A composite hydrogel for local oxygen-producing drug delivery, its preparation method and uses

Abstract

The present invention belongs to the technical field of wound dressings for wound repair, and particularly relates to a composite hydrogel for local oxygen production and drug delivery, a preparation method thereof, and uses thereof. The present invention provides a composition made from raw materials including the following components in parts by weight: 90-230 parts of natural polyphenol, 5000-10000 parts of EPBA, 5000-10000 parts of oxidized sodium alginate, 5-10 parts of an angiogenesis-promoting drug, and 10-50 parts of MnO2 nanoparticles; the EPBA is polylysine grafted with phenylboronic acid. The composition of the present invention can be made into a hydrogel wound dressing, especially a dressing for chronic wounds. By carrying different drugs and regulating their release order, the hydrogel wound dressing has various biological activities such as oxygen production, antibacterial, antioxidant, anti-inflammatory, and angiogenesis-promoting effects, and has a good promoting effect on the healing of chronic wounds. Therefore, the present invention has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of wound dressings for wound repair, and particularly relates to a composite hydrogel capable of locally generating oxygen and delivering drugs, a preparation method thereof, and uses thereof. Background Art

[0002] As the first line of defense of the human body, the skin is the main defense system of the internal structure and is also the organ most prone to injury. The wound repair of the skin is a highly programmed process, involving four stages: hemostasis, inflammation, proliferation, and remodeling. Damage to this process may form chronic non-healing wounds. Diabetes, vascular diseases, and aging are the main inducements for the formation of chronic non-healing wounds, which can lead to diabetic foot ulcers, venous leg ulcers, and pressure ulcers respectively. With the increasing annual incidence of diabetes and the rapid development of population aging, the proportion of the treatment cost of chronic non-healing wounds in medical expenditure is getting larger and larger, causing a huge burden on the patient's family, society, and national medical system. Although there are currently various intervention measures, such as growth factors, extracellular matrix, artificial skin, etc., the curative effects of these treatment methods have large individual differences and cannot meet the clinical needs. There is an urgent need for new, simpler, and more effective treatment methods.

[0003] In the microenvironment of chronic wounds, the excessive recruitment of inflammatory cells, the increased expression of pro-inflammatory factors, the accumulated reactive oxygen species (ROS), and persistent hypoxia will lead to the aggravation of the inflammatory response. In addition, the ischemia secondary to angiogenesis disorder will further aggravate hypoxia, resulting in the release of more ROS from the wound tissue, further aggravating the inflammatory response, and entering a vicious cycle. Therefore, promoting chronic wound healing is crucial by continuously generating oxygen and sequentially clearing bacterial infections, reducing the ROS level, inhibiting the inflammatory response, and inducing the formation of new blood vessels.

[0004] Wound dressings are the most commonly used method for treating chronic wounds clinically. As a wound dressing, hydrogel can endow hydrogel wound dressings with many biological functions through chemical modification or drug loading, including antibacterial activity, conductivity, anti-inflammatory / antioxidant activity, adhesion, drug controlled / sustained release delivery, angiogenesis-promoting activity, and stimulus responsiveness, etc. Local oxygen-producing materials, antibacterial agents, anti-inflammatory drugs, drugs for promoting tissue repair, etc. can be further introduced into the hydrogel. For example, MnO2 can decompose hydrogen peroxide (H2O2), one of the ROS, into oxygen (O2) and water, thereby relieving local oxygen pressure. The MnO2 nanostructures commonly used in the field of biomaterials can be degraded into manganese ions and excreted through the kidneys without producing biological toxicity, so it is very suitable for application in chronic wounds; Salvianolic acid B (SAB) is often used to treat myocardial infarction due to its strong angiogenesis-promoting ability. A number of studies have shown that SAB promotes the formation of new blood vessels by promoting the expression of NO, HIF-1α, and VEGF in vascular endothelial cells, and has the potential to accelerate the repair of chronic wounds. These drugs or materials can be added to the hydrogel to improve the wound healing effect.

[0005] However, there are many factors interfering with the delayed healing of the wound surface. Delivering the same drug in different environments cannot effectively play a therapeutic role. Therefore, sequential drug delivery is also equally important for the repair of chronic wounds. Thus, for the treatment of chronic wounds, how to design the composite method of the hydrogel to achieve sequential release of different drugs by the hydrogel wound dressing at the wound site through stepwise response and programmed acceleration of wound surface healing at different stages is the key to treating chronic wounds. Summary of the Invention

[0006] Aiming at the problems of the existing technology, the present invention provides a composite hydrogel for local oxygen-producing drug delivery, its preparation method and its use, aiming to provide a composite hydrogel with good repair effect on chronic wounds.

[0007] A composition for promoting wound healing is prepared from raw materials including the following components in parts by weight:

[0008] 80 - 180 parts of natural polyphenols,

[0009] 5000 - 10000 parts of EPBA,

[0010] 5000 - 10000 parts of oxidized sodium alginate,

[0011] 5 - 10 parts of angiogenesis-promoting drug,

[0012] 10 - 50 parts of MnO2 nanoparticles;

[0013] The EPBA is polylysine grafted with phenylboronic acid.

[0014] Preferably, it is made from raw materials comprising the following components in parts by weight:

[0015] 138 parts of natural polyphenol,

[0016] 5000 parts of EPBA,

[0017] 5000 parts of oxidized sodium alginate,

[0018] 5 parts of angiogenesis-promoting drug,

[0019] 15 parts of MnO2 nanoparticles.

[0020] Preferably, the molecular weight of the polylysine is 5000 - 10000, and the grafting rate of the phenylboronic acid is 30 - 35%;

[0021] And / or, the natural polyphenol is selected from at least one of tea polyphenols, tannic acid or procyanidins;

[0022] And / or, the angiogenesis-promoting drug is selected from at least one of salvianolic acid B, mangiferin or ginsenoside.

[0023] Preferably, the MnO2 nanoparticles are honeycomb-like MnO2 nanoparticles, the angiogenesis-promoting drug is loaded in the honeycomb-like MnO2 nanoparticles, and the honeycomb-like MnO2 nanoparticles are secondarily encapsulated with polydopamine.

[0024] Preferably, the composition is a hydrogel prepared by mixing the raw materials in water.

[0025] Preferably, in the hydrogel, the concentration of natural polyphenol is 2 - 5 mM, the concentration of polylysine is 5% - 10% w / v, the concentration of angiogenesis-promoting drug is 50 - 100 μg / mL, and the concentration of MnO2 nanoparticles is 100 - 500 μg / mL.

[0026] The present invention also provides a preparation method of the above composition, comprising the following steps:

[0027] Step 1, graft 4-formylphenylboronic acid onto the side chain of polylysine to prepare EPBA;

[0028] Step 2, blend natural polyphenol and EPBA and react to form polymer EPEG;

[0029] Step 3, load the angiogenesis-promoting drug into the honeycomb-like MnO2 nanoparticles by solution blending method, and secondarily encapsulate the honeycomb-like MnO2 nanoparticles loaded with the angiogenesis-promoting drug with polydopamine to obtain PHMS nanoparticles;

[0030] Step 3: Blend polymer EPEG with sodium alginate oxide, and add PHMS nanoparticles before gel formation, then form a gel to obtain the product.

[0031] The present invention also provides the use of the above composition in the preparation of a drug for wound repair.

[0032] The present invention also provides a drug for wound repair, which is prepared from the above composition as an active ingredient and pharmaceutically acceptable excipients or auxiliary components.

[0033] Preferably, the drug is for promoting the healing of chronic wounds.

[0034] Through the design of the hydrogel formulation and the design of the component composite method, the present invention provides a composite hydrogel for local oxygen production and drug delivery. Specifically, MnO2 nanoparticles can decompose hydrogen peroxide, one of the ROS, into oxygen and water at the wound, thereby providing continuous local oxygen delivery to the wound. At the same time, drugs such as angiogenesis-promoting drugs and natural polyphenols are sequentially released through stepwise response to meet the treatment needs at different stages of the wound healing process. The continuous oxygen production capacity and drug spatiotemporal controlled release behavior of the composite hydrogel constructed by the present invention can effectively meet the needs of wound repair at all stages of chronic wounds, promote the healing of chronic wounds, and have good application prospects.

[0035] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0036] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Description of the Drawings

[0037] Figure 1 is the preparation route of OA and EPEG polymers.

[0038] Figure 2 is the characterization of HM and PHMS nanoparticles. Among them, a is the SEM image characterization result, b is the X-ray photoelectron spectroscopy analysis result, c is the size distribution and Zeta potential of the nanoparticles, d is the transmission electron microscope image, e is the UV-Vis spectroscopy characterization result, f is the particle size distribution of the nanoparticles, g is the oxygen generation curve, and h is the SAB release curve of the nanoparticles.

[0039] Figure 3Physicochemical characterization results of the hydrogel and the polymer raw materials and products during its preparation. Among them, a is the 1H nuclear magnetic resonance spectrum of EPL, EPBA, and EPEG, b is the FTIR spectrum of EPBA and EPEG, c is the nuclear magnetic resonance spectrum of the OA polymer, d is the FITR spectrum of the OA polymer, e is the photo of the hydrogel preparation process, f is the SEM image of the hydrogel, g is the frequency sweep of the rheological properties of the hydrogel, h is the self-healing scan of the rheological properties of the hydrogel, i is the self-healing situation of the hydrogel, j is the injectability of the hydrogel, k is the viscosity of the hydrogel, l is the response performance of the hydrogel, m is the adhesion principle of the hydrogel, n is the release curve of PHMS in the hydrogel, o is the release curve of SAB in the hydrogel

[0040] Figure 4 Detection results of the antibacterial properties of the hydrogel. Among them, a is the antibacterial property of the hydrogel against Staphylococcus aureus, b is the antibacterial property of the hydrogel against Pseudomonas aeruginosa, c is the result of studying the antibacterial activity of the hydrogel by the agar plate culture method, d is the live / dead staining of bacteria, e is the SEM image of bacteria, f is the schematic diagram of the antibacterial principle of the hydrogel

[0041] Figure 5 Experimental results of the antioxidant property, oxygen generation activity, and biocompatibility of the hydrogel. a is the experimental results of the scavenging rates of the hydrogel against H2O2, O2· - and ·OH, b is the experimental results of the scavenging rates of the hydrogel against DPPH and ABTS, c is the oxygen generation curve, d is the intracellular ROS scavenging activity, e is the experimental results of the production level of intracellular O2, f is the image of the intracellular ROS scavenging experiment, g is the image of the production of intracellular O2, h is the CCK-8 detection results of the cytotoxicity of the hydrogel against L929 cells, i is the CCK-8 detection results of the cytotoxicity of the hydrogel against HUVECs, j is the hemolysis experiment results, k is the 3D cell culture image of the hydrogel

[0042] Figure 6 Experimental results of the anti-inflammatory mechanism of the hydrogel. Among them, a is the cell scratch healing rate, b is the ELISA detection results of the expression level of TNF-α, c is the ELISA detection results of the expression level of IL-10, d is the WB experimental results, e is the immunofluorescence staining images of TNF-α and IL-10, f is the proportion of M1 macrophages, g is the proportion of M2 macrophages, h is the representative image of flow cytometry, i is the schematic diagram of the anti-inflammatory principle of the hydrogel, j is the immunofluorescence image display

[0043] Figure 7Experimental results of the angiogenesis-promoting mechanism of the hydrogel. Among them, a is the ELISA detection result of bFGF expression level, b is the ELISA detection result of VEGF expression level, c is the WB experimental result of bFGF and VEGF expression levels, d is the immunofluorescence detection result, e is the WB detection result of Akt phosphorylation level, f is the WB detection quantitative graph, g is the angiogenesis image of HUVEC, h is the number of HUVEC angiogenesis, i is the length of HUVEC angiogenesis, and j is the schematic diagram of the angiogenesis mechanism.

[0044] Figure 8 Gross and pathological analysis results of the in vivo experiment of the hydrogel. Among them, a is the schematic diagram of the animal experiment process, b is the appearance photo of wound healing, c is the simulation graph of wound contraction rate, d is the wound healing rate, e is the in vivo antibacterial plating experiment image, f is the HE and Masson staining of the wound tissue, g is the quantitative analysis of antibacterial performance, h is the hair follicle regeneration speed after treatment, i is the diameter of the wound edge, and j is the collagen deposition amount.

[0045] Figure 9 Experimental results of the cytokine level analysis of the regenerated skin of rats after hydrogel treatment. Among them, a is the immunofluorescence images of iNOS, CD206, VEGF, CD31, and DHE in the wound section, b is the experimental result of immunohistochemical analysis, c is the expression level of VEGF, d is the expression level of CD31, e is the expression level of TNF-α, and f is the expression level of IL-10. Detailed implementation manners

[0046] The materials and reagents used in the following examples and experimental examples are all commercially available products unless otherwise specified.

[0047] Example 1 Composite hydrogel for local oxygen production and drug delivery

[0048] This example provides a hydrogel for use as a wound dressing, and its preparation method and characterization are as follows:

[0049] I. Preparation and material characterization of EPEG, OA polymers, and PHMS nanoparticles

[0050] (1) Preparation and synthesis characterization of OA and EPEG polymers

[0051] Dissolve polylysine EPL (1.0 g) in distilled water (DW, 10 mL), then add 5 mL of FPBA solution (200 mg / mL) dropwise to the EPL solution and stir for 2 h. After that, add NaBH3CN (3 M) to the mixture and stir for 18 h. The product EPBA is obtained by dialysis against DW for 72 h and freeze-drying. Before gelation, add EGCG (3 mM) to the EPBA solution (5%, w / v) and stir for 6 h. The two are synthesized into the polymer EPEG through borate ester bond and Michael addition reaction.

[0052] Disperse sodium alginate (10 g) in ethanol (50 mL), then add it to 50 mL of aqueous NaIO4 (3 g) solution. After stirring at room temperature for 6 h, add 20 mL of ethylene glycol and stir for another 2 h to terminate the reaction. OA is obtained by dialysis against DW for 5 days and freeze-drying.

[0053] The synthesis route is shown in Figure 1 。

[0054] (2) Synthesis and characterization of PHMS nanoparticles

[0055] Completely dissolve KMnO4 (0.1 g) in 10 mL of distilled water while continuously stirring for 30 min, then add oleic acid (1.0 mL) to the solution. Carry out the emulsion reaction for 6 h until the formation of a brownish-black precipitate is observed. After centrifuging at 2000 rpm for 20 min, wash it several times with distilled water and methanol to remove the residual reactants. Disperse 10 mL of the above solution (2 mg / mL) in 2 mg / mL SAB aqueous solution and stir the resulting mixture at room temperature for 2 h. Centrifuge the solution at 12,000 rpm for 10 min, wash it three times with water, stir the separated sample for 6 h, and then suspend it in 10 mL of Tris-HCl (pH = 8.5, 10 mM) supplemented with dopamine hydrochloride (10 mg). Then collect PHMS by centrifugation and wash it twice. Use UV-Vis ultraviolet spectroscopy to detect its drug loading rate and encapsulation rate, and combine TEM, SEM and DLS particle size potential to observe the synthesis situation. Explore its continuous oxygen production ability, ROS responsiveness, antioxidant property and SAB sustained release characteristics in an environment with different concentrations of H2O2. The characterization of PHMS nanoparticles is shown in Figure 2 。

[0056] Figure 2a shows a typical honeycomb-mesoporous structure. The particle size of HM nanoparticles is uniform (about 120 nm) as observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The SEM images indicate that the dispersibility and uniformity of PHMS nanoparticles remain unchanged after loading SAB. The TEM photographs show an obvious shell structure of PHMS nanoparticles, confirming the successful coating of PDA. The TEM photographs confirm the response activity of HM and PHMS nanoparticles to H2O2. After incubation at 37 °C for 3 h in PBS with a pH of 5.5 containing 100 μM H2O2, the honeycomb structure of the nanoparticles changes to irregular flakes or clusters. Among them, the dissociation degree of PHMS nanoparticles is significantly lower than that of HM, which may be due to the protective effect of the PDA shell;

[0057] Figure 2 The results of X-ray photoelectron spectroscopy analysis in b show that the constituent elements of HM are Mn and O. The characteristic peaks at 642.39 eV and 653.8 eV are Mn(IV)2p1 / 2 and Mn(IV)2p2 / 3, respectively. Compared with the XPS measurement results of HM, a new characteristic peak appears at 409.65 eV for PHMS nanoparticles, which belongs to N1s of PDA.

[0058] Figure 2 The size distribution and Zeta potential of the nanoparticles in c. After loading SAB, the hydrodynamic diameter of the nanoparticles hardly changes (about 180 nm). After loading SAB, the Zeta potential of the nanoparticles changes from -21.4 mV (HM) to -15.4 mV (PHMS).

[0059] Figure 2 The TEM atlas in d confirms the coexistence of C, N, O, and Mn elements in PHMS nanoparticles.

[0060] Figure 2 e shows the UV-Vis spectra of HM, SAB, and PHMS nanoparticles. Compared with SAB, PHMS nanoparticles have a similar absorption peak at 285 nm, further confirming the successful encapsulation of SAB. A standard curve of SAB was established based on the absorption peaks of different concentrations of SAB, and the loading efficiency of SAB was calculated to be about 28.6%.

[0061] Figure 2 f shows the particle size distribution of PHMS nanoparticles. A new peak was also observed in the particle size distribution of PHMS in PBS with a pH of 5.5 containing 100 μM H2O2, and its PDI value changes from 0.016 to 0.364.

[0062] Figure 2g is the detection result of the oxygen generation curve of PHMS, and the result shows that a large amount of O2 is generated after PHMS nanoparticles are incubated with H2O2.

[0063] Figure 2 h is the detection result of the drug release performance. In PBS (pH 7.4) without H2O2, a small amount of SAB is released from the PHMS nanoparticles. In PBS (pH 5.5) containing 100 μM H2O2, 48.2% of SAB is released from the nanoparticles after 12 h. While incubating in PBS (pH 5.5) containing 100 μM HO2 for 12 h, the release amount can reach up to 93.3%, indicating that the PDA shell layer of the nanoparticles controls the release efficiency of SAB.

[0064] From the above characterization results, it can be seen that in this example, PHMS nanoparticles with the ability to produce oxygen and release the drug SAB in the wound microenvironment are synthesized.

[0065] II. Characterization of the EPEG-OA@PHMS composite hydrogel and its physicochemical properties and biological characteristics

[0066] (1) Construction and physicochemical property characterization of the EPEG-OA@PHMS oxygen generation - drug delivery system

[0067] Add the same volume of 5% w / v EPEG solution to the 5% w / v OA solution and observe the gelation situation. Add 200 μg / mL of PHMS to the OA solution before gelation to construct a multi-responsive EPEG-OA@PHMS oxygen generation - drug delivery system. Observe the microstructure and drug loading of the hydrogel by SEM and EDS; verify its adhesion, super-intimate skin contact characteristics, injectability, swelling property and degradability through in vitro experiments; dynamically observe its gelation time, elastic modulus, loss modulus and other rheological properties with a rheometer; put the hydrogel into environments with different pH and the presence of H2O2 to verify its pH / ROS multi-responsiveness and drug release performance. The physicochemical characterization of the hydrogel is shown in Figure 3 .

[0068] In the finally prepared hydrogel, the dosage weight ratio of each component and its final concentration in the hydrogel are as follows:

[0069] EGCG: 138 parts, final concentration: 3 mM;

[0070] EPBA: 5000 parts, final concentration: 2.5% w / v;

[0071] Oxidized sodium alginate: 5000 parts, final concentration: 2.5% w / v;

[0072] SAB: 5 parts, final concentration: 50 μg / mL;

[0073] 15 parts of MnO2 nanoparticles, with a final concentration of 150 μg / mL.

[0074] Note: The weight ratio and final concentration of SAB and MnO2 nanoparticles were calculated from the dosage of PHMS nanoparticles and their drug loading. Among them, the thickness of the polydopamine shell layer on the surface of PHMS nanoparticles was relatively thin, and its weight was negligible compared with that of SAB and MnO2 nanoparticles.

[0075] The 1H nuclear magnetic resonance spectra of EPL, EPBA, and EPEG are as Figure 3 shown in a. The benzene matrix proton peaks at 7.3 - 7.7 ppm were observed in EPBA and EPEG, confirming the successful grafting of 4-formylphenylboronic acid. Compared with the nuclear magnetic resonance spectrum of EPBA, the new peaks at 6.3 ppm and 6.8 ppm belong to the aromatic ring protons of EGCG, indicating the successful introduction of EGCG.

[0076] As Figure 3 shown in b, in the FTIR spectra of EPBA and EPEG, the peaks at 1342 cm-1 and 1085 cm-1 were initially attributed to the stretching vibrations of B-O and C-B groups.

[0077] As Figure 3 shown in c, for the OA polymer, the new peaks in the nuclear magnetic resonance spectrum were located at 5.3 - 5.6 ppm, indicating the successful oxidation of sodium alginate.

[0078] As Figure 3 shown in d, a new absorption peak (1735 cm-1) of C=O stretching vibration was visible in the FITR spectrum of OA, indicating the successful oxidation of sodium alginate.

[0079] As Figure 3 e is a photo of the hydrogel preparation process, and the process is as follows: at room temperature, an equal volume of EPEG aqueous solution (containing 3 mM EGCG) and OA solution (containing 400 μg / mL PHMS) were mixed to prepare EPEGOA@PHMS hydrogel. EPBA solution or EPEG solution was mixed with OA solution to prepare EPBA-OA and EPEG-OA hydrogels.

[0080] As Figure 3 shown in f, the porous morphology of the hydrogels was observed by scanning electron microscopy. All the hydrogels had interconnected cavities and a uniform porous structure. The coexistence of C, N, O, B, and Mn elements was determined by energy spectrum analysis of the EPEG-OA@PHMS hydrogel, further verifying the formation of a homogeneous hydrogel structure.

[0081] As Figure 3 shown in g, the frequency scan showed that G′ exceeded G″, and the hydrogel showed frequency dependence, indicating the formation of an elastic network.

[0082] As Figure 3 shown in h, after applying 1000% strain, the G′ and G″ of the hydrogel decreased significantly, and G′ was higher than G″. After applying 1% strain twice, it quickly recovered, indicating that the hydrogel has extraordinary self-healing ability.

[0083] As Figure 3 shown in i, the hydrogel was dyed with different colors to observe its macroscopic self-healing ability. The hydrogel was cut into two pieces to simulate the complete separation of the dressing. After the two pieces were in contact for 3 min, they were completely spliced together with a blurred cutting line, indicating that the hydrogel has good self-healing ability.

[0084] As Figure 3 shown in j, the EPEG-OA@PHMS hydrogel was extruded with a syringe to form the letter "wy", indicating that the hydrogel has good injectability.

[0085] As Figure 3 shown in k, the adhesion strength of EPEG-OA (36.9 kPa) was higher than that of EPBA-OA, indicating that the introduction of EGCG effectively improved the adhesion ability of the hydrogel.

[0086] As Figure 3 shown in l, the hydrogel showed different degrees of disintegration in acidic and ROS-rich environments.

[0087] As Figure 3 shown in m, the unreacted catechol moiety in EGCG can interact with amine, imidazole, and thiol groups on the surface of biological substrates through covalent and non-covalent bonds;

[0088] As Figure 3 shown in n and Figure 3 o, the in vitro drug release curves showed that the release curves of PHMS nanoparticles and SAB were similar, both showing pH and redox dual-responsive release properties. The release of SAB was later than that of PHMS nanoparticles. This is because the hydrogel first dissociates and releases PHMS nanoparticles under acidic and oxidative conditions. Then, the PHMS nanoparticles react with hydrogen peroxide to decompose and release SAB. This can improve the bioavailability of SAB. Compared with the control group (pH 7.4), after incubation for 48 h, in PBS solution containing 100 μM H2O2, PBS solution at pH 5.5, and PBS solution at pH 5.5 containing 100 μM H2O2, the release amounts of SAB increased by 27.0%, 37.6%, and 51.1%, respectively, indicating that pH and ROS are the intelligent switches for EPEG-OA@PHMS hydrogel to control the release of SAB. In addition, SAB can be continuously released for more than 72 h to continuously promote angiogenesis.

[0089] The above experimental results indicate that a composite hydrogel with uniformity, injectability, good rheological properties, and the ability to locally produce oxygen and deliver drugs was prepared in this example.

[0090] Composite Hydrogel for Local Oxygen Production and Drug Delivery in Example 2

[0091] This example provides a hydrogel for use as a wound dressing. Its preparation method is the same as that of Example 1, except that the amounts of each raw material are adjusted so that in the resulting composite hydrogel, the weight ratio of the amounts of each component and its final concentration in the hydrogel are as follows:

[0092] EGCG: 90 parts, final concentration: 2 mM;

[0093] EPBA: 5000 parts, final concentration: 2.5% w / v;

[0094] Oxidized sodium alginate: 5000 parts, final concentration: 2.5% w / v;

[0095] SAB: 10 parts, final concentration: 100 μg / mL;

[0096] MnO2 nanoparticles: 50 parts, final concentration: 500 μg / mL.

[0097] Composite Hydrogel for Local Oxygen Production and Drug Delivery in Example 3

[0098] This example provides a hydrogel for use as a wound dressing. Its preparation method is the same as that of Example 1, except that the amounts of each raw material are adjusted so that in the resulting composite hydrogel, the weight ratio of the amounts of each component and its final concentration in the hydrogel are as follows:

[0099] EGCG: 230 parts, final concentration: 5 mM;

[0100] EPBA: 10000 parts, final concentration: 5% w / v;

[0101] Oxidized sodium alginate: 10000 parts, final concentration: 5% w / v;

[0102] SAB: 5 parts, final concentration: 50 μg / mL;

[0103] MnO2 nanoparticles: 10 parts, final concentration: 100 μg / mL.

[0104] The technical solution of the present invention is further illustrated by the following experiments. Unless otherwise specified, the hydrogels used in the following experimental examples were prepared according to the method of Example 1.

[0105] Experimental Example 1 Biological Characterization of the EPEG-OA@PHMS Oxygen Production-Drug Delivery System

[0106] I. Experimental Method

[0107] (1) Experimental grouping: Different drugs were loaded into the hydrogel drug delivery system to verify the antibacterial activity, antioxidant activity, and biocompatibility of different hydrogels. The groups included:

[0108] Blank group: Hydrogel was not used.

[0109] EPEG-OA group: The blank EPEG-OA hydrogel was used for intervention.

[0110] PHMS group: PHMS nanoparticles were used for intervention.

[0111] EPEG-OA@PHMS group: The EPEG-OA hydrogel loaded with PHMS nanoparticles (i.e., the EPEG-OA@PHMS hydrogel of Example 1) was used for intervention.

[0112] (2) Antibacterial activity: Using S. aureus as a representative of Gram-positive bacteria and P. aeruginosa as a representative of Gram-negative bacteria, after co-culturing the hydrogel with bacteria, the broad-spectrum antibacterial activity of different hydrogels was detected by the OD600 method, bacterial plating method, and bacterial live / dead fluorescence staining method.

[0113] (3) Antioxidant activity: The scavenging abilities of different hydrogels against reactive oxygen species such as DPPH, O2 ·- , ·OH, etc. were detected by DPPH kits, superoxide anion detection kits, and hydroxyl radical activity determination kits respectively; the H2O2 scavenging ability of the hydrogel was detected by the ammonium molybdate colorimetric method; the O2 generation ability of the hydrogel was detected by a portable dissolved oxygen meter. The in vivo antioxidant ability and oxygen generation ability were evaluated using L929 cells.

[0114] (4) Biocompatibility: After co-culturing the hydrogel with fibroblasts (L929) and vascular endothelial cells (HUVECs), its cell compatibility was evaluated by a CCK-8 kit; after co-culturing the hydrogel with rat red blood cells, its blood compatibility was evaluated by observing the appearance and the OD540 method.

[0115] II. Experimental results

[0116] The results of antibacterial activity are shown in Figure 4 . As Figure 4 a and Figure 4 b show, the EPEG-OA@PHMS hydrogel exhibited good broad-spectrum antibacterial activity against Staphylococcus aureus (94.8%) and Pseudomonas aeruginosa (89.7%). As Figure 4 c shows, the antibacterial activity of the hydrogel was further studied by the agar plate culture method, and it was found that after co-culturing with the EPEG-OA@PHMS hydrogel, a small number of colonies formed on the agar plate. As Figure 4As shown in Figure 4 d, a large number of dead bacteria (red) were observed in the EPEG-OA@PHMS group under a fluorescence microscope. As Figure 4 shown in

[0117] e, after incubation with Staphylococcus aureus and Pseudomonas aeruginosa in the EPEG-OA@PHMS hydrogel, the physiological structure of the membrane surface was lost, and depressions and ruptures occurred. These results indicate that the EPEG-OA@PHMS hydrogel has good antibacterial activity. Figure 5 .

[0118] As Figure 5 shown in - a, the introduction of PHMS nanoparticles effectively eliminated H2O2, and the H2O2 scavenging rate reached 78.6% in the EPEG-OA@PHMS group. In addition to H2O2, the EPEG-OA@PHMS hydrogel also had strong scavenging ability for O2· Figure 5 (74.5%) and ·OH (67.0%). As + shown in Figure 5 b, the DPPH scavenging rate of the EPEG-OA@PHMS group was the highest, reaching 94.5%, significantly higher than that of the EPEG-OA group (72.6%) and the PHMS group (24.9%). The ABTS· Figure 5 scavenging rate of the EPEG-OA@PHMS group was 97.7%, higher than that of the PHMS group (14.7%). As Figure 5 shown in

[0119] c, as the reaction time extended, the dissolved O2 concentration continuously increased and was dissociated into O2 by PHMS nanoparticles. As Figure 5 shown in Figure 5 d, the EPEG-OA@PHMS hydrogel effectively alleviated intracellular oxidative stress. As Figure 5 shown in Figure 5As shown in [[i]], the CCK-8 assay results indicated that the EPEG-OA@PHMS hydrogel had no obvious cytotoxicity to either L929 cells or HUVECs cells. As Figure 5 As shown in [[j]], the hemocompatibility of the EPEG-OA@PHMS hydrogel was evaluated using a hemolysis test, and no obvious hemolytic reaction was observed, with a hemolysis rate of 3.4%. As Figure 5 As shown in [[k]], L929 cells tolerated 3D embedding and continued to proliferate within the 3D hydrogel, which might be due to the fact that the glycopeptide hydrogel mimicked the glycoprotein composition and nanofiber structure of the skin ECM. The above experimental results indicated that the EPEG-OA@PHMS hydrogel had low toxicity and good biocompatibility.

[0120] Experimental Example 2: In vitro cell experiments to explore the possible mechanism of action of the EPEG-OA@PHMS oxygen-producing and drug-delivering system in promoting chronic wound healing

[0121] I. Experimental methods

[0122] (1) Exploring the ability of the hydrogel to promote the migration of inflammatory damaged cells based on fibroblasts

[0123] Taking L929 as an example, a 100 μmol H2O2 stimulation was used to simulate an inflammatory microenvironment. A sterile pipette tip was used to scratch the monolayer cells to form a cell scratch to simulate the formation of a single-cell chronic wound. The experimental groups were set up in the same way as in Experimental Example 1. After co-culturing the hydrogel with the cells (the culture medium was DMEM), the healing of the cell scratch was recorded at 6, 12, 24, and 48 hours to evaluate the ability of the hydrogel to promote the migration of inflammatory damaged cells.

[0124] (2) Exploring the anti-inflammatory mechanism of the hydrogel based on macrophages

[0125] ① Detection of macrophage anti-inflammatory activity: Taking macrophage RAW 264.7 as an example, lipopolysaccharide (LPS) was used to induce inflammatory damage. After intervention with hydrogels of different groups, the expression results of anti-inflammatory and pro-inflammatory factors IL-10 and TNF-α were detected by Western-blot, ELISA, and immunofluorescence.

[0126] ② Detection of the polarization of M1 / M2 macrophages: Taking RAW 264.7 as an example, LPS was used to induce its differentiation into the M1 subset. After intervention with hydrogels of different groups, the cell morphology was observed, and the cell subsets were identified by flow cytometry (the M1 subset was labeled with CD86, and the M2 subset was labeled with CD206).

[0127] ③ Taking macrophage RAW 264.7 as an example, LPS was used to induce inflammatory damage. After intervention with hydrogels of different groups, Western-blot was used to detect the effects of different intervention methods on the expression of downstream marker proteins and genes related to the IKK / NF-κB signaling pathway.

[0128] (3) Exploring the mechanism of hydrogel promoting neovascularization based on vascular endothelial cells

[0129] Taking HUVECs as the representative, explore the mechanism of action of the oxygen-producing and drug-delivering system hydrogel of H-mMnO2@SABEPEG-OA@PHMS in exerting pro-angiogenic activity based on the PI3K / Akt / eNOS signaling pathway. Select LY294002 as the PI3K inhibitor. After adding it to the hydrogel for co-culture, detect its effects on the expression of downstream marker proteins and genes of the PI3K / Akt signaling pathway by Western-blot, and detect the expression of pro-angiogenic factors bFGF and VEGF by Western-blot, ELISA and immunofluorescence.

[0130] II. Experimental results

[0131] The experimental results of the anti-inflammatory mechanism of the hydrogel are shown in Figure 6 . As Figure 6 shown in a, after adding LPS, the migration of L929 cells was significantly inhibited. At the same time, the EPEG-OA@PHMS hydrogel has a strong ability to promote cell migration, which may be due to the polysaccharides in the hydrogel and the antioxidant ability of EGCG and PHMS nanoparticles. As Figure 6 shown in b and Figure 6 c, the ELISA experiment showed that after LPS activated RAW264.7 cells, the expression of TNF-α in the cells increased significantly, while after co-culture with the EPEG-OA@PHMS hydrogel, the expression of TNF-α in RAW264.7 cells decreased significantly; after co-culture with the EPEG-OA@PHMS hydrogel, the expression of macrophage IL-10 was significantly higher than that of macrophages activated by LPS. As Figure 6 shown in d, the WB experiment showed that the expression of TNF-α in the cells increased significantly after co-culture with the hydrogel, and the expression of IL-10 was significantly higher than that of the control group. As Figure 6 shown in e, the immunofluorescence staining images of TNF-α and IL-10 also showed that the EPEG-OA@PHMS hydrogel reduced the expression of TNF-α in inflammatory cells and up-regulated the expression of IL-10. As Figure 6 shown in f, after adding LPS, the proportion of M1 macrophages (CD86+) increased significantly. After intervention with the EPEG-OA@PHMS hydrogel, a significant decrease in the proportion of M1 macrophages (CD86+) was observed. As Figure 6 shown in g, after intervention with the EPEG-OA@PHMS hydrogel, the proportion of M2 macrophages (CD206+) increased. Figure 6h is a representative image of flow cytometry, showing that the proportion of M1 macrophages decreased while the proportion of M2 cells increased after the intervention of EPEG-OA@PHMS hydrogel. Figure 6 i is a schematic diagram of the anti-inflammatory mechanism of the hydrogel, that is, EPEG-OA@PHMS hydrogel exerts an anti-inflammatory effect by inducing M2 polarization of macrophages, upregulating the expression of anti-inflammatory factors and downregulating the expression level of pro-inflammatory factors. As Figure 6 shown in j, the immunofluorescence image shows that the fluorescence signal of iNOS (M1 marker) is significantly weakened after treatment with EPEG-OA@PHMS, while the fluorescence signal of CD206 is enhanced.

[0132] The experimental results of the angiogenesis mechanism of the hydrogel are shown in Figure 7 . As Figure 7 a and Figure 7 b shown, ELISA detection indicates that the expressions of bFGF and VEGF in the EPEG-OA@PHMS group are significantly higher than those in the control group. As Figure 7 shown in c, WB detection indicates that the expressions of bFGF and VEGF in the EPEG-OA@PHMS group are significantly higher than those in the control group. As Figure 7 shown in d, immunofluorescence detection also indicates that the expressions of bFGF and VEGF in the EPEG-OA@PHMS group are significantly higher than those in the control group. As Figure 7 shown in e, WB detection indicates that the expression of phosphorylated Akt is significantly increased after treatment with EPEG-OA@PHMS hydrogel. As Figure 7 shown in g, the angiogenesis experiment shows that treatment with EPEG-OA@PHMS hydrogel significantly stimulates in vitro angiogenesis. As Figure 7 h and Figure 7 i shown, more vascular connections and longer tubule lengths are observed in HUVECs treated with EPEG-OA@PHMS hydrogel. Figure 7 j is a schematic diagram of the angiogenesis mechanism, that is, EPEG-OA@PHMS hydrogel promotes neovascularization by activating the PI3k / Akt pathway..

[0133] The above results indicate that EPEG-OA@PHMS hydrogel has good cell compatibility. Cell experiments prove that the hydrogel can regulate the expression level of inflammatory factors by affecting macrophage polarization to exert an anti-inflammatory effect, and can promote neovascularization by activating the PI3k / Akt pathway.

[0134] Experimental Example 3 Establish an animal model of chronic infectious full-thickness skin defect based on diabetic rats to verify the in vivo chronic wound repair effect of the EPEG-OA@PHMS oxygen-producing and drug-delivering system

[0135] I. Experimental methods

[0136] (1) Establishment of animal model

[0137] A rat diabetes model was established by injecting streptozotocin into the tail vein. On this basis, a full-thickness skin wound with a diameter of 10 mm was created on the back of the rat using a skin punch, and 100 μL of S. aureus (1.0×10 8 CFU / mL) was dropped to establish a chronic infected wound model.

[0138] (2) Verification of chronic wound repair effect

[0139] The experimental procedure was as Figure 8 shown in a. After successful modeling, the rats were grouped in the same manner as in Experimental Example 1, and a commercial hydrogel Hydrosorb (B. Braun Melsungen AG, Germany) control group was added. Subsequently, different hydrogels were implanted at the wound site for intervention treatment. 200 μL was injected into each wound to cover the surface, and it was changed every 2 days. The healing of the wounds on the rats was observed and recorded within 14 days. On the 3rd day, after the rats were euthanized by inhaling excessive isoflurane, the wound tissues were taken for the following treatments: ① After the tissues were frozen and homogenized, the antibacterial activity in the body of different treatment groups was detected by the bacterial plate method; ② After the tissues were cooled with liquid nitrogen, frozen section DHE staining was used to evaluate the antioxidant activity in the body.

[0140] On the 7th day and the 14th day, the rats were euthanized, and the wound tissues were taken for HE and Masson staining to evaluate the wound healing degree, angiogenesis and collagen deposition. Immunofluorescence staining and ELISA were used to detect the expression of angiogenesis-related cytokines (VEGF, bFGF) and inflammation-related cytokines (IL-10, TNF-α) in the wound tissues, and the in vivo wound repair efficacy was comprehensively evaluated.

[0141] II. Experimental results

[0142] Experimental results Figure 8 and Figure 9 .

[0143] The appearance photos of wound healing were as Figure 8 shown in b, the simulation diagram of wound contraction rate was as Figure 8 shown in c. The wound contraction rate was quantified, and the results were as Figure 8 shown in d. The EPEG-OA@PHMS group had a higher wound contraction rate on the 7th day, which was 78.6%. In contrast, the Hydrosorb group and the blank group were 60.5% and 51.9% respectively. After 14 days of treatment, the wounds in the EPEG-OA@PHMS group were almost completely healed, while the wound repair in other groups was delayed. As Figure 8 shown in e, EPEG-OA@PHMS had obvious in vivo antibacterial activity. As Figure 8As shown in Figure f, the HE staining images showed that more new blood vessels (red arrows) and hair follicles (green arrows) were formed in the regenerated skin of the EPEG-OA@PHMS group. The Masson's trichrome staining showed that compared with other groups, the wound edge (black arrow) was significantly reduced after treatment with the EPEG-OA@PHMS hydrogel, and a large amount of dense and orderly collagen deposition was observed under the epidermis. The antibacterial performance is as Figure 8 shown in Figure g. After treatment with the EPEG-OA@PHMS hydrogel, Staphylococcus aureus at the wound site was effectively cleared, showing good antibacterial performance. As Figure 8 shown in Figures h, 8i, and 8j, the hair follicle regeneration rate was faster after treatment with the EPEG-OA@PHMS hydrogel, and the amount of collagen deposition was significantly better than that of the blank control group and the hydrosorb group.

[0144] As Figure 9 shown in Figure a, compared with the control group, on the 14th day after application of the EPEG-OA@PHMS hydrogel, the expressions of VEGF and CD31 were both significantly increased, the proportion of M1 cells decreased, the M2 cells increased, and the ROS level was significantly reduced. As Figure 9 shown in Figure b, immunohistochemical analysis showed that the expression of the pro-inflammatory factor TNF-α in the EPEG-OA@PHMS hydrogel group was significantly lower than that in the control group, and the expression of IL-10 was significantly higher than that in the control group. As Figure 9 c- Figure 9 shown in Figure f, ELISA analysis on the 14th day showed that in the EPEG-OA@PHMS hydrogel group, the expressions of VEGF and CD31 were significantly higher than those in the control group, the expression of TNF-α was significantly lower than that in the control group, and the expression of IL-10 was significantly higher than that in the control group.

[0145] The above results indicate that the EPEG-OA@PHMS hydrogel provided in Example 1 has good anti-inflammatory and angiogenesis-promoting effects, and has a better wound repair effect compared with other hydrogels in the prior art.

[0146] From the above examples and experimental examples, it can be seen that the present invention provides a composite hydrogel. By loading different drugs and regulating their release order, it has multiple biological activities such as oxygen production, antibacterial, antioxidant, anti-inflammatory, and angiogenesis-promoting, which has a good promoting effect on the healing of chronic wounds.

Claims

1. A composition for promoting wound healing, characterized in that, It is prepared from raw materials comprising the following components in parts by weight: 90 - 230 parts of natural polyphenols, 5000 - 10000 parts of EPBA, 5000 - 10000 parts of oxidized sodium alginate, 5 - 10 parts of angiogenesis - promoting drug, 10 - 50 parts of MnO₂ nanoparticles; The natural polyphenols are selected from at least one of tea polyphenols, tannic acid or procyanidins; The EPBA is polylysine grafted with phenylboronic acid; the molecular weight of the polylysine is 5000 - 10000, and the grafting rate of the phenylboronic acid is 30 - 35%; The angiogenesis - promoting drug is selected from at least one of salvianolic acid B, mangiferin or ginsenoside; the MnO₂ nanoparticles are honeycomb - shaped MnO₂ nanoparticles, the angiogenesis - promoting drug is loaded in the honeycomb - shaped MnO₂ nanoparticles, and the honeycomb - shaped MnO₂ nanoparticles are secondarily encapsulated with polydopamine; The preparation method of the composition comprises the following steps: Step 1: Graft 4 - formylphenylboronic acid onto the side chain of polylysine to prepare EPBA; Step 2: Blend and react natural polyphenols and EPBA to form polymer EPEG; Step 3: Load the angiogenesis - promoting drug into the honeycomb - shaped MnO₂ nanoparticles by solution blending method, and secondarily encapsulate the honeycomb - shaped MnO₂ nanoparticles loaded with the angiogenesis - promoting drug with polydopamine to obtain PHMS nanoparticles; Step 4: Blend polymer EPEG and oxidized sodium alginate, add PHMS nanoparticles before gelling, and gel to obtain the product.

2. The composition according to claim 1, characterized in that, It is prepared from raw materials comprising the following components in parts by weight: 138 parts of natural polyphenols, 5000 parts of EPBA, 5000 parts of oxidized sodium alginate, 5 parts of angiogenesis - promoting drug, 15 parts of MnO₂ nanoparticles.

3. The composition according to claim 1 or 2, characterized in that: The composition is a hydrogel prepared by mixing the above raw materials in water.

4. The composition according to claim 3, characterized in that: In the hydrogel, the concentration of natural polyphenols is 2 - 5 mM, the concentration of polylysine is 5% - 10% w / v, the concentration of the angiogenesis - promoting drug is 50 - 100 μg / mL, and the concentration of MnO₂ nanoparticles is 100 - 500 μg / mL.

5. A method for preparing the composition according to any one of claims 1 to 4, characterized in that, Comprising the following steps: Step 1: Graft 4 - formylphenylboronic acid onto the side chain of polylysine to prepare EPBA; Step 2: Blend and react natural polyphenols and EPBA to form polymer EPEG; Step 3: Load the angiogenesis - promoting drug into the honeycomb - shaped MnO₂ nanoparticles by solution blending method, and secondarily encapsulate the honeycomb - shaped MnO₂ nanoparticles loaded with the angiogenesis - promoting drug with polydopamine to obtain PHMS nanoparticles; Step 4: Blend polymer EPEG and oxidized sodium alginate, add PHMS nanoparticles before gelling, and gel to obtain the product.

6. Use of the composition according to any one of claims 1 - 4 in the preparation of a drug for wound repair.

7. A drug for wound repair, characterized in that: It is prepared with the composition according to any one of claims 1 - 4 as the active ingredient, plus pharmaceutically acceptable excipients or auxiliary components.

8. The drug according to claim 7, characterized in that: The drug is a drug for promoting chronic wound healing.

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