A composite hydrogel and a preparation method and application thereof

Abstract

The present application relates to the technical field of biomaterials for eyes, in particular to a composite hydrogel and a preparation method and application thereof. The composite hydrogel is prepared from an extracellular vesicle solution, a rhein solution, a calcium chloride solution and a sodium hyaluronate solution; the concentration of the rhein in the hydrogel is 0.7mM-24.6mM, the concentration of the calcium chloride is 0.10mM-0.5mM, the final concentration of the sodium hyaluronate is 1.0%-1.5% by mass percentage, and the concentration of the extracellular vesicle is 1.9x10 6 particles / mL-1.9x10 11 particles / mL; the extracellular vesicle is a notoginseng-derived extracellular vesicle prepared from notoginseng. The composite hydrogel of the present application delivers the notoginseng-derived extracellular vesicle to the cornea through the slow-release effect of the notoginseng-derived extracellular vesicle, and the notoginseng-derived extracellular vesicle is uniformly embedded and slowly released in the gel pores, thereby prolonging the residence time of the notoginseng-derived extracellular vesicle on the surface of the cornea.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a composite hydrogel, its preparation method, and its applications. Background Technology

[0002] As the eye's first physical and biochemical barrier, the cornea is frequently exposed to various harmful environmental factors, making it susceptible to damage from ultraviolet radiation, chemicals, pathogenic microorganisms, and other harmful influences. Oxidative stress is a common mechanism in many pathological states of the cornea. Excessive production or impaired clearance of reactive oxygen species such as hydrogen peroxide in corneal epithelial cells can lead to lipid peroxidation, protein denaturation, and DNA damage, ultimately causing apoptosis and tissue damage. Long-term oxidative damage can lead to corneal ulcers, corneal opacity, and even decreased vision or blindness.

[0003] Currently, there are several limitations to medications used clinically to treat corneal oxidative damage. First, the drugs have a short residence time on the corneal surface and low bioavailability. Traditional eye drops only remain on the ocular surface for a few minutes, with most of the drug being rapidly flushed out with tears. To maintain effective drug concentrations, patients need to administer the medication multiple times daily, leading to poor adherence and potential local irritation. Second, there is a lack of multi-target synergistic drug delivery systems for corneal damage repair. Specific treatments for oxidative stress are limited and have single mechanisms of action. Existing antioxidants, such as N-acetylcysteine ​​eye drops and vitamin E eye drops, typically target a single antioxidant pathway, making it difficult to address the complex pathological processes of oxidative damage.

[0004] In addition, hydrogel delivery systems for the eye have received widespread attention in recent years. However, the components of these hydrogels are mostly synthetic polymers with limited biocompatibility, and some of them have complex preparation processes and high costs.

[0005] In summary, existing medications for treating corneal oxidative damage have a short residence time on the corneal surface. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a composite hydrogel, its preparation method, and its application, thereby solving the problem of short drug delivery time in the prior art.

[0007] The technical solution adopted in this invention is: a composite hydrogel, which is made of extracellular vesicle solution, rhein solution, calcium chloride solution and sodium hyaluronate solution; The hydrogel contains rhein at concentrations of 0.7 mM to 24.6 mM, calcium chloride at concentrations of 0.10 mM to 0.5 mM, sodium hyaluronate at a final concentration of 1.0% to 1.5% by mass, and extracellular vesicles at a concentration of 1.9 × 10⁻⁶. 6 Particles / mL ~1.9×10 11 Particles / mL; The extracellular vesicles are Panax notoginseng-derived extracellular vesicles obtained from Panax notoginseng.

[0008] Preferably, the method for preparing the Panax notoginseng-derived extracellular vesicles is as follows: the supernatant is obtained by juicing the roots of Panax notoginseng; the supernatant is extracted with sucrose solution to obtain a crude extract; and the crude extract is purified to obtain the Panax notoginseng extracellular vesicles.

[0009] Preferably, when extracting the supernatant, a gradient sucrose solution with mass fractions of 27% and 68% is used.

[0010] Preferably, during purification, a gradient of sucrose solutions with mass fractions of 8%, 30%, 45%, and 60% are used.

[0011] Furthermore, the present invention also provides a method for preparing the composite hydrogel, wherein rhein solution and CaCl2 solution are mixed and stirred evenly, Panax notoginseng extracellular vesicle suspension is added and mixed evenly, and the mixture is slowly added to sodium hyaluronate solution under stirring conditions and mixed evenly to obtain composite hydrogel.

[0012] Preferably, the rhein solution is obtained by dissolving rhein in PBS.

[0013] Furthermore, the present invention also provides the use of the composite hydrogel in the preparation of medicaments for protecting the cornea or treating corneal damage.

[0014] Preferably, the drug relieves corneal ulcers or corneal opacity.

[0015] Furthermore, the present invention also provides a medicament for protecting the cornea or treating corneal damage, the medicament comprising the aforementioned composite hydrogel.

[0016] Furthermore, the present invention also provides a composition comprising the composite hydrogel or the drug.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes a composite hydrogel composed of rhein, calcium ions, and sodium hyaluronate to deliver Panax notoginseng-derived extracellular vesicles to the cornea via a sustained-release effect. The Panax notoginseng-derived extracellular vesicles are uniform in size and spherical in shape. The rhein hydrogel exhibits a three-dimensional porous network structure with uniformly connected pores, which facilitates the uniform embedding and slow release of the Panax notoginseng-derived extracellular vesicles within the gel channels, thereby prolonging the residence time of the vesicles on the corneal surface.

[0018] Furthermore, rhein hydrogel has high viscoelasticity, which facilitates the formation of a stable adhesion layer on the corneal surface to prevent the extracellular capsules of Panax notoginseng from being lost with tears, thereby improving its bioavailability.

[0019] The rhein hydrogel not only continuously releases vesicles but also promotes the antioxidant activity of Panax notoginseng-derived extracellular vesicles. As an anthraquinone compound, rhein possesses free radical scavenging and metal chelating abilities, providing a favorable antioxidant microenvironment. This environment helps protect Panax notoginseng-derived extracellular vesicles from oxidative inactivation, maintaining vesicle activity. Simultaneously, it works synergistically with the antioxidant components inherent in the Panax notoginseng-derived extracellular vesicles themselves, producing an enhanced antioxidant protective effect. Therefore, the hydrogel system of this invention achieves the comprehensive therapeutic advantages of "delayed delivery + functional synergy" without increasing the frequency of drug administration. Attached Figure Description

[0020] Figure 1 This is a particle size distribution diagram of PDNs; Figure 2 Morphological results for PDNs; Figure 3 Fourier transform infrared spectrum of rhein hydrogel; Figure 4 The morphological results are for rhein hydrogel; Figure 5 The rheological characteristics of rhein hydrogel are shown in Figure 1. In Figure 2, A represents the low-frequency oscillation frequency scan result, B represents the high-frequency oscillation frequency scan result, and C represents the steady-state shear test result. Figure 6 Analysis of PDNs release from PDNs@rhein composite hydrogel; Figure 7 The results are for ROS level detection. Figure 8 The results show the cell apoptosis results. A is a schematic diagram of the apoptosis rate measured by flow cytometry in the control group; B is a schematic diagram of the apoptosis rate measured by flow cytometry in the H2O2-damaged group; C is a schematic diagram of the apoptosis rate measured by flow cytometry in the PDNs@rhein composite hydrogel group; and D is a statistical analysis of the apoptosis rates in the three groups. Figure 9 This refers to the cell viability test results; Figure 10 The results are for the detection of the inflammatory factor IL-1β. Figure 11 The results show the detection results of the inflammatory factor TNF-α. Detailed Implementation

[0021] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0022] Example 1: Preparation of Panax notoginseng-derived extracellular vesicles (PDNs) Extracellular vesicles were extracted from Panax notoginseng using density gradient centrifugation. The specific steps are as follows: Primary separation: After washing the roots of Panax notoginseng, put them into a juicer to collect the fresh juice. Filter the juice through gauze to remove large pieces of plant debris. Centrifuge the filtered solution at 2000g for 20 minutes and collect the supernatant. Then centrifuge at 10000g and 4℃ for 1 hour using an ultracentrifuge to obtain a dark brown, transparent supernatant for later use.

[0023] Sucrose gradient separation: 2 mL of 27% and 68% sucrose solutions were sequentially placed into 38.6 mL centrifuge tubes, followed by the addition of 34 mL of the supernatant prepared from the primary separation, resulting in a mixed solution with a distinct stratified interface. The mixed solution was centrifuged at 100,000 g and 4°C for 1.5 hours, and the band exceeding the 68% sucrose layer was collected to obtain the crude extract.

[0024] Purification: 2 mL of 8% sucrose, 2 mL of 30% sucrose, 2 mL of 45% sucrose, and 2 mL of 60% sucrose were placed sequentially into centrifuge tubes to form a continuous gradient layer, yielding a sucrose gradient solution. 29 mL of the crude extract was slowly added to the sucrose gradient solution, and the mixture was centrifuged at 200,000 g and 4°C for 1.5 hours. The precipitate was collected to obtain Panax notoginseng-derived extracellular vesicles, denoted as PDNs.

[0025] Choosing polarization densities of 27% and 68% allows for rapid trapping of target vesicles near the interface between the two layers, facilitating one-time enrichment and transfer and reducing the volumetric burden in subsequent fine gradients. The isodensity band of exosomes / vesicles generally appears in the 30%-45% sucrose range. By setting four density segments of 8%, 30%, 45%, and 60% for purification, the target PDNs are enriched at the 30%-45% interface, which best matches their buoyancy density. At the same time, lighter or heavier contaminants remain at the upper or lower interface, respectively, resulting in a higher purity vesicle population.

[0026] Characterization of PDNs: PDNs were resuspended in PBS and mixed thoroughly to prepare a homogeneous PDNs solution. The size of the vesicles was then measured using a nanoparticle size analyzer. The results are as follows: Figure 1 As shown, the size of the PDNs is 153nm.

[0027] Morphological observation was performed using transmission electron microscopy to observe the morphological characteristics of PDNs.

[0028] The precipitate was resuspended in PBS to obtain a concentration of approximately 1.9 × 10⁻⁶. 11PDNs solution with particles / mL. Add 10 μL to a copper grid, let stand for 1 min for adsorption, and blot off excess liquid with filter paper. Add 10 μL of uranium acetate to the copper grid for precipitation, let stand for 1 min, and blot off any remaining liquid with filter paper. Let stand for a few minutes to dry, and observe using a transmission electron microscope. Results are as follows: Figure 2 As shown, PDNs are uniform in size and have a spherical structure.

[0029] Example 2: Preparation of hydrogel containing rhein, calcium ions, and sodium hyaluronate First, prepare a 24.6 mM rhein solution, a 100 mM CaCl2 solution, and a 3% (w / w) sodium hyaluronate solution. Mix 4.27 mL of the rhein solution with 0.03 mL of the CaCl2 stock solution and stir until homogeneous. Then, while stirring, slowly add the mixture to 5 mL of the 3% (w / w) sodium hyaluronate solution. Finally, add PBS to bring the volume to 10 mL. The resulting rhein hydrogel is then obtained.

[0030] The 24.6 mM rhein solution was prepared by weighing 70.0 mg, adding PBS, sonicating to dissolve, and then bringing the volume to 10.0 mL. 100mM CaCl2 solution: 111mg CaCl2 dissolved in 10mL PBS; 3% sodium hyaluronate: Dissolve 150 mg in an appropriate amount of PBS and bring the volume to 5.0 mL.

[0031] 1. Fourier Transform Infrared Spectroscopy (FTIR) To analyze the gelation mechanism of rhein hydrogel and the roles of calcium ions and sodium hyaluronate in gelation, Fourier transform infrared spectroscopy (FTIR) was used to test three groups of samples: Group 1: 10 mM rhein + 0.3 mM calcium ions; Group 2: 10 mM rhein + 1.5% sodium hyaluronate; and Group 3: 10 mM rhein + 0.3 mM calcium ions + 1.5% sodium hyaluronate. The scanning range was 400-4000 cm⁻¹. -1 KBr was mixed with the powder sample to prepare a thin film, which was used as a blank control.

[0032] The results are as follows Figure 3 As shown, group 1 exhibits characteristic peaks of carboxylate, with broadening of the O–H stretching band and a shift in the C=O region with decreased intensity, suggesting deprotonation of the carboxyl / phenolic hydroxyl groups of rhein and their interaction with Ca. 2+ Coordination occurs. Group 2 shows a significant broadening of the O–H / N–H band and enhanced C–O-related absorption, indicating the formation of a hydrogen bond network between rhein and sodium hyaluronate. Group 3 comprehensively presents and reinforces the above changes: the O–H / N–H band further broadens, the C=O region continues to shift, the C–O region is enhanced, and the carboxylate absorption separation is clearer, indicating that Ca… 2+A bridging coordination is formed between the carboxylate site of sodium hyaluronate and the oxygen-containing group of rhein, and a three-dimensional cross-linked network is constructed in synergy with the hydrogen bonds between rhein and sodium hyaluronate.

[0033] In summary, this invention utilizes Ca 2+ Coordination crosslinking and multi-point hydrogen bonding between rhein and sodium hyaluronate are used to construct a stable three-dimensional hydrogel structure and endow it with controlled drug release properties.

[0034] 2. Morphological characterization 10 μL of rhein hydrogel was freeze-dried, and its microstructure was then observed using a scanning electron microscope. The results are as follows: Figure 4 As shown, the rhein hydrogel exhibits a three-dimensional porous network structure with uniformly connected pores.

[0035] 3. Rheological properties The rheological behavior of rhein hydrogels was evaluated using a rheometer. The state of the rhein hydrogel was represented by measuring the relationship between the storage modulus and the loss modulus, denoted as G' and G”. When G' > G”, the sample was in a gel state; when G' < G”, the sample was in a solution state.

[0036] The results are as follows Figure 5 As shown, the rhein hydrogel exhibited a G'>G'' ratio across the entire testing frequency range, demonstrating an elastic-dominant characteristic and indicating the formation of a stable three-dimensional gel network. With increasing frequency, both G' and G'' increased synchronously, and their difference widened, indicating that the network maintains good structural integrity under high-frequency perturbations, which is beneficial for long-term retention on the ocular surface. Steady-state shear testing showed that the apparent viscosity decreased significantly with increasing shear rate, exhibiting typical shear-thinning characteristics. This indicates that the rhein hydrogel has good flowability during high-shear processes such as dripping or extrusion. Under low-shear static conditions, it can maintain high viscoelasticity, facilitating the formation of a stable adhesion layer on the corneal surface, reducing drug loss, and improving drug bioavailability.

[0037] The above experiments show that rhein hydrogel has properties such as elasticity dominance and shear thinning, making it suitable for ocular drug delivery and achieving retention and sustained release on the ocular surface.

[0038] Example 3: Preparation of PDNs@Rhein composite hydrogel and its protective effect against corneal oxidative damage. 1. Preparation of PDNs@Rhein composite hydrogel First, prepare a 24.6 mM rhein solution, a 100 mM CaCl2 solution, a 3% (w / w) sodium hyaluronate stock solution, and 1.9 × 10⁻⁶ ppm sodium hyaluronate solution. 11 PDNs with particle size per mL are prepared as follows: 24.6mM rhein solution: Weigh 70.0mg, add PBS, sonicate to dissolve, and bring the volume to 10.0mL; 100mM CaCl2 solution: 111mg CaCl2 dissolved in 10mL PBS; 3% sodium hyaluronate: Dissolve 150 mg in an appropriate amount of PBS and bring the volume to 5.0 mL.

[0039] 4.27 mL of rhein solution and 0.03 mL of CaCl2 stock solution were mixed and stirred until homogeneous. Then, 0.1 mL of PDNs suspension was added and mixed thoroughly. Next, under stirring, the mixture was slowly added to 5 mL of 3% (w / w) sodium hyaluronate solution. Finally, 0.6 mL of PBS was added to bring the volume to 10 mL, and the mixture was stirred to obtain the PDNs@rhein composite hydrogel. In the PDNs@rhein composite hydrogel, the concentration of rhein was 10.5 mM, the concentration of CaCl2 solution was 0.3 mM, the concentration of sodium hyaluronate was 1.5% (w / w), and the concentration of PDNs was 1.9 × 10⁻⁶. 9 Particles / mL.

[0040] 2. PDNs Release Analysis One mL of PDNs@rhein composite hydrogel was placed in a dialysis bag, sealed, and then placed in a beaker containing artificial tears. The bag was shaken at a constant speed on a horizontal shaker at 37°C. Samples were then collected at 0, 4, 8, 12, 24, 48, and 72 h and analyzed using a BCA kit to assess the release of PDNs.

[0041] The results are as follows Figure 6 As shown, the PDNs encapsulated in this PDNs@rhein composite hydrogel exhibit long-term sustained-release characteristics, which is beneficial to the long-lasting effect of PDNs.

[0042] 3. Establish a model of H2O2-induced oxidative damage in corneal epithelial cells. Human corneal epithelial cells (HCE-T) were cultured at 37°C in a 5% CO2 incubator. Once the cells reached 80% confluence, an oxidative damage model was established by treating the HCE-T cells with 400 μM H2O2.

[0043] The experiment was set up with the following groups: Normal control group: No treatment was given; H2O2 damage group: treated with 400μM H2O2 for 6h; PDNs@rhein composite hydrogel: PDNs@rhein composite hydrogel loaded with 10 μM PDNs was subjected to protective treatment while being treated with H2O2.

[0044] 4. Cell viability detection Cell viability was assessed using a CCK-8 assay kit. After treatment as described above, 10 μL of CCK-8 solution was added to each well, and the absorbance was then measured using a microplate reader.

[0045] The results are as follows Figure 9 As shown, compared with the control group, the cell survival rate of the H2O2-damaged group was significantly reduced, while the PDNs@rhein composite hydrogel treatment group showed significant recovery, indicating that it has a significant protective effect against H2O2-induced cell damage.

[0046] 5. ROS level detection Intracellular ROS levels were detected using a ROS detection kit. Three groups of cells were treated with the DCFH-DA fluorescent probe and incubated at 37°C in the dark for 30 min. Intracellular ROS levels in different groups were then analyzed using flow cytometry.

[0047] like Figure 7 As shown, the results indicate that H2O2 treatment significantly increased intracellular ROS levels compared to the control group. In contrast, PDNs@rhein composite hydrogel treatment significantly reduced intracellular ROS levels, demonstrating a good antioxidant effect.

[0048] 6. Inflammatory factor detection Cell culture supernatants from each group were collected, and the expression levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected using ELISA kits.

[0049] like Figure 10 and Figure 11 As shown, the results indicate that hydrogel treatment significantly reduced the levels of TNF-α and IL-1β, suggesting that the PDNs@rhein composite hydrogel can effectively inhibit oxidative stress-induced inflammatory responses.

[0050] 7. Apoptosis detection Apoptosis was detected using the Annexin V-FITC / PI kit. Cells from each group were treated separately, digested with trypsin to collect cells, washed with PBS, and then incubated with 5 μL Annexin V-FITC and 5 μL PI at room temperature in the dark for 30 min. Flow cytometry was then used for analysis.

[0051] Experimental results are as follows Figure 8 As shown, compared with the control group, the apoptosis rate in the H2O2-damaged group was significantly increased, while the apoptosis rate was significantly reduced after treatment with PDNs@rhein composite hydrogel. This confirms the anti-apoptotic protective effect of PDNs@rhein composite hydrogel on corneal epithelial cells.

[0052] In recent years, plant-derived extracellular vesicles (PDNs) have attracted widespread attention due to their abundant sources, low immunogenicity, and good therapeutic effects. Panax notoginseng is a traditional and precious Chinese medicine with a long history of use in treating eye diseases. Panax notoginseng-derived PDNs, as a novel plant-derived nanodelivery carrier, contain various effective active ingredients and exhibit low immunogenicity and good therapeutic effects. However, research on the application of PDNs in ophthalmology, especially in the prevention and treatment of corneal oxidative damage, is still relatively limited. How to effectively deliver PDNs to the cornea and exert their biological activity is a key scientific problem that urgently needs to be solved. This invention delivers PDNs to the cornea mainly due to the sustained-release effect of a rhein hydrogel composed of rhein, calcium ions, and sodium hyaluronate on extracellular vesicles. The rhein hydrogel not only continuously releases vesicles but also possesses antioxidant properties.

[0053] Rhein is an anthraquinone compound isolated from rhubarb. Rhein not only significantly reduces corneal inflammation and oxidative damage, but it also possesses self-assembly properties, allowing it to form hydrogels with various other components.

[0054] To design hydrogels for on-demand treatment, this invention develops a hydrogel delivery system that integrates the synergistic effects of Panax notoginseng-derived extracellular vesicles and rhein, possessing suitable properties for ocular drug delivery. This system not only delivers Panax notoginseng-derived extracellular vesicles to the cornea but also prolongs the drug's residence time, which is of great significance for improving the therapeutic effect on corneal oxidative damage.

[0055] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0056] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0057] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A composite hydrogel, characterized in that, The hydrogel is made from extracellular vesicle solution, rhein solution, calcium chloride solution, and sodium hyaluronate solution; The hydrogel contains rhein at concentrations of 0.7 mM to 24.6 mM, calcium chloride at concentrations of 0.10 mM to 0.5 mM, sodium hyaluronate at a final concentration of 1.0% to 1.5% by mass, and extracellular vesicles at a concentration of 1.9 × 10⁻⁶. 6 Particles / mL ~1.9×10 11 Particles / mL; The extracellular vesicles are Panax notoginseng-derived extracellular vesicles obtained from Panax notoginseng; the preparation method of the Panax notoginseng-derived extracellular vesicles is as follows: Primary separation: After washing the roots of Panax notoginseng, put them into a juicer, collect the fresh juice, and filter it through gauze to remove large pieces of plant debris; centrifuge the filtered solution and collect the supernatant; then centrifuge at 10000g and 4℃ for 1 hour to obtain a dark brown transparent supernatant for later use. Sucrose gradient separation: 27% and 68% sucrose solutions were placed sequentially in centrifuge tubes; then the supernatant prepared from the primary separation was added to form a mixed solution with a clear layered interface; the mixed solution was centrifuged at 100,000g and 4℃ for 1.5 hours, and the bands with more than 68% sucrose layer were collected to obtain the crude extract; Purification: Use a gradient of sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%; slowly add the crude extract to the gradient of sucrose solutions and centrifuge at 200,000g and 4°C for 1.5 hours, collect the precipitate to obtain Panax notoginseng-derived extracellular vesicles.

2. The composite hydrogel according to claim 1, characterized in that, Rhein solution and CaCl2 solution were mixed and stirred evenly. Panax notoginseng-derived extracellular vesicle suspension was added and mixed evenly. Then, the mixture was slowly added to sodium hyaluronate solution under stirring and mixed evenly to obtain composite hydrogel.

3. The composite hydrogel according to claim 1, characterized in that, The rhein solution was obtained by dissolving rhein in PBS.

4. The use of the composite hydrogel according to claim 1 in the preparation of a medicament for treating corneal damage.

5. The application according to claim 4, characterized in that, The drug relieves corneal ulcers or corneal opacity.

6. A drug for treating corneal damage, characterized in that, It includes the composite hydrogel according to claim 1.

7. A composition, characterized in that, It comprises the composite hydrogel of claim 1 or the drug of claim 6.

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