A multi-active hydrogel patch

Abstract

The application belongs to the field of medicine and cosmetics, and provides a multi-activity hydrogel, which comprises water and the following raw materials in mass parts: gel matrix forming agent 10-250 parts, crosslinking agent 0.5-5 parts, crosslinking regulator 0.5-5 parts, stabilizer 1-5 parts, zwitterion protective agent 1-5 parts, humectant 200-800 parts, and active ingredient 0.1-600 parts. The active ingredient is not easy to be inactivated, has good storage stability, and has controllable release rhythm. The hydrogel can achieve good functions when used externally, does not need to be injected, has low cytotoxicity, has no skin irritation and sensitization, and is suitable for long-term care of sensitive skin around the eyes; can significantly promote collagen synthesis of fibroblasts, inhibit expression of matrix metalloproteinase, and effectively improve tear secretion and corneal repair of an xerophthalmia model mouse.

Description

Technical Field

[0001] This invention belongs to the fields of medicine and cosmetics, and relates to a hydrogel for use in the eye area. Background Technology

[0002] With the widespread use of electronic devices and increased eye strain, eye fatigue syndrome, dry eye syndrome, and aging of the skin around the eyes have become global health challenges. Currently, commercially available eye care products mainly fall into two categories: eye drops, which act directly on the surface of the eye but suffer from drawbacks such as rapid drug loss, short duration of action, and the need for frequent application; and eye patches, which are applied to the outer eyelids. However, existing products primarily focus on simple moisturizing, lacking bioactive ingredients and having a relatively singular function. Furthermore, the skin around the eyes lacks the support of elastin and collagen, and the frequency of blinking—approximately 15,000 to 20,000 times per day, often exceeding 10,000—results in repeated stretching and fine lines and wrinkles, accelerating the aging process. Current treatments for eye wrinkles mainly include topical anti-aging serums (such as those containing vitamin A and peptides), physical radiofrequency beauty devices, and invasive injection treatments (such as botulinum toxin and hyaluronic acid fillers). However, traditional topical preparations are limited by the stratum corneum barrier, resulting in low transdermal absorption of active ingredients and difficulty in effectively reaching the dermis to stimulate collagen and elastin regeneration. While physical methods such as radiofrequency ablation have some effect, the equipment is expensive, the operation is complex, and there is a risk of thermal damage. Injection treatments are invasive, require professional physicians, are costly, and may cause side effects such as local swelling or facial stiffness. Therefore, developing a non-invasive, safe, effective, and sustained anti-wrinkle solution for the skin around the eyes has urgent clinical need and market value.

[0003] Extracellular vesicles (EVs), especially exosomes (Exos), have shown great potential in tissue repair and anti-inflammation due to their nanoscale size (30-150 nm) and ability to carry various bioactive molecules (such as proteins, mRNA, miRNA, etc.). However, current applications of exosomes in skin anti-aging largely rely on injection or microneedling, which have limitations due to invasive risks and high technical barriers. Eye patches, as a classic dosage form in transdermal drug delivery, can promote hydration and softening of the periocular stratum corneum through closed application, prolonging the retention time of active ingredients locally, and have the advantages of convenient use and good patient compliance. Loading exosomes into an eye patch carrier, relying on their nanoscale penetration ability and excellent biological activity, can efficiently deliver collagen-regenerating signaling molecules to dermal fibroblasts without invasiveness, thereby achieving continuous repair of wrinkles around the eyes.

[0004] Hydrogel patches have been used in transdermal drug delivery, but existing hydrogel matrix formulations are mainly designed for small molecule drugs. For example, Chinese patent CN116370350A discloses a hydrogel patch that uses sodium polyacrylate as the backbone, aluminum hydroxyl as the crosslinking agent, and disodium EDTA and tartaric acid as crosslinking regulators to obtain a gel with good viscosity and cohesive strength. However, when such traditional hydrogel systems are used to load extracellular vesicles (Exos), conditioned medium (CM), and other protein / vesicle macromolecular active substances, the following technical defects exist: (1) Traditional crosslinking regulators (such as EDTA / tartaric acid) cannot effectively protect the Exos membrane structure, and the aluminum ion crosslinking system may induce Exos aggregation or rupture; (2) The release behavior of the gel network of active ingredients is uncontrollable, and there is a burst release phenomenon, resulting in a short action time and difficulty in achieving time-sequential regulation of the repair process; (3) Exos are easily inactivated during gel storage, resulting in a short product shelf life.

[0005] Furthermore, exosome-only therapy still has certain limitations: although exosomes carry abundant bioactive molecules, their composition is affected by factors such as the state of donor cells and extraction processes, leading to fluctuations in the concentration of active ingredients between different batches. Moreover, simply using exosomes to promote collagen regeneration often requires high doses or multiple administrations to achieve the desired effect. Conditioned culture media, collected during cell culture, contain cell secretions and, in addition to exosomes, are rich in various soluble active ingredients such as growth factors, cytokines, metabolites, and extracellular matrix proteins. Existing studies have shown that conditioned culture media can synergistically regulate fibroblast function through their complex paracrine network; however, traditional conditioned culture media have low concentrations of active ingredients and contain many impurities, limiting their direct application effectiveness. Summary of the Invention

[0006] To address the problems of short duration of action, weak bioactivity, and limited application scenarios of current eye care products, this invention provides a hydrogel patch containing multiple active ingredients, which are not easily deactivated, have good storage stability, and controllable release rhythm.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A hydrogel containing multiple active ingredients, comprising water and the following raw materials in parts by weight: 10-250 parts of gel matrix forming agent Crosslinking agent 0.5-5 parts, Crosslinking regulator 0.5-5 parts, Stabilizer 1-5 parts, 1-5 parts of zwitterionic protectant 200-800 parts of moisturizer Active ingredient: 0.1-600 parts.

[0009] The gel matrix forming agent is sodium polyacrylate, which can be selected from one or more of commercially available brands P46NH, P55N, NP-700, NP-600, and NP-800. Preferably, the gel matrix forming agent is a combination of sodium polyacrylate P46NH and sodium polyacrylate P55N, with a mass ratio of (20-1):1, more preferably (3-5):1.

[0010] The crosslinking agent is selected from aluminum salts or calcium salts, such as aluminum hydroxyl and calcium chloride.

[0011] The crosslinking regulator is selected from a combination of a hydroxycarboxylic acid and a salt of the corresponding hydroxycarboxylic acid; the salt is a sodium or potassium salt. The hydroxycarboxylic acid is selected from one or more of tartaric acid, citric acid, malic acid, gluconic acid, lactic acid, and glycolic acid. Preferably, the mass ratio of the hydroxycarboxylic acid to the corresponding hydroxycarboxylic acid is (1-3):(1-2). The ratio can be adjusted according to the target crosslinking rate or the acidity or alkalinity of the reaction system.

[0012] The stabilizer is a metal ion complexing agent, such as one or more of disodium edetate, calcium sodium edetate, tetrasodium edetate, diethylenetriaminepentaacetic acid (DTPA), pentasodium diethylenetriaminepentaacetic acid (DTPA-5Na), nitrotriacetic acid (NTA) and its salts, and ethylenediamine-N,N'-disuccinic acid (EDDS) and its salts; preferably disodium edetate and / or calcium sodium edetate.

[0013] The zwitterionic protectant is selected from at least two of betaine, proline, and trehalose.

[0014] The moisturizer is selected from polyols, such as ethylene glycol and glycerin.

[0015] The active ingredient is selected from a combination of extracellular vesicles and conditioned medium. The cell source is at least one of animal cells, plant cells, or probiotics, wherein the plant cells are plant cell cultures or plant callus tissue. Conditioned medium includes, for example, mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), immune cells, corneal epithelial cells, fibroblasts, and precursor cells of the above cells, as well as carrots, onions, goji berries, blueberries, grapes, licorice, chrysanthemum, ginger, goji berries, cassia seeds, buddleja flowers, bifidobacteria, galactosomal molds, etc. The extracellular vesicles can be naturally occurring or engineered. Engineered extracellular vesicles loaded with active ingredients, such as small molecule compounds, nucleic acids, and proteins, through methods such as electroporation, co-incubation, ultrasound, or extrusion, can enhance the specific technical effects. Preferably, the mass fraction of the extracellular vesicles is 0.1-100 parts, calculated based on the total protein content of the extracellular vesicles. The amount of conditioned medium added is 50-500 parts. To increase the content of effective components in conditioned medium, a concentrated solution can be prepared by separating cells from the culture medium after cell culture and concentrating it 20-100 times.

[0016] The hydrogel may also be supplemented with other active ingredients according to its function. These other active ingredients are one or more of plant extracts or clinical drugs with antimicrobial, antioxidant or anti-inflammatory activities; such as hyaluronic acid and its salts (e.g. sodium hyaluronate), chondroitin sulfate and its salts (e.g. magnesium chondroitin sulfate), chitosan and its derivatives (e.g. quaternary ammonium chitosan), gelatin and its derivatives (e.g. methacrylated gelatin), sodium alginate, tannic acid, asiaticoside, gallic acid or antimicrobial peptides.

[0017] The hydrogel has a pH of 6.0-7.5 and an osmotic pressure of 250-350 mOsm / L.

[0018] The above-mentioned multi-element active hydrogels can be prepared by mixing all raw materials evenly, or by dissolving them in batches and then mixing them, depending on their solubility properties.

[0019] One of the above-mentioned multi-functional active hydrogels can be used for eye care and the preparation of drugs for treating eye diseases.

[0020] A product comprising the above-mentioned hydrogel. The product may be a pharmaceutical, medical device, or cosmetic.

[0021] The above-mentioned hydrogel can be encapsulated in a container to prepare a topical product or coated on a base fabric to prepare a patch product.

[0022] The present invention has the following advantages: Significantly improves Exos stability: By introducing a composite crosslinking regulator (hydroxycarboxylic acid and its sodium salt) and a zwitterionic protectant, the damage to the Exos membrane structure caused by the aluminum ion crosslinking system is effectively reduced. After storage at 4°C for 28 days, the Exos particle retention rate is ≥85%, while existing technologies cannot completely preserve exosomes.

[0023] Achieving controlled sustained release: The composition of the present invention achieves stable sustained release of the active ingredient, with a cumulative release rate of 91% over 24 hours, no burst release phenomenon, and an appropriate duration of action, avoiding the phenomenon of incomplete Exo action due to excessively slow release rate.

[0024] Effective protection of biological activity: zwitterionic protectants form a protective microenvironment around Exos / CM through the hydration layer effect and the principle of "preferential exclusion", which can maintain the cell proliferation activity and anti-inflammatory activity of Exos.

[0025] Synergistic effect: The combination of Exos and CM produces a synergistic effect, which can significantly promote collagen synthesis in fibroblasts, inhibit matrix metalloproteinase expression, and effectively improve tear secretion and corneal repair in mice with dry eye syndrome.

[0026] Safe and non-invasive: The composition of this invention can achieve good function when applied topically without injection, and has low cytotoxicity, no skin irritation or sensitization, making it suitable for long-term care of sensitive skin around the eyes. Attached Figure Description

[0027] Figure 1 It represents the Exos release rate of different samples at different time points; Figure 2 This shows the effect of different samples on cell proliferation rate; in the figure, This indicates that p < 0.05 compared to the blank control. ## indicates p < 0.001 compared to the blank control, and ## indicates p < 0.01 compared to sample 2; Figure 3 This shows the expression of Col I in different treatment groups (ELISA); in the figure, This indicates that p < 0.05 compared to the blank control. # indicates p < 0.001 compared to the blank control, and # indicates p < 0.05 compared to sample 2; Figure 4 This shows the expression of MMP1 in different treatment groups (ELISA); in the figure, This indicates that p < 0.05 compared to the blank control. # indicates p < 0.001 compared to the blank control, and # indicates p < 0.05 compared to sample 2; Figure 5 SA-β-gal staining is used to detect the proportion of senescent cells; Figure 6 Masson staining of skin tissue from photoaging models under different treatment interventions; Figure 7 The expression of Col I in different treatment groups (immunohistochemistry); Figure 8 This represents the relative expression levels of inflammatory factors IL-6 and IL-8 mRNA; in the figure, This indicates that p < 0.05 compared to the model group. This indicates that p < 0.01 compared to the model group. # indicates p < 0.001 compared to the model group, # indicates p < 0.05 compared to sample 7, and ## indicates p < 0.01 compared to sample 7. Figure 9 It represents the relative expression level of miR-21 in the tissue. Detailed Implementation

[0028] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0029] Example 1: Preparation of hydrogel patch 1. Preparation of conditioned medium (CM) for MSCs Human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultured to 80-90% confluence using conventional methods, and then replaced with serum-free medium and cultured for another 48 hours. The supernatant was collected, centrifuged at 3000g for 15 min to remove cell debris, and then filtered through a 0.22μm filter membrane for sterilization. The supernatant was concentrated 50 times using a tangential flow process (30kDa molecular weight cutoff) to obtain CM, which was stored at 4℃ for later use.

[0030] 2. Preparation of hydrogel patches Weigh the different raw materials according to the formula in Table 1, and prepare the hydrogel patch according to the following method: (1) Tartaric acid and sodium tartrate were mixed in a molar ratio of 2:1.5 as a composite crosslinking regulator; betaine and trehalose were mixed in a mass ratio of 1:1 as a zwitterionic protectant; (2) First add glycerin, then add sodium polyacrylate P46NH, sodium polyacrylate P55N, aluminum hydroxyl, and disodium edetate in sequence, stir for 30 minutes to form a uniform paste semi-fluid with no visible particles, and obtain phase A; (3) Add crosslinking regulator and zwitterion protectant to water for injection and stir until dissolved; slowly add sodium hyaluronate while stirring, and finally turn on mechanical stirring to completely dissolve to obtain phase B; (4) Slowly add phase A to phase B while stirring with a mechanical stirrer for 10 minutes to form a uniform viscous gel; let the gel stand at 25°C and 60% humidity for 45 minutes to complete cross-linking and obtain the gel matrix. (5) Cool the gel matrix to 4-10℃, add hUC-MSC exosomes and 50× concentrated CM, mix gently; then coat it on the base fabric, cut it into appropriate shapes according to the application site, package it, and store it at 4℃.

[0031] Table 1 Different formulations 3. Stability of Exos in hydrogel dressings The prepared patch samples 1, 3, and 4 were stored at 4℃ for 0, 7, 14, and 28 days, respectively, and Exos were extracted from the gel. Method for extracting exosomes: Patches stored for different times (0 days, 7 days, 14 days, 28 days) were cut into small pieces of approximately 1 mm³ and added to pre-cooled PBS at a ratio of 1:10 (w / v). The mixture was then dispersed by shaking at 4°C. Hyaluronidase was added to the dispersion to a final concentration of 20 U / mL, and the mixture was incubated at 37°C for 30 minutes to degrade the sodium hyaluronate matrix and release exosomes. The supernatant was collected and centrifuged sequentially at 300×g (10 minutes), 2000×g (20 minutes), and 10000×g (30 minutes), collecting the supernatant each time and removing gel fragments and large particulate impurities. The supernatant was then ultracentrifuged at 100000×g for 70 minutes, and the precipitate was resuspended in PBS to obtain a purified exosome suspension.

[0032] The exosome suspension was used for nanoflow cytometry to detect the number of Exos particles (with the initial particle number set at 100%), and the retention rate was calculated.

[0033] Table 2. Exos retention rates in different samples As shown in the table above, sample 1 significantly improved the storage stability of Exo in sodium polyacrylate hydrogel through the synergistic effect of the composite crosslinking regulator and the zwitterionic protectant. After 28 days of storage, even at low temperatures, Exos was virtually undetectable in samples 3 and 4, indicating that the gel system had a destructive effect on exosomes, with extremely low detection values. This demonstrates that the introduction of the composite crosslinking regulator and the zwitterionic protectant significantly improved the storage stability of Exo in the gel matrix.

[0034] 4. Exos release behavior during hydrogel patch application Take equal weights (approximately 0.2-0.5 g) of patch samples 1, 3, and 4, and place them in 2 mL centrifuge tubes. Add 1 mL of PBS (pH 7.4). Fix the centrifuge tubes in a constant-temperature shaker at 32±1℃ and a shaking speed of 100 rpm for different incubation times (0.5, 1, 4, 8, 12, and 24 hours). Use a separate sample tube for each time point (i.e., use different EP tubes for different time points to avoid volume changes or contamination caused by repeated sampling). After incubation, collect all the released liquid and directly determine the number of exosome particles using a nanoflow cytometer. Calculate the release rate.

[0035] Sample 1 achieved the protection of Exos in the formulation through the synergistic effect of the composite crosslinking regulator and the zwitterionic protectant (4h release rate > 50%, 12-hour continuous release rate > 80%, no burst release). Compared with Sample 3 and Sample 4, as mentioned above, without the presence of the crosslinking compound and zwitterionic protectant, the gel system cannot protect Exos, leading to its rupture or degradation. Therefore, a high concentration of Exos could not be detected throughout the detection period.

[0036] Example 2: Application of hydrogel patches in improving skin Each group of patches prepared in Example 1 was immersed in PBS, incubated at 37°C for 24 hours, and centrifuged at 3000 rpm for 10 minutes to collect the extract as the test sample.

[0037] 1. Promotes cell proliferation HaCaT cells were cultured normally to 90% confluence, digested with trypsin, counted, and then the cell density was adjusted to 2 × 10⁶ cells / year. 4 Cells / mL. Add 100 μL of cell suspension and corresponding test samples to a 96-well plate, with 6 replicates per sample. Incubate at 37°C and 5% CO2 for 48 h. Then add 10 μL of CCK8 to each well and continue incubating for 4 h. Measure the absorbance at 450 nm using a microplate reader and calculate the proliferation rate (%) according to the following formula: Cell proliferation rate (%) = .

[0038] Cell proliferation rate was detected using the CCK-8 assay. Figure 2 The results showed that, with the blank control as the baseline, all groups except sample 5 promoted cell proliferation to some extent. Compared with the blank control, samples 1 and 2 had a significant proliferative capacity. ), and sample 1 showed the best results.

[0039] 2. Effects on collagen expression Human skin fibroblasts (HSF cells) were cultured to 60%-80% confluence, triedpsinized, and seeded into 6-well flat-bottomed cell culture plates. Cells were treated with extracts of different samples for 48 hours, and then the supernatant was collected for ELISA detection of Col I and MMP1. Separate blank wells (containing an equal volume of PBS), standard wells, and sample wells were prepared, with two parallel replicates for each group. After sample addition, incubation, washing, color development, and termination were performed, and the sample was immediately measured at a wavelength of 450 nm. OD Value. Different concentrations of the standard were used as... X Axis, corresponding OD Value Y The standard curve was plotted on the axis, and the logisic curve was selected as the fitting model to obtain the standard equation. The content of ColⅠ and MMP-1 was calculated by substituting the detected values ​​into the equation.

[0040] Sample 5 contained only the gel matrix, and this treatment group had the lowest Col I content, close to the blank group. Samples 1 and 2 showed a significant increase in Col I content, with Sample 1 showing the best effect. Matrix metalloenzyme 1 (MMP1) can degrade collagen. Samples 3-5 showed almost no inhibitory effect on MMP1, while samples 1 and 2 showed inhibitory effects on MMP1, with Sample 1 showing the best effect.

[0041] 3. SA-β-gal staining to detect the proportion of senescent cells HSF cells were at 10 5 Cells were seeded at a density of [number] cells / mL into 6-well plates. Once cell confluence reached 90% or higher, a senescence model was established by treating with 1 mM hydrogen peroxide for 3 hours. Cells were then washed with PBS and fixed with fixative at room temperature for 6-7 minutes. After PBS washing, X-gal staining working solution was added, and the plates were sealed with sealing film and incubated overnight at 37°C (CO2-free). The proportion of blue-positive cells (senescent cells) to the total cell count was observed and counted under a microscope.

[0042] The aging model revealed that the blank treatment group had the most senescent cells, while sample 5 (simple gel matrix) showed almost no significant ability to inhibit senescent cells. Although samples 3 and 4 inhibited the number of senescent cells to some extent, their effects were not as good as those of samples 1 and 2.

[0043] The above in vitro experiments demonstrate that when applied to the skin, the patch of the present invention can effectively promote fibroblast vitality, collagen synthesis, and delay cell aging, and is suitable for improving skin, especially fine lines around the eyes and skin laxity.

[0044] 4. Repair of photoaged skin in a mouse model A mouse dorsal skin photoaging model was established: nude mice were fixed, and a 5cm × 5cm area of ​​their backs was exposed, while the rest of the body was covered for protection; the minimum erythema dose (150mJ·(cm × cm)) was used to determine the photoaging effect of the mice. -1 Four ultraviolet lamps [TL20W / 12RS; 290-320nm] were used to irradiate the backs of mice at the same time every day, once a day, with a one-day interval every six days. During the modeling process, mice were randomly divided into groups, and different samples were applied to the irradiated areas after each irradiation for four consecutive weeks; nude mice that were not irradiated and did not have samples applied served as a control group. After the experiment, the skin elasticity of the different treatment groups was measured, and then the mice were sacrificed, and samples were taken from the irradiated areas for Masson staining to observe the arrangement of collagen fibers; the expression level of type I collagen (Col I) was observed by immunohistochemical staining.

[0045] Table 3 Comparison of skin elasticity parameters of mice in different groups using the skin analyzer (x̄ ± SD, n=10) Note: This indicates a significant difference compared to sample 5 (p<0.05). The value indicates a highly significant difference compared to sample 5 (p<0.01). # indicates a significant difference compared to the blank group (p<0.05), and ## indicates a highly significant difference compared to the blank group (p<0.01).

[0046] Analysis of the data in the table above shows that the total elasticity, net elasticity, and bioelasticity of sample 5 were significantly lower than those of the control group, decreasing by 31.1%, 33.3%, and 52.9%, respectively. The decrease in bioelasticity was particularly large, indicating successful model establishment. Meanwhile, sample 3 did not show a significant improvement, suggesting a compatibility issue between Exos and the comparative gel, leading to functional loss or degradation. Sample 2 showed a significantly better repair effect than sample 3, but it did not fully recover to the control group level, especially in bioelasticity. Sample 1 exhibited the best recovery ability across all elasticity indices, with total elasticity and net elasticity even surpassing the control group, and bioelasticity closest to the control group, showing a highly significant difference (p<0.01).

[0047] Figure 6 The results showed that after using sample 5, photoaging damage led to disordered collagen fiber arrangement and epidermal thickening, consistent with the histological changes in the photoaging mouse model: the epidermal thickness was significantly increased compared to the control group (P<0.05); this was manifested as reduced epidermal thickness and collagen fiber breakage. No significant increase in collagen was observed after using sample 3. In contrast, after using samples 1 and 2, the epidermal thickening was improved, the collagen fiber arrangement tended to normalize, and the number of collagen fibers increased; among them, sample 1 showed the most significant effect.

[0048] Depend on Figure 7 It can be seen that the blank group showed abundant brownish-yellow positive granules with high staining intensity; the positive expression of samples 5 and 3 was significantly reduced, and the staining was light. Combined with the results of related studies showing that the dermal collagen fiber bundles of photoaging model mice had structural disorder and abnormal arrangement with significant differences from the control group, it indicates that photoaging leads to a significant decrease in Col I content; sample 2 can promote Col I expression, with a significant increase in positive signal and partial recovery of fiber structure; sample 1 has a more significant effect, with dense brownish-yellow granules and staining intensity close to or even exceeding that of the blank group, and the collagen fiber arrangement tends to be more regular.

[0049] The above animal model results demonstrate that the patch of the present invention can improve skin laxity caused by photoaging through local application, and is suitable for anti-aging care around the eyes.

[0050] Example 3: Preparation of hydrogel patch 1. Preparation of exosomes loaded with exogenous nucleic acids (miR-21 Exos) Take purified exosomes (approximately 10) 11 The particles were resuspended in 400 μL of electroporation buffer (formulation: 1.15 mM KH2PO4, 25 mM K2HPO4, 2 mM MgCl2, pH 7.2), and 5 nmol of chemically synthesized miR-21 mimic (5′-UAGCUUAUCAGACUGAUGUUGA-3′) was added. After mixing, electroporation was performed using a Gene Pulser Xcell (Bio-Rad) at 250 V, 200 μF, and single-pulse mode, with a pulse duration of approximately 20 ms. After electroporation, the samples were placed on ice for 5 min to allow the membrane structure to recover, and RNase A (final concentration 10 μg / mL) was added and incubated at 37 °C for 30 min to remove unencapsulated free nucleic acids. Centrifuge at 100,000 × g at 4 °C for 70 min, discard the supernatant, resuspend the precipitate with PBS, centrifuge again at 100,000 × g for 70 min, wash, and finally resuspend with an appropriate amount of PBS and filter through a 0.22 μm filter membrane for sterilization.

[0051] 2. Preparation of conditioned medium (CM) for MSCs Human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultured to 80-90% confluence using conventional methods, and then replaced with serum-free medium and cultured for another 48 hours. The supernatant was collected, centrifuged at 3000g for 15 min to remove cell debris, and then filtered through a 0.22μm filter membrane for sterilization. The supernatant was concentrated 50 times using a tangential flow process (30kDa molecular weight cutoff) to obtain CM, which was stored at 4℃ for later use.

[0052] 3. Preparation of hydrogel patches Weigh the different raw materials according to the formula in Table 3' and prepare the hydrogel patch according to the following method: (1) Tartaric acid and sodium tartrate were mixed in a molar ratio of 2:1.5 as a composite crosslinking regulator; betaine and trehalose were mixed in a mass ratio of 1:1 as a zwitterionic protectant; (2) First add glycerin, then add sodium polyacrylate P46NH, sodium polyacrylate P55N, aluminum hydroxyl, and disodium edetate in sequence, stir for 30 minutes to form a uniform paste semi-fluid with no visible particles, and obtain phase A; (3) Add crosslinking regulator and zwitterion protectant to water for injection and stir until dissolved; slowly add sodium hyaluronate while stirring, and finally turn on mechanical stirring to completely dissolve to obtain phase B; (4) Slowly add phase A to phase B while stirring with a mechanical stirrer for 10 minutes to form a uniform viscous gel; let the gel stand at 25°C and 60% humidity for 45 minutes to complete cross-linking and obtain the gel matrix. (5) Cool the gel matrix to 4-10℃, add hUC-MSC exosomes and 50× concentrated CM, mix gently; then coat it on the base fabric, cut it into appropriate shapes according to the application site, package it, and store it at 4℃.

[0053] Table 3' Different Formulas Example 4: Application of hydrogel patches in the treatment of dry eye syndrome 1. Anti-inflammatory effect Each group of patches prepared in Example 3 was immersed in PBS, incubated at 37°C for 24 h, and centrifuged at 3000 rpm for 10 min to collect the extract as the test sample.

[0054] An inflammation model was induced using human meibomian gland epithelial cells (HMGEC) with LPS (1 μg / mL): Human meibomian gland epithelial cells (HMGEC) in the logarithmic growth phase were harvested, digested with trypsin, counted, and the cell density was adjusted to an appropriate concentration (e.g., 2 × 10⁻⁶). 4 HMGEC inflammation model cells were seeded into culture plates and cultured at 37°C in a 5% CO2 incubator until the cells reached 70%-80% confluence. The original culture medium was discarded, and fresh culture medium containing lipopolysaccharide (LPS, final concentration 1 μg / mL) was added. The cells were then incubated for another 24 hours to obtain the HMGEC inflammation model cells.

[0055] After induction, the cells were lysed and total RNA was extracted using a kit. Then, the mRNA expression levels of inflammatory factors IL-6 and IL-8 were detected by qRT-PCR.

[0056] pass Figure 8It was found that samples 8 and 9 did not inhibit the important pro-inflammatory factors IL-6 and IL-8; their lack of therapeutic effect is likely due to impaired or ineffective Exos function in the formulation. Samples 6 and 7 significantly inhibited the expression of IL-6 and IL-8, with sample 6 showing the best effect.

[0057] 2. Therapeutic effect on mouse model of dry eye syndrome A mouse model of dry eye was induced using benzalkonium chloride (BAC): BAC was prepared into a 0.2% (w / v) solution with physiological saline, and 2 μL was instilled into each eye once at 9:00 AM and 5:00 PM daily for 21 consecutive days. Tear secretion and corneal fluorescein staining were performed on day 7 of modeling. A successful model was defined as a ≥50% decrease in tear secretion from baseline and a corneal staining score ≥3 points (out of 12).

[0058] Starting from day 8 of modeling, mice that successfully developed the model were randomly divided into groups. After being lightly anesthetized with isoflurane, the eye patch samples 6-9 prepared in Example 3 were applied to the corneas and ocular surfaces of both eyes with sterile forceps, and gently pressed to adhere. The patches were removed after 30 minutes. The treatment was administered once daily for 14 consecutive days. The blank control group consisted of healthy mice that only had PBS added to keep their corneas moist.

[0059] On day 7 (day 0) of modeling, day 7 of treatment, and day 14 of treatment, tear secretion was measured using the phenol red cotton thread method. A phenol red cotton thread was placed at the outer canthus of the mouse eyelid, left for 30 seconds, and then removed. The length (mm) of the thread soaked in tear fluid and turned red was measured, and the average value was taken for each eye. At the same time points, 1 μL of 0.1% sodium fluorescein solution was instilled into both eyes, rinsed after 1 minute, and the corneal epithelial defects were observed under cobalt blue slit lamp light. The cornea was divided into four quadrants and scored according to the degree of staining: 0 points = no staining; 1 point = scattered punctate staining; 2 points = dense but non-fused punctate staining; 3 points = patchy fused staining. The total score ranged from 0 to 12 points, with higher scores indicating more severe damage. After the experiment, the mice were sacrificed, the corneal tissue was dissected, and total RNA was extracted for qRT-PCR to detect the miR-21 content.

[0060] Table 4. Tear secretion and corneal fluorescein staining scores of mice in each group (Mean±SD, n=10) Note: Compared with the blank group, P<0.05, P < 0.01, P < 0.001; compared with sample group 6, #P < 0.05.

[0061] On day 7 of modeling, the tear secretion of each modeling group was significantly lower than that of the control group (P<0.001), and the fluorescein staining score was significantly higher than that of the control group (P<0.001). Furthermore, the tear secretion decreased by ≥50% from the baseline and the corneal staining score was ≥3 points, indicating that the dry eye model was successfully established.

[0062] In the Sample 6 treatment group, tear secretion (5.6±0.3 mm) and fluorescein staining score (1.1±0.3 points) both recovered to levels not significantly different from the control group (P≥0.05), and were significantly better than the Sample 7 group in both indicators (P<0.05), indicating that Sample 6 has excellent therapeutic effects on dry eye syndrome. In the Sample 7 treatment group, after 14 days of administration, tear secretion was 4.9±0.4 mm, showing a highly significant difference compared to the control group (P<0.01); the fluorescein staining score was 3.5±0.6, also showing a highly significant difference compared to the control group (P<0.01), indicating that the therapeutic effect was inferior to that of Sample 6.

[0063] After 14 days of drug administration, the tear secretion volume (3.2±0.3 mm and 3.5±0.4 mm, respectively) in both sample 8 and sample 9 treatment groups was significantly different from that in the control group (P<0.05), and the fluorescein staining scores (8.9±1.2 and 7.8±0.9, respectively) were also significantly different from those in the control group (P<0.05), indicating that both treatments have limited therapeutic effects on dry eye syndrome.

[0064] The above animal experiments demonstrated that exosome eye patches without amphoteric protective agents were essentially ineffective against dry eye syndrome, with no difference in tear secretion (3.2±0.3 mm) compared to the model group (3.5±0.4 mm). Eye patches containing only single cross-linked exosomes showed significantly lower tear secretion (3.5±0.4 mm) and corneal staining scores (7.8±0.9 points) compared to those containing a composite cross-linked agent.

[0065] qRT-PCR results ( Figure 9 The results showed that the relative expression level of miR-21 in the corneal tissue of sample group 6 (6.2±0.7) was significantly higher than that in the blank control group (1.0±0.1), sample group 9 (1.5±0.2), sample group 8 (1.2±0.2) (P<0.001) and sample group 7 (3.5±0.3) (P<0.05), proving that miR-21 Exos in the eye patch can effectively deliver miR-21 to the corneal tissue and exert targeted anti-inflammatory and repair-promoting effects.

[0066] The above results indicate that the exosome eye patch prepared using the gel matrix (sample 7) has a synergistic effect, with all indicators significantly superior to those of sample 8. The combination of the gel matrix and exosomes further amplifies the therapeutic advantages of Exos, achieving a synergistic effect. The exosome eye patch with added CM on the gel matrix (sample 6) showed that tear secretion recovered to 5.6±0.3 mm, and the corneal staining score decreased to 1.1±0.3 points, close to the blank control group. The expression level of miR-21 in corneal tissue (6.2±0.7) was significantly higher than that in other groups (P<0.05, P<0.0001), confirming its significant anti-inflammatory and repair-promoting advantages.

[0067] The above results demonstrate that the eye patch of the present invention can improve meibomian gland dysfunction and treat dry eye syndrome through its anti-inflammatory effect.

[0068] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A hydrogel containing multiple active ingredients, comprising water and the following raw materials in parts by weight: 10-250 parts of gel matrix forming agent Crosslinking agent 0.5-5 parts, Crosslinking regulator 0.5-5 parts, Stabilizer 1-5 parts, 1-5 parts of zwitterionic protectant 200-800 parts of moisturizer Active ingredient: 0.1-600 parts; The gel matrix forming agent is sodium polyacrylate; The crosslinking agent is selected from aluminum salts or calcium salts; The crosslinking regulator is selected from a combination of hydroxycarboxylic acids and their corresponding salts; the salt is a sodium salt or a potassium salt; the stabilizer is a metal ion complexing agent. The zwitterionic protectant is selected from at least two of betaine, proline and trehalose; The humectant is a polyol; The active ingredient is selected from a combination of extracellular vesicles and conditioned medium.

2. The hydrogel according to claim 1, characterized in that, The extracellular vesicles or conditioned medium are derived from at least one of animal cells, plant cells, and probiotics; preferably, mesenchymal stem cells, induced pluripotent stem cells, immune cells, corneal epithelial cells, fibroblasts, and precursor cells of the above cells, cells of carrots, onions, wolfberries, blueberries, grapes, licorice, chrysanthemums, ginger, wolfberries, cassia seeds, and buddleja flowers, and at least one of bifidobacteria and galactomyces.

3. The hydrogel according to claim 1, characterized in that, The extracellular vesicles are naturally occurring or engineered extracellular vesicles; the engineered extracellular vesicles are extracellular vesicles loaded with small molecule compounds, nucleic acids or proteins by means of electroporation, co-incubation, sonication or extrusion.

4. The hydrogel according to claim 1, characterized in that, The extracellular vesicles are 0.1-100 parts by weight, and the mass fraction is calculated based on the total protein content of the extracellular vesicles; the amount of the conditioned medium added is 50-500 parts.

5. The hydrogel according to claim 4, characterized in that, The conditioned medium is obtained by concentrating the cells 20-100 times after separating them from the culture medium following cell culture.

6. The hydrogel according to claim 1, characterized in that, The grade of the sodium polyacrylate is selected from one or more of P46NH, P55N, NP-700, NP-600, and NP-800; preferably, it is a combination of sodium polyacrylate P46NH and sodium polyacrylate P55N. The crosslinking agent is aluminum hydroxyl or calcium chloride; The hydroxycarboxylic acid is selected from one or more of tartaric acid, citric acid, malic acid, gluconic acid, lactic acid, and glycolic acid; the mass ratio of the hydroxycarboxylic acid to the corresponding hydroxycarboxylic acid is (1-3):(1-2); The metal ion complexing agent is selected from one or more of disodium edetate, calcium sodium edetate, tetrasodium edetate, diethylenetriaminepentaacetic acid, pentasodium diethylenetriaminepentaacetic acid, nitrotriacetic acid and its salts, and ethylenediamine-N,N'-disuccinic acid and its salts; preferably disodium edetate and / or calcium sodium edetate. The polyol is selected from ethylene glycol and / or glycerol; The hydrogel has a pH value of 6.0-7.5; The hydrogel has an osmotic pressure of 250-350 mOsm / L.

7. The hydrogel according to claim 6, characterized in that, The mass ratio of sodium polyacrylate P46NH to sodium polyacrylate P55N is (20-1):1 or (3-5):

1.

8. The hydrogel according to claim 1, characterized in that, The hydrogel also includes other active ingredients; the other active ingredients are one or more of plant extracts or clinical drugs with antimicrobial, antioxidant or anti-inflammatory activities; the other active ingredients are preferably hyaluronic acid and its salts, chondroitin sulfate and its salts, chitosan and its derivatives, gelatin and its derivatives, sodium alginate, tannic acid, asiaticoside, gallic acid or antimicrobial peptides.

9. The use of the hydrogel as described in any one of claims 1-8 in eye care and the preparation of medicaments for treating eye diseases, and products thereof.

10. The product according to claim 9, characterized in that, The product is a pharmaceutical, medical device, or cosmetic; the product is a topical or patch product.

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