Temperature-sensitive in-situ hydrogel loaded with ferroptosis inhibitor and antifungal drug as well as preparation method and application of temperature-sensitive in-situ hydrogel

Abstract

The invention relates to the technical field of external medicines for eyes, in particular to temperature-sensitive in-situ hydrogel loaded with a ferroptosis inhibitor and an antifungal medicine as well as a preparation method and application of the temperature-sensitive in-situ hydrogel. The hydrogel can remarkably prolong the residence time of the medicine on the ocular surface, in-situ gelling repair of corneal defects and targeted synergistic release of the medicine are achieved, and an effective medicine delivery system and a therapeutic scheme basis are provided for efficient synergistic treatment of fungal keratitis.

Description

Technical Field

[0001] This invention relates to the field of topical ocular medication technology, specifically to temperature-sensitive in-situ hydrogels loaded with ferroptosis inhibitors and antifungal drugs, their preparation methods, and applications. Background Technology

[0002] Fungal keratitis (FK) is an inflammatory corneal disease caused by fungal infection and is one of the leading causes of infectious blindness worldwide. Its ocular pathological features include corneal edema, ulceration, and opacity. In severe cases, disease progression can lead to corneal stromal infiltration and endothelial perforation, resulting in endophthalmitis or vision-threatening complications such as corneal transplantation. Studies have confirmed that ferroptosis is a key pathogenic factor in fungal keratitis, and while ferroptosis inhibitors (Ferrostatin-1, Fer-1) possess targeted cytoprotective effects, their efficacy in vivo is limited due to poor water solubility and short ocular retention time.

[0003] Currently, the only clinically approved antifungal drug for ocular treatment is 5% natamycin. However, natamycin suffers from poor water solubility, weak penetration into intraocular tissues, and the tendency to induce resistance when used alone, limiting its clinical efficacy. Another broad-spectrum antifungal drug, 1% voriconazole, exerts its antifungal effect by inhibiting cytochrome P450-mediated ergosterol biosynthesis and has high permeability. However, voriconazole also faces the challenge of poor water solubility, requiring frequent dosing and fresh reconstitution, which not only reduces patient compliance but also exacerbates the development of resistance. To improve drug utilization efficiency, some studies have attempted to use novel delivery systems to enhance antifungal efficacy, such as co-loading voriconazole with anthocyanin nanozymes in hydrogel coatings, or using materials such as silver nanoparticles and metal-phenol nanoflowers to enhance its antifungal ability. However, these studies mostly focus on enhancing the antifungal activity itself, with insufficient attention paid to the protective mechanisms of host cells during the infection process.

[0004] Currently, the treatment of fungal keratitis still faces three major bottlenecks: (1) Short drug retention time: Antifungal drugs have a short retention time on the surface of the eye, requiring frequent administration to maintain an effective concentration. This not only easily leads to drug resistance, but also often results in insignificant effects from single antifungal treatment. Patients often have large corneal scars after healing, and their vision recovery is poor. (2) Poor targeting: Traditional drug delivery systems lack specificity and cannot achieve targeted release of drugs at the site of corneal lesions. This results in indiscriminate distribution of drugs in healthy and diseased tissues, reducing drug utilization and potentially increasing the risk of local toxicity. (3) Single therapy: Current treatment strategies mainly rely on single antifungal drugs and do not combine treatments based on the disease mechanism. Although chemical synthesis technology has been used to improve drug utilization, research has mostly focused on enhancing antifungal function and has failed to achieve the synergistic therapeutic goal of reducing corneal tissue damage while fighting fungi.

[0005] Improving drug utilization through chemical techniques while simultaneously achieving targeted combined therapy to combat fungal infections and reduce corneal damage has become a critical issue in the treatment of fungal keratitis. Therefore, there is an urgent need to develop a novel drug delivery system that can significantly prolong drug retention time on the ocular surface and possess targeted release capabilities. This would allow for effective antifungal treatment while simultaneously providing synergistic protection for host corneal cells, ultimately improving patient visual outcomes. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a temperature-sensitive in-situ hydrogel loaded with ferroptosis inhibitors and antifungal drugs. The temperature-sensitive hydrogel prepared by the present invention can overcome the shortcomings of poor water solubility and short ocular retention time of ferroptosis inhibitors and antifungal drugs, realize in-situ gelation to repair corneal defects and target synergistic drug release, and provide an effective drug delivery system and treatment plan basis for the efficient synergistic treatment of fungal keratitis.

[0007] The present invention provides a composition comprising a ferroptosis inhibitor and an antifungal drug, wherein the mass ratio of the ferroptosis inhibitor to the antifungal drug is (0.0015:1) to (0.01:1); for example, the mass ratio of the ferroptosis inhibitor to the antifungal drug is (0.0015:1), (0.002:1), (0.003:1), (0.004:1), (0.005:1), (0.006:1), (0.007:1), (0.008:1), (0.009:1), or (0.01:1).

[0008] In some embodiments, the antifungal drug includes at least one of natamycin, voriconazole, amphotericin B, itraconazole, fluconazole, posaconazole, caspofungin, micafungin, anidoxane, clotrimazole, bifonazole, econazole, ketoconazole, terbinafine, or butenafine.

[0009] In some embodiments, the ferroptosis inhibitor includes at least one of Ferrostatin-1 (Fer-1), deferoxamine, deferoxone, Liproxstatin-1, N-acetylcysteine, glutathione ethyl ester, selenium compounds, α-tocopherol, Trolox, cyclohexenone, or rosiglitazone.

[0010] In some embodiments, the mass ratio of the ferroptosis inhibitor to the antifungal drug in the composition is (0.0015:1) to (0.006:1). For example, the mass ratio of the ferroptosis inhibitor to the antifungal drug in the composition is (0.0015:1), (0.002:1), (0.003:1), (0.004:1), (0.005:1), or (0.006:1). Preferably, the mass ratio of the ferroptosis inhibitor to the antifungal drug in the composition is 0.006:1. This composition treats fungal keratitis through multiple synergistic mechanisms, not only more effectively inhibiting or killing pathogenic fungi, but also repairing damaged corneal tissue by inhibiting ferroptosis, reducing the risk of scarring.

[0011] In some embodiments, the antifungal drug is voriconazole. The ferroptosis inhibitor is the antioxidant Ferrostatin-1. Specifically, the mass ratio of Ferrostatin-1 to voriconazole in the composition is 0.006:1.

[0012] This invention does not limit the presence or combination of the components in the composition, and its scope of protection covers all physical forms. This includes, but is not limited to, the components existing independently or in mixtures. Furthermore, the composition can be further mixed with other components, and can be presented in basic forms such as powder or solution, or loaded into delivery systems such as hydrogels, microspheres, and nanoparticles. Any physical form and combination that contains the core active ingredient of this invention or utilizes the core concept of this invention falls within the scope of this invention.

[0013] The present invention also provides a temperature-sensitive hydrogel, comprising a gel matrix and the composition as described above.

[0014] In some embodiments, the gel matrix comprises poloxamer and hyaluronic acid, wherein the poloxamer comprises at least one of poloxamer 407 or poloxamer 188.

[0015] In some embodiments, the mass ratio of poloxamer 407, poloxamer 188, and hyaluronic acid in the gel matrix is ​​(16-20):(3-13):(0.1-2). For example, the mass ratio of poloxamer 407, poloxamer 188, and hyaluronic acid in the gel matrix is ​​(16:3:0.1), (17:4:0.5), (18:3:1.2), (18:5:1), (19:8:1), (19:10:1.5), or (20:13:2). Preferably, the mass ratio of poloxamer 407, poloxamer 188, and hyaluronic acid in the gel matrix is ​​18:5:1. This ratio can significantly enhance the adhesion and retention time of the gel to the corneal injury site and ensure rapid in-situ gelation at the physiological temperature of the cornea, thereby achieving efficient local delivery and long-lasting repair of the drug.

[0016] In some embodiments, the temperature-sensitive hydrogel comprises, by weight percentage:

[0017] 16%~20% Poloxamer 407;

[0018] 3%~13% Poloxamer 188;

[0019] 0.1%~2% hyaluronic acid;

[0020] 0.0015%~0.006% ferroptosis inhibitor;

[0021] It contains 0.6% to 1% antifungal drugs, with the remainder being water.

[0022] Preferably, by weight percentage, the temperature-sensitive hydrogel comprises:

[0023] 18% Poloxamer 407;

[0024] 5% Poloxamer 188;

[0025] 1% hyaluronic acid;

[0026] 0.006% ferroptosis inhibitor;

[0027] And 1% antifungal drug, the remainder being water;

[0028] The temperature-sensitive hydrogel effectively combats fungi while inhibiting host cell ferroptosis, thereby reducing corneal scarring, promoting tissue healing, and achieving multiple therapeutic effects.

[0029] As a feasibility example, the temperature-sensitive hydrogel, by weight percentage, comprises:

[0030] 18% Poloxamer 407;

[0031] 5% Poloxamer 188;

[0032] 1% hyaluronic acid;

[0033] 0.006% Ferrostatin-1;

[0034] Add 1% voriconazole, with the remainder being water.

[0035] Furthermore, the present invention also provides a method for preparing the temperature-sensitive hydrogel as described above, comprising:

[0036] Poloxamer 407, poloxamer 188, ferroptosis inhibitors and antifungal drugs were mixed with anhydrous ethanol and evaporated to obtain a drug-loaded film.

[0037] The drug-loaded film is hydrated, ethanol is removed, and the solution is reconstituted to obtain a micelle solution;

[0038] The mixture is prepared by mixing hyaluronic acid with the micelle solution.

[0039] In some embodiments, the evaporation temperature is 35-40°C. For example, the evaporation temperature is 35, 36, 37, 38, 39, or 40°C; specifically, the evaporation temperature is 37°C.

[0040] In some embodiments, the ethanol removal can be achieved through drying, vacuum drying, freeze drying, spray drying, microwave drying, or far-infrared drying. All of these methods can be used in the preparation method described in this invention, and they can be used individually or in combination; this invention does not limit their use. In a specific embodiment, the drying method is freeze-vacuum drying.

[0041] Furthermore, the present invention also provides the use of the composition as described above, the temperature-sensitive hydrogel as described above, or the temperature-sensitive hydrogel obtained by the preparation method as described above, in the preparation of a medicament for treating fungal keratitis.

[0042] In some embodiments, the drug has functions including at least one of the following: anti-inflammatory, anti-corneal damage, reducing host cell ferroptosis, inhibiting corneal scarring, and / or promoting corneal healing.

[0043] Furthermore, the present invention also provides a medicament for treating fungal keratitis, comprising the composition as described above, the thermosensitive hydrogel as described above, or the thermosensitive hydrogel obtained by the preparation method as described above, and pharmaceutically acceptable excipients.

[0044] In some embodiments, the excipients include, but are not limited to, the following types:

[0045] pH adjusters used to adjust the pH of formulations, such as phosphate buffer pairs, borate buffers, or Tris-HCl;

[0046] Isotonic agents used to adjust the osmotic pressure of formulations, such as glycerol, mannitol, or sodium chloride;

[0047] Preservatives used to inhibit microbial growth, such as benzalkonium chloride or potassium sorbate;

[0048] Stabilizers or antioxidants used to prevent the oxidation or degradation of components, such as disodium edetate, ascorbic acid, or sodium bisulfite;

[0049] Thickeners or rheology modifiers used to adjust the viscosity and ocular retention properties of formulations, such as hydroxyethyl cellulose or carbomer.

[0050] The present invention provides a method for treating fungal keratitis, comprising applying an effective dose of the active ingredient (including the composition described in the present invention, the thermosensitive hydrogel described in the present invention, the thermosensitive hydrogel prepared according to the method of the present invention, or the medicament for treating fungal keratitis described in the present invention) to the site of treatment.

[0051] This invention relates to a thermosensitive in-situ hydrogel loaded with ferroptosis inhibitors and antifungal drugs, its preparation method, and its application. The thermosensitive hydrogel can significantly prolong the drug's residence time on the ocular surface, achieving responsive drug release at the corneal lesion site, and simultaneously exerting a dual function of potent antifungal activity and inhibition of corneal cell ferroptosis. This provides a novel solution for the synergistic treatment of fungal keratitis by eradicating pathogens while reducing tissue damage. Attached Figure Description

[0052] Figure 1 The figure shows the effect of different concentrations of poloxamer 407, poloxamer 188 and hyaluronic acid on the temperature change of the hydrogel in Example 1.

[0053] Figure 2 The results of hydrogel rheology testing in Example 2 are as follows;

[0054] Figure 3 This is a schematic diagram illustrating the preparation process of the temperature-sensitive hydrogel in Example 2 and its application in treating fungal keratitis.

[0055] Figure 4The results of antifungal testing in Example 3 are as follows: (a, b: images and statistical analysis of surviving fungi on SDA plates after different treatments; c, d: images and statistical analysis of surviving fungi on SDA plates after different treatments; e, f: images and quantitative analysis of biofilms stained with crystal violet after different treatments; g, h: three-dimensional confocal images and fluorescence intensity of Candida albicans biofilms stained with SYTO 9 after different treatments; i: transmission electron microscopy images of Candida albicans after different treatments; control refers to the normal fungal group (i.e., untreated group); PHF refers to poloxamer / hyaluronic acid / ferroptosis inhibitor hydrogel; PHFV refers to poloxamer / hyaluronic acid / ferroptosis inhibitor / voriconazole hydrogel; VOR refers to voriconazole eye drops (also serving as a clinical positive control group). Data are expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA and Tukey multiple comparison test)).

[0056] Figure 5 Example 4: Evaluation of the therapeutic effects of different treatment methods on a mouse model of fungal keratitis (a) Schematic diagram of the FK mouse model, intervention measures, and experimental evaluation; b) Anterior segment photographs, fluorescein staining, and corneal fungal load measurement on day 3 of different treatments; c) Clinical scores of the anterior segment of mice after different treatments (n = 5); d) Quantification of corneal defect area after different treatments (n = 3); e) Quantification of corneal fungal load on day 3 of different treatments (n = 3); f, g) Representative thermograms of mouse movement trajectories after different treatments and quantitative analysis of dwell time in light / dark chambers (n = 3); h) Microscopic images of corneal tissue PAS staining after different treatments (n = 3); i) Representative images of corneal sections stained with H&E after different treatments (n = 3); j, k) Immunofluorescence staining showing IL-1β (red), IL-10 (green), and cell nuclei counterstained with DAPI (blue) (n = 3, scale bar = ). 50 micrometers); data are expressed as mean ± standard deviation, ns: no significant difference, **p < 0.01, ****p < 0.0001 (c, two-way ANOVA, one-way ANOVA and Tukey multiple comparison test); control refers to the fungal keratitis model group (i.e., the untreated group)). Detailed Implementation

[0057] This invention provides a temperature-sensitive in-situ hydrogel loaded with ferroptosis inhibitors and antifungal drugs, its preparation method, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0058] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0059] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0060] When used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0061] The term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0062] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0063] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0064] Characteristics of each drug component:

[0065] Poloxamer 407 and Poloxamer 188 are triblock copolymers (a mixture of polyethylene oxide and polypropylene oxide, PEO-PPO-PEO) used to develop multifunctional and temperature-sensitive ophthalmic drops. This sol-gel transition property allows the formulation to be used in liquid form, gelling at the site of corneal injury to fill the defect and achieve sustained drug release. However, poloxamer alone exhibits poor adhesion after gelation, limiting its retention on the corneal surface. Given the high blinking frequency (10–20 times per minute), enhanced adhesion and prolonged retention time are crucial for achieving sustained drug delivery.

[0066] Hyaluronic acid (HA) has strong adhesive properties; incorporating it into poloxamer hydrogel can enhance adhesion to corneal injuries. The ideal gelation temperature range for ophthalmic formulations is room temperature to 30±2℃.

[0067] Ferraphobia inhibitors have been shown in our in vitro primary cell experiments to reduce fungal-induced corneal oxidative damage and fibrosis.

[0068] Voriconazole is a candidate drug for the treatment of fungal keratitis due to its broad-spectrum antifungal properties and high permeability; however, it has the disadvantage of low bioavailability.

[0069] This invention reveals a close relationship between the pathogenesis of fungal keratitis and ferroptosis, and proposes the application of ferroptosis inhibitors in the treatment of this disease. By combining ferroptosis inhibitors with the antifungal drug voriconazole, a synergistic therapeutic effect can be achieved, enhancing efficacy. Based on this, we further constructed a multifunctional hydrogel drug delivery system: utilizing the amphiphilic loading of poloxamer with the hydrophobic drugs ferroptosis inhibitors and voriconazole, achieving the goal of "one drug, multiple functions"; simultaneously, by forming a temperature-sensitive hydrogel with poloxamer and hyaluronic acid, a hydrogel is formed at the site of fungal corneal ulcers under physiological corneal conditions, achieving targeted retention and long-term release of the drug at the lesion site.

[0070] In this invention, all test materials are commercially available.

[0071] The present invention will be further illustrated below with reference to the embodiments:

[0072] Example 1: Screening of Gel Matrix

[0073] To obtain a thermosensitive hydrogel capable of undergoing a sol-gel transition at physiological corneal temperatures, we systematically evaluated the effects of the concentrations and ratios of poloxamer 407 (P407), poloxamer 188 (P188), and hyaluronic acid (HA) on the phase transition temperature. Using a Box-Behnken design, we used 16%–20% poloxamer 407, 3%–13% poloxamer 188, and 0.1%–2% hyaluronic acid as independent variables, with the solution-gel transition temperature as the dependent variable. The target temperature was 30 ± 2 °C (in-situ gelation at physiological corneal temperatures). The optimized formulation obtained was 18% (mass / volume ratio) of P407, 5% (mass / volume ratio) of P188, and 1% (mass / volume ratio) of HA. This formulation achieved in-situ gelation at physiological corneal temperatures. Figure 1 ).

[0074] Example 2: Preparation of a poloxamer / hyaluronic acid / ferroptosis inhibitor / voriconazole (PX / HA / Fer-1 / VOR, PHFV) thermosensitive hydrogel

[0075] Poloxamer hyaluronic acid thermosensitive hydrogel loaded with ferroptosis inhibitor and voriconazole was prepared by thin-film hydration method. First, 0.6 mg Fer-1, 100 mg voriconazole, 1800 mg poloxamer 407 and 500 mg poloxamer 188 were weighed, and then the above substances were dissolved in a flask containing 20 ml of anhydrous ethanol. Under ultrasonic assistance, the solid material was completely dissolved in anhydrous ethanol. After forming a homogeneous solution, the anhydrous ethanol was rotary evaporated under vacuum (at 37°C) until a homogeneous thin film was visible in the flask. 10 ml of deionized water was then added to the flask, and the sample was transferred to a glass petri dish and frozen at -20°C for 24 hours. The sample was then vacuum-dried to completely remove moisture. Finally, the lyophilized sample was weighed again and dissolved in deionized water to form micelles. Hyaluronic acid was added to prepare a PX / HA / Fer-1 / VOR hydrogel (18% P407, 5% P188, 200 µM Fer-1, 1% VOR). The solution-gel transition temperature was determined to be 29.8°C using rheological methods. It can gel in situ at the physiological temperature of the cornea (e.g., ...). Figure 2The successful encapsulation of Fer-1 and VOR was confirmed by bulk 1H NMR and Fourier transform infrared spectroscopy. Light absorption was measured at 324 nm and 256 nm using UV spectroscopy to calculate the concentrations of Fer-1 and VOR (encapsulation efficiency was calculated using the following formula: EE% = (loaded mFer-1 / VOR) / (added mFer-1 / VOR) × 100%). The encapsulation efficiencies of Fer-1 and VOR were measured to be 95% and 88%, respectively. Retention and adhesion of PX / HA / Fer-1 / VOR eye drops in rabbit corneal lesions were evaluated using anterior segment optical coherence tomography. Anterior segment images were acquired at 0, 30, 60, and 90 minutes after eye drop administration. Using the IVIS Lumina XRMS Series III imaging system, the ocular retention time of the PHFV hydrogel in a rabbit corneal defect model was confirmed to be at least 90 minutes. In vitro antifungal experiments demonstrated that the antifungal activity of the PX / HA / Fer-1 / VOR hydrogel was superior to that of the traditional drug voriconazole. A mouse model of fungal keratitis showed that it was superior to single-drug therapy or the clinical control group in anti-inflammatory, anti-corneal damage, anti-corneal scarring, and corneal healing effects. A schematic diagram of the preparation process of the PHFV temperature-sensitive hydrogel and its application in the treatment of fungal keratitis is shown below. Figure 3 As shown.

[0076] Example 3 Antifungal Detection

[0077] This study assessed the effects of the negative control group, PX / HA group, PX / HA / Fer-1 group, PX / HA / Fer-1 / VOR group, and VOR group on the survival rate of *Candida albicans* by counting colony-forming units after treating the fungal suspensions for 24 hours and then culturing them on SDA for 24 hours. The effects on biofilm formation ability were evaluated by counting colony-forming units. After treating the fungal suspensions for 48 hours, the effects on biofilm formation ability were assessed by crystal violet staining. After treating the fungal suspensions on confocal dishes as described above for 48 hours, SYTO 9 biofilm dye was added, and the three-dimensional structure of the biofilms in each treatment group was observed using confocal microscopy. After stimulating the fungal suspensions with the aforementioned drugs for 24 hours, the fungal suspensions were fixed and immersed in 4% glutaraldehyde for 20 minutes, and the ultramorphological changes inside the fungal cells were observed using transmission electron microscopy.

[0078] like Figure 4 As shown, the results indicate that the PX / HA / Fer-1 / VOR temperature-sensitive hydrogel prepared in this study releases the drug Fer-1 and the antifungal drug VOR at the lesion site. PX / HA / Fer-1 / VOR significantly inhibits fungal growth and disrupts fungal biofilms, exhibiting superior antifungal activity compared to the clinical control group VOR. (The treatment times for the negative control group (PBS control group, PX / HA group, PX / HA / Fer-1 group, PX / HA / Fer-1 / VOR group, and VOR group) are as described in the steps above.

[0079] The Combination Index (CI) was calculated based on the Bliss independent model. CI > 1 indicates a synergistic effect, CI = 1 indicates an additive effect, and CI < 1 indicates an antagonistic effect. When Fer-1 was used in combination with the antifungal drug VOR, the fungal survival rate (Table 1): CI ≈ 1.1, biofilm disruption (Table 3): CI ≈ 1.2, and fluorescence intensity of the Candida albicans biofilm (Table 4): CI ≈ 1.3, demonstrating a significant synergistic effect. The combination therapy group was significantly superior to both the Fer-1 monotherapy group and the voriconazole monotherapy group in reducing fungal survival rate, disrupting fungal biofilm structure, and reducing total biofilm volume.

[0080] Table 1. Statistical analysis of survival rate on SDA plates after different treatments.

[0081]

[0082] Table 2. Statistical analysis of survival on SDA plates after different treatments (diameter)

[0083]

[0084] Table 3. Quantitative analysis of crystal violet staining of biofilms after different treatments (volume)

[0085]

[0086] Table 4. Fluorescence intensity of Candida albicans biofilms stained with SYTO 9 after different treatments.

[0087]

[0088] Example 4: Clinical Inflammation Control and Epithelial Repair Detection

[0089] The therapeutic effect of PX / HA / Fer-1 / VOR hydrogel on fungal keratitis: In a mouse model of fungal keratitis, PX / HA / Fer-1, PX / HA / Fer-1 / VOR hydrogel and VOR eye drops were used to treat the patients. The anterior segment clinical score, corneal defect staining, corneal fungal count, corneal pathological staining and inflammatory factor detection were used to comprehensively evaluate the improvement effect of different treatment methods on corneal clinical symptoms, fungal load and inflammatory response.

[0090] like Figure 5As shown, the experimental results indicated that the PX / HA / Fer-1 / VOR hydrogel significantly reduced anterior segment scores, corneal inflammation, and fibrosis levels. The PX / HA / Fer-1 / VOR hydrogel group treated once daily showed better therapeutic effects on fungal keratitis than the clinical control group treated six times daily. (Control group: model group, PBS once daily; PH group: PX / HA hydrogel once daily; PHF group: PX / HA / Fer-1 hydrogel once daily; PHFV group: PX / HA / Fer-1 / VOR hydrogel once daily; VOR group: VOR suspension six times daily, treated for 8 days, with measurements taken on days 0, 3, 5, and 8).

[0091] The Combination Index (CI) was calculated based on the Bliss independent model. CI > 1 indicates a synergistic effect, CI = 1 indicates an additive effect, and CI < 1 indicates an antagonistic effect. When Fer-1 was used in combination with the antifungal drug VOR, the anterior segment clinical score (Table 5): CI ≈ 1.10, corneal defect area (Table 6): CI ≈ 1.24, and corneal fungal load (Table 7): CI ≈ 1.02, showing a significant synergistic effect. The combination therapy group showed significant advantages in reducing anterior segment clinical score, shrinking corneal defect area, and reducing corneal fungal load, and its efficacy was significantly better than that of Fer-1 or voriconazole monotherapy groups.

[0092] Table 5 Clinical scores of the anterior segment of mice after different treatments (n = 5)

[0093]

[0094] Table 6. Quantification of corneal defect area in mice after different treatments (n = 3)

[0095]

[0096] Table 7. Quantification of corneal fungal load on day 3 after different treatments (n = 3) (fungal survival rate)

[0097]

[0098] Table 8. Quantitative analysis of the time mice spent in the light / dark chamber after different treatments (n = 3)

[0099]

[0100] The above results demonstrate that the PX / HA / Fer-1 / VOR thermosensitive in situ hydrogel achieves dual efficacy of antifungal and corneal protection through the synergistic effect of Fer-1 and VOR. This hydrogel system not only exhibits significant antifungal activity but also effectively reduces ferroptosis in host cells, thereby reducing corneal scarring and promoting corneal healing.

[0101] The temperature-sensitive hydrogel described in this invention has the following advantages:

[0102] 1. Reduced medication frequency with better efficacy; the medication remains in the simulated corneal defect for at least 90 minutes, compared to the shorter retention time of traditional eye drops; enhanced in vitro antifungal activity; in animal models of fungal keratitis, the clinical control group (voriconazole) required 6 applications per day, while the hydrogel only required 1 application per day. The hydrogel treatment significantly reduced scarring in mice with fungal keratitis, while the clinical group still had larger corneal scars. The clinical score was 2.2 ± 0.447, and the corneal defect areas were 10.33 ± 2.517 and 61.00 ± 5.292, respectively.

[0103] 2. The multifunctional hydrogel has the function of "one drug for multiple purposes". The antifungal drugs in the clinical control group only have antifungal effects, while the PX / HA / Fer-1 / VOR temperature-sensitive in situ hydrogel has the characteristics of reducing host cell ferroptosis, reducing corneal scar formation and promoting corneal healing while having antifungal effects.

[0104] This invention provides a temperature-sensitive composite multifunctional hydrogel (poloxamer / hyaluronic acid / ferroptosis inhibitor / voriconazole, PX / HA / Fer-1 / VOR eye drops), which utilizes the pathological characteristics of fungal keratitis—corneal ulceration and defects. This composite hydrogel can form a hydrogel in situ at the physiological temperature of the cornea, compensating for corneal defects in situ while serving as a local drug reservoir, thereby significantly prolonging the drug retention time and simultaneously targeting the release of ferroptosis inhibitors and voriconazole for synergistic effects.

[0105] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition, characterized in that, It consists of ferroptosis inhibitors and antifungal drugs, wherein the mass ratio of ferroptosis inhibitors to antifungal drugs is (0.0015:1) to (0.01:1). The antifungal drugs include at least one of natamycin, voriconazole, amphotericin B, itraconazole, fluconazole, posaconazole, caspofungin, micafungin, anidoxane, clotrimazole, bifonazole, econazole, ketoconazole, terbinafine, or butenafine. The ferroptosis inhibitors include at least one of Ferrostatin-1, deferoxamine, deferoxone, Liproxstatin-1, N-acetylcysteine, glutathione ethyl ester, selenium compounds, α-tocopherol, Trolox, cyclohexenone, or rosiglitazone.

2. The composition according to claim 1, characterized in that, The mass ratio of ferroptosis inhibitor to antifungal drug is (0.0015:1) to (0.006:1); preferably, the mass ratio of ferroptosis inhibitor to antifungal drug in the composition is 0.006:

1.

3. The composition according to claim 1 or 2, characterized in that, The antifungal drug is voriconazole, and the ferroptosis inhibitor is the antioxidant Ferrostatin-1.

4. A temperature-sensitive hydrogel, characterized in that, It includes a gel matrix and the composition according to any one of claims 1 to 3.

5. The temperature-sensitive hydrogel according to claim 4, characterized in that, The gel matrix comprises poloxamer and hyaluronic acid, wherein the poloxamer comprises at least one of poloxamer 407 or poloxamer 188.

6. The temperature-sensitive hydrogel according to claim 4 or 5, characterized in that, The mass ratio of poloxamer 407, poloxamer 188 and hyaluronic acid in the gel matrix is ​​(16~20):(3~13):(0.1~2).

7. The temperature-sensitive hydrogel according to any one of claims 4 to 6, characterized in that, In terms of mass percentage, it includes: 16%~20% Poloxamer 407; 3%~13% Poloxamer 188; 0.1%~2% hyaluronic acid; 0.0015%~0.006% ferroptosis inhibitor; And 0.6%~1% antifungal drug, the remainder being water; Preferably, the following are included in the mass percentage: 18% Poloxamer 407; 5% Poloxamer 188; 1% hyaluronic acid; 0.006% ferroptosis inhibitor; And 1% antifungal drug, the remainder being water.

8. The method for preparing the temperature-sensitive hydrogel according to any one of claims 4 to 7, characterized in that, include: Poloxamer 407, poloxamer 188, ferroptosis inhibitors and antifungal drugs were mixed with anhydrous ethanol and evaporated to obtain a drug-loaded film. The drug-loaded film is hydrated, ethanol is removed, and the solution is reconstituted to obtain a micelle solution; The mixture is prepared by mixing hyaluronic acid with the micelle solution.

9. The composition according to any one of claims 1 to 3, the thermosensitive hydrogel according to any one of claims 4 to 7, or the thermosensitive hydrogel obtained by the preparation method according to claim 8, in the preparation of a medicament for treating fungal keratitis; wherein the medicament has at least one of the following functions: anti-inflammatory, anti-corneal damage, reducing host cell ferroptosis, inhibiting corneal scarring and / or promoting corneal healing.

10. A medicament for treating fungal keratitis, comprising the composition according to any one of claims 1 to 3, the thermosensitive hydrogel according to any one of claims 4 to 7, or the thermosensitive hydrogel obtained by the preparation method according to claim 8, and pharmaceutically acceptable excipients.

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