Dual-crosslinking hydrogel microsphere as well as preparation method and application thereof

Abstract

The invention discloses a double-crosslinking hydrogel microsphere as well as a preparation method and application thereof. The preparation method of the double cross-linked hydrogel microspheres comprises the following steps: mixing methacrylated silk fibroin and phenylboronic acid-low molecular weight heparin, adding (OC-6-44)-tricarbonyl chloride (glycinyl) ruthenium and a photoinitiator, and carrying out ultraviolet irradiation to form the double cross-linked hydrogel microspheres; wherein the phenylboronic acid-low molecular weight heparin is obtained by carrying out condensation reaction on low molecular weight heparin and aminated phenylboronic acid. The double-crosslinked hydrogel microspheres have the functions of inhibiting bacterial growth and promoting corneal repair, and are suitable for various bacterial keratitis, especially refractory keratitis caused by drug-resistant bacterial infection.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a double cross-linked hydrogel microsphere and a preparation method and application thereof. Background Art

[0002] Refractory keratitis is considered one of the leading causes of visual impairment and blindness worldwide because of the complexity and drug resistance of its pathogens, which makes corneal infection difficult to control and corneal inflammation persists. As bacteria become more resistant to traditional antibiotics, the efficacy of conventional antibiotics is gradually weakening. In addition, when bacterial infection is controlled, corneal tissue enters a repair period, and existing drugs have limited ability to eliminate multiple pro-inflammatory factors such as IL-6 and reduce persistent corneal inflammatory responses, making the cure of refractory keratitis still a difficult problem. Summary of the invention

[0003] In order to solve the deficiencies in the prior art, the present invention aims to provide a double cross-linked hydrogel microsphere and its preparation method and use. The double cross-linked hydrogel microsphere of the present invention mainly comprises three components: methacrylylated silk fibroin (SFMA), phenylboronic acid (PBA), low molecular weight heparin (LWMH), and (OC-6-44)-tricarbonyl chloride (glycine) ruthenium (CORM-3). Low molecular weight heparin contains abundant carboxyl groups and can be bonded with amino PBA through condensation reaction; CORM-3 is water-soluble and can finally form double cross-linked hydrogel microspheres through self-assembly and ultraviolet cross-linking under the operation of a microfluidic device.

[0004] The specific technical solutions of the present invention are as follows:

[0005] The invention provides a method for preparing double cross-linked hydrogel microspheres, comprising the steps of: mixing methacrylylated silk fibroin and phenylboronic acid-low molecular weight heparin, adding (OC-6-44)-tricarbonyl chloride (glycine) ruthenium and a photoinitiator, and irradiating with ultraviolet light to form double cross-linked hydrogel microspheres; wherein the phenylboronic acid-low molecular weight heparin is obtained by condensation reaction of low molecular weight heparin and aminated phenylboronic acid.

[0006] Furthermore, the concentration ratio of the methacrylylated silk fibroin to phenylboronic acid-low molecular weight heparin is 1:1.

[0007] Further, the concentration of the methacrylylated silk fibroin is 10-90% (W / V);

[0008] The concentration of the phenylboronic acid-low molecular weight heparin is 10-90% (W / V).

[0009] Further, the concentration of the (OC-6-44)-tricarbonyl chloride (glycinyl) ruthenium is 1-10 mM;

[0010] And / or, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

[0011] Furthermore, the preparation method of the methacrylylated silk fibroin comprises: dissolving the degummed silk fibroin in a lithium bromide solution, then slowly adding glycidyl methacrylate at 200-400 rpm and 60° C., and continuing the reaction for 3-4 hours; after dialyzing with double distilled water, using PEG reverse dialysis to obtain the methacrylylated silk fibroin.

[0012] Furthermore, after the degummed silk fibroin is dissolved in a lithium bromide solution, the concentration of the silk fibroin is 1%-10% (W / V);

[0013] The concentration of the glycidyl methacrylate is 0.1-0.5M.

[0014] Furthermore, the preparation method of phenylboronic acid-low molecular weight heparin includes: mixing 3-aminophenylboronic acid, low molecular weight heparin and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride dissolved in phosphate buffer, reacting at 200-400rpm and 37°C for 1-2 days, dialysis with double distilled water, and using PEG reverse dialysis to obtain phenylboronic acid-low molecular weight heparin.

[0015] Furthermore, the concentration of the 3-aminophenylboronic acid is 15 mM, the concentration of the low molecular weight heparin is 3-5 M, and the concentration of the 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is 0.09-0.1 mM.

[0016] The present invention also provides double cross-linked hydrogel microspheres prepared by the preparation method.

[0017] The present invention also provides the use of the double cross-linked hydrogel microspheres in preparing the drug described in any one of (1) to (3):

[0018] (1) Drugs for preventing and / or treating bacterial keratitis;

[0019] (2) Drugs for preventing and / or treating refractory keratitis caused by drug-resistant bacterial infection;

[0020] (3) Drugs that inhibit bacterial growth.

[0021] The beneficial effects of the present invention are:

[0022] The double cross-linked hydrogel microspheres provided by the present invention are formed by double cross-linking of phenylboronic acid, low molecular weight heparin and methacrylylated silk fibroin, and have the functions of inhibiting bacterial growth and promoting corneal repair, and are suitable for various bacterial keratitis, especially refractory keratitis caused by drug-resistant bacterial infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the antibacterial properties of double cross-linked hydrogel;

[0024] Figure 2 CO release for hydrogel microspheres with different concentrations;

[0025] Figure 3 Hydrogel microspheres promote healing of keratitis in mice. DETAILED DESCRIPTION

[0026] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0027] Example 1

[0028] This embodiment provides a method for preparing double cross-linked hydrogel microspheres, comprising the following steps:

[0029] (1) Preparation of methacrylylated silk fibroin (SFMA)

[0030] The cocoons were cut into pieces, boiled in 0.02M sodium carbonate solution at 98°C for 1h, repeated twice to degummed the silk, and then soaked and washed in double distilled water (Millipore Milli-Q system) for 5 times; the degummed silk fibroin (SF) was dissolved in 9.3M lithium bromide solution at 65°C for 2h, and the silk fibroin concentration was adjusted to 10% (W / V); 0.5M glycidyl methacrylate was slowly added dropwise at 300rpm and 60°C, and the reaction was continued for 3h. After dialyzing with double distilled water for 3 days, a high concentration of methacrylylated silk fibroin aqueous solution (concentration greater than 8%) was obtained using PEG reverse dialysis.

[0031] (2) Preparation of phenylboronic acid-low molecular weight heparin (PBA-LWMH)

[0032] 15 mM 3-aminophenylboronic acid, 5 M LWMH and 0.1 mM 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) dissolved in phosphate buffer were mixed and reacted at 300 rpm and 37°C for 1 day. After dialysis against double distilled water for 2 days, a high concentration PBA-LWMH solution (concentration of 15%) was obtained using PEG reverse dialysis.

[0033] (3) Preparation of double cross-linked hydrogel microspheres

[0034] A 15% (W / V) SFMA solution was mixed with a PBA-LWMH solution of the same concentration in a volume ratio of 7:3, and then 1.7 mM (OC-6-44)-tricarbonyl chloride (glycine) ruthenium (CORM-3) and 0.4% weight percent photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) were added, and the mixture was irradiated with UV light for 45 s under the operation of a microfluidic device to form double-cross-linked hydrogel microspheres (SFMA / PBA-LWMH@CORM-3).

[0035] Example 2

[0036] The double cross-linked hydrogel microspheres of the present invention contain CORM-3 and have good antibacterial efficacy. This example proves that the double cross-linked hydrogel microspheres containing CORM-3 prepared in Example 1 have good antibacterial effects through bacterial culture experiments. The specific experimental steps are: take 1mL of hydrogel, add 5mL of Pseudomonas aeruginosa bacterial solution in the logarithmic growth phase, culture overnight on a shaker at 37°C, take the upper bacterial solution for plate coating, and calculate the number of colonies after 6 hours.

[0037] The results are as follows Figure 1 As shown, H@C represents the double cross-linked hydrogel microspheres prepared in Example 1, C represents CORM-3 alone, and H represents the double cross-linked hydrogel microspheres without CORM-3.

[0038] Example 3

[0039] The double cross-linked hydrogel microspheres of the present invention are uniform, monodisperse, have a designed size and a porous microstructure, and the encapsulated CORM-3 molecules can effectively release CO and prolong its residence time on the ocular surface, thereby improving the therapeutic effect. This embodiment adopts the myoglobin detection method, and a certain amount of double cross-linked hydrogel microsphere freeze-dried powder prepared by the preparation method of Example 1 is added to the deoxy horse skeletal myoglobin (Deoxy-Mb) buffer reduced by an excess of sodium dithionite, and a small amount of mineral oil is added to seal the liquid. The conversion process is reflected by a spectrophotometer, and the absorbance of the solution is tested multiple times at a fixed time in the range of 500-600nm to draw a CO release curve.

[0040] The results are as follows Figure 2 As shown, the meanings of 9:1, 7:3, and 5:5 in the figure are: different hydrogel prepolymers formed by mixing SFMA solution of the same concentration (15% w / v) and PBA-LWMH solution in volume ratios of 9:1, 7:3, and 5:5 respectively are used to encapsulate CORM-3.

[0041] Example 4

[0042] The double cross-linked hydrogel microspheres of the present invention have excellent biocompatibility and significantly promote the fight against keratitis caused by multidrug-resistant Staphylococcus aureus (MASR) infection. Taking the double cross-linked hydrogel microspheres (SFMA / PBA-LWMH@CORM-3) prepared by the preparation method of Example 1 as an example, its promoting effect on the healing of mouse keratitis was verified. Construction and treatment of bacterial keratitis in mice: 10 μL (3×10 8 CFU / mL) of MRSA suspension was inoculated on the mechanically damaged mouse cornea to construct bacterial keratitis in mice. After 24 hours of infection, the corneal infection was observed under diffuse light of a slit lamp, and the mice that were successfully infected were screened. The mice with bacterial keratitis were then randomly divided into 5 groups (NC group, H group, C group, ToBrex group, and H@C group). The NC group used sterile PBS, the H group used hydrogel microspheres (SFMA / PBA-LWMH) suspended in sterile PBS, the C group used CORM-3 (1 mg / mL) dissolved in sterile PBS, the ToBrex group used commercially available drugs: tobramycin eye drops (Tobrex), and the H@C group used hydrogel microspheres (SFMA / PBA-LWMH@CORM-3) encapsulated with CORM-3 (1 mg / ml) suspended in sterile PBS. All patients were treated with topical eye drops, twice a day, 5 μL of the corresponding group of eye drops per eye each time, for a total of 7 days, and the corneal infection was observed and graded using the diffuse light of a slit lamp microscope on days 0, 1, 3, 5, and 7 of topical eye drops. The rating criteria are as follows: turbid area: grade 1 (1-25% of the total corneal area), grade 2 (26-50%), grade 3 (51-75%), and grade 4 (76-100%). Turbidity: grade 1 (mild turbidity, relatively clear pupil and iris), grade 2 (shallow corneal opacity, see), grade 3 (uneven turbidity of the entire corneal layer), and grade 4 (dense opacity). Hypopyon: grade 1 (not reaching the paracentral cornea) and grade 2 (reaching the central cornea).

[0043] The results are as follows Figure 3 As shown in the figure, the double cross-linked hydrogel (SFMA / PBA-LWMH@CORM-3) treatment group basically achieved the cure of keratitis (more than 95%) in about 7 days, which was significantly better than the single hydrogel microspheres (SFMA / PBA-LWMH) and traditional commercial antibiotics (ToBreX). However, the PBS-treated control group did not show significant improvement in keratitis after 7 days of treatment.

[0044] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing double cross-linked hydrogel microspheres, characterized in that: The method comprises the following steps: mixing methacrylylated silk fibroin and phenylboronic acid-low molecular weight heparin, adding (OC-6-44)-tricarbonyl chloride (glycine) ruthenium and a photoinitiator, and irradiating with ultraviolet light to form double cross-linked hydrogel microspheres; wherein the phenylboronic acid-low molecular weight heparin is obtained by condensation reaction of low molecular weight heparin and amino phenylboronic acid.

2. The preparation method according to claim 1, characterized in that: The concentration ratio of the methacrylylated silk fibroin to phenylboronic acid-low molecular weight heparin is 1:

1.

3. The preparation method according to claim 1, characterized in that: The concentration of the methacrylylated silk fibroin is 10-90% (W / V); The concentration of the phenylboronic acid-low molecular weight heparin is 10-90% (W / V).

4. The preparation method according to claim 1, characterized in that: The concentration of the (OC-6-44)-tricarbonyl chloride (glycinyl)ruthenium is 1-10 mM; And / or, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt.

5. The preparation method according to claim 1, characterized in that: The preparation method of the methacrylylated silk protein comprises: dissolving the degummed silk protein in a lithium bromide solution, then slowly adding glycidyl methacrylate at 200-400 rpm and 60° C., and continuing the reaction for 3-4 hours; after dialyzing with double distilled water, using PEG reverse dialysis to obtain the methacrylylated silk protein.

6. The preparation method according to claim 5, characterized in that: After the degummed silk fibroin is dissolved in a lithium bromide solution, the concentration of the silk fibroin is 1%-10% (W / V); The concentration of the glycidyl methacrylate is 0.1-0.5M.

7. The preparation method according to claim 1, characterized in that: The preparation method of phenylboronic acid-low molecular weight heparin comprises: mixing 3-aminophenylboronic acid, low molecular weight heparin and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride dissolved in phosphate buffer, reacting at 200-400 rpm and 37° C. for 1-2 days, dialyzing with double distilled water, and then reverse dialysis with PEG to obtain phenylboronic acid-low molecular weight heparin.

8. The preparation method according to claim 7, characterized in that: The concentration of the 3-aminophenylboronic acid is 15 mM, the concentration of the low molecular weight heparin is 3-5 M, and the concentration of the 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is 0.09-0.1 mM.

9. Double cross-linked hydrogel microspheres prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the double cross-linked hydrogel microspheres according to claim 9 in the preparation of the drug according to any one of (1) to (3): (1) Drugs for preventing and / or treating bacterial keratitis; (2) Drugs for preventing and / or treating refractory keratitis caused by drug-resistant bacterial infection; (3) Drugs that inhibit bacterial growth.

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