Application of black phosphorus nanosheet hydrogel in preparation of medicine for treating dry eye syndrome

Abstract

The invention relates to the technical field of biology, in particular to application of black phosphorus nanosheet hydrogel to preparation of medicine for treating dry eye syndrome. The black phosphorus nanosheet hydrogel can promote tear film healing, treat dry corneal injury and remove active oxygen and free radicals, has an anti-inflammatory effect, inhibits squamous metaplasia, inhibits expression of IL-1beta, K10 and MMP-9 and the like, and provides a new direction for treatment of the dry eye syndrome.

Description

TECHNICAL FIELD

[0001] The present specification relates to the field of biotechnology, in particular to the use of black phosphorus nanosheet hydrogel in the preparation of a drug for treating dry eye syndrome. BACKGROUND

[0002] Dry eye syndrome (DED) is a common ocular surface disease with complex pathogenesis, mainly driven by multiple factors such as inflammatory response and oxidative stress. During the disease progression, ocular surface tissues produce excessive reactive oxygen species (ROS) and inflammatory mediators, which exacerbate the damage to ocular surface cells, disrupt the stability of the tear film, and form a vicious cycle, thereby significantly promoting the progression of ocular surface disease, leading to a series of symptoms such as ocular dryness, pain, blurred vision, etc.

[0003] Currently, the treatment methods for dry eye syndrome in clinical practice, such as artificial tears and anti-inflammatory drugs, can only temporarily relieve symptoms and are difficult to fundamentally interrupt the vicious cycle of oxidative stress and inflammation. These traditional therapies have limitations such as single mode of action, short ocular residence time, and the need for frequent administration, and their therapeutic effect and patient compliance are not ideal.

[0004] Therefore, developing a new treatment strategy that can efficiently eliminate multiple reactive oxygen species, fundamentally block the oxidative stress-inflammation pathway, and achieve long-term residence on the ocular surface is of great significance for effectively controlling the progression of dry eye syndrome, relieving symptoms, and promoting ocular surface tissue repair. This is also a technical problem that needs to be broken through in the current dry eye syndrome treatment field. SUMMARY

[0005] To solve the above technical problems, the present application provides the use of black phosphorus nanosheet hydrogel in the preparation of a product with one or more of the following functions: 1) treating dry eye syndrome; 2) promoting tear film healing; 3) treating dry corneal injury.

[0006] The beneficial effects brought by the present application include but are not limited to: the present application discovers a new drug for treating dry eye syndrome, black phosphorus nanosheet hydrogel, which can promote tear film healing, treat dry corneal injury, eliminate reactive oxygen and free radicals, has anti-inflammatory effect, inhibits squamous metaplasia, inhibits the expression of IL-1β, K10 and MMP-9, etc., providing a new direction for the treatment of dry eye syndrome. BRIEF DESCRIPTION OF DRAWINGS

[0007] The present application will be further illustrated in the form of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein:

[0008] Figure 1 Synthesis and Identification of BPNS@Gel Composites. (A) Schematic diagram of the BPNS@Gel preparation procedure. (B, C) Typical images of BPNS produced from transmission electron microscopy (TEM) and scanning electron microscopy (SEM). (D) Hydrodynamic size distribution of BPNS determined by dynamic light scattering (DLS). (E) Energy dispersive X-ray (EDX) spectra of BPNS, showing its elemental composition after culture following body fluid simulation. (F) Atomic force microscopy (AFM) images of BPNS captured with matched height profile phase. Rheological measurements of BPNS@Gel are (G) stress scan and (H) time scan. (I) Strain amplitude dependence of stored (G) and lost (G) moduli. (J) Visual representation of the sol-gel transition. (K) Ability of BPNS to quench superoxide (O). (LM) Quantitative photosimulation activity and (OP) ability of BPNS to scavenge free radicals at different concentrations. Data are shown as mean ± SD (n = 3).

[0009] Figure 2 Cellular uptake, apoptosis, and in vitro cell compatibility of BPNS. (AC) Viability of HCECs, (B) hRMECs, and (C) ARPE-19 cells after 24 hours of exposure to increased BPNS concentrations (n=6). (D) Fluorescence images (dead, red) and calcein-AM (live, green) of HCECs after BPNS incubation (n=3). (E) Flow cytometry study of V-FITC / PI staining in HCECs after BPNS treatment (n=3). (F) Representative fluorescence images of CM-H unk DCFDA (green) under hyperosmolar pressure, measuring cellular ROS levels in HCECs (n=3). (G) Viability of HCECs under hyperosmolar pressure after BPNS incubation. (H) Detection of apoptosis in HCECs treated with BPNS in hyperosmolar medium (n=3). (I) JC-1 labeling and flow cytometry were used to quantify mitochondrial membrane potential (red / green fluorescence ratio indicates m-health) (n=3). Secretion levels of IL-1β (K), IL-6 (L), and IL-10 (M) in cell culture were obtained from all 6 experimental groups. P<0.05, **P<0.01, **P<0.001, mean values ​​are ±SD.

[0010] Figure 3Anti-inflammatory properties of BPNS@Gel and macrophage remodeling. (AD) Secretion levels of TNF-α, IL-1β, IL-6, and IL-10 in HCEC cells, from each of the 6 experimental groups. (E, F) Quantitative fluorescence intensity analysis (n=3) and (E) typical ethyl immunofluorescence staining are indicators of M1 polarization. (G, H) Quantitative analysis (n=3) and (G) typical G-1 immunofluorescence staining are indicators of M2 polarization. P<0.05, **P<0.01, **P<0.001 are data mean ± SD.

[0011] Figure 4 Macrophage-HCEC interaction mediates the protective effect of BPNS@Gel. (A) Volcano plot of different expressed genes (DEGs) in HCECs treated with lps-stimulated macrophages (cmlps) and bpns@ger-modified macrophages (cmp-bpns@gel). (B) Top 20 DEG-condensed KEGG pathways. (C) Heatmap showing DEG expression patterns in several treatment groups. (D) IL-6 has been identified as a major center of the protein-protein interaction (PPI) functional gene network. (E) A molecular docking model predicting IGJ and IL-6 interaction. (F, G) Relative expression ratios of IGJ and IL-6 in different groups. (H) Quantitative and representative immunofluorescence staining of HCECs with p-JAK and p-STAT. Data are ±SD (n=6); *P<0.05, **P<0.01, **P<0.001.

[0012] Figure 5 The therapeutic effects of BPNS@Gel on the ocular surface of mice. (A) Typical photographs of corneal fluorescence in different treatment groups. (B) Tear volume and (C) clinical evaluation in mice receiving various treatments. (D) Statistical analysis of corneal epithelial thickness. (E) Typical anterior segment optical coherence tomography (AS-OCT) at 3 days post-treatment (n=5). (F) Quantitative evaluation of conjunctival cup cell density stained in PAS. Representative immunofluorescence staining and quantitative data for (G) cytokeratin K10, (H) TUNEL (apoptosis), (I) ROS, (J) MMP-9, (K) IL-6, and (L) IGJ. Data are ±SD (n=6); *P<0.05, **P<0.01, **P<0.001.

[0013] Figure 6The therapeutic effects of BPNS@Gel on the ocular surface of mice. (A) Typical photographs of corneal fluorescence in different treatment groups. (B) Tear volume and (C) clinical evaluation in mice receiving various treatments. (D) Statistical analysis of corneal epithelial thickness. (E) Typical anterior segment optical coherence tomography (AS-OCT) at 3 days post-treatment (n=5). (F) Quantitative evaluation of conjunctival cup cell density stained in PAS. Representative immunofluorescence staining and quantitative data for (G) cytokeratin K10, (H) TUNEL (apoptosis), (I) ROS, (J) MMP-9, (K) IL-6, and (L) IGJ. Data are ±SD (n=6); *P<0.05, **P<0.01, **P<0.001.

[0014] Figure 7 Long-term in vivo biosafety evaluation of BPNS. (A, B) IGJ and (C) MMP-9 expression in corneal tissue after IL-6 silencing and subsequent BPNS treatment. (D) Partial staining of cornea and retina 14 days after local administration. (E) Statistical analysis of corneal and (F) retinal thickness. (G, H) Quantification of (G) a-wave and (H) b-wave amplitudes in electronic language (ERG) recordings. (IL) Complete blood count results: (I) WBC, (J) erythrocytes, (K) HGB, (L) PLT. (M) Representative H&E staining of major organs (heart, liver, spleen, lung, kidney) from BPNS-treated and controlled animals (n=3). Data are ±SD (n=6); *P<0.05, **P<0.01, **P<0.001. Detailed Implementation

[0015] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0016] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0017] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0018] This application provides the use of black phosphorus nanosheet hydrogels in the preparation of products having one or more of the following functions:

[0019] 1) Treatment of dry eye syndrome;

[0020] 2) Promotes tear film healing;

[0021] 3) Treatment of dry corneal damage.

[0022] In some embodiments, the black phosphorus nanosheet hydrogel is capable of scavenging reactive oxygen species.

[0023] In some embodiments, the black phosphorus nanosheet hydrogel is capable of scavenging free radicals.

[0024] In some embodiments, the black phosphorus nanosheet hydrogel has anti-inflammatory properties.

[0025] In some embodiments, the black phosphorus nanosheet hydrogel can protect cells from oxidative stress-induced cell death.

[0026] In some embodiments, the black phosphorus nanosheet hydrogel can promote the transformation of macrophages into M2 macrophage phenotypes.

[0027] In some embodiments, the black phosphorus nanosheet hydrogel has an anti-inflammatory effect on LPS-stimulated HCECs.

[0028] In some embodiments, the black phosphorus nanosheet hydrogel can reverse the inflammatory response and aging process of HCECs.

[0029] In some embodiments, the black phosphorus nanosheet hydrogel can inhibit the IL-6 / IGJ pathway by modulating crosstalk between macrophages and HCECs, thereby reversing the inflammatory response and aging process of HCECs.

[0030] In some embodiments, the black phosphorus nanosheet hydrogel can promote the increase of goblet cells.

[0031] In some embodiments, the black phosphorus nanosheet hydrogel has antioxidant, anti-inflammatory, anti-apoptotic and anti-keratinization effects on corneal tissue.

[0032] In some embodiments, the black phosphorus nanosheet hydrogel can reduce the number of TUNEL-positive cells.

[0033] In some embodiments, the black phosphorus nanosheet hydrogel can reduce IL-6 expression levels.

[0034] In some embodiments, the black phosphorus nanosheet hydrogel can inhibit squamation.

[0035] In some embodiments, the black phosphorus nanosheet hydrogel can enhance IGJ expression levels.

[0036] In some embodiments, the black phosphorus nanosheet hydrogel can inhibit the expression of IL-1β, K10, and MMP-9.

[0037] In some embodiments, the black phosphorus nanosheet hydrogel can eradicate MMP-9 in individuals with dry eye syndrome.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0039] The method used in the embodiments of this application is as follows:

[0040] Preparation of black phosphorus nanosheets (BPNSs):

[0041] This step aims to prepare the key nanomaterials required for the gel.

[0042] Prepare ultrapure water: Boil the water, cool it, and then pass nitrogen gas through it for deoxygenation.

[0043] Dispersing black phosphorus powder: Disperse 100 mg of black phosphorus crystal powder into 100 mL of the above-treated ultrapure water.

[0044] Ultrasonic treatment: The mixture was ultrasonically treated at low temperature for 20 hours using a cell disruption ultrasonic homogenizer to exfoliate the nanosheets. Differential centrifugation purification:

[0045] First, centrifuge at 2000 rpm for 5 minutes to remove large, unpeeled black phosphorus crystals (precipitate).

[0046] Then, the supernatant was centrifuged at 12,000 rpm for 15 minutes to collect the exfoliated black phosphorus nanosheets (BPNSs) (precipitate).

[0047] Preparation of F127 hydrogel:

[0048] This step prepared pure F127 hydrogel and composite hydrogel loaded with BPNSs.

[0049] Preparation of pure F127 hydrogel:

[0050] Under low-temperature stirring, different masses (16%, 18%, 20% w / v) of F127 powder were gradually added to deionized water. Stirring continued until a transparent solution was formed, thus obtaining F127 hydrogels of different concentrations.

[0051] Preparation of BPNSs-F127 composite hydrogel:

[0052] F127 powder (concentration of 18% w / v) was gradually added to the BPNSs aqueous dispersion prepared in the first part under continuous stirring at low temperature.

[0053] Continue stirring until a transparent, uniform, light yellow aqueous dispersion is formed, which yields the final BPNSs hydrogel.

[0054] Evaluation of the free radical scavenging ability of black phosphorus nanosheet gel (BPNSs@Gel):

[0055] To identify the free radical scavenging ability of black phosphorus nanosheets (BPNSs), this study employed multiple methods. Common antioxidant activity tests, including DPPH, ABTS, PTIO, and hydroxyl radical (·OH) scavenging experiments, were performed using UV-Vis spectroscopy. The reaction between BPNSs and hydrogen peroxide (H₂O₂) was detected using Raman spectroscopy. Furthermore, electron spin resonance (ESR) spectroscopy was used to identify its activity against ·OH and superoxide anion (O₂··OH). - The scavenging capacity of BPNSs and singlet oxygen (1O2) was measured. DPPH experiment: A 0.04 mg / mL DPPH ethanol solution was prepared beforehand. 2 mL of BPNSs solutions of different concentrations were mixed with 2 mL of DPPH solution. After reacting for half an hour, the absorbance was measured at 519 nm using a UV-Vis spectrometer.

[0056] ABTS experiment: 0.2 mL of potassium persulfate solution (2.6 mmol / L) was mixed with 0.2 mL of ABTS solution (7.4 mmol / L), and the mixture was reacted in the dark for 24 hours to generate ABTS. + Free radicals. The solution was then diluted 50-fold with PBS buffer for later use. Take 2.4 mL of the diluted ABTS. + The solution was mixed with 0.6 mL of BPNSs solutions of different concentrations, and after reacting for 10 minutes, its absorbance was measured at a wavelength of 734 nm.

[0057] PTIO assay: A PTIO solution with a concentration of approximately 0.05 mg / mL was prepared using PBS buffer. 2 mL of the PTIO solution was mixed with 2 mL of BPNSs solutions of different concentrations, and after standing at room temperature for 2 hours, the absorbance of the PTIO was measured at 557 nm. ·OH scavenging assay: The procedure was performed according to the kit instructions. The ·OH content was quantified by measuring the absorbance at 550 nm. Before UV-Vis spectroscopy detection, all samples were centrifuged at 10,000 rpm for 10 minutes to remove residual BPNSs.

[0058] Each measurement was repeated three times for quantitative analysis.

[0059] The formula for calculating the free radical scavenging rate (inhibition rate) is: Inhibition rate (%) = [(A0-A) / A0] × 100%, where A0 is the absorbance without BPNSs and A is the absorbance with BPNSs.

[0060] To evaluate the scavenging ability of BPNSs for H2O2, 500 μg / mL of BPNSs was mixed with 10% H2O2 (2.9 M) at room temperature. The mixture was then rapidly added dropwise onto a clean glass slide, and the reaction process was monitored using Raman spectroscopy over 6 minutes.

[0061] ESR spectroscopy was used to identify the effects of BPNSs on ·OH and O2· - And the scavenging effect of 1O2.

[0062] ·OH: Produced through the Fenton reaction of FeSO4 and H2O2.

[0063] 1 O2: Produced through the photocatalytic reaction of titanium dioxide (TiO2).

[0064] O2· - Produced through the xanthine / xanthine oxidase reaction.

[0065] 5,5-Dimethyl-1-pyrrolidone-N-oxide (DMPO) was chosen as the ·OH and O2· - For the spin trapping agent, 2,2,6,6-tetramethylpiperidine (TEMP) was selected as the spin trapping agent for 1O2.

[0066] Cell viability assay (CCK-8 assay):

[0067] Cell viability was determined using the Cell Counting Kit-8 (CCK-8). Mouse chondrocytes (1×10⁻⁶) were used. 4(Each well) was seeded into a 96-well plate and incubated for 24 and 48 hours, respectively. After the experimental treatment, 10 μL of CCK-8 solution was added to each well, and the plates were incubated at room temperature for another 2 hours before detection.

[0068] Cytotoxicity assessment:

[0069] Human retinal epithelial cells (ARPE-19), human retinal microvascular endothelial cells (hRMECs) and human corneal epithelial cells (HCECs) were all purchased from the BeNa cell bank, and the cell culture method was the same as our previous study

[37] .

[0070] Brief description of the procedure: To assess in vitro cytotoxicity, firstly, 1×10 4 HCECs were seeded in 96-well plates and cultured at 37°C for 24 hours to promote cell adhesion. The medium was then replaced with fresh medium containing different concentrations of BPNSs. Each concentration group was divided into at least six replicates for assay. After another 24 hours of culture, cell viability was assessed using the CCK-8 assay kit (APExBIO). The same experimental procedure was used for ARPE-19 cells and hRMECs as for HCECs.

[0071] Calcein-AM / PI double staining method:

[0072] To investigate the effects of BPNSs on HCECs in more detail, cell viability was assessed using the Calcein-AM / PI double staining method. Following previous studies, cells were incubated for 24 hours with BPNSs at concentrations ranging from 80 to 640 μg / mL, after which Calcein-AM / PI staining solution was added to the medium. Incubation was continued for another 15 minutes, and the distribution of live cells (green) and dead cells (red) was observed and photographed using a DMI8 inverted fluorescence microscope (n=3 per group).

[0073] Apoptosis assessment:

[0074] Following the previous method, HCECs were seeded in 96-well plates and cultured at 37°C for 24 hours to promote adhesion. The medium was then replaced with fresh medium containing different concentrations of BPNSs. After another 24 hours of culture, cells were collected by trypsin digestion, washed with PBS, and resuspended in 300 μL of 1× binding buffer. 4 μL of Annexin V-FITC and 1.5 μL of propidium iodide (PI) were added sequentially to the sample, and after reacting in the dark for 15 minutes, the cells were analyzed using an Accuri C6 flow cytometer (n=6 per group). Intracellular reactive oxygen species (ROS) scavenging activity:

[0075] To assess the ability of BPNSs to scavenge intracellular ROS, HCECs were incubated in a hypertonic solution (500 mosmol / L; containing 90 mM NaCl) to induce oxidative stress. The ROS probe CM-H2DCFDA was used for detection.

[0076] Steps: Take 1×10 4 HCECs were seeded in 96-well plates and cultured for 24 hours to allow them to adhere. After discarding the supernatant, 100 μL of serum-free medium containing 90 mM NaCl was added to each well (n=6 per group). This medium contained different concentrations (80 to 320 μg / mL) of BPNSs or the conventional antioxidant NAC (10 mmol / L). Cells were co-incubated for another 18 hours. The hyperosmolar group (HS) was given 1 mL of serum-free medium containing 90 mM NaCl, while the control group received no treatment. After co-incubation, the supernatant was discarded, and 500 μL of BPNSs-H2DCFDA working solution (5 μg / mL) was added to each well. After incubation for 20 minutes to allow the probes to internalize, the cells were carefully washed 2-3 times with PBS and observed using an inverted fluorescence phase-contrast microscope.

[0077] In vitro evaluation of its efficacy against oxidative stress damage:

[0078] The protective effect of BPNSs on cell viability and their ability to resist oxidative stress-induced damage were investigated using the CCK-8 assay and the Annexin V-FITC / PI apoptosis detection kit. Oxidative stress and apoptosis were induced by incubating HCECs in a hypertonic solution (500 mosmol / L; containing 90 mM NaCl).

[0079] Steps: Take 1×10 4 HCECs were seeded per well in 96-well plates and cultured for 24 hours until adherence. After discarding the supernatant, 100 μL of serum-free medium containing 90 mM NaCl was added to each well (n=6 per group), which contained different concentrations of BPNSs or NAC (10 mmol / L). After co-incubation for another 18 hours, cell viability was assessed using a CCK-8 assay kit, and apoptosis was analyzed using an Annexin V-FITC / PI apoptosis assay kit. The hyperosmolar group (HS) received 1 mL of serum-free medium containing 90 mM NaCl (n=3 per group), while the control group received no treatment.

[0080] 8-Hydroxyguanosine (8-OHdG) immunofluorescence staining:

[0081] First, HCECs were treated with different solutions (normal culture medium, hypertonic solution, and hypertonic solution containing NAC or BPNSs). Then, cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes, followed by washing three times with PBS. Cells were permeabilized with 0.5% Triton X-100 for 20 minutes, washed with PBS, and then blocked with goat serum at room temperature for 30 minutes. Anti-8-OHdG primary antibody (1:200, Abcam) was added, and the cells were incubated overnight at 4°C. After washing three times with PBS, anthocyanin 3 (Cy3)-labeled donkey anti-mouse IgG (H+L) secondary antibody was added, and the cells were incubated at 37°C for 1 hour. Finally, cell nuclei were counterstained with DAPI, and images were observed and acquired under a fluorescence microscope (n=3 per group).

[0082] Cytokine assessment:

[0083] Cytokine levels were detected using a commercially available kit via enzyme-linked immunosorbent assay (ELISA). The simplified procedure is as follows: HCECs were stimulated with LPS for 24 hours, and the cell culture supernatant was collected. A standard curve was plotted according to the manufacturer's instructions, and the absorbance was measured at 450 nm using a microplate reader. Cytokine concentrations were calculated based on the standard curve.

[0084] Macrophage phenotype analysis:

[0085] The expression of CD86 (M1 marker) and CD206 (M2 marker) in HCEC cells after treatment was detected by flow cytometry.

[0086] Procedure: Cells were treated with LPS and IL-4 for 24 hours to induce polarization, and single-cell suspensions were prepared. Subsequently, cells were incubated for 30 minutes at room temperature in the dark with APC-labeled anti-F4 / 80 antibody, FITC-labeled anti-CD86 antibody, and PE-labeled anti-CD206 antibody. Finally, flow cytometry analysis was performed.

[0087] Macrophage phenotypic characteristics:

[0088] Macrophage polarization was studied by analyzing the expression of CD86 (M1 marker) and CD206 (M2 marker) in HCEC cells using flow cytometry.

[0089] Brief procedure: After cell polarization by LPS and IL-4 treatment for 24 hours, single-cell suspensions were prepared. Cells were stained with APC-labeled anti-F4 / 80 (1:200), FITC-labeled anti-CD86 (1:100), and PE-labeled anti-CD206 (1:100) antibodies at 25°C in the dark for 30 minutes. Data were analyzed using a BD FACSCelesta flow cytometer and processed using FlowJo v10.9 software.

[0090] Real-time quantitative PCR (qRT-PCR):

[0091] Total RNA was extracted from treated HCECs using RNA-easy Isolation Reagent (Novizan, Nanjing, China). Reverse transcription was then performed using HiScript IV RT Super Mix (Novizan) according to the manufacturer's instructions. Quantitative PCR (qPCR) was performed using AceQqPCR SYBR Green Master Mix (Novizan) on a Bio-Rad CFX Connect RT system (Bio-Rad, Hercules, USA) to assess mRNA levels, with all procedures strictly following the manufacturer's instructions. Data were analyzed using the 2-ΔΔCt method and normalized using the ACTB gene as an internal control.

[0092] JC-1 method for detecting mitochondrial membrane potential (MMP):

[0093] The mitochondrial membrane potential of HCECs was detected using the JC-1 method to assess the material's ability to prevent mitochondrial oxidative stress damage induced by hyperosmolar stimulation.

[0094] Steps: Take 1×10 5 HCECs were seeded in 24-well plates and cultured overnight at 37°C. The supernatant was discarded and replaced with fresh medium containing either the traditional antioxidant NAC (10 mmol / L) or different concentrations of BPNSs (80–320 μg / mL). Cells were co-incubated for 4 hours in medium containing BPNSs / NAC and hypertonic medium (130 mM, 570 mosmol / L). After treatment, cells were collected, stained with JC-1 dye at 37°C in the dark for 20 minutes, and then washed twice with buffer to remove excess dye. Cells were resuspended, and changes in mitochondrial membrane potential were detected using flow cytometry (Agilent) (n=3 per group). In vitro antioxidant stress characteristics were assessed.

[0095] To evaluate in vitro the antioxidant capacity of BPNSs and their role in protecting HCECs from ROS damage.

[0096] Model establishment: HCECs were seeded in culture plates and treated with 400 μM H2O2 or 10 ng / mL IL-1β for 24 hours to establish an oxidative stress model. The experimental groups were simultaneously treated with BPNSs (10 or 20 μg / mL) along with IL-1β or H2O2.

[0097] Cell viability: Cells were treated with 800 μM H2O2 and their viability was assessed by the CCK-8 assay and Calcein-AM / PI live / dead cell double staining assay.

[0098] ROS detection: DCFH-DA was used as the ROS fluorescent probe to detect ROS in cells treated with different methods after incubation for 30 minutes. After incubation and washing with PBS, ROS fluorescence and live / dead staining signals were observed and captured using a fluorescence microscope, and quantitative analysis was performed using ImageJ software.

[0099] SOD activity and MDA levels: SOD activity in chondrocytes subjected to H2O2-induced oxidative stress was assessed using a complete WST-8 SOD assay kit. Lipid peroxidation levels in HCECs under different treatments were investigated using a lipid peroxidation MDA assay kit.

[0100] RNA-seq data analysis:

[0101] We conducted in-depth downstream analysis of the batch RNA-seq data.

[0102] Principal Component Analysis (PCA): PCA analysis was performed using data from 9 samples (3 replicates) to visualize overall expression differences between groups and within groups. Par-scaling was used for data preprocessing to improve the reliability and intuitiveness of the results. Automated model fitting analysis was used to determine the optimal number of principal components, ensuring the most reliable mathematical model and generating PCA score plots. The analysis included expression levels (FPKM values) for all genes.

[0103] Differentially expressed genes (DEGs) analysis: Using the Limma R package, significantly differentially expressed genes were systematically screened with an adjusted P-value < 0.05 and |log2FC| > 1.5 as thresholds.

[0104] Functional enrichment analysis: The ClusterProfiler R package was used to perform GO function and KEGG pathway enrichment analysis on DEGs to further investigate their biological functions. Particular attention was paid to the top 10 genes with the most significant upregulation and downregulation (adjusted P-value < 0.05) to identify potentially affected biological processes and molecular functions.

[0105] Expression pattern clustering and PPI analysis: Gene expression patterns were clustered using the Mfuzz method in the ClusterGVis R package to gain deeper insights into the complexity of gene regulatory networks and potential associations among DEGs. Finally, protein-protein interaction (PPI) analysis was performed using the String database, providing new contextual information for the RNA-seq results.

[0106] Molecular simulation and docking:

[0107] As previously reported, molecular docking studies of IGJ and IL-6 were conducted in our work. The crystal structures of IGJ and IL-6 were obtained from the ChEMBL database. The docking studies were completed using the online tool CB-Dock, and the docking conformations were viewed using its visualization function.

[0108] Mouse dry eye disease (DED) model:

[0109] Female C57BL / 6 mice (6 - 8 weeks old, 18 - 20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (License number: SCXK(Beijing)2021 - 0006). All animal experiments were conducted in accordance with the "Guide for the Care and Use of Laboratory Animals" of Shanghai Tongren Hospital and approved by the Animal Ethics Committee of the hospital.

[0110] Housing and grouping: Male C57BL / 6 mice at 6 - 8 weeks old were adaptively housed in a ventilated environment for one week. The inclusion criteria were healthy mice without old white spots or ulcers in the eyes.

[0111] Model establishment: A DED model was established by instilling 5 μL of 0.2% benzalkonium chloride (BAC) solution into the conjunctival sacs of both eyes twice a day (at 10 am and 5 pm) for 14 days.

[0112] Index detection: The body weight, corneal fluorescein staining score, tear secretion volume, and tear film break-up time (TBUT) of the mice were measured on days 0, 3, 7, 10, and 14. Each measurement was repeated three times.

[0113] Slit-lamp imaging and local drug administration:

[0114] Local drug treatment was performed on the mouse dry eye model by instilling 5 μL of different eye drops (PBS, BPNSs solution, BPNSs@Gel) twice a day without anesthesia for 5 days.

[0115] Operation: The researchers carefully grasped the mice and tried their best to make them relax for stress-free and painless subcutaneous injection. All researchers were trained to operate correctly to avoid injuring the mice.

[0116] Observation: After instilling the eye drops and manually blinking to distribute the drug solution, the corneal conditions were observed and photographed using a slit-lamp microscope to show the hydrogel on the ocular surface.

[0117] Tear secretion test:

[0118] The tear secretion test was performed after the treatment. The brief steps were as follows: A phenol red thread was placed in the conjunctival sac of the lower eyelid. After keeping the mouse still for 15 seconds, the tear secretion rate was calculated by measuring the length (millimeters) of the wetting front of the tear fluid.

[0119] Corneal fluorescein staining:

[0120] Corneal fluorescein staining was performed by instilling 5% Oregon green dextran (OGD) solution into the ocular surface of mice for 1 minute, followed by several manual blinks. After euthanizing the mice, the cornea was flushed five times with 1 mL of physiological saline. The cornea was imaged using a fluorescence stereomicroscope (Leica, Wetzlar, Germany) under 470 nm excitation light. Using NIS Elements software, a 200,000-pixel circular region of the central cornea was selected for quantitative analysis of the fluorescence intensity of the OGD staining.

[0121] Histological staining (H&E and PAS):

[0122] Mouse eyeballs and periorbital tissues were isolated and collected, fixed in 4% paraformaldehyde for at least 24 hours, and then embedded in paraffin. Following the manufacturer's instructions, the paraffin-embedded samples were cut into 5 μm thick sagittal sections, mounted on glass slides, and stained using hematoxylin-eosin (H&E) and periodic acid-Scheffler (PAS) staining kits. All stained sections were observed and photographed using an optical microscope (Nikon Eclipse E400 with DS-Fi1). For PAS staining, goblet cells in the upper and lower palpebral conjunctiva were quantitatively counted using NIS Elements software.

[0123] Immunofluorescence staining:

[0124] After sampling, the tissue was embedded in OCT embedding agent and stored at -80℃.

[0125] Procedure: Frozen samples were prepared into 5 μm thick sagittal sections and mounted on glass slides. After fixation with acetone and blocking with 2% bovine serum albumin (BSA), the sections were incubated overnight at 4°C with anti-matrix metalloproteinase-3 (MMP-3), anti-matrix metalloproteinase-9 (MMP-9), and anti-CD4 antibodies. The next day, the sections were washed three times with PBS, and then incubated with Alexa Fluor 488-labeled donkey anti-goat IgG or donkey anti-rabbit IgG secondary antibodies at room temperature in the dark for 1 hour. Finally, the sections were observed and photographed using a fluorescence microscope (Leica DM2500), and mounted with DAPI.

[0126] Example 1

[0127] Characterization and synthesis of BPNSs@gel

[0128] The layered structure of BPNS was demonstrated using transmission electron microscopy and scanning electron microscopy images, such as... Figure 1 As shown. BPNS is obtained by modifying liquid glass with ultrasound. The purity and concentration of BPNS were measured by centrifugation. Figure 1B, C). According to dynamic light scattering experiments, the average size of BPNS is 422.65 ± 120.02 nm (B, C). Figure 1 D). The in-situ biomineralization of BPNS in simulated body fluid (SBF) was further investigated using elemental mapping and transmission electron microscopy (TEM) combined with energy dispersive spectroscopy (EDX). EDX spectral analysis showed that biodegradable BPNSs are mainly composed of P, C, O, and Ca. Figure 1 E). In aqueous solution, BPNS exhibits a zeta potential of -21.6 mV. We used high-resolution electron microscopy to demonstrate the high crystal structure of BPNS for further investigation. Similar to previous results, the atomic arrangement in the z-direction is 0.22 nm. The ultrathin nature of BPNS (approximately 3-5 nm thick) is clearly visible in typical atomic force microscopy (AFM) images. Figure 1 F). Subsequently, the viscoelastic characteristics of the BPNS@gel were investigated by rheological testing. At a constant frequency of 1.0 Hz, such as... Figure 1 As shown in Figure GI, when the strain is less than 10%, the storage modulus (G') is greater than the loss modulus (G"), indicating a gel state. However, when the strain exceeds 10%, G' decreases to below G", indicating a transition from a gel state to a sol state. Furthermore, the stress scan curve of the BPNS@gel at a frequency of 1.0 Hz was also recorded. Figure 1 J).

[0129] To elucidate the ability of BPNS to scavenge reactive oxygen species (ROS), we evaluated its superoxide dismutase (SOD)-like activity using the nitroblue tetrazolium (NBT) technique. NBT is a probe for superoxide anion O2, which has a peak absorbance at 560 nm and can be converted into blue methane. The SOD enzyme prevents methane formation by eliminating O2. Dynamic curve analysis at 560 nm showed that the SOD-like activity of BPNS was positively correlated with its concentration. Furthermore, the SOD-like activity of BPNS was assessed using a WST-based total SOD activity assay. Based on WST data, the enzyme activity of BPNS was estimated to be approximately 4858.25 U / mg. The overall antioxidant capacity of BPNS was assessed using a total antioxidant capacity assay kit to determine its antioxidant potential. In this process, molecule 2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was used as a chromogenic agent, which oxidizes to produce green ABTS+ in the presence of an oxidant. Since the presence of antioxidants inhibits the formation of ABTS+, antioxidant capacity can be determined by measuring ABTS+ uptake at 756 nm. The inhibition rate of ABTS+ increases with increasing BPNS concentration, exceeding 60% at 150 μg / mL, indicating that BPNS may scavenge reactive oxygen species (ROS). Figure 1K, L, M). The location of BPNS in human corneal epithelial cells was then assessed, such as... Figure 1 As shown in N.

[0130] The 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·) assay and the 2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid) radical ion (ABTS·+) assay are two widely used methods for assessing antioxidant activity, initially used to evaluate the reactive nitrogen scavenging ability of BPNS. In both the DPPH· and ABTS·+ assays, BPNS demonstrated strong free radical scavenging capabilities. For example, at concentrations as low as 7 μg / mL, BPNS achieved an efficiency of 88.62% in scavenging reactive oxygen species (ROS). In the ABTS assay, after reaction with 0.8 μg / mL BPNS, its scavenging efficiency was 89.8%, also demonstrating excellent scavenging ability. Figure 1 (O, P, Q). The results of this part of the study indicate that BPNS has antioxidant functions.

[0131] BNPS@Gel Biocompatibility Assessment

[0132] Calcein-AM / PI double staining further validated the biosafety of HCEC. When the BNPS concentration was less than 20 μg / ml, such as... Figure 2 As shown in AC, no dead cells were observed in the PI plot (detected by red fluorescence), indicating that BNPS has high cell safety. Furthermore, the survival rate, early cell death, late apoptosis, and necrosis rates of HCECs were detected after 24 hours of exposure to different doses of BPNS using the Annexin V-FITC / PI apoptosis detection kit. The excellent cell safety of BPNS further demonstrates that even at a dose of 20 μg / ml, the viable cell rate remained above 96.2%. Figure 2 As shown in DE.

[0133] like Figure 2In our study, we evaluated the biocompatibility of BNPS@Gel with different doses of BNPS in HCECs using the CCK-8 assay and the annexin V-FITC / PI apoptosis assay to further verify the tissue compatibility of the BPNSs with the hydrogel complex. According to the CCK-8 test, BNPS@Gel exhibited excellent biocompatibility, comparable to that of the BPNSs themselves. Even at a BPNS aqueous solution concentration of 20 μg / ml, cell viability remained stable at approximately 100%. Furthermore, the results of the annexin V-FITC / PI cell death assay further demonstrated the superior biocompatibility of the sample. After 24 hours of incubation with BNPS@Gel containing BPNS (20 μg / ml), the viable cell rate of HCECs remained above 91.8%, indicating that BNPS@Gel has good cell safety. Moreover, cell viability was not significantly affected by prolonged incubation.

[0134] Assessment of the mechanisms and capacity of BPNS cells to withstand oxidative stress damage

[0135] An oxidative stress model of HCECs was established using a hypertonic medium (500 mol / L) containing 90 mM sodium chloride (NaCl) to induce oxidative stress and lead to cell death, in order to further investigate the antioxidant properties of intracellular BPNS. N-acetyl-1-L-cysteine ​​(NAC) is a therapeutic antioxidant known for its ability to reduce ROS, and was used at a concentration of 10 mM.

[0136] This study used the CM-H2DCFDA fluorescence sensor to measure intracellular ROS levels. First, the effectiveness of intracellular BPNS in reducing oxidative stress damage was assessed by analyzing fluorescence images obtained from an inverted contrast fluorescence microscope. Figure 4 As shown in Figure F, the control group showed no green fluorescence signal, while the hypertonic medium-induced oxidative stress (HS) group exhibited strong green fluorescence, confirming the effective establishment of the model. Compared with the HS group, the fluorescence intensity was significantly reduced when high doses of NAC or BPNSs (10 μg / ml–20 μg / ml) were added, indicating the effect of BPNSs in reducing ROS levels. Furthermore, the gradual decrease in ROS-induced green fluorescence intensity was related to the increase in BPNS concentration. When the BPNS concentration was 20 μg / ml, its fluorescence intensity was even lower than that of NAC, indicating that BPNS has a stronger ROS scavenging ability. Flow cytometry analysis of the average fluorescence intensity of the CM-H2DCFDA probe in HCECs under hypertonic medium further supports this conclusion. Figure 2 F).

[0137] To further assess cell viability in each group, CCK-8 assay and annexin V-FITC / PI staining were used. Compared to the control group, the cell viability in the HS group decreased sharply to 47%. However, the addition of NAC or BPNS significantly improved cell viability, suggesting that BPNS may reduce ROS levels and protect cells from apoptosis. In particular, when the BPNS concentration was maintained at 5 μg / ml, the cell viability in the BPNS group was comparable to that in the NAC group. Cell viability gradually increased with increasing BPNS levels. Notably, at a BPNS dose of 20 μg / ml, the cell viability was comparable to that in the control group, indicating that BPNS has superior antioxidant properties. Figure 2 G).

[0138] Flow cytometry was used to detect the percentage of viable cells and the percentage of viable cells at each apoptotic phase after adding BPNS to hypertonic medium. After 24 hours of incubation, only 75.62% of cells survived due to apoptosis caused by hypertonic oxidative stress. Figure 4 (H and I) are shown. However, this ratio increases when NAC or BPNS are added. Interestingly, at a BPNS dose of 20 μg / ml, cell viability increases to 92%, very similar to the 91.02% viability in the NAC group. This demonstrates the superior antioxidant properties of BPNSs, as well as their strong ability to scavenge intracellular ROS and protect cells from oxidative stress-induced cell death. Figure 4 H). Statistical data showed that the pattern of early apoptotic cell reduction was similar after the introduction of BPNS, which is consistent with our results. Notably, the 320 μg / ml BPNS group showed a significant reduction in early apoptotic cells, indicating that BPNS has a protective effect against apoptosis. Figure 2 HI).

[0139] As is well known, a decrease in mitochondrial membrane potential is an important biomarker in the early stages of apoptosis and a major signal of cellular energy status and stress environment. This study first investigated the mitochondrial membrane potential of cells cultured under different conditions to explore the possible sources of intracellular oxidative stress in BPNSs. A fluorescent probe commonly used to identify mitochondrial membrane potential, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carboxycyanoiodide), was used for this purpose. Under typical conditions, JC-1 aggregates and emits red fluorescence when the mitochondrial membrane potential is high. On the other hand, JC-1 dissociates into monomers and emits green fluorescence when cells are subjected to stress environments such as oxidative stress, thereby reducing the mitochondrial membrane potential. When intracellular antioxidant levels are low or impaired, cells are more susceptible to oxidative stress, which further reduces the potential of the mitochondrial membrane. Compared to the proportion of JC-1 monomers in HS (22.06 ± 2.36%), the presence of NAC or CD significantly reduced this proportion, as shown in the results.Figure 5 As shown in N. Interestingly, when the BPNS concentration was 20 μg / ml, the proportion of JC-1 monomer decreased to 7.02%, even lower than the 8.62% in the NAC group. Figure 2 Anti-inflammatory properties of JN)BPNS@Gel

[0140] Since DED is also associated with M1 polarization of macrophages, we further investigated the antioxidant and anti-inflammatory effects of BPNSs on M1 macrophages. Intracellular ROS levels were then detected using the ROS fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). After incubating M1 macrophages stimulated with different concentrations of BPNS, the antioxidant and anti-inflammatory effects of BPNS were assessed using enzyme-linked immunosorbent assays (ELISA) and DCFH-DA assays of representative inflammatory cytokines (such as IL-6, TNF-α, and IL-1β). Macrophage polarization towards the M1 phenotype was manifested by the development of pseudopodia in LPS-treated HCECs, as well as an increase in intracellular ROS levels and pro-inflammatory cytokines. BPNSs demonstrated their antioxidant and anti-inflammatory effects by significantly reducing the production of inflammatory cytokines, eliminating excessive ROS, and restoring the round morphology of M1 macrophages. HCECs were treated with H2O2 (800 μM) for 24 hours, followed by double staining of live / dead cells using a calcein-acetoxymethyl ester / propidiumiodiine (calcein-AM / PI) double staining kit to verify the protective effect of BPNSs against ROS damage. Although H2O2 almost completely killed cells without BPNS protection, no dead cells were found in the untreated group. However, the addition of BPNSs increased the number of live cells, and this number increased with increasing BPNS concentration, suggesting that BPNSs can effectively protect chondrocytes from H2O2-induced cell death. This study used HCECs generated by IL-1β (10 ng / mL) and oxidative stress chondrocytes induced by H2O2 (400 μM). Both HCECs and H2O2-induced HCECs showed enhanced fluorescence, indicating increased intracellular ROS levels. However, IL-1β or H2O2 significantly reduced ROS levels, indicating that BPNSs were involved in (…). Figure 3 AE).

[0141] Crucially, it is well known that elevated ROS levels in the macrophage microenvironment lead to polarization of the pro-inflammatory M1 phenotype, thereby exacerbating the inflammatory cascade. Next, we used immunofluorescence analysis to assess the ability of BPNS@Gel to modulate polarization. Figure 3As shown in the EF analysis, the intensity of nitric oxide synthase (iNOS) induced by the M1 marker decreased by 28% (LPS + BPNS@Gelvs.LPS), while the expression of M2-associated arginase-1 (Arg-1) increased by 80%, indicating that macrophages treated with BPNS@Gel exhibited a characteristic M2 morphological transformation. Western blot (WB) analysis of iNOS and Arg-1 expression also confirmed these findings. According to the results, 40.25% of the control group and 69.62% of the LPS treatment group had CD86, a marker of M1 macrophages. The proportion of M1 macrophages in the BPNS@Gel-treated cells was significantly lower than that in the LPS group, at 30.25%. Furthermore, when labeling the M2 macrophage marker CD206, we found that the frequency of M2 macrophages in the BPNS@Gel group (39.68%) was higher than that in the IL-4 treatment group (30.25%). Based on these results, BPNS@Gel was more successful in promoting the conversion to the M2 macrophage phenotype. Figure 3 FG).

[0142] The development of BPNS@Gel-mediated macrophage-HCEC crosstalk DED exacerbates the disease and harms eye health by interfering with the normal functioning of HCECs. We investigated the molecular mechanism by which BPNS@Gel protects HCECs through macrophages, given its ability to clear RONS and promote M2 macrophage polarization. HCECs were co-cultured with conditioned medium containing LPS-activated macrophages (CMs) and macrophages containing BPNS@Gel (BP.-gel), and RNA sequencing was performed. Volcano plot analysis showed ( Figure 4 A) Significant differences were observed in 4562 genes between the cm treatment group and the cm gel treatment group, with 2524 genes upregulated and 2038 genes downregulated, such as... Figure 4As shown in BC. Heatmap analysis of differentially expressed genes (DEGs) further revealed these expression patterns. Based on these findings, CM-gel-treated HCECs significantly affected the expression of HCEC-related genes compared to CM-treated HCECs. Gene Ontology (GO) analysis showed significant changes in immune-related regulatory genes in CM-gel-treated HCECs, particularly those related to immune responses and inflammatory processes. Kyoto Encyclopedia of Genetics and Genomes (KEGG) pathway analysis also revealed a significant enrichment of immune-inflammatory pathways, with significant activation of the immune network involving TNF-mediated signal transduction, NF-κB transduction, and IgA manufacturing cascades. Pro-inflammatory mediators (IL-6 and IGJ) were downregulated in CM-gel-treated HCECs, as shown by the expression profiles of pathway-related DEGs. We used pathway-specific protein-protein interaction (PPI) network analysis to determine the functional networks of these DEGs. In the interactome, IL-6 appeared as a pivot gene ( Figure 4 D).

[0143] Furthermore, through molecular modeling and docking, we created a hypothetical IL-6:IGJ model to determine the precise binding site between these two proteins. Figure 4 E). Studies of docking and protein-protein interfaces revealed a cavity size of 5489 and an IL-6:IGJ vina score of -6.8. Previous research indicates that IL-6 is a key cytokine promoting B cell differentiation into plasma cells, with plasma cells particularly expressing IGJ to complete the final assembly of antibodies.40,41 Quantitative studies found that compared to the LPS intervention group, BPNS intervention significantly increased IGJ levels and significantly decreased IL-6 levels. Compared to BPNS intervention, subsequent BPNS@Gel intervention significantly reduced IL-6 levels. Figure 4 (F, G). Further examination of STAT and JAK data using simultaneous immunofluorescence analysis revealed that IL-6 may activate JAK kinase by attaching to the cell membrane receptor complex, leading to STAT phosphorylation. Additionally, data showed that BPNS@Gel intervention significantly reduced JAK and STAT expression. Figure 4 (H) This further demonstrates that BPNS@Gel has an anti-inflammatory effect on LPS-stimulated HCECs. Therefore, our results indicate that BPNS@Gel mainly inhibits the IL-6 / IGJ pathway by regulating crosstalk between macrophages and HCECs, thereby reversing the inflammatory response and senescence process of HCECs.

[0144] Effects of BPNS@Gel on in vivo DED mouse model

[0145] At BPNS@Gel concentrations of 20 and 50 μg / mL, it was found that BPNS@Gel, when diluted with pseudotear fluid, gelled at approximately 36°C. The T2 should be around 34°C, but studies have shown that the average ocular surface temperature in healthy individuals is 33.7°C. Injecting 5 μL of blank in situ gel and BPNS@Gel (20 μg / mL and 50 μg / mL) into the conjunctival sac of Balb / c mice resulted in rapid gel formation, approximately 3–4 seconds. Figure 5 (AB). Ocular surface temperatures exceeded those of BPNS@Gel 20 and 50 μg / mL, reaching 36°C and 36°C, respectively. However, in mouse eyes, both doses of hydrogel formed rapidly. The difference between the T2 data and the actual ocular surface temperature in mice (34°C) may be due to certain conditions preventing the drug from being fully replicated on the mouse ocular surface in vitro.

[0146] Benzocyanate (BAC), a common preservative found in many eye drops, has been shown in clinical practice to play an important role in the unpleasant sensations associated with depigmentation edema (DED). An experimental mouse model of DED was established by topical administration of 0.2% BAC to the ocular surface for 14 consecutive days. The histological changes and clinical symptoms of BAC-induced DED were very similar to those in human DED, leading to corneal epithelial lesions, tear film instability, and ocular surface damage. Tear volume (SIT), corneal fluorescein staining score (FL), and total turbidity ulceration (TBUT) were the main indicators for evaluating the established DED model. Results showed that the DED group had higher FL and lower SIT and TBUT compared to the control group. The DED model was consistent, and there were no statistically significant differences in SIT, TBUT, and FL between the left and right eyes of mice in each group (P<0.05). There was no significant difference in body weight change between the two groups (P>0.05), indicating that BAC had no effect on mouse body weight. Figure 5 CF). One of the main symptoms of BAC is decreased tear volume, which is also a key factor in the clinical diagnosis of DED. Therefore, the effect of BPNS and BPNS@Gel treatment on tear volume changes in DED model mice is the main focus of our study. Compared with the DED group, the tear volume of BPNS and BPNS@Gel mice was significantly increased (P<0.01). Figure 5 These results indicate that BPNS and BPNS@Gel have a restorative effect on the reduction in tear volume in bac-induced DED mice.

[0147] Studies have found that using eye drops containing the preservative BAC shortens tear film breakup time, a key criterion for diagnosing tear film edema (DED), and leads to tear film instability. Both treatment groups showed prolonged tear film breakup times compared to the DED group. However, from... Figure 4As shown in Figure 1, the difference between the DED group and the BPNS@Gel group was statistically significant (P<0.05). Tear film stability increased with prolonged breakup time. These results indicate that BPNS@Gel promotes tear film healing in DED mice. Corneal fluorescein staining is an effective method for assessing corneal epidermal lesions and is crucial for the clinical diagnosis of DED.

[0148] Compared with the DED group, the corneal fluorescence staining score of BPNS@Gel mice was significantly lower on day 7 after drug administration (P<0.05). Figure 6 A, B). The results showed that BPNS@Gel was superior to BPNS and successfully promoted the healing of eye injuries in DED mice. Analysis of the corneal fluorescent staining area at different time points (days 0, 1, 4, and 7) showed that the green fluorescent area of ​​the cornea in mice treated with BPNS and BPNS@Gel decreased to varying degrees over time, indicating that corneal tissue damage gradually recovered after administration. Figure 6 C).

[0149] To assess the effects of the drugs, the corneal and conjunctival tissues of mice in each group were pathologically observed using an optical microscope. In the DED group, the corneal tissue exhibited vacuolar structures, thinning of the superficial layer, reduction and amorphization of the cell layer, and absence of epithelium. Furthermore, the stroma was loosely arranged, with inflammatory cell infiltration and cell loss. Symptoms subsided after 7 days of treatment with BPNS and BPNS@Gel. In particular, the BPNS@Gel group showed thickened epithelial cells and orderly arranged stroma cells, while the BPNS group showed thinned epithelial cells and a limited number of vacuoles. Since BPNS@Gel was superior to BPNS, these results indicate that both BPNS and BPNS@Gel have therapeutic effects on dry corneal damage. Figure 6 E).

[0150] Goblet cells in the conjunctiva lubricate and protect the ocular surface by secreting mucin. The progression of dry eye syndrome (DED) is characterized by a decrease in goblet cells. In contrast, the control group had a dense conjunctival epithelial cell layer with diffuse goblet cells. The DED group showed a significant decrease in goblet cells and a significant increase in the epithelial cell layer. After 7 days of treatment with BPNS and BPNS@Gel, goblet cells increased in both groups. Compared to the BPNS group, the BPNS@Gel group had more goblet cells. In dry eye syndrome, studies have found that BPNS has a restorative effect on the reduction of conjunctival goblet cells, and BPNS@Gel is more effective than point C. Figure 6 F).

[0151] Evaluation of the antioxidant, anti-inflammatory, anti-apoptotic and anti-keratinization properties of BPNS@Gel in corneal tissue

[0152] This study aimed to comprehensively evaluate the antioxidant, anti-inflammatory, anti-apoptotic, and anti-keratinization effects of the prepared BPNS@Gel on corneal tissue to better understand its anti-derivative properties. Exogenous BAC stressors may disrupt the precise balance maintained by corneal tissue, leading to excessive ROS production and increased expression of matrix metallopeptidase 9 (MMP-9), cytokeratin 10 (K10), and IL-1β. The appearance of a green luminescent signal indicates that prolonged exposure to BAC increases ROS levels in ocular epithelial cells, which persisted even 4 days after treatment with BPNS and BPNS@Gel. This result suggests that the ROS scavenging ability of BPNS and BPNS@Gel is limited. Simultaneously, the TUNEL assay was used to evaluate the degree of corneal cell growth and apoptosis after different treatments. Both BPNS aqueous dispersion and BPNS@Gel significantly reduced the number of TUNEL-positive cells, indicating that both treatments successfully prevented cell death. Figure 6 H). In this system, IL-6, a key pro-inflammatory cytokine associated with the pathogenesis of DED, has been thoroughly examined. Figure 6 Figure 1 shows the results, revealing significant changes. DED animals treated with PBS and BPNS@Gel showed reduced IL-6 red luminescence signal, consistent with the pattern observed in ROS. This significant reduction underscores the superior anti-inflammatory properties of BPNS@Gel, which greatly contributes to alleviating DED conditions. Furthermore, the red luminescence signal indicates the presence of K10 (a biomarker of keratinization) in the superficial epithelial layer of DED mice treated with PBS or BPNS@Gel. However, the signal intensity was significantly reduced upon administration of BPNS aqueous dispersion or BPNS@Gel, indicating effective inhibition of squamous metaplasia. Figure 6 G). Our results indicate that BPNS aqueous dispersion or BPNS@Gel can successfully eradicate MMP-9 in DED mice. MMP-9 is a protease associated with corneal barrier dysfunction, surface abnormalities, and apoptosis. It has recently been identified as a diagnostic biomarker for DED. Figure 6 L). Prolonged exposure to BAC leads to the production of ROS and upregulates the expression of IL-1β, K10, and MMP-9, thereby disrupting the structure and morphology of the corneal epithelium, resulting in corneal epithelial desquamation and weakening. Even after 4 days of treatment, neither PBS nor BPNS@Gel significantly alleviated DED symptoms; however, BPNS hydrate dispersion and BPNS@Gel both showed inhibition or elimination of ( Figure 5 In addition to inhibiting the expression of IL-1β, K10, and MMP-9, it also has a strong ability to neutralize various free radicals, such as OH·, O2-·, and DPPH. It provides a comprehensive therapeutic approach, demonstrating significant antioxidant, anti-inflammatory, and anti-cell transplantation properties.

[0153] Following BPNS and BPNS@Gel interventions, IGJ levels were significantly higher than in the DED intervention group. To confirm downstream IGJ expression, we conducted an IL-6 silencing experiment. We found that IGJ levels significantly increased after IL-6 silencing, with the increase being more pronounced after BPNS and BPNS@Gel interventions. However, when we measured MMP-9 expression, we found that MMP-9 expression decreased significantly after IL-6 knockout, and the decrease was even greater after BPNS intervention. Figure 7 AC).

[0154] Long-term in vivo biocompatibility of BPNS

[0155] Mice that received long-term BPNS were used as a control group to observe its effects on blood, internal organs, and the anterior and posterior parts of the eye. H&E staining showed no abnormal changes in corneal and retinal structures two weeks after BPNS injection. Notably, the upper left corner of the retina in the BPNS group, stained with H&E, represents the superficial retinal capillaries, which are naturally located between the ganglion cell layer and the nerve fiber layer. Figure 7 ).

[0156] Furthermore, according to electroretinography (ERG) data, there were no significant changes in the amplitudes of the a and b waves between mice treated with and untreated with BPNS, indicating that frequent use of BPNS does not interfere with retinal electrical activity. These results suggest that BPNS eye drops have broad application prospects and show no signs of ocular toxicity. In addition, untreated mice were used as controls for standard blood analysis and biochemical tests, and blood samples were collected. Blood values ​​showed no statistically significant differences compared to the control group. All these findings indicate that mice treated with BPNS dispersant showed almost no infection or inflammation, which greatly enhances the product's safety and potential therapeutic use. Simultaneously, the heart, liver, spleen, lungs, and kidneys of both treated and untreated mice were removed. H&E staining showed no significant pathological changes. In particular, no necrosis was observed in any histological samples, kidney sections showed clear glomerular structures, liver samples showed normal hepatocyte appearance, and lung samples showed no pulmonary fibrosis. Figure 7 These findings provide compelling evidence of a low incidence of adverse reactions, further supporting the safety of BPNS dispersants.

[0157] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0158] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0159] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0160] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. Uses of black phosphorus nanosheet hydrogels in the preparation of products with one or more of the following functions: 1) Treatment of dry eye syndrome; 2) Promotes tear film healing; 3) Treatment of dry corneal damage.

2. The application as described in claim 1, characterized in that, The black phosphorus nanosheet hydrogel can scavenge reactive oxygen species. And / or, the black phosphorus nanosheet hydrogel is capable of scavenging free radicals; And / or, the black phosphorus nanosheet hydrogel has anti-inflammatory properties.

3. The application as described in claim 1, characterized in that, The black phosphorus nanosheet hydrogel protects cells from oxidative stress-induced cell death.

4. The application as described in claim 1, characterized in that, The black phosphorus nanosheet hydrogel promotes the transformation of macrophages into M2 macrophage phenotypes.

5. The application as described in claim 1, characterized in that, The black phosphorus nanosheet hydrogel has an anti-inflammatory effect on LPS-stimulated HCECs.

6. The application as described in claim 1, characterized in that, The black phosphorus nanosheet hydrogel reverses the inflammatory response and aging process of HCECs.

7. The application as described in claim 1, characterized in that, The black phosphorus nanosheet hydrogel inhibits the IL-6 / IGJ pathway by modulating crosstalk between macrophages and HCECs, thereby reversing the inflammatory response and aging process of HCECs.

8. The application as described in claim 1, characterized in that, The black phosphorus nanosheet hydrogel promotes the increase of goblet cells; And / or, the black phosphorus nanosheet hydrogel has antioxidant, anti-inflammatory, anti-apoptotic and anti-keratinization effects on corneal tissue.

9. The application as described in claim 1, characterized in that, The black phosphorus nanosheet hydrogel reduces the number of Tunnel-positive cells; And / or, the black phosphorus nanosheet hydrogel reduces IL-6 expression levels; And / or, the black phosphorus nanosheet hydrogel inhibits squamation.

10. The application as described in claim 1, characterized in that, The black phosphorus nanosheet hydrogel enhances IGJ expression levels; And / or, the black phosphorus nanosheet hydrogel inhibits the expression of IL-1β, K10 and MMP-9; And / or, the black phosphorus nanosheet hydrogel eradicates MMP-9 in individuals with dry eye syndrome.

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