Hydrogel beads for treating keratitis and use thereof for treatment during sleep

Abstract

The present invention discloses hydrogel beads for treating keratitis and their use during sleep. Polyvinyl alcohol (PVA) is dissolved in water and heated, followed by heating water-soluble short-chain chitosan (CS) and stirring until dissolved to obtain a PVA / CS mixed solution. Tobramycin is dissolved in water, followed by addition of terephthalic acid and baicalin, and ultrasonication until completely dissolved to obtain a TOB / BA mixed solution. The PVA / CS mixed solution is then added dropwise to the TOB / BA mixed solution, where the droplets form hydrogel beads under gravity, resulting in hydrogel beads for treating keratitis. The hydrogel microspheres continuously degrade and release TOB and BA during sleep. TOB exhibits broad-spectrum antimicrobial activity, while BA promotes the transformation of macrophages from M1 to M2 morphology and scavenges reactive oxygen species, achieving simultaneous antibacterial, anti-inflammatory, and antioxidant effects. In vivo experiments in a rabbit corneal S. aureus infection model further confirmed that the PCBT hydrogel microspheres exhibit superior therapeutic efficacy and clinical potential compared to existing commercially available eye drops, making them suitable for the treatment and management of BK during sleep.

Description

Technical Field

[0001] The present invention relates to a hydrogel based on polyvinyl alcohol (PVA), water-soluble chitosan (CS), tobramycin (TOB) and baicalin (BA), which is used for treating bacterial keratitis and belongs to the technical field of medical materials. Background Art

[0002] Bacterial keratitis (BK) is a major cause of corneal ulcers. Its high prevalence and the high risk of corneal perforation, intraocular infection, and ultimately irreversible visual impairment and blindness make it a serious threat to human visual health worldwide. Clinically, the treatment of BK is challenging, primarily due to four key challenges: (i) low drug bioavailability; (ii) bacterial resistance; (iii) poor patient compliance; and (iv) prolonged corneal wound healing. While numerous advanced technologies and biomaterials have been developed to address these challenges, a significant shortcoming has long been overlooked: the inability to manage BK during sleep. Most currently available strategies are either inapplicable or have diminished efficacy during sleep. For example, antibiotic eye drops require frequent instillation to enhance drug bioavailability, which is clearly unavailable during sleep. Photothermal or photocatalytic therapy requires prolonged light exposure, while ultrasound dynamic therapy requires noisy ultrasound stimulation, all of which can severely impact patients' sleep quality. Because BK progresses rapidly and may worsen rapidly if not treated properly, finding ideal alternatives to prevent BK from worsening during sleep is crucial, but this issue has not received widespread attention.

[0003] To date, some strategies have shown some promise for treating BK during sleep, but these efforts remain far from ideal. For example, Luo et al. injected gelatin-coated silver nanoparticles into the corneal stroma, creating a localized, sustained antimicrobial microenvironment. However, intrastromal delivery is a complex therapeutic procedure and can exacerbate corneal trauma. Similar challenges also exist in Jiang et al., who used a microneedle patch to deliver Ag@ZIF-8 nanoparticles into the corneal stroma. Another promising biomaterial is contact lenses, which can create a long-term antimicrobial microenvironment on the corneal surface through controlled release of therapeutic molecules or post-modification with antimicrobial coatings. However, wearing contact lenses during sleep can cause various ocular discomforts, exacerbate infection, and lead to invasive events. Therefore, hydrogel eye drops, in situ forming hydrogels, thermosensitive hydrogels, and supramolecular hydrogels may represent more reasonable alternatives due to their higher patient compliance and reduced side effects. Traditional hydrogels used for BK treatment primarily focus on rapid and efficient sterilization. However, bacterial infections are often accompanied by severe inflammatory responses and oxidative stress mediated by reactive oxygen species (ROS). Although ROS can inhibit bacterial growth, they can also impair normal cellular function and induce cell death. Therefore, the key to managing BK during sleep lies in finding a balance between bacterial clearance and inflammation relief.

[0004] Tobramycin (TOB) is an aminoglycoside antibiotic with broad-spectrum antimicrobial activity and has been widely used in the treatment of BK. Furthermore, TOB has good water solubility, and its aqueous solution is weakly alkaline due to the protonation of pendant amino groups. Macrophages (Mφ), the primary immune cells in BK wounds, maintain a proinflammatory M1 phenotype, secrete inflammatory cytokines, and produce ROS, thereby impairing inflammation resolution and wound healing. Baicalin (BA), a bioactive flavonoid containing a catechol structure, exhibits multiple anti-inflammatory effects. Studies have shown that it can induce the transformation of M1 macrophages to M2 phenotypes and inhibit ROS production by M1 macrophages. Water-soluble short-chain chitosan (CS) is a cationic polyelectrolyte with excellent biocompatibility and antimicrobial activity. High-concentration aqueous CS solutions rapidly transform into high-strength crystalline hydrogels upon treatment with alkaline solutions. Polyvinyl alcohol (PVA), a neutral linear polymer with abundant hydroxyl pendant groups, has been widely used as a primary component of eye drops. The phenylboronic acid group can react with catechol structures or diol structures to form a reversible phenylboronic acid ester bond. Summary of the Invention

[0005] Based on this, the present invention developed millimeter-sized hydrogel microspheres for the management of BK during sleep. These hydrogel microspheres are composed of PVA, CS, terephthaloylboronic acid (1,4-BDBA), BA, and TOB. Specifically, a PVA / CS solution is slowly dripped into a 1,4-BDBA / BA / TOB solution via a syringe. Subsequently, PVA / CS / BA / TOB (PCBT) hydrogel microspheres are obtained. In this formulation, the hydrogel microspheres are shaped by pH-induced crystallization of CS and further strengthened by reversible crosslinking mediated by interphenylboronic acid bonds within the PVA chains. Furthermore, BA molecules are reversibly attached to the PVA chains, and TOB molecules are encapsulated within the hydrogel microspheres. For BK treatment during sleep, PCBT hydrogel microspheres are placed on the inner side of the lower eyelid. The hydrogel microspheres remain in this area for an extended period, during which time the hydrogel microspheres gradually degrade and the TOB and BA molecules are continuously released, thereby continuously inhibiting bacterial growth and alleviating inflammatory responses and ROS damage.

[0006] In this study, the composition, structure, mechanical properties, structural stability, and drug release behavior of PCBT hydrogel microspheres were studied in detail. The antibacterial activity of the hydrogel microspheres was evaluated using Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). In vitro cell co-culture experiments were used to assess the immunomodulatory ability of the hydrogel microspheres to induce M1 macrophage to M2 transformation and scavenge ROS. Furthermore, a rat BK model combined with immunohistochemical staining was used to explore the potential of these hydrogel microspheres for managing BK during sleep. This study not only reports an effective strategy for managing BK during sleep but also provides new insights for related research.

[0007] The present invention aims to address the low utilization rate, short duration of action, and poor patient compliance of antibiotic eye drops for treating keratitis. This invention utilizes dynamic cross-linking technology to construct a PVA / CS / TOB / BA hydrogel, enabling pH-responsive drug release. This hydrogel combines long-lasting antibacterial and anti-inflammatory properties with corneal repair-promoting properties.

[0008] To achieve the above object, the technical solution adopted by the present invention is: a tobramycin baicalin hydrogel bead with antibacterial effect and a preparation method thereof, comprising the following steps:

[0009] (1) Preparation of PVA / CS mixed solution: Add polyvinyl alcohol (PVA) to water, raise the temperature to dissolve, then add water-soluble short-chain chitosan (CS), and stir until dissolved to obtain the PVA / CS mixed solution;

[0010] (2) Preparation of a TOB / BA mixed solution: Tobramycin was dissolved in water, followed by addition of terephthalic acid and baicalin, and ultrasonication was performed until completely dissolved to obtain a TOB / BA mixed solution;

[0011] (3) Preparation of hydrogel beads: The PVA / CS mixed solution of step (1) is added dropwise to the TOB / BA mixed solution of step (2), and the droplets form hydrogel beads under the action of gravity to obtain hydrogel beads for treating keratitis.

[0012] In the step (1), the concentration of the polyvinyl alcohol solution is 5-10% (w / w), the dissolution temperature is 95°C±2°C; the addition temperature of the water-soluble chitosan is 50°C±5°C, and the stirring time is 30-60 minutes; the mass ratio of polyvinyl alcohol to water-soluble chitosan is 5-20:1.

[0013] In the step (2), the concentration of the tobramycin solution is 0.1-0.3% (w / w), the concentration of terephthalic acid is 0.02-0.12% (w / w), the concentration of baicalin is 0.01-0.04% (w / w), and the ultrasonic treatment conditions are a power of 200-300 W and a time of 10-30 minutes.

[0014] In step (3), the PVA / CS mixed solution is added dropwise to the TOB / BA mixed solution by means of a syringe, the syringe needle diameter is 0.23-0.6 mm, the dropping height is 10 cm ± 2 cm; the diameter of the hydrogel beads is 0.2-5 mm, and the standing time is 30 minutes ± 5 minutes.

[0015] In some preferred embodiments, the PVA / CS mixed solution prepared in step (1) is placed 10 cm above the TOB / BA mixed solution prepared in step (2) using a syringe, and then slowly dripped into the mixed solution drop by drop. Under the action of gravity, the dripping liquid droplets will completely enter the solution and form hydrogel beads. By changing the diameter of the syringe needle, the size of the prepared hydrogel beads can be controlled. The hydrogel beads can be prepared by standing for 30 minutes.

[0016] The hydrogel beads for treating keratitis prepared by the above method have BA and TOB uniformly dispersed in the PVA / CS network structure in an amorphous form in the hydrogel beads. The crystal structure of the hydrogel beads is jointly constructed by PVA and CS, and the two interact through hydrogen bonds to form a stable composite crystal system.

[0017] The drug loading in the hydrogel beads is 0.5-5 wt%, and the particle size of the hydrogel beads is 200 μm-5 mm.

[0018] PVA is a neutral linear polymer with abundant hydroxyl side groups and a cis-diol structure.

[0019] P-terephthalic acid undergoes configurational transformations in different acidic and alkaline environments, making it a dynamic crosslinker. In acidic media, p-terephthalic acid exists as an uncharged, relatively hydrophobic triangle, significantly reducing its reactivity with cis-vicinal diol groups. However, in weakly alkaline conditions, it exists as a tetrahedral structure, exposing highly reactive boronic acid groups, which readily form reversible coordination complexes with compounds containing cis-diol structures, such as boronic acid.

[0020] The free amino groups on the CS molecular chains will be attracted by the hydroxyl groups in the PVA molecules to achieve hybrid cross-linking, and self-assemble in alkaline and monovalent anion salt solutions to form physical microcrystalline networks and chain entanglement networks with both biodegradability and antibacterial activity.

[0021] Cross-linking of PVA and CS molecules through hydrogen bonding and electrostatic interactions can significantly improve the mechanical properties of the composites.

[0022] TOB is easily soluble in water, combines with protons in water, and releases hydroxide ions to make the solution alkaline.

[0023] BA has a cis-diol structure and will form a phenylboronic acid ester bond with phenylboronic acid to prevent the activity of baicalin from being destroyed.

[0024] TOB is dissolved in water to provide an alkaline environment. P-terephthalic acid is added to the TOB solution with a pH of about 8.5, so that phenylboronic acid forms a tetrahedral active structure, which then combines with the cis-diol structure to form a more stable charged complex.

[0025] The beneficial effects of the present invention are as follows: the present invention provides a tobramycin baicalin hydrogel bead with antibacterial effect and a preparation method thereof. Through the pH-responsive self-assembly mechanism, the CS molecular chain is induced to be oriented under weak alkaline conditions. Secondly, terephthalic acid is introduced as a dynamic crosslinker, and its tetrahedral configuration forms a reversible boronate bond with the cis-vicinal diol group of PVA under alkaline conditions to construct a three-dimensional network with self-repairing properties. At the same time, the BA molecule also has a cis-vicinal diol structure, so it can also form a reversible boronate bond with terephthalic acid, and the TOB molecule is wrapped in the hydrogel bead to form a drug-carrying system. When the drug-releasing gel is placed in an environment of pH 6.4-pH 7.7, the TOB in the hydrogel is continuously released, the pH is continuously reduced, and the PVA / CS double network crosslinking structure is transformed from a stable tetrahedral structure to an unstable planar structure. The BA molecule also detaches from the terephthalic acid, and the CS reverse folding crystals are restored to the original irregular linear form in this process until the gel is degraded. This patent solves the problems of single drug action and uncontrollable release in the treatment of bacterial keratitis through dynamic cross-linking and dual-drug collaborative design, providing a new strategy for the research and development of ophthalmic anti-infection materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Schematic diagram of the preparation of PCBT hydrogel beads in Example 1.

[0027] Figure 2 (A) is a physical picture of hydrogel beads with different diameters during the preparation process of Example 1, and (B, C) are pictures of the surface morphology of hydrogel beads during the preparation process of Example 1.

[0028] Figure 3 (A, BC) are Fourier transform infrared spectra of the hydrogel beads prepared in Example 1.

[0029] Figure 4 This is the XRD pattern of the hydrogel beads prepared in Example 1.

[0030] Figure 5 This is the ultraviolet absorption spectrum of the hydrogel beads prepared in Example 1.

[0031] Figure 6 (A, B) are rheological analyses of the hydrogel beads prepared in Example 1.

[0032] Figure 7 This is the in vitro drug release curve of the hydrogel beads prepared in Example 1.

[0033] Figure 8 The TOB (H) and BA delivery model (I) of PCBT hydrogel beads in Example 1.

[0034] Figure 9 This is a graph showing the effects of the hydrogel beads prepared in Example 1 on mouse mononuclear macrophages (RAW264.7) (A) and human corneal epithelial cells (HECE) (B).

[0035] Figure 10 Representative fluorescence images (A) and corresponding quantitative statistical graphs (B) of iNOS and CD206 after the hydrogel beads prepared in Example 1 were incubated with RAW264.7 cells for 1 day.

[0036] Figure 11 These are H&E, Masson and immunohistochemical staining images of the hydrogel beads prepared in Example 1.

[0037] Figure 12 Corneal staining and immunohistochemical staining (iNOS and IL-10) on day 7 (A), and their quantitative analysis (B). DETAILED DESCRIPTION

[0038] Example 1

[0039] This embodiment provides a method for preparing tobramycin baicalin hydrogel beads with antibacterial effect. Figure 1The preparation method is carried out according to the principle and process of the present invention, comprising the following steps:

[0040] (1) Weigh a certain amount of polyvinyl alcohol (PVA) and water-soluble chitosan (CS) (purchased from Qingdao Bozhihuili Biotechnology Co., Ltd.) in a ratio of 10:1. Dissolve the polyvinyl alcohol in pure water (95°C) and stir at 600 r / min for 1 hour to prepare a 10% polyvinyl alcohol solution. Cool the solution to 50°C and add water-soluble chitosan. Continue stirring at a constant temperature under magnetic stirring at 600 r / min for 1 hour to completely dissolve it. The concentrations of PVA and CS are maintained at 0.085 g / mL and 0.008 g / mL, respectively. Cool the prepared PVA / CS solution to room temperature (25°C) to obtain a PVA / CS aqueous solution.

[0041] (2) Dissolve 0.06 g of 1,4-diboronic acid (1,4-BDBA) and 0.02 g of baicalin (BA) in 2 mL of ethanol and sonicate for 10 min. Dissolve 0.15 g of tobramycin (TOB) in 50 mL of pure water to a pH of approximately 8.5. Then, using a drop pipette (0.25 mL / drop, 10 drops / min), slowly drip the 1,4-BDBA / BA / TOB aqueous solution into the mixed ethanol solution.

[0042] (3) Prepare PCBT hydrogel beads. Use a syringe to draw up the PVA / CS aqueous solution. Use a 2 mm diameter needle and place it 10 cm above the 1.4-BDBA / BA / TOB aqueous solution. Then slowly drip it drop by drop. Under the action of gravity, the dripping liquid beads will completely enter the solution and form individual hydrogel beads. By changing the diameter of the syringe needle, the size of the prepared hydrogel beads can be controlled. After standing for 30 minutes, the hydrogel beads PCBT are obtained.

[0043] Example 2

[0044] This example detects the surface morphology of the PCBT hydrogel beads prepared in Example 1.

[0045] Surface morphology detection of hydrogel beads:

[0046] (1) Macroscopic changes of hydrogel beads in each group are as follows Figure 2 As shown in A, it is a spherical shape with a smooth surface and a yellow color, indicating that baicalin is loaded into it. The diameter of the prepared hydrogel beads can be changed by changing the diameter of the syringe needle. Using a 0.18mm diameter needle can produce Figure 2 The smallest diameter hydrogel beads shown are 200 μm, which can be prepared using a 0.6 mm diameter needle. Figure 2 The 2 mm diameter hydrogel beads shown can be prepared using a 2 mm diameter needle. Figure 25 mm diameter hydrogel beads shown.

[0047] (2) Figure 2 Scanning electron microscopy (SEM) was used to characterize the sample surface morphology (B and C). The hydrogel beads exhibit a sparse, porous structure with pore sizes ranging from 50 to 200 nm. The TOB loaded onto the hydrogel beads is primarily located on the surface and shallow layers of the beads. This porous structure facilitates the preferential release of TOB during pH-responsive degradation of BA, PVA, and CS.

[0048] Hydrogel structure detection:

[0049] (3) Figure 3 The FTIR spectra of CS, PVA, TOB, BA and PCBT beads at different wavelengths are shown in Figure 2. In the FTIR spectrum of PCBT beads, the representative peaks of CS (axial -OH and -NH stretching at 3439 cm -1 , -COC in the glycosidic bond is 1096 cm -1 ), PVA (-OH47 is 3439 cm -1 , -CC stretching is 1260 cm -1 ), BA (-COOH is 1727 cm -1 , -C=O is 1664 cm -1 and 1613 cm -1 ), and TOB (-NH2 at 1582 cm -1 ), indicating that BA and TOB molecules were successfully encapsulated into the PVA / CS dropper to form PCBT gel beads. In addition, the 3552 cm -1 、3491 cm -1 and 1368 cm -1 The band at 1363 cm disappeared in the FTIR spectrum of PCBT beads, indicating that a phenylboronic acid ester bond was formed between 1,4-BDBA and BA. This conclusion can also be seen from the FTIR spectrum of PCBT beads. -1 In addition, the present invention also calculated and compared the peaks at 1260 cm of pure PVA and PCBT beads. -1 (-CC-) and 3437 cm -1 (-OH)(I 3034 / I 1260 ) The intensity ratio of pure PVA and PCBT beads is 3034 / I 1260The ratios were 0.983 and 0.937, respectively, which proved to some extent that phenylboronic acid ester bond was formed between 1,4-BDBA and PVA.

[0050] (4) In order to further study the microstructure of PBCT hydrogel beads, XRD ( Figure 4 ). No diffraction peaks of TOB and BA were found in the XRD spectrum of PCBT hydrogel beads, indicating that they were evenly distributed in the PCTB hydrogel microbeads. CS was 12.3 o and 24.2 o The diffraction peak at 9.6 disappeared in the XRD spectrum of PCBT hydrogel beads, but o The peak at 9.6° still exists. The peak at 9.6° corresponds to the crystal plane (020) of the CS aggregates. Here, the crystallinity index (CrI) is used to study the crystallization behavior of the CS chains.

[0051]

[0052] Among them I 020 and Iamor are peaks (020) and amorphous peaks (2θ = 16 o ) has a diffraction intensity of 55. Calculations show that the Crl of CS aggregates in PCBT hydrogel beads (0.271) is much higher than that of pure CS (0.054), which means that the alkaline TOB solution induces the crystallization of CS chains in PCBT hydrogel beads, and the CS aggregates are mainly arranged in the direction perpendicular to the crystal plane (020) (Figure 4). As for PVA in PCBT hydrogel beads, its representative diffraction peaks (intermolecular and intramolecular hydrogen bonds) range from 19.5 o Moving to 20.1 o Here, the apparent crystal size Dapp is calculated with the aid of the Scherrer equation:

[0053]

[0054] where β is the half-width of the diffraction peak, k is a constant with a value of 1, λ is the wavelength of the X-ray (0.89 nm), and θ is half the Bragg angle. 55 Calculations show that the Dapp of PVA in PCBT hydrogel beads decreases from 24.41 nm to 11.59 nm compared to the original PVA, which may be due to the destruction of the ordered arrangement of hydrogen bonds by the cross-linking reaction ( Figure 4 ).

[0055] (5) Figure 5 shows the UV spectra of 1,4-BDBA, BA, and PCBT hydrogels. When BA was incorporated into PCBT hydrogel, the absorbance peak of BA at 316 nm shifted to 306 nm, further verifying the formation of a phenylboronic acid ester bond between 1,4-BDBA and BA.

[0056] (6) Figure 6 shows the rheological behavior of the PVA / CS solution and PCBT hydrogel beads. The prepared hydrogel beads were placed on the bottom plate of the rotational rheometer, and then a frequency sweep was performed. The variable vibration stress was controlled, and the frequency sweep was set to a logarithmic mode of angular frequency (0.1-100). The results showed that the loss modulus (G'') of the PVA / CS solution was higher than its storage modulus (G'), indicating that the PVA / CS solution was viscous. For the PCBT hydrogel beads, obvious elasticity was observed (tan δ < 1).

[0057] (7) Dynamic dialysis was used to determine the release of BAN and TOB drugs from PVA / CS / BAN / TOB hydrogel beads. The molecular weight cutoff of the dialysis bag was 1KD. Twenty PVA / CS / BAN / TOB hydrogel beads with a diameter of 2 mm were placed in the dialysis bag, and then placed in 10 mL of PBS buffer with pH 7.4 and 5.5. The beads were shaken at 100 rpm on a 37°C constant temperature shaker. 1 mL of release medium was taken every 0.08, 0.25, 0.5, 0.75, 1, 5, 9, 12, 20, and 28 seconds, and an equal amount of fresh release medium was added to maintain a constant volume within the predetermined time intervals. The UV absorption peak areas of BAN and TOB drugs in the release medium were measured respectively. The content of BAN / TOB and its cumulative release over a period of time were calculated according to the established standard curve. The in vitro drug release curve was plotted with time as the horizontal axis and cumulative release as the vertical axis. The cumulative release calculation formula is as follows:

[0058] Cumulative release amount =

[0059] Where V is the total volume of the release medium (ml), V0 is the amount of release medium taken out each time, C i is the drug concentration at the time of sampling for the i-th time.

[0060] Since the microenvironment of a normal eye is neutral while that of an infected eye is weakly acidic, the cumulative release curves of TOB and BA in PCBT hydrogel beads in PBS with a pH of 7.5 or 5.5 were plotted in detail to demonstrate the possible TOB / BA release behavior of PCBT hydrogel beads at the lower eyelid (Figure 7). Apparently, the influence of the acidic environment on the release behavior of TOB in PCBT hydrogel beads is negligible, and TOB can be continuously released for more than 30 h. However, in weakly acidic PBS, the release amount of BA from PCBT hydrogel beads is greater than that in neutral PBS, which may be due to the oxidation of the catechol group of BA into benzoquinone in the neutral microenvironment. The release period of BA from PCBT hydrogel beads is also about 30 h. From the correlation coefficient (R 2 ), it was found that the delivery of TOB and BA from PCBT hydrogel beads conformed to the Weibull model ( Figure 8 H-I). The corresponding fitting data are summarized in Table 1. The b value of the Weibull equation is an important indicator of the drug transport mechanism through the matrix. 0.69 < b < 0.75 indicates the diffusion of the drug in normal Euclidean space; 0.75 < b < 1 indicates the diffusion of the drug in normal Euclidean substrates with the contribution of another release mechanism; b > 1 indicates a complex release mechanism. Apparently, the release of TOB from PCBT hydrogel beads is mainly based on physical diffusion, but the crystalline CS network of PCBT hydrogel beads in the neutral microenvironment may affect the release of TOB to a certain extent (b = 0.797). As for BA, in the neutral microenvironment, its release behavior generally conforms to physical diffusion, but the reversible phenylborate ester bond affects its release kinetics (b = 0.821); in the weakly acidic microenvironment, the release behavior becomes quite complex. Here, several factors may contribute: phenylborate ester bond, conformational transformation of CS microcrystals, physical diffusion, degradation, etc.

[0061] Table 1 Mathematical modeling and related parameters based on release data

[0062]

[0063] Example 3

[0064] This test example detected the biocompatibility of the tobramycin baicalin hydrogel beads with antibacterial effect prepared in Example 1 and its influence on the polarization state of macrophages.

[0065] (1) CCK-8 assay and live / dead cell staining were used to evaluate the cytotoxicity of hydrogel extracts on mouse mononuclear macrophages and human corneal epithelial cells. HCEC (third passage) or original 264.7 cells (2×10 3 Cells were cultured in 96-well plates supplemented with DMEM containing 10% FBS and 1% penicillin / streptomycin (37°C, 5% CO2) at 37°C. After the confluency reached 70%, four different culture media (10 μL) were added to the wells: (1) control, fresh complete culture medium; (2) conditioned medium 1, complete culture medium supplemented with 0.3% (w / v) tobramycin and 0.8% (w / v) baicalin; and (3) conditioned medium 2, complete culture medium treated with UV-sterilized PCBT hydrogel beads (5 beads with a diameter of approximately 2 mm were immersed in 5 mL of culture medium for 24 hours). After 24 hours of co-culture, cell viability was assessed using a CCK-8 kit (absorbance at 450 nm, Varioskan LUX microplate reader). Live / dead staining was performed using calcein-AM / PI, and images were captured using a confocal microscope (Leica DMI3000B).

[0066] CCK-8 results showed that there was no difference in cell proliferation among different groups ( Figure 9 Live / dead cell results are shown in Figure 2B. Figure 9 As shown in (A). Each group emitted mainly green fluorescence, with little red fluorescence, indicating that a large number of living cells and a small number of dead cells were observed. The results show that the material has good biocompatibility.

[0067] (2) Raw 264.7 cells were cultured in 96-well plates until all cells adhered and the fusion rate reached 70%, and then they were divided into three groups: (1) control group, which did not receive any treatment; (2) lipopolysaccharide (LPS) group (10 ng / mL LPS was added to induce macrophage M1 polarization); (3) experimental group, in which macrophages were first treated with LPS and then treated with conditioned medium of PCBT hydrogel beads. The treated Raw264.7 cells were fixed and immunostained with anti-iNOS (M1 surface marker, Proteintech Group, Inc) and anti-CD206 (M2 surface marker, Proteintech Group, Inc). Cell nuclei were stained with DAPI. Fluorescence intensity was quantified using ImageJ (NIH). Figure 10As shown in the figure, the expression of iNOS (M1 marker) and CD206 (M2 marker) was detected by cell immunofluorescence staining to study the effect of hydrogel on RAW264.7 cell polarization. The results showed that the expression of iNOS (M1 marker) was reduced and the proportion of CD206 was increased in the PCBT hydrogel leaching solution group.

[0068] Example 4

[0069] This test evaluated the in vivo antibacterial and corneal repair-promoting properties of the antibacterial tobramycin baicalin hydrogel beads prepared in Example 1. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were cultured overnight in Luria-Bertani (LB) broth at 37°C. Bacterial counts were assessed using a microplate reader by optical density at 600 nm. 150 μL of E. coli or S. aureus bacterial suspension (10 8 CFU / mL) were plated on agar plates. Four equidistant wells (5 mm diameter) were created per plate. No other treatment was applied to the control group. Three experimental groups were set up: PVA / CS solution, PVA / CS / TOB hydrogel beads, and PCBT hydrogel beads. These materials (equal volumes) were added to the wells and incubated with bacteria at 37°C for 12 hours. Finally, zones of inhibition (ZOIs) were measured. To investigate the sustained antibacterial activity of PCBT hydrogel beads, ZOIs were recorded at 12, 24, 48, 72, 96, and 120 hours.

[0070] To evaluate the keratitis after the application of hydrogel beads, H&E, Masson and immunohistochemical staining were performed on the samples on day 4, and iNOS and IL-10 were selected as inflammatory markers. The PCBT group had the lowest iNOS positive level, while the control group showed significant iNOS positive ( Figure 11 ). It can be seen from the H&E and Masson staining results ( Figure 12 Both the control and PC groups showed significant inflammatory cell infiltration, necrosis of corneal collagen fibers, and localized corneal thickening. In the PCBT group, the corneal stroma showed minimal inflammatory cells, abundant and orderly arranged collagen fibers, and intact tissue morphology. These results confirm the excellent antibacterial, anti-inflammatory, and pro-repair capabilities of PCBT hydrogel.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing hydrogel beads for treating keratitis, characterized in that: The steps include: (1) Preparation of PVA / CS mixed solution: Add polyvinyl alcohol (PVA) to water, heat and dissolve, then add water-soluble chitosan (CS), and stir until dissolved to obtain the PVA / CS mixed solution; (2) Preparation of a TOB / BA mixed solution: Tobramycin was dissolved in water, followed by addition of terephthalic acid and baicalin, and ultrasonication was performed until completely dissolved to obtain a TOB / BA mixed solution; (3) Preparation of hydrogel beads: The PVA / CS mixed solution of step (1) is added dropwise to the TOB / BA mixed solution of step (2), and the droplets form hydrogel beads under the action of gravity to obtain hydrogel beads for treating keratitis.

2. The preparation method according to claim 1, characterized in that In the step (1), the concentration of the polyvinyl alcohol solution is 5-10% w / w, the dissolution temperature is 95°C ± 2°C; the addition temperature of the water-soluble chitosan is 50°C ± 5°C, and the stirring time is 30-60 minutes; The mass ratio of polyvinyl alcohol to water-soluble chitosan is 5-20:

1.

3. The preparation method according to claim 1, characterized in that In the step (2), the concentration of the tobramycin solution is 0.1-0.3% w / w, the concentration of terephthalic acid is 0.02-0.12% w / w, the concentration of baicalin is 0.01-0.04% w / w, and the ultrasonic treatment conditions are a power of 200-300 W and a time of 10-30 minutes.

4. The preparation method according to claim 1, characterized in that In step (3), the PVA / CS mixed solution is added dropwise to the TOB / BA mixed solution by means of a syringe, the syringe needle diameter is 0.23-0.6 mm, the dropping height is 10 cm ± 2 cm; the diameter of the hydrogel beads is 0.2-5 mm, and the standing time is 30 minutes ± 5 minutes.

5. The hydrogel beads for treating keratitis prepared by the preparation method according to any one of claims 1 to 4, characterized in that: In the hydrogel beads, BA and TOB are uniformly dispersed in the PVA / CS network structure in an amorphous form. The crystal structure of the hydrogel beads is jointly constructed by PVA and CS, and the two interact through hydrogen bonds to form a stable composite crystal system.

6. The hydrogel beads for treating keratitis according to claim 5, characterized in that The drug loading in the hydrogel beads is 0.5-5 wt%, and the particle size of the hydrogel beads is 200 μm-5 mm.

7. Use of the hydrogel beads for treating keratitis according to claim 5 or 6 in the preparation of a drug for treating keratitis.

8. The use according to claim 7, wherein the treatment of keratitis is performed during sleep.

9. The use according to claim 7, wherein the hydrogel beads for treating keratitis have pH-responsive drug release characteristics at a tear pH of 7.4-7.

6.

10. The use according to claim 9, characterized in that The keratitis treatment is performed by placing the device on the lower eyelid during sleep, and suppressing keratitis caused by infection of one or more bacteria including Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa through sustained release of TOB / BA.

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