Collagen anchored temperature-sensitive gel as well as preparation method and application thereof

Abstract

The invention provides a collagen anchoring type temperature-sensitive gel. The collagen anchoring type temperature-sensitive gel is prepared from amphotericin B, poloxamer and tannic acid. The temperature-sensitive gel provided by the invention is composed of two materials with excellent biocompatibility, namely poloxamer and tannic acid, and the drug retention time is prolonged through non-invasive temperature-induced phase change and a collagen specific anchoring mechanism, so that the potential damage of a physical delivery mode to cornea tissues is completely avoided, and the safety advantage is obvious. Besides, tannic acid is used as a collagen specific anchoring agent, and also has various beneficial biological activities including anti-inflammatory, anti-oxidation, antibacterial and metal ion chelating functions and the like. By prolonging the residence time of the tannic acid at the focus part, the biological effect of the tannic acid is systematically and remarkably expanded, the dual advantages of drug delivery and biological activity synergistic effect are realized, and an innovative solution is provided for comprehensive treatment of fungal keratitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a collagen-anchored thermosensitive gel, a preparation method thereof, and an application thereof. Background Art

[0002] Fungal keratitis (FK) is a common blinding corneal infectious disease. Its main symptoms include pain, tearing, photophobia, and vision loss, which are closely related to corneal ulcers. Corneal ulcers are not only the main pathological feature of the disease but also accelerate the progression of symptoms.

[0003] The pathogenic mechanism of FK begins with the invasion of microorganisms through the damaged epithelial barrier. Subsequently, the pathogen secretes collagenase and triggers the infiltration of inflammatory cells, ultimately leading to matrix degradation and the formation of characteristic ulcer pits, which may progress to corneal perforation. Compared with other infections, corneal ulcers caused by FK are more likely to perforate, often leading to endophthalmitis and irreversible blindness.

[0004] Currently, the first-line drugs for the clinical treatment of FK are amphotericin B and natamycin. Among them, amphotericin B (AmB), as the "gold standard" drug for antifungal treatment, its mechanism of action is mainly based on the specific binding to ergosterol in the fungal cell membrane, forming transmembrane pores and causing cell apoptosis. However, AmB faces multiple challenges in the treatment of FK. From the perspective of the molecular mechanism of action, AmB may also interact with cholesterol in the mammalian cell membrane. This non-selective binding is the main source of its cytotoxicity, so precise control of the dose is crucial. Especially in the case of the formation of fungal biofilms, the treatment difficulty is further increased. The biofilm formed by Candida albicans (C. albicans) by attaching to the surface of the corneal epithelium or medical devices (such as contact lenses) not only enhances its survival ability in diverse environments but also provides a protective barrier against antifungal drugs and the body's immune system. From the perspective of drug delivery, the clinical application of AmB is severely restricted by its physicochemical properties:

[0005] (1) Solubility limitation: Currently, there is no commercial ophthalmic preparation. The specially prepared AmB eye drops in hospitals can only dissolve low-concentration drugs (0.15%) and have poor stability (the shelf life is only 1 week), which significantly increases the treatment cost;

[0006] (2) Influence of the drug aggregation state: Although the monomer form of AmB has a high bioavailability, it also shows strong toxicity and can be used for local administration through cyclodextrin inclusion. In contrast, the aggregate form of AmB shows higher selectivity for ergosterol, but its permeability is limited;

[0007] (3)Short corneal residence time: The rapid clearance of conventional eye drops from the ocular surface significantly reduces the local drug concentration, especially in the case of biofilm infections, making it difficult to maintain an effective therapeutic concentration. Amphotericin B mixed micelles (Fungizone) with sodium deoxycholate as a solubilizer have certain efficacy, but due to the toxicity of deoxycholic acid itself, it is not suitable for ophthalmic topical administration.

[0008] These limitations highlight the importance and urgency of developing an ophthalmic formulation of AmB with sustained release ability.

[0009] In the current treatment strategies for FK, although significant progress has been made in novel drug delivery systems (such as hydrogels, ointments, and drug-loaded therapeutic contact lenses) in the past decade, topical eye drops still dominate. Clinically, eye drops are the main dosage form for ocular administration in fungal keratitis and the most commonly used method for treating anterior segment diseases, accounting for more than 90% of all dosage forms. This situation reflects both the inherent advantages of the eye drop administration method (such as convenience of use, cost-effectiveness, good patient acceptance, etc.), while FK has many complications and poor treatment outcomes under the current treatment means, and also exposes the major challenges faced by existing treatment regimens in clinical applications. Especially in the application of first-line antifungal drugs such as amphotericin B (AmB) and natamycin, these limitations are particularly prominent. This is mainly due to the unique physiological and anatomical characteristics of the eye: The corneal epithelium is composed of tightly connected cells, forming a strong physical barrier that can effectively block the penetration of external harmful substances and drug molecules. Its highly hydrophilic nature makes it difficult for most drugs, especially lipophilic drugs, to pass through this layer. Although the stromal layer allows partial penetration of water-soluble drugs, due to its rich collagen fiber structure, the diffusion rate of drugs in this layer is slow. Although the endothelium does not have a significant barrier function like the epithelium, it can still regulate the hydration of the cornea and affect the permeability of drugs. In addition, tears stay on the ocular surface before flowing into the nasolacrimal duct, and frequent blinking actions will remove debris and excess fluid on the ocular surface, resulting in less than 5% of the drugs administered through eye drops being absorbed. Therefore, in the acute phase of FK, multiple administrations are required daily, and even once every 15 minutes, which not only leads to poor patient compliance, but also increases the hospitalization rate and may promote the generation of antifungal drug resistance. In addition, due to the significant toxic side effects associated with the blood-eye barrier and systemic administration, systemic antifungal treatment is usually only used as an adjunct to local treatment in severe cases. Therefore, there is an urgent need to develop a new type of ocular drug delivery platform that is convenient to use, has a good experience, and has a long-lasting effect to increase drug absorption on the ocular surface and improve the management of fungal keratitis.

[0010] To address the key challenge of rapid pre - corneal clearance (short corneal drug residence time), researchers have developed a series of innovative drug delivery strategies. For example, the microneedle delivery system represents a minimally invasive drug administration solution. Its core design consists of a degradable polymer microneedle array with micron - scale precise geometric dimensions, achieving directional drug delivery through controlled mechanical penetration. Studies on corneal drug delivery have shown that this system can extend the residence time of drugs in corneal tissue to more than 2 hours, mainly attributed to its unique dual - action mechanism: First, the physical penetration of microneedles can cross the corneal epithelial barrier and directly deliver drugs to the stromal layer; second, the direct mechanical contact between microneedles and corneal tissue enhances the local retention ability of the formulation. However, in the treatment of infectious keratitis, this method faces significant challenges. Due to the significant damage to the corneal structure in the diseased state (manifested as disrupted epithelial integrity, stromal layer degradation, etc.), the mechanical action of microneedles may further exacerbate tissue damage, increasing the risk of infection spread and perforation.

[0011] Mucoadhesive polymers offer another non - invasive solution. Such materials are usually composed of functionalized hydrophilic polymers (such as hyaluronic acid, carboxymethyl cellulose, chitosan, etc.), whose surfaces carry a large number of functional groups that can specifically interact with ocular surface mucins. In theory, this adhesion at the mucin molecular level can significantly extend the corneal contact time of drugs. However, in - depth studies have found that its clinical application effect is affected by multiple factors: First, the corneal mucin layer has the characteristic of dynamic renewal; second, the mechanical shear force generated by blinking movements (frequency about 15 - 20 times per minute) and the flushing effect of tears significantly limit the surface residence time of the material. More importantly, in the inflammatory state, changes in the ocular surface microenvironment (such as pH changes, increased protease activity, etc.) may further reduce the adhesion efficiency. The limitations of these technological explorations highlight the importance of developing new drug delivery systems. An ideal drug delivery system not only needs to overcome the physiological clearance mechanism but also should have the ability to adapt to changes in the tissue microenvironment under pathological conditions. This requires considering multiple dimensions such as biocompatibility, long - acting drug properties, and pathological adaptability in material design to optimize drug delivery efficiency. At the same time, the development of new delivery systems should also fully consider their clinical translation potential, including factors such as the scalability of the preparation process, ease of use, and cost - effectiveness.

[0012] Therefore, for the long - term management of FK, existing treatments show limitations such as limited corneal drug residence time, poor treatment effect, and many treatment outcome complications. There is an urgent need to develop a new ocular drug delivery platform that is easy to use, provides a good experience, and has a long - lasting effect to increase drug absorption on the ocular surface and improve the management of fungal keratitis. Summary of the Invention

[0013] In view of this, the technical problem to be solved by the present invention is to provide a collagen-anchored thermosensitive gel, its preparation method and application. The thermosensitive gel provided by the present invention is convenient to use, has a good experience and a long-lasting effect, and can be used to prepare drugs for treating fungal keratitis.

[0014] The present invention provides a collagen-anchored thermosensitive gel, which is prepared from amphotericin B, poloxamer and tannic acid.

[0015] Preferably, the mass ratio of tannic acid to poloxamer is 0.07-0.24:1.

[0016] Preferably, the poloxamer is selected from poloxamer 407 and poloxamer 188;

[0017] Preferably, the mass ratio of poloxamer 407 to poloxamer 188 is 1.06-4:1.

[0018] Preferably, in the thermosensitive gel, the content of amphotericin B is 0-0.20 wt%.

[0019] Preferably, by mass percentage, the preparation raw materials include:

[0020] 16%-20% of poloxamer 407;

[0021] 5%-15% of poloxamer 188;

[0022] 2.5%-5% of tannic acid;

[0023] 0-0.2% of amphotericin B;

[0024] The balance is water.

[0025] The present invention also provides a preparation method of the above-mentioned collagen-anchored thermosensitive gel, which includes the following steps:

[0026] A) Prepare AmB-loaded micelles from amphotericin B and poloxamer by the thin film hydration method;

[0027] B) Dissolve the dried AmB-loaded micelles and tannic acid in water to obtain a collagen-anchored thermosensitive gel.

[0028] Preferably, step A) includes:

[0029] A1) Dissolve amphotericin B and poloxamer in a solvent to obtain a mixed solution;

[0030] A2) Remove the solvent from the mixed solution to obtain a thin film;

[0031] A3) Mix the film with water to obtain AmB-loaded micelles.

[0032] Preferably, the solvent is selected from N,N-dimethylformamide;

[0033] The method for removing the solvent from the mixed solution includes rotary evaporation.

[0034] Preferably, in step B), the temperature of the dissolution is lower than 4°C.

[0035] The present invention also provides an application of the above collagen-anchored thermosensitive gel in the preparation of a drug for treating fungal keratitis.

[0036] Compared with the prior art, the present invention provides a collagen-anchored thermosensitive gel, which is prepared from amphotericin B, poloxamer and tannic acid. The thermosensitive gel provided by the present invention is composed of two materials with excellent biocompatibility, poloxamer and tannic acid. By means of non-invasive temperature-induced phase change and collagen-specific anchoring mechanism, the residence time of the drug is prolonged, and potential damage to corneal tissue caused by physical delivery methods is completely avoided, with obvious safety advantages. In addition, in addition to being a collagen-specific anchoring agent, tannic acid itself also has a variety of beneficial biological activities, including anti-inflammatory, antioxidant, antibacterial and metal ion chelating functions. By prolonging its residence time at the lesion site, the system significantly expands the biological effects of tannic acid, realizing the dual advantages of the synergistic effect of drug delivery and biological activity, and providing an innovative solution for the comprehensive treatment of fungal keratitis. Description of the Drawings

[0037] Figure 1 It is a schematic diagram of the preparation process and application of the collagen-anchored thermosensitive gel provided by the present invention;

[0038] Figure 2 It is the Fourier transform infrared spectrum (A), nuclear magnetic resonance hydrogen spectrum (B) of PX-TA, TA and PX, and the particle size distribution diagram (C) of dynamic laser light scattering;

[0039] Figure 3 It is the real image (A) of PX-TA at room temperature (24°C) and corneal physiological temperature (30.5°C), the rheological property diagram (B) of different preparations with temperature change, and the viscosity change diagram (C) of different preparations with temperature change;

[0040] Figure 4 It is to evaluate the corneal adhesion of different formulations to intact corneal epithelium and scraped corneal epithelium;

[0041] Figure 5 It is the anterior segment optical coherence tomography result;

[0042] Figure 6For the test results of in vivo imaging system;

[0043] Figure 7 For the representative colony images of Candida albicans after treatment with different preparations (A) and the corresponding quantitative analysis of fungal survival rate (B);

[0044] Figure 8 For the representative images of Kirby - Bauer diffusion test of Candida albicans after treatment with different preparations (A) and the statistical analysis of the diameter of the corresponding inhibition zone (B);

[0045] Figure 9 For the establishment and treatment plan of the mouse FK model (A), the clinical manifestations of mouse FK under different treatments (B), the clinical scores of mouse FK under different treatments (C), and the corneal epithelial healing of mouse FK under different treatments (D);

[0046] Figure 10 For the multiplex detection results of 23 inflammatory cytokines and chemokines in the mouse cornea on the 6th day after different treatments;

[0047] Figure 11 For the establishment and treatment plan of the corneal injury model of New Zealand white rabbits (A), the clinical manifestations of rabbit corneal injury under different treatments (B), the corneal epithelial healing of rabbit corneal injury under different treatments (C), the mRNA expression level of α - SMA under different treatments (D), and the mRNA expression level of LOX under different treatments (E);

[0048] Figure 12 For the Masson staining (A) and H&E staining (B) of the rabbit corneal injury under different treatments;

[0049] Figure 13 For the results of H&E staining (A), corresponding PAS staining (B), and corresponding CFW staining (C) of the corneal tissues of FK mice after different treatments;

[0050] Figure 14 For the in vitro cytotoxicity assay results after co - incubation of human corneal epithelial cells (HCECs) with PX - TA - AmB;

[0051] Figure 15 For the chicken embryo chorioallantoic membrane after different treatments (A) and the corresponding stimulation score (B). Detailed implementation manners

[0052] The present invention provides a collagen - anchored thermosensitive gel, which is prepared from amphotericin B, poloxamer, and tannic acid.

[0053] To solve the problems of short corneal retention time and frequent dosing frequency of drugs for current infectious keratitis including fungal keratitis, based on in-depth understanding of pathological characteristics and innovative application of material science principles, the present invention provides a collagen-anchored thermosensitive gel for FK treatment. The design concept of the thermosensitive gel provided by the present invention stems from the microscopic analysis of pathological changes in corneal ulcers: during the infection process, pathogen invasion and inflammatory responses lead to the loss of corneal epithelial barrier function, resulting in the pathological exposure of collagen that would not be exposed under physiological conditions in the stroma. This histological change provides a molecular basis for the construction of a targeted delivery system. In terms of material selection, the present invention adopts a two-component synergistic design strategy: First, tannic acid (TA) is selected as a functional ligand. The catechol and gallol groups rich in its molecular structure can form stable binding with the exposed collagen through multiple non-covalent interactions (including hydrophobic interactions and hydrogen bond networks, etc.). The multiple biological functions of TA molecules themselves (including anti-inflammatory, antioxidant, and metal chelation effects, etc.) may further enhance the therapeutic effect through synergistic effects. Second, poloxamer (PX) with thermosensitive properties is selected as an intelligent response carrier. Its amphiphilic molecular structure enables it to exhibit controllable phase change behavior at physiological temperature and has the ability to stably load hydrophobic drugs. The present invention designs an ophthalmic thermosensitive gel by introducing TA. The long-term retention also expands the biological activity of TA, which is manifested as the inhibition of inflammatory factors and the reduction of corneal scars in the treatment of fungal keratitis.

[0054] Specifically, the collagen-anchored thermosensitive gel provided by the present invention includes poloxamer. In some specific embodiments of the present invention, the poloxamer is selected from poloxamer 407 and poloxamer 188; preferably, the mass ratio of poloxamer 407 to poloxamer 188 is 1.06 - 4:1, and can be 1.06:1, 1.1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, or any value between 1.06 - 4:1.

[0055] The collagen-anchored thermosensitive gel provided by the present invention further includes tannic acid, and the mass ratio of tannic acid to poloxamer is 0.07 - 0.24:1, and can be 0.07:1, 0.09:1, 0.1:1, 0.12:1, 0.13:1, 0.15:1, 0.16:1, 0.18:1, 0.2:1, 0.22:1, 0.24:1, or any value between 0.07 - 0.24:1.

[0056] The present invention is based on tannic acid and the temperature-sensitive material poloxamer, designs a collagen-anchored temperature-sensitive gel, and loads the antifungal drug amphotericin B, named PX-TA-AmB. PX-TA-AmB binds to collagen at the corneal defect site and exerts its drug effect for a long time, solving the key problems in clinical medication such as the need for patients to instill eye drops frequently for a long time, poor treatment effect, and poor prognosis of the disease.

[0057] In the temperature-sensitive gel, the content of amphotericin B is 0 to 0.20 wt%, and can be 0 wt%, 0.001 wt%, 0.01 wt%, 0.05 wt%, 0.1 wt%, 0.15 wt%, 0.2 wt%, or any value between 0 and 0.20 wt%.

[0058] In some specific embodiments of the present invention, the raw materials for preparing the collagen-anchored temperature-sensitive gel include, by mass percentage:

[0059] 16% to 20% of poloxamer 407;

[0060] 5% to 15% of poloxamer 188;

[0061] 2.5% to 5% of tannic acid;

[0062] 0 to 0.2% of amphotericin B;

[0063] The balance of water.

[0064] The raw materials for preparing the collagen-anchored temperature-sensitive gel provided by the present invention include 16% to 20% of poloxamer 407, and can be 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, or any value between 16% and 20%.

[0065] The raw materials for preparing the collagen-anchored temperature-sensitive gel provided by the present invention further include 5% to 15% of poloxamer 188, and can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or any value between 5% and 15%.

[0066] The raw materials for preparing the collagen-anchored temperature-sensitive gel provided by the present invention further include 2.5% to 5% of tannic acid, and can be 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or any value between 2.5% and 5%.

[0067] The raw materials for preparing the collagen-anchored temperature-sensitive gel provided by the present invention further include 0 to 0.2% of amphotericin B, and can be 0 wt%, 0.001 wt%, 0.01 wt%, 0.05 wt%, 0.1 wt%, 0.15 wt%, 0.2 wt%, or any value between 0 and 0.20 wt%.

[0068] The raw materials for preparing the collagen-anchored thermosensitive gel provided by the present invention further include the balance of water.

[0069] A method for preparing the above-mentioned collagen-anchored thermosensitive gel of the present invention includes the following steps:

[0070] A) Amphotericin B and poloxamer are prepared into AmB-loaded micelles by the thin-film hydration method;

[0071] B) The dried AmB-loaded micelles are dissolved in tannic acid in water to obtain the collagen-anchored thermosensitive gel.

[0072] The present invention first prepares AmB-loaded micelles, which specifically includes the following steps:

[0073] A1) Amphotericin B and poloxamer are dissolved in a solvent to obtain a mixed solution;

[0074] A2) The solvent in the mixed solution is removed to obtain a thin film;

[0075] A3) The thin film is mixed with water to obtain AmB-loaded micelles.

[0076] Specifically, in the present invention, amphotericin B and poloxamer are dissolved in a solvent to obtain a mixed solution. Among them, the solvent is selected from N,N-dimethylformamide (DMF). The dissolution method is preferably ultrasonic dissolution.

[0077] Then, the solvent in the mixed solution is removed to obtain a thin film. In the present invention, the method for removing the solvent in the mixed solution includes rotary evaporation. After rotary evaporation, a thin film is formed on the inner wall surface of the rotary evaporation device.

[0078] Then, water is added to the device, and the thin film is mixed with water to obtain AmB-loaded micelles. Among them, the mixing is preferably ultrasonic mixing.

[0079] After obtaining the AmB-loaded micelles, the AmB-loaded micelles are dried. Among them, the drying is preferably freeze-drying to obtain a lyophilized powder.

[0080] The lyophilized powder is mixed with tannic acid, and then added to pre-cooled deionized water. Then, mixing and dissolution are carried out. The mixing and dissolution are preferably magnetic stirring. The dissolution temperature is lower than 4°C.

[0081] The present invention also provides an application of the above-mentioned collagen-anchored thermosensitive gel in the preparation of a drug for treating fungal keratitis.

[0082] Figure 1The schematic diagram of the preparation process and application of the collagen-anchored thermosensitive gel provided by the present invention. Figure 1 As shown, the triblock copolymers poloxamer P407 and P188 are used as thermosensitive gel-forming materials. PX and TA can be uniformly compounded by a simple aqueous phase mixing method with TA at 4°C. Amphotericin B is loaded in the gel to obtain a collagen-anchored thermosensitive gel. The collagen-anchored thermosensitive gel provided by the present invention quickly forms a gel network at the physiological temperature of the cornea, which not only fills the corneal ulcer defect, but also realizes long-term drug retention by specific binding with the exposed collagen in the matrix layer.

[0083] The present invention uses corneal ulcer, a specific pathological feature of fungal keratitis, as a target anchor for drug delivery. During the formation of corneal ulcer, the corneal epithelium is destroyed and a large number of collagen fibers in the matrix are exposed. This pathological change provides an ideal target for the specific collagen-binding delivery system. This design concept realizes the innovative strategy of "treating disease with disease": on the one hand, the pathological features generated during the disease process are used to enhance the efficiency of drug delivery, and on the other hand, the biological activity and drug loading capacity of the delivery system itself are used to effectively treat the primary disease.

[0084] The collagen-anchored thermosensitive gel provided by the present invention quickly forms a gel network at the physiological temperature of the cornea, which not only fills the corneal ulcer defect, but also realizes long-term drug retention through specific binding with the exposed collagen in the matrix layer.

[0085] The thermosensitive gel provided by the present invention is composed of two materials with excellent biocompatibility, poloxamer and tannic acid. It prolongs the drug retention time through non-invasive temperature-induced phase change and collagen-specific anchoring mechanism, completely avoiding the potential damage to corneal tissue by physical delivery methods, and has obvious safety advantages. In addition, in addition to being a collagen-specific anchoring agent, tannic acid itself also has a variety of beneficial biological activities, including anti-inflammatory, antioxidant, antibacterial and metal ion chelating functions. By prolonging its retention time at the lesion site, the system significantly expands the biological effect of tannic acid, achieves the dual advantages of drug delivery and biological activity synergy, and provides an innovative solution for the comprehensive treatment of fungal keratitis.

[0086] Collagen, as the main structural protein of the corneal stroma, is exposed in large quantities during the formation of fungal keratitis ulcers, providing abundant and stable anchoring sites for drug delivery systems. This delivery strategy based on disease-specific pathological characteristics significantly enhances the targeting and retention efficiency of the system, is more targeted, and fundamentally solves the problem that traditional mucin-binding preparations are limited by the expression and metabolism of target molecules.

[0087] The present invention solves the problem of short drug residence time through corneal collagen anchoring. Collagen exists in the corneal stroma, which is covered by corneal epithelium outside. Only when the cornea is damaged (such as during the onset of fungal keratitis), a large amount of collagen will be exposed, thus showing collagen-binding anchor points. This design enables the thermosensitive gel to achieve continuous corneal surface retention (greater than 90 minutes), showing great advantages compared with traditional preparations (15 minutes), so that the efficacy of once-daily administration is better than that of amphotericin B administered six times a day.

[0088] To further understand the present invention, the following examples are used to illustrate the collagen-anchored thermosensitive gel provided by the present invention, its preparation method and application. The protection scope of the present invention is not limited by the following examples.

[0089] Example 1

[0090] 1. The preparation of the collagen-anchored thermosensitive gel loaded with amphotericin B (Amphotericin B, AmB) (i.e., PX-TA-AmB) adopts the film hydration method, and the specific steps are as follows:

[0091] (1) Preparation of poloxamer-amphotericin B micelles:

[0092] Dissolve 20 mg of AmB, 1800 mg of poloxamer 407 (Poloxamer 407, P407) and 500 mg of poloxamer 188 (Poloxamer 188, P188) (the final target concentrations are 18% P407, 5% P188, 0.2% AmB) in 10 mL of N,N-dimethylformamide (N,N-dimethylformamide, DMF). After the solid substances are completely dissolved by strong ultrasonic treatment, rotate and evaporate DMF under high vacuum to form a film in the flask. Subsequently, add 30 mL of deionized water to the flask, and form AmB-loaded micelles after strong ultrasonic treatment.

[0093] (2) Preparation of tannic acid-poloxamer-amphotericin B collagen-anchored thermosensitive gel:

[0094] Lyophilize the above micelles, accurately weigh 2320 mg of micelle lyophilized powder and 250 mg of tannic acid (Tannic acid, TA). After mixing the above two powders, gradually add pre-cooled deionized water, and finally fix the volume to 10 mL. Stir at 600 rpm on a magnetic stirrer at 4°C for 6 h to prepare a collagen-anchored thermosensitive gel (PX-TA-AmB) eye drops loaded with 0.2% AmB. The proportion of each component is 18% P407, 5% P188, 2.5% TA, 0.2% AmB.

[0095] 2. Preparation of the Carrier Poloxamer-Tannic Acid (PX-TA)

[0096] To compare the necessity of loading AmB in subsequent evaluations, the formulation of the carrier (PX-TA) was set as a control. The specific method is as follows: Weigh 1800 mg of P407 and 500 mg of P188 and dissolve them in 9 mL of pre-cooled deionized water. Stir continuously at 600 rpm for 2 h at 4 °C. Weigh 250 mg of TA and slowly add it to the above solution, and then stir continuously at 600 rpm for 4 h at room temperature. Finally, make up the volume to 10 mL with deionized water to obtain 10 mL of PX-TA solution.

[0097] 3. Characterization Experiments

[0098] (1) Characterization of the thermosensitive gel of collagen-anchored poloxamer-tannic acid-amphotericin B:

[0099] Freeze-dry the thermosensitive gel (PX-TA-AmB) eye drops of collagen-anchored poloxamer-tannic acid-amphotericin B, dissolve it in deuterated dimethyl sulfoxide, and then perform proton nuclear magnetic resonance ( 1 H nuclear magnetic resonance, 1 H NMR) and Fourier transform infrared spectroscopy (Fourier transform infrared, FT-IR) characterizations.

[0100] The results are shown in Figure 2 , Figure 2 which are the Fourier transform infrared spectra (A), proton nuclear magnetic resonance spectra (B) of PX-TA, TA and PX, and the number distribution diagram of the particle size distribution after micelles encapsulate AmB (C).

[0101] 1 1H NMR analysis confirmed the characteristic proton peaks of PX and TA in the PX-TA complex ( Figure 2 B). Through the calculation of the integral area of the characteristic peaks, the actual ratio of the two is highly consistent with the feeding ratio, indicating that PX and TA have formed a stable intermolecular interaction. FT-IR further revealed the intermolecular interaction of PX-TA: the carbonyl vibration peak of TA has a significant red shift (from 1697 cm -1 shifted to 1730 cm -1 ). From this shift, it can be speculated that a hydrogen bond network has been formed between the oxygen atom in the PX molecule and the phenolic hydroxyl group of TA, and this network has also weakened the original hydrogen bond strength between the hydroxyl group and the carbonyl group within the TA molecule ( Figure 2 A).

[0102] (2) Testing the gel transition temperature of the carrier by the vial inversion method

[0103] PX usually exhibits excellent sol-gel transition characteristics at 37 °C, and at room temperature, PX molecules can self-assemble to form nano-micelles; when the temperature rises to the transition temperature, the micelles further aggregate to form a hydrogel with a specific network structure. When the natural adhesion molecule TA rich in phenolic hydroxyl groups is introduced, the gel transition temperature (T gel ) will be significantly reduced after adding TA to the thermosensitive system. The T gel of PX-TA was tested by the vial inversion method, and the results are shown in Figure 3 A. Figure 3 Figure A shows the actual images of PX-TA at room temperature (24 °C) and corneal physiological temperature (30.5 °C). It can be inferred from Figure 3 Figure A that the thermosensitive gel (PX-TA) of tannic acid-poloxamer collagen anchorage type prepared in the present invention gels at 30.5 °C and maintains good fluidity at 24 °C ( Figure 3 Figure A).

[0104] (3) Rheological characterization of the gel

[0105] The rheological properties of PX, PX-TA and PX-TA-AmB eye drops were measured using an MCR 302 rheometer (equipped with a 25 mm parallel plate). The rheological tests were carried out in two parts: First, in the temperature scanning mode, with a heating rate of 1 °C / min (temperature range 25-40 °C), the changes of the storage modulus (G') and loss modulus (G”) of each formulation with temperature were measured under the conditions of 5% strain and 1 Hz frequency; Subsequently, the apparent viscosities of the three formulations were measured at a shear rate of 5 s-1. The results are shown in Figure 3 , Figure 3 Figure B shows the rheological behaviors of the three formulations at different temperatures. It can be inferred and verified that the thermosensitive gel (PX-TA-AmB) of tannic acid-poloxamer-amphotericin B collagen anchorage type prepared in the present invention and its carrier tannic acid-poloxamer (PX-TA) gel at 30.5 °C, and there is no significant effect on its T gel before and after loading AmB. In addition, Figure 3 Figure C shows the viscosities of the three formulations at different temperatures. It can be inferred that the addition of TA significantly enhances the viscosity of the system.

[0106] (4) Verification of the encapsulation of poloxamer-amphotericin B micelles:

[0107] The particle size of AmB-loaded poloxamer micelles was measured by Dynamic Light Scattering (DLS), and the drug encapsulation efficiency was determined by detecting the AmB concentration using an ultraviolet-visible spectrophotometer method. The DLS results are shown in Figure 2 Figure C. Figure 2C is the number distribution diagram of the particle size distribution of poloxamer-amphotericin B micelles encapsulating AmB. It can be inferred that the average particle size of PX-TA-AmB micelles after drug loading is 65.5 ± 6.2 nm (n = 3). In addition, the ultraviolet-visible absorption spectrum of AmB was recorded using a Shimadzu UV-1800 ultraviolet-visible spectrophotometer. The detection wavelength of AmB was set at 410 nm. The standard curve was plotted by measuring the ultraviolet absorption values of a series of AmB (dissolved in DMF) with concentrations ranging from 0.05 μg / mL to 50 μg / mL at this wavelength. The drug encapsulation efficiency of the micelles was calculated according to "Encapsulation efficiency (%) = (mass of AmB actually encapsulated / total mass of AmB input) × 100%", and the encapsulation rate of poloxamer-amphotericin B micelles was 95%.

[0108] Comparative Example 1

[0109] To judge the importance of tannic acid as a ligand and the advantage of collagen anchoring, chitosan (CS), a commonly used excipient for ophthalmic gels, was used as a control group, and in vivo imaging system (IVIS) fluorescence imaging was performed on ex vivo porcine corneas with intact corneal epithelium (ICE) and debrided corneal epithelium (DCE) to judge the corneal retention time of different formulations.

[0110] Chitosan-poloxamer-amphotericin B thermosensitive gel (PX-CS-AmB, 18% P407, 5% P188, 2.5% CS, 0.2% AmB) was prepared in a similar method. Specifically, about 90 mL of deionized water was taken, 1 mL of acetic acid was added, and they were mixed evenly to make about 1% dilute acetic acid aqueous solution. 2.5 g of chitosan powder was weighed and slowly added to the above dilute acetic acid solution, stirred until it was completely dissolved, and placed in a refrigerator or ice bath (about 4 °C) for cooling and standby. 2320 mg of the above-mentioned poloxamer-amphotericin B micelle lyophilized powder was gradually added to the cooled chitosan solution, and finally made up to 100 mL with deionized water to prepare it.

[0111] The results show Figure 4 , Figure 4 To evaluate the corneal adhesion of different formulations to intact corneal epithelium and debrided corneal epithelium. Among them, Figure 4 A is the IVIS fluorescence images of the corneas in the ICE group and DCE group after using PX-TA-AmB and PX-CS-AmB; Figure 4 B is the corresponding average fluorescence intensity analysis. DCE: Debrided corneal epithelium; ICE: Intact corneal epithelium.

[0112] From Figure 4It can be seen that regardless of whether the porcine corneal epithelium is scraped off (i.e., whether collagen is exposed), the corneal residence time of PX-TA-AmB with TA as the ligand is better than that of PX-TA-AmB with CS as the ligand. More importantly, PX-TA-AmB shows a significant increase in corneal residence time in the DCE group after collagen exposure( Figure 4 B), which also confirms the good collagen anchoring ability brought by TA as the ligand to the whole system.

[0113] Test Example 1 - Evaluation of the Gelation and Retention Behaviors of Collagen-Anchored Thermosensitive Gels on the Corneal Surface through Collagen Anchoring

[0114] Anterior segment optical coherence tomography (AS-OCT) and in vivo imaging system (IVIS) were used to evaluate the gelation and retention behaviors of 50 μL of different eye drops on the corneal surface. The specific steps are as follows:

[0115] (1) Healthy New Zealand white rabbits were selected as experimental animals. Before the experiment, sodium pentobarbital (dose: 30 mg / kg) was injected into the marginal ear vein for general anesthesia to ensure that the animals remained stable during the imaging process.

[0116] (2) For AS-OCT, after dropping the corresponding eye drop preparations on the surface of the rabbit eyes in the control group (50 μL of ophthalmic ointment (tobramycin ophthalmic ointment, ), the PX group, and the PX-TA-AmB group, AS-OCT (Optovue, Inc., USA, anterior segment mode) was used for scanning and imaging to evaluate the gelation and retention of the preparations on the corneal surface. The AS-OCT imaging time points were 0 min (immediately), 30 min, and 90 min after administration.

[0117] (3) To more intuitively observe the retention and diffusion of the eye drops on the surface of the rabbit eyes, the fluorescent marker rhodamine B (RhB, final concentration: 0.3 mg / mL) was added to all the preparations. After eye drop administration, the IVIS (PerkinElmer, Inc., USA, fluorescence mode, excitation / emission wavelength: 530 nm / 600 nm) system was immediately used to perform continuous fluorescence imaging on the rabbit head area. The specific imaging time points were set as 0 s (immediately), 90 s, 3 min, 10 min, 20 min, 45 min, and 90 min after administration to dynamically observe the changes in the fluorescence signals of each preparation on the ocular surface.

[0118] (4) For all the images obtained by the above AS-OCT and IVIS, the ImageJ image processing software was used for analysis and processing to obtain quantitative data on the gel formation and the change of fluorescence intensity over time, so as to evaluate the in vivo gelation and retention performance of different preparations.

[0119] (5) See Figure 5 , Figure 5 is the anterior segment optical coherence tomography result. Figure 5 (A) is a representative AS-OCT image, Figure 5 (B) is the representative pseudo-color AS-OCT of PX-TA-AmB eye drops, Figure 5 (C) shows the line graph of the remaining hydrogel area on the corneal surface changing over time.

[0120] AS-OCT data showed that 90 minutes after administration, the PX-TA-AmB group showed significant gel retention (56.14 ± 2.25%) at the corneal defect site, and the retention amount was 5.58 times that of the PX group (10.06 ± 2.07%) and 25.17 times that of the control group of ophthalmic ointment (tobramycin eye ointment, ) ( Figure 5 ). This result indicates that PX-TA-AmB can maintain a long-term retention at the corneal injury site, which is significantly better than traditional drug preparations. In contrast, the PX hydrogel showed rapid clearance (17.39 ± 3.04%) within 30 minutes, and its retention rate was lower than that of conventional ophthalmic ointment (28.27 ± 3.14%), suggesting that the retention time of the PX hydrogel is short and it is difficult to provide long-term therapeutic benefits. According to the pseudo-color AS-OCT images, over time, the reflection density of the PX-TA-AmB hydrogel increased ( Figure 5 B). It is suggested that this preparation achieves progressive concentration through dehydration, and at the same time, the mechanical strength is significantly improved. This characteristic enables it to form a stable drug reservoir at the corneal injury site.

[0121] See Figure 6 , Figure 6 is the test result of the in vivo imaging system. Figure 6 are the representative IVIS images after administration of RhB, PX, PX-TA-AmB (to the damaged cornea) and PX TA-AmB (to the intact cornea) (the white dotted circle indicates the effective corneal area). IVIS fluorescence analysis using rhodamine B (RhB) labeling further verified the above results ( Figure 6 ). At 90 minutes, the fluorescence signal was only detected in the defective corneas treated with PX-TA-AmB (0.572 ± 0.06), while when PX-TA-AmB was applied to the intact cornea, the fluorescence signal almost disappeared at 90 minutes (0.005 ± 0.0004) ( Figure 6)。The traditional RhB eye drops could hardly detect the fluorescence signal after 10 minutes due to tear clearance, while the PX preparation accumulated on the eyelids after 45 minutes and did not directly contact the cornea, resulting in limited therapeutic effect. These IVIS results further confirmed the collagen-binding ability of PX-TA-AmB in the damaged cornea, highlighting its unique advantages in the treatment of corneal diseases.

[0122] Test Example 2 - Evaluation of in vitro and in vivo therapeutic effects of fungal keratitis

[0123] TA not only enhanced the adhesion performance of the hydrogel system, but its unique polyphenol structure also endows the material with strong broad-spectrum antibacterial activity, which is of great significance in the design of antibacterial biomaterials. To comprehensively evaluate the antifungal effect of PX-TA-AmB, a variety of complementary evaluation methods such as colony counting method (CFU), fungal viability assay, and fungal inhibition zone experiment were used to evaluate the in vitro antifungal activity of each formulation.

[0124] For the determination of colony counting method and fungal viability, the specific steps are as follows:

[0125] (1) After restoring the cryopreserved strain of Candida albicans, inoculate it onto Sabouraud dextrose agar to culture single colonies, and then transfer it to Sabouraud dextrose broth liquid medium to culture for 24 h to prepare a spore suspension (1×10 6 CFU / mL). Subsequently, add 1 μL of PBS, AmB (pure AmB suspension, 0.2%), PX-TA, and PX-TA-AmB preparation to 1 mL of the spore suspension respectively, and incubate at 30 °C for 24 h. Then measure the absorbance value of the mixture at 600 nm using a microplate reader, which is the fungal viability.

[0126] (2) In addition, take 1 μL of the above mixture and culture it on a Sabouraud dextrose agar plate to count the number of viable bacteria (CFUs). After 24 h, take pictures of the growth status of fungal colonies to evaluate the antifungal activity.

[0127] The results are shown in Figure 7 , Figure 7 which are the representative colony images of Candida albicans after treatment with different formulations (A) and the corresponding quantitative analysis of fungal viability (B). The colony counting results showed that each treatment group showed different degrees of inhibitory effects on Candida albicans ( Figure 7 A). In the fungal viability assay, although PX-TA showed excellent antibacterial activity, its antifungal effect was relatively weak, and the fungal viability was still as high as more than 70%. In contrast, the drug-loaded PX-TA-AmB almost completely inhibited fungal growth, and its curative effect was significantly better than that of the free AmB group. It should be noted that even as a commonly used antifungal drug in clinical practice, there was still about 50% fungal survival in the free AmB group ( Figure 7 B).

[0128] For the evaluation of the inhibition zone, the specific steps are as follows:

[0129] (1) Sterile filter paper disks (6 mm in diameter) were respectively soaked in 0.2 wt% AmB, PX-TA or PX-TA-AmB solution for 1 min and set aside for use.

[0130] (2) A fungal suspension with a concentration of 1×10 7 CFU / mL was evenly spread on the surface of a Sabouraud dextrose agar plate, and the above-mentioned soaked filter paper disks were placed on it. After incubation at 30 °C for 24 h, the diameter of the inhibition zone of each group was measured, and the obtained images were analyzed by ImageJ software to process the inhibition diameter.

[0131] The results are shown in Figure 8 , Figure 8 are the representative images (A) of the Kirby-Bauer diffusion test of Candida albicans after treatment with different preparations and the statistical analysis of the diameter of the corresponding inhibition zone (B). It can be seen from the results of the inhibition zone experiment that the diameter of the inhibition zone of the PX-TA-AmB group is about twice that of the AmB group, fully confirming the excellent antifungal activity of PX-TA-AmB.

[0132] To evaluate the therapeutic effect of PX-TA-AmB in prolonging the ocular surface retention in FK treatment, a FK animal model was established and systematically studied according to the flow chart. The specific method steps are as follows:

[0133] (1) Mice were anesthetized systemically by intraperitoneal injection of tribromoethanol (200 mg / kg). Under the assistance of a portable surgical microscope, the right cornea of each mouse was scratched in a "cross" shape with a sterile 30G needle, with a total of 20 scratches to damage the corneal epithelial barrier. Subsequently, 5 μL of Candida albicans suspension (concentration of 1×10 6 CFU / mL) was immediately and evenly dropped on the scratched corneal surface to prepare a mouse model of fungal keratitis.

[0134] (2) 24 h after modeling, the mice with keratitis models were randomly divided into four treatment groups: control group (PBS, administered once a day), AmB group (0.2% AmB suspension, administered six times a day), PX-TA group (PX-TA eye drops, administered once a day), and PX-TA-AmB group (PX-TA-AmB eye drops, administered once a day). The volume of each administration for all treatment groups was 5 μL.

[0135] (3) The anterior segment images of mice in each group were recorded every 2 days using a slit lamp microscope, and the severity of inflammatory changes was evaluated according to the established scoring criteria (4-point method for mice). At the same time, on the 10th day of treatment, corneal surface fluorescein sodium staining images were taken using a cobalt blue light slit lamp to evaluate corneal epithelial integrity.

[0136] See Figure 9 , Figure 9 for the establishment and treatment plan of the FK model in mice (A), the clinical manifestations of FK in mice under different treatments (B), the clinical scores of FK in mice under different treatments (C), and the corneal epithelial healing of FK in mice under different treatments (D).

[0137] As shown in the results, 24 hours (i.e., day 0) after Candida albicans infected the corneas of mice, significant corneal infection-related symptoms were observed, including corneal edema and opacity ( Figure 9 B). The symptoms in the control group and the PX group deteriorated progressively, and the disease scores reached their peaks on day 4 (11.40 ± 0.55 in the control group and 11.00 ± 0.71 in the PX group), showing severe corneal edema, opacity, and perforation, and no significant remission was observed during the entire 10-day observation period. While the other three groups (AmB, PX-TA, PX-TA-AmB) showed varying degrees of improvement in corneal inflammation control. The AmB group, as the positive control of the clinical protocol, was administered six times a day, but the treatment effect was limited, and corneal inflammation progressed to significant opacity and edema (7.40 ± 1.14) by day 10. The PX-TA group administered once a day significantly improved the treatment effect, effectively controlling corneal inflammation on day 4 (7.00 ± 0.71), and the residual corneal opacity was significantly reduced on day 10 (5.20 ± 0.45)( Figure 9 C). The PX-TA-AmB loaded with antifungal agents further enhanced the treatment effect. After once-daily administration, inflammation was almost controlled on day 10 (2.00 ± 0.71), with only very little residual opacity remaining, and its treatment effect was significantly better than all other groups. The treatment effect was further evaluated by fluorescein sodium staining, and the results showed that PX-TA-AmB and PX-TA significantly repaired the corneal epithelial defect, especially the PX-TA-AmB group, in which there was almost no fluorescein sodium staining of the corneal epithelium( Figure 9 B, 9D).

[0138] Test Example 3 - Anti-inflammatory and anti-scar effects of collagen-anchored thermosensitive gel

[0139] Inflammation is an important part of fungal keratitis. To deeply analyze its anti-inflammatory molecular mechanism, a high-throughput Luminex multiplex detection platform was used to quantitatively analyze the protein levels of 23 cytokines in corneal tissues collected from each group on day 6. The specific steps are as follows:

[0140] (1) Prepare samples: On day 6 after treatment, the corneal tissues collected from mice in all groups were homogenized and ultrasonically treated, proteins were extracted with RIPA lysis buffer, and 45 μg was taken for on-machine detection.

[0141] (2) Preparation of standards: Add 500 μl of standard diluent to the standard vial, vortex and then incubate on ice for 30 min, and then dilute the standard curves S1 - S8.

[0142] (3) Chip detection operation: The detection kit is Bio - Plex Pro Mouse Cytokine Grp IPanel 23 - plex. Incubate sequentially with microbeads (1 h), detection antibodies (30 min), and PE - labeled streptavidin (10 min).

[0143] (4) Feed it into a calibrated Luminex machine (Luminex 200 platform) for reading values.

[0144] (5) Use the fluorescence detection values obtained from the standards to fit the standard curve in multi - parameter mode to obtain the standard curve and its equation. Calculate the concentration of the samples according to the curve equation, with the unit pg / ml. Further use GraphPad Prism (version 9.0) software for data normalization and heat map plotting.

[0145] The results are shown in Figure 10 , Figure 10 which are the multiplex detection results of 23 inflammatory cytokines and chemokines in the corneas of mice on the 6th day after different treatments. As can be seen from Figure 10 it, the PX - TA and PX - TA - AmB treatment groups showed a broad - spectrum anti - inflammatory effect, significantly down - regulating the expression of 22 inflammatory and chemotactic factors except TNF - α, making their levels close to the physiological state. This comprehensive regulation of inflammatory factors is in sharp contrast to the AmB group, which only inhibited a few factors such as IL - 6, IL - 17A, G - CSF, and eotaxin, while most pro - inflammatory factors continued to maintain a high expression state, indicating that the cytokine storm was not effectively controlled.

[0146] Corneal scar (i.e., corneal nebula) formation is the main pathological endpoint of FK progression. Even with active antifungal treatment during the treatment, it may still lead to final permanent vision loss. The aforementioned studies have shown that the PX - TA - AmB preparation exhibits significant therapeutic potential in promoting corneal healing and reducing scar formation. This potential may stem from two mechanisms: (1) timely clearance of fungi prevents progressive corneal tissue damage, and (2) TA - based bioactive materials promote healing and prevent scar formation by regulating the microenvironment at the injury site. To systematically evaluate the wound - healing ability of PX - TA - AmB independent of its antifungal effect, a corneal injury model of New Zealand rabbits was established to evaluate its comprehensive in - vivo corneal wound - healing effect. The evaluation was carried out according to the following method steps:

[0147] (1) The rabbits were anesthetized with sodium pentobarbital (50 mg / kg) via the marginal ear vein, and topical surface anesthesia was performed by instilling 0.5% proxymetacaine into the eyes.

[0148] (2) A lamellar keratoplasty knife combined with a surgical blade was used to perform lamellar cutting in the center of the cornea. The depth of the defect was approximately 1 / 3 of the full thickness of the cornea to prepare corneal epithelial and stromal defects. Immediately after the operation, the cornea was stained with sodium fluorescein, and images were taken under cobalt blue light to record the defect situation.

[0149] (3) After modeling, the experimental animals were divided into four groups, and corresponding drugs were immediately instilled into the eyes for treatment according to the grouping, and evaluations were carried out at different time points. The control group was given PBS once a day; the PX group was given PX eye drops (PX poloxamer eye drops without TA) once a day; the PX-TA group was given PX-TA eye drops once a day; the PX-TA-AmB group was given PX-TA-AmB eye drops once a day.

[0150] (4) Every two days after the operation, the anterior segment inflammatory reaction and defect healing of each group of rabbits were observed and recorded. On the 28th day after treatment, some rabbit corneal tissues were removed and HE staining kit and Masson staining kit were used for HE staining and Masson staining to evaluate the corneal tissue repair. All experimental images were analyzed and processed using ImageJ software.

[0151] See Figure 11 , Figure 11 for the establishment and treatment plan of the corneal injury model in New Zealand white rabbits (A), the clinical manifestations of corneal injury in rabbits under different treatments (B), the corneal epithelial healing of corneal injury in rabbits under different treatments (C), the mRNA expression levels of α-SMA under different treatments (D), and the mRNA expression levels of LOX under different treatments (E). Sodium fluorescein staining analysis showed different epithelial healing patterns in each treatment group ( Figure 11 B, 11C). On the 3rd day after injury, significant epithelial defects still existed in the control group and the PX group (32.18 ± 2.39% and 29.38 ± 3.23% of the initial wound area, respectively), while the fluorescent staining areas in the PX-TA group and the PX-TA-AmB group were significantly reduced (5.43 ± 1.86% and 7.32 ± 2.86%, respectively), indicating enhanced epithelial healing ability. Anterior segment photography of the rabbit cornea on the 28th day showed that obvious turbidity appeared in the center of the cornea in the control group and the PX group (marked by white stars in the figure), while the PX-TA and PX-TA-AmB treatment groups maintained better transparency.

[0152] Pathological histological analysis was carried out according to the corresponding kits. Specifically:

[0153] (1) Euthanize the rabbits in each group on the 28th day after treatment, remove the eyeballs, and fix them in 4% paraformaldehyde for 24 h. After fixation, dehydrate them successively in ethanol solutions with different concentrations of 75%, 85%, 95%, 100%, and 100%. Subsequently, place the tissue in an embedding mold box, pour liquid paraffin into the embedding mold box, and trim off the excess paraffin after the paraffin has completely solidified.

[0154] (2) Fix the wax block on a paraffin slicer, ensure that the wax block is parallel to the blade, and perform continuous sectioning at a thickness of 4 μm. After spreading, air-drying, and baking, it is ready for further staining.

[0155] (3) HE staining: Remove the paraffin with xylene until it is hydrated, and wash the paraffin sections through gradient alcohol and water. Subsequently, stain them successively with hematoxylin (3 min) and eosin (4 min), then dehydrate them through gradient alcohol, clear them with xylene, and finally seal them with a resin mounting medium for photography. Masson staining: Stain the tissue sections with Weigert iron hematoxylin staining solution for 5 min. Differentiate with acidic ethanol differentiating solution for 1 min and wash with water. Blue with Masson bluing solution for 1 min and wash with water. Stain with Ponceau fuchsin staining solution for 5 min. Prepare a weak acid working solution (dH2O: weak acid solution = 2:1 ratio), wash the sections with the weak acid working solution for 1 min. Place them in aniline blue staining solution and stain for 1 min. After washing with the above weak acid working solution for 1 min, quickly dehydrate (95% ethanol), then dehydrate with absolute ethanol 3 times, 10 s each time. After clearing with xylene, seal the sections.

[0156] See Figure 12 , Figure 12 for the corneal Masson staining (A) and corneal H&E staining (B) of rabbit corneal injuries under different treatments. The histopathological results further verified the above clinical observations. The HE and Masson staining results showed that the collagen arrangement at the corneal injury sites in the control group and the PX group was disordered and loose, accompanied by obvious myofibroblast differentiation, showing typical pathological features of slow corneal healing and stromal fibrosis. However, PX-TA and PX-TA-AmB treatments made the collagen arrangement at the injury sites more regular, and there was no obvious myofibroblast differentiation, showing the normal healing characteristics of the corneal structure. On the 28th day, there were significant differences in α-smooth muscle actin (α-SMA, also known as ACTA2) in the cornea, with the control group being 5.88 times that of PX-TA-AmB and 3.54 times that of PX-TA. Similarly, the mRNA expression of lysyl oxidase (LOX) was significantly downregulated in the PX-TA and PX-TA-AmB groups, with the control group being 1.95 times that of PX-TA-AmB and 2.29 times that of PX-TA. These results further supported these multifunctional findings of the TA-based collagen-anchored thermosensitive gel ( Figure 11 D, 11E).

[0157] Test Example 4

[0158] To better reveal the differences in the treatment effects of each treatment group and the comparison with the existing first-line clinical drug AmB, the histopathological changes of the corneas of mice with fungal keratitis after different treatments were comprehensively evaluated by hematoxylin-eosin (H&E), periodic acid-Schiff (PAS) and calcofluor white (CFW) staining. The specific steps are as follows:

[0159] (1) 24 hours after the establishment of the fungal keratitis mouse model, the keratitis model mice were randomly divided into four treatment groups: control group (PBS, administered once a day), AmB group (0.2% AmB suspension, administered six times a day), PX-TA group (PX-TA eye drops, administered once a day), PX-TA-AmB group (PX-TA-AmB eye drops, administered once a day). The volume of each administration for all treatment groups was 5 μL. Corneas of mice in each group at the acute stage of the disease (day 4) were fixed with 4% paraformaldehyde for 24 hours, and after fixation, they were successively dehydrated in gradient ethanol solutions with different concentrations of 75%, 85%, 95%, and 100%. Subsequently, the tissue was placed in an embedding mold box, and liquid paraffin was poured into the embedding mold box. After the paraffin was completely solidified, the excess paraffin was trimmed off.

[0160] (2) The wax block was fixed on a paraffin slicer to ensure that the wax block was parallel to the blade, and serial sections were made at a thickness of 4 μm. After spreading, air-drying, and baking, it was ready for further staining.

[0161] (3) HE staining: After dewaxing and rehydrating the sections, they were stained with hematoxylin (3 min) and eosin (4 min) at 20 μL / section in sequence, dehydrated, cleared, and sealed with resin for observing the tissue structure. PAS staining: After oxidation with periodic acid for 15 min, Schiff reagent was added for color development for 15 min, followed by treatment with sodium sulfite for 1 min and hematoxylin counterstaining for 30 s. After sealing, the tissue was examined under a microscope to evaluate the fungal distribution in the tissue. CFW staining: After dewaxing and rehydrating the sections, they were stained with Calcofluor white fluorescence at 20 μL / section for 3 min, washed with PBS, sealed with neutral resin, and observed for the fungal fluorescence distribution in the tissue under a fluorescence confocal microscope.

[0162] See Figure 13 , Figure 13 for the H&E staining (A), corresponding PAS staining (B), and corresponding CFW staining (C) results of the corneal tissues of FK mice after different treatments.

[0163] HE staining results ( Figure 13A shows that the corneal stroma of the control group presented typical inflammatory pathological changes: a large number of inflammatory cell infiltrations caused stromal structural disorders, accompanied by significant edematous thickening and damage to the integrity of the epithelial layer. The histopathological manifestations of each treatment group were significantly different: Although the AmB group reduced the degree of stromal damage through antifungal effects, it failed to effectively improve inflammatory infiltration and corneal edema; the PX-TA group significantly inhibited the infiltration of inflammatory cells through its anti-inflammatory properties and rapidly reconstructed the integrity of the epithelial structure after injury, but there was significant stromal damage; the PX-TA-AmB group achieved the optimal treatment effect through synergistic effects, manifested as a significant reduction in inflammatory cell infiltration, no obvious corneal edema thickening, and intact epithelial structure.

[0164] The superiority of PX-TA-AmB in fungal clearance was further verified by PAS staining and CFW staining ( Figure 13 B and 13C). It is particularly noteworthy that in the results of PAS staining, it was found that PX-TA-AmB not only effectively inhibited fungal infection but also promoted the physiological repair process of corneal epithelium. Both PX-TA and PX-TA-AmB maintained the integrity of the anterior elastic lamina, avoiding abnormal proliferative changes as seen in the AmB group. CFW staining ( Figure 13 C) further enhanced the visualization effect of fungal structure through specific fluorescence labeling. The results showed that almost all fungi in the PX-TA-AmB group were killed, while a small amount of fungi remained in the AmB and PX-TA groups. It is worth noting that the fungi in the PX-TA group had invaded the anterior chamber, showing a fungal distribution similar to that of the control group.

[0165] Test Example 5

[0166] The evaluation of the safety and tolerance of ophthalmic preparations is crucial for ensuring the safety and comfort of patients during medication. To compare the safety differences with existing products, in vitro experiments were conducted to measure the in vitro cytotoxicity by co-incubating human corneal epithelial cells (HCECs) with PX-TA-AmB. The specific steps were as follows:

[0167] (1) Human corneal epithelial cells (HCECs) were inoculated into DMEM medium containing 10% fetal bovine serum (FBS) and routinely cultured in a cell culture incubator at 37°C and 5% CO2. When the cell confluence reached 80%-90%, they were digested with 0.25% trypsin-EDTA and prepared into a cell suspension with a concentration of 1×10 6 cells / mL. Subsequently, 1 mL of the cell suspension was evenly inoculated into each well of a 6-well plate and continued to be cultured at 37°C and 5% CO2 overnight.

[0168] (2) Add 1 μL of PBS (control), AmB (0.2%), PX-TA (18% P407, 5% P188, 2.5% TA), or PX-TA-AmB (18% P407, 5% P188, 2.5% TA, 0.2% AmB) respectively, and incubate for 24 h;

[0169] (3) After fluorescence staining with Calcein-AM / PI staining solution for 20 min, examine under a microscope. Green represents live cells and red represents dead cells.

[0170] The results are shown in Figure 14 , Figure 14 which is the result of in vitro cytotoxicity assay after co-incubation of human corneal epithelial cells (HCECs) with PX-TA-AmB. Green represents viable cells and red represents dead cells. The results show a very low level of propidium iodide (PI) staining for PX-TA-AmB, similar to the control group (PBS treatment), and almost all cells are positive for calcein (AM) ( Figure 14 ). In contrast, the PI staining in the AmB treatment group is significantly increased, indicating its potential cytotoxicity.

[0171] Test Example 6

[0172] By using the vascular network of the choroid and vitelline membrane of the chicken embryo to simulate the stimulation response of the human eye, different preparations are evaluated for whether they can cause eye irritation or toxicity ( Figure 15 ). The chicken embryo chorioallantoic membrane assay (HET-CAM) further verifies the above findings. The specific steps are as follows:

[0173] (1) Use SPF-grade fertilized eggs and incubate them at 38 ± 0.5 °C. On the 10th day of incubation, irradiate the eggs with an egg candler to locate the air chamber, and then carefully remove the 2×2 cm 2 portion of the eggshell at the air chamber site with forceps. After wetting the inner membrane with 0.5 mL of normal saline, gently peel off the inner membrane, avoiding damage to the blood vessels.

[0174] (2) Apply 0.3 mL of the test preparations (NaCl, SDS, NaOH, AmB, and PX-TA-AmB) to the chorioallantoic membrane (CAM). 0.9% w / v NaCl is used as the negative control, and 1 M NaOH and 1% (w / v) SDS are used as the positive controls respectively, which are used to induce bleeding and vasoconstriction.

[0175] (3) Observe and record the changes of the chorioallantoic membrane within 5 minutes. The irritation score is carried out according to the import and export industry standard SN / T2329-2009.

[0176] The results are shown in Figure 15 , Figure 15Chorioallantoic membranes of chicken embryos after different treatments (A) and corresponding irritation scores (B). The results showed that neither PX-TA-AmB nor the negative control group (0.9% w / v NaCl solution) caused obvious irritation reactions, while it was noteworthy that AmB treatment led to mild irritation. Different positive control groups showed the irritation reactions of different strong irritants to the eyes. The positive control 0.1 M NaOH (positive control for vascular hemorrhage) caused severe vascular hemorrhage and coagulation, and 1% w / v sodium dodecyl sulfate (SDS, positive control for vasoconstriction) resulted in vasoconstriction, hemorrhage and coagulation. The irritation scores (ISs) of NaOH and SDS were 16.78 ± 0.62 and 12.95 ± 0.25 respectively, indicating extremely strong irritation. However, neither PX-TA-AmB nor NaCl treatment caused irritation reactions. The irritation score of AmB treatment was 2.413 ± 0.98, showing mild irritation. Figure 15 B). Cytotoxicity and irritation are important factors limiting the clinical application of AmB, and the above results indicate that PX-TA-AmB has good biosafety and is suitable for topical administration.

[0177] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A collagen-anchored thermosensitive gel, characterized in that, It is prepared from amphotericin B, poloxamer and tannic acid.

2. The thermosensitive gel according to claim 1, wherein The mass ratio of the tannic acid to the poloxamer is 0.07 - 0.24:

1.

3. The thermosensitive gel according to claim 1, wherein The poloxamer is selected from poloxamer 407 and poloxamer 188; Preferably, the mass ratio of the poloxamer 407 to the poloxamer 188 is 1.06 - 4:

1.

4. The thermosensitive gel according to claim 1, wherein In the thermosensitive gel, the content of amphotericin B is 0 - 0.20 wt%.

5. The thermosensitive gel according to claim 1, characterized in that, By mass percentage, the preparation raw materials include: 16% - 20% of poloxamer 407; 5% - 15% of poloxamer 188; 2.5% - 5% of tannic acid; 0 - 0.2% of amphotericin B; The balance of water.

6. A preparation method of the collagen-anchored thermosensitive gel according to any one of claims 1 to 5, characterized in that, It includes the following steps: A) Prepare amphotericin B-loaded micelles from amphotericin B and poloxamer by the thin-film hydration method; B) Dissolve the dried amphotericin B-loaded micelles and tannic acid in water to obtain a collagen-anchored thermosensitive gel.

7. The preparation method according to claim 6, characterized in that, Step A) includes: A1) Dissolve amphotericin B and poloxamer in a solvent to obtain a mixed solution; A2) Remove the solvent from the mixed solution to obtain a thin film; A3) Mix the thin film with water to obtain amphotericin B-loaded micelles.

8. The preparation method according to claim 7, characterized in that, The solvent is selected from N,N-dimethylformamide; The method for removing the solvent from the mixed solution includes rotary evaporation.

9. The preparation method according to claim 6, characterized in that, In step B), the temperature of the dissolution is lower than 4°C.

10. Use of a collagen-anchored thermosensitive gel as described in any one of claims 1 - 5 in the preparation of a drug for treating fungal keratitis.

Images