Hydrogel bead for treating keratitis and application of hydrogel bead in sleep treatment

Abstract

The invention discloses a hydrogel bead for treating keratitis and application of the hydrogel bead in sleep treatment. The preparation method comprises the following steps: adding water into polyvinyl alcohol (PVA), heating for dissolving, heating water-soluble short-chain chitosan (CS), and stirring until dissolving to obtain a PVA / CS mixed solution; the preparation method comprises the following steps: dissolving tobramycin in water, then adding p-phenylenediboronic acid and baicalin, and carrying out ultrasonic treatment until the tobramycin and the baicalin are completely dissolved to obtain a TOB / BA mixed solution; the PVA / CS mixed solution is dropwise added into the TOB / BA mixed solution, liquid drops form hydrogel beads under the action of gravity, and the hydrogel beads for treating keratitis are obtained. The hydrogel microspheres can be continuously degraded to release TOB and BA in the sleep stage, TOB has broad-spectrum antibacterial activity, BA can promote macrophage to be converted from M1 type to M2 type and remove active oxygen, and antibacterial, anti-inflammatory and anti-oxidation effects are achieved at the same time. In-vivo experiments of a rabbit cornea S. aureus infection model further prove that compared with conventional commercially available eye drops, the PCBT hydrogel microspheres show a better treatment effect and better clinical application potential, and are suitable for treatment and management of BK in a sleep stage.

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) for the treatment of bacterial keratitis, belonging to the technical field of medical materials. Background Art

[0002] Bacterial keratitis (BK) is the main causative factor of corneal ulcer. Its high incidence in daily life and the high risk of corneal perforation, intraocular infection, and ultimately irreversible vision loss and blindness make it a serious threat to human visual health globally. Clinically, the treatment of BK is quite challenging, and the main difficulties are reflected in four aspects: (i) low drug bioavailability; (ii) bacterial drug resistance; (iii) poor patient compliance; (iv) long corneal wound healing cycle. Although a large number of advanced technologies and biomaterials have been developed to solve the above problems, an obvious defect has long been ignored, that is, the lack of ability to manage BK during the sleep stage. During the sleep stage, most of the currently available strategies are either inapplicable or have impaired efficacy. For example, antibiotic eye drops need to be instilled frequently to improve drug bioavailability, which is obviously impossible during the sleep stage. Photothermal or photocatalytic therapy requires long-term exposure to light, and sonodynamic therapy requires noisy ultrasonic stimulation, which may seriously affect the patient's sleep quality. Since BK progresses rapidly and may deteriorate rapidly if not treated properly, it is crucial to find an ideal alternative to prevent the deterioration of BK during the sleep stage, but this problem has not attracted wide attention.

[0003] So far, some strategies have shown certain potential in the treatment of BK during the sleep phase, but they are still far from achieving the ideal effect. For example, Luo et al. locally constructed a continuously antibacterial microenvironment by injecting gelatin-coated silver nanoparticles into the corneal stroma. However, intravitreal administration is a complex treatment process that may exacerbate corneal trauma. The same problem also exists in the study of 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 form a long-term antibacterial microenvironment on the corneal surface by controllably releasing therapeutic molecules or post-modifying antibacterial coatings. However, wearing contact lenses during the sleep phase may cause various eye discomforts, exacerbate infections, and lead to invasive events. Therefore, hydrogel eye drops, in-situ forming hydrogels, thermosensitive hydrogels, and supramolecular hydrogels may be more reasonable alternatives because they have higher patient compliance and weaker side effects. Traditional hydrogels used for BK treatment mainly target rapid and efficient bactericidal effects. However, bacterial infections are usually accompanied by severe inflammatory responses and oxidative stress damage mediated by reactive oxygen species (ROS). Although ROS can inhibit bacterial growth, they can also damage normal cell functions and induce cell death. Therefore, the key to managing BK during the sleep phase lies in finding a balance between bacterial clearance and inflammation alleviation.

[0004] Tobramycin (TOB) is an aminoglycoside antibiotic with broad-spectrum antibacterial activity and has been widely used in the treatment of BK. In addition, TOB has good water solubility, and its aqueous solution is weakly alkaline due to the protonation of the side amino groups. Macrophages (Mφ) are the main immune cells at the BK wound site. They maintain a pro-inflammatory M1 phenotype, secrete inflammatory cytokines, and produce ROS, thus hindering inflammation resolution and wound healing. Baicalin (BA) is a bioactive flavonoid drug containing a catechol structure and has various anti-inflammatory effects. Studies have shown that it can induce the transformation of M1 macrophages into M2 macrophages and inhibit the production of ROS by M1 macrophages. Water-soluble short-chain chitosan (CS) is a cationic polyelectrolyte with excellent biocompatibility and antibacterial activity. A high-concentration CS aqueous solution can rapidly transform into a high-strength crystalline hydrogel after treatment with an alkaline solution. Polyvinyl alcohol (PVA) is a neutral linear polymer with abundant hydroxyl side groups and has been widely used as the main component of eye drops. Phenylboronic acid groups can react with catechol structures or diol structures to form reversible phenylborate ester bonds. Summary of the Invention

[0005] Based on this, the present invention has developed a millimeter-sized hydrogel microsphere for managing BK during the sleep stage. This hydrogel microsphere is composed of PVA, CS, 1,4-benzenediboronic acid (1,4-BDBA), BA, and TOB. Specifically, the PVA / CS solution is slowly dropped into the 1,4-BDBA / BA / TOB solution through a syringe. Subsequently, PVA / CS / BA / TOB (PCBT) hydrogel microspheres are obtained. In this formulation, the hydrogel microspheres form their shape through pH-induced CS crystallization and are further strengthened by reversible crosslinking mediated by phenylboronic acid ester bonds between PVA chains. In addition, BA molecules are reversibly linked to the PVA chains, and TOB molecules are encapsulated in the hydrogel microspheres. During the treatment of BK in the sleep stage, the PCBT hydrogel microspheres are placed inside the lower eyelid. The hydrogel microspheres can stay in this area for a long time, during which the hydrogel microspheres gradually degrade, and TOB and BA molecules are continuously released, thereby continuously inhibiting bacterial growth, alleviating the inflammatory response and ROS damage.

[0006] In the present invention, the composition, structure, mechanical properties, structural stability, and drug release behavior of the 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). The immunomodulatory ability of the hydrogel microspheres to induce the transformation of M1 macrophages into M2 macrophages and scavenge ROS was evaluated through in vitro cell co-culture experiments. In addition, a rat BK model was used in combination with immunohistochemical staining and other methods to explore the potential of the hydrogel microspheres to manage BK in the sleep state. The present invention not only reports an effective strategy for managing BK during the sleep stage but also provides new inspiration for related research.

[0007] The objective of the present invention is to solve the problems of low drug utilization rate, short action time, and poor patient compliance in the treatment of keratitis with antibiotic eye drops. The present invention constructs a hydrogel of PVA / CS / TOB / BA through a dynamic crosslinking technique of glue to achieve pH-responsive drug release, which has the functions of long-acting antibacterial, anti-inflammatory, and promoting corneal repair.

[0008] To achieve the above objective, the technical solution adopted by the present invention is: a tobramycin baicalin hydrogel bead with antibacterial effects and its preparation method, comprising the following steps: (1) Prepare a PVA / CS mixed solution: Add water to polyvinyl alcohol PVA and heat it to dissolve, then heat water-soluble short-chain chitosan CS, and stir until dissolved to obtain a PVA / CS mixed solution; (2) Prepare a TOB / BA mixed solution: Dissolve tobramycin in water, then add 1,4-benzenediboronic acid and baicalin, and ultrasonically treat until completely dissolved to obtain a TOB / BA mixed solution; (3) Preparation of hydrogel beads: The PVA / CS mixed solution from step (1) was dropped into the TOB / BA mixed solution from step (2), and the droplets formed hydrogel beads under the action of gravity to obtain hydrogel beads for treating keratitis.

[0009] In step (1), the concentration of the polyvinyl alcohol solution was 5 - 10% (w / w), and the dissolution temperature was 95°C ± 2°C; the addition temperature of the water-soluble chitosan was 50°C ± 5°C, and the stirring time was 30 - 60 minutes; the mass ratio of polyvinyl alcohol to water-soluble chitosan was 5 - 20:1.

[0010] In step (2), the concentration of the tobramycin solution was 0.1 - 0.3% (w / w), the concentration of terephthalic boric acid was 0.02 - 0.12% (w / w), the concentration of baicalin was 0.01 - 0.04% (w / w), and the ultrasonic treatment conditions were a power of 200 - 300 W and a time of 10 - 30 minutes.

[0011] In step (3), the PVA / CS mixed solution was dropped into the TOB / BA mixed solution by means of a syringe. The diameter of the syringe needle was 0.23 - 0.6 mm, and the dropping height was 10 cm ± 2 cm; the diameter of the hydrogel beads was 0.2 - 5 mm, and the standing time was 30 minutes ± 5 minutes.

[0012] In some preferred embodiments, the PVA / CS mixed solution prepared in step (1) was placed 10 cm above the TOB / BA mixed solution prepared in step (2) using a syringe, and then slowly dropped drop by drop. The droplets dropping under the action of gravity would completely enter the solution and form individual hydrogel beads. By changing the diameter of the syringe needle, the size of the prepared hydrogel beads could be controlled, and the hydrogel beads could be prepared after standing for 30 minutes.

[0013] The hydrogel beads for treating keratitis prepared by the above method, in which BA and TOB were uniformly dispersed in the PVA / CS network structure in an amorphous form, and the crystal structure of the hydrogel beads was jointly constructed by PVA and CS, and the two formed a stable composite crystal system through hydrogen bond interaction.

[0014] The drug loading amount in the hydrogel beads was 0.5 - 5 wt%, and the particle size of the hydrogel beads was 200 μm - 5 mm.

[0015] PVA is a neutral linear polymer with rich hydroxyl side groups and contains a cis-diol structure.

[0016] Phenylenediboronic acid can undergo configurational transformation in different acidic and basic environments and can be used as a dynamic crosslinking agent. When in an acidic medium, phenylenediboronic acid exists as a relatively hydrophobic triangle without charge, and its reactivity with cis - diol groups is significantly reduced; while under weakly basic conditions, it exists in a tetrahedral structure, exposing highly reactive boronic acid groups, which can easily form reversible coordination complexes with compounds containing cis - diol structures.

[0017] The free amino groups on the CS molecular chain will be attracted by the hydroxyl groups in the PVA molecule to achieve hybrid cross - linking, and self - assemble in alkaline and monovalent anion salt solutions to form a physical microcrystalline network and chain entanglement network with both biodegradability and antibacterial activity.

[0018] Cross - linking PVA and CS molecules through hydrogen bonding and electrostatic interactions can significantly improve the mechanical properties of the composite material.

[0019] TOB is easily soluble in water, binds protons in water, and releases hydroxide ions to make the solution alkaline.

[0020] BA has a cis - diol structure and will form phenylboronic acid ester bonds with phenylboronic acid to prevent the destruction of the activity of baicalin.

[0021] TOB dissolves in water to provide an alkaline environment. Phenylenediboronic acid is added to the TOB solution with a pH of about 8.5, causing phenylboronic acid to form a tetrahedral active structure, which then binds to the cis - diol structure to form a more stable charged complex.

[0022] The beneficial effects of the present invention are as follows: A tobramycin baicalin hydrogel bead with antibacterial effects and its preparation method provided by the present invention. Through a pH - responsive self - assembly mechanism, the CS molecular chain is induced to align directionally under weakly basic conditions. Secondly, phenylenediboronic acid is introduced as a dynamic cross - linking agent, and its tetrahedral configuration forms reversible boronic acid ester bonds with the cis - diol groups of PVA under alkaline conditions to construct a three - dimensional network with self - repair characteristics. At the same time, the BA molecule also has a cis - diol structure, so it can also form reversible phenylboronic acid ester bonds with phenylenediboronic acid, and the TOB molecule is encapsulated in the hydrogel bead to form a drug - loading system. When the drug - releasing gel is placed in an environment with a pH of 6.4 - 7.7, TOB in the hydrogel is continuously released, the pH continuously decreases, the PVA / CS double - network cross - linked structure changes from a stable tetrahedral structure to an unstable planar structure, the BA molecule also detaches from phenylenediboronic acid, and CS refolds and crystallizes to return to its original random linear form until the gel degrades. 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 synergistic design, providing a new strategy for the research and development of ophthalmic anti - infective materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Schematic diagram for the preparation of PCBT hydrogel beads in Example 1.

[0024] Figure 2 (A) is a physical image of hydrogel beads with different diameters during the preparation process of Example 1, and (B, C) are the surface morphology results of hydrogel beads during the preparation process of Example 1.

[0025] Figure 3 (A, B, C) are Fourier transform infrared spectra of the hydrogel beads prepared in Example 1.

[0026] Figure 4 XRD pattern of the hydrogel beads prepared in Example 1.

[0027] Figure 5 Ultraviolet absorption spectrum of the hydrogel beads prepared in Example 1.

[0028] Figure 6 (A, B) are rheological analyses of the hydrogel beads prepared in Example 1.

[0029] Figure 7 In vitro drug release curve of the hydrogel beads prepared in Example 1.

[0030] Figure 8 TOB(H) and BA delivery models (I) of the PCBT hydrogel beads in Example 1.

[0031] Figure 9 Results of the effects of the hydrogel beads prepared in Example 1 on mouse mononuclear macrophages (RAW264.7) (A) and human corneal epithelial cells (HECE) (B).

[0032] Figure 10 Representative fluorescence images (A) and corresponding quantitative statistical charts (B) of iNOS and CD206 after the hydrogel beads prepared in Example 1 were incubated with RAW264.7 cells for 1 day.

[0033] Figure 11 H&E, Masson, and immunohistochemical staining images of the hydrogel beads prepared in Example 1.

[0034] Figure 12 Corneal staining and immunohistochemical staining (iNOS and IL-10) on the 7th day (A), and their quantitative analysis (B). Detailed implementation mode

[0035] Example 1 This example provides a preparation method of tobramycin baicalin hydrogel beads with antibacterial effects according to the present invention, and the following method is prepared by using the Figure 1 principle and process, including the following steps: (1) Weigh a certain mass of polyvinyl alcohol PVA and water-soluble chitosan CS (purchased from Qingdao Bozhihuil Biotechnology Co., Ltd.) in a ratio of 10:1. First, dissolve polyvinyl alcohol in pure water (95 °C), stir (600 r / min) for 1 hour to prepare a 10% polyvinyl alcohol solution. Cool the solution to 50 °C, add water-soluble chitosan, and continue to stir magnetically at a constant temperature (600 r / min) for 1 h until it is completely dissolved. 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; (2) Dissolve 0.06 g of 1,4-benzenediboronic 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 with a pH of about 8.5. Then, using a dropping pipette (0.25 mL / drop, 10 drops / min), slowly drop the mixed ethanol solution of 1,4-benzenediboronic acid into the TOB aqueous solution to obtain a 1.4-BDBA / BA / TOB aqueous solution; (3) Prepare PCBT hydrogel beads. Use a syringe to suck up the PVA / CS aqueous solution, use a 2 mm diameter needle, place it 10 cm above the 1.4-BDBA / BA / TOB aqueous solution, and then slowly drop it drop by drop. The liquid beads dropping under the action of gravity 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, hydrogel beads PCBT are obtained.

[0036] Example 2 In this example, the surface morphology of the PCBT hydrogel beads prepared in Example 1 was detected.

[0037] Detection of the surface morphology of hydrogel beads: (1) The macroscopic photographs of each group of hydrogel beads changed as shown in Figure 2 A, which was a smooth spherical shape on the surface, showing yellow, indicating that baicalin was loaded into it. By changing the diameter of the syringe needle, the diameter of the prepared hydrogel beads can be changed. Using a 0.18 mm diameter needle, hydrogel beads with a minimum diameter of 200 μm as shown in Figure 2 can be prepared. Using a 0.6 mm diameter needle, 2 mm diameter hydrogel beads as shown in Figure 2 can be prepared. Using a 2 mm diameter needle, 5 mm diameter hydrogel beads as shown in Figure 2 can be prepared.

[0038] (2) Figure 2In B and C, the surface morphology of the sample was characterized using a scanning electron microscope (SEM). The surface of the hydrogel beads was a sparse porous structure with pore sizes between 50 - 200 nm. Most of the TOB loaded on the hydrogel beads was located on the surface and in the shallow layer of the hydrogel beads. The loose porous structure facilitated the preferential release of TOB during the pH-responsive degradation and release of BA from the hydrogel beads, as well as the release processes of PVA and CS.

[0039] Hydrogel structure detection: (3) Figure 3 In it, the FTIR spectra of CS, PVA, TOB, BA, and PCBT beads in different wavelength ranges are shown. In the FTIR spectrum of the PCBT microbeads, the representative peaks of CS (axial -O-H and -N-H stretching at 3439 cm -1 , -C-O-C in the glycosidic bond at 1096 cm -1 ), PVA (-OH47 at 3439 cm -1 , -C-C stretching at 1260 cm -1 ), BA (-COOH at 1727 cm -1 , -C=O at 1664 cm -1 and 1613 cm -1 ), and TOB (-NH2 at 1582 cm -1 ) are shown, indicating that BA and TOB molecules have been successfully encapsulated into the PVA / CS droppers to form PCBT gel beads. In addition, the bands at 3552 cm -1 , 3491 cm -1 and 1368 cm -1 corresponding to the catechol group of BA disappeared in the FTIR spectrum of the PCBT beads, proving the formation of a phenylboronic acid ester bond between 1,4-BDBA and BA. This conclusion can also be verified by the newly emerged peak at 1363 cm -1 in the FTIR spectrum of the PCBT microbeads. In addition, the present invention also calculated and compared the intensity ratios of the peaks at 1260 cm -1 (-CC-) and 3437 cm -1 (-OH) (I 3034 / I 1260 ) of pure PVA and PCBT beads. The I 3034 / I 1260 ratios of pure PVA and PCBT beads were 0.983 and 0.937 respectively, which proved to a certain extent the formation of a phenylboronic acid ester bond between 1,4-BDBA and PVA.

[0040] (4) To further investigate the microstructure of PBCT hydrogel beads, XRD ( Figure 4 ) was applied. No diffraction peaks of TOB and BA were found in the XRD spectrum of the PCBT hydrogel beads, indicating their uniform distribution in the PCTB hydrogel microbeads. The diffraction peaks of CS at 12.3 o and 24.2 o disappeared in the XRD spectrum of the PCBT hydrogel beads, but the peak at 9.6 o remained. The peak at 9.6° corresponds to the crystal plane (020) of the CS aggregates. Here, the crystallinity index (CrI) was used to study the crystallization behavior of the CS chains.

[0041]

[0042] where I 020 and Iamor are the diffraction intensities of the peak (020) and the amorphous peak (2θ = 16 o ), respectively55. It was found by calculation that the Crl (0.271) of the CS aggregates in the PCBT hydrogel beads was much higher than that of pure CS (0.054), which means that the alkaline TOB solution induced the crystallization of the CS chains in the PCBT hydrogel beads, and the CS aggregates were mainly arranged perpendicular to the crystal plane (020) (Figure 4). As for PVA in the PCBT hydrogel beads, its representative diffraction peaks (intermolecular and intramolecular hydrogen bonds) shifted from 19.5 o to 20.1 o . Here, the apparent crystal size Dapp was calculated with the Scherrer equation:

[0043] 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 of the Bragg angle55. It was found by calculation that the Dapp of PVA in the PCBT hydrogel beads decreased from 24.41 nm to 11.59 nm compared with the original PVA, which may be due to the crosslinking reaction destroying the ordered arrangement of hydrogen bonds ( Figure 4 ).

[0044] (5) Figure 5 shows the UV spectra of 1,4-BDBA, BA, and PCBT hydrogels. When BA was incorporated into the PCBT hydrogel, the absorbance peak of BA at 316 nm shifted to 306 nm, further verifying the formation of phenylboronic ester bonds between 1,4-BDBA and BA.

[0045] (6) Figure 6 shows the rheological behaviors of PVA / CS solutions and PCBT hydrogel beads. The prepared hydrogel beads were placed on the bottom plate of a rotational rheometer, and then frequency scanning was carried out while controlling the variable vibration stress. The results obtained from the logarithmic mode of angular frequency (0.1 - 100) 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 (tanδ < 1) was observed.

[0046] (7) The drug release of BAN and TOB in PVA / CS / BAN / TOB hydrogel beads was determined by dynamic dialysis. The molecular weight cut-off of the dialysis bag was 1 KD. Twenty PVA / CS / BAN / TOB hydrogel beads with a diameter of 2 mm were placed in the dialysis bag and then put into 10 mL of PBS buffer solution with pH 7.4 and 5.5, and shaken on a constant temperature shaker at 37°C at a speed of 100 rpm. 1 mL of the release medium was taken at 0.08, 0.25, 0.5, 0.75, 1, 5, 9, 12, 20, and 28, and an equal amount of fresh release medium was added to keep the volume constant at the predetermined time intervals. The UV absorption peak areas of BAN and TOB drugs in the release medium were measured respectively, and the contents of BAN / TOB were calculated according to the established standard curve and the cumulative release amounts over a period of time were calculated. The drug in vitro release curve was plotted with time as the abscissa and the cumulative release amount as the ordinate. The formula for calculating the cumulative release amount is as follows: Cumulative release amount =

[0047] where V is the total volume of the release medium (ml), V0 is the volume of the release medium taken each time, and C i is the drug concentration at the i-th sampling.

[0048] Since the microenvironment of the normal eye is neutral while that of the 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 in 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.

[0049] Table 1 Mathematical modeling and related parameters based on release data

[0050] Example 3 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.

[0051] (1) The CCK-8 method and live / dead cell staining were used to evaluate the cytotoxicity of the hydrogel leachate on mouse mononuclear macrophages and human corneal epithelial cells. HCEC (the third passage) or raw 264.7 cells (2×10 3 / holes) were cultured in a 96-well plate supplemented with DMEM containing 10% FBS and 1% penicillin / streptomycin (37 °C, 5% CO2). After the confluence rate reached 70%, four different types of media (10 μL) were added to the wells: (1) control group, fresh complete medium; (2) conditioned medium 1, complete medium supplemented with 0.3% (w / v) tobramycin and 0.8% (w / v) baicalin; (3) conditioned medium 2, complete medium treated with UV-sterilized PCBT hydrogel beads (5 beads with a diameter of about 2 mm were soaked in 5 mL of medium for 24 hours). After co-culturing for 24 hours, cell viability was evaluated using a CCK-8 kit (absorbance at 450 nm, Varioskan LUX microplate reader). Live / dead staining was performed using calcein-AM / PI, and photos were captured using a confocal microscope (Leica DMI3000B).

[0052] The CCK-8 results showed that there was no difference in cell proliferation among different groups ( Figure 9 in B). The results of live / dead cells were as Figure 9 shown in (A). Each group mainly emitted green fluorescence and rarely emitted red fluorescence, indicating that a large number of live cells and a small number of dead cells were observed. The results showed that the material had good biocompatibility.

[0053] (2) Raw 264.7 cells were cultured in a 96-well plate until all cells adhered and the confluence rate reached 70%, and then they were divided into three groups: (1) control group, without any treatment; (2) lipopolysaccharide (LPS) group (10 ng / mL LPS was added to induce macrophage M1 polarization); (3) experimental group, macrophages were first treated with LPS and then with the 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). The cell nuclei were stained with DAPI. The fluorescence intensity was quantified using ImageJ (NIH). As Figure 10 shown, cell immunofluorescence staining was used to detect the expression of iNOS (M1 marker) and CD206 (M2 marker) to study the effect of the hydrogel on the polarization of RAW264.7 cells. The results showed that the expression of iNOS (M1 marker) decreased and the proportion of CD206 increased in the PCBT hydrogel leachate group.

[0054] Example 4 This test example detected the in vivo antibacterial and corneal repair-promoting performance evaluation of the tobramycin baicalin hydrogel beads with antibacterial effect prepared in Example 1. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were cultured overnight in Luria-Bertani (LB) broth at 37 °C. The bacterial count was evaluated by the optical density at 600 nm using a microplate reader. 150 μL of the Escherichia coli or Staphylococcus aureus bacterial suspension (10 8 CFU / mL) was spread on the agar plate. Four equally spaced holes (5 mm in diameter) were created on each plate. No other treatment was applied to the control group. A total of 3 experimental groups were set up: PVA / CS solution, PVA / CS / TOB hydrogel beads, and PCBT hydrogel beads. These materials (the same volume) were added to the holes and co-cultured with the bacteria at 37 °C for 12 hours. Finally, the inhibition zones (ZOIs) were measured. To study the sustained antibacterial ability of the PCBT hydrogel beads, the ZOIs at 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h were recorded.

[0055] To evaluate the condition of keratitis after the application of the hydrogel beads, the samples on the 4th day were subjected to H&E, Masson, and immunohistochemical staining, and iNOS and IL-10 were selected as inflammatory markers. The PCBT group had the lowest positive level of iNOS, while obvious iNOS positivity was visible in the control group ( Figure 11 ). As can be seen from the results of H&E and Masson staining ( Figure 12 ), obvious inflammatory cell infiltration and necrosis of corneal collagen matrix fibers were visible in both the control group and the PC group, and the local corneal tissue was thickened. There were very few inflammatory cells in the corneal stroma of the PCBT group, the collagen fibers were abundant / arranged orderly, and the tissue morphology was intact. All these results confirmed that the PCBT hydrogel had good antibacterial, anti-inflammatory, and repair-promoting abilities.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Preparation method of hydrogel beads for treating keratitis, characterized in that, It includes the following steps: (1) Prepare a PVA / CS mixed solution: Add water to polyvinyl alcohol (PVA) and heat it to dissolve. Then add water-soluble chitosan (CS), and stir until dissolved to obtain a PVA / CS mixed solution; (2) Prepare a TOB / BA mixed solution: Dissolve tobramycin in water, then add terephthalic boric acid and baicalin, and perform ultrasonic treatment until completely dissolved to obtain a TOB / BA mixed solution; (3) Prepare hydrogel beads: Drop the PVA / CS mixed solution in step (1) into the TOB / BA mixed solution in 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, wherein In step (1), the concentration of the polyvinyl alcohol solution is 5 - 10% (w / w), and 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 step (2), the concentration of the tobramycin solution is 0.1 - 0.3% (w / w), the concentration of terephthalic boric 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 dropped into the TOB / BA mixed solution by using a syringe. The diameter of the syringe needle is 0.23 - 0.6 mm, and 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 1-4, 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 form a stable composite crystal system through hydrogen bond interaction.

6. The hydrogel beads for treating keratitis according to claim 5, characterized in that, The drug loading amount 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. According to the use of claim 7, the treatment of keratitis is carried out in a sleeping state.

9. According to the use of claim 7, the hydrogel beads for treating keratitis have a pH-responsive drug release property in the tear pH environment (7.4 - 7.6).

10. The use according to claim 9, characterized in that, The treatment of keratitis is to place it under the lower eyelid during sleep, and continuously release TOB / BA through slow release to inhibit keratitis caused by the infection of one or more bacteria including Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa.

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