Pharmaceutical composition based on two-dimensional halogen bond organic framework XOFs and application of pharmaceutical composition in treatment of bacterial keratitis

Abstract

The invention provides a pharmaceutical composition based on a two-dimensional halogen bond organic framework XOFs and application of the pharmaceutical composition in treatment of bacterial keratitis, and the pharmaceutical composition forms a composite drug based on the two-dimensional halogen bond organic framework XOFs by loading new indocyanine green. The compound medicine has a good bacteriostatic effect and a good treatment effect on the bacterial keratitis, provides a new choice for treatment of the bacterial keratitis, can avoid drug resistance caused by long-term use of fixed medicines such as antibiotics and the like, and has a remarkable application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of keratitis treatment drugs, in particular to a drug composition based on two-dimensional halogen bond organic frameworks (XOFs) and an application thereof in treating bacterial keratitis. Background Art

[0002] Bacterial keratitis, especially infection caused by Pseudomonas aeruginosa (Pseudomonas aeruginosa), is a serious corneal disease that poses a significant threat to vision. As an opportunistic pathogen, Pseudomonas aeruginosa causes keratitis that is characterized by rapid onset and progression. If not intervened in time, it can lead to corneal perforation within 2 to 3 days, and in severe cases, enucleation 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, there is an urgent need to develop potent antibiotics to deal with Pseudomonas aeruginosa keratitis.

[0003] Two-dimensional halogen-bonded organic frameworks (XOFs), as a new type of organic framework material, not only exhibit good crystallinity, but also remain stable in various organic solvents and aqueous solutions with a wide pH range. It is a new type of material with broad application prospects, but its medicinal properties have not yet been developed.

[0004] It is necessary to develop a new pharmaceutical composition based on two-dimensional halogen-bonded organic frameworks (XOFs) to provide new options for the treatment of bacterial keratitis. Summary of the invention

[0005] The present invention proposes a pharmaceutical composition based on a two-dimensional halogen bond organic framework XOFs, which is used to improve the therapeutic effect of bacterial keratitis, solve the defect of the prior art that relies on antibiotic treatment and is prone to drug resistance, and provide a new option for the treatment of bacterial keratitis.

[0006] The technical solution of the present invention is achieved in this way:

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

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

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

[0010] Preferably, the two-dimensional halogen-bonded organic framework XOFs is firstly ultrasonically dispersed in the solution and then allowed to stand for adsorption for 0.5-2 hours.

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

[0012] The second aspect of the present invention is to propose the use of the pharmaceutical composition described in the first aspect in antibacterial treatment, wherein the use is for non-therapeutic purposes.

[0013] Furthermore, the antibacterial activity is the inhibition of Pseudomonas aeruginosa.

[0014] The third aspect of the present invention is to propose the use of the pharmaceutical composition described in the first aspect in the preparation of a drug for treating bacterial keratitis.

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

[0016] Furthermore, the drug for treating bacterial keratitis also includes a pharmaceutically acceptable adjuvant.

[0017] Furthermore, the drug for treating bacterial keratitis is a solid preparation, a liquid preparation or a semisolid preparation, preferably an eye drop, an eye cream or an eye gel.

[0018] Furthermore, when the content of the drug composition in the therapeutic drug is 20 mg / mL, it has a better therapeutic effect.

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

[0020] The pharmaceutical composition of the present invention forms a composite drug based on a two-dimensional halogen bond organic framework XOFs by loading new indocyanine green. The composite drug has good antibacterial effect and can have a good therapeutic effect on bacterial keratitis, providing a new option 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 invention forms a composite drug based on a two-dimensional halogen bond organic framework XOFs by loading new indocyanine green, has high biocompatibility and safety, and has significant application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

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

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

[0026] Figure 4 This is the EDS element mapping spectrum of IR820@XOF(Br)-TPPA in the embodiment.

[0027] Figure 5 This is the infrared thermal imaging image of IR820@XOF(Br)-TPPA in the embodiment under 808nm LED light.

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

[0029] Figure 7 The scoring results of the micrographs of the mouse ocular surface recorded after treatment with XOF(Br)-TPPA in the experimental example.

[0030] Figure 8 This is an experimental example to evaluate the therapeutic effects of various drugs on Pseudomonas aeruginosa keratitis. (a) Schematic diagram of XOF's in vivo experiment on bacterial keratitis. (b) Images of corneal ulcers at different stages observed with cobalt blue light after fluorescein staining. Using sodium fluorescein staining, a standard clinical ophthalmology method, corneal ulcers are quickly and accurately identified. When exposed to blue light in the range of 485-500nm, the stained ulcers show bright green fluorescence. (c) Bar graph of corneal opacity scores under different conditions and time points. The opacity scores were evaluated according to previous studies, and higher scores indicate more severe corneal opacity. Data error bars represent standard error means. * P < 0.05; ** P < 0.01; *** P<0.001 (n=3-4).

[0031] Fig. 9Scoring results for micrographs of the mouse ocular surface recorded after treatment with XOF(I,Br)-TPPA and XOF(I,Br)-TPPE in the experimental example. (a) IHC staining of the cornea reveals different patterns of protein expression, with green indicating positive staining. TUNEL detection highlights areas of apoptosis, while the presence of inflammatory cells is identified by staining with Iba-1, GFAP, VEGF, and HIF-1. The scale bar represents 200 μm. (b) Accompanying the images is a diagram illustrating the fluorescence intensity measurement of TUNEL, IBA-1, GFAP, VEGF, and HIF-1 IHC staining on the cornea to quantify the effects of treatment. The data error bars represent the standard error of the mean (SEM). * P<0.05 ** P<0.01 *** P<0.001. (n=3).

[0032] Fig.10 Representative micrographs of ocular surface and keratitis stained with sodium fluorescein of mice treated with IR820@XOF(Br)-TPPA in the experimental example.

[0033] Fig.11 These are the scoring results of the micrographs of the mouse eye surface recorded after treatment with IR820@XOF(Br)-TPPA in the experimental example.

[0034] Fig.12 These are the scoring results of the micrographs of the mouse eye surface recorded after treatment with IR820@XOF(Br)-TPPA in the experimental example.

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

[0036] Fig.14 The bar graph is a graph showing the average thickness at five equidistant locations in the cornea and the fluorescence intensity of CD31 fluorescent staining of the cornea in the experimental example.

[0037] Fig.15The results of the inhibition of corneal tissue cell apoptosis and inflammation by IR820@XOF(Br)-TPPA treatment in the experimental example. One day after type induction, corneal tissues were collected from different treatment groups and then subjected to immunohistochemistry (MHC) analysis to evaluate changes in protein expression. (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. The scale bar represents 200 μm. (b) Accompanying the images is a diagram illustrating the fluorescence intensity measurement of TUNEL, IBA-1, GFAP, VEGF, and HIF-1 IHC staining on the cornea to quantify the effects of treatment. Data error bars represent standard error of the mean (SEM). *P<0.05**P<0.01***P<0.001. (n=3).

[0038] Fig.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 the experimental example.

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

[0040] Fig.18 The bar graphs are the average thickness of 5 equally spaced locations in the cornea and the fluorescence intensity of corneal tunnel fluorescence staining in the experimental example. The data error bars represent the standard error of the mean (SEM), n=3.

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

[0042] Fig. 20 The weight changes of mice during the drug administration period in the experimental example.

[0043] Fig.21 These are the biochemical examination results of liver and kidney function of mice during drug administration in the experimental example. DETAILED DESCRIPTION

[0044] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0046]

[0047] Wherein, the element X in formula I and formula II is a halogen, and each is independently selected from Br or I. That is, four types of two-dimensional halogen bond organic frameworks XOFs are included: XOF(Br)-TPPA, XOF(Br)-TPPE, XOF(I)-TPPA, and XOF(I)-TPPE.

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

[0049] In the above embodiment, the two-dimensional halogen bond organic framework XOFs is prepared with TPPA and TPPE as ligands and has good crystallinity. The preparation process of the organic framework can be obtained by using TPPA / TPPE as ligands and undergoing a silver-halogen exchange reaction. The preparation can be referred to the document https: / / doi.org / 10.1002 / anie.202408428.

[0050] In a preferred embodiment, the preparation of XOF(Br)-TPPA is taken as an example, specifically: AgBF in MeOH (1 mL) is added to 4 (29.0 mg, 0.15 mmol) was added dropwise to a solution of TPPA (47.6 mg, 0.1 mmol) in methanol (3 mL). After the solution was stirred at room temperature for 1 hour, a solution of bromine (23.6 mg, 0.15 mmol) in methanol (68.2 μL) was added. Then, the solution was degassed under vacuum-argon for three cycles, and the mixture was reacted at 40° C. for 3 hours. Then, the solvent was removed under reduced pressure, and the precipitate was dried under vacuum for 5 hours to obtain.

[0051] In a preferred embodiment, the preparation of XOF(Br)-TPPE is taken as an example, specifically: AgBF in MeOH (1 mL) is added to 4 (27.1 mg, 0.14 mmol) was added dropwise to a mixed 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 halogen X as Br as an example, the specific structural formulas of XOF(Br)-TPPA and XOF(Br)-TPPE are shown in Formula III and Formula IV, respectively.

[0053]

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

[0055] In a preferred embodiment, the loading amount of IR820 on XOF(Br)-TPPA or XOF(Br)-TPPE can be achieved by physical intervention, such as stirring, shaking, etc. Preferably, the feeding mass ratio of XOF(Br)-TPPA or XOF(Br)-TPPE to IR820 put into the solution for adsorption is (1-10):1. Taking the IR820 solution concentration of 0.03 mg / mL as an example, after fully adsorbing the load according to the above method, the maximum adsorption amount of XOF(Br)-TPPA detected by UV-visible spectrophotometer is 141.1 mg / g, and the mass ratio of XOF(Br)-TPPA to IR820 in the adsorbed composition 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 effects. Those skilled in the art can adjust the feed ratio and determine the maximum adsorption amount.

[0057] In a preferred embodiment, the pharmaceutical composition is used to prepare a drug for treating bacterial keratitis, which may include pharmaceutically acceptable adjuvants. Those skilled in the art select appropriate adjuvants to prepare the desired dosage form according to treatment needs. The adjuvants include common pharmaceutical diluents, excipients, carriers, etc.

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

[0059] The following are preferred implementation examples. If the manufacturer of the reagent or instrument is not specified in the examples, conventional products can be purchased from the market. If the specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer can be used.

[0060] Example

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

[0062] Taking 6:1 as an example, the preparation method of the drug complex IR820@XOF(Br)-TPPA is illustrated: 3.0 mg IR820 is weighed and dissolved in 100 mL ultrapure water, 10.0 mg XOF(Br)-TPPA is added thereto and ultrasonically dispersed evenly, stirred at room temperature for 1 hour to promote adsorption, filtered and washed three times with 20 mL ultrapure water, and then vacuum dried at room temperature for 6 hours to obtain a drug composition of XOF(Br)-TPPA and IR820 in a ratio of 6:1.

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

[0064] 1) The formation of IR820@XOF(Br)-TPPA was characterized by UV-visible absorption spectroscopy. Figure 1 The absorption spectrum of IR820@XOF(Br)-TPPA is shown to have a characteristic wavelength of 843 nm, while the absorption spectrum of free IR820 in water has a characteristic wavelength of 814 nm. In addition, compared with IR820 in water, the IR820 loaded in IR820@XOF(Br)-TPPA exhibits broadening and significant red shift in the absorption spectrum.

[0065] 2) Characterize IR820@XOF(Br)-TPPA using Zeta potential diagram, Figure 2The potential of IR820@XOF(Br)-TPPA is shown to be 1.97 ± 0.38 mV, which is significantly decreased compared with that of XOF(Br)-TPPA, indicating the formation of IR820@XOF(Br)-TPPA.

[0066] 3) X-ray photoelectron spectroscopy (XPS) was used to analyze the elemental changes of IR820@XOF(Br)-TPPA. Figure 3 Shown is the X-ray photoelectron spectroscopy (XPS) graph of the prepared IR820@XOF(Br)-TPPA. From the graph, it can be observed that the appearance of high-intensity peaks of S2s, S2p and Cl 2p means that IR820 is successfully loaded into XOF(Br)-TPPA.

[0067] 4) Use EDS element mapping spectrum to analyze the elements of IR820@XOF(Br)-TPPA. Figure 4 Shown is the EDS element mapping spectrum of the prepared IR820@XOF(Br)-TPPA. The characteristic elements S and Cl corresponding to IR820 can be found in the element mapping, confirming the successful formation of IR820@XOF(Br)-TPPA.

[0068] 5) Use infrared thermal imaging to characterize the photothermal performance of IR820@XOF(Br)-TPPA. Figure 5 Shown is the infrared thermal imaging of the prepared IR820@XOF(Br)-TPPA under 808nm LED light, indicating that IR820@XOF(Br)-TPPA has good photothermal properties.

[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 housed with a standard light–dark cycle (L:D = 12 h:12 h) at appropriate temperature and humidity. Food and water were replenished in time and provided to mice free of charge. Mice were usually anesthetized intraperitoneally with 1% sodium pentobarbital (40–50 mg / kg), and the entire corneal epithelium of the mice was scraped under a microscope after anesthesia. The corneal wound was then infected with a suspension of P. aeruginosa (1.5 × 108 CFU / mL, 10 μL). Five minutes after infection, mice were treated with XOF(I, Br)-TPPA, XOF(I, Br)-TPPE, or saline. Mice were treated daily throughout the treatment period, and microscopic images of the ocular surface of each group were recorded, and the area of ​​epithelial defect was evaluated by fluorescein staining.

[0072] 1) H&E staining

[0073] After 7 days of treatment, the eyes were removed, fixed with 4% PFA, embedded in paraffin and cut into sections. After the paraffin sections were dewaxed into water, they were stained with hematoxylin, differentiated with differentiation solution and re-blued with re-blue solution. Then, they were dehydrated with 85% and 95% ethanol in sequence. After dehydration, they were stained with eosin. Finally, the sections were dehydrated with anhydrous ethanol and xylene in sequence and sealed with neutral paraffin.

[0074] 2) Immunofluorescence

[0075] Eyes were removed after 7 and 1 day of treatment and fixed with 4% PFA. Eyes were embedded in optimal cutting temperature compound, frozen and cryoprotected to a thickness of 10 μm. Eye sections were permeabilized with 0.1% Triton for 15 min, then blocked with 5% BSA for 30 min, and then incubated overnight 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). After washing three times with PBS, sections were incubated with secondary antibodies conjugated with Alexa Fluor 488 or 594. Dapi staining was performed, sections were blocked, and observed with a fluorescence microscope.

[0076] 3) TUNEL

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

[0078] 4) In vivo toxicology analysis

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

[0080] 2. Data Analysis

[0081] All experiments were performed at least three times. GraphPad Prism 7.0 (GraphPad Software., Inc.) was used to perform statistical analysis and plot the data. Results are expressed as mean ± standard deviation (mean ± SEM) of continuous variables. The mean values ​​of the two groups were compared using Student's t test. Statistics were considered significant at P < 0.05.

[0082] When infected with Pseudomonas aeruginosa, small blisters are first formed. Then, when corneal epithelial cells fall off from the stromal layer as blisters accumulate, corneal ulcers occur. Due to the lack of an epithelial layer, the water-soluble dye fluorescein is used to mark epithelial defects, also known as corneal ulcers. Observed with cobalt blue light, the ulcerated area is detected as green. Using XOF(Br)-TPPA as a model, the dose of XOFs(I / Br) for treating Pseudomonas aeruginosa keratitis model mice was optimized. Three concentration gradients, i.e., 10 mg / mL, 20 mg / mL and 50 mg / mL of the obtained XOF(Br)-TPPA were administered at a dose of 20 μL / day 5 minutes after infection. It is noteworthy that the therapeutic effect of 20 mg / mL XOF(Br)-TPPA was observed after 7 days of treatment (clinical score of 6.3±1.5), which was statistically significant compared with 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 efficacy of both groups was better than that of the untreated group (clinical score of 13.0±1.4).

[0083] Among the treatment groups, mice treated with 20 mg / mL of XOF(Br)-TPPA had the mildest symptoms, with only mild corneal opacity, pupil penetration, and significantly less corneal ulcer area and neovascularization than the remaining groups. In the control group, the ulcer spread to almost the entire corneal surface within 24 hours. The corneal damage slowly recovered within about 1 week ( Figure 6 The clinical evaluation scores of 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 for bacterial keratitis, 20 mg / mL of a topical suspension of each sample was applied daily to a mouse model exhibiting Pseudomonas aeruginosa-induced keratitis. Analysis showed that mice treated with XOF(Br)-TPPA (clinical score of 5.0±0.0 on day 3) showed a significantly favorable response and were superior to 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 deterioration in corneal condition. This deterioration was characterized by increased turbidity, hypopyon, significant neovascularization, and in some cases corneal perforation.

[0085] The treatment group showed a significant reduction in corneal haze and turbidity, as well as a significant reduction in neovascularization. In addition, as highlighted, corneal ulcer healing was significantly improved, with faster recovery ( Figure 8 c and Fig. 9 These findings strongly support the potential of XOF(Br)-TPPA as a promising treatment option for bacterial keratitis. However, further studies are needed to optimize the treatment, elucidate the mechanism of action, and validate its efficacy in human clinical trials.

[0086] To explore whether the treatment effect was improved after the addition of IR820, we initially sought to determine the optimal duration of light exposure. After the administration of the turbid IR820@XOF(Br)-TPPA solution, mice were illuminated with 808 nm LED light for 5, 10, and 20 minutes ( Figure 8 a). Compared with the untreated group (clinical score of 9.0±0.8 on day 3), which suffered severe corneal opacity, neovascularization and even corneal perforation after infection, mice treated with IR820@XOF(Br)-TPPA showed significantly fewer symptoms, and those irradiated with LED light for 5 minutes (clinical score of 2.8±0.9 on day 3) (p<0.0001) had the mildest symptoms ( Fig.10 and Fig.11 By the third day of treatment, the cornea was completely clear and almost healed ( Figure 8 b). This was confirmed by clinical scoring of representative images of mouse corneas ( Figure 8 c and Fig.12 ).

[0087] The therapeutic efficacy of IR820@XOF(Br)-TPPA was further investigated by comparing the therapeutic efficacy of IR820@XOF(Br)-TPPA with that of 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 min), or IR820@XOF(Br)-TPPA (exposed to 808 nm LED light for 5 min). Figure 8 a). Although symptom improvement was observed in mice treated with XOF(Br)-TPPA (with a clinical score of 4.3±1.5 on day 6), ocular surface opacity persisted 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) almost recovered transparency on day 2 and were nearly completely healed on day 6, with no visible corneal opacity, 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 efficacy of bacterial keratitis and has great potential in the treatment of bacterial keratitis.

[0088] After 7 days of treatment, corneal sections were then stained with H&E and CD31 ( Fig.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 showed more regular structures and fewer inflammatory cells. In addition, the thickness of the corneas of mice that received 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 ( Fig.14 ). This further indicates that IR820@XOF(Br)-TPPA can significantly inhibit the formation of corneal neovascularization in keratitis.

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

[0090] To further investigate the therapeutic mechanism, corneal tissues were collected one day after IR820@XOF(Br)-TPPA treatment. TUNEL staining showed that corneal epithelial cell apoptosis was significantly reduced. In addition, the decreased expression of inflammatory factors GFAP and Iba-I indicated that IR820@XOF(Br)-TPPA could reduce corneal epithelial cell apoptosis by alleviating inflammatory response ( Fig.15 a. Fig.15b). In addition to inflammation, corneal neovascularization is another factor contributing to visual impairment in bacterial keratitis. After 7 days of treatment, a significant decrease in CD31 expression was observed in the cornea, indicating a reduction 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 ( Fig.15 a. Fig.15 b). In addition, decreased VEGF expression was noted, indicating that IR820@XOF(Br)-TPPA treatment reduced HIF-1 expression, prevented neovascularization, and ultimately improved corneal transparency. IR820@XOF(Br)-TPPA treatment significantly alleviated the inflammatory response in the post-infection cornea and reduced the expression of neovascularization-related factors, leading to reduced corneal epithelial cell apoptosis and neovascularization.

[0092] The ultimate design of the drug is to develop an effective medical product for clinical transformation, for which the biosafety of the drug is particularly important. In order to evaluate the biocompatibility of IR820@XOF(Br)-TPPA to determine its potential for clinical transformation, 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 the treatment period were recorded. After 7 consecutive days of treatment, the mouse cornea was transparent, without conjunctival redness, edema, or increased secretions, and there was no obvious coloration of fluorescein sodium staining ( Fig.16 ). H&E staining of the corneas of treated mice 7 days later also showed that the structure of the corneas of the mice was normal, without obvious inflammatory cell infiltration. Tunel staining of the corneas of treated mice also showed no significant cell apoptosis ( Fig.17 and Fig.18 ). This indicates that IR820@XOF(Br)-TPPA has no significant cytotoxic effect on mouse cornea. During the entire administration period, the liver and kidney functions (ALT, AST, BUN and CREA) of the four groups of mice remained normal ( Fig.19 ). It also proved that there was no significant difference in body weight ( Fig. 20 H&E staining of the main organs of mice also showed no obvious lesion areas ( Fig.21 ). All of these indicate the high biocompatibility of IR820@XOF(Br)-TPPA.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A pharmaceutical composition based on two-dimensional halogen-bonded organic frameworks (XOFs), characterized in that: The method is obtained by loading new indocyanine green on a two-dimensional halogen bond organic framework XOFs; the two-dimensional halogen bond organic framework XOFs is selected from at least one of the compounds of the structural formulas shown in Formula I and Formula II: Wherein, the element X in Formula I and Formula II is a halogen, each independently selected from Br or I.

2. The pharmaceutical composition according to claim 1, characterized in that The drug composition is obtained by adsorbing new indocyanine green and two-dimensional halogen bond organic framework XOFs in a solution and removing the solvent.

3. The pharmaceutical composition according to claim 2, characterized in that The feed mass ratio of the two-dimensional halogen bond organic framework XOFs to the new indocyanine green is (1-10):

1.

4. The pharmaceutical composition according to claim 2, characterized in that The two-dimensional halogen bond organic framework XOFs is firstly ultrasonically dispersed in a solution and then allowed to stand for adsorption for 0.5-2 hours.

5. The pharmaceutical composition according to claim 1, characterized in that The mass ratio of the two-dimensional halogen bond organic framework XOFs to the loaded new indocyanine green is 6:

1.

6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in antibacterial treatment, wherein the use is for non-therapeutic purposes.

7. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a drug for treating bacterial keratitis.

8. The use according to claim 7, characterized in that The bacterial keratitis is keratitis caused by Pseudomonas aeruginosa.

9. The use according to claim 7, characterized in that The drug for treating bacterial keratitis also includes a pharmaceutically acceptable adjuvant.

10. The use according to claim 7, characterized in that The bacterial keratitis treatment drug is eye drops, eye cream or eye gel.

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