Gel skin wound dressing capable of loading medicine and exosome

Abstract

The invention relates to a drug-loadable exosome-loadable gel skin wound dressing, the gel material is prepared from biomass macromolecules, modified polyvinyl alcohol, an ionic macromonomer, an ionic micromolecule monomer and a hydrogen-bond interaction monomer as raw materials, and the gel dressing can load a large amount of cationic drugs and stem cell exosomes. The exosome can survive for a long time, and the loaded exosome and the medicine can be gradually released when the exosome covers the wound surface of the skin of the human body. In addition, the gel dressing has good mechanical properties, mechanical fatigue resistance, biocompatibility, moderate skin adhesiveness and water absorption, and can be used as a dressing for treating wound surfaces difficult to heal.

Description

Technical Field

[0001] The present invention relates to a skin wound dressing, and in particular to a wound dressing preparation which can be used alone, has good biocompatibility, adhesion, wound exudate absorption, water absorption and retention, mechanical properties, and can load a large amount of stem cell exosomes and cationic small molecule drugs. Background Art

[0002] The surface of open wounds in the human body is often prone to infection, especially some special wounds. For example, when there is an underlying disease such as diabetes, the wound surface is difficult to recover due to the influence of high sugar, causing pain to the patient. How to effectively reduce the probability of wound infection and promote wound recovery and tissue refunctionalization is a hot and difficult issue that needs to be solved in the medical field.

[0003] Dressing materials are essential tools for wound treatment, which can protect wounds, independently or collaboratively treat wounds, and promote recovery. Dressing materials have undergone many years of evolution. Traditional dressings such as gauze and bandages are made of dry fabrics, which have limited absorption of exudate and are easy to damage newly generated tissues when removed, causing secondary damage. Newly developed dressings such as foam dressings, polymer dressings, and biomass dressing films can enhance the synergistic treatment effect, but it is difficult to load drugs and achieve independent treatment effects. Hydrogels have similar properties to biomass tissues. Due to their own swelling, water retention, and easy removal properties, they are a good choice for wound dressings. However, as dressings, hydrogels need to meet a variety of comprehensive performance requirements, such as good mechanical properties to achieve skin-like adaptability, good water absorption and water retention to absorb wound exudate and reduce the number of material changes, and good biocompatibility and moderate adhesion. At the same time, in order to achieve better therapeutic effects, the dressing itself should preferably be able to load different drugs and achieve gradual release.

[0004] In recent years, regenerative medicine has made great progress. Regenerative medicine represented by stem cells and their derivatives has made rapid progress in tissue repair. In 2015, the successful treatment of a child patient with 80% burns by epidermal stem cell gene therapy at Ruhr University in Bochum, Germany, became a milestone. Exosomes are a kind of cell derivatives. After the multivesicular bodies of cells fuse with the plasma membrane, they are released into the extracellular environment by exocytosis as small vesicles wrapped by a lipid molecular membrane. Exosomes contain abundant mRNA, microRNA and proteins. After these substances fuse with recipient cells, they can regulate the biological functions of recipient cells. Exosomes can be obtained from almost all cultured cells and are almost non-immunogenic. Therefore, they are expected to become important biopharmaceuticals for tissue repair and organ reconstruction. After years of research, it has been found that various stem cell exosomes can play a huge role in tissue regeneration, immune regulation, anti-inflammatory and anti-fibrotic effects, etc., and have great potential in tissue injury repair. However, if exosomes are used alone, they often quickly lose their activity, thereby affecting the treatment effect.

[0005] The hydrogel is filled with an aqueous medium and can theoretically load various drugs or biological macromolecules to achieve sustained release, so as to achieve better treatment effects. In recent years, a large number of examples have also confirmed this property of the gel. However, when used as a dressing, first, the amount of drug loading and sustained release need to be considered, especially when loading stem cell exosomes, the internal environment, especially the elastoplasticity of the polymer chain, is suitable for the survival and gradual release of exosomes; second, its comprehensive performance needs to meet the requirements of dressing-type medical devices, such as adhesiveness, water retention, mechanical properties, biocompatibility, etc. all need to be suitable. And this is still a great challenge for the construction of gel dressings. This invention patent aims at the above problems and develops a dressing made of a hydrogel with excellent performance that can be used alone and can load drugs and exosomes to meet the application requirements. Summary of the Invention

[0006] The present invention is committed to overcoming one or several defects in the loading amount, loading activity, mechanical properties, water absorption and water retention, adhesiveness and other properties of current hydrogel wound dressings for stem cell exosomes and drugs, and provides a gel dressing that can be used alone and can load drugs and exosomes.

[0007] The object of the present invention is achieved by the following technical solutions: A gel skin wound dressing that can load drugs and exosomes is prepared from the following substances and the corresponding mass ratios: Biomacromolecule 100 Modified polyvinyl alcohol 30 - 120 Ionic macromonomer 15 - 40 Ionic small molecule monomer 4 - 12 Hydrogen bond monomer 20 - 60 Furthermore, the biomass macromolecule is a water-soluble biomass macromolecule having ionic groups and macromolecules capable of hydrogen bonding, including gelatin, agar, chitosan, and modified substances of the above biomass. Preferably, it is gelatin modified with methacrylic acid, chitosan modified with quaternary ammonium salt, and agar modified with maleic anhydride.

[0008] Furthermore, the gelatin, agar, and chitosan all have a large number of hydroxyl groups, amino groups, carboxyl groups, etc., which can provide interactions with polymers and sites for drug loading.

[0009] Furthermore, the gelatin modified with methacrylic acid has double bonds introduced on its surface, which can react with monomers through photopolymerization and then be directly incorporated into the polymer.

[0010] Furthermore, the chitosan modified with quaternary ammonium salt introduces cationic sites that can provide anion drug loading through quaternary ammonium salt modification, which can strengthen the binding between the biomass-based macromolecule and the polymer and further enhance the antibacterial properties of chitosan.

[0011] Furthermore, the agar modified with maleic anhydride can further improve the water solubility of agar and the flexibility of the gel prepared therefrom.

[0012] Furthermore, the degree of substitution of the gelatin modified with methacrylic acid is between 15 - 25% calculated based on the weight of the modified substance and gelatin.

[0013] Furthermore, the degree of substitution of the chitosan modified with quaternary ammonium salt is between 10 - 20% calculated based on the weight of the modified substance and chitosan.

[0014] Furthermore, the degree of substitution of the agar modified with maleic anhydride is between 8 - 16% calculated based on the weight of the modified substance and agar Furthermore, the modified polyvinyl alcohol, ionic macromonomer, ionic small molecule monomer, and hydrogen bonding monomer all have carbon-carbon double bonds, and can achieve free radical reactions under photocatalysis to realize the polymerization connection of various monomers.

[0015] Furthermore, the modified polyvinyl alcohol is polyvinyl alcohol modified with N-(2,2-dimethoxyethyl)-2-acrylamide or N-(methoxymethyl)-2-acrylamide. The methoxy group in the modifier and the hydroxyl group in polyvinyl alcohol are condensed under acidic conditions to achieve modification, and the proportion of the hydroxyl group in polyvinyl alcohol replaced by the modifier is greater than 90%; the molecular weight of the polyvinyl alcohol is between 4000 - 16000, and the degree of alcoholysis is between 88 - 98%; Furthermore, the modification process of the polyvinyl alcohol is as follows: Put a certain amount of polyvinyl alcohol into deionized water, heat it to 95 °C and stir to dissolve it, with the concentration between 12 - 20 g / mL. Add a small amount of hydrochloric acid to adjust the pH value to between 6 - 4.5. Take a certain amount of modifier and put it into the above solution, continuously stir and react for 10 - 16 h to prepare a modified polyvinyl alcohol solution. Add pure water to cool it and precipitate the product, and obtain the final modified product after repeated rinsing; among them, the addition amount of the modifier is between 10 - 24 μL / g of the mass of the polyvinyl alcohol; the final degree of modification is calculated based on the mass ratio of the grafted substance to the polyvinyl alcohol and is between 12 - 24%.

[0016] Furthermore, taking the modification with N-(2,2-dimethoxyethyl)-2-acrylamide as an example to illustrate the reaction that occurs:

[0017] Furthermore, the ionic macromolecules are zwitterionic monomer [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide (DMAPS) and anionic monomer 2-acrylamide-2-methylpropanesulfonic acid (AMPS).

[0018] Furthermore, the ionic small molecule monomers include acrylic acid and acrylate esters.

[0019] Furthermore, preferably, the mass ratio of the ionic macromolecule to the ionic small molecule monomer is between 3:1 - 4:1.

[0020] Furthermore, the hydrogen bond acting monomer is one of N-vinylpyrrolidone, hydroxyethyl acrylamide, and N-isopropylacrylamide.

[0021] Furthermore, the preparation process of the drug-loading and exosome-loading gel skin wound dressing is as follows: Add the biomass macromolecule, modified polyvinyl alcohol, ionic macromolecule monomer, ionic small molecule monomer, and hydrogen bond acting monomer into pure water or weakly acidic water with a pH value of 5.5 - 6.5 to prepare an aqueous solution with a mass concentration of 8 - 12%. After stirring evenly, add a water-soluble photoinitiator with a mass of 0.2 - 0.4% of the modified polyvinyl alcohol, stir evenly, and irradiate with ultraviolet light at the maximum absorption wavelength corresponding to the photoinitiator for 6 - 12 hours to obtain a pre-product of the dressing gel; Soak the pre-product in excess water and repeatedly rinse to remove unreacted small molecules and acid ions therein, and adjust the pH value to neutral to obtain the final dressing product.

[0022] Furthermore, the drug-loading and exosome-loading gel skin wound dressing can be used directly or can be stored by drying and dehydrating. Before use, soak it in pure water to balance swelling, or use it after reaching a swelling rate of 3 - 5 times the weight.

[0023] Furthermore, when the drug-loaded and exosome-loaded gel skin wound dressing is used, it can be applied on the wound surface and then a bonding substance can be covered at the edge to fit the skin more tightly.

[0024] Furthermore, the exosomes are stem cell exosomes, which can be mesenchymal stem cell exosomes, adipose-derived stem cell exosomes, pluripotent mesenchymal stem cell exosomes, etc.

[0025] Furthermore, the cationic small molecule drugs can be antibacterial drugs such as polyhexamethylene biguanide hydrochloride and levofloxacin hydrochloride, and their molecular weights do not exceed 1500. If the molecular weight is too large, it will affect the penetration of the drugs.

[0026] Furthermore, this gel dressing can also load non-ionic small molecule drugs. As long as the drug has certain hydrogen bond-binding groups such as levofloxacin containing hydroxyl groups, efficient loading can be achieved.

[0027] Furthermore, the dressing can achieve efficient loading of exosomes by being immersed in the exosome solution after equilibrium swelling and freeze-drying. The loading concentration can reach 8×10 4 cells / ml. The dressing can be freeze-dried after equilibrium swelling, or directly immersed in the drug solution in a swollen state to achieve efficient loading of small molecule cationic drugs, and the loading concentration can reach 140 mg / ml.

[0028] Furthermore, for the loading of the stem cell exosomes, a high-concentration exosome culture medium solution can be prepared and the loading can be achieved by the immersion method. After loading, a certain concentration of culture medium solution can be continuously infiltrated for a certain period of time to promote the infiltration of the culture medium into the gel to increase the survival days of the stem cells.

[0029] Furthermore, in the gel dressing, the biopolymeric macromolecules mainly provide strength and elastic modulus. The ratios of the ionic macromolecules, ionic small molecule monomers, and hydrogen bond interaction monomers have an important impact on the elastoplasticity of the dressing. Among them, the dosages of these three monomers jointly determine the swelling property of the gel, as well as the elastoplasticity in the swollen state, the adhesiveness to the skin, and the water absorption and water retention properties, etc.

[0030] Furthermore, appropriate elastoplasticity can make the gel have mechanical properties similar to those of the skin, and is also beneficial to the loading and survival of exosomes in the gel network. Among them, increasing the mass fraction of the biopolymeric macromolecules and the concentration of the modified polyvinyl alcohol will significantly improve the rigidity and elasticity of the dressing. The hydrogen bond interaction monomers, ionic macromolecules, and ionic small molecule monomers will achieve an appropriate range of elasticity and plasticity within a certain ratio range.

[0031] Furthermore, the increase of ionic groups in the system is beneficial to the improvement of the swelling degree. An appropriate swelling ratio is beneficial to the loading and survival of exosomes, but too many ionic groups will cause the swelling ratio of the gel to be too large and the mechanical properties to decline, affecting the use.

[0032] Furthermore, the ratio of various macromolecules and monomers has a certain impact on the survival of exosomes; in principle, a relatively larger swelling ratio and a softer network structure are more conducive to the survival of exosomes. Of course, this is also related to the groups within the network to a certain extent.

[0033] Furthermore, the increase of ionic groups in the system is beneficial to the loading of counterion drugs; the increase of hydrogen bond - acting groups is beneficial to the loading of drugs containing corresponding hydrogen bond - acting groups.

[0034] Furthermore, when the hydrogen - bond - acting monomer used is N - isopropylacrylamide, due to the critical phase transition behavior of N - isopropylacrylamide at about 37 °C, after the dressing covers the skin, it will produce a certain contraction, the adhesion force will decrease slightly, and at the same time, it will promote the release of drugs on the wound surface, and this effect is proportional to the monomer concentration.

[0035] Furthermore, the gel is tested in the following way: The test methods for the tensile strength, elongation at break, and toughness of the gel are as follows: The gel is prepared into dumbbell - shaped specimens with a width of 10 mm and a thickness of 1 mm in the middle of the dumbbell. It is detected by a universal mechanical testing instrument with a tensile rate of 10 mm / min. The toughness is the area under the stress - strain curve, and the unit is MJ / m 3 。

[0036] The test methods for the storage modulus and loss modulus of the gel are as follows: The gel is tested with a rotational rheometer. Rheological tests are carried out by preparing cylindrical hydrogel blocks (diameter 15 mm, thickness 8 mm), the interval is set to 20 mm, a strain of 1% is set, and the scanning frequency is set from 0.1 to 100 rad / s −1 ,and the storage modulus and loss modulus within the linear viscoelastic region in the test range are taken.

[0037] The test method for the equilibrium swelling property of the gel is as follows: The prepared gel is freeze - dried and then immersed in pure water. After reaching weight equilibrium, the weight is recorded. The swelling ratio = (equilibrium weight - dry gel weight) / dry gel weight.

[0038] The test method for the skin adhesion of the gel is as follows: An 80 mm×15 mm×1.5 mm gel is adhered to the surface of pig skin and peeled off by a universal mechanical tester at a rate of 10 mm / min. The skin adhesion strength is calculated by the formula G = F / W for estimation, where F is the average force in the steady - state region during the peeling process and W is the width of the tested hydrogel sheet.

[0039] Loading and concentration test of exosomes: Stem cell exosomes in serum - free medium are diluted to 5×10 5A solution with a concentration of / ml. The freeze-dried gel was immersed in it, and after reaching the swelling equilibrium, the exosome loading was completed. The freeze-dried exosome-loaded gel was observed by electron microscopy, and the number of exosomes in the field of view was observed and counted by Image J software, and then the overall exosome loading concentration was calculated.

[0040] Average survival days of exosomes: According to the observation by electron microscopy after slicing and freeze-drying the gel within N days, the number of days when more than 50% of the exosomes with a complete spherical or quasi-spherical shape and intact structural morphology were present within a 1μm field of view.

[0041] Exosome release concentration: The gel was immersed in 25 o C neutral PBS buffer solution, and the concentration of exosome particles in the solution was measured by dynamic light scattering. During the immersion, the water was changed once at a fixed time every day, and the release concentration values on the 1st, 3rd, and 5th days were measured respectively.

[0042] Drug loading and concentration test: The gel was immersed in an aqueous drug solution with a concentration of 100mg / ml for 24h, and the drug concentration in the aqueous solution after absorption was measured by ultraviolet spectroscopy, and the loading concentration was calculated from the ultraviolet spectroscopy absorbance intensity.

[0043] Drug release characteristics: The gel was immersed in 25 o C neutral PBS buffer solution, samples were taken at a given time, and the drug concentration in the PBS buffer solution was measured by ultraviolet spectroscopy, and the release concentration was calculated from the spectral absorbance intensity. Description of the Drawings

[0044] Figure 1 Fluorescence image of the dressing prepared in Example 1 after loading exosomes; Figure 2 Release curve of the dressing prepared in Example 1 after loading exosomes in neutral PBS solution; Figure 3 Release curve of the dressing prepared in Example 5 after loading levofloxacin hydrochloride in neutral PBS solution. Detailed Description of the Invention

[0045] The following will describe in detail the exemplary implementation methods of the present invention. However, these implementation methods are only for illustrative purposes, and the present invention is not limited thereto. Example 1

[0046] A drug-loaded and exosome-loaded gel skin wound dressing includes the following combination of substances and components Biomacromolecule 100 Modified polyvinyl alcohol 85 Ionic macromonomer 32 Ionic small molecule monomer 6 Hydrogen bond - acting monomer 24 The biomass macromolecule is gelatin.

[0047] The modified polyvinyl alcohol is PVA modified with N-(2,2 - dimethoxyethyl)-2 - acrylamide. The proportion of substituted PVA hydroxyl groups is 94%. The molecular weight of the PVA is 8000, and the degree of alcoholysis is 95%. The ionic macromolecule is zwitterionic monomer [2-(methacryloyloxy)ethyl]dimethyl-(3 - sulfopropyl) ammonium hydroxide (DMAPS).

[0048] The ionic small - molecule monomer is acrylic acid.

[0049] The hydrogen bond - acting monomer is hydroxyethyl acrylamide.

[0050] The preparation process of the drug - loaded and exosome - loaded gel skin wound dressing is as follows: (1) Modification of PVA: Dissolve a certain amount of PVA in deionized water with a concentration of 16 g / mL. Heat it to 95 °C and stir to dissolve. Add a small amount of hydrochloric acid to adjust the pH value to 5.5. Put a certain amount of modifier into the above solution and react for 12 h to prepare a modified PVA solution. Add pure water to cool and precipitate the product, and repeatedly rinse to obtain the final modified product. Among them, the addition amount of the modifier is calculated as 16 μL / g according to the mass ratio of PVA, and the final degree of modification is calculated as 16% based on the mass ratio of the grafted substance and polyvinyl alcohol, and the final degree of modification is calculated as 14.5% based on the mass ratio of the grafted substance and polyvinyl alcohol; (2) Preparation of the dressing: Add gelatin, modified PVA, ionic macromolecule, ionic small - molecule monomer, and hydrogen bond - acting monomer into pure water, heat it to 60 °C, and prepare an aqueous solution with a mass concentration of 10%. After stirring evenly, add 1.5% of photoinitiator 2959 based on the mass of the modified polyvinyl alcohol. After stirring evenly, irradiate with ultraviolet light at a wavelength of 365 nm for 8 h to obtain a pre - product of the dressing; The pre - product is soaked in excess water and repeatedly rinsed to remove unreacted small molecules to obtain the final dressing product.

[0051] The dressing loaded with mesenchymal stem cell exosomes is used for treatment.

[0052] The loading method of the exosomes is as follows: Dilute the mesenchymal stem cell exosomes with serum - free medium to a solution with a concentration of 5×10 5 cells / ml. Immerse the prepared gel in it for 12 h. After reaching swelling equilibrium, the exosome loading is completed. As Figure 1 seen from the fluorescence image of the cross - section of the exosome - loaded dressing, the red - stained exosomes are widely distributed in the gel.

[0053] The dressing loaded with stem cell exosomes is directly used for wound treatment; AsFigure 2 As shown, the loaded exosomes can achieve effective release for 7 days in neutral PBS solution.

[0054] The performance of the prepared gel dressing is shown in Table 1.

[0055] Example 2 A drug-loaded and exosome-loaded gel skin wound dressing comprises a combination of the following substances and components Biomacromolecule 100 Modified polyvinyl alcohol 65 Ionic macromonomer 20 Ionic small molecule monomer 8 Hydrogen bond monomer 40 The biomacromolecule is agar.

[0056] The modified polyvinyl alcohol is PVA modified with N-(2,2-dimethoxyethyl)-2-acrylamide, and the proportion of substituted PVA hydroxyl groups is 92%. The molecular weight of the PVA is 12000, and the degree of alcoholysis is 96% The ionic macromolecule is anionic monomer 2-acrylamido-2-methylpropanesulfonic acid (AMPS).

[0057] The ionic small molecule monomer is methyl acrylate.

[0058] The hydrogen bond monomer is hydroxyethyl acrylamide.

[0059] The preparation process of the drug-loaded and exosome-loaded gel skin wound dressing is as follows: (1) Modification of PVA: Dissolve a certain amount of PVA in deionized water at a concentration of 14 g / mL, heat to 95 °C and stir to dissolve, add a small amount of hydrochloric acid to adjust the pH value to 6, add a certain amount of modifier to the above solution, react for 12 h to prepare a modified PVA solution, add pure water to cool and precipitate the product, and obtain the final modified product after repeated washing. Among them, the addition amount of the modifier is calculated as 18 μL / g according to the mass ratio of PVA, and the final modification degree is calculated as 16.2% based on the mass ratio of the grafted substance and polyvinyl alcohol; (2) Preparation of the dressing: Add agar, modified PVA, ionic macromolecule, ionic small molecule monomer, and hydrogen bond monomer to pure water, heat to 60 °C, and configure into an aqueous solution with a mass concentration of 9%, stir evenly, add 2959 photoinitiator at 1.8% of the mass of the modified polyvinyl alcohol, stir evenly, and irradiate with ultraviolet light at a wavelength of 365 nm for 10 h to obtain a pre-product of the dressing; The pre-product is soaked in excess water and repeatedly rinsed to remove unreacted small molecules to obtain the final dressing product.

[0060] The dressing loaded with adipose-derived stem cell exosomes is used for treatment.

[0061] The loading method of the exosomes is as follows: Dilute mesenchymal stem cell exosomes into a solution with a concentration of 5×10 5 cells / ml with serum-free medium, immerse the prepared gel in it for 16 h, and complete the exosome loading after reaching swelling equilibrium.

[0062] The dressing loaded with stem cell exosomes is directly used for wound treatment.

[0063] The performance of the prepared gel dressing is shown in Table 1.

[0064] Example 3 A drug-loaded and exosome-loaded gel skin wound dressing includes the following combination of substances and components Biomacromolecule 100 Modified polyvinyl alcohol 100 Ionic macromonomer 35 Ionic small molecule monomer 8.5 Hydrogen bond monomer 55 The biomacromolecule is methacrylic acid-modified gelatin, and its degree of substitution is between 15-25% calculated by the weight of gelatin.

[0065] The modified polyvinyl alcohol is PVA modified with N-(methoxymethyl)-2-acrylamide, the proportion of substituted PVA hydroxyl groups is 95%, the molecular weight of the PVA is 6000, and the degree of alcoholysis is 99% The ionic macromolecule is anionic monomer 2-acrylamido-2-methylpropanesulfonic acid (AMPS).

[0066] The ionic small molecule monomer is acrylic acid.

[0067] The hydrogen bond monomer is N-vinylpyrrolidone.

[0068] The preparation process of the drug-loaded and exosome-loaded gel skin wound dressing is as follows: (1) Modification of PVA: Dissolve a certain amount of PVA in deionized water with a concentration of 18 g / mL, heat it to 95 °C and stir to dissolve, add a small amount of hydrochloric acid to adjust the pH value to 5.5, add a certain amount of modifier into the above solution, react for 16 h to prepare a modified PVA solution, add pure water to cool and precipitate the product, and obtain the final modified product after repeated washing. Among them, the addition amount of the modifier is calculated as 20 μL / g according to the mass ratio of PVA, and the final degree of modification is calculated as 18.4% based on the mass ratio of the grafted substance and polyvinyl alcohol; (2) Preparation of the dressing: Add methacrylic acid-modified gelatin, modified PVA, ionic macromolecules, ionic small molecule monomers, and hydrogen bond monomers to pure water, heat to 60 °C, and prepare an aqueous solution with a mass concentration of 12%. After stirring evenly, add 2% of the photoinitiator 2959 based on the mass of the modified polyvinyl alcohol. After stirring evenly, irradiate with ultraviolet light at a wavelength of 365 nm for 12 h to obtain a pre-product of the dressing; the pre-product is soaked in excess water and repeatedly rinsed to remove unreacted small molecules to obtain the final dressing product.

[0069] The dressing loaded with mesenchymal stem cell exosomes is used for treatment.

[0070] The loading method of the exosomes is as follows: Dilute mesenchymal stem cell exosomes with serum-free medium to a solution with a concentration of 5×10 5 cells / ml, immerse the prepared gel in it for 16 h, and the exosome loading is completed after reaching the swelling equilibrium.

[0071] The dressing loaded with stem cell exosomes is directly used for wound treatment.

[0072] The performance of the prepared gel dressing is shown in Table 1.

[0073] Example 4 A drug-loaded and exosome-loaded gel skin wound dressing includes the following combination of substances and components Biomass macromolecule 100 Modified polyvinyl alcohol 50 Ionic macromolecule monomer 40 Ionic small molecule monomer 5.5 Hydrogen bond monomer 50 The biomass macromolecule is quaternary ammonium salt-modified chitosan, and its degree of substitution is 18% calculated based on the weight of chitosan.

[0074] The modified polyvinyl alcohol is PVA modified with N-(methoxymethyl)-2-acrylamide, and the proportion of PVA hydroxyl groups replaced is 92%. The molecular weight of the PVA is 8000, and the degree of alcoholysis is 95% The ionic macromolecule is the zwitterionic monomer [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide (DMAPS).

[0075] The ionic small molecule monomer is acrylic acid.

[0076] The hydrogen bond monomer is N-isopropylacrylamide.

[0077] The preparation process of the drug-loaded and exosome-loaded gel skin wound dressing is as follows: (1)Modification of PVA: A certain amount of PVA was dissolved in deionized water at a concentration of 12 g / mL. After heating to 95 °C and stirring for dissolution, a small amount of hydrochloric acid was added to adjust the pH value to 6. A certain amount of modifier was added to the above solution, and after reacting for 14 h, a modified PVA solution was prepared. After adding pure water to cool, the product was precipitated, and the final modified product was obtained after repeated rinsing. Among them, the addition amount of the modifier was calculated as 14 μL / g based on the mass ratio of PVA, and the final modification degree was calculated as 12.6% based on the mass ratio of the grafted substance and polyvinyl alcohol; (2)Preparation of the dressing: Quaternary ammonium salt-modified chitosan, modified PVA, ionic macromolecules, ionic small molecule monomers, and hydrogen bond-forming monomers were added to pure water, heated to 60 °C, and configured into an aqueous solution with a mass concentration of 10%. After stirring evenly, 1% of photoinitiator 2959 based on the mass of modified polyvinyl alcohol was added. After stirring evenly, it was irradiated with ultraviolet light at a wavelength of 365 nm for 12 h to obtain a pre-product of the dressing; the pre-product was soaked in excess water and repeatedly rinsed to remove unreacted small molecules to obtain the final dressing product.

[0078] The prepared dressing is loaded with the anti-inflammatory drug polyhexamethylene biguanide hydrochloride for treatment use.

[0079] The drug loading method is as follows: The gel was soaked in an aqueous solution of the drug with a concentration of 100 mg / ml for 24 h to complete drug loading.

[0080] The dressing after loading the drug is directly used for wound treatment.

[0081] The performance of the prepared gel dressing is shown in Table 1.

[0082] Example 5 A drug-loaded and exosome-loaded gel skin wound dressing includes the following combination of substances and components Biomacromolecule 100 Modified polyvinyl alcohol 42 Ionic macromolecule monomer 22 Ionic small molecule monomer 8 Hydrogen bond-forming monomer 56 The biomacromolecule is chitosan.

[0083] The modified polyvinyl alcohol is PVA modified with N-(methoxymethyl)-2-acrylamide, and the proportion of substituted PVA hydroxyl groups is 94%. The molecular weight of the PVA is 12,000, and the degree of alcoholysis is 96% The ionic macromolecule is the zwitterionic monomer [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide (DMAPS).

[0084] The ionic small molecule monomer is ethyl acrylate.

[0085] The hydrogen bond monomer is hydroxyethyl acrylamide.

[0086] The preparation process of the drug-loaded and exosome-loaded gel skin wound dressing is as follows: (1) Modification of PVA: Dissolve a certain amount of PVA in deionized water at a concentration of 15 g / mL, heat it to 95 °C and stir to dissolve, add a small amount of hydrochloric acid to adjust the pH value to 5, add a certain amount of modifier into the above solution, react for 12 h to prepare a modified PVA solution, add pure water to cool and precipitate the product, and obtain the final modified product after repeated washing. Among them, the addition amount of the modifier is calculated as 22 μL / g according to the mass ratio of PVA, and the final degree of modification is calculated as 19.4% based on the mass ratio of the grafted substance to polyvinyl alcohol; (2) Preparation of the dressing: Add chitosan, modified PVA, ionic macromolecules, ionic small molecule monomers, and hydrogen bond monomers into pure water, add oxalic acid to adjust the pH value to 5.5, heat it to 50 °C, and prepare an aqueous solution with a mass concentration of 8%, stir evenly, add 1% of photoinitiator 2959 based on the mass of the modified polyvinyl alcohol, stir evenly, and irradiate with ultraviolet light at a wavelength of 365 nm for 12 h to obtain a pre-product of the dressing; the pre-product is soaked in excess water and repeatedly washed to remove unreacted small molecules to obtain the final dressing product.

[0087] The dressing is loaded with the anti-inflammatory drug levofloxacin hydrochloride for treatment use.

[0088] The drug loading method is as follows: Soak the gel in an aqueous solution of the drug with a concentration of 100 mg / ml for 24 h to complete drug loading, and the drug loading concentration reaches 63.4 mg / ml. As Figure 3 shown, it is immersed in a neutral PBS solution, and there is a significant release in the first 6 days, and there is still a sustained release effect within 8 - 10 days.

[0089] The dressing loaded with the drug is directly used for wound treatment.

[0090] The performance of the prepared gel dressing is shown in Table 1.

[0091] Comparative Example 1 Compared with Example 1, the modified polyvinyl alcohol is replaced with unmodified pure polyvinyl alcohol raw material, and the raw material characteristics are the same as those in Example 1. Other preparation processes and the process of loading exosomes are the same as those in Example 1.

[0092] The performance of the prepared gel dressing is shown in Table 2.

[0093] Overall, since polyvinyl alcohol was not modified to incorporate double bonds, the added polymers did not polymerize into a whole, resulting in a significant decrease in the toughness of the gel. At the same time, due to the complex entanglement and wrapping state of the polymer segments, the ability to load exosomes decreased significantly, and the viability of exosomes decreased significantly.

[0094] Comparative Example 2 Compared with Example 1, no ionic small molecule monomer was added, and the other preparation processes and the exosome-loading process were the same as those in Example 1.

[0095] The properties of the prepared gel dressing are shown in Table 2.

[0096] Overall, since no ionic small molecule monomer was added, the concentration of ionic groups in the system decreased significantly, resulting in a significant decrease in the swelling rate of the gel dressing, a decrease in the exosome-loading concentration, and a decrease in the survival days.

[0097] Comparative Example 3 Compared with Example 1, no ionic macromolecule was added, and the other preparation processes and the exosome-loading process were the same as those in Example 1.

[0098] The properties of the prepared gel dressing are shown in Table 2.

[0099] Overall, since no ionic macromolecule was added, the concentration of ionic groups in the system decreased significantly, and the flexibility of the whole molecular chain decreased, resulting in an increase in the strength of the gel dressing, a significant decrease in toughness, a significant decrease in the swelling rate, a decrease in the exosome-loading concentration, and a decrease in the survival days.

[0100] Comparative Example 4 Compared with Example 1, no hydrogen-bonding monomer was added, and the other preparation processes and the exosome-loading process were the same as those in Example 1.

[0101] The properties of the prepared gel dressing are shown in Table 2.

[0102] Overall, since no hydrogen-bonding monomer was added, the flexibility of the system decreased significantly, the swelling rate decreased significantly, the exosome-loading concentration decreased, and the survival days decreased.

[0103] Comparative Example 5 Compared with Example 3, the methacrylic acid-modified gelatin was replaced with unmodified pure gelatin, and the other preparation and exosome-loading processes were exactly the same.

[0104] The properties of the prepared gel dressing are shown in Table 2.

[0105] Overall, due to the lack of modification, gelatin can only be dispersed in the gel network in the form of independent macromolecules. The strength and modulus of the gel change little, but the elongation at break and toughness decrease significantly. At the same time, the swelling rate decreases slightly, the loading capacity for exosomes decreases, and the secretion rate decreases.

[0106] Comparative Example 6 Compared with Example 4, no ionic macromolecules or ionic small molecule monomers are added during the preparation process, and the other preparation and drug loading processes are exactly the same.

[0107] The properties of the prepared gel dressing are shown in Table 2.

[0108] Overall, due to the lack of addition of ionic monomers, the concentration of ionic groups in the system decreases significantly, the flexibility of the whole molecular chain decreases, it is very easy to break, the swelling rate decreases significantly, and the concentration of the loaded drug decreases significantly.

[0109] Table 1. Summary of the properties of the gel dressings prepared in the examples: Example 1 Example 2 Example 3 Example 4 Example 5 Tensile strength (MPa) 3.24 2.6 3.63 2.1 1.9 <![CDATA[Toughness (MJ / m 3 ).]]> 3.87 3.49 2.87 4.62 4.76 Elongation at break (%) 424.7 517.7 296.3 714.6 672.4 Elastic modulus (MPa) 1.04 0.95 1.14 0.88 0.76 Storage modulus / Loss modulus (KPa) <![CDATA[2.8*10 3 / 1.4*10 3 > <![CDATA[2.5*10 3 / 4.2*10 2 > <![CDATA[3.2*10 3 / 5.3*10 2 > <![CDATA[1.7*10 3 / 8.2*10 2 > <![CDATA[1.4*10 3 / 6.7*10 2 > Skin adhesion strength (kPa) 183.5 192.7 287.4 214.5 325.6 Equilibrium swelling ratio 4.32 4.65 5.61 6.26 5.28 <![CDATA[Exosome loading concentration (10 4 cells / ml)]]> 4.96 4.53 5.41 / / Average survival days of exosomes 2.4 3.5 3.9 / / <![CDATA[Accumulative release concentration of exosomes (10 4 cells / ml) (1 / 3 / 5 days)]]> 0.65 / 1.13 / 1.75 1.41 / 2.12 / 2.46 1.67 / 2.43 / 2.92 / / Drug loading concentration (mg / ml) / / / 83.9 63.4 Drug release concentration (mg / ml) (Day 1 / 3 / 5 / 7) / / / 14.1 / 8.7 / 3.8 / 2.8 11.5 / 8.4 / 4.1 / 2.8 Table 2. Summary of the properties of the gel dressings prepared in the comparative examples: Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Comparative Example 6 Tensile strength (MPa) 2.78 3.14 2.46 2.71 3.18 3.51 <![CDATA[Toughness (MJ / m 3 )]]> 1.46 2.76 1.38 0.54 0.51 0.42 Elongation at break (%) 94.5 349.2 104.2 67.2 58.1 49.2 Elastic modulus (MPa) 0.92 1.18 1.24 0.88 0.96 1.28 Storage modulus / Loss modulus (KPa) <![CDATA[2.3*10 3 / 1.4*10 2 > <![CDATA[3.1*10 3 / 1.2*10 2 > <![CDATA[2.8*10 3 / 1.7*10 2 > <![CDATA[3.2*10 3 / 0.9*10 2 > <![CDATA[2.4*10 3 / 1.3*10 2 > <![CDATA[4.6*10 3 / 2.1*10 2 > Skin adhesion strength (kPa) 82.4 138.5 145.3 79.2 167.8 42.7 Equilibrium swelling ratio 3.73 3.67 3.92 3.85 4.12 2.46 <![CDATA[Exosome loading concentration (10 4 cells / ml)]]> 0.84 0.92 1.05 0.89 2.05 / Average survival days of exosomes 2.2 2.1 3.4 1.9 2.3 / <![CDATA[Accumulative release concentration of exosomes (10 4 cells / ml) (1 / 3 / 5 days)]]> 0.14 / 0.33 / 0.46 0.12 / 0.31 / 0.54 0.28 / 0.41 / 0.52 0.21 / 0.42 / 0.46 0.62 / 1.14 / 1.51 / Drug loading concentration (mg / ml) / / / / / 24.6 Drug release concentration (mg / ml) (1 / 3 / 5 / 7 days) / / / / / 7.2 / 2.1 / 1.4 / 1.1

Claims

1. A drug-loaded and exosome-loaded gel skin wound dressing, prepared from the following substances and their corresponding mass ratios: Biomass Macromolecules 100 Modified polyvinyl alcohol 30-120 Ionic macromonomer 15-40 Ionic small molecule monomer 4-12 Hydrogen bonding monomer 20-60 in, The biomass macromolecule is a water-soluble biomass macromolecule having an ionic group and a hydrogen-bonding group; The modified polyvinyl alcohol, ionic macromolecular monomer, ionic small molecule monomer and hydrogen bonding monomer all have double bonds and can be polymerized and connected to each other through free radical polymerization.

2. The drug-loaded and exosome-loaded gel skin wound dressing according to claim 1, characterized in that: The biomass macromolecules include gelatin, agar, chitosan and water-soluble modified products of the above biomass macromolecules; preferably, the modified products are methacrylic acid modified gelatin, quaternary ammonium salt modified chitosan, and maleic anhydride modified agar.

3. The drug-loaded and exosome-loaded gel skin wound dressing according to claim 1, characterized in that: The modified polyvinyl alcohol is polyvinyl alcohol modified with N-(2,2-dimethoxyethyl)-2-acrylamide or N-(methoxymethyl)-2-acrylamide or N-(methoxymethyl)-2-acrylamide as a modifier, wherein the methoxy group in the modifier and the hydroxyl group in the polyvinyl alcohol are condensed under acidic conditions to achieve modification, and the proportion of the hydroxyl group in the polyvinyl alcohol replaced by the modifier is greater than 90%; the molecular weight of the polyvinyl alcohol is between 4000 and 16000, and the alcoholysis degree is between 88 and 98%; It is also characterized in that the modification process of the polyvinyl alcohol is as follows: a certain amount of polyvinyl alcohol is added into deionized water, heated to 95°C and stirred to dissolve, the concentration is between 12-20 g / mL, a small amount of hydrochloric acid is added to adjust the pH value to between 6-4.5, a certain amount of modifier is added into the above solution, the reaction is continuously stirred for 10-16 hours to prepare a modified polyvinyl alcohol solution, pure water is added and cooled to precipitate the product, and the final modified product is obtained after repeated rinsing; wherein, the amount of the modifier added is between 10-24 μL / g of the mass of the polyvinyl alcohol; and the final degree of modification is calculated based on the mass ratio of the accession and the polyvinyl alcohol and is between 12-24%.

4. The drug-loaded and exosome-loaded gel skin wound dressing according to claim 1, characterized in that: The ionic macromonomer is one of the zwitterionic monomer [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonatepropyl)ammonium hydroxide (DMAPS) or the anionic monomer 2-acrylamide-2-methylpropanesulfonic acid (AMPS).

5. The drug-loaded and exosome-loaded gel skin wound dressing according to claim 1, characterized in that: The ionic small molecule monomer is acrylic acid or an acrylic ester monomer.

6. The drug-loaded and exosome-loaded gel skin wound dressing according to claim 1, characterized in that: The hydrogen bonding monomer is one of N-vinyl pyrrolidone, hydroxyethyl acrylamide and N-isopropyl acrylamide.

7. The drug-loaded and exosome-loaded gel skin wound dressing according to claim 1, characterized in that: The gel dressing is immersed in an exosome culture solution after freeze-drying to achieve exosome loading, and the loading concentration is between 0-8×10 4 Between pc / ml.

8. The drug-loaded and exosome-loaded gel skin wound dressing according to claim 1, characterized in that: The gel dressing can realize efficient loading of cationic small molecule drugs by immersing the swollen or freeze-dried gel in a drug solution, and the loading concentration is between 0-140 mg / ml.

9. The drug-loaded and exosome-loaded gel skin wound dressing according to claim 1, characterized in that: The preparation process of the drug-carrying and exosome-loaded gel skin wound dressing is as follows: biomass macromolecules, modified polyvinyl alcohol, ionic macromolecular monomers, ionic small molecule monomers, and hydrogen bonding monomers are added to pure water or weakly acidic water with a pH value of 5.5-6.5 to prepare an aqueous solution with a mass concentration of 8-12%, and after stirring evenly, a water-soluble photoinitiator with a mass of 0.2-0.4% of modified polyvinyl alcohol is added, and after stirring evenly, ultraviolet light with a maximum absorption wavelength corresponding to the photoinitiator is irradiated for 6-12 hours to obtain a dressing gel pre-product; the pre-product is soaked in excess water and repeatedly rinsed to remove unreacted small molecules and acid ions therein, and the pH value is adjusted to neutral to obtain a final dressing product.

10. The drug-loaded and exosome-loaded gel skin wound dressing according to claim 1, characterized in that: If the drug-loaded and exosome-loaded gel skin wound dressing needs to be loaded with exosomes before use, it needs to be freeze-dried and dehydrated for storage after preparation, and soaked in the culture medium solution of exosomes before use to achieve sufficient swelling so that the exosomes enter the gel and reach the required concentration before use.

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