Process for the preparation of degradable bandages based on poly-lactic acid blend formulations

Abstract

The application relates to the technical field of functional medical dressing, and discloses a preparation process of a degradable bandage based on a polylactic acid blending formula, which comprises the following steps: dissolving polylactic acid and polyethylene glycol in an organic solvent to obtain a polymer base solution; dispersing pre-prepared sodium bicarbonate-urea inclusion compound particles and PLGA-wrapped citric acid sustained-release microsphere particles in the polymer base solution to obtain a spinning precursor suspension; and performing electrostatic spinning on the suspension to collect a fiber membrane. By immobilizing the two functional particles in the electrostatic spinning fiber membrane matrix, the rate of the whole acid-base neutralization reaction is controlled by the acid sustained-release step, so that the inherent explosive gas production reaction is changed into a long-acting and stable process. The application solves the technical problems of unstable gas production components and uncontrollable reaction rate, and obtains a degradable bandage which can continuously and stably generate gas in an application environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of functional medical dressing, in particular to a preparation process of a degradable bandage based on a polylactic acid blending formula. BACKGROUND

[0002] In the field of medical materials, a dressing capable of generating a specific gas (such as carbon dioxide) in situ and continuously at the use site has attracted attention due to its potential application in improving local microcirculation and regulating local acid-base environment. The technical approach to achieve this function is usually to simultaneously load a solid acid and a solid carbonate or bicarbonate in an inert carrier material. When the composite material is in contact with an aqueous environment (such as wound exudate), the acid and base dissolve and undergo a neutralization reaction, releasing carbon dioxide gas.

[0003] However, the preparation method in the prior art by directly physically blending powders of acid and base in the matrix material has two inherent technical defects. First, sodium bicarbonate and other substances as base sources have certain hygroscopicity and are chemically unstable. In a porous dressing matrix, they are prone to premature decomposition or reaction by absorbing moisture from the environment during the storage and transportation of the product, resulting in a decrease or complete failure of the gas production performance of the product when it is actually used, affecting the shelf life and functional reliability of the product.

[0004] Secondly, when such directly blended materials come into contact with a liquid, the solid acid and base will dissolve quickly at the same time and immediately trigger a violent and uncontrolled chemical reaction. This reaction kinetic characteristic results in almost all the gas being explosively generated in a very short time window. This instantaneous gas release behavior makes it difficult to meet the needs of long-acting and stable action on the application site, limiting the actual application range of this type of material. Therefore, the prior art has not yet provided a technical solution that can simultaneously ensure the storage stability of the gas-producing components and achieve the sustained and controllable release of gas. SUMMARY

[0005] The technical problem to be solved by the present application is how to overcome the technical defects that the gas-producing functional components are prone to failure due to unstable chemical properties during storage, and the gas is released instantaneously due to uncontrollable reaction rate in the application environment.

[0006] To solve the above problems, the present application provides the following technical solutions:

[0007] In a first aspect, the present application provides a preparation process of a degradable bandage based on a polylactic acid blending formula, which adopts the following technical solutions:

[0008] A preparation process of a degradable bandage based on a polylactic acid blending formula, comprising the following steps:

[0009] a) dissolving polylactic acid and polyethylene glycol in an organic solvent to obtain a polymer base solution;

[0010] b) dispersing the pre-prepared sodium bicarbonate-urea inclusion compound and the pre-prepared PLGA-encapsulated citric acid sustained-release microspheres in the polymer base solution of step a) to obtain a spinning precursor suspension;

[0011] c) electrospinning the spinning precursor suspension of step b) to collect a fibrous membrane to obtain the degradable bandage.

[0012] By adopting the technical scheme, the process realizes control of in-situ chemical reaction in time dimension by structurally combining two functional components and immobilizing them in a degradable fiber matrix. The mechanism is as follows:

[0013] First, during storage and transportation, the chemically active alkali source sodium bicarbonate is co-crystallized with urea to form an inclusion compound, and the urea crystal lattice structure physically encapsulates the sodium bicarbonate molecules. This structure effectively blocks the contact between external water molecules and internal sodium bicarbonate, inhibits the decomposition of sodium bicarbonate due to moisture absorption under storage conditions, and ensures the chemical stability of the alkali source before application.

[0014] Second, when the bandage contacts with an aqueous medium (such as wound exudate), the two functional components respond in time sequence:

[0015] 1) The host (urea) of the sodium bicarbonate-urea inclusion compound has high water solubility, and its crystal lattice structure rapidly dissolves under the action of water molecules, causing the encapsulated guest molecule sodium bicarbonate to be immediately and completely released into the medium, providing sufficient bicarbonate ions (HCO3 - ).

[0016] 2) The PLGA-encapsulated citric acid sustained-release microspheres respond at a controlled rate. Water molecules penetrate into the PLGA polymer matrix, or the PLGA matrix itself undergoes slow hydrolytic degradation, causing the encapsulated citric acid molecules to be continuously released into the external medium at a lower flux.

[0017] 3) The continuously released citric acid (C6H8O7) undergoes neutralization reaction with the bicarbonate ions already existing in the medium, as shown in the chemical formula:

[0018] 3HCO3 - +C6H8O7→C6H5O7 3- +3H2O+3CO2(g)↑;

[0019] Since the rate of this reaction is limited by the slow release rate of citric acid, carbon dioxide gas is continuously and smoothly generated, avoiding the instantaneous explosive reaction caused by directly mixing the two reactants.

[0020] Therefore, the present process decomposes a fast chemical reaction into a sustainable reaction process controlled by a slow-release step by differentiating the release behaviors of the acid source and the base source, thereby achieving the technical effect of long-term and stable generation of carbon dioxide in an application environment.

[0021] Preferably, in the total mass of the polylactic acid, polyethylene glycol, sodium bicarbonate-urea inclusion compound and PLGA-coated citric acid sustained-release microspheres, the mass percentage of each component is as follows: polylactic acid 60%-75%; polyethylene glycol 15%; the sum of sodium bicarbonate-urea inclusion compound and PLGA-coated citric acid sustained-release microspheres 10%-25%.

[0022] By adopting the above technical solution, within this component allocation ratio range, it can not only ensure sufficient functional component content to obtain the expected total amount of gas generation, but also maintain the spinning precursor suspension with suitable viscosity and electrical conductivity, while ensuring that the final prepared fiber membrane has necessary mechanical properties and structural integrity.

[0023] Preferably, the sodium bicarbonate-urea inclusion compound is prepared by dissolving urea and sodium bicarbonate in a hot methanol solution, and then crystallizing the inclusion compound by programmed cooling; wherein the molar ratio of urea to sodium bicarbonate is 5:1-10:1.

[0024] By adopting the above technical solution, the preparation method can stably obtain a crystal structure with good inclusion effect. The selected molar ratio range ensures that there are enough urea molecules to form the host lattice, thereby achieving high-efficiency physical packaging of sodium bicarbonate guest molecules.

[0025] Preferably, the PLGA-coated citric acid sustained-release microspheres are prepared by a double emulsion solvent evaporation method, which includes forming an inner water phase containing an aqueous citric acid solution, an oil phase containing PLGA, and an outer water phase containing polyvinyl alcohol, preparing a W1 / O / W2 type double emulsion, and then evaporating the solvent in the oil phase to solidify and form microspheres; wherein the mass concentration of PLGA in the oil phase is 5%(w / v)-15%(w / v).

[0026] By adopting the above technical solution, the double emulsion method is an effective way to prepare polymer microspheres of water-soluble core substances (citric acid). The concentration of PLGA in the oil phase is a key parameter for regulating the density and degradation rate of the microsphere wall. Within this concentration range, microspheres with different sustained-release curves can be prepared, thereby realizing the controllability of the gas generation rate of the final product.

[0027] Preferably, the process parameters of electrospinning are as follows: applied voltage 15kV-25kV, solution propulsion speed 0.5mL / h-1.5mL / h, and needle tip to receiver distance 15cm-25cm.

[0028] By adopting the technical scheme, Taylor cones can be stably formed, and nanoscale fibers with uniform morphology and concentrated diameter distribution can be obtained by spinning in the process parameter range, so that a fiber membrane with high porosity and high specific surface area can be finally prepared.

[0029] In a second aspect, the application provides a degradable bandage, which adopts the following technical scheme:

[0030] The degradable bandage is composed of a fiber membrane matrix made of polylactic acid and polyethylene glycol blended fibers, and sodium bicarbonate-urea inclusion compound particles and PLGA-coated citric acid sustained-release microsphere particles dispersed in the fiber membrane matrix.

[0031] By adopting the technical scheme, the degradable bandage of the application has a structure in which two functionally designed particles with different physical and chemical properties are solid-loaded in a solid dispersion form in a porous fiber membrane matrix, thereby constructing an environmentally responsive time-sequential reaction system.

[0032] The sodium bicarbonate-urea inclusion compound particles isolate sodium bicarbonate molecules from the external environment by using the physical encapsulation effect of the urea lattice, thereby ensuring the chemical stability of the alkali source in a dry state. The PLGA-coated citric acid sustained-release microsphere particles use a degradable PLGA polymer matrix as a physical barrier to achieve sustained release of the internal encapsulated acid source through a combination of bulk degradation and molecular diffusion mechanisms.

[0033] When the bandage contacts an aqueous medium, the high porosity structure of the fiber membrane matrix promotes rapid penetration of the medium and simultaneously triggers time-sequential responses of the two particles: the sodium bicarbonate-urea inclusion compound instantaneously releases all bicarbonate ions due to the rapid dissolution of urea; and the PLGA microspheres continuously release citric acid at a slow rate determined by their own degradation rate. As a result, a sustainable acid-base neutralization reaction determined by the slow-release step is established, enabling carbon dioxide to be continuously and smoothly generated within a period of several hours. At the same time, the continuously released citrate ions can chelate calcium ions, and the generated carbon dioxide and protons can reduce the local pH, triggering the Bohr effect and promoting the release of oxygen from oxygenated hemoglobin.

[0034] Preferably, in the total mass of the polylactic acid, polyethylene glycol, sodium bicarbonate-urea inclusion compound, and PLGA-coated citric acid sustained-release microspheres, the mass percentage of each component is as follows: polylactic acid 60% to 75%; polyethylene glycol 15%; and the sum of sodium bicarbonate-urea inclusion compound and PLGA-coated citric acid sustained-release microspheres 10% to 25%.

[0035] By adopting the technical scheme, the component distribution ratio ensures that the fiber membrane substrate has sufficient mechanical support strength, and sufficient functional particles are loaded to achieve the expected technical effect.

[0036] Preferably, in the sodium bicarbonate-urea inclusion compound, the molar ratio of urea to sodium bicarbonate is 5:1 to 10:1.

[0037] By adopting the technical scheme, the molar ratio range ensures that the urea molecules can form a complete crystal lattice channel structure, thereby realizing efficient inclusion of sodium bicarbonate guest molecules and structural stability.

[0038] Preferably, in the preparation of the PLGA-coated citric acid sustained-release microspheres, the mass concentration of PLGA in the oil phase used is 5% (w / v) to 15% (w / v).

[0039] By adopting the technical scheme, the concentration range is a key parameter for adjusting the density and degradation rate of the microsphere polymer wall, directly determines the release curve of citric acid, and thereby provides a technical means for regulating the gas generation rate of the final product.

[0040] In summary, the present application has at least one of the following beneficial technical effects:

[0041] 1. The preparation process of the present application significantly improves the chemical stability of the gas generating functional component during storage and transportation by preparing chemically unstable sodium bicarbonate into sodium bicarbonate-urea inclusion compound. The mechanism is that the crystal lattice structure formed by urea molecules provides a physical barrier for the internal sodium bicarbonate molecules, effectively preventing their contact with external water vapor, thereby inhibiting the premature decomposition of the material due to moisture absorption before application, and ensuring the functional reliability of the product within the shelf life.

[0042] 2. The present application realizes effective control of the in-situ gas generation reaction rate by structurally combining the fast-release alkali source (sodium bicarbonate-urea inclusion compound) with the slow-release acid source (PLGA-coated citric acid sustained-release microspheres). When the bandage is activated, the rate of the entire acid-base neutralization reaction is determined by the slow release rate of citric acid from the PLGA microspheres, thereby converting the inherent instantaneous and explosive reaction into a sustainable and smooth gas generation process lasting for several hours, and realizing the regulation of the gas release behavior in the time dimension.

[0043] 3. The present application adopts electrospinning technology to uniformly disperse and immobilize two kinds of pre-prepared functional particles in the polylactic acid blended fiber network, forming a structural integrated functional composite material. This process not only ensures the stable existence and spatial proximity of the two kinds of functional particles in the matrix, but also the high porosity and high specific surface area of the formed fiber membrane, which is beneficial to the rapid absorption of liquid and the activation of internal time sequence chemical reaction in application, successfully integrating the complex two-component controlled release system into a single, easy-to-use bandage product. DETAILED DESCRIPTION

[0044] The main raw materials and reagents used in the following examples and comparative examples are as follows, and the reagents not specifically mentioned are commercially available analytical pure or higher grade products.

[0045] Polylactic acid, CAS number: 26100-51-6, the weight average molecular weight of polylactic acid used in the present application is 80000-120000 g / mol.

[0046] Polyethylene glycol, CAS number: 25322-68-3, the number average molecular weight of polyethylene glycol used in the present application is 4000-8000 g / mol.

[0047] Polylactic acid-glycolic acid copolymer is a copolymer obtained by random copolymerization of lactic acid and glycolic acid monomers, and the molar ratio of lactic acid units to glycolic acid units of the PLGA used in the present application is 75:25, and its intrinsic viscosity in chloroform is 0.50-0.70 dL / g.

[0048] Polyvinyl alcohol, CAS number: 9002-89-5, the alcoholysis degree of polyvinyl alcohol used in the present application is 87-89%, and the average molecular weight is 30000-70000 g / mol.

[0049] Preparation Examples 1-6:

[0050] Preparation Example 1:

[0051] The present preparation example provides a preparation method of sodium bicarbonate-urea inclusion compound (A1), the steps are as follows:

[0052] Take 90.09 g (1.5 mol) of urea and add it to 1000 mL of methanol, heat to 55°C and magnetically stir to completely dissolve. Take 25.20 g (0.3 mol) of sodium bicarbonate powder and slowly add it to the above urea-methanol hot solution, at this time the molar ratio of urea to sodium bicarbonate is 5:1.

[0053] The stirring was continued at 55°C for 1 hour. The heating was stopped, and the mixed solution was programmed to decrease the temperature to 5°C at a rate of 0.8°C / min, and was left to crystallize at this temperature for 6 hours. The precipitated crystals were collected by vacuum filtration, and were washed twice with 100 mL of pre-cooled anhydrous methanol. The obtained crystals were dried in a vacuum drying oven at 45°C for 20 hours to obtain the sodium bicarbonate-urea clathrate (Al) in the form of white crystal powder, which was sealed and stored in a desiccator for later use.

[0054] Preparation Example 2:

[0055] This preparation example provides a method for preparing sodium bicarbonate-urea clathrate (A2), the steps of which are as follows:

[0056] 90.09 g (1.5 mol) of urea was weighed and added to 1000 mL of methanol, which was heated to 55°C and magnetically stirred to completely dissolve. 12.60 g (0.15 mol) of sodium bicarbonate powder was slowly added to the above-mentioned hot urea-methanol solution, at which time the molar ratio of urea to sodium bicarbonate was 10:1.

[0057] The subsequent operation steps, including stirring, temperature decrease, crystallization, collection, washing, and drying, were all identical to those of Preparation Example 1. Finally, the sodium bicarbonate-urea clathrate (A2) was obtained in the form of white crystal powder, which was sealed and stored in a desiccator for later use.

[0058] Preparation Example 3:

[0059] This preparation example provides a method for preparing sodium bicarbonate-urea clathrate (A3), the steps of which are as follows:

[0060] 90.09 g (1.5 mol) of urea was weighed and added to 1000 mL of methanol, which was heated to 55°C and magnetically stirred to completely dissolve. 16.80 g (0.2 mol) of sodium bicarbonate powder was slowly added to the above-mentioned hot urea-methanol solution, at which time the molar ratio of urea to sodium bicarbonate was 7.5:1.

[0061] The subsequent operation steps, including stirring, temperature decrease, crystallization, collection, washing, and drying, were all identical to those of Preparation Example 1. Finally, the sodium bicarbonate-urea clathrate (A3) was obtained in the form of white crystal powder, which was sealed and stored in a desiccator for later use.

[0062] Preparation Example 4:

[0063] This preparation example provides a method for preparing PLGA-coated citric acid sustained-release microspheres (B1), the steps of which are as follows:

[0064] Preparation of the inner water phase (W1): 4.0 g of anhydrous citric acid was weighed and dissolved in 10 mL of deionized water to obtain a citric acid solution with a mass concentration of 40% (w / v).

[0065] Preparation of oil phase (O): 5.0 g PLGA (lactic acid / ethanol acid = 75:25) was weighed and dissolved in 100 mL dichloromethane to obtain a PLGA solution with a mass concentration of 5% (w / v).

[0066] Preparation of external water phase (W2): 1.0 g polyvinyl alcohol (PVA) was weighed and dissolved in 100 mL deionized water to obtain a PVA solution with a mass concentration of 1% (w / v).

[0067] The inner water phase (W1) was added to the oil phase (O) and emulsified using a high-speed shearing homogenizer at a speed of 12,000 rpm for 1 minute to form a primary emulsion (W1 / O). The primary emulsion was then added to the external water phase (W2) and homogenized at a speed of 2000 rpm for 2 minutes to form a multiple emulsion (W1 / O / W2). The multiple emulsion was transferred to a beaker and stirred magnetically at room temperature for 4 hours to volatilize the dichloromethane. The microspheres were collected by centrifugation (4000 rpm, 8 minutes) and washed repeatedly with deionized water and centrifuged three times.

[0068] The obtained microspheres were freeze-dried for 48 hours to obtain PLGA-encapsulated citric acid sustained-release microspheres (B1) in the form of white powder, which were sealed and stored in a desiccator for future use.

[0069] Preparation Example 5:

[0070] This preparation example provides a method for preparing PLGA-encapsulated citric acid sustained-release microspheres (B2), the steps of which are as follows:

[0071] Preparation of inner water phase (W1): 2.0 g of anhydrous citric acid was weighed and dissolved in 10 mL of deionized water to obtain a citric acid solution with a mass concentration of 20% (w / v).

[0072] Preparation of oil phase (O): 15.0 g of PLGA (lactic acid / ethanol acid = 75:25) was weighed and dissolved in 100 mL of dichloromethane to obtain a PLGA solution with a mass concentration of 15% (w / v). The preparation of the external water phase (W2) was the same as in Preparation Example 4.

[0073] The subsequent emulsification, solvent evaporation, collection, washing, and drying steps were all exactly the same as in Preparation Example 4. Finally, PLGA-encapsulated citric acid sustained-release microspheres (B2) in the form of white powder were obtained, which were sealed and stored in a desiccator for future use.

[0074] Preparation Example 6:

[0075] This preparation example provides a method for preparing PLGA-encapsulated citric acid sustained-release microspheres (B3), the steps of which are as follows:

[0076] Preparation of inner water phase (W1): 3.0 g of anhydrous citric acid was weighed and dissolved in 10 mL of deionized water to obtain a citric acid solution with a mass concentration of 30% (w / v).

[0077] Preparation of oil phase (O): 10.0 g of PLGA (lactic acid / ethanol acid = 75:25) was weighed and dissolved in 100 mL of dichloromethane to obtain a PLGA solution with a mass concentration of 10% (w / v). The preparation of outer water phase (W2) was the same as that in Preparation Example 4.

[0078] The subsequent emulsification, solvent evaporation, collection, washing, and drying steps were all exactly the same as in Preparation Example 4. Finally, PLGA-encapsulated citric acid sustained-release microspheres (B3) in the form of white powder were obtained and stored in a desiccator for future use.

[0079] Examples 1-6:

[0080] Example 1: This example provides a preparation process for a degradable bandage based on a poly-lactic acid blending formula, which uses the functional components prepared in Preparation Example 3 and Preparation Example 6. The specific steps are as follows:

[0081] 1. Preparation of spinning precursor suspension:

[0082] 13.0 g of poly-lactic acid (PLA) and 3.0 g of polyethylene glycol (PEG) were weighed and added to a mixed solvent composed of 106.7 mL of dichloromethane (DCM) and 26.7 mL of N,N-dimethylformamide (DMF), and magnetically stirred until completely dissolved to obtain a polymer base solution. Subsequently, 2.3 g of sodium bicarbonate-urea inclusion compound (A3) obtained in Preparation Example 3 and 1.7 g of PLGA-encapsulated citric acid sustained-release microspheres (B3) obtained in Preparation Example 6 were added to the base solution. At this time, the mass percentage of each component in the composition was PLA 65%, PEG 15%, and functional components 20%. After the mixture was continuously magnetically stirred for 1 hour, intermittent ultrasonic treatment was performed under an ice water bath for 20 minutes to obtain a uniform suspension with a total solid concentration of 15% (w / v).

[0083] 2. Electrospinning forming:

[0084] The suspension prepared in step 1 was loaded into a syringe and installed on a microsyringe pump. The process parameters were set as follows: applied voltage 20 kV, solution advancing speed 1.0 mL / h, and distance between needle tip and drum receiver 20 cm. The device was started for continuous spinning, and a fiber membrane was collected on the drum receiver.

[0085] 3. Post-treatment and packaging:

[0086] The collected fiber membrane was vacuum dried at 45°C for 24 hours. After drying, the fiber membrane was cut into a predetermined size and vacuum sealed packaging under nitrogen protection, and finally sterilized by cobalt-60 irradiation to obtain the final product.

[0087] Example 2: This example provides a preparation process of a degradable bandage, the steps are as follows:

[0088] 1. Preparation of spinning precursor suspension:

[0089] 12.0 g of PLA and 3.0 g of PEG were weighed and added to the same volume of mixed solvent as in Example 1. Then, 2.9 g of A3 obtained in Preparation Example 3 and 2.1 g of B3 obtained in Preparation Example 6 were added. At this time, the mass percentage of each component in the composition was PLA 60%, PEG 15%, and functional component 25%. The subsequent dissolution and dispersion operations were exactly the same as in Example 1, and a uniform suspension with a total solid concentration of 15% (w / v) was obtained.

[0090] 2. Electrospinning forming and post-treatment:

[0091] The steps and parameters used in electrospinning forming, post-treatment and packaging were exactly the same as in Example 1.

[0092] Example 3: This example provides a preparation process of a degradable bandage, the steps are as follows:

[0093] 1. Preparation of spinning precursor suspension:

[0094] 15.0 g of PLA and 3.0 g of PEG were weighed and added to the same volume of mixed solvent as in Example 1. Then, 1.2 g of A3 obtained in Preparation Example 3 and 0.8 g of B3 obtained in Preparation Example 6 were added. At this time, the mass percentage of each component in the composition was PLA 75%, PEG 15%, and functional component 10%. The subsequent dissolution and dispersion operations were exactly the same as in Example 1, and a uniform suspension with a total solid concentration of 15% (w / v) was obtained.

[0095] 2. Electrospinning forming and post-treatment:

[0096] The steps and parameters used in electrospinning forming, post-treatment and packaging were exactly the same as in Example 1.

[0097] Example 4: This example provides a preparation process of a degradable bandage, the steps are as follows:

[0098] 1. Preparation of spinning precursor suspension:

[0099] The polymer base solution was prepared as in Example 1. To the base solution, 2.3 g of A1 obtained in Preparation Example 1 and 1.7 g of B1 obtained in Preparation Example 4 were added. The composition ratio was the same as in Example 1. The subsequent dissolving and dispersing operations were the same as in Example 1, and a uniform suspension having a total solid concentration of 15% (w / v) was obtained.

[0100] 2. Electrospinning and post-treatment:

[0101] The steps of electrospinning, post-treatment and packaging and the parameters used were the same as in Example 1.

[0102] Example 5: This example provides a preparation process of a degradable bandage, the steps are as follows:

[0103] 1. Preparation of the spinning precursor suspension:

[0104] The preparation of the suspension, including the raw materials used and the composition ratio of each component, were the same as in Example 1.

[0105] 2. Electrospinning:

[0106] The suspension prepared in step 1 was electrospun. The process parameters were set as follows: applied voltage was 15 kV, solution propelling speed was 0.5 mL / h, and the distance between the needle tip and the drum receiver was 15 cm.

[0107] 3. Post-treatment and packaging:

[0108] The steps of post-treatment and packaging were the same as in Example 1.

[0109] Example 6: This example provides a preparation process of a degradable bandage, the steps are as follows:

[0110] 1. Preparation of the spinning precursor suspension:

[0111] The preparation of the suspension, including the raw materials used and the composition ratio of each component, were the same as in Example 1.

[0112] 2. Electrospinning:

[0113] The suspension prepared in step 1 was electrospun. The process parameters were set as follows: applied voltage was 25 kV, solution propelling speed was 1.5 mL / h, and the distance between the needle tip and the drum receiver was 25 cm.

[0114] 3. Post-treatment and packaging:

[0115] The steps of post-treatment and packaging were the same as in Example 1.

[0116] Comparative Examples 1-6:

[0117] Comparative Example 1:

[0118] Compared with Example 1, the difference is that no functional component is contained in its composition, i.e. no sodium bicarbonate-urea inclusion compound (A3) and PLGA-encapsulated citric acid sustained-release microspheres (B3) are added. Its spinning solution is only composed of polylactic acid and polyethylene glycol, and the mass ratio of the two is the same as that of the polymer matrix in Example 1, and the rest of the preparation conditions are the same.

[0119] Comparative Example 2:

[0120] Compared with Example 1, the difference is that only the alkali source functional component is contained in its composition, i.e. only sodium bicarbonate-urea inclusion compound (A3) is added, and no PLGA-encapsulated citric acid sustained-release microspheres (B3) are added. The addition amount of component A3 is the same as that of Example 1, and the rest of the preparation conditions are the same.

[0121] Comparative Example 3:

[0122] Compared with Example 1, the difference is that only the acid source functional component is contained in its composition, i.e. only PLGA-encapsulated citric acid sustained-release microspheres (B3) are added, and no sodium bicarbonate-urea inclusion compound (A3) is added. The addition amount of component B3 is the same as that of Example 1, and the rest of the preparation conditions are the same.

[0123] Comparative Example 4:

[0124] Compared with Example 1, the difference is the form of addition of the functional component. This comparative example does not use pre-treated inclusion compound (A3) and sustained-release microspheres (B3), but directly adds the physical mixture of sodium bicarbonate powder and anhydrous citric acid powder of the same mass as Example 1 without any treatment into the polymer base solution, and the rest of the preparation conditions are the same.

[0125] Comparative Example 5:

[0126] Compared with Example 1, this comparative example uses PLGA-encapsulated tartaric acid sustained-release microspheres instead of PLGA-encapsulated citric acid sustained-release microspheres (B3). The preparation method of the tartaric acid microspheres is exactly the same as that of Preparation Example 6, except that the citric acid is replaced by an equal molar amount of tartaric acid. The addition amount is the same as the mass of B3 in Example 1, and all other components and preparation conditions are the same.

[0127] Comparative Example 6:

[0128] The difference compared with Example 1 is the selection of the base source, aiming to verify the necessity of gas generation. This comparative example uses a non-gas-producing base, sodium phosphate tribasic (Na3P04), to replace sodium bicarbonate, and uses a similar method to Preparation Example 6 to prepare PLGA-coated sodium phosphate tribasic microspheres, which replace sodium bicarbonate-urea clathrate (A3). The molar ratio of added sodium phosphate tribasic microspheres to citric acid microspheres (B3) is 1:1 to ensure that the acid is neutralized. All other components and preparation conditions are the same.

[0129] Test Examples 1-5:

[0130] Test Example 1:

[0131] This test example aims to verify the chemical stability of functional component A prepared by the clathration method in a humid environment and the responsive release characteristics in an aqueous medium.

[0132] 1. Stability evaluation

[0133] Weigh 1 g of sodium bicarbonate-urea clathrate obtained in Preparation Examples 1, 2 and 3 (samples A1, A2, A3, respectively), and 1 g of untreated analytical pure sodium bicarbonate powder as a control group. Place the four samples in open weighing bottles and place them in a constant temperature and humidity chamber with a temperature setting of 25°C and a relative humidity setting of 75%, and expose them for 24 hours.

[0134] 2. Effective component retention rate determination

[0135] After 24 hours, the four samples are removed from the constant temperature and humidity chamber. Transfer the entire amount of each sample to a 250 mL conical flask, and use a pipette to add 100.00 mL of 0.1000 mol / L hydrochloric acid standard solution, and magnetically stir until the sample is completely dissolved. Add 2 drops of phenolphthalein indicator to the solution, and then use 0.1000 mol / L sodium hydroxide standard solution to back-titrate the excess hydrochloric acid in the conical flask until the solution changes from colorless to pink and does not fade within 30 seconds. Record the volume of sodium hydroxide standard solution consumed. Calculate the mass of the remaining effective component (sodium bicarbonate) in each sample based on the titration results, and calculate the effective component retention rate.

[0136] 3. Responsive release evaluation

[0137] Weigh 1.000 g of the clathrate obtained in Preparation Example 3 (A3) and quickly add it to a beaker containing 100.00 mL of deionized water at a temperature of 37°C, while starting the magnetic stirrer and timer. Immerse a pre-calibrated conductivity probe into the solution, and record the conductivity value of the solution every 30 seconds from the time the sample is added, for a continuous monitoring period of 10 minutes.

[0138] The test results are shown in Table 1 and Table 2:

[0139] Table 1. Stability test results of various samples in a humid environment

[0140]

[0141] Table 2. Conductivity data of responsive release test of functional component A3

[0142]

[0143] The test results of Table 1 show that the samples obtained from Preparation Examples 1 to 3 all maintain a retention rate of active ingredient above 99% after 24 hours of exposure to a humid environment. In contrast, the retention rate of active ingredient of the untreated sodium bicarbonate control group is significantly reduced to 8.2%. This data confirms that by containing sodium bicarbonate molecules in the crystal lattice structure of urea to form a clathrate, direct contact between external water molecules and internal sodium bicarbonate molecules can be effectively blocked. This steric hindrance effect inhibits the decomposition reaction of sodium bicarbonate due to moisture absorption under storage conditions, thereby ensuring its chemical stability and functional integrity before application.

[0144] The data of Table 2 show that when functional component A3 is in contact with an aqueous medium, the conductivity of the solution rapidly increases from the initial value to 2550.1 μS / cm within the first 120 seconds, and reaches a relatively stable plateau region (about 3710 μS / cm) after about 360 seconds, with little subsequent change. This rapid change in conductivity curve characterizes the rapid disintegration process of the clathrate: the urea host lattice dissolves under the action of water molecules, causing the clathrated sodium bicarbonate guest molecules to be rapidly released and dissociated into high-mobility Na + and HCO3 - ions, resulting in a significant increase in solution conductivity. This data confirms the instant responsiveness of the clathrate structure in an aqueous environment.

[0145] In summary, the bicarbonate-urea clathrate used in the present technical solution achieves a dual technical effect in structure. Firstly, in anhydrous or low-humidity environments, by physically encapsulating guest molecules with host molecules, it provides effective isolation and chemical passivation of the active alkali source component, solving the technical problem of its easy degradation during storage. Secondly, when in contact with an aqueous trigger medium, it can achieve instant and complete release of the internal active component through rapid disintegration of the host structure.

[0146] Test Example 2:

[0147] This test example aims to quantitatively characterize the in vitro cumulative release characteristics of functional component B prepared by the multiple emulsion solvent evaporation method in a simulated body fluid environment.

[0148] 1. Sample Preparation

[0149] PLGA-encapsulated citric acid sustained-release microspheres (Sample B1, B2, B3) obtained in Preparation Example 4, Preparation Example 5 and Preparation Example 6 were weighed 100.0 mg each. Each sample was placed in a 50 mL centrifuge tube, and 40.0 mL of phosphate buffer solution (PBS, pH = 7.4) was added.

[0150] 2. Release Experiment

[0151] After all the centrifuge tubes were sealed, they were fixed horizontally in a constant temperature water bath shaker. The shaker temperature was set to 37°C, the shaking rate was 100 rpm, and the timer was started.

[0152] 3. Sample Collection and Treatment

[0153] At the set time points, 2, 4, 8, 12, 24, 48, and 72 hours, the centrifuge tubes were taken out of the shaker. The centrifuge tubes were centrifuged at 4000 rpm for 5 minutes to allow the microspheres to settle. A 2.0 mL liquid was aspirated from the supernatant using a pipette as the sample to be tested, and immediately 2.0 mL of fresh PBS buffer solution preheated to 37°C was added to the centrifuge tube, and then the centrifuge tube was returned to the shaker for continued testing. This operation aimed to maintain the total volume of the release medium essentially constant.

[0154] 4. Concentration Determination and Data Calculation

[0155] The mass concentration of citric acid in the collected 2.0 mL sample was determined using a high-performance liquid chromatography (HPLC) system. According to the measured concentration and the known volume of the release medium, the mass of citric acid released at each time point was calculated. By cumulative calculation, the cumulative release mass at each time point was obtained, and divided by the total mass of citric acid theoretically loaded in the initial microspheres, and finally the cumulative release percentage was calculated.

[0156] The test results are shown in Table 3:

[0157] Table 3: In vitro cumulative release percentage (%) of functional component B:

[0158]

[0159] The experimental data in Table 3 objectively show that the three kinds of microsphere samples all exhibit sustained release behavior in the simulated body fluid environment. During the entire 72-hour test period, the cumulative release percentage of sample B1 (PLGA concentration 5%) is the highest, reaching 84.9%; the cumulative release percentage of sample B2 (PLGA concentration 15%) is the lowest, being 60.7%; and the cumulative release percentage of sample B3 (PLGA concentration 10%) is between the two. The data also show that the release rate of all samples is relatively fast in the initial stage (0-12 hours), and then gradually slows down, which constitutes a typical sustained-release characteristic curve.

[0160] The differences in these release curves are directly related to the physical structure of the microspheres. The release process of citric acid is mainly controlled by two physical and chemical processes: one is the penetration of water molecules into the PLGA matrix and the dissolution of internal citric acid, and then the diffusion of the dissolved citric acid molecules to the outside through the pore network of the matrix; the other is the hydrolytic cleavage of the PLGA polymer chain itself (i.e., bulk degradation), which causes the gradual erosion and disintegration of the matrix structure, thereby releasing the encapsulated citric acid. Sample B2 has the highest PLGA concentration, forming a more dense and more hydrophobic polymer matrix, which increases the resistance to water molecule penetration and citric acid molecule diffusion, and its degradation rate is also relatively slow, so its overall release rate is the slowest. Conversely, the low PLGA concentration of sample B1 forms a relatively loose and porous structure, resulting in the fastest release rate.

[0161] Therefore, by adjusting the PLGA concentration of the oil phase during the preparation process, the release rate of the acid source component can be precisely controlled. This technical means solves the technical problem of instantaneous reaction and difficult control caused by directly introducing a highly water-soluble small molecule acid into the system. By encapsulating citric acid in degradable PLGA microspheres, the acid is supplied continuously and predictably.

[0162] Test Example 3:

[0163] This test example aims to quantitatively compare the in vitro gas generation capacity and generation rate of the bandage samples prepared in Examples 1-6 and Comparative Examples 1-6 in a simulated body fluid environment.

[0164] 1. Sample preparation

[0165] 100.0 mg of dry bandage samples prepared in Examples 1-6 and Comparative Examples 1-6 were weighed using an electronic balance. To ensure the uniformity of the reaction contact area, each sample was cut into small pieces of about 2 mm x 2 mm using surgical scissors.

[0166] 2. Experimental device setup and reaction initiation

[0167] A prepared sample piece was placed in a 100 mL conical flask fitted with a stopper. The side neck of the conical flask was connected to a horizontal placed, 20 mL range precision gas syringe via a gas tight tubing. The whole setup (conical flask and syringe) was fixed in a 37 °C constant temperature water bath and allowed to equilibrate for 10 minutes. 50.0 mL of pre-heated (to 37 °C) simulated body fluid (SBF, pH 7.4) was quickly injected into the conical flask through its top stopper using the syringe and the timer was immediately started.

[0168] 3. Data collection

[0169] At pre-set time points, 0.5, 1, 2, 4, 8, 12, 24 hours, the cumulative gas volume indicated by the piston movement of the gas syringe was visually read and recorded. Each sample was run in triplicate and the final result was taken as the arithmetic mean.

[0170] The test results are shown in Table 4:

[0171] Table 4: Cumulative gas generation volume (mL) of each sample in vitro:

[0172]

[0173] The experimental data in Table 4 shows that all the samples of Examples 1 to 6 exhibit a sustained gas generation behavior after being immersed in the simulated body fluid, with a steady growth trend in the cumulative gas volume within 24 hours. Among them, Example 2 (functional component content 25%) has the highest total gas production, reaching 17.9 mL; Example 3 (functional component content 10%) has the lowest total gas production, which is 8.9 mL, showing a clear correspondence between the total gas production and the content of functional components. In contrast, the samples of Comparative Examples 1, 2, 3 and 6 do not show obvious gas generation during the entire test period, and their readings are close to the experimental background value. Comparative Example 4 (direct physical mixing) shows a completely different behavior, with gas generation reaching 11.2 mL within the first 0.5 hours, and approaching its maximum value of 12.9 mL after 1 hour, after which the volume no longer increases. Comparative Example 5 (using tartaric acid) also shows sustained gas generation, with a total amount similar to that of Example 1.

[0174] The differences in behavior and rate of the above gas generation are rooted in the accessibility of the reactant species and the control of the reaction rate. The sustained gas generation of the sample of Example is due to the time sequence control of the internal acid-base reaction by the structure. When the sample is wetted, the PLGA microspheres (functional component B) slowly release citric acid through bulk degradation and molecular diffusion at a controlled rate. The released protons (H + ) immediately react with the bicarbonate ions (HCO3 -) a neutralization reaction occurs, generating carbon dioxide gas. The rate-limiting step of the entire process is the slow release of acid from the PLGA matrix, which ensures that gas can be continuously generated at a steady rate for hours or even longer.

[0175] The results of the comparative examples inversely verify the above mechanism. Comparative Examples 1, 2, 3 cannot generate gas due to the absence of reactants (acid and base, acid, base, respectively). Comparative Example 6 contains both acid and base, but the neutralization reaction of its base source (trisodium phosphate) does not generate gas, confirming the necessity of bicarbonate as a gas precursor. In Comparative Example 4, unprotected citric acid and sodium bicarbonate dissolve and react vigorously upon contact with the liquid, resulting in a burst release of gas, which directly proves that without the two-component time-controlled structure of the present application, sustained gas generation cannot be achieved.

[0176] Test Example 4:

[0177] This test example aims to quantitatively evaluate the chelation and removal ability of calcium ions in the in vitro environment of the bandage samples prepared in Examples 1-6 and Comparative Examples 1-6.

[0178] 1. Preparation of standard solution

[0179] Prepare a Tris-HCl buffer solution (50 mM, pH 7.4) and dissolve analytical pure calcium chloride (CaCl2) in it, so that the initial concentration of calcium ions in the solution is exactly 5.00 mmol / L.

[0180] 2. Sample incubation

[0181] Weigh 100.0 mg of dry bandage samples prepared in Examples 1-6 and Comparative Examples 1-6 respectively into separate 50 mL centrifuge tubes. Accurately add 20.0 mL of the above prepared calcium ion standard solution to each centrifuge tube. Seal all centrifuge tubes and fix them horizontally in a constant temperature water bath shaker at 37°C, and oscillate at a speed of 100 rpm for 24 hours of incubation.

[0182] 3. Sample processing and analysis

[0183] After 24 hours of incubation, remove all centrifuge tubes and centrifuge at a speed of 5000 rpm for 5 minutes to sediment the bandage fragments and any insoluble matter. Carefully aspirate the supernatant and dilute it with deionized water as appropriate. Use an inductively coupled plasma optical emission spectrometer (ICP-OES) to determine the concentration of remaining calcium ions in the diluted supernatant.

[0184] 4. Data calculation

[0185] The calcium ion removal rate of each sample was calculated by the following formula according to the measured residual calcium ion concentration after incubation and the known initial concentration (5.00 mmol / L):

[0186] Calcium ion removal rate (%) = [(initial concentration - residual concentration) / initial concentration] x 100%;

[0187] The test results are shown in Table 5:

[0188] Table 5: In vitro calcium ion removal rate (%) of each sample:

[0189]

[0190] The experimental data in Table 5 show that the samples of Examples 1 to 6 all exhibit significant calcium ion removal ability, with removal rates ranging from 42.1% to 88.7%. In contrast, Comparative Examples 1 and 2, which do not contain any functional components, and Comparative Example 2, which contains only an alkali source, both have calcium ion removal rates of less than 3%, which can be attributed to non-specific physical adsorption on the surface of the material. At the same time, Comparative Examples 3, 4, 5, and 6, which contain acid source components, also show varying degrees of calcium ion removal effect.

[0191] The internal mechanism of this calcium ion removal phenomenon is the chemical chelation of citrate ions. The functional component B (PLGA-encapsulated citric acid sustained-release microspheres) in the examples of the present application continuously releases citric acid in an aqueous environment. Citric acid is a polycarboxylic acid that deprotonates to form citrate ions in a pH 7.4 environment. The multiple carboxyl oxygen atoms in this anion structure can act as electron pair donors, forming multiple coordination bonds with a divalent calcium ion (Ca 2+ ), forming a stable, water-soluble ring structure, i.e., a chelate. This chemical process converts free calcium ions in solution into chelated calcium ions, resulting in a significant decrease in the concentration of free calcium ions that can be detected by ICP-OES. The trend of the data also indicates that the size of the calcium ion removal rate is directly related to the initial content of the acid source functional component in the sample, as the removal rate of Example 2 is significantly higher than that of Examples 1 and 3.

[0192] The present technical solution achieves controlled release of the chelating agent in the time dimension by encapsulating citric acid in PLGA sustained-release microspheres, as demonstrated by the data of Test Example 2. This structural design ensures that the chelation is not a one-time instantaneous process, but a continuous process throughout the functional period of the material. The results of Comparative Example 5 (using tartaric acid) show that other polycarboxylic acids also have chelation ability, but there are differences in efficiency compared to citric acid, confirming that the selection of a specific chelating agent molecule will affect the final technical effect.

[0193] Test Example 5:

[0194] This test example aims to quantitatively evaluate the ability of the bandage samples prepared in Examples 1-6 and Comparative Examples 1-6 to promote oxygen release from oxyhemoglobin by affecting the local microenvironment pH and CO2 concentration in situ through chemical reactions in an in vitro hemoglobin model.

[0195] 1. Preparation of Oxyhemoglobin Solution

[0196] Weigh the bovine hemoglobin and prepare a solution with a concentration of 5.0 g / L in physiological saline (0.9% NaCl) at 4°C. Place the solution in a sealed container with a gas inlet tube, slowly introduce a standard gas containing 5% O2, 5% CO2 and 90% N2, and continue for 30 minutes to allow the hemoglobin to reach a stable oxygen saturation. This is the oxyhemoglobin solution to be tested.

[0197] 2. Test system setup and baseline stabilization

[0198] Transfer 100.0 mL of the prepared oxyhemoglobin solution to a sealed, double-jacketed reaction cell with a high-precision dissolved oxygen (DO) electrode and a magnetic stirrer. Maintain the reaction cell temperature at 37°C by external circulation water bath. Start the magnetic stirring, and when the dissolved oxygen electrode reading fluctuates by no more than 0.02 mg / L within 5 minutes, record the dissolved oxygen concentration at this moment as the initial baseline value.

[0199] 3. Sample addition and data monitoring

[0200] Weigh 50.0 mg of dry bandage samples prepared in Examples 1-6 and Comparative Examples 1-6, respectively. Through the sealed sample inlet at the top of the reaction cell, quickly add one sample to the stirring oxyhemoglobin solution and immediately start timing. Continuously monitor and record the change in dissolved oxygen concentration for 30 minutes.

[0201] 4. Data processing

[0202] From the monitoring data of each sample, find the highest dissolved oxygen concentration reading within 30 minutes. Subtract the initial baseline value from this highest reading to obtain the maximum dissolved oxygen increment (ΔDO) caused by the sample, which is used as an indicator to evaluate its ability to promote oxygen release.

[0203] The test results are shown in Table 6:

[0204] Table 6 Effect of each sample on the dissolved oxygen concentration of the oxyhemoglobin solution:

[0205]

[0206] The experimental data in Table 6 show that the samples of Examples 1 to 6 all result in a significant increase in the dissolved oxygen concentration in the oxygenated hemoglobin solution, with the maximum increase ranging from 1.29 mg / L to 2.85 mg / L. In contrast, the samples of Comparative Examples 1, 2, 6 (which lack the acid-base component, the acid source, and the gas-producing base source, respectively) result in negligible changes in the dissolved oxygen concentration, which are within the experimental error. Comparative Examples 3, 4, 5, which contain the acid source, also result in an increase in the dissolved oxygen concentration.

[0207] The increase in the dissolved oxygen concentration is based on the Bohr effect of hemoglobin. After the sample of the example is infiltrated, the in-situ acid-base reaction (as confirmed in Test Example 3) generates carbon dioxide and releases protons (H + ). The increase in the CO2 partial pressure and the decrease in the pH value in the local microenvironment act together as allosteric effectors on the oxygenated hemoglobin molecules. These effectors promote a conformational change in the hemoglobin, which reduces its binding affinity for oxygen molecules, resulting in the dissociation and release of the bound oxygen into the solution, which is macroscopically manifested as an increase in the dissolved oxygen concentration that can be detected by the electrode.

[0208] The technical effects achieved by the technical solution are not only to induce the Bohr effect, but also to time-regulate the induction process. Although Comparative Example 4 (direct physical mixing) also produces a high increase in the dissolved oxygen, the reaction process is instantaneous and explosive. By encapsulating the acid source in a slow-release carrier, the sample of the example achieves the continuous and stable generation of protons and carbon dioxide. This controlled reaction process allows the allosteric regulation of hemoglobin to be maintained for a longer period of time, avoiding negative effects such as protein denaturation that can be caused by a transiently low local pH.

[0209] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A process for the preparation of a degradable bandage based on a poly lactic acid blend formulation, characterized by, The preparation method comprises the following steps: a) dissolving polylactic acid and polyethylene glycol in an organic solvent to obtain a polymer base solution; b) dispersing pre-prepared sodium bicarbonate-urea inclusion complex and pre-prepared PLGA-coated citric acid sustained-release microspheres in the polymer base solution of step a) to obtain a spinning precursor suspension; c) electrospinning the spinning precursor suspension of step b) to collect a fiber membrane to obtain the degradable bandage. The sodium bicarbonate-urea inclusion complex is prepared by the following steps: dissolving urea and sodium bicarbonate in a hot methanol solution, and then crystallizing the inclusion complex by programmed cooling.

2. The process for preparing a degradable bandage based on polylactic acid blend formulation as claimed in claim 1 wherein, In step b), the mass percentage of each component in the total mass of polylactic acid, polyethylene glycol, sodium bicarbonate-urea inclusion complex and PLGA-coated citric acid sustained-release microspheres is as follows: Polylactic acid: 60% to 75%; Polyethylene glycol: 15%; The sum of sodium bicarbonate-urea inclusion complex and PLGA-coated citric acid sustained-release microspheres: 10% to 25%.

3. The process for preparing a degradable bandage based on polylactic acid blend formulation as claimed in claim 1 wherein, When preparing the sodium bicarbonate-urea inclusion complex, the molar ratio of urea to sodium bicarbonate is 5:1 to 10:

1.

4. The process for preparing a degradable bandage based on polylactic acid blend formulation as claimed in claim 1 wherein, The PLGA-coated citric acid sustained-release microspheres are prepared by a double emulsion solvent evaporation method, which comprises: forming an inner aqueous phase containing an aqueous citric acid solution, an oil phase containing PLGA, and an outer aqueous phase containing polyvinyl alcohol to prepare a W1 / O / W2 double emulsion, and then evaporating the solvent in the oil phase to solidify and form microspheres.

5. The process for preparing a degradable bandage based on polylactic acid blend formulation as claimed in claim 4 wherein, When preparing the PLGA-coated citric acid sustained-release microspheres, the mass concentration of PLGA in the oil phase is 5% w / v to 15% w / v.

6. The process for preparing a degradable bandage based on polylactic acid blend formulation as claimed in claim 1, wherein, In step b), to uniformly disperse the inclusion complex and the microspheres, the step of magnetically stirring and ultrasonic treating the mixture is further included.

7. The process for preparing a biodegradable bandage based on polylactic acid blend formulation as claimed in claim 1, wherein, In step c), the process parameters of electrospinning are as follows: applied voltage 15 kV to 25 kV, solution propelling speed 0.5 mL / h to 1.5 mL / h, and distance between needle tip and receiver 15 cm to 25 cm.

8. A degradable bandage prepared by the preparation process of any one of claims 1-7.

9. The degradable bandage of claim 8, wherein, The structure is a fiber membrane matrix composed of polylactic acid and polyethylene glycol blended fibers, and sodium bicarbonate-urea inclusion complex particles and PLGA-coated citric acid sustained-release microsphere particles dispersed in the fiber membrane matrix.

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