A protein composite fiber film material having wet adhesion and delivering a drug and a method for preparing the same

Abstract

This invention discloses a protein composite fiber membrane material with wet adhesion and drug delivery capabilities, and its preparation method. Protein, functional active ingredients, and a volatile conformational stabilizer are mixed and treated with a disulfide reducing agent to obtain a spinning solution. Electrospinning causes the conformational stabilizer to volatilize, while the protein maintains its untransformed conformation. Subsequently, a bioadhesive polymer interlayer is applied to the fiber membrane surface, followed by secondary spinning to form a sandwich-structured composite fiber membrane material. The material obtained by this invention undergoes a controllable conformational transition in a wet environment. The β-sheets and hydrophobic residues at the interface, in conjunction with the interlayer, disrupt the hydration barrier at the interface, achieving strong adhesion through multiple mechanisms and effectively resisting interference from dynamic physiological environments. Simultaneously, the co-spinning technology enables flexible loading and controlled release of functional molecules, providing a scalable technical platform for constructing composite patches with both physical isolation and active therapeutic functions, showing promising applications in tissue repair and drug delivery.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a protein composite fiber membrane material with wet adhesion and drug delivery capability, and its preparation method. Background Technology

[0002] In the biomedical field, achieving stable adhesion at moist biological interfaces such as the oral cavity, gastrointestinal tract, nasal cavity, and skin wounds is a key challenge driving the development of precision medicine and emerging medical technologies. Reliable wet adhesion not only provides a foundation for physical isolation but is also an important prerequisite for achieving local drug delivery, real-time physiological monitoring, and active promotion of tissue repair. It has significant scientific value and application prospects for developing next-generation high-end dressings, mucosal drug delivery systems, and wearable bioelectronic devices.

[0003] However, due to the persistent hydration layer at the biological interface and the influence of physiological activities such as chewing, peristalsis, respiration, and movement, traditional adhesive materials are prone to overhydration, mechanical property degradation, and interfacial instability, leading to adhesion failure and premature detachment, making it difficult to meet the long-term use requirements in humid dynamic environments. Currently, adhesive materials applied to humid biological interfaces still have significant limitations. For example, patches based on hydrogels or biopolymers such as chitosan and hyaluronic acid often fail due to excessive water absorption, insufficient mechanical strength, or a rapid decline in adhesion in humid environments. Although improvement strategies such as dopamine biomimetic modification and photocrosslinking enhancement have emerged, these methods still generally face the following problems: adhesion stability is significantly affected by environmental factors (such as pH, ionic strength, and redox state); it is difficult to achieve high-strength adhesion while maintaining good biocompatibility; the materials are often non-degradable or have drawbacks such as complex preparation processes and high costs. In addition, most existing materials have single functions, making it difficult to integrate multiple biological functions such as antibacterial and anti-inflammatory effects while achieving long-lasting interfacial adhesion.

[0004] Inspired by the mechanism by which barnacles achieve strong underwater adhesion by secreting proteins to form amyloid fibers, protein-based biomimetic wet adhesion strategies have shown significant potential. However, translating this biomimetic principle into macroscopic functional materials suitable for biomedical applications still faces significant challenges, especially in processing proteins with amyloid structures into practical materials. Electrospinning, as a method for scalable preparation of nanofiber materials, can precisely control fiber morphology and size, and achieves efficient loading of active ingredients due to its ultra-high specific surface area, showing promising application prospects. However, the poor solubility of amyloid proteins and their tendency to undergo uncontrolled aggregation and misfolding during processing make it difficult to directly electrospin them without the assistance of carrier polymers. This contradiction severely restricts the development of pure protein-based electrospun nanofiber materials that combine biomimetic wet adhesion properties with drug loading capabilities. Summary of the Invention

[0005] This invention addresses the shortcomings of existing bioadhesive materials, such as insufficient adhesion, limited protective effect, and single function in humid dynamic environments, as well as the difficulty in large-scale processing of protein-based biomimetic adhesive materials. It provides a protein composite fiber membrane material with wet adhesion and capable of drug delivery, along with its preparation method. This method is simple to operate, highly controllable, and has broad applicability.

[0006] The protein composite fiber membrane material with wet adhesion and drug loading and delivery provided by the present invention is prepared by the following steps:

[0007] Step 1: Mix the protein, functional active ingredient and conformation stabilizer to obtain a mixed solution; adjust the pH of the disulfide bond reducing agent aqueous solution to 5.0-9.0 with a pH adjuster, mix it evenly with the mixed solution, and let it stand at room temperature to obtain a spinning solution; wherein, the conformation stabilizer is selected from any one or more of trifluoroethanol, difluoroethanol, trifluoroacetic acid and hexafluoroisopropanol.

[0008] Step 2: The spinning solution is processed into a protein nanofiber membrane using an electrospinning process. During the spinning process, the conformation stabilizer evaporates, allowing the protein to maintain its untransformed conformation in the fiber membrane.

[0009] Step 3: Apply the bioadhesion polymer uniformly to the surface of the protein nanofiber membrane by coating or spraying to form an intermediate layer; wherein the bioadhesion polymer is a polymeric material that can produce adhesion in a humid environment.

[0010] Step 4: Using the electrospinning process described in Step 2, the spinning solution is processed into a protein nanofiber membrane on the intermediate layer to obtain a protein composite fiber membrane material with a sandwich structure; wherein, when the protein composite fiber membrane material is exposed to water or a wet environment, the protein conformation changes and wet adhesion is generated.

[0011] Furthermore, in step 1 above, the protein is selected from any one or more of the following: lysozyme, bovine serum albumin, lactoferrin, pepsin, trypsin, catalase, hemoglobin, human serum albumin, whey albumin, insulin, α-lactalbumin, fibrinogen, ribonuclease A, cytochrome c, α-amylase, horseradish peroxidase, myoglobin, actin, silk fibroin, myosin, collagen, keratin, ferritin, and casein.

[0012] Furthermore, in step 1 above, the functional active ingredient is selected from any one or more of antibacterial active ingredients, anti-inflammatory active ingredients, soluble inorganic salts, traditional Chinese medicine extracts, polypeptide active ingredients, protein active ingredients, growth factors, cytokines, and nanoparticles.

[0013] Furthermore, the aforementioned antibacterial active ingredient is selected from at least one of antibiotics, antimicrobial peptides, quaternary ammonium salt antimicrobial agents, and biguanide antimicrobial agents. The antibiotic is selected from at least one of β-lactam antibiotics, macrolide antibiotics, tetracycline antibiotics, aminoglycoside antibiotics, glycopeptide antibiotics, quinolone antibiotics, lincosamide antibiotics, polypeptide antibiotics, oxazolidinone antibiotics, lipopeptide antibiotics, glycylcycline antibiotics, and antifungal drugs; the antimicrobial peptide is a cationic antimicrobial peptide containing 10 to 100 amino acid residues and having a net positive charge; the quaternary ammonium salt antimicrobial agent is selected from at least one of benzalkonium chloride, hexadecylpyridinium halide, alkyltrimethylammonium bromide, benzyltriethylammonium chloride, dialcyldimethylammonium chloride, dialcyldimethylammonium bromide, tetradecyl-2-methylpyridinium bromide, dialcylmethylhydroxyethylammonium chloride, and dialcylmethylhydroxypropylammonium chloride; the biguanide antimicrobial agent is selected from at least one of guanidine dodecyl acetate, alixidine, chlorhexidine acetate, chlorhexidine gluconate, polyhexylene biguanide, polyaminopropyl biguanide, and polyhexamethylene biguanide.

[0014] Furthermore, the aforementioned anti-inflammatory active ingredients are selected from at least one of ibuprofen, diclofenac sodium, piroxicam, meloxicam, ketorolac, aminopyrine, parecoxib, celecoxib, nimesulide, flurbiprofen ester, dexamethasone, resveratrol, and epigallocatechin gallate.

[0015] Furthermore, the aforementioned soluble inorganic salt is selected from at least one of silver nitrate, silver acetate, copper sulfate, sodium iodide, calcium chloride, calcium lactate, calcium gluconate, ferric chloride, and magnesium chloride.

[0016] Furthermore, the above-mentioned Chinese herbal extracts are selected from at least one of glycyrrhizic acid, andrographolide, tripterygium wilfordii, berberine, matrine, tetrandrine, quercetin, baicalin, puerarin, ginsenosides, notoginsenosides, astragaloside A, astragalus polysaccharide, lentinan, rhein, tanshinone, aescinol, schisandrin A, menthol, eugenol, camphor, chlorogenic acid, gallic acid, and tannic acid.

[0017] Furthermore, the aforementioned polypeptide active ingredients are compounds composed of 2 to 100 amino acids linked by peptide bonds, and possess biological activity, selected from one or more of the following functional categories: (a) tissue regeneration promoters: epidermal growth factor mimic peptides, fibroblast growth factor mimic peptides, collagen synthesis promoting peptides; (b) angiogenesis promoters: vascular endothelial growth factor mimic peptides, RGD sequence peptides, laminin-derived peptides; (c) cell migration and adhesion regulation promoters: cell membrane penetration peptides, targeting integrin peptides, fibronectin-derived peptides; (d) matrix metalloproteinase regulation promoters: matrix metalloproteinase inhibitory peptides, procollagen C-terminal protease inhibitory peptides; (e) neuromodulation and analgesia promoters: nerve growth factor mimic peptides, opioid analgesic peptides;

[0018] Furthermore, the aforementioned protein-based active ingredients are selected from one or more of the following functional categories: (a) structural proteins: collagen, silk fibroin, elastin, gelatin, keratin; (b) globular functional proteins: insulin, enzymes, plant-derived functional proteins; (c) antibodies: antibodies or their antigen-binding fragments.

[0019] Furthermore, the aforementioned growth factors are selected from at least one of epidermal growth factor, fibroblast growth factor, vascular endothelial growth factor, platelet-derived growth factor, transforming growth factor-β, insulin-like growth factor, and nerve growth factor.

[0020] Furthermore, the aforementioned cytokines are selected from at least one of interleukins, interferons, tumor necrosis factors, chemokines, and colony-stimulating factors;

[0021] Furthermore, the aforementioned nanoparticles are selected from at least one of silver nanoparticles, gold nanoparticles, zinc oxide nanoparticles, silica nanoparticles, iron oxide nanoparticles, hydroxyapatite nanoparticles, and bioglass nanoparticles.

[0022] Furthermore, in step 1 above, the disulfide bond reducing agent is selected from any one or more of tris(2-carboxyethyl)phosphine hydrochloride, glutathione, cysteine, dithiothreitol, dithiothreitol isomer, β-mercaptoethanol, tris(3-hydroxypropyl)phosphine, and tributylphosphine.

[0023] Furthermore, in step 1 above, the pH adjuster is selected from any one or more of the following: tris(hydroxymethyl)aminomethane, 4-hydroxyethylpiperazine ethanesulfonic acid, N,N-dihydroxyethylglycine, piperazine-1,4-diethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid, 3-(N-morpholino)-2-hydroxypropanesulfonic acid, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, 3-(cyclohexylamine)-1-propanesulfonic acid, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium benzoate, and sodium citrate.

[0024] Furthermore, in step 1 above, the concentration of protein in the spinning solution is 10–300 mg / mL, the concentration of disulfide bond reducing agent is 10–200 mmol / L, and the volume fraction of conformation stabilizer is 50%–95%. Preferably, the concentration of protein in the spinning solution is 100–200 mg / mL, the concentration of disulfide bond reducing agent is 15–100 mmol / L, and the volume fraction of conformation stabilizer is 70%–90%.

[0025] Furthermore, in step 2 above, during electrospinning, the DC high voltage intensity between the nozzle and the roller receiver is set to 3–50 kV, and the flow rate is 0.2–5 mL / h. A planar grounded receiver or a high-speed rotating grounded receiver can be selected, and the distance between the nozzle and the receiver is 5–30 cm. Preferably, the DC high voltage intensity between the nozzle and the roller receiver is 3–25 kV, the flow rate is 0.5–2 mL / h, and the distance between the nozzle and the receiver is 5–20 cm.

[0026] Furthermore, in step 3 above, the bioadhesive polymer is selected from any one of acrylic polymers, cellulose derivatives, natural polysaccharides and their derivatives, modified starch, and other water-absorbing polymers. The acrylic polymers are selected from any one or more of carbomer (such as Carbopol 934P, 971P, 974P), polycarbophil, and acrylic / acrylate copolymers; the cellulose derivatives are selected from any one or more of sodium carboxymethyl cellulose (CMC-Na), hydroxypropyl methylcellulose (HPMC), and hydroxyethyl cellulose (HEC); the natural polysaccharides and their derivatives are selected from any one or more of chitosan, hyaluronic acid, sodium alginate, agarose, carrageenan, guar gum, xanthan gum, gellan gum, and pectin; and the other water-absorbing polymers are selected from any one or more of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and polyethylene oxide (PEO).

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. This invention proposes and verifies a "folding-stabilization" strategy, which achieves precise control and dynamic maintenance of protein molecular conformation through the synergistic effect of volatile conformation stabilizers and disulfide bond reducers. Specifically, disulfide bond reducers moderately open disulfide bonds within protein molecules, increasing chain segment flexibility and intermolecular entanglement, while conformation stabilizers stabilize the protein's native conformation through hydrogen bonding, hydrophobic interactions, and other means, preventing premature folding to form insoluble aggregates. During electrospinning, the conformation stabilizers volatilize rapidly, and the protein maintains a "folded-intermediate" conformation during the critical window of fiber formation, thus achieving good spinnability. This strategy solves the inherent problem of "unspinning" or "difficulty in continuous spinning" caused by poor solubility and spontaneous aggregation and folding of amyloid proteins (such as lysozyme, bovine serum albumin, and zein) at the molecular level, achieving for the first time pure protein electrospinning without relying on any carrier polymer. Meanwhile, by adjusting parameters such as the type and ratio of conformation stabilizers and the concentration of reducing agents, the diameter, morphology and mechanical properties of nanofibers can be precisely controlled, providing a new process route for biomimetic adhesive materials that can be mass-produced and have a designable structure, simulating natural amyloid proteins such as barnacle cement protein and spider silk protein.

[0029] 2. The protein composite fiber membrane material prepared in this invention undergoes a controllable conformational transformation when applied (in contact with moist biological tissues or mucous membranes), gradually forming an amyloid structure rich in β-sheets from the initial untransformed state, accompanied by the exposure of hydrophobic regions. This dynamic conformational evolution has the following key advantages: (1) The exposed hydrophobic regions work synergistically with the bioadhesive polymer in the intermediate layer to effectively displace and destroy the dense hydration layer covering the surface of moist biological interfaces (such as mucous membranes and damaged tissues), establishing a direct contact basis for adhesion; (2) Through the synergy of multiple intermolecular forces such as physical chain segment interpenetration, hydrogen bond network reconstruction, electrostatic attraction, and hydrophobic interaction, a strong and durable anchoring with the tissue interface is achieved; (3) The formation of the β-sheet structure gives the material a certain rigid skeleton, enabling it to resist physiological dynamic interferences such as liquid scouring, chewing peristalsis, and respiratory movements, significantly reducing the risk of interface deadhesion. Experimental data show that the wet adhesion strength of the material of this invention can reach 6 to 50 times that of commercially available medical adhesive products (such as fibrin glue and cyanoacrylates), and it can maintain stable adhesion performance under different pH, ionic strength and dynamic mechanical environments, solving the core problems of existing materials being easy to fall off and having rapid adhesion strength decay in humid and dynamic physiological environments.

[0030] 3. This invention fully utilizes the inherent high compatibility and designability of electrospinning technology, and has significant advantages in multifunctional integration: (1) Through one-step co-spinning technology, various functional active ingredients such as antibiotics, anti-inflammatory agents, growth factors, antimicrobial peptides, traditional Chinese medicine extracts, and inorganic nanoparticles can be directly and uniformly embedded into the protein nanofiber matrix, avoiding the problems of easy detachment and uncontrollable release in traditional coating methods; the degradation or conformational transformation process of the fiber matrix can drive the programmed release of functional molecules, realizing local drug delivery and long-term treatment; (2) The upper and lower protein fiber layers provide physical barrier protection (resisting external bacterial invasion). (3) The preparation method is simple, the conditions are mild, it is easy to scale up, and the protein sources are wide (including animal sources, plant sources, and recombinant proteins). The functional active ingredients can be flexibly selected and modularly designed and customized for different indications (such as oral ulcers, skin trauma, gastrointestinal mucosal damage, surgical wound closure, etc.). It has broad application prospects in tissue engineering, wound repair, drug delivery, wearable devices and other fields. Attached Figure Description

[0031] Figure 1 These are photographs (a), scanning electron microscope images (b), and surface morphology diagrams (c) of bovine serum albumin electrospun fiber membrane-carbomer composite material.

[0032] Figure 2 This section presents the adhesion strength and adhesion stability of bovine serum albumin electrospun fiber membrane-carbomer composite material. Section a compares the adhesion strength of the composite material with various commercially available oral ulcer patches obtained through tensile testing. Section b shows images of the composite material tightly adhering to tissue under different deformation states. Section c reflects the adhesion stability of various commercially available ulcer patches and the composite material on the surface of an isolated liver using the rotating disk method. From left to right, these are: Zizhu propolis oral membrane, Yiketie dexamethasone acetate oral patch, Kanghua chlorhexidine dexamethasone membrane, Boyijie oral ulcer healing gauze, Youchidi oral ulcer membrane, and the composite material.

[0033] Figure 3 Scanning electron microscope images (a) and a statistical graph of fiber diameters (b) of bovine serum albumin (BSA) electrospun fiber membranes prepared with different concentrations of bovine serum albumin (BSA).

[0034] Figure 4 These are scanning electron microscope images of bovine serum albumin electrospun fiber membranes prepared using cysteine ​​(a) and glutathione (b) as disulfide bond reducing agents, respectively.

[0035] Figure 5Scanning electron microscope images (a) and fiber diameter statistics (b) of bovine serum albumin electrospun fiber membranes prepared from tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solutions at different pH values.

[0036] Figure 6 The surface morphology (a) of the composite material prepared using sodium carboxymethyl cellulose as an intermediate layer and its adhesion effect on the skin surface (b) are shown.

[0037] Figure 7 The images show the appearance of the spinning solution (a), bovine serum albumin in the spinning solution (b), the Fourier transform infrared spectroscopy of the secondary structure transformation of the freshly spun electrospun fiber membrane (c), and the electrospun fiber membrane after being moistened with water (d), specifically the amide I band (1600–1700 cm⁻¹). -1 ) and amide II band (1500–1600 cm) -1 Gaussian deconvolution fitting results.

[0038] Figure 8 The antibacterial properties (a), in vitro glycyrrhetinic acid release curve (b), and effect on promoting the healing of oral ulcers in rats are shown in Example 1.

[0039] Figure 9 The degradation results (a) of bovine serum albumin electrospun fiber membrane, bovine serum albumin electrospun fiber membrane-carbomer composite material, and glycyrrhetinic acid-loaded composite patch in Example 1 after immersion in simulated gastric fluid for 20 minutes, and the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry analysis results (b) after immersion in simulated gastric fluid for 20 minutes, are shown below.

[0040] Figure 10 The results show the cell compatibility of bovine serum albumin electrospun fiber membrane, bovine serum albumin electrospun fiber membrane-carbomer composite material, and glycyrrhetinic acid-loaded composite patch in Example 1.

[0041] Figure 11 This refers to the antibacterial properties of the polylysine-loaded composite patch in Example 2.

[0042] Figure 12 This is the surface morphology of the human epidermal growth factor composite patch in Example 3.

[0043] Figure 13 This is the surface morphology of the gallic acid-loaded composite patch in Example 5.

[0044] Figure 14 This is the surface morphology of the glycyrrhetinic acid-loaded composite patch prepared using glutathione as a reducing agent in Example 6.

[0045] Figure 15This is the surface morphology of the oriented glycyrrhetinic acid-loaded composite patch obtained by electrospinning using a high-speed rotating receiver in Example 9. Detailed Implementation

[0046] This invention provides a protein composite fiber membrane with wet adhesion and drug delivery capability, and its preparation method. Employing a "unfolding-stabilization" strategy, the method involves mixing protein, functional active ingredients, and a volatile conformational stabilizer, followed by treatment with a disulfide reducing agent to obtain a spinning solution. Electrospinning causes the conformational stabilizer to volatilize, while the protein retains its untransformed conformation. A bioadhesive polymer interlayer is then applied to the fiber membrane surface, followed by secondary spinning to obtain a sandwich-structured protein composite fiber membrane material. To demonstrate the universality of this method, experiments were conducted by varying key parameters such as protein concentration, type of disulfide reducing agent, pH of the reducing agent solution, and type of bioadhesive polymer.

[0047] 1. Preparation and performance testing of bovine serum albumin composite fiber membrane materials

[0048] Step 1: Bovine serum albumin was dissolved in trifluoroethanol as a conformational stabilizer to obtain a protein solution. Separately, an aqueous solution of tris(2-carboxyethyl)phosphonate was prepared, and its pH was adjusted to 9.0 using sodium hydroxide. This solution was then thoroughly mixed with the protein solution and allowed to stand at room temperature for 3 hours to obtain the spinning solution. The spinning solution contained bovine serum albumin at a concentration of 120 mg / mL, tris(2-carboxyethyl)phosphonate at a concentration of 25 mmol / L, and trifluoroethanol at a volume fraction of 90%.

[0049] Step 2: Draw the above spinning solution into a 5 mL syringe equipped with a 20G dispensing needle. Connect the positive terminal of the high-voltage DC power supply to the needle. Lay aluminum foil as the receiving substrate flat on a grounded flat receiver, and ground the receiver. Set the electrospinning parameters as follows: DC high voltage intensity between the nozzle and the roller receiver is 23 kV, the distance between the nozzle and the receiver is 8 cm, and the flow rate is 2 mL / h. After spinning is completed, remove the aluminum foil from the receiver to obtain a bovine serum albumin electrospun fiber membrane that can be peeled off from the surface of the aluminum foil.

[0050] Step 3: Mix carbomer powder at a concentration of 2 mg / cm³ 2 The density is uniformly coated on the surface of the bovine serum albumin electrospun fiber membrane prepared in step 2 to form a carbomer intermediate layer.

[0051] Step 4: Repeat the electrospinning operation of Step 2 to coat the bovine serum albumin electrospun fiber membrane onto the carbomer interlayer. Peel the resulting composite material from the foil surface to obtain the bovine serum albumin electrospun fiber membrane-carbomer composite material with a sandwich structure (hereinafter referred to as "composite material").

[0052] The microstructure of the composite material was observed using field emission scanning electron microscopy, and the results are as follows: Figure 1 As shown. Figure 1 Image a is an optical photograph of the composite material, showing that the material has a uniform and intact appearance. Figure 1 b is a cross-sectional scanning electron microscope image of the composite material, clearly showing a sandwich-like structure: the upper and lower layers are bovine serum albumin electrospun fiber layers, and the middle layer is a carbomer bioadhesion polymer layer. Figure 1 c represents the surface morphology of the bovine serum albumin electrospun fiber membrane, showing that the fiber diameter is uniform and the morphology is good, proving that the method of the present invention can successfully prepare pure protein nanofiber membranes.

[0053] To evaluate the adhesion performance of composite materials at a moist biological interface, fresh porcine oral mucosa was used as a simulated substrate, and a 90° peel test was conducted, comparing it with several commercially available oral ulcer patches. The selected control samples included: Kanghua chlorhexidine dexamethasone film, Zizhu propolis oral film, Yiketie dexamethasone acetate oral patch, and Youchidi oral ulcer film. All samples were cut to the same size for use. The fresh porcine oral mucosa was washed with physiological saline and fixed to the lower clamp of a tensile testing machine. An 8 mm diameter titanium sheet was fixed to the upper clamp to define the peel area. During the test, the sample was attached to the moist mucosa surface, and 3 μL of bio-adhesive was applied to the back of the titanium sheet for fixation. The upper clamp was lowered to contact the sample surface, and a pressure of 2 N was applied for 10 seconds. Then, the sample was peeled vertically upwards at a constant speed until it was completely detached, and the peel force-displacement curve was recorded. The maximum peel force was calculated based on the titanium sheet area (50.24 mm²). 2 The peel strength is calculated using the ratio of ( ). The results are as follows: Figure 2 As shown in Figure a, the peel strength of Kanghua Chlorhexidine Dexamethasone Film was 0.45 kPa, that of Zizhu Propolis Oral Film was 0.84 kPa, that of Yiketie Dexamethasone Acetate Oral Patch was 0.90 kPa, and that of Youchidi Oral Ulcer Film was 3.89 kPa. The peel strength of the composite material reached 22.67 kPa, which was significantly higher than all commercially available control products.

[0054] To further evaluate the adhesion stability of the composite material in a dynamic environment, the following two tests were conducted:

[0055] (1) Skin dynamic deformation test: The composite material was attached to the surface of fresh pig skin, and the skin tissue was subjected to torsion, stretching, and bending. For example... Figure 2 As shown in b, even if the skin undergoes significant deformation, the composite material remains in place without displacement or detachment, demonstrating its excellent flexibility and interface following properties.

[0056] (2) Water erosion durability test: A rotating disk method was used to simulate a dynamic wet environment. The composite material and various commercially available control samples were attached to the surface of isolated pig liver tissue and placed in a circulating water flow at a speed of 300 rpm. The time it took for the samples to completely detach from the tissue surface was recorded. The results are as follows: Figure 2 As shown in c, all commercially available control samples disintegrated or detached within 10 minutes to 3 hours, while the composite material adhered stably for more than 24 hours, with an adhesion duration at least 8 times longer than the control products, demonstrating its excellent durability in dynamic wet environments.

[0057] 2. Verification of the universality of key process parameters

[0058] (1) Adjustability of protein concentration

[0059] Following the above preparation method, the concentration of bovine serum albumin in the spinning solution of step 1 was adjusted to 150 mg / mL, while other conditions remained unchanged, to prepare the composite material. The surface morphology and fiber diameter of the obtained fiber membrane are statistically shown below. Figure 3 As shown, the results indicate that fibrous membranes with good morphology can be obtained over a wide concentration range, demonstrating that this method has good adaptability to protein concentration.

[0060] (2) Universality of disulfide bond reducing agents

[0061] Cysteine ​​and glutathione were used respectively to replace tris(2-carboxyethyl)phosphonic acid hydrochloride as disulfide bond reducing agents, and their concentrations in the spinning solution were controlled at 25 mmol / L. Other conditions were the same as in the previous experiments to prepare composite materials. The surface morphology of the resulting fiber membrane is shown in the figure. Figure 4 As shown, bovine serum albumin electrospun fiber membranes with intact structures and uniform fiber morphology can be successfully prepared under both reducing agent conditions. This result demonstrates that the method of this invention is not particularly dependent on the type of disulfide bond reducing agent, possesses good universality, and provides a flexible technical solution for regulating protein conformation and fiber formation.

[0062] (3) The pH regulation effect of reducing agent solution

[0063] Following the above preparation method, the pH of the tris(2-carboxyethyl)phosphonate hydrochloride aqueous solution in step 1 was adjusted to 3.0, 5.0, 7.0, and 11.0, respectively, while keeping other conditions unchanged, to prepare the composite material. The surface morphology of the resulting fiber membrane is as follows. Figure 5 As shown in the figure. The results indicate that the diameter of electrospun fiber membranes can be effectively controlled by adjusting the pH value of the reducing agent solution, providing a reliable approach for the customized preparation of protein-based fiber materials of different specifications.

[0064] (4) Universality of bioadhesion polymers

[0065] Following the above preparation method, the bioadhesion polymer in step 3 was replaced with sodium carboxymethyl cellulose, while maintaining the applied density and other conditions unchanged, to prepare the composite material. The surface morphology and adhesion effect of the resulting fiber membrane on the skin surface are as follows: Figure 6 As shown in the figure. The results indicate that the method of the present invention is not particularly dependent on the type of bioadhesion polymer. Composite fiber materials with good adhesion properties can also be successfully prepared using other bioadhesion polymers such as sodium carboxymethyl cellulose, further demonstrating the universality and scalability of the technical solution.

[0066] The above experimental system verified that the technical solution of the present invention successfully achieved pure protein electrospinning without relying on carrier polymers. The prepared fiber membranes had uniform morphology and complete structure. The resulting composite material exhibited excellent adhesion strength (22.67 kPa, which is 6 to 50 times that of commercially available products) and adhesion stability under dynamic conditions (stable adhesion for more than 24 hours under water flow). By changing key parameters such as protein concentration, type of disulfide bond reducing agent, pH of reducing agent solution, and type of bioadhesive polymer, composite fiber materials with good morphology and adhesion properties could be successfully prepared, proving that the method has good process adaptability and wide application value.

[0067] The present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0068] Example 1

[0069] Step 1: Bovine serum albumin was dissolved in trifluoroethanol, and glycyrrhetinic acid was added. After mixing, a protein solution was obtained. Separately, a 250 mmol / L aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride was prepared, and its pH was adjusted to 9.0 using sodium hydroxide. This solution was then thoroughly mixed with the protein solution and allowed to stand at room temperature for 3 hours to obtain the spinning solution. The spinning solution contained bovine serum albumin at a concentration of 120 mg / mL, tris(2-carboxyethyl)phosphine hydrochloride at a concentration of 25 mmol / L, glycyrrhetinic acid at a concentration of 10 mg / mL, and trifluoroethanol at a volume fraction of 90%.

[0070] Step 2: Draw the above spinning solution into a 5 mL syringe equipped with a 20G dispensing needle. Connect the positive terminal of the high-voltage DC power supply to the needle. Lay aluminum foil as the receiving substrate flat on a grounded flat receiver, and ground the receiver. Set the electrospinning parameters as follows: DC high voltage intensity between the nozzle and the roller receiver is 23 kV, receiving distance is 8 cm, and flow rate is 2 mL / h. After spinning is completed, remove the aluminum foil from the receiver to obtain a bovine serum albumin fiber membrane loaded with glycyrrhetinic acid that can be peeled off from the surface of the aluminum foil.

[0071] Step 3: Mix carbomer powder at a concentration of 2 mg / cm³2 The density is coated onto the surface of the fiber membrane prepared in step 2 to form a carbomer intermediate layer.

[0072] Step 4: Repeat the electrospinning operation of Step 2 to spin the glycyrrhetinic acid-loaded bovine serum albumin fiber membrane onto the carbomer interlayer, forming a sandwich structure. Peel the resulting composite material from the tin foil surface to obtain the glycyrrhetinic acid-loaded bovine serum albumin electrospun fiber membrane-carbomer composite material (hereinafter referred to as glycyrrhetinic acid-loaded composite patch).

[0073] The appearance of the spinning solution prepared in step 1 is as follows: Figure 7 As shown in Figure a, it exhibits a uniform, pale yellow appearance. The infrared spectra of the spinning solution, the freshly spun and dried bovine serum albumin electrospun fiber membrane obtained in steps 1 and 2, and the fiber membrane after being moistened with water are shown below. Figure 7 As shown in b to 7d. Bovine serum albumin in the spinning solution showed no significant difference from the dried electrospun fiber membrane after infrared peak separation. Figure 7 (b, c) Conformation stabilizers such as trifluoroethanol can effectively stabilize the unfolded protein conformation. During spinning, the conformation stabilizers evaporate rapidly, and the protein molecules remain in the unfolded state. The electrospun fiber membrane was wetted, dried, and then subjected to infrared spectroscopy again. Figure 7 d and Figure 7 A comparison of b and 7c shows a significant change in the infrared peak shape: at 1515 cm⁻¹ -1 1623cm -1 and 1689 cm -1 A distinct β-sheet characteristic peak was observed, with a significantly enhanced peak intensity compared to the dry state. These results indicate that proteins in the electrospun fiber membrane remain unfolded in the dry state, undergoing a conformational change upon contact with water, resulting in a significant increase in β-sheet content. This wet-induced β-sheet enrichment disrupts the hydration barrier on the protein molecule surface, promoting the formation of multiple intermolecular forces between the protein molecules and the adhesion interface, thus providing a molecular structural basis for the strong adhesion of the material in a wet environment.

[0074] The antibacterial properties, in vitro release behavior, and effects on promoting oral ulcer healing of the above-mentioned glycyrrhetinic acid-loaded composite patch are as follows: Figure 8 As shown. This glycyrrhetinic acid-loaded composite patch exhibits significant inhibitory effects against common bacteria, including Gram-positive and Gram-negative bacteria (see...). Figure 8 a), and possesses continuous and controllable drug release characteristics (see Figure 8 b). In animal studies, compared with the blank control group and common commercially available patches, the glycyrrhetinic acid-loaded composite patch significantly accelerated the healing process of oral ulcer lesions (see [link]). Figure 8 c).

[0075] To verify the degradability and biocompatibility of the protein electrospun fiber material described in this invention, 20 mg of bovine serum albumin electrospun fiber membrane, bovine serum albumin electrospun fiber membrane-carbomer composite material, and glycyrrhetinic acid-loaded composite patch were placed in 20 mL of simulated gastric fluid and incubated in a constant-temperature shaker at 37°C for 0, 10, and 20 minutes, respectively, and photographs were taken to record the changes of each sample over time. After 20 minutes, the simulated gastric fluid was tested using a MALDI-TOF mass spectrometer to obtain matrix-assisted laser desorption / ionization time-of-flight mass spectra of each sample, as shown below. Figure 9 As shown. All materials were completely degraded within 20 minutes in simulated gastric fluid, with no obvious solid residue observed. Further analysis of the degradation products using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry revealed that none of the degradation products exhibited a characteristic peak corresponding to the molecular weight (66 kDa) of bovine serum albumin. Figure 9 (b) confirmed the complete enzymatic digestion of bovine serum albumin molecules.

[0076] The biocompatibility of the materials was assessed using cytotoxicity testing. The MTT assay was used to test the cytotoxicity of bovine serum albumin electrospun fiber membranes, bovine serum albumin electrospun fiber membrane-carbomer composites, and glycyrrhetinic acid-loaded composite patches. Sterilized samples were placed at 6 cm intervals. 2 The L929 cells were seeded at a density of 2700 cells per well in 96-well plates. After 24 hours of culture and adhesion, the medium was replaced with different concentrations of the extract (1 / 2, 1 / 4, and 1 / 8), and cultured for 24, 48, and 72 hours, respectively. At each time point, the medium was discarded, and the cells were incubated with 0.5 mg / mL MTT solution for 3 hours. Then, DMSO was added to dissolve formazan crystals, and the absorbance at 490 nm was measured using a microplate reader to evaluate cell viability. The results are shown below. Figure 10 As shown, after culturing L929 cells with the above sample extract for 24 hours, 48 ​​hours, and 72 hours, the cell viability was above 95%, indicating that this series of materials has good cell compatibility.

[0077] Example 2

[0078] The preparation method is the same as in Example 1, except that glycyrrhetinic acid is replaced with polylysine, with concentrations in the spinning solution of 1, 5, 10, and 30 mg / mL, respectively. The final product is a bovine serum albumin electrospun fiber membrane-carbomer composite material loaded with polylysine (referred to as the polylysine-loaded composite patch), whose antibacterial properties are as follows: Figure 11As shown, this method successfully loads polylysine into the fiber structure. Therefore, as the concentration of polylysine in the spinning solution increases, the antibacterial properties of the resulting polylysine-loaded composite patch show a corresponding increasing trend. This demonstrates that the loading amount of functional active ingredients can be flexibly controlled through the method of this invention, thereby effectively endowing the final product with the required biological properties.

[0079] Example 3

[0080] The preparation method was the same as in Example 1, except that glycyrrhetinic acid was replaced with human epidermal growth factor (HGF) to achieve a concentration of 20 μg / mL in the spinning solution. This resulted in a bovine serum albumin electrospun fiber membrane-carbomer composite material loaded with HGF (human epidermal growth factor-loaded composite patch). The surface morphology of the above-mentioned human epidermal growth factor-loaded composite patch was observed using FESEM, as shown below. Figure 12 As shown in the figure. This result demonstrates that this method can successfully load biomolecules such as epidermal growth factor while maintaining a uniform and intact fiber structure, further proving that this method has broad compatibility with the types of loaded molecules and can be used for the effective loading and fiber preparation of various functional active ingredients.

[0081] Example 4

[0082] The preparation method was the same as in Example 1, except that glycyrrhetinic acid was replaced with dexamethasone to achieve a dexamethasone concentration of 5 mg / mL in the spinning solution. The final product was a bovine serum albumin electrospun fiber membrane-carbomer composite material loaded with dexamethasone (dexamethasone-loaded composite patch).

[0083] Example 5

[0084] The preparation method is the same as in Example 1, except that gallic acid is used instead of glycyrrhetinic acid to make the concentration of gallic acid in the spinning solution 1 mg / mL. Finally, a gallic acid-loaded bovine serum albumin electrospun fiber membrane-carbomer composite material (gallic acid-loaded composite patch) is obtained, with the surface morphology as shown in the figure. Figure 13 As shown.

[0085] Example 6

[0086] The preparation method is the same as in Example 1, except that glutathione is used instead of tris(2-carboxyethyl)phosphine hydrochloride to make the concentration of glutathione in the spinning solution 25 mmol / L. Finally, a bovine serum albumin electrospun fiber membrane-carbomer composite material loaded with glycyrrhetinic acid (glycyrrhetinic acid-loaded composite patch) is obtained, and its surface morphology is as follows. Figure 14 As shown.

[0087] Example 7

[0088] The preparation method is the same as in Example 1, except that sodium carboxymethyl cellulose powder is used instead of carbomer powder, at a concentration of 2 mg / cm³. 2 The density of the coating is applied to the surface of the fiber membrane prepared in step 2. Finally, a bovine serum albumin electrospun fiber membrane-carboxymethyl cellulose sodium composite material loaded with glycyrrhetinic acid is obtained (glycyrrhetinic acid-loaded composite patch).

[0089] Example 8

[0090] The preparation method is the same as in Example 4, except that whey albumin is used instead of bovine serum albumin to make the concentration of whey albumin in the spinning solution 150 mg / mL. Finally, a whey albumin electrospun fiber membrane-carbomer composite material loaded with dexamethasone (dexamethasone-loaded composite patch) is obtained.

[0091] Example 9

[0092] The preparation method is the same as in Example 1, except that tin foil is used as the receiving substrate and wound around the surface of a high-speed rotating drum receiver, which is grounded. The final product is an oriented bovine serum albumin electrospun fiber membrane-carbomer composite material loaded with glycyrrhetinic acid (glycyrrhetinic acid-loaded composite patch), with a surface morphology as shown in Example 1. Figure 15 As shown, compared with the material prepared by the planar receiver in Example 1, the use of the rotating receiver can yield an electrospun fiber membrane material with an oriented arrangement structure.

[0093] Example 10

[0094] The preparation method is the same as in Example 1, except that hexafluoroisopropanol is used instead of trifluoroethanol to make the volume fraction of hexafluoroisopropanol in the spinning solution 90%. Finally, a bovine serum albumin electrospun fiber membrane-carbomer composite material loaded with glycyrrhetinic acid (glycyrrhetinic acid-loaded composite patch) is obtained.

Claims

1. A method for preparing a protein composite fiber membrane material with wet adhesion and capable of loading and delivering drugs, characterized in that: The preparation method consists of the following steps: Step 1: Mix the protein, functional active ingredient, and conformation stabilizer to obtain a mixed solution; adjust the pH of the disulfide bond reducing agent aqueous solution to 5.0-9.0 with a pH adjuster, mix it evenly with the mixed solution, and let it stand at room temperature to obtain a spinning solution; wherein, the conformation stabilizer is selected from any one or more of trifluoroethanol, difluoroethanol, trifluoroacetic acid, and hexafluoroisopropanol; Step 2: The spinning solution is processed into a protein nanofiber membrane using an electrospinning process. During the spinning process, the conformation stabilizer evaporates, allowing the protein to maintain its untransformed conformation in the fiber membrane. Step 3: Apply the bioadhesion polymer uniformly to the surface of the protein nanofiber membrane by coating or spraying to form an intermediate layer; wherein the bioadhesion polymer is a polymeric material capable of producing adhesion in a humid environment; Step 4: Using the electrospinning process described in Step 2, the spinning solution is processed into a protein nanofiber membrane on the intermediate layer to obtain a protein composite fiber membrane material with a sandwich structure; wherein, when the protein composite fiber membrane material is exposed to water or a wet environment, the protein conformation changes and wet adhesion is generated.

2. The method for preparing the protein composite fiber membrane material with wet adhesion and drug delivery according to claim 1, characterized in that: The protein is selected from any one or more of the following: lysozyme, bovine serum albumin, lactoferrin, pepsin, trypsin, catalase, hemoglobin, human serum albumin, whey albumin, insulin, α-lactalbumin, fibrinogen, ribonuclease A, cytochrome c, α-amylase, horseradish peroxidase, myoglobin, actin, silk fibroin, myosin, collagen, keratin, ferritin, and casein.

3. The method for preparing the protein composite fiber membrane material with wet adhesion and drug delivery according to claim 1, characterized in that: The functional active ingredients are selected from any one or more of the following: antibacterial active ingredients, anti-inflammatory active ingredients, soluble inorganic salts, traditional Chinese medicine extracts, polypeptide active ingredients, protein active ingredients, growth factors, cytokines, and nanoparticles. The antibacterial active ingredient is selected from at least one of antibiotics, antimicrobial peptides, quaternary ammonium salt antimicrobial agents, and biguanide antimicrobial agents; wherein the antibiotic is selected from at least one of β-lactam antibiotics, macrolide antibiotics, tetracycline antibiotics, aminoglycoside antibiotics, glycopeptide antibiotics, quinolone antibiotics, lincosamide antibiotics, polypeptide antibiotics, oxazolidinone antibiotics, lipopeptide antibiotics, glycylcycline antibiotics, and antifungal drugs; the antimicrobial peptide contains 10 to 100 amino acid residues and has a net positive charge. The cationic antimicrobial peptide; the quaternary ammonium salt antimicrobial agent is selected from at least one of benzalkonium chloride, hexadecylpyridinium halide, alkyltrimethylammonium bromide, benzyltriethylammonium chloride, dialcyldimethylammonium chloride, dialcyldimethylammonium bromide, tetradecyl-2-methylpyridinium bromide, dialcylmethylhydroxyethylammonium chloride, and dialcylmethylhydroxypropylammonium chloride; the biguanide antimicrobial agent is selected from at least one of guanidine dodecyl acetate, aricetin, chlorhexidine acetate, chlorhexidine gluconate, polyhexylene biguanide, polyaminopropyl biguanide, and polyhexamethylene biguanide. The anti-inflammatory active ingredient is selected from at least one of ibuprofen, diclofenac sodium, piroxicam, meloxicam, ketorolac, aminopyrine, parecoxib, celecoxib, nimesulide, flurbiprofen ester, dexamethasone, resveratrol, and epigallocatechin gallate. The soluble inorganic salt is selected from at least one of silver nitrate, silver acetate, copper sulfate, sodium iodide, calcium chloride, calcium lactate, calcium gluconate, ferric chloride, and magnesium chloride. The herbal extracts are selected from at least one of the following: glycyrrhizic acid, andrographolide, tripterygium wilfordii, berberine, matrine, tetrandrine, quercetin, baicalin, puerarin, ginsenosides, notoginsenosides, astragaloside A, astragalus polysaccharide, lentinan, rhein, tanshinone, aescin, schisandrin A, menthol, eugenol, camphor, chlorogenic acid, gallic acid, and tannic acid. The polypeptide active ingredients are compounds composed of 2 to 100 amino acids linked by peptide bonds, and possess biological activity, selected from one or more of the following functional categories: (a) tissue regeneration promoters: epidermal growth factor mimic peptides, fibroblast growth factor mimic peptides, collagen synthesis promoting peptides; (b) angiogenesis promoters: vascular endothelial growth factor mimic peptides, RGD sequence peptides, laminin-derived peptides; (c) cell migration and adhesion regulators: cell membrane-penetrating peptides, targeting integrin peptides, fibronectin-derived peptides; (d) matrix metalloproteinase regulators: matrix metalloproteinase inhibitory peptides, procollagen C-terminal protease inhibitory peptides; (e) neuromodulation and analgesia promoters: nerve growth factor mimic peptides, opioid analgesic peptides; The protein-based active ingredients are selected from one or more of the following functional categories: (a) structural proteins: collagen, silk fibroin, elastin, gelatin, keratin; (b) globular functional proteins: insulin, enzymes, plant-derived functional proteins; (c) antibodies: antibodies or their antigen-binding fragments. The growth factor is selected from at least one of epidermal growth factor, fibroblast growth factor, vascular endothelial growth factor, platelet-derived growth factor, transforming growth factor-β, insulin-like growth factor, and nerve growth factor. The cytokines are selected from at least one of interleukin, interferon, tumor necrosis factor, chemokine, and colony-stimulating factor. The nanoparticles are selected from at least one of silver nanoparticles, gold nanoparticles, zinc oxide nanoparticles, silica nanoparticles, iron oxide nanoparticles, hydroxyapatite nanoparticles, and bioglass nanoparticles.

4. The method for preparing the protein composite fiber membrane material with wet adhesion and drug delivery according to claim 1, characterized in that: The disulfide bond reducing agent is selected from any one or more of tris(2-carboxyethyl)phosphine hydrochloride, glutathione, cysteine, dithiothreitol, dithiothreitol isomer, β-mercaptoethanol, tris(3-hydroxypropyl)phosphine, and tributylphosphine.

5. The method for preparing the protein composite fiber membrane material with wet adhesion and drug delivery according to claim 1, characterized in that: The pH adjuster is selected from any one or more of the following: tris(hydroxymethyl)aminomethane, 4-hydroxyethylpiperazine ethanesulfonic acid, N,N-dihydroxyethylglycine, piperazine-1,4-diethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid, 3-(N-morpholino)-2-hydroxypropanesulfonic acid, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, 3-(cyclohexylamine)-1-propanesulfonic acid, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium benzoate, and sodium citrate.

6. The method for preparing the protein composite fiber membrane material with wet adhesion and drug delivery according to claim 1, characterized in that: In step 1, the concentration of protein in the spinning solution is 10–300 mg / mL, the concentration of disulfide bond reducing agent is 10–200 mmol / L, and the volume fraction of conformation stabilizer is 50%–95%.

7. The method for preparing the protein composite fiber membrane material with wet adhesion and drug delivery according to claim 6, characterized in that: In step 1, the concentration of protein in the spinning solution is 100–200 mg / mL, the concentration of disulfide bond reducing agent is 15–100 mmol / L, and the volume fraction of conformation stabilizer is 70%–90%.

8. The method for preparing the protein composite fiber membrane material with wet adhesion and drug delivery according to claim 1, characterized in that: In step 2, during electrospinning, the DC high voltage intensity between the nozzle and the roller receiver is set to 3-50 kV, the flow rate is 0.2-5 mL / h, a planar grounded receiver or a high-speed rotating grounded receiver is selected, and the distance between the nozzle and the receiver is 5-30 cm.

9. The method for preparing the protein composite fiber membrane material with wet adhesion and drug delivery according to claim 1, characterized in that: The bioadhesive polymer is selected from any one of acrylic polymers, cellulose derivatives, natural polysaccharides and their derivatives, modified starch, and other water-absorbing polymers; The acrylic polymer is selected from any one or more of carbomer, polycarbofer, and acrylic / acrylate copolymers; The cellulose derivative is selected from any one or more of sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, and hydroxyethyl cellulose. The natural polysaccharides and their derivatives are selected from any one or more of chitosan, sodium alginate, hyaluronic acid, agarose, carrageenan, guar gum, xanthan gum, gellan gum, and pectin. The modified starch is selected from any one of hydroxypropyl starch and carboxymethyl starch, or a mixture of both. The other water-absorbing polymers are selected from any one or more of polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene oxide.

10. The protein composite fiber membrane material with wet adhesion and drug loading and delivery obtained by the preparation method according to any one of claims 1 to 9.

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