Pharmaceutical compositions based on two-dimensional halogen-bonded organic frameworks xofs and their use in the treatment of bacterial keratitis

Abstract

The application provides a drug composition based on a two-dimensional halogen bond organic framework XOF and application thereof in treating bacterial keratitis, the drug composition is formed by loading new indocyanine green to form a composite drug based on the two-dimensional halogen bond organic framework XOF, the composite drug has good bacteriostatic effect and can have good treatment effect on bacterial keratitis, and provides a new choice for the treatment of bacterial keratitis, can avoid drug resistance caused by long-term use of fixed drugs such as antibiotics, and has a significant application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of keratitis treatment drugs, in particular to a drug composition based on two-dimensional halogen bond organic framework XOFs and application thereof in treating bacterial keratitis. BACKGROUND

[0002] Bacterial keratitis, especially the infection caused by Pseudomonas aeruginosa, is a serious corneal disease that poses a significant threat to vision. As an opportunistic pathogen, Pseudomonas aeruginosa causes keratitis with the characteristics of rapid onset and rapid progression. If not intervened in time, it can lead to corneal perforation within 2 to 3 days, and in severe cases, enucleation of the eyeball is required. At present, the treatment of bacterial keratitis mainly relies on antibiotics, but the increase in drug resistance poses a challenge to the treatment effect. Therefore, it is urgent to develop a powerful antibiotic to cope with Pseudomonas aeruginosa keratitis.

[0003] Two-dimensional halogen bond organic framework XOFs, as a new type of organic framework material, not only exhibits good crystallinity, but also remains stable in various organic solvents and aqueous solutions with a wide pH range, making it a new type of material with wide application prospects. However, its performance in the pharmaceutical field has not been developed.

[0004] It is necessary to develop a new drug composition based on two-dimensional halogen bond organic framework XOFs to provide a new choice for the treatment of bacterial keratitis. SUMMARY

[0005] The present application proposes a drug composition based on two-dimensional halogen bond organic framework XOFs to improve the treatment effect of bacterial keratitis, solve the defect of existing technology that relies on antibiotic treatment to easily produce drug resistance, and provide a new choice for the treatment of bacterial keratitis.

[0006] The technical solution of the present application is as follows:

[0007] The first aspect of the present application is to propose a drug composition based on two-dimensional halogen bond organic framework XOFs, which is obtained by loading new indocyanine green on two-dimensional halogen bond organic framework XOFs; the two-dimensional halogen bond organic framework XOFs are selected from at least one of XOF(X)-TPPA and XOF(X)-TPPE, X is halogen selected from I or Br.

[0008] Further, the drug composition is obtained by adsorbing new indocyanine green on two-dimensional halogen bond organic framework XOFs in solution and removing the solvent.

[0009] Preferably, the mass ratio of two-dimensional halogen bond organic framework XOFs to new indocyanine green is (1-10): 1.

[0010] Preferably, the two-dimensional halogen-bonding organic framework XOFs are first ultrasonically dispersed in solution, and then adsorbed and allowed to stand for 0.5-2h.

[0011] Further, in the pharmaceutical composition, the mass ratio of the two-dimensional halogen-bonding organic framework XOFs to the loaded new indocyanine green is 6:1.

[0012] The second aspect of the present application is the use of the pharmaceutical composition of the first aspect above in bacteriostasis, which is for non-therapeutic purposes.

[0013] Further, the bacteriostasis is the inhibition of Pseudomonas aeruginosa.

[0014] The third aspect of the present application is the use of the pharmaceutical composition of the first aspect above in the preparation of a therapeutic drug for bacterial keratitis.

[0015] Further, the bacterial keratitis is keratitis caused by Pseudomonas aeruginosa.

[0016] Further, the therapeutic drug for bacterial keratitis further comprises a pharmaceutically acceptable auxiliary agent.

[0017] Further, the therapeutic drug for bacterial keratitis is a solid preparation, a liquid preparation or a semi-solid preparation, preferably an eye drop, an eye cream or an eye gel.

[0018] Further, when the content of the pharmaceutical composition in the therapeutic drug is 20mg / mL, the therapeutic effect is better.

[0019] Compared with the prior art, the beneficial effects of the present application are not limited to:

[0020] The pharmaceutical composition of the present application forms a composite drug based on two-dimensional halogen-bonding organic framework XOFs by loading new indocyanine green, the composite drug has good bacteriostatic effect and can have good therapeutic effect on bacterial keratitis, providing a new choice for the treatment of bacterial keratitis and avoiding drug resistance caused by long-term use of fixed drugs such as antibiotics.

[0021] The pharmaceutical composition of the present application forms a composite drug based on two-dimensional halogen-bonding organic framework XOFs by loading new indocyanine green, which has high biocompatibility and safety, and has significant application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0023] Figure 1 UV absorption (UV) analysis spectrum of IR820@XOF(Br)-TPPA in the example.

[0024] Figure 2 Zeta potential map of IR820@XOF(Br)-TPPA in the example.

[0025] Figure 3 X-ray photoelectron spectroscopy (XPS) of IR820@XOF(Br)-TPPA in the example.

[0026] Figure 4 EDS elemental mapping spectrum of IR820@XOF(Br)-TPPA in the example.

[0027] Figure 5 Infrared thermal imaging of IR820@XOF(Br)-TPPA in the example under 808 nm LED light irradiation.

[0028] Figure 6 Representative microscopic photographs of the ocular surface and fluorescein sodium staining of keratitis in mice treated with XOF(Br)-TPPA in the experimental example.

[0029] Figure 7 Score results of microscopic photographs of the ocular surface of mice recorded after treatment with XOF(Br)-TPPA in the experimental example.

[0030] Figure 8 Evaluation of the therapeutic effect of various drugs on Pseudomonas aeruginosa keratitis in the experimental example. (a) Schematic diagram of the in vivo experiment of XOF on bacterial keratitis. (b) Images of corneal ulcers at different stages observed with cobalt blue light after fluorescein staining. Using fluorescein sodium staining, a standard clinical ophthalmic method, corneal ulcers are quickly and accurately determined. When exposed to blue light in the range of 485-500 nm, the stained ulcers show bright green fluorescence. (c) Bar graph of corneal opacity scores at different conditions and time points. Opacity scores were assessed according to previous studies, with higher scores indicating more severe corneal opacity. Error bars represent standard error of the mean. * P < 0.05; ** P < 0.01; *** P < 0.001 (n = 3-4).

[0031] Figure 9Scored results for the microscopic photographs of the ocular surface of mice recorded after treatment with XOF(I, Br)-TPPA and XOF(I, Br)-TPPE in the experimental example. (a) IHC staining of the cornea revealed different patterns of protein expression, with green indicating positive staining. TUNEL detection highlighted areas of apoptosis, while the presence of inflammatory cells was identified by Iba-1, GFAP, VEGF, and HIF-1 staining. Scale bar represents 200 pm. (b) Accompanying the images are plots that illustrate the fluorescence intensity measurements of TUNEL, IBA-1, GFAP, VEGF, and HIF-1 IHC staining on the cornea to quantify the effects of treatment. Data error bars represent the standard error of the mean (SEM). * P < 0.05 ** P < 0.01 *** P < 0.001. (n = 3).

[0032] Figure 10 Representative microscopic photographs of the ocular surface and corneal fluorescein sodium staining of mice treated with IR820@XOF(Br)-TPPA in the experimental example.

[0033] Figure 11 Scored results for the microscopic photographs of the ocular surface of mice recorded after treatment with IR820@XOF(Br)-TPPA in the experimental example.

[0034] Figure 12 Scored results for the microscopic photographs of the ocular surface of mice recorded after treatment with IR820@XOF(Br)-TPPA in the experimental example.

[0035] Figure 13 Representative H&E staining and CD31 immunofluorescence staining of corneal paraffin sections of mice 7 days after infection in the experimental example. Scale bar: 200 pm.

[0036] Figure 14 Bar graph of the average thickness at 5 equidistant locations in the cornea and the fluorescence intensity of CD31 fluorescence staining of the cornea in the experimental example.

[0037] Figure 15To apoptosis and inflammation inhibition results of IR820@XOF(Br)-TPPA treatment on corneal tissue in experimental example. One day after the type induction, corneal tissues were collected from different treatment groups and then subjected to immunohistochemistry (IHC) analysis to assess the changes in protein expression. (a) IHC staining of corneas revealed different patterns of protein expression, with green color indicating positive staining. TUNEL detection highlighted the areas of apoptosis, while the presence of inflammatory cells was identified by Iba-1, GFAP, VEGF, and HIF-1 staining. Scale bar represents 200 μm. (b) Accompanying images are graphs illustrating the fluorescence intensity measurements of TUNEL, IBA-1, GFAP, VEGF, and HIF-1 IHC staining on corneas to quantify the effects of treatment. Data error bars represent the standard error of the mean (SEM). *P < 0.05 **P < 0.01 ***P < 0.001. (n = 3).

[0038] Figure 16 Representative micrographs of the ocular surface and sodium fluorescein staining of mice treated with IR820@XOF(Br)-TPPA (20 mg / mL, 808 nm, 5 min) in experimental example.

[0039] Figure 17 HE staining and TUNEL assay of mice treated with IR820@XOF(Br)-TPPA (20 mg / mL, 808 nm, 5 min) after 7 days in experimental example. Scale bar: 200 μm.

[0040] Figure 18 Bar graph of the average thickness of 5 equidistant locations in the cornea and bar graph of the fluorescence intensity of the tunnel fluorescence staining of the cornea in experimental example. Data error bars represent the standard error of the mean (SEM), n = 3.

[0041] Figure 19 H&E staining of the main organs of mice after treatment with IR820@XOF(Br)-TPPA in experimental example. Scale bar: 200 μm.

[0042] Figure 20 Body weight changes during drug administration in mice in experimental example.

[0043] Figure 21 Biochemical examination results of liver and kidney function during drug administration in mice in experimental example. DETAILED DESCRIPTION

[0044] The technical solutions of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present application.

[0045] In one embodiment, a drug composition based on two-dimensional halogen-bonding organic framework XOFs is proposed, which is obtained by loading new indocyanine green (IR820) on two-dimensional halogen-bonding organic framework XOFs; the two-dimensional halogen-bonding organic framework XOFs are selected from at least one of XOF(X)-TPPA (Formula I) and XOF(X)-TPPE (Formula II).

[0046]

[0047] In the formula I and the formula II, the element X is halogen, and each is independently selected from Br or I. That is, the four types of two-dimensional halogen-bonding organic framework XOFs include XOF(Br)-TPPA, XOF(Br)-TPPE, XOF(I)-TPPA, and XOF(I)-TPPE.

[0048] In the above embodiments, the two-dimensional halogen-bonding organic framework XOFs load IR820 to form a composite drug IR820@XOF(X)-TPPA and / or IR820@XOF(X)-TPPE through electrostatic adsorption. The obtained two-dimensional halogen-bonding organic framework XOFs composite drug has good antibacterial effect and can have good therapeutic effect on bacterial keratitis.

[0049] In the above embodiments, the two-dimensional halogen-bonding organic framework XOFs are prepared by taking TPPA and TPPE as ligands, and have good crystallinity. The preparation process of the organic framework can be obtained by taking TPPA / TPPE as ligands and through silver-halogen exchange reaction. The preparation can be referred to the literature https: / / doi.org / 10.1002 / anie.202408428.

[0050] In a preferred embodiment, taking the preparation of XOF(Br)-TPPA as an example, specifically: AgBF4(29.0 mg, 0.15 mmol) in MeOH (1 mL) is added dropwise to a solution of TPPA (47.6 mg, 0.1 mmol) in methanol (3 mL). After the solution is stirred at room temperature for 1 hour, a solution of bromine (23.6 mg, 0.15 mmol) in methanol (68.2 μL) is added. Then, the solution is degassed under vacuum-argon for three cycles, and the mixture is reacted at 40°C for 3 hours. Then, the solvent is removed under reduced pressure, and the precipitate is dried under vacuum for 5 hours to obtain.

[0051] In preferred embodiments, taking the preparation of XOF(Br)-TPPE as an example, specifically: AgBF4(27.1 mg, 0.14 mmol) in MeOH (1 mL) was added dropwise to a mixture solution of TPPE (44.8 mg, 0.07 mmol) in methanol (2 mL). After the solution was stirred at room temperature for 1 hour, a solution of bromine (22.1 mg, 0.14 mmol) in methanol (1 mL) was added. Then, the solution was degassed under vacuum-argon for three cycles, and the mixture was reacted at 90°C for 3 hours. Then, the solvent was removed under reduced pressure, and the precipitate was dried under vacuum for 6 hours to obtain.

[0052] In some embodiments, taking Br as an example, the specific structural formula of XOF(Br)-TPPA and XOF(Br)-TPPE is shown in formula III and formula IV, respectively.

[0053]

[0054] In preferred embodiments, IR820@XOF(X)-TPPA and IR820@XOF(X)-TPPE are obtained by adsorbing and removing solvent from new indocyanine green and two-dimensional halogen-bonded organic framework XOFs in solution; two-dimensional halogen-bonded organic framework XOFs are ultrasonically dispersed in aqueous solution and adsorb IR820 in the aqueous solution, and the successful loading of IR820 is achieved through electrostatic interaction.

[0055] In preferred embodiments, the loading amount of IR820 on XOF(Br)-TPPA or XOF(Br)-TPPE can be achieved by physical intervention, such as stirring, oscillation, etc. As preferred, the mass ratio of XOF(Br)-TPPA or XOF(Br)-TPPE and IR820 in the solution is (1-10):1. Taking the IR820 solution concentration of 0.03 mg / mL as an example, after sufficient adsorption and loading according to the above method, the maximum adsorption amount of XOF(Br)-TPPA is 141.1 mg / g detected by ultraviolet-visible spectrophotometer, and the mass ratio of XOF(Br)-TPPA and IR820 in the composition after adsorption is about 6:1.

[0056] It can be understood that XOF(X)-TPPE and XOF(X)-TPPA have similar properties and both have electrostatic adsorption effect, and those skilled in the art can adjust the feeding ratio and determine the maximum adsorption amount.

[0057] In a preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating bacterial keratitis, which can comprise a pharmaceutically acceptable adjuvant, and the person skilled in the art can select a suitable adjuvant according to the treatment needs to prepare a desired dosage form, and the adjuvant comprises common pharmaceutical diluents, excipients, carriers, etc.

[0058] In a preferred embodiment, the medicament for treating bacterial keratitis is a solid preparation, a liquid preparation or a semi-solid preparation, preferably an eye drop, an eye cream or an eye gel; it has been proved by animal experiments that the IR820@XOF(X)-TPPA pharmaceutical composition has the best therapeutic effect when the content is 20 mg / mL. Similarly, IR820@XOF(X)-TPPE also has excellent effect on treating bacterial keratitis.

[0059] The following are preferred examples, and the reagents or instruments not indicated by the manufacturer can be purchased from the market. The specific conditions not indicated can be carried out according to the conventional conditions or the recommended conditions of the manufacturer.

[0060] Examples

[0061] 1. Preparation of IR820@XOF(Br)-TPPA

[0062] The preparation method of the drug compound IR820@XOF(Br)-TPPA is illustrated by taking 6:1 as an example: 3.0 mg of IR820 is dissolved in 100 mL of ultrapure water, 10.0 mg of XOF(Br)-TPPA is added and uniformly dispersed by ultrasonic, and then stirred at room temperature for 1 hour to promote adsorption. After filtration and washing with 20 mL of ultrapure water for three times, vacuum drying at room temperature for 6 hours, the drug composition of XOF(Br)-TPPA and IR820 with a ratio of 6:1 is obtained.

[0063] 2. Characterization of IR820@XOF(Br)-TPPA

[0064] 1) The formation of IR820@XOF(Br)-TPPA is characterized by using ultraviolet-visible absorption spectrum, Figure 1 The characteristic wavelength of the absorption spectrum of IR820@XOF(Br)-TPPA is 843 nm, while the characteristic wavelength of the absorption spectrum of free IR820 in water is 814 nm. In addition, compared with IR820 in water, the loaded IR820 in IR820@XOF(Br)-TPPA shows broadening and significant red shift in the absorption spectrum.

[0065] 2) The characterization of IR820@XOF(Br)-TPPA is carried out by using Zeta potential map, Figure 2The potential of IR820@XOF(Br)-TPPA was 1.97±0.38 mV, which was significantly decreased compared with XOF(Br)-TPPA, indicating the formation of IR820@XOF(Br)-TPPA.

[0066] 3) The elemental changes of IR820@XOF(Br)-TPPA were analyzed using X-ray photoelectron spectroscopy (XPS), Figure 3 The X-ray photoelectron spectroscopy (XPS) of prepared IR820@XOF(Br)-TPPA was shown, and the appearance of high-intensity peaks of S2s, S2p and Cl 2p in the figure indicated that IR820 was successfully loaded into XOF(Br)-TPPA.

[0067] 4) The elements of IR820@XOF(Br)-TPPA were analyzed using EDS elemental mapping spectrum, Figure 4 The EDS elemental mapping spectrum of prepared IR820@XOF(Br)-TPPA was shown, and the characteristic elements S and Cl corresponding to IR820 could be found in the element mapping, which confirmed the successful formation of IR820@XOF(Br)-TPPA.

[0068] 5) The photothermal performance of IR820@XOF(Br)-TPPA was characterized using infrared thermal imaging, Figure 5 The infrared thermal imaging of prepared IR820@XOF(Br)-TPPA under 808 nm LED light irradiation was shown, indicating that IR820@XOF(Br)-TPPA had good photothermal performance.

[0069] Experimental Example

[0070] 1. Animal experiments

[0071] All in vivo animal experiments were approved by the Experimental Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. C57 mice (male, 7-8 weeks old) were used in this experiment. All animals were raised under appropriate temperature and humidity with a standard light-dark cycle (L:D=12h:12h). Food and water were replenished in time, and mice were provided free of charge. Mice were usually anesthetized with 1% sodium pentobarbital (40-50 mg / kg) intraperitoneally, and the entire corneal epithelium of the mice was scraped under a microscope after anesthesia. Then the corneal wound was infected with a Pseudomonas aeruginosa suspension (1.5x108 CFU / mL, 10 μL). Five minutes after infection, the mice were treated with XOF(I, Br)-TPPA, XOF(I, Br)-TPPE or normal saline. The mice were treated every day during the entire treatment period, and the microscopic images of the ocular surface of each group were recorded, and the epithelial defect area was evaluated by fluorescein staining.

[0072] 1) H&E staining

[0073] After 7 days of treatment, the eyes were removed, fixed with 4% PFA, paraffin-embedded and sectioned. After deparaffinization of the paraffin sections into water, hematoxylin staining, differentiation with differentiation fluid and re-staining with re-staining solution were performed. Then, they were dehydrated with 85% and 95% ethanol, respectively. After dehydration, they were stained with eosin. Finally, the sections were dehydrated with absolute ethanol and xylene, respectively, and sealed with neutral paraffin.

[0074] 2) Immunofluorescence

[0075] After 7 days and 1 day of treatment, the eyes were removed and fixed with 4% PFA. The eyes were embedded in an optimal cutting temperature compound, frozen and frozen to a thickness of 10 pm. The eye sections were permeabilized with 0.1% Triton for 15 minutes, then blocked with 5% BSA for 30 minutes, then incubated with primary antibodies against CD31 (1:200, GB113151-100, Servicebio), Iba-1 (1:800, LEE6425, WAKO), GFAP (1:800, 3670S, CST), VEGF (1:200, AF5131, Affinity), HIF-1 (1:200), BF8002, Affinite) overnight. After three washes with PBS, the sections were incubated with Alexa Fluor 488 or 594 conjugated secondary antibodies. Dapi staining was performed, the sections were mounted and observed with a fluorescence microscope.

[0076] 3) TUNEL

[0077] TUNEL was performed according to the manufacturer's instructions using ApopTag Fluorescein In Situ Apoptosis Detection Kit (KTA2010, Abkine).

[0078] 4) In vivo toxicology analysis

[0079] The control and XOF-TPPA(Br) groups (n=3) were weighed daily during the seven days of treatment. At the end of the 7-day treatment, orbital blood was taken for biochemical analysis. Their heart, lung, liver, spleen, kidney were taken, fixed with 5% paraformaldehyde, paraffin-embedded and sectioned. The sections were used for H&E staining.

[0080] 2. Data analysis

[0081] All experiments were performed at least in triplicate. The resulting data were statistically analyzed and plotted using GraphPad Prism 7.0 (GraphPad Software. Inc.) for this study. The results are expressed as the mean ± standard deviation (mean ± SEM) of continuous variables. The mean values of the two groups were compared using the Student t test. Statistics were considered significant at P<0.05.

[0082] When infected with P. aeruginosa, small blebs are formed first. Then, corneal ulceration occurs when the corneal epithelial cells are sloughed off from the stromal layer as the blebs accumulate. Due to the lack of epithelial layer, a water-soluble dye, fluorescein, is used to mark the epithelial defect, also known as corneal ulceration. With cobalt blue light observation, the ulcerated area is detected as green. Using XOF(Br)-TPPA as a model, the dosage of XOFs(I / Br) in treating P. aeruginosa keratitis model mice was optimized. Three concentration gradients, i.e., 10 mg / mL, 20 mg / mL and 50 mg / mL of XOF(Br)-TPPA prepared by the example were administered at a dose of 20 μL / day after 5 minutes of infection. Notably, 20 mg / mL XOF(Br)-TPPA observed therapeutic effect after 7 days of treatment (clinical score of 6.3 ± 1.5), with statistical difference compared to 50 mg / mL XOF(Br)-TPPA (clinical score of 12.0 ± 1.7) and 10 mg / mL XOF(Br)-TPPA (clinical score of 8.7 ± 0.6). The therapeutic effect of both groups was superior to the untreated group (clinical score of 13.0 ± 1.4).

[0083] In the treatment group, the symptoms of mice treated with 20 mg / mL XOF(Br)-TPPA had the mildest symptoms, with only slight corneal opacity, pupil penetration, and significantly less corneal ulcer area and neovascularization than the rest of the groups. In the control group, the ulcer almost spread to the entire corneal surface within 24 hours. The corneal damage slowly recovered in about 1 week Figure 6 ) The clinical evaluation score results of the representative images of mouse corneas were also consistent Figure 7 ) This further confirmed the in vivo antimicrobial efficacy of XOF(Br)-TPPA, and the best therapeutic effect was observed in the 20 mg / mL group.

[0084] To determine the therapeutic efficacy of XOF(I, Br)-TPPA and XOF(I, Br)-TPPE against bacterial keratitis, topical suspensions of 20 mg / mL of each sample were applied daily to a murine model exhibiting P. aeruginosa-induced keratitis. Analysis revealed that mice treated with XOF(Br)-TPPA (clinical score of 5.0 ± 0.0 on day 3) exhibited a significantly favorable response and outperformed mice treated with XOF(Br)-TPPE (clinical score of 7.0 ± 0.0 on day 3). In contrast, control mice treated with saline (clinical score of 9.8 ± 1.5 on day 3), XOF(I)-TPPA (clinical score of 8.7 ± 0.6 on day 3), and XOF(I)-TPPE (clinical score of 9.0 ± 1.0 on day 3) experienced a worsening of corneal conditions. This worsening was characterized by increased haze, hypopyon, significant neovascularization, and in some cases corneal perforation.

[0085] This treatment group exhibited a significant reduction in corneal haze and turbidity, as well as a marked decrease in neovascularization. Furthermore, as highlighted, corneal ulcer healing was significantly improved, with a faster rate of recovery Figure 8 c and Figure 9 ). These findings strongly support the potential of XOF(Br)-TPPA as a promising therapeutic option for bacterial keratitis. However, further research is needed to optimize the treatment regimen, elucidate the mechanism of action, and validate its efficacy in human clinical trials.

[0086] To explore whether the treatment effect is improved upon the addition of IR 820, it was initially sought to determine the optimal illumination duration. After the administration of the hazy IR820@XOF(Br)-TPPA solution, mice were illuminated with an 808 nm LED for 5 minutes, 10 minutes, and 20 minutes Figure 8 a). Compared to the untreated group (clinical score of 9.0 ± 0.8 on day 3), which suffered from severe corneal haze, neovascularization, and even corneal perforation after infection, mice treated with IR820@XOF(Br)-TPPA showed significantly fewer symptoms, with those illuminated for 5 minutes (clinical score of 2.8 ± 0.9 on day 3) having the lightest symptoms Figure 10 and Figure 11 ). By the third day of treatment, the corneas were completely transparent and almost healed Figure 8 b). This was confirmed by the clinical scores of representative images of the mice's corneas Figure 8 c and Figure 12 ).

[0087] The therapeutic effect of IR820@XOF(Br)-TPPA was further investigated by comparing the therapeutic effect of IR820@XOF(Br)-TPPA with XOF(Br)-TPPA. After infection, mice were treated with saline, 20 mg / mL XOF(Br)-TPPA, IR 820 (exposed to 808 nm LED light for 5 minutes), or IR820@XOF(Br)-TPPA (exposed to 808 nm LED light for 5 minutes) Figure 8 a). Although improvement of symptoms was observed in mice treated with XOF(Br)-TPPA (which had a clinical score of 4.3 ± 1.5 on day 6), ocular surface turbidity still existed and corneal transparency was not restored. In contrast, the corneas of mice treated with IR820@XOF(Br)-TPPA (clinical score of 0.8 ± 0.5 on day 6) were almost transparent on day 2 and close to complete healing on day 6, with no visible corneal turbidity, neovascularization, or sodium fluorescein staining Figure 8 b and Figure 8 c). The above experimental results show that the addition of IR820 greatly improves the therapeutic effect of bacterial keratitis and has great potential in the treatment of bacterial keratitis.

[0088] After 7 days of treatment, the corneal sections were then subjected to H&E staining and CD31 staining Figure 13 ). H&E staining showed that the corneas of the untreated group were significantly thickened and contained more inflammatory cells, while the corneas of the group treated with IR820@XOF(Br)-TPPA exhibited a more regular structure and had fewer inflammatory cells. In addition, the corneal thickness of mice receiving 5 minutes of irradiation was significantly reduced. Corneal neovascularization was labeled with CD31, and CD31 fluorescent staining of mouse corneas showed a significant increase in CD31 expression in untreated corneas Figure 14 ). This further indicates that IR820@XOF(Br)-TPPA can significantly inhibit the formation of corneal neovascularization in keratitis.

[0089] After bacterial invasion, keratitis occurs, which exacerbates corneal inflammation by secreting various virulence factors and activating pattern recognition receptors. Reducing the corneal inflammatory response is crucial for effective treatment of keratitis.

[0090] To further investigate the treatment mechanism, corneal tissue was collected after one day of IR820@XOF(Br)-TPPA treatment. TUNEL staining showed a significant decrease in corneal epithelial cell apoptosis. In addition, the reduced expression of inflammatory factors GFAP and Iba-I indicates that IR820@XOF(Br)-TPPA can reduce corneal epithelial cell apoptosis by reducing the inflammatory response Figure 15 a, Figure 15b). In addition to inflammation, corneal neovascularization is another factor that leads to visual impairment in bacterial keratitis. After 7 days of treatment, a significant decrease in CD31 expression was observed in the cornea, indicating a decrease in neovascularization.

[0091] To explore its potential mechanism, the expression of HIF-1, a key factor in neovascularization, was detected. Compared with the untreated group, a significant decrease in HIF-1 expression was observed in the treated group Figure 15 a、 Figure 15 b). In addition, a decrease in VEGF expression was noted, indicating that IR820@XOF(Br)-TPPA treatment decreased HIF-1 expression, prevented neovascularization, and ultimately improved corneal transparency. IR820@XOF(Br)-TPPA treatment significantly reduced the inflammatory response in the cornea after infection and reduced the expression of neovascularization-related factors, leading to a decrease in corneal epithelial cell apoptosis and neovascularization.

[0092] The final design of a drug is to develop an effective medical product for clinical translation, for which the biological safety of the drug is particularly important. To evaluate the biocompatibility of IR820@XOF(Br)-TPPA to determine its potential for clinical translation, we conducted a series of toxicity studies. First, blank mice were treated with XOF(Br)-TPPA, and representative microscopic images of the mouse ocular surface during treatment were recorded. After 7 days of continuous treatment, the mouse cornea was transparent, with no conjunctival redness, edema, increased secretion, and no significant coloration with fluorescein sodium staining Figure 16 ). H&E staining of the cornea of mice treated for 7 days also showed that the mouse corneal structure was normal, with no obvious inflammatory cell infiltration. Tunel staining of the treated mouse cornea also showed no significant cell apoptosis Figure 17 and Figure 18 ). This indicates that IR820@XOF(Br)-TPPA has no obvious cytotoxic effect on the mouse cornea. During the entire drug administration period, the liver and kidney functions (ALT, AST, BUN, and CREA) of the four groups of mice remained normal Figure 19 ). It was also demonstrated that there was no significant difference in body weight Figure 20 ). H&E staining of the main organs of mice also showed no obvious lesion area Figure 21 ). All of this indicates the high biocompatibility of IR820@XOF(Br)-TPPA.

[0093] The above merely provides the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a pharmaceutical composition in the preparation of a bacterial keratitis treatment drug, wherein the pharmaceutical composition is obtained by loading new indocyanine green on two-dimensional halogen-bonded organic frameworks (XOFs); the two-dimensional halogen-bonded organic frameworks (XOFs) are selected from at least one of the compounds represented by the structural formulae of formula I and formula II: formula I and formula II; wherein the element X in the formulae I and II is Br. ； wherein The pharmaceutical composition is obtained by adsorbing new indocyanine green on the two-dimensional halogen-bonded organic frameworks (XOFs) in a solution and removing the solvent.

2. Use according to claim 1, wherein The mass ratio of the two-dimensional halogen-bonded organic frameworks (XOFs) to new indocyanine green is (1-10):

1.

3. Use according to claim 2, wherein the compound is ###0002### The two-dimensional halogen-bonded organic frameworks (XOFs) are first ultrasonically dispersed in a solution, and then adsorbed for 0.5-2 h.

4. The use according to claim 2, wherein The mass ratio of the two-dimensional halogen-bonded organic frameworks (XOFs) to the loaded new indocyanine green is 6:

1.

5. The use according to claim 1, wherein The bacterial keratitis is caused by Pseudomonas aeruginosa.

6. The use according to claim 1, wherein The bacterial keratitis treatment drug further comprises a pharmaceutically acceptable auxiliary agent.

7. The use according to claim 1, wherein The bacterial keratitis treatment drug is an eye drop, an eye cream or an eye gel.

8. The use according to claim 1, wherein ​

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