydride and chemically cross-linked dextran hydrogel microspheres The molar ratio of C=N double bond is 2:1~10:1.