Double crosslinked hydrogel microspheres, methods of making and uses thereof

Abstract

This invention discloses a double-crosslinked hydrogel microsphere, its preparation method, and its applications. The preparation method of the double-crosslinked hydrogel microsphere includes: mixing methacrylamide silk fibroin and phenylboronic acid-low molecular weight heparin, adding (OC-6-44)-tricarbonyl chloride (glycine-based)ruthenium and a photoinitiator, and irradiating with ultraviolet light to form double-crosslinked hydrogel microspheres; wherein, the phenylboronic acid-low molecular weight heparin is obtained by a condensation reaction between low molecular weight heparin and aminated phenylboronic acid. The double-crosslinked hydrogel microspheres of this invention 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 infections.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a double cross-linked hydrogel microsphere, its preparation method, and its uses. Background Technology

[0002] Refractory keratitis, due to the complexity and drug resistance of its pathogens, makes corneal infection difficult to control and causes persistent corneal inflammation, and is considered one of the leading causes of vision impairment and blindness worldwide. As bacteria become increasingly resistant to traditional antibiotics, the efficacy of conventional antibiotics is gradually diminishing. Furthermore, even after bacterial infection is controlled, corneal tissue enters a repair phase, but existing drugs have limited ability to eliminate various pro-inflammatory factors such as IL-6 and reduce persistent corneal inflammation, making the cure of refractory keratitis a challenging problem. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention aims to provide a double-crosslinked hydrogel microsphere, its preparation method, and its applications. The double-crosslinked hydrogel microspheres of this invention mainly comprise three components: methacryloyl silk fibroin (SFMA), phenylboronic acid (PBA), low molecular weight heparin (LWMH), and (OC-6-44)-tricarbonyl chloride (glycine-3)ruthenium (CORM-3). Low molecular weight heparin contains abundant carboxyl groups, which can bind with aminated PBA through a condensation reaction; while CORM-3 is water-soluble and can ultimately form double-crosslinked hydrogel microspheres through self-assembly under ultraviolet light crosslinking in a microfluidic device.

[0004] The specific technical solution of the present invention is as follows:

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

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

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

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

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

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

[0011] Furthermore, the preparation method of the methacrylamide silk fibroin includes: dissolving the degummed silk fibroin in a lithium bromide solution, then slowly adding glycidyl methacrylate dropwise at 200-400 rpm and 60°C, and continuing the reaction for 3-4 hours; after double distillation and dialysis, obtaining the methacrylamide silk fibroin by PEG reverse dialysis.

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

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

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

[0015] Further, the concentration of 3-aminophenylboronic acid is 15 mM, the concentration of low molecular weight heparin is 3-5 M, and the concentration of 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 described above.

[0017] This invention also provides the use of the double cross-linked hydrogel microspheres in the preparation of any one of the drugs described in (1)-(3):

[0018] (1) Medications for the prevention and / or treatment of bacterial keratitis;

[0019] (2) Medications for the prevention and / or treatment of refractory keratitis caused by drug-resistant bacterial infections;

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

[0021] The beneficial effects of this invention are as follows:

[0022] The double cross-linked hydrogel microspheres provided by this invention are formed by double cross-linking of phenylboronic acid, low molecular weight heparin and methacrylamide silk fibroin. They 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. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the antibacterial properties of a double cross-linked hydrogel.

[0024] Figure 2 CO was released from hydrogel microspheres of different concentrations;

[0025] Figure 3 Hydrogel microspheres promote the healing of keratitis in mice. Detailed Implementation

[0026] To better understand the present invention, it is now further described with reference to the following embodiments and accompanying drawings. The embodiments are for illustrative purposes only and do not limit the invention in any way. In the embodiments, all original reagents and materials are commercially available, and experimental methods not specifically specified are conventional methods and 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, including the following steps:

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

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

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

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

[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 at a volume ratio of 7:3. Then, 1.7 mM of (OC-6-44)-tricarbonyl chloride (glycinyl)ruthenium (CORM-3) and 0.4% (w / w) of the photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP) were added. The mixture was then irradiated with UV light for 45 s under microfluidic control to form double cross-linked hydrogel microspheres (SFMA / PBA-LWMH@CORM-3).

[0035] Example 2

[0036] The double-crosslinked hydrogel microspheres of this invention contain CORM-3 and exhibit good antibacterial effects. This embodiment demonstrates, through bacterial culture experiments, that the double-crosslinked hydrogel microspheres containing CORM-3 prepared in Example 1 have good antibacterial effects. The specific experimental steps are as follows: 1 mL of hydrogel was taken, and 5 mL of logarithmic-phase *Pseudomonas aeruginosa* bacterial suspension was added. The mixture was incubated overnight at 37°C in a shaker. The upper bacterial suspension was then plated, and the colony count was calculated 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 double cross-linked hydrogel microspheres without CORM-3.

[0038] Example 3

[0039] The double-crosslinked hydrogel microspheres of this invention are uniform, monodisperse, and possess a designed size and porous microstructure. The encapsulated CORM-3 molecules can effectively release CO and prolong its residence time on the ocular surface, thereby improving the therapeutic effect. In this embodiment, the myoglobin detection method was used. A certain amount of the lyophilized powder of the double-crosslinked hydrogel microspheres prepared according to the method of Example 1 was added to a deoxy-horse skeletal myoglobin (Deoxy-Mb) buffer solution that had been reduced with excess sodium dithionite, and a small amount of mineral oil was added for sealing. The conversion process was reflected by spectrophotometry. The absorbance of the solution was measured multiple times at fixed times within the 500-600 nm range to plot the CO release curve.

[0040] The results are as follows Figure 2 As shown in the figure, the meanings of 9∶1, 7∶3, and 5∶5 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-crosslinked hydrogel microspheres of this invention exhibit excellent biocompatibility and significantly promote keratitis caused by multidrug-resistant Staphylococcus aureus (MASR) infection. Using the double-crosslinked hydrogel microspheres (SFMA / PBA-LWMH@CORM-3) prepared by the method in Example 1 as an example, their promoting effect on the healing of keratitis in mice was verified. Construction and treatment of bacterial keratitis in mice: 10 μL (3 × 10⁻⁶) of the microspheres were prepared... 8 A suspension of MRSA (CFU / mL) was inoculated onto the mechanically damaged corneas of mice to induce bacterial keratitis. Corneal infection was observed under slit-lamp diffuse light 24 hours after infection, and successfully infected mice were selected. The mice with bacterial keratitis were then randomly divided into five groups (NC, H, C, ToBrex, and H@C). The NC group used sterile PBS, the H group used sterile PBS-suspended hydrogel microspheres (SFMA / PBA-LWMH), the C group used sterile PBS-dissolved CORM-3 (1 mg / mL), the ToBrex group used commercially available tobramycin eye drops (Tobramycin), and the H@C group used sterile PBS-suspended hydrogel microspheres (SFMA / PBA-LWMH@CORM-3) encapsulated with CORM-3 (1 mg / mL). All patients received topical eye drops twice daily, 5 μL of the corresponding group of eye drops per eye each time, for a total of 7 days. Corneal infection was observed and graded using a slit-lamp microscope under diffuse light on days 0, 1, 3, 5, and 7 of the topical eye drop treatment. The grading criteria are as follows: Opacity area: Grade 1 (1-25% of total corneal area), Grade 2 (26-50%), Grade 3 (51-75%), and Grade 4 (76-100%); Opacity: Grade 1 (mild opacity, relatively clear pupil and iris), Grade 2 (superficial corneal opacity, see), Grade 3 (uniform opacity throughout the 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, the double cross-linked hydrogel (SFMA / PBA-LWMH@CORM-3) treatment group achieved near-complete cure of keratitis (over 95%) within approximately 7 days, significantly superior to hydrogel microspheres alone (SFMA / PBA-LWMH) and conventional commercially available antibiotics (ToBreX). In contrast, the PBS-treated control group showed no significant improvement in keratitis after 7 days of treatment.

[0044] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing double cross-linked hydrogel microspheres, characterized in that, The steps include: mixing methacrylamide silk fibroin and phenylboronic acid-low molecular weight heparin, adding (OC-6-44)-tricarbonyl chloride (glycine-based)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 a condensation reaction of low molecular weight heparin and aminated phenylboronic acid; The method for preparing the methacrylamide silk fibroin includes: dissolving degummed silk fibroin in lithium bromide solution, then slowly adding glycidyl methacrylate dropwise at 200-400 rpm and 60°C for 3-4 hours; after double distillation and dialysis, obtaining methacrylamide silk fibroin by PEG reverse dialysis. The method for preparing phenylboronic acid-low molecular weight heparin includes: mixing 3-aminophenylboronic acid dissolved in phosphate buffer, low molecular weight heparin, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, reacting at 200-400 rpm and 37°C for 1-2 days, dialysis with double-distilled water, and then obtaining phenylboronic acid-low molecular weight heparin by reverse dialysis with PEG.

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

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

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

6. The preparation method according to claim 1, characterized in that, The concentration of 3-aminophenylboronic acid is 15 mM, the concentration of low molecular weight heparin is 3-5 M, and the concentration of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is 0.09-0.1 mM.

7. The double cross-linked hydrogel microspheres prepared by the preparation method according to any one of claims 1-6.

8. Use of the double cross-linked hydrogel microspheres of claim 7 in the preparation of any one of (1)-(2) of the drug: (1) Medications for the prevention and / or treatment of bacterial keratitis; (2) Drugs that inhibit the growth of Pseudomonas aeruginosa.

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