Injectable hydrogel containing CD-PEI as well as preparation method and application of injectable hydrogel in treatment of xerophthalmia

Abstract

The invention belongs to the technical field of eye disease treatment, and discloses injectable hydrogel containing CD-PEI, a preparation method of the injectable hydrogel and application of the injectable hydrogel to treatment of xerophthalmia. The carbon dot nano enzyme is designed in the hydrogel so as to effectively treat xerophthalmia. The injectable hydrogel is named as OHACDgel, and the injectable hydrogel is formed by combining oxidized hyaluronic acid containing an aldehyde group (OHA), carbon dots (CD-PEI) and gelatin (gel) through dynamic covalent bonds. The potential mechanism of treating xerophthalmia by OHACDgel may involve removal of reactive oxygen species (ROS) and regulation of antioxidant related genes. The nano-enzyme hydrogel has the functions of removing excessive ROS, reducing oxidative damage, inhibiting expression of inflammatory factors and prolonging the retention time of ocular surfaces. The nano-enzyme hydrogel eye drops can provide effective anti-oxidation and anti-inflammatory treatment for xerophthalmia.

Description

Technical Field

[0001] The invention belongs to the technical field of eye disease treatment, and more specifically, relates to an injectable hydrogel containing CD-PEI, a preparation method thereof, and an application thereof in treating dry eye. Background Art

[0002] Dry eye disease (DED) is a common ocular disease that causes ocular discomfort and impaired vision, adversely affecting an individual’s quality of life. The widespread use of electronic devices and a sedentary lifestyle are believed to be contributing factors to the increasing incidence of DED. Although the pathogenesis of DED has not been fully elucidated, inflammation caused by innate and adaptive immune responses is the main driver of the vicious cycle of dry eye disease. In addition to inflammation, reactive oxygen species (ROS) play a vital role in regulating the inflammatory process. Excessive accumulation of ROS in ocular tissues cannot be effectively cleared, leading to the activation of inflammatory pathways, resulting in the loss of goblet cells, decreased corneal wettability, abnormal corneal epithelial cells, and impaired immune responses. The cornea is located in the front part of the eye and can be damaged when exposed to intense ultraviolet radiation, high levels of ionizing radiation, air pollution, and excessive use of electronic devices for a long time. This exposure can lead to oxidative stress and subsequent damage to the ocular surface, which may contribute to the development of DED by inducing inflammation and the formation of hyperosmolarity in the tear film.

[0003] Nanozymes are a new class of biomaterials that have attracted much attention due to their advantages in scavenging ROS to aid wound healing and reduce inflammation. These nanozymes have multiple enzymatic activities, such as catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) properties, which can effectively regulate the reactive oxygen species in the body. Carbon dots (CDs) have also attracted people's interest due to their biocompatibility, high catalytic activity, and ease of surface functionalization, making them valuable in biomedical and environmental applications. As a new type of nanofluorescent material, fluorescent carbon dots (CDs) use carbon as the main structural unit, can be dispersed in water or solvents, have a diameter of less than 10 nanometers, and can be used for the treatment of dry eye.

[0004] In recent years, the treatment of DED has focused on relieving symptoms, reducing ocular inflammation, and improving tear film stability. However, these drugs can only temporarily relieve symptoms by moistening and lubricating the ocular surface, but cannot fundamentally solve the problem of DED. Many drugs are difficult to dissolve in eye drops and are quickly washed away by tears, thus limiting their therapeutic effects. In order to achieve the desired therapeutic effect, sufficient doses of eye drops are required, and frequent medication is usually required (four to five times a day), which increases the risk of adverse reactions. Eye drop preparations should keep high concentrations of soluble drugs in contact with the ocular epithelium for as long as possible. However, tears and frequent blinking limit the retention of drugs on the ocular surface. Therefore, the development of new drug delivery systems is crucial to prolong their residence time on the ocular surface. Summary of the invention

[0005] In order to make up for the deficiencies in the prior art, the present invention provides a method for preparing an injectable hydrogel containing CD-PEI.

[0006] The second object of the present invention is to provide the use of injectable hydrogel containing CD-PEI.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The method for preparing an injectable hydrogel containing CD-PEI comprises the following steps:

[0009] (1) Preparation of CD-PEI: Glycerol and PBS solution were mixed, and polyethyleneimine was added; then, the mixture was heated in a microwave oven, and after the reaction was completed, CD-PEI powder was obtained by dialyzing and freeze-drying;

[0010] (2) Preparation of OHA: Hyaluronic acid is oxidized with sodium periodate solution. After the reaction is completed, OHA is obtained by dialysis and freeze-drying.

[0011] (3) Preparation of OHACDgel: 1-10 wt% OHA solution, 5-80 wt% gelatin solution and 0.5-5 mg / mL CD-PEI were mixed at room temperature to obtain OHACDgel, i.e., an injectable hydrogel containing CD-PEI; wherein the volume ratio of the OHA solution to the gelatin solution was 1:1.

[0012] The present invention develops a nanozyme hydrogel eye drops by encapsulating carbon dot nanozymes with hydrogels. This injectable hydrogel is named OHACDgel, which is formed by hyaluronic acid (OHA) containing aldehyde groups, PEI-modified carbon dots (CD-PEI) and gelation (gel) through dynamic covalent bonding. The combination of these elements forms a network in the hydrogel, and the aldehyde-amino cross-linking promotes the enhancement of ocular retention. OHACDgel has antioxidant and anti-inflammatory properties and is beneficial for the treatment of dry eye. In addition, OHACDgel can also interact with various cells in the corneal microenvironment, affect the polarization of pro-inflammatory macrophages, and promote the regeneration of corneal epithelial cells. Nanozyme hydrogel can remove excess ROS, reduce oxidative damage, inhibit the expression of inflammatory factors, and prolong the residence time on the surface of the eye.

[0013] Therefore, the present invention also provides the use of the OHACDgel obtained by the method in the preparation of drugs for promoting corneal cell migration and proliferation.

[0014] The present invention also provides application of the OHACDgel obtained by the method in preparing a drug for removing ROS in human corneal endothelial cells and macrophages.

[0015] The present invention also provides the use of the OHACDgel obtained by the method in preparing a medicine for reducing the incidence of corneal ulcer.

[0016] The present invention also provides the use of the OHACDgel obtained by the method in preparing a medicine for reducing cell inflammation.

[0017] Preferably, the OHACDgel can transform macrophages from the pro-inflammatory M1 subtype to the anti-inflammatory M2 subtype, and reduce the levels of inflammatory factors IL-6 and IL-1β.

[0018] The present invention also provides the use of the OHACDgel obtained by the method in preparing a drug for treating / alleviating dry eye.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention encapsulates carbon dot nanozymes in hydrogels to effectively treat DED. This injectable hydrogel is named OHACDgel, which is composed of oxidized aldehyde-containing hyaluronic acid (OHA), carbon dots (CD) and gelatin (gel) bonded by dynamic covalent bonds. The potential mechanism of OHACDgel in treating DED may involve the removal of reactive oxygen species (ROS) and the regulation of antioxidant-related genes. Nanozyme hydrogel has the function of removing excess ROS, reducing oxidative damage, inhibiting the expression of inflammatory factors, and prolonging the retention time on the ocular surface. Nanozyme hydrogel eye drops will provide effective antioxidant and anti-inflammatory treatment for DED. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Preparation and characterization of OHACDgel; (A) TEM image of CD, scale bar is 20 nm, inset is HRTEM image of CD, scale bar is 1 nm; (B) Raman spectrum of CD; (C) FTIR spectrum of CD; (D) Zeta potential of CD; (E) UV-Vis spectrum of CD; (F) 1H-NMR spectra of HA and OHA, indicating the presence of aldehyde groups in modified HA; (H) Gelation of OHA hydrogel; (I) FTIR spectrum analysis of OHAgel; (J) Frequency-dependent rheological properties of OHAgel; (K) Changes in storage modulus (G′) and loss modulus (G″) with increasing shear strain, showing a gel-liquid transition when the shear strain is ≈60%;

[0022] Figure 2 The effects of different ratios of gelatin and oxidized hyaluronic acid on the gelation of CD hydrogels;

[0023] Figure 3 The effects of different ratios of gelatin and oxidized hyaluronic acid on the mechanical properties of CD hydrogels;

[0024] Figure 4 CD, OHAgel, and OHACDgel's retention time in the eye;

[0025] Figure 5 Show the biocompatibility of OHACDgel; (A) Cell viability of HCEC cells treated with different concentrations of CD; (B) Cell viability of HCEC cells treated with different concentrations of OHAgel leaching solution; (C) Cell viability of HCEC cells treated with different concentrations of OHACDgel filtrate; (D) Calcium-AM / PI staining of HCEC cells treated with PBS, CD, OHAgel and OHACDgel; (E) Effect of different concentrations of CD on J774.1A cells; (F) Cell viability of J774.1A cells treated with different concentrations of OHAgel leaching solution; (G) Effect of different concentrations of OHACDgel leaching solution on J774.1A cells; (H) Calcium-AM / PI staining of J774.1A cells treated with PBS, CD, OHAgel and OHACDgel; (I) HCEC cell wound healing under different treatments; The scale size is 400μm;

[0026] Figure 6 The in vitro antioxidant effect of OHACDgel is shown; (A) Fluorescence images show that after treatment with CD, OHA and OHACDgel, ROS in HCEC cells is reduced by DCFH-DA probe staining, and the scale bar is 100 μm; (B) Flow cytometry shows that ROS in HCEC cells is reduced; (C) ROS staining of LPS-induced J774.1A cells with DCFH-DA probe, and fluorescence images show that ROS in cells is reduced, and the scale bar is 100 μm; (D) Flow cytometry shows that ROS in LPS-induced J774.1A cells is reduced;

[0027] Figure 7 Showing the in vitro anti-inflammatory activity of OHACDgel; (AC) Representative flow cytometry images of CD86 and CD206 expression detected by flow cytometry and corresponding statistical analysis; (D) ELISA method was used to detect the expression levels of IL-6 and IL-1β in J774.1A macrophages;

[0028] Figure 8Figure 2 shows gene transcriptome analysis (A) Volcano plot analysis of HCEC cells treated with OHACDgel, highlighting differentially expressed genes (DEGs); green indicates downregulated genes, red indicates upregulated genes; blue dots indicate genes with differential expression less than 2-fold; (B) Three basic functional categories of gene ontology for DEGs analysis: biological process (green), cellular component (red), and molecular function (blue); (C) Heat map of several differentially expressed genes related to oxidative stress and iron metabolism (**p<0.001); (D) Functional analysis of HCEC cells after CD treatment, including GO and KEGG analysis;

[0029] Fig. 9 Showing the in vivo therapeutic effect of OHACDgel on DED; (A) Schematic diagram of the establishment, treatment and subsequent evaluation of the DED model in mice; (B, C, D) Corneal fluorescence staining, Schirmer test and body weight during the establishment of the DED model; slit lamp observation of the mouse cornea; representative images of the cornea (E); (F, G) Representative images of corneal fluorescein staining (F) and statistical analysis of fluorescein scores (G); (H, I) Paired comparison of the 20s Schirmer test line length (H) and body weight (I) of mice in different groups on consecutive days; (J) H&E and PAS staining showed the conjunctival structure and goblet cell number, scale bar = 100μm;

[0030] Fig.10 The injectable properties of OHACDgel were demonstrated. DETAILED DESCRIPTION

[0031] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with specific drawings and embodiments. In the embodiments, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0032] Example 1 Preparation of OHACDgel

[0033] Materials: PEI, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and glycerol were obtained from Sigma-Aldrich (USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo, Japan. MEM, DMEM, fetal bovine serum, penicillin, streptomycin and phosphate buffered saline (PBS) were all from Gibco (USA). Calcium AM / PI live-dead cell double staining kit was from Jiangsu KeyGen Biotech Co., Ltd.

[0034] Preparation of CD-PEI: CD was synthesized by microwave method. The first step was to mix 25 mL of glycerol with 6 mL of PBS solution. Subsequently, 0.5 g of polyethyleneimine (PEI, 25 kMW) was added to the mixture. Next, the mixture was placed in a microwave oven and heated for 10 minutes. Then, the mixture was dialyzed to obtain a substance with a molecular weight of less than 500 Da. Finally, the mixture was dialyzed for two days and freeze-dried to obtain CD-PEI powder.

[0035] Preparation of OHAgel: (1) Hyaluronic acid was dissolved in 150 ml of deionized water. The mixture was reacted in a sodium periodate solution for 3 hours. The reaction was terminated after adding ethylene glycol for one hour. The mixture was then dialyzed with a molecular weight cutoff of 3.5 kDa. The mixture was then dialyzed for three days and then freeze-dried to prepare hyaluronic acid (OHA) containing an aldehyde group; (2) OHA solution (4 wt%) and gelatin (type B, 20 wt%) were mixed at room temperature to obtain OHAgel.

[0036] Preparation of OHACDgel: (1) Hyaluronic acid was dissolved in 150 ml of deionized water. The mixture was reacted in sodium periodate solution for 3 hours. The reaction was terminated after adding ethylene glycol for one hour. The mixture was then dialyzed with a molecular weight cutoff of 3.5 kDa. The mixture was then dialyzed for three days and then freeze-dried to prepare hyaluronic acid (OHA) containing an aldehyde group; (2) OHA solution (4 wt%), gelatin (type B, 20 wt%), and CD-PEI (1 mg / mL) were mixed at room temperature to obtain OHACDgel.

[0037] Characterization: Transmission electron microscopy (TEM) images of CD-PEI were obtained using a JEOL JEM-F200 microscope with accompanying mapping images. The crystal structure of CD-PEI was determined by x-ray diffraction (XRD). Zeta potential and particle size were determined using a zetasizer (zetasizerNano-ZS, Malvern). UV-visible absorbance of the samples was measured using a UV-visible spectrophotometer (thermal).

[0038] 1. Characterization of OHACDgel

[0039] Synthetic CDs such as Figure 1 As shown in A. CD was prepared by a previously reported method. In addition, CD-PEI was prepared by a unidirectional microwave method.

[0040] High-resolution transmission electron microscopy (HRTEM) images show that CD has a uniform spherical structure with a diameter of about 4nm. The particle size of carbon dots is between 2 and 10nm, and its condensed carbon core is surrounded by a polymer PEI layer. The interplanar spacing of CD-PEI is about 0.32nm. Figure 1As shown in A. The Raman spectrum of CD shows characteristic peaks of carbon dots, including G peak and D peak ( Figure 1 B) FTIR spectrum of CD at 3230 cm -1 The absorption peak observed at 3603 cm can be attributed to the overlap of hydroxyl (OH) and amino (NH) vibrations. -1 The peak at 1623cm belongs to the hydroxyl (OH) vibration mode, while the peak at 1623cm -1 The peak at is mainly related to the stretching vibration peak of hydroxyl group (C=O) ( Figure 1 C). In addition, CD-PEI showed a positive zeta potential of 10 mV, which was attributed to the exposure of amino groups on the PEI surface ( Figure 1 D) Preliminary identification of CD by UV-visible spectrum: Figure 1 As shown in E. OHACDgel has no absorption peak in the range of 600-900 nm.

[0041] The injectable property of the prepared OHA hydrogel was demonstrated to be the result of dynamic covalent bonds ( Fig.10 ). The hydrogels formed by dynamic covalent bonding are usually homogeneous and show good mechanical properties. The initial step is to synthesize oxidized aldehyde-containing hyaluronic acid (OHA) by oxidation with sodium periodate (NaIO4). The purified products of OHA, OHAgel and gelatin were confirmed by Fourier transform infrared spectroscopy (FTIR) and 1H nuclear magnetic resonance spectroscopy (1H NMR).

[0042] Figure 1 F shows the 1H NMR spectrum, which shows the chemical structures of HA and OHA. The NMR spectrum shows that the black squares indicate the presence of new peaks corresponding to the aldehyde groups in OHA. Figure 1 FTIR spectrum of OHA in I at 1640 cm -1 A unique peak associated with the aldehyde group was shown at , indicating the presence of this functional group. 1H N4MR and FTIR spectra confirmed the successful synthesis of OHA. Figure 1 As shown in I, at 3000~3750cm -1 and 2930cm -1 The peaks in the range belong to -OH and -CH stretching vibrations. -1 The characteristic peak of aldehyde group at 1633cm -1 The appearance of the amine group at the OHA site demonstrated the formation of an imine bond between the aldehyde group of OHA and the amino group of gelatin. This observation confirmed the successful preparation of the OHA hydrogel. Due to the successful modification of OHA, the mixture of OHA and gelatin could be easily gelated ( Figure 1 H).

[0043] Subsequently, the OHA aqueous solution was mixed to prepare the hydrogel. The effective aldehyde-amino covalent cross-linking reaction allowed the formation of the hydrogel. Considering the overall rotation frequency scale, it can be observed that G' always exceeds G", indicating that the structure of OHAgel is solid. After the OHA and gelatin solutions were mixed, the storage modulus (G') and loss modulus (G") increased with time, reaching an equal point of about 60% ( Figure 1 K).

[0044] 2. Performance control and exploration of OHACDgel

[0045] The ratio of gelatin to OHA has a significant effect on the gelation time and mechanical properties of the hydrogel. As the ratio of OHA increases, the gelation time of OHAgel hydrogel gradually increases ( Figure 2 ). In fact, in the experiment, we found that the higher the OHA content, the longer the gelation time of the hydrogel. When the ratio of gelatin to OHA is 1:2 or 1:3, the gelation time can be as long as 1 to 2 days. At the same time, when the OHA content is high, the prepared hydrogel is easy to liquefy again and cannot stably maintain the gel-like morphology of the hydrogel. Therefore, when preparing OHACDgel, the proportion of OHA should not be too high.

[0046] On the other hand, the prepared OHAgel hydrogel is used for ocular drug delivery, so the mechanical properties of the prepared OHAgel hydrogel must also be considered. The hydrogel needs to form a highly uniform and clear thin layer to cover the ocular surface, and at the same time have stronger adhesion to the cornea, which can continuously and slowly release drugs and protect corneal epithelial cells from long-term exposure to a tear-deficient environment. If it is too hard, it is not easy to evenly cover the corneal surface, and if it is too soft, the hydrogel will not cover evenly, and the continuous and slow release of drugs cannot be guaranteed. Therefore, taking comprehensive considerations, the present invention selects a ratio of gelatin to OHA of 1:1 (v:v).

[0047] After mixing OHA and gelatin solutions, the storage modulus (G') and loss modulus (G") increased with time and reached an equal point at about 40-80% ( Figure 3 ). This confirmed that the gel is an injectable hydrogel.

[0048] Example 2 Biocompatibility and Antioxidant Properties of OHACDgel

[0049] Cell culture media used: Human corneal epithelial cells (HCEC) and mouse macrophages were purchased from Guangzhou Genio Biotechnology Co., Ltd. HCEC cells were cultured in minimum essential medium (MEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U / mL penicillin and 100 μg / mL streptomycin (Solarbio, Beijing, China) at 37°C, 5% CO2 incubator. HCEC cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U / mL penicillin and 100 μg / mL streptomycin (Gibco, USA) at 37°C, 5% CO2 incubator.

[0050] Biocompatibility of OHACDgel: The biocompatibility of OHACDgel was evaluated by cell viability assessment and live / dead cell staining. Cell viability after contact with OHACDgel and CD was assessed by using CCK8 assay. CCK8 assay measured absorbance at 450 nm using a microplate reader. HCEC cells and J774.1A cells were seeded in 6-well plates, CD and OHACDgel extracts were added to the culture medium and incubated for 24 hours. Live / dead cell assay was performed using propidium iodide (PI) and Calcium AM kits, and the results were imaged using confocal laser scanning microscopy.

[0051] Anti-inflammatory ability of OHACDgel: J774.1A cells were seeded in 6-well plates and stimulated with LPS (1 μg / mL) for 24 hours to polarize them toward M1 macrophages. Subsequently, hydrogel extract was added and incubated for 24 hours. Subsequently, cells were collected and the expression of macrophage polarization markers was analyzed by flow cytometry. In addition, the effect of oacdgel intervention on LPS-induced IL-1β and IL-6 expression levels was detected by ELISA.

[0052] Antioxidant capacity of OHACDgel: HCEC cells were seeded in 24-well plates and stimulated with high-pressure culture medium to induce the accumulation of ROS. Subsequently, the hydrogel extract was added and incubated for 24 hours. J774.1A cells were seeded in 6-well plates and stimulated with LPS (1 μg / mL) for 24 hours to polarize them to M1 macrophages. Subsequently, the hydrogel extract was added and incubated for 24 hours. The antioxidant capacity of OHACDgel was then evaluated by DCFH-DA staining and laser confocal scanning microscopy imaging. The amount of ROS was analyzed by flow cytometry, and the data obtained were expressed as mean fluorescence intensity.

[0053] 1. Biocompatibility study

[0054] The biocompatibility of OHACDgel is essential for promoting cell proliferation and accelerating corneal cell healing. First, to evaluate cytotoxicity, HCEC cells and J774.1A cells were treated with OHACDgel and analyzed using the Cell Counting Kit-8 (CCK-8) assay. The results showed that both the CD group and the OHACDgel group exhibited higher cell viability compared with the control group, indicating that at concentrations up to 50 μg / mL, OHACDgel had negligible cytotoxicity against HCEC and J774.1A cells ( Figure 5 A~5G). In addition, at a concentration of 100 μg / mL, the cell viability of CD, OHAgel, and OHACDgel was slightly higher than 90% ( Figure 5 A~5C). Live / dead staining was performed under a confocal laser scanning microscope to evaluate the biocompatibility of CD, OHAgel, and OHACDgel after 24 hours of action. Strong green fluorescence and negligible red fluorescence showed that OHACDgel had good biocompatibility ( Figure 5 D). OHACDgel is considered a promising option for anti-DED treatment due to its enhanced catalytic capacity, enhanced ROS inhibition, and reduced cytotoxicity, as demonstrated by the above findings ( Figure 5 H). The results of the wound healing test showed that the hydrogel had a good promoting effect on cell proliferation and migration. The migration of cells in the CD-modified hydrogel group was accelerated within 48 hours. The healing rate of the OHACDgel group was 55.8%, and that of the PBS group was 45.7% ( Figure 5 I). In conclusion, these findings suggest that OHACDgel can effectively promote cell proliferation, potentially promote tissue regeneration, and successfully assist in corneal tissue reconstruction.

[0055] 2. Study on Antioxidant Properties

[0056] Corneal epithelial cells and macrophages play a crucial role in the development and treatment of DED. Notably, corneal epithelial cells are central to the pathological process of DED as they directly respond to adverse environmental conditions and trigger innate immune responses. Previous studies have found that the microenvironment of DED exhibits accumulation of ROS and a chronic inflammatory state. Oxidative stress is an important factor in the pathogenesis of DED. To determine the ability of OHACDgel to scavenge ROS in vitro, we used 2',7'-dichlorofluorescein 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe to quantify ROS production in human corneal epithelial cells (HCEC) under hyperosmotic conditions (HOM, 500mOsM), which induces ROS overproduction and inflammation. The high osmotic pressure of the tear film has been shown to induce elevated ROS levels, thereby exacerbating the inflammatory response. Therefore, effective elimination of excess ROS can help regulate the inflammatory response and stop the vicious cycle of ocular diseases. The hyperosmotic group (HOM) showed the highest green fluorescence, indicating significant ROS accumulation due to the effect of the hyperosmotic medium. Compared with the HOM group, the green fluorescence of the CD, OHA, and OHACDgel-treated groups was significantly reduced, indicating that OHACDgel has a special ability to scavenge ROS in HCEC cells ( Figure 6 A-6B). Notably, OHACDgel treatment resulted in the lowest level of ROS detected, which was observed to be almost comparable to that in normal cells.

[0057] Macrophages are an important component of the immune regulatory system and play a crucial role in the pathogenesis of DED. In addition to their powerful phagocytic and antigen-presenting abilities, macrophages secrete a variety of proteins, inflammatory mediators, and chemokines when activated, which can exacerbate inflammation and accelerate the progression of DED. Regulating the inflammatory response of these cells may be an effective strategy to reduce cell death and corneal tissue damage associated with DED. To determine the potential therapeutic effects of the proposed treatment for DED, we performed in vitro studies using lipopolysaccharide (LPS)-induced oxidative stress stimulation and inflammation in J774.1A macrophages. Confocal laser scanning microscopy data showed that after 24 h of macrophage stimulation with 1 μg / mL LPS, the intracellular ROS level was significantly increased ( Figure 6 C). Of particular interest, it was observed that CD, OHAgel, and OHACDgel treatments exhibited a significant inhibitory effect on LPS-induced ROS levels. Figure 6 As shown in D, compared with the LPS group, the DCF fluorescence in the OHACDgel group was significantly reduced, indicating that OHACDgel can effectively inhibit the overexpression of ROS in J774.1A cells induced by LPS. OHACDgel has significant antioxidant activity.

[0058] 3. Research on anti-inflammatory properties

[0059] Dry eye disease (DED) is increasingly recognized as a multifactorial disease that is closely related to inflammatory activity on the ocular surface, triggered by factors such as environmental stress, autoimmune diseases, and prolonged screen time, which disrupt tear production and stability. Inflammatory mediators, including cytokines and chemokines, aggravate symptoms by damaging the ocular surface and altering glandular function. This bidirectional relationship not only amplifies patient discomfort and visual impairment but also emphasizes the importance of targeting inflammation in the management and treatment of DED. Next, to evaluate the anti-inflammatory effect of OHACDge, we measured the expression of CD206 (a marker of m2-type anti-inflammatory macrophages) and CD86 (a producer of m1-type pro-inflammatory macrophages) by flow cytometry (FCM). After 1 μg / mL LPS stimulation for 24 h, ROS levels were significantly increased and CD86 levels were significantly upregulated, suggesting that LPS induces macrophage polarization toward the m1 type. CD inhibited the expression of CD86 while enhancing the expression of CD206. OHAgel showed a stronger ability to inhibit CD86 and promote CD206 expression ( Figure 7 A~7C). In addition, CD effectively reduced the increase of IL-6 and IL-1β induced by LPS, and OHACDgel showed a stronger inhibitory effect ( Figure 7 D). These results indicate that OHACDgel can transform macrophages from the pro-inflammatory M1 subtype to the anti-inflammatory M2 subtype and significantly reduce the levels of inflammatory factors IL-6 and IL-1β.

[0060] 4. Mechanism Exploration

[0061] RNA sequencing and analysis: HCEC cells were treated with OHACDgel and control groups for 24 h, respectively. After treatment, cells were detached with trypsin and washed twice with cold PBS. Cell pellets were collected by centrifugation, quickly frozen in liquid nitrogen, and sent to IGE Biotechnology Co., Ltd. (Guangzhou, China) for transcriptome sequencing. The process included sample testing, library construction, and sequencing. After successful quality control of the library, sequencing was performed using IlluminaPE150 (Illumina, USA). DESeq2 was used to analyze the differential expression between the two groups. To identify the key pathways affected by OHACDgel in HCEC cells, gene ontology (GO) enrichment analysis was performed. To describe the main pathways affected by OHACDgel in HCEC cells, gene ontology (GO) enrichment analysis was performed. The goana function in the limmaBioconductor software package was used to identify the most enriched GO terms in the deg list, including biological process, cellular component, and molecular function. The false discovery rate (FDR) was set to 0.05, and the significance of GO terms was determined using the topGO function, comparing the sets of top 20, 50, 100, 200, and 300 GO terms.

[0062] RNA sequencing was used to further explore the potential anti-DED mechanism of OHACDgel and identify and analyze the differentially expressed genes (DEGs) in HCEC cells after OHACDgel treatment. Compared with the control group, a total of 5298 DEGs were detected, including 2825 up-regulated genes and 2473 down-regulated genes ( Figure 8 A). Gene Ontology (GO) analysis showed that deg was enriched in different categories, including biological process (10 GO terms), cellular component (10 GO terms), and molecular function (10 GO terms) ( Figure 8 B). Among the three process categories, oxidoreductase activity, stress response, and mitochondria were the most represented. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis ( Figure 8 C) showed that DED had a significant impact on the KEGG oxidative stress pathway, which may be because oxidative stress can disrupt the mechanism of DED progression. Figure 8 D shows that antioxidant-related genes were significantly upregulated, indicating that oxidative stress may occur after OHACDgel treatment.

[0063] Example 3 Animal Experiment

[0064] Animal experiments were approved by the Animal Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University. Adult female C57BL / 6 mice (6–8 weeks old, weighing 20–25 g) were used for animal experiments, and were placed under a 12-h light / dark cycle at room temperature and given food and water. A 0.2% benzalkonium chloride (BAC) solution was used to establish a mouse DED experimental model. Briefly, 5 μL of 0.2% BAC eye drops (w / v) were administered to the right eye of the mice twice a day for 7 consecutive days. The mice were randomly divided into 4 groups (6 mice in each group) and treated with 5 μL of eye drops. The control group was treated with 0.9% sterile saline, and the remaining groups were treated with PBS, 100 μg / mL CD, OHAgel, and OHACDgel, respectively. These treatments were performed twice a day for 7 days. The body weight of the test mice was recorded every day. The above treatments were performed as indicated.

[0065] Tear secretion was quantified using cotton thread according to the instructions. The thread was placed on the inferior palpebral conjunctiva at one-third of the distance from the lateral canthus. The length of the wet cotton thread was measured in millimeters within 20 seconds without anesthesia. After 3 days of treatment, the mice were anesthetized and killed. The removed organs were then fixed for histological examination. Paraffin-fixed immunohistochemical analysis was also performed on the tumor tissue. Corneal epithelial damage was evaluated by corneal fluorescein staining. Sodium fluorescein was injected into the conjunctival sac and observed under a slit lamp microscope with a cobalt blue filter. The cornea was divided into 4 areas, and each area was graded according to the following criteria: The scoring criteria for evaluating corneal epithelial damage were as follows: The scoring criteria for evaluating corneal epithelial damage were as follows: 0 = no staining, 1 = mild punctate staining, 2 = marked punctate or mild light macular staining, 3 = marked light macular or mild macular staining, and 4 = marked macular staining.

[0066] After anesthesia overdose, the eyes and adnexa were fixed with FAS eye fixative (Servicebio, China) and embedded in paraffin. Then, sagittal sections were cut at 5 μm thickness and stored at room temperature. Eye sections were subjected to routine histological analysis, including hematoxylin and eosin (H&E) staining. Periodic acid-Schiff (PAS) staining was used to observe and quantify the number of conjunctival goblet cells.

[0067] Fluorescent immunostaining: Immunofluorescence assays were performed according to the prior art. In summary, cells were fixed and permeabilized with Triton X-100. The designated primary antibodies were incubated with cells at 4°C for 12 to 16 hours. Subsequently, after washing with the primary antibody, the cells were incubated with a fluorescently conjugated secondary antibody. Subsequently, the cells were observed using a confocal microscope. Subsequently, the cells were detected by flow cytometry.

[0068] 1. Experimental results

[0069] To evaluate the therapeutic effect of OHACDgel on DED in vivo, we established a mouse DED model. All animal experiments were performed in accordance with the guidelines of the Animal Ethics Committee of Sun Yat-sen University. Fig. 9As shown in A. The DED model was established using 0.2% benzalkonium chloride (BAC) twice a day for 7 consecutive days. BAC is a commonly used drug used to induce DED with acute corneal epithelial damage, excessive ROS accumulation and corneal inflammation. The established DED model was mainly evaluated by Schirmer test and corneal fluorescein staining score. Compared with the control group, the Schirmer test in the DED group decreased and the fluorescein staining increased, confirming that the DED model was successfully established. There was no statistically significant difference in the Schirmer test and fluorescein staining scores between the left and right eyes of the mice in each group (P>0.05), indicating the consistency of the establishment of the DED model. After 7 days of BAC induction, corneal edema, epithelial defects and insufficient tear secretion occurred in all groups, confirming the establishment of the DED model. There was no statistically significant difference in the weight changes of mice between the two groups (P>0.05), indicating that BAC had no effect on the weight of mice.

[0070] After the DED model was established, the in vivo anti-inflammatory and antioxidant effects of OHACDgel were subsequently evaluated. Mice with scores >12 were randomly divided into 4 groups and treated with PBS, CD, OHA, and OHACDgel, respectively, twice a day for 3 consecutive days. The therapeutic effects were evaluated by clinical observation, Schirmer test, and histopathological analysis. The degree of corneal epithelial damage and tear deficiency was monitored every 0, 1, 2, and 3 days. The clinical characteristics of DED are corneal surface damage and tear deficiency, which occur every day. Slit lamp images showed that the treatment groups (CD, OHAgel, and OHACDgel) showed minimal discomfort compared with the control group, thus verifying the in vivo antioxidant effect of OHACDgel. After 3 days of treatment, corneal defects (green fluorescent staining) persisted in the DED and CD groups. During the 3-day treatment, plaque staining was observed in the control group, while OHACDgel showed minimal micro-dot staining. It was found that OHACDgel significantly reduced the incidence of corneal ulcers compared with the CD or OHAgel groups. The Schirmer test was used to evaluate tear secretion. The measurement of average tear volume is an important clinical indicator for diagnosing DED symptoms. The data showed that the tear volume in the treatment group was higher than that in the control group, and the tear volume in the OHACDgel group was high, almost indistinguishable from that in normal eyes.

[0071] To further investigate the recovery of BAC-induced corneal epithelial damage, we fixed the corneal epithelium, and the results of histological staining showed that the corneal epithelial damage was obvious in the DED group. After treatment, the ocular surface epithelium of the entire treatment group was multi-layered, and the interstitial layer was well organized, indicating that the ocular surface repair effect of the animal model was good. H&E staining was used to evaluate the biocompatibility of OHACDgel in mice. Compared with the control group, no signs of pathological damage were found in the organs of mice in the drug-treated group, suggesting that CD, OHA, and OHACDgel did not show any systemic toxicity. Hemolysis in the three groups was within the normal range and was not statistically significant. These results collectively indicate that CD and OHACDgel exhibit enhanced biocompatibility in vivo.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing an injectable hydrogel containing CD-PEI, characterized in that: The following steps are involved: (1) Preparation of CD-PEI: Glycerol and PBS solution were mixed, and polyethyleneimine was added; then, the mixture was heated in a microwave oven, and after the reaction was completed, CD-PEI powder was obtained by dialyzing and freeze-drying; (2) Preparation of OHA: Hyaluronic acid is oxidized with sodium periodate solution. After the reaction is completed, OHA is obtained by dialysis and freeze-drying. (3) Preparation of OHACDgel: 1-10 wt% OHA solution, 5-80 wt% gelatin solution and 0.5-5 mg / mL CD-PEI were mixed at room temperature to obtain OHACDgel, i.e., an injectable hydrogel containing CD-PEI; wherein the volume ratio of the OHA solution to the gelatin solution was 1:

1.

2. Use of the OHACDgel obtained by the method of claim 1 in the preparation of a drug for promoting corneal cell migration and proliferation.

3. Use of the OHACDgel obtained by the method of claim 1 in the preparation of a drug for removing ROS in human corneal endothelial cells and macrophages.

4. Use of the OHACDgel obtained by the method of claim 1 in the preparation of a drug for reducing the incidence of corneal ulcers.

5. Use of the OHACDgel obtained by the method of claim 1 in the preparation of a drug for reducing cellular inflammation.

6. The use according to claim 5, characterized in that: The OHACDgel can transform macrophages from the pro-inflammatory M1 subtype to the anti-inflammatory M2 subtype, and reduce the levels of inflammatory factors IL-6 and IL-1β.

7. Use of the OHACDgel obtained by the method of claim 1 in the preparation of a drug for treating / alleviating dry eye.

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