A preparation method of photo-crosslinking chitosan-based composite hydrogel microspheres based on microfluidic technology

Abstract

The application discloses a preparation method of photocrosslinking chitosan-based composite hydrogel microspheres based on microfluidic technology and belongs to the technical field of biomedical materials. The method comprises the following steps: obtaining double-bonded chitosan by reacting methacrylic anhydride with chitosan; obtaining lipoic acid modified chitosan by reacting lipoic acid with chitosan; mixing the double-bonded chitosan and the lipoic acid modified chitosan to obtain an aqueous solution of chitosan, adding tris(2-carboxyethyl) phosphonium hydrochloride, poloxamer F-127, a photoinitiator, dopamine and methyl orange glycoside, and then performing stirring, centrifugal defoaming treatment to obtain a composite hydrogel precursor solution; and using microfluidic technology and photocuring technology to prepare composite hydrogel microspheres from the composite hydrogel precursor solution. The prepared hydrogel microspheres can effectively promote cell adhesion, and have good biocompatibility, water retention and mechanical compressibility.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and more specifically, to a method for preparing photocrosslinked chitosan-based composite hydrogel microspheres based on microfluidic technology. Background Technology

[0002] Currently, hydrogel microspheres are highly promising materials in the biomedical field, but problems still exist in cell adhesion, biocompatibility, water retention, and mechanical compressibility: 1) Cells are difficult to adhere, spread, and grow effectively on the surface of hydrogel microspheres, leading to problems such as decreased cell survival rate, slow proliferation, and inhibited functional expression, thus reducing their effect on promoting tissue regeneration; 2) The introduction of cross-linking agents to improve mechanical properties can cause biotoxicity; 3) Water in hydrogel microspheres easily evaporates in the environment, leading to dehydration and shrinkage, and a decrease in their flexibility and swelling capacity; 4) Traditional hydrogel microspheres cannot withstand large pressures or deformations, directly affecting their service life. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing photocrosslinked chitosan-based composite hydrogel microspheres based on microfluidic technology. The composite hydrogel microspheres prepared by this method can effectively promote cell adhesion and have good biocompatibility, water retention and mechanical compressibility.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A method for preparing photocrosslinked chitosan-based composite hydrogel microspheres based on microfluidic technology includes the following steps:

[0006] (1) Double-bonded chitosan is obtained by reacting methacrylic anhydride with chitosan;

[0007] (2) By reacting lipoic acid with chitosan, lipoic acid-modified chitosan is obtained;

[0008] (3) Mix double-bonded chitosan with thioctic acid-modified chitosan to obtain an aqueous solution of chitosan. Add tris(2-carbonylethyl)phosphohydrochloride, poloxamer F-127, photoinitiator, dopamine, and methyl hesperidin to react. After stirring and centrifugation to defoam, a composite hydrogel precursor solution is obtained.

[0009] (4) The composite hydrogel precursor liquid was made into composite hydrogel microspheres using microfluidic technology and photocuring technology.

[0010] Further, step (1) specifically involves dissolving chitosan in glacial acetic acid solution to obtain a first chitosan glacial acetic acid solution, adding methanol and methacrylic anhydride to react, and then purifying by dialysis and freeze-drying to obtain double-bonded chitosan.

[0011] Furthermore, the concentration of the glacial acetic acid solution is 0.1-0.2 mol / L, the mass-volume fraction of chitosan in the first chitosan glacial acetic acid solution is 0.5-5%, the volume ratio of methanol to the first chitosan glacial acetic acid solution is (1-5):1, the mass ratio of methacrylic anhydride to chitosan is 1:(1-5), and the reaction time is 12-24 h.

[0012] Further, step (2) specifically involves dissolving chitosan in glacial acetic acid solution to obtain a second chitosan glacial acetic acid solution, then sequentially adding anhydrous ethanol solution of thioctic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide for reaction. After the reaction, the chitosan is purified by dialysis and freeze-dried to obtain thioctic acid modified chitosan.

[0013] Furthermore, the volume fraction of glacial acetic acid in the glacial acetic acid solution is 1-2%, and the mass volume fraction of chitosan in the second chitosan glacial acetic acid solution is 1-5%; the volume ratio of the second chitosan glacial acetic acid solution to the anhydrous ethanol solution of lipoic acid is (3-1):1; the reaction time is 12-24 h; and the mass ratio of chitosan, lipoic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 6:6:4:1.

[0014] Furthermore, in step (3), the mass ratio of double-bonded chitosan to thioctic acid-modified chitosan is 3:7; tris(2-carbonylethyl)phosphohydrochloride accounts for 0.15 wt% of the chitosan aqueous solution; poloxamer F-127 accounts for 2 wt% of the chitosan aqueous solution; the photoinitiator is one or more of phenyl(2,4,6-trimethylbenzoyl)phosphite, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone and 2,4,6-trimethylbenzoyl-diphenylphosphine oxide, accounting for 0.4 wt% of the chitosan aqueous solution; dopamine accounts for 2 wt% of the chitosan aqueous solution; and methyl hesperidin accounts for 1 wt% of the chitosan aqueous solution.

[0015] Further, step (4) specifically involves: using the composite hydrogel precursor liquid as the dispersed phase and oleic acid as the continuous phase, passing the dispersed phase and the continuous phase into a microfluidic device to mix and shear the dispersed phase and the continuous phase into droplets, curing the droplets by UV irradiation, and washing and centrifuging the cured droplets to obtain composite hydrogel microspheres.

[0016] Furthermore, the flow rate ratio of the continuous phase to the dispersed phase is (2~5):2; the shear orifice size is 50~200μm.

[0017] Furthermore, the ultraviolet lamp has a wavelength of 365nm.

[0018] In summary, the present invention has the following beneficial effects:

[0019] The hydrogel microspheres prepared by this invention can effectively promote cell adhesion, thereby promoting efficient cell proliferation and functional expression. The hydrogel microspheres prepared by this invention have good biocompatibility, high mechanical compressibility and water retention, further improving the comprehensive performance of hydrogel microspheres in biomedical applications. Attached Figure Description

[0020] Figure 1 This is a reaction diagram of the process in Example 1 to obtain double-bonded chitosan from chitosan using methacrylic anhydride;

[0021] Figure 2 This is a reaction diagram of obtaining lipoic acid modified chitosan from chitosan using lipoic acid in Example 1;

[0022] Figure 3 This is a microscope image of the microfluidic device continuously shearing the dispersed phase in Example 1.

[0023] Figure 4 This is a microscope image of the sheared droplets flowing in the microchannel of the microfluidic device in Example 1.

[0024] Figure 5 The image shows an electron microscope image of the composite hydrogel microspheres prepared in Example 1.

[0025] Figure 6 The 1H NMR spectra of chitosan, double-bonded chitosan, and thioctic acid-modified chitosan in Example 1 are shown.

[0026] Figure 7 The Fourier transform infrared spectra of chitosan, double-bonded chitosan, and thioctic acid-modified chitosan in Example 1 are shown. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] A method for preparing a photocrosslinked chitosan-based composite hydrogel based on microfluidic technology includes the following steps:

[0029] (1) Chitosan powder (CS) was added to a glacial acetic acid solution with a concentration of 0.1~0.2mol / L to obtain a first chitosan glacial acetic acid solution with a chitosan mass volume fraction of 0.5~5%. Methanol (the volume ratio of methanol to the first chitosan glacial acetic acid solution is (1~5):1) and methacrylic anhydride (the mass ratio of methacrylic anhydride to chitosan powder is 1:(1~5)) were added and reacted for 12-24h. The mixture was then placed in a dialysis bag and dialyzed in deionized water (the volume ratio of deionized water to the first chitosan glacial acetic acid solution is 1~10:10~50) for 5 days. After freeze-drying, double-bonded chitosan (MC) was obtained.

[0030] (2) Chitosan powder is dispersed and dissolved in 1-2% volume fraction of glacial acetic acid solution. After stirring and dissolving, a second chitosan glacial acetic acid solution with a mass volume fraction of 1-5% is obtained. Anhydrous ethanol solution of lipoic acid is added sequentially (the volume ratio of the second chitosan glacial acetic acid solution to the anhydrous ethanol solution of lipoic acid is (1-3):1), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide are reacted (the mass ratio of chitosan, lipoic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 6:6:4:1). After stirring and reacting for 12-24 hours, the mixture is placed in a dialysis bag and dialyzed in deionized water (the volume ratio of deionized water to the second chitosan glacial acetic acid solution is 1-10:10-100) for 5 days. After freeze-drying, lipoic acid modified chitosan (LACS) is obtained.

[0031] (3) Add MC and LACS (mass ratio of MC to LACS is 3:7) to deionized water to obtain a chitosan aqueous solution with a total concentration of 7 wt% of MC and LACS. Add tris(2-carbonylethyl)phosphohydrochloride (TCEP·HCl; accounting for 0.15 wt% of the chitosan aqueous solution) and stir for 15-20 minutes. Add poloxamer F-127 (accounting for 2 wt% of the chitosan aqueous solution) and stir slowly until completely dissolved to prevent excessive foaming. Then add the photoinitiator [phenyl(2,4,6-trimethyl)-2-methyl-2-ethyl ... One or more of the following are selected: lithium benzoyl phosphate (LAP blue light initiator), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (photoinitiator 2959), and 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (photoinitiator 819); the photoinitiator accounts for 0.4 wt% of the chitosan aqueous solution. Dopamine (accounting for 2 wt% of the chitosan aqueous solution) and methyl hesperidin (accounting for 1 wt% of the chitosan aqueous solution) are added, and the mixture is stirred evenly and then centrifuged to remove foam, thereby obtaining the composite hydrogel precursor solution.

[0032] (4) Using oleic acid as the continuous phase and the composite hydrogel precursor liquid as the dispersed phase, the dispersed phase and the continuous phase are pushed into the microfluidic device (the flow rate ratio of the continuous phase and the dispersed phase is (2~5):2; the shearing orifice size is 50~200μm). The continuous phase is sheared with the dispersed phase, and the microchannel outlet of the microfluidic device is irradiated with a 365nm ultraviolet lamp to solidify the product. The solidified droplet-like product is collected with a centrifuge tube, and ethanol (the volume ratio of ethanol to the solidified product is 2:1) is added for washing and centrifugation to obtain microsphere precipitate. After washing with deionized water, the composite hydrogel microspheres are obtained.

[0033] Example 1

[0034] (1) 1g of chitosan powder (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number C105799) was added to 50mL of 0.175mol / L glacial acetic acid solution to obtain a first chitosan glacial acetic acid solution with a chitosan mass-volume fraction of 2%. 50mL of methanol and 0.42g of methacrylic anhydride were added, and the solution was reacted for 18h. The solution was then placed in a dialysis bag and dialyzed in 2000mL of deionized water for 5 days. After freeze-drying, double-bonded chitosan (MC) was obtained. Figure 1 The diagram shows the reaction scheme for obtaining double-bonded chitosan from chitosan using methacrylic anhydride.

[0035] (2) 1.2 g of chitosan powder was dispersed and dissolved in 60 mL of 1.5% glacial acetic acid solution. After stirring and dissolving, a second chitosan glacial acetic acid solution with a mass-volume fraction of 2% was obtained. 40 mL of anhydrous ethanol solution containing 1.2 g of lipoic acid, 0.8 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.2 g of N-hydroxysuccinimide were added sequentially. After stirring for 18 h, the solution was placed in a dialysis bag and dialyzed in 2000 mL of deionized water for 5 days. After freeze-drying, lipoic acid-modified chitosan (LACS) was obtained. Figure 2 The diagram shows the reaction formula for obtaining lipoic acid-modified chitosan from chitosan using lipoic acid.

[0036] (3) Add MC and LACS (the mass ratio of MC and LACS is 3:7) to deionized water to obtain a chitosan aqueous solution with a total concentration of 7wt% of MC and LACS. Add tris(2-carbonylethyl)phosphohydrochloride (0.15wt% of the chitosan aqueous solution) and stir for 15 minutes. Add poloxamer F-127 (2wt% of the chitosan aqueous solution) and stir slowly until completely dissolved to prevent excessive foaming. Then add LAP blue light initiator (0.4wt% of the chitosan aqueous solution), methyl hesperidin (1wt% of the chitosan aqueous solution; purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number B20593) and dopamine (2wt% of the chitosan aqueous solution; purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S72977). After stirring evenly, centrifuge to defoam and obtain the composite hydrogel precursor solution.

[0037] (4) Using oleic acid as the continuous phase and the composite hydrogel precursor as the dispersed phase, the dispersed phase and the continuous phase are respectively pushed into a microfluidic device (the flow rates of the continuous phase and the dispersed phase are 5:2; the shearing aperture size is 100 μm), and the continuous phase is sheared against the dispersed phase (e.g., ...). Figure 3 The image shown is a microscope image taken during shearing; as shown Figure 4 The diagram shows the flow of sheared droplets in the microchannel of a microfluidic device. The product was solidified by irradiating the outlet of the microchannel with a 365nm UV lamp. The solidified droplet-like product was collected in a centrifuge tube, and ethanol (2:1 volume ratio of ethanol to solidified product) was added for washing and centrifugation to obtain microsphere precipitate. After washing with deionized water, composite hydrogel microspheres (such as...) were obtained. Figure 5 (As shown).

[0038] Nuclear magnetic resonance (NMR) 1H spectra of chitosan, double-bonded chitosan, and lipoic acid-modified chitosan were analyzed (e.g.) Figure 6 As shown in the image): Compared to chitosan (CS), the NMR spectrum of double-bonded chitosan (MC) shows a distinct new peak in the chemical shift range of 5.0-6.0 ppm, a region exclusive to olefinic hydrogen (=CH2). In the CS spectrum, this region only has a flat baseline, while the MC spectrum shows a clear peak signal, indicating successful double bond grafting.

[0039] Compared to chitosan (CS), the NMR spectrum of lipoic acid-modified chitosan (LACS) showed new, resolvable characteristic peaks in the characteristic chemical shift range (δ 4.0–4.5 ppm). These new signals are attributed to proton resonances between the disulfide bonds and protons adjacent to the carboxyl group in the lipoic acid molecule. The chemical shifts of the methylene protons (-O-CO-CH2-) attached to the carboxyl group and the methine protons (-CH-SS-) on the dithiopentyl ring typically fall within the 4.0–4.5 ppm range. Due to the deshielding effect of the neighboring highly electronegative oxygen and sulfur atoms, the resonance signals of these protons shift to a lower field (i.e., higher ppm values). Therefore, it can be concluded that lipoic acid has been successfully grafted onto chitosan.

[0040] Fourier transform infrared spectroscopy (FTIR) was performed on chitosan, double-bonded chitosan, and thioctic acid-modified chitosan (e.g. Figure 7 As shown): Double-bonded chitosan (MC) at 1656.33 cm⁻¹ -1 The absorption peak at this point may correspond to the stretching vibration of a carbon-carbon double bond (C=C) or the amide I band (C=O stretching). If the grafting involves unsaturated groups (such as acryloyl groups), this peak indicates the introduction of a double bond. 1590.53 cm⁻¹ -1 The absorption peak at 1656.33 cm⁻¹ may be related to the amide II band (NH bending and CN stretching) or double bond-related vibrations, further indicating the presence of double bond functional groups. The shifts of these new or existing peaks suggest the introduction of double-bonded groups into the chitosan molecular chain. -1 and 1580.53cm -1 The characteristic peaks confirmed the successful grafting of the double bond.

[0041] Lipoic acid-modified chitosan (LACS) at 1764.62 cm⁻¹ -1 A completely new absorption peak appears at this position, while CS shows no characteristic peak at this location. 1764.62cm -1 The absorption peak at this point is usually attributed to the stretching vibration of the carbonyl group (C=O), which is a typical characteristic of ester bonds (-COOR) or carboxylic acids (-COOH). Since lipoic acid contains a carboxyl group, the appearance of this new peak indicates that lipoic acid has been successfully grafted onto the hydroxyl (-OH) or amino (-NH2) groups of chitosan via esterification, forming an ester bond structure.

[0042] Analysis using 1H NMR spectroscopy confirmed the successful grafting of lipoic acid onto the chitosan molecular chain. Therefore, both 1H NMR and Fourier transform infrared spectroscopy confirmed the successful modification of double-bonded chitosan with lipoic acid.

[0043] The performance of the composite hydrogel microspheres obtained in Example 1 was tested:

[0044] Biocompatibility testing:

[0045] Biocompatibility (expressed as cell viability) was tested using the CCK-8 kit (purchased from Beijing Lanbolide Trading Co., Ltd., catalog number CK001) by in vitro culture of L929 mouse fibroblast suspension with hydrogel microsphere extract.

[0046] The biocompatibility test was divided into three groups: experimental group, control group, and blank group.

[0047] The biocompatibility test of the experimental group includes the following steps:

[0048] (1) Add 100 μL of L929 mouse fibroblast suspension to each well of a 96-well plate (L929 mouse fibroblasts were added to DMEM medium to prepare a cell concentration of 1×10⁻⁶ cells). 4 L929 mouse fibroblast suspension (cells / mL) was used to pre-culture the culture plate in an incubator (at 37°C and 5% CO2) for 24 hours.

[0049] (2) Add 100 μL of hydrogel microsphere extract (incubate hydrogel microspheres at 37°C for 24 h in DMEM medium at an extraction rate of 20 mg / mL, then sterilize through a 0.22 μm filter and add 10% fetal bovine serum) to the culture plate after pre-culture and incubate in an incubator (at 37°C and 5% CO2) for 96 h;

[0050] (3) Add 10 μL of CCK-8 solution to each well (add slowly to prevent air bubbles from affecting the experiment), and incubate the culture plate in an incubator (at 37°C and 5% CO2) for 4 hours;

[0051] (4) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0052] The control group was tested in the same way as the experimental group, except that it did not undergo step (2).

[0053] The test procedure for the blank group was the same as that for the control group, except that in step (1), the same volume of L929 mouse fibroblast suspension was replaced with DMEM culture medium; and the treatment in step (2) was not performed.

[0054] Biocompatibility is evaluated by cell viability, and the formula for calculating cell viability is as follows:

[0055] Cell viability = [(A 实验组 -A 空白组 ) / (A 对照组 -A 空白组 )]×100%.

[0056] The calculation showed that the cell survival rate of the experimental group was 100%, indicating that the composite hydrogel microspheres prepared in Example 1 have high biocompatibility.

[0057] Mechanical compressibility test:

[0058] a. Add MC and LACS (mass ratio of MC to LACS is 3:7) to deionized water to obtain a chitosan aqueous solution with a total concentration of 7wt% of MC and LACS. Then add tris(2-carbonylethyl)phosphohydrochloride (0.15wt% of chitosan aqueous solution) to obtain MC / LACS test solution.

[0059] b. Mix the prepared composite hydrogel microspheres with MC / LACS test solution (the mass ratio of composite hydrogel microspheres to MC / LACS test solution is 1:2), pour the mixture into a cylindrical mold, and cure it under a 420nm ultraviolet lamp to obtain a hydrogel sample.

[0060] c. Place the hydrogel sample in the middle of the table of the high and low temperature dual column tester, set the compression deformation to 90%, the compression speed to 2 mm / min, start the compression test when the compression clamp is close to the upper and lower surfaces of the hydrogel, and stop the test when the hydrogel sample is completely broken. Each group of samples is tested 3 times and the average value is taken.

[0061] The test results are as follows: the compressive modulus of hydrogel sample 1 is 43.2 kPa.

[0062] Comparative Example 1

[0063] The operation steps are the same as in Example 1, except that methyl hesperidin is not added in step (3).

[0064] Comparative Example 2

[0065] The operation steps are the same as in Example 1, except that dopamine is not added in step (3).

[0066] Comparative Example 3

[0067] The operation steps are the same as in Example 1, except that methyl hesperidin and dopamine are not added in step (3).

[0068] Cell adhesion properties of the composite hydrogel microspheres obtained in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were tested.

[0069] The composite hydrogel microspheres obtained from Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were grafted into 12-well plates (3 wells each for Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3, 100 μL per well). Mouse embryonic fibroblast cell line (purchased from Wuhan Shangen Biotechnology Co., Ltd., catalog number SNL-025) was added at 5 × 10⁻⁶ cells / well. 4Cells were seeded at a density of 1 / mL in 12-well plates containing hydrogel microspheres. DMEM cell culture medium (containing 1.5 g / L sodium bicarbonate, 100 U / mL penicillin, 10% fetal bovine serum, and 100 μg / mL streptomycin) was then added to each well to a final volume of 2 mL. The 12-well plates were incubated at 37°C with 5% CO2 for 24 hours. The number of floating cells in the culture medium was counted, and the results are as follows: In Example 1, the number of floating cells was 1.9 × 10⁻⁶. 3 The number of floating cells in control group 1 was 2.5 × 10⁶; the number of floating cells in control group 1 was 2.5 × 10⁶. 3 The number of floating cells in the control group was 3.6 × 10⁶; the number of floating cells in the control group 2 was 3.6 × 10⁶. 3 The number of floating cells in the three control groups was 1.8 × 10⁶; the number of floating cells in the control group was 1.8 × 10⁶. 4 indivual.

[0070] The water retention properties of the composite hydrogel microspheres obtained in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were tested.

[0071] a. Add MC and LACS (MC to LACS in a mass ratio of 3:7) to deionized water to obtain an aqueous solution of silk fibroin and hyaluronic acid with a total MC and LACS concentration of 7 wt%. Then add tris(2-carbonylethyl) phosphate hydrochloride (0.15 wt% of the aqueous solution of silk fibroin and hyaluronic acid) to obtain MC / LACS test solution 1. Follow the steps in a to obtain MC / LACS test solution 2, MC / LACS test solution 3, MC / LACS test solution 4, and MC / LACS test solution 5.

[0072] b. Pour MC / LACS test solution 1 into a cylindrical mold and cure it under a 420nm UV lamp to obtain hydrogel sample 1;

[0073] The composite hydrogel microspheres prepared in Example 1 were mixed and stirred with MC / LACS test solution 2 (the mass ratio of composite hydrogel microspheres to MC / LACS test solution 1 was 1:2), poured into a cylindrical mold, and cured under a 420nm ultraviolet lamp to obtain hydrogel sample 2.

[0074] The composite hydrogel microspheres prepared in Comparative Example 1 were mixed and stirred with MC / LACS test solution 3 (the mass ratio of composite hydrogel microspheres to MC / LACS test solution 1 was 1:2), poured into a cylindrical mold, and cured under a 420nm ultraviolet lamp to obtain hydrogel sample 3.

[0075] The composite hydrogel microspheres prepared in Comparative Example 2 were mixed and stirred with MC / LACS test solution 4 (the mass ratio of composite hydrogel microspheres to MC / LACS test solution 1 was 1:2), poured into a cylindrical mold, and cured under a 420nm ultraviolet lamp to obtain hydrogel sample 4.

[0076] The composite hydrogel microspheres prepared in Comparative Example 3 were mixed and stirred with MC / LACS test solution 5 (the mass ratio of composite hydrogel microspheres to MC / LACS test solution 1 was 1:2), poured into a cylindrical mold, and cured under a 420nm ultraviolet lamp to obtain hydrogel sample 5.

[0077] c. Remove moisture from hydrogel samples 1 to 5, weigh them separately, record their weights, and then place them at room temperature for 4 days, recording their weights again.

[0078] The test results are as follows: hydrogel sample 1 can retain 33% of its mass, hydrogel sample 2 can retain 69% of its mass, hydrogel sample 3 can retain 51% of its mass, hydrogel sample 4 can retain 47% of its mass, and hydrogel sample 5 can retain 38% of its mass. This indicates that hydrogel microspheres can improve water retention performance, and hydrogel microspheres with added dopamine and methyl hesperidin are even more effective in water retention.

[0079] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing photocrosslinked chitosan-based composite hydrogel microspheres based on microfluidic technology, characterized in that, Includes the following steps: (1) Double-bonded chitosan is obtained by reacting methacrylic anhydride with chitosan; (2) By reacting lipoic acid with chitosan, lipoic acid-modified chitosan is obtained; (3) Mix double-bonded chitosan with thioctic acid-modified chitosan to obtain an aqueous solution of chitosan. Add tris(2-carbonylethyl)phosphohydrochloride, poloxamer F-127, photoinitiator, dopamine, and methyl hesperidin to react. After stirring and centrifugation to defoam, a composite hydrogel precursor solution is obtained. The mass ratio of double-bonded chitosan to lipoic acid-modified chitosan was 3:7; tris(2-carbonylethyl)phosphohydrochloride accounted for 0.15 wt% of the chitosan aqueous solution; poloxamer F-127 accounted for 2 wt% of the chitosan aqueous solution; the photoinitiator was one or more of phenyl(2,4,6-trimethylbenzoyl)phosphite, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, and 2,4,6-trimethylbenzoyl-diphenylphosphine oxide, accounting for 0.4 wt% of the chitosan aqueous solution; dopamine accounted for 2 wt% of the chitosan aqueous solution; and methyl hesperidin accounted for 1 wt% of the chitosan aqueous solution. (4) Composite hydrogel microspheres were prepared from the composite hydrogel precursor solution using microfluidic technology and photocuring technology; Step (4) specifically involves: using the composite hydrogel precursor liquid as the dispersed phase and oleic acid as the continuous phase, introducing the dispersed phase and the continuous phase into a microfluidic device to mix and shear the dispersed phase and the continuous phase into droplets, curing the droplets by UV irradiation, and washing and centrifuging the cured droplets to obtain composite hydrogel microspheres.

2. The method for preparing photocrosslinked chitosan-based composite hydrogel microspheres based on microfluidic technology according to claim 1, characterized in that, The specific steps (1) are as follows: Chitosan is dissolved in glacial acetic acid solution to obtain a first chitosan glacial acetic acid solution, methanol and methacrylic anhydride are added to react, and after the reaction, the chitosan is purified by dialysis and freeze-dried to obtain double-bonded chitosan.

3. The method for preparing photocrosslinked chitosan-based composite hydrogel microspheres based on microfluidic technology according to claim 2, characterized in that, The concentration of the glacial acetic acid solution is 0.1-0.2 mol / L, and the mass-volume fraction of chitosan in the first chitosan glacial acetic acid solution is 0.5-5%; the volume ratio of methanol to the first chitosan glacial acetic acid solution is (1-5):1; the mass ratio of methacrylic anhydride to chitosan is 1:(1-5), and the reaction time is 12-24 h.

4. The method for preparing photocrosslinked chitosan-based composite hydrogel microspheres based on microfluidic technology according to claim 1, characterized in that, The specific steps (2) are as follows: Chitosan is dissolved in glacial acetic acid solution to obtain a second chitosan glacial acetic acid solution, and anhydrous ethanol solution of thioctic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added in sequence to react. After the reaction, the chitosan is purified by dialysis and freeze-dried to obtain thioctic acid modified chitosan.

5. The method for preparing photocrosslinked chitosan-based composite hydrogel microspheres based on microfluidic technology according to claim 4, characterized in that, The volume fraction of glacial acetic acid in the glacial acetic acid solution is 1-2%, and the mass-volume fraction of chitosan in the second chitosan glacial acetic acid solution is 1-5%; the volume ratio of the second chitosan glacial acetic acid solution to the anhydrous ethanol solution of thioctic acid is (3-1):1; the reaction time is 12-24 h; the mass ratio of chitosan, thioctic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 6:6:4:

1.

6. The method for preparing photocrosslinked chitosan-based composite hydrogel microspheres based on microfluidic technology according to claim 1, characterized in that, The flow rate ratio of the continuous phase to the dispersed phase is (2~5):2; the shear orifice size is 50~200μm.

7. The method for preparing photocrosslinked chitosan-based composite hydrogel microspheres based on microfluidic technology according to claim 1, characterized in that, The wavelength of the ultraviolet lamp is 365nm.

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