Chitosan-based dressing with multi-layer gradient structure and preparation method of chitosan-based dressing

Abstract

The invention belongs to the technical field of medical dressings, and relates to a multi-layer gradient structure chitosan-based dressing and a preparation method thereof, the multi-layer gradient structure chitosan-based dressing is formed by compounding an internal porous functional matrix and an external protective layer; the internal porous functional matrix comprises at least two layers of chitosan-based fiber aggregates, and the two adjacent layers of chitosan-based fiber aggregates are compounded by spraying a liquid treating agent, needling and drying; during preparation, at least two layers of chitosan-based fiber assemblies are laid layer by layer according to a preset sequence, in the laying process, after each layer is laid, a liquid treating agent is evenly sprayed to the outer side face of the layer, then the next layer continues to be laid till laying is completed, and after the overlapped chitosan-based fiber assemblies are obtained, needling treatment is conducted, and the chitosan-based fiber assemblies are obtained. And uniformly spraying a liquid treating agent on the outer side surface of the treated product, aligning and overlapping the treated product with an external protective layer prepared in advance, applying pressure, and drying at room temperature to obtain the product. The method is efficient and mild; the product performance is excellent.

Description

Technical Field

[0001] This invention belongs to the field of medical dressing technology, and relates to a multi-layered gradient structure chitosan-based dressing and its preparation method. Background Technology

[0002] As the largest organ in the human body, the skin is the first line of defense against external physical, chemical, and biological aggressors. When the skin is damaged due to trauma, burns, or chronic diseases (such as diabetes), effective wound management is crucial for preventing infection, accelerating healing, and reducing scar formation. An ideal wound dressing should have multiple functions, including: effectively absorbing and managing wound exudate, providing a moderately moist healing environment, having good breathability, effectively blocking the invasion of external microorganisms, not adhering to newly formed tissue, and actively participating in and promoting the healing process.

[0003] Traditional dressings, such as gauze and cotton pads, while inexpensive, have several inherent drawbacks. They tend to adhere to the wound, causing secondary damage and severe pain during changes; after absorbing exudate, they easily spread laterally, soaking the healthy skin around the wound; and they lack antibacterial or healing-promoting bioactivity. To overcome these problems, modern wound dressing research has shifted towards bioactive polymers, among which chitosan has attracted considerable attention due to its excellent biocompatibility, biodegradability, natural broad-spectrum antibacterial activity, and hemostatic properties.

[0004] However, chitosan dressings alone have also revealed new problems in practical applications. As disclosed in patent application CN117018264A, chitosan fibers swell significantly after absorbing exudate, causing blockage of the microporous structure inside the dressing and a sharp decrease in breathability. This can lead to fluid accumulation under the dressing, creating conditions for bacterial growth and increasing the risk of infection. In addition, single-component chitosan dressings struggle to balance the contradiction between moisture absorption and retention, failing to provide dynamic support throughout the entire wound healing process.

[0005] To address these issues, researchers have proposed constructing multilayered composite or gradient-structured dressings to mimic the layered structure of skin and achieve functional zoning. For example, patent CN201610857216.9 discloses a chitosan-based multilayered nanofiber membrane dressing that aims to achieve phased regulation of the healing process through the combination of different functional layers. While this design concept is advanced, its implementation faces new technical bottlenecks: the functional layers of multilayer dressings typically rely on physical stacking or simple lamination, resulting in weak bonding and a tendency for delamination during use, leading to structural damage and functional failure of the dressing.

[0006] To enhance interlayer bonding, existing technologies often employ chemical cross-linking methods (such as using glutaraldehyde) or hot pressing. However, these processes are demanding and potentially harmful. Chemical cross-linking agents are typically cytotoxic, and their residues can negatively impact wound healing. High-temperature treatments like hot pressing can damage the porous structure of chitosan, reducing its permeability and responsiveness. More seriously, high temperatures can inactivate heat-sensitive bioactive substances such as growth factors and exosomes, causing the dressing to lose its core function of actively promoting healing. Furthermore, while methods like layer-by-layer electrospinning, as used in patent CN201610857216.9, can construct fine structures, the process is complex, time-consuming, and difficult to scale up for industrial production, resulting in high costs.

[0007] Therefore, researching a multi-layered gradient chitosan-based dressing and its preparation method is of great significance. It can be achieved through a simple and efficient process under mild ambient temperature conditions to firmly integrate chitosan-based fiber layers with different functions into an integrated multi-layered structure, while fully preserving the porous characteristics and bioactive substances of the material. Summary of the Invention

[0008] The purpose of this invention is to solve the problems existing in the prior art and to provide a multi-layered gradient structure chitosan-based dressing and its preparation method.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A multi-layered gradient structure chitosan-based dressing is composed of an internal porous functional matrix and an external protective layer.

[0011] The internal porous functional matrix comprises at least two layers of chitosan-based fiber assemblies. Adjacent layers are composited by spraying a liquid treatment agent followed by needle punching and drying. The two chitosan-based fiber assemblies constituting the internal porous functional matrix can have the same or different functions, and can be flexibly constructed according to requirements. For example, when the chitosan-based fiber assembly is used as a hydrophilic anti-adhesion layer, it requires a water contact angle of less than 65° and a water absorption ratio of more than 18 times; when used as an antibacterial layer, it requires an antibacterial rate of >95%; when used as a flow-guiding layer, it requires a large pore size (50–200 μm); and when used as a liquid-locking layer, it requires a high basis weight (≥200 g / m³). 2 ")", to enhance water retention capacity;

[0012] The composite of the internal porous functional matrix and the external protective layer is achieved by spraying a liquid treatment agent onto the outer surface of the internal porous functional matrix and then drying it.

[0013] The liquid treatment agent is a mixture of a solvent and an aqueous ethanol solution. The solvent is a mixture of one or more of trifluoroethanol, hexafluoroisopropanol, dichloromethane, chloroform, toluene, tetrahydrofuran, and acetone with an organic acid, wherein the organic acid is one or more of acetic acid and formic acid.

[0014] The aqueous ethanol solution contains 60–90 wt% anhydrous ethanol;

[0015] The mass ratio of solvent to aqueous ethanol solution is 5–10:100;

[0016] The outer protective layer is made of hydrophobically modified chitosan-based material.

[0017] As a preferred technical solution:

[0018] As described above, a multi-layered gradient structure chitosan-based dressing, the chitosan-based fiber assembly refers to a chitosan-based needle-punched nonwoven fabric obtained by needle-punching nonwoven fabric of one or two chitosan-based fibers; the chitosan-based fibers refer to pure chitosan fibers, silanized chitosan fibers, carboxymethyl chitosan fibers, carboxyethyl chitosan fibers, quaternized chitosan fibers, acylated modified chitosan fibers, thiolated chitosan fibers, or sulfonated chitosan fibers; the needle-punching process parameters are: needle-punching density 200 needles / cm². 2 3cm deep, 1Hz frequency (60 times / min).

[0019] As described above, a multi-layered gradient chitosan-based dressing has a chitosan-based fiber aggregate thickness of 1–10 mm and a basis weight of 100–300 g / m². 2 The chitosan-based fibers have a diameter of 1.2–3.6 dtex and a length of 30–60 mm.

[0020] As described above, a multi-layered gradient chitosan-based dressing has at least one layer of chitosan-based fiber assembly that is a chitosan-based fiber assembly loaded with bioactive substances. The preparation process of the chitosan-based fiber assembly loaded with bioactive substances is as follows: the chitosan-based fiber assembly is immersed in a solution containing bioactive substances, and the bioactive substances are loaded in the chitosan-based fiber assembly through electrostatic adsorption. The bioactive substances are negatively charged bioactive substances, such as exosomes, glycosaminoglycans, and negatively charged polysaccharides from natural plants.

[0021] As described above, a multilayer gradient structure chitosan-based dressing uses a hydrophobically modified chitosan-based material, which is a chitosan-based nanofiber membrane or a chitosan-based spunlace nonwoven fabric. The thickness of the chitosan-based nanofiber membrane is 20–100 μm; the thickness of the chitosan-based spunlace nonwoven fabric is 0.2–0.5 mm, and the basis weight is 40–150 g / m². 2 .

[0022] As described above, the chitosan-based dressing with a multi-layered gradient structure has an organic acid content of 30-50 wt% in the solvent.

[0023] The chitosan-based dressing with a multi-layered gradient structure, as described above, has a peel strength ≥1.0 N / cm.

[0024] A method for preparing a multi-layered gradient chitosan-based dressing as described in any of the preceding claims, characterized by comprising the following steps:

[0025] (1) At least two layers of chitosan-based fiber assemblies constituting the internal porous functional matrix are laid layer by layer in a preset order. During the laying process, after each layer is laid, a liquid treatment agent is evenly sprayed on the outer surface of the layer by a spraying device. Then the next layer is laid until the laying is completed, and the stacked chitosan-based fiber assemblies are obtained.

[0026] (2) The stacked chitosan-based fiber aggregates are needle-punched using a needle punching machine; the needle punching density is 180–220 cm³. 2 The needle penetration depth is 2.5–3.5 cm, and the needle penetration frequency is 0.8–1.2 Hz (50–70 times / min). The needle penetration density and depth affect the interlayer peeling force, while the frequency affects the needle penetration uniformity. Experiments have shown that when the needle penetration process is set within the range given in this invention, a tight connection between multiple layers can be achieved.

[0027] (3) Spray the liquid treatment agent evenly on the outer surface of the product after the needle punching treatment in step (2), then align and overlap it with the pre-prepared outer protective layer and apply pressure. After drying at room temperature, the chitosan molecular chains at the interface interpenetrate and fuse to form a strong bond, thus forming a multi-layer gradient structure chitosan-based dressing.

[0028] In step (2) above, the mechanical puncture of the needle machine forces the fibers of each layer to become three-dimensionally entangled (macroscopic bonding). At the same time, the liquid treatment agent (containing a specific solvent and an aqueous ethanol solution) activates the molecular chains on the surface of the fibers, and under the needle pressure, the molecular chains of adjacent layers are encouraged to interpenetrate and diffuse. Subsequently, in step (3), the fibers are dried and cured to form a physical cross-linking network dominated by hydrogen bonds (microscopic bonding), thereby achieving a strong "welding" composite between the layers, resulting in a structurally complete internal porous functional matrix with strong interlayer bonding. The internal porous functional matrix is ​​then combined with the external protective layer to obtain a multi-layered gradient structure chitosan-based dressing.

[0029] It should be noted that when spraying liquid treatment agents, the functional core layer area already loaded with bioactive substances (such as exosomes and bFGF) should be actively avoided to prevent sensitive components from being dissolved or inactivated by the liquid treatment agent. When loading bioactive substances, these substances adhere to the surface of the functional core layer fibers through specific loading methods (such as electrostatic adsorption, where the chitosan-based fiber aggregate to be loaded is immersed in a solution containing bioactive substances, and the bioactive substances are loaded onto the fibers through electrostatic adsorption), forming relatively specific loading areas. These loading areas can be roughly identified by microscopic observation (optical or electron microscopy), and areas rich in bioactive substances should be selectively avoided. For example, adjustable nozzles can be used, or masking materials such as plastic film can be used and removed after spraying.

[0030] In the preparation method of the multi-layer gradient chitosan-based dressing described above, the amount of liquid treatment agent sprayed in step (1) is ≤0.1 g / cm³. 2 .

[0031] In the preparation method of the multi-layer gradient chitosan-based dressing described above, the spraying amount of the liquid treatment agent in step (3) is ≤0.05 g / cm³. 2 The standard is to avoid damaging its dense structure.

[0032] Invention principle:

[0033] The core of the composite process proposed in this invention lies in an innovative "dual-mode physical integration mechanism," which achieves high-strength integration of multilayer heterogeneous structural materials under mild conditions through the "synergistic effect of macroscopic mechanical locking and microscopic physical cross-linking" and "controllable interpenetrating fusion of interface molecules."

[0034] 1. The "synergistic integration" mechanism of internal porous functional matrices:

[0035] Macroscopic Mechanical Bonding: As a mature nonwoven technology, needle punching plays a crucial role at the "macro" level. During high-speed puncture, barbed needles forcibly carry some fibers from the upper fiber assembly into the lower layer, forming fiber bundles perpendicular to the plane between layers. These fiber bundles act like "physical stitches," firmly "nailing" the layers together on a macroscopic scale, providing the matrix with basic structural integrity and resistance to delamination.

[0036] Interfacial micro-physical cross-linking: This is the key innovation of this invention. When the liquid treatment agent is selectively sprayed onto the interlayer interface, it does not simply act as an adhesive, but rather as an "interfacial activator." Its mechanism of action is as follows: the strong solvent (such as trifluoroethanol) in the liquid treatment agent works synergistically with the organic acid to effectively break the strong hydrogen bond network between chitosan molecular chains, allowing the molecular chains on the fiber surface to acquire a certain degree of mobility, entering a limited, flowable "plasticized" state. The aqueous ethanol solution in the formulation (especially 60–90 wt% ethanol) plays a crucial role as a "reaction regulator" and "non-solvent." It dilutes the concentration of the strong solvent to a precise threshold, ensuring that its effect is limited to the micron-level depth of the fiber surface without excessively dissolving or damaging the fiber's main structure. This concentration window is crucial: if the ethanol content is below 60 wt%, the system has a high water content, which enhances the corrosiveness of the acid; if it is above 90 wt%, it excessively inhibits the movement of molecular chains, which is detrimental to subsequent adhesion. Under the mechanical pressure of needle punching, the "activated" fiber surfaces on two adjacent interfaces are tightly pressed together, allowing the chitosan molecular chains on the surface to diffuse and entangle with each other. As the solvent evaporates rapidly, the molecular chains lose their mobility again and are "frozen" in an entangled state, forming a strong, "welded" physical cross-linked network at the interface.

[0037] Therefore, the robust bonding of the internal porous functional matrix is ​​the result of the synergistic effect of macroscopic mechanical interlocking and microscopic physical cross-linking. Needling provides the physical conditions for close contact, while the liquid treatment agent creates a chemical environment for molecular-level fusion. The two complement each other, resulting in interlayer peeling strength far exceeding that of simple physical stacking or needle punching.

[0038] 2. The "interface fusion" mechanism of the external high-density protective layer:

[0039] When combining a pre-fabricated dense protective layer (such as an electrospun film) with a porous functional matrix, the needle punching process, which would damage its structure, cannot be used. In this case, the present invention utilizes a "controllable interfacial molecular interpenetration fusion" mechanism to achieve precise surface activation: by spraying a trace amount (≤0.05 g / cm³) onto the outer surface of the matrix. 2 The liquid treatment agent also precisely and superficially plasticizes the chitosan fibers on the surface. When the dense protective layer comes into contact with the activated substrate surface and a slight pressure is applied, the interfacial molecular chains interpenetrate. Because the amount of liquid treatment agent used is extremely small, it does not penetrate and damage the dense structure of the protective layer. During the room temperature drying process, the solvent evaporates, and the interpenetrating molecular chains form a strong physical bond at the interface. This bonding method is similar to "solvent welding" of polymer materials; the interface formed is a seamless fusion zone, rather than a simple adhesive layer, resulting in high and durable bonding strength.

[0040] Beneficial effects:

[0041] (1) The preparation method of a multi-layer gradient structure chitosan-based dressing of the present invention has a unique step-by-step composite process of “internal needle-punching integration + external interface fusion”, which not only ensures a strong physical bond between the internal functional layers, but also completely preserves the dense antibacterial structure of the external protective layer, thus solving the technical contradiction that traditional single process cannot take into account both porosity and density.

[0042] (2) The preparation method of a multi-layer gradient structure chitosan-based dressing of the present invention firmly integrates different functional layers into a whole. The dressing has high peel strength (≥1.0N / cm) and is not easy to delaminate, thus achieving a high degree of synergy of multiple functions such as efficient one-way moisture wicking, anti-adhesion, intelligent drug release and physical barrier.

[0043] (3) The preparation method of the multi-layer gradient structure chitosan-based dressing of the present invention is carried out at room temperature, which avoids the destruction of heat-sensitive bioactive substances by high temperature; the physical method is used to replace the toxic chemical crosslinking agent, with no risk of chemical residue, ensuring the excellent biocompatibility and safety of the dressing.

[0044] (4) The preparation method of the multi-layer gradient structure chitosan-based dressing of the present invention, through precise control of the formulation and dosage of liquid treatment agent, and combined with different mechanical processes (needle punching or micro-pressure), successfully realizes the strong, non-destructive and safe composite integration of chitosan-based materials with different structures (porous and dense) and different functions (hydrophilic and hydrophobic) at room temperature and pressure, thereby preparing a high-performance multifunctional gradient dressing.

[0045] (5) The multi-layer gradient structure chitosan-based dressing of the present invention has high application flexibility. It can be flexibly customized into complex structure dressings with 3, 4, 5 or even more layers according to different clinical needs. It is suitable for a variety of application scenarios from acute trauma to chronic difficult-to-heal wounds and has broad market prospects.

[0046] (6) The present invention provides a multi-layer gradient chitosan-based dressing in which all layers are tightly connected through physical cross-linking networks or interface fusion, with a peel strength ≥1.0N / cm, achieving efficient one-way management of wound exudate, preventing wound adhesion, intelligently releasing bioactive substances according to the healing cycle, and possessing excellent biocompatibility and safety. Attached Figure Description

[0047] Figure 1 A multi-layer gradient structure chitosan-based dressing integrated molding machine;

[0048] Among them, 1-top layer feeder, 2-transfer device, 3-needle punching machine, 4-outer protective layer feeding area, 5-pressing device, 6-drying channel. Detailed Implementation

[0049] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0050] The test methods involved in the performance indicators in the embodiments and comparative examples of this invention are as follows:

[0051] Peel strength: Refer to FZT 60011-2016 Test method for peel strength of composite fabrics; When testing the peel strength between any two adjacent layers of a multilayer dressing, it is necessary to peel 50mm from the target two layers on the finished dressing, keeping the two target layers in a tight fit except for the target two layers. Clamp the target two layers that have been peeled 50mm apart in two clamps respectively, and make the clamping surfaces of the two clamps on the same plane of the tensile axis. Turn on the tester, start peeling, stretch the sample until it is completely separated, and record the corresponding peel strength.

[0052] Antibacterial rate: Tested according to FZ / T64077.2-2019.

[0053] Moisture permeability: Tested according to GB / T12704.1-2009 "Textiles - Test methods for moisture permeability - Part 1: Moisture absorption method".

[0054] Contact angle: Cut to a size of 1cm×1cm and measure using a DSA30 video optical contact angle meter (Kruss, Germany).

[0055] Hemostasis test: The test was conducted according to YY / T1477.5-2020 "Standard test model for evaluating the performance of contact wound dressings Part 5: In vitro model for evaluating hemostasis performance". The blood absorption and hemostasis time of the dressing were calculated by conducting a rat test on the dressing. The tail artery incision (2 mm in length) of SD rats (200±20g) was made, and the dressing was applied under pressure for 10s, and the hemostasis time was recorded.

[0056] Absorption capacity: The ability of the dressing to absorb simulated wound exudate (95wt% saline + 5wt% bovine serum albumin). Test procedure: The test was conducted according to YY / T 0471.1-2004 "Test methods for contact wound dressings - Part 1: Liquid absorbability". A 5cm × 5cm sample dressing was taken at a mass ratio of 1:40 and placed in simulated wound exudate for 30 min. It was then transferred to a drying oven at 37±1℃ for 30 min. After draining the liquid by holding one corner with tweezers, it was weighed again.

[0057] The sources of some of the chitosan-based fibers in this invention are as follows:

[0058] Pure chitosan fiber: 1.67dtex×38mm, degree of deacetylation >90%, molecular weight 100kDa, from Wenzhou Jiayuan Biotechnology Co., Ltd., China.

[0059] Silanized chitosan fiber: After vacuum drying 1g of pure chitosan fiber for 4 hours, it was immersed in 50ml of ethanol / water mixture (volume ratio 4:1), and 2ml of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was stirred at 60℃ for 12 hours (pH = 5.0, adjusted with acetic acid). The reaction product was ultrasonically washed 3 times with anhydrous ethanol (10 minutes each time) and then vacuum dried at 60℃ for 6 hours to obtain silanized chitosan fiber. The diameter of the silanized chitosan fiber was 1.7±0.1 detx and the length was 38±1mm.

[0060] Carboxymethyl chitosan fiber: 1g of pure chitosan fiber was immersed in 20ml of 40wt% sodium hydroxide aqueous solution and shaken at 50℃ for 1h. Then, 2g of chloroacetic acid was added and reacted at 50℃ for 6h. After the reaction was completed, the pH was adjusted to 7.0 with glacial acetic acid. The product was repeatedly washed with anhydrous ethanol / water (volume ratio 1:1) and vacuum dried for 8h to obtain carboxymethyl chitosan fiber. The diameter of the carboxymethyl chitosan fiber was 1.6±0.2detx and the length was 40±2mm.

[0061] Carboxyethyl chitosan fiber: 1 g of chitosan fiber was immersed in 20 mL of 40 wt% NaOH solution and swollen at 50 °C for 1 hour. 3 mL of acrylonitrile (Aladdin, 99.5%) was added, and the reaction was continued at 65 °C for 4 hours. The reaction was then stopped by adding 50 mL of deionized water. After the reaction, the pH was adjusted to 7.0 with 0.1 M HCl. The fiber was dialyzed against 80% ethanol and then deionized water for 48 hours, followed by vacuum drying for 8 hours to obtain carboxyethyl chitosan fiber. The diameter of the carboxyethyl chitosan fiber was 1.75 ± 0.2 detx, and the length was 39 ± 2 mm.

[0062] Quaternized chitosan fibers: 1g of pure chitosan fibers were immersed in PBS buffer (pH=8.5) and swollen at 50℃ for 1h. 1.6g of glycidyltrimethylammonium chloride (GTA) (from Sigma) was added and stirred at 60℃ for 12h. The pH was adjusted to 7.0 with 0.1M hydrochloric acid. The product was repeatedly washed with anhydrous ethanol and vacuum dried for 8h to obtain quaternized chitosan fibers. The diameter of the quaternized chitosan fibers was 1.9±0.1detx and the length was 38±1mm.

[0063] Acylated modified chitosan fiber: 1g of pure chitosan fiber was immersed in an anhydride / pyridine solution (3mL of anhydride:acetic anhydride / propionic anhydride (1mol:1mol) + 20mL of pyridine), and stirred at 25℃ for 6h. After stirring, 50ml of ice-cold ethanol was added to terminate the reaction. The product was repeatedly washed with anhydrous ethanol and vacuum dried for 8h to obtain acylated modified chitosan fiber. The diameter of the acylated modified chitosan fiber was 2.0±0.3detx and the length was 40±2mm.

[0064] Thiolized chitosan fibers: 1g of pure chitosan fibers were immersed in 1.2g of thioglycolic acid + EDC / NHS (0.3g + 0.1g) in MES buffer (pH = 5.5) for 30min. The product was washed repeatedly with PBS and vacuum dried for 8 hours to obtain thiolated chitosan fibers. The diameter of the thiolated chitosan fibers was 1.6±0.2detx and the length was 35±1mm.

[0065] Sulfonated chitosan fiber: 1g of pure chitosan fiber was immersed in 3ml chlorosulfonic acid / 1ml pyridine solution and reacted at 25℃ for 4h. It was then neutralized with saturated sodium bicarbonate. The product was repeatedly washed with dichloromethane and vacuum dried for 8 hours to obtain sulfonated chitosan fiber. The diameter of the sulfonated chitosan fiber was 2.2±0.2detx and the length was 42±1mm.

[0066] The above-mentioned chitosan-based fibers are needle-punched nonwoven fabric / nanofiber membrane through electrospinning, i.e., chitosan-based fiber aggregate;

[0067] The parameters for electrospinning silanized chitosan nanofiber membranes are as follows: spinning positive and negative voltages are +25kV and -5kV, respectively; the distance between the spinning needle and the collecting plate is 15cm; and the spinning speed is 3mL / h.

[0068] Multi-layer gradient structure chitosan-based dressings are prepared using an integrated molding machine for multi-layer gradient structure chitosan-based dressings, such as... Figure 1 As shown, the multi-layer gradient structure chitosan-based dressing integrated molding machine includes a top feeder 1, a conveying device 2, a needle punching machine 3, an outer protective layer feeding area 4, a pressing device 5, and a drying channel 6.

[0069] From left to right, the conveying device 2, the needle punching machine 3, the pressing device 5, and the drying channel 6 are arranged in sequence; the needle punching machine 3 is located downstream of the conveying device 2; the drying channel 6 is located downstream of the pressing device 5.

[0070] The top feeder 1 is the starting section, which stores chitosan-based fiber assemblies with different functions. Then, liquid treatment agent is sprayed through the conveying device 2. The sprayed raw material rolls are laid in a preset order. The outer protective layer fed by the outer protective layer feeding area 4 and the internal porous functional matrix formed by the needle punching machine 3 are compounded in the pressing device 5. The pressing device 5 is adjacent to the 3 needle punching area and can guide the composite material that has been needle punched into the next process and apply slight pressure to make them bond more tightly. After the composite is completed, the material is wound up after passing through the drying channel 6, and finally a multi-layer gradient structure chitosan-based dressing is obtained.

[0071] Example 1

[0072] A method for preparing a multi-layered gradient chitosan-based dressing, the specific steps of which are as follows:

[0073] (1) Preparation of raw materials:

[0074] Organic acid: acetic acid;

[0075] Solvent: Trifluoroethanol, with an organic acid content of 30 wt% in the solvent;

[0076] Liquid treatment agent: a mixture of solvent and aqueous ethanol solution; the aqueous ethanol solution contains 80 wt% anhydrous ethanol; the mass ratio of solvent to aqueous ethanol solution is 8:100;

[0077] Chitosan-based fiber aggregate I: Pure chitosan fiber needle-punched nonwoven fabric, weight 200 g / m² 2 Thickness 2.0mm;

[0078] Chitosan-based fiber aggregate II: Carboxymethyl chitosan fiber needle-punched nonwoven fabric, 1.5 mm thick, 150 g / m² weight. 2 ;

[0079] Bioactive Substance I: Hyaluronic Acid, Bloomage Biotech, FCH001, Mw = 1.5 MDa;

[0080] Bioactive substance II: Exosomes derived from mesenchymal stem cells (extraction method refers to patent CN 110252440 A an exosome filtration extraction device and exosome extraction method);

[0081] External protective layer material: silanized chitosan electrospun nanofiber membrane, basis weight 30 g / m 2 Thickness 50μm;

[0082] (2) Preparation of chitosan-based fiber assemblies loaded with bioactive substances:

[0083] Chitosan-based fiber aggregate I was immersed in a solution containing bioactive substance I at a concentration of 10 mg / mL (PBS with pH = 5.5 as solvent). Bioactive substance I was loaded into chitosan-based fiber aggregate I through electrostatic adsorption.

[0084] Chitosan-based fiber aggregate II was immersed in a solution with a concentration of 1×10⁻⁶. 7 In a solution containing bioactive substance II (solvent: PBS pH = 5.5) at a particle / mL concentration, bioactive substance II is loaded into chitosan-based fiber aggregate II via electrostatic adsorption.

[0085] (3) The two layers of chitosan-based fiber assemblies constituting the internal porous functional matrix are laid layer by layer in the order of chitosan-based fiber assembly I and chitosan-based fiber assembly ⅠⅠ. During the laying process, after each layer is laid, a liquid treatment agent is evenly sprayed on the outer surface of the layer using a spraying device. Then, the next layer is laid until the laying is completed, resulting in a stacked chitosan-based fiber assembly. The amount of liquid treatment agent sprayed each time is 0.08 g / cm³. 2 ;

[0086] (4) The stacked chitosan-based fiber aggregates are needle-punched using a needle punching machine; the needle punching process parameters are: needle punching density of 200 needles / cm. 2 The depth is 3cm and the frequency is 1Hz (60 times / min);

[0087] (5) Evenly spray the liquid treatment agent onto the outer surface of the product treated in step (4), then align and overlap it with the pre-prepared outer protective layer and apply a pressure of 0.01 MPa. After maintaining this pressure for 5 minutes, allow it to dry at room temperature to obtain a multi-layered gradient structure chitosan-based dressing; the spraying amount of the liquid treatment agent is 0.04 g / cm³. 2 .

[0088] The final multi-layered gradient chitosan-based dressing is composed of an inner porous functional matrix and an outer protective layer. Chitosan-based fiber assembly I serves as the absorbent layer, and chitosan-based fiber assembly II serves as the contact layer. The peel strength between chitosan-based fiber assembly I and chitosan-based fiber assembly II is 1.5 N / cm, and the peel strength between chitosan-based fiber assembly II and the outer protective layer is 1.2 N / cm. The contact angle of the outer layer of the multi-layered gradient chitosan-based dressing is 75°, and the moisture permeability is 2800 g / (m²). 2 • 24h), absorption capacity is 18g / g; the multi-layer gradient structure chitosan-based dressing has a 48-hour inhibition rate of 96% against Staphylococcus aureus and a 48-hour inhibition rate of 95% against Escherichia coli; blood absorption is 4.2g, and hemostasis time is 45s.

[0089] Comparative Example 1

[0090] A method for preparing a multi-layer gradient chitosan-based dressing is basically the same as in Example 1, except that the solvent is replaced with methanol.

[0091] In the final multi-layered gradient chitosan-based dressing, the chitosan-based fiber assembly II and the outer protective layer, as well as the two chitosan-based fiber assemblies, could not achieve a tight connection through a physical cross-linking network. The peel strength between chitosan-based fiber assembly II and the outer protective layer was 0.3 N / cm, and the peel strength between chitosan-based fiber assembly I and chitosan-based fiber assembly II was 0.7 N / cm.

[0092] Comparing Comparative Example 1 and Example 1, it can be found that the peel strength of Comparative Example 1 is significantly reduced. This is because methanol has insufficient swelling capacity for chitosan on the fiber surface, which cannot form an effective physical cross-linking network and results in poor interfacial bonding.

[0093] Comparative Example 2

[0094] A method for preparing a multi-layer gradient chitosan-based dressing is basically the same as in Example 1, except that the aqueous ethanol solution contains 58 wt% anhydrous ethanol.

[0095] The final multi-layered gradient chitosan-based dressing exhibits a peel strength of 1.1 N / cm between chitosan-based fiber aggregate I and chitosan-based fiber aggregate II, and a peel strength of 1.0 N / cm between chitosan-based fiber aggregate II and the outer protective layer. The multi-layered gradient chitosan-based dressing demonstrates an 85% inhibition rate against Staphylococcus aureus and Escherichia coli within 48 hours. It also shows a blood absorption capacity of 3.5 g and a hemostasis time of 58 seconds.

[0096] Comparing Comparative Example 2 with Example 1, it can be found that the antibacterial performance of Comparative Example 2 is worse and the blood absorption is lower. This is because the anhydrous ethanol content is less than 60 wt%, which is insufficient to protect chitosan. The chitosan fiber structure is excessively corroded and destroyed by the organic acid in the liquid treatment agent, and the surface -NH2 groups are lost due to fiber breakage or dissolution, resulting in a significant decrease in antibacterial ability and hemostatic performance.

[0097] Comparative Example 3

[0098] A method for preparing a multi-layer gradient chitosan-based dressing is basically the same as in Example 1, except that the aqueous ethanol solution contains 92 wt% anhydrous ethanol.

[0099] In the final multi-layered gradient chitosan-based dressing, the peel strength between chitosan-based fiber aggregate I and chitosan-based fiber aggregate II is 0.7 N / cm, and the peel strength between chitosan-based fiber aggregate II and the outer protective layer is 0.5 N / cm. The multi-layered gradient chitosan-based dressing has an antibacterial rate of 82% against Staphylococcus aureus and 80% against Escherichia coli after 48 hours.

[0100] Comparing Comparative Example 3 with Example 1, it can be found that the peel strength and antibacterial properties of Comparative Example 3 are significantly reduced. This is because the high ethanol content partially shields the -NH2 groups of chitosan, weakening its ability to electrostatically adsorb negatively charged bacteria. Furthermore, the movement of chitosan molecular chains is hindered, resulting in a decrease in swelling capacity and interfacial bonding force, which in turn affects the antibacterial properties.

[0101] Comparative Example 4

[0102] A method for preparing a multi-layer gradient chitosan-based dressing is basically the same as in Example 1, except that the mass ratio of solvent to aqueous ethanol solution is 4:100.

[0103] In the final multi-layered gradient chitosan-based dressing, the peel strength between chitosan-based fiber assembly I and chitosan-based fiber assembly II is 0.9 N / cm, and the peel strength between chitosan-based fiber assembly II and the outer protective layer is 0.6 N / cm.

[0104] Comparing Comparative Example 4 with Example 1, it can be found that the peel strength of Comparative Example 4 is weaker. This is because the amount of solvent in the liquid treatment agent is insufficient, the swelling effect is weak, and the interpenetration of the interface molecular chains is insufficient.

[0105] Comparative Example 5

[0106] A method for preparing a multi-layer gradient chitosan-based dressing is basically the same as in Example 1, except that the mass ratio of solvent to aqueous ethanol solution is 11:100.

[0107] The final multi-layered gradient chitosan-based dressing had a moisture permeability of 1600 g / (m²). 2 •24h).

[0108] Comparing Comparative Example 5 with Example 1, it can be found that the moisture permeability of Comparative Example 5 decreased and the chitosan-based fiber aggregate structure collapsed. This is because excessive solvent caused the fiber to dissolve, the pores to become blocked, and the moisture permeability deteriorated accordingly.

[0109] Comparative Example 6

[0110] A method for preparing a multi-layer gradient chitosan-based dressing is basically the same as in Example 1, except that the needling treatment in step (4) is omitted.

[0111] In the final multi-layered gradient chitosan-based dressing, the peel strength between chitosan-based fiber assembly I and chitosan-based fiber assembly II is 0.5 N / cm, and the peel strength between chitosan-based fiber assembly II and the outer protective layer is 1.1 N / cm.

[0112] Comparing Comparative Example 6 with Example 1, it can be found that the bonding force (peel strength) between chitosan-based fiber aggregate I and chitosan-based fiber aggregate II in Comparative Example 6 is significantly reduced. The dressing is prone to delamination during use. This is because the needle punching process is omitted, and the key role of macroscopic mechanical interlocking (fiber entanglement) is lacking. Relying only on weak spray adhesion and possible limited physical cross-linking, a strong overall structure cannot be formed, resulting in a serious lack of interlayer bonding strength.

[0113] Comparative Example 7

[0114] A method for preparing a multi-layer gradient chitosan-based dressing is basically the same as in Example 1, except that: no liquid treatment agent is sprayed in step (3).

[0115] In the final multi-layered gradient chitosan-based dressing, the peel strength between chitosan-based fiber assembly I and chitosan-based fiber assembly II is 0.4 N / cm, and the peel strength between chitosan-based fiber assembly II and the outer protective layer is 0.3 N / cm.

[0116] Comparing Comparative Example 7 with Example 1, it can be found that the interlayer bonding force (peel strength) of Comparative Example 7 is almost completely lost. The layers of the dressing are only physically stacked and are very easy to separate. This is because no liquid treatment agent was sprayed in step (3), resulting in the lack of the key "interfacial molecular interpenetration fusion" process between the internal porous functional matrix and the external protective layer. Without the activation effect of the liquid treatment agent on the matrix surface, the chitosan molecular chains cannot diffuse and entangle with each other, and cannot form a strong physical bonding interface. As a result, the two layers of materials are only bonded by weak van der Waals forces and are very easy to delaminate and fall off.

[0117] Example 2

[0118] A method for preparing a multi-layered gradient chitosan-based dressing, the specific steps of which are as follows:

[0119] (1) Preparation of raw materials:

[0120] Organic acid: acetic acid;

[0121] Solvent: Trifluoroethanol, with an organic acid content of 30 wt% in the solvent;

[0122] Liquid treatment agent: a mixture of solvent and aqueous ethanol solution; the aqueous ethanol solution contains 80 wt% anhydrous ethanol; the mass ratio of solvent to aqueous ethanol solution is 8:100;

[0123] Chitosan-based fiber aggregate I: Quaternized chitosan needle-punched nonwoven fabric, basis weight 250 g / m² 2 Thickness 2.5mm;

[0124] Chitosan-based fiber aggregate II: Pure chitosan fiber needle-punched nonwoven fabric, weight 120 g / m² 2 Thickness 1.2mm, pore size 70μm;

[0125] Chitosan-based fiber aggregate III: Carboxyethyl chitosan fiber needle-punched nonwoven fabric, 1.5 mm thick, 150 g / m² weight. 2 ;

[0126] Bioactive Substance I: Astragalus Polysaccharide: Shaanxi Jiahe Biotechnology, AGP-01;

[0127] Bioactive Substance II: Sodium Heparin: Sigma-Aldrich;

[0128] Bioactive Substance III: Collagen, Sigma-Aldrich, C9879;

[0129] External protective layer material: silanized chitosan electrospun nanofiber membrane, basis weight 30 g / m 2 Thickness 50μm;

[0130] (2) Preparation of chitosan-based fiber assemblies loaded with bioactive substances:

[0131] Chitosan-based fiber aggregate I was immersed in a solution containing bioactive substance I at a concentration of 5 mg / mL (PBS with pH = 5.5) and bioactive substance I was loaded into chitosan-based fiber aggregate I through electrostatic adsorption.

[0132] Chitosan-based fiber aggregate II was immersed in a solution containing bioactive substance II at a concentration of 8 mg / mL (PBS with pH = 5.5 as solvent). Bioactive substance II was loaded into chitosan-based fiber aggregate II through electrostatic adsorption.

[0133] Chitosan-based fiber aggregate III was immersed in a solution containing bioactive substance III at a concentration of 5 mg / mL (PBS with pH = 5.5 as solvent). Bioactive substance III was loaded into chitosan-based fiber aggregate III through electrostatic adsorption.

[0134] (3) The three-layer chitosan-based fiber assembly constituting the internal porous functional matrix is ​​laid layer by layer in the order of chitosan-based fiber assembly I, chitosan-based fiber assembly ⅠⅠ, and chitosan-based fiber assembly III. During the laying process, after each layer is laid, a liquid treatment agent is evenly sprayed on the outer surface of the layer using a spraying device. Then, the next layer is laid until the laying is completed, resulting in a stacked chitosan-based fiber assembly. The amount of liquid treatment agent sprayed each time is 0.08 g / cm³. 2 ;

[0135] (4) The stacked chitosan-based fiber aggregates are needle-punched using a needle punching machine; the needle punching process parameters are: needle punching density of 180 needles / cm. 2 The depth is 2.5cm, and the frequency is 0.8Hz (50 times / min);

[0136] (5) Evenly spray the liquid treatment agent onto the outer surface of the product treated in step (4), then align and overlap it with the pre-prepared outer protective layer and apply a pressure of 0.01 MPa. After maintaining this pressure for 5 minutes, allow it to dry at room temperature to obtain a multi-layered gradient structure chitosan-based dressing; the spraying amount of the liquid treatment agent is 0.04 g / cm³. 2 .

[0137] The final multi-layered gradient chitosan-based dressing is composed of an inner porous functional matrix and an outer protective layer. Chitosan-based fiber assembly I serves as the liquid-locking layer, chitosan-based fiber assembly II as the flow-guiding layer, and chitosan-based fiber assembly III as the contact layer. The peel strength between chitosan-based fiber assembly I and chitosan-based fiber assembly II is 1.6 N / cm, the peel strength between chitosan-based fiber assembly II and chitosan-based fiber assembly III is 1.7 N / cm, and the peel strength between chitosan-based fiber assembly III and the outer protective layer is 1.3 N / cm. The contact angle of the outer layer of the multi-layered gradient chitosan-based dressing is 72°, and the moisture permeability is 2650 g / (m²). 2 • 24h), absorption capacity is 25g / g; the multi-layer gradient structure chitosan-based dressing has a 48-hour inhibition rate of 99% against Staphylococcus aureus and 98-hour inhibition rate against Escherichia coli; blood absorption is 5g, and hemostasis time is 38s.

[0138] Example 3

[0139] A method for preparing a multi-layered gradient chitosan-based dressing, the specific steps of which are as follows:

[0140] (1) Preparation of raw materials:

[0141] Organic acid: acetic acid;

[0142] Solvent: Trifluoroethanol, with an organic acid content of 30 wt% in the solvent;

[0143] Liquid treatment agent: a mixture of solvent and aqueous ethanol solution; the aqueous ethanol solution contains 80 wt% anhydrous ethanol; the mass ratio of solvent to aqueous ethanol solution is 8:100;

[0144] Chitosan-based fiber aggregate I: Pure chitosan fiber needle-punched nonwoven fabric, weight 200 g / m² 2 Thickness 2mm;

[0145] Chitosan-based fiber aggregate II: Pure chitosan fiber needle-punched nonwoven fabric, weight 120 g / m² 2 Thickness 1.2mm;

[0146] Chitosan-based fiber aggregate III: Pure chitosan fiber needle-punched nonwoven fabric, weight 120 g / m² 2 Thickness 1.2mm;

[0147] Chitosan-based fiber aggregate IV: Carboxymethyl chitosan fiber needle-punched nonwoven fabric, 1.5 mm thick, 150 g / m² weight. 2 ;

[0148] Bioactive Substance I: Sodium Heparin: Sigma-Aldrich;

[0149] Bioactive substance II: Exosomes derived from mesenchymal stem cells (extraction method refers to patent CN 110252440 A an exosome filtration extraction device and exosome extraction method);

[0150] Bioactive Substance III: Chondroitin Sulfate: Sigma, extracted from shark cartilage;

[0151] Bioactive Substance IV: Sodium Heparin: Sigma-Aldrich;

[0152] External protective layer material: silanized chitosan electrospun nanofiber membrane, basis weight 30 g / m 2 Thickness 50μm;

[0153] (2) Preparation of chitosan-based fiber assemblies loaded with bioactive substances:

[0154] Chitosan-based fiber aggregate I was immersed in a solution containing bioactive substance I at a concentration of 15 mg / mL (PBS with pH = 5.5 as solvent). Bioactive substance I was loaded into chitosan-based fiber aggregate I through electrostatic adsorption.

[0155] Chitosan-based fiber aggregate II was immersed in a solution with a concentration of 1×10⁻⁶. 7In a solution containing bioactive substance II (solvent: PBS pH = 5.5) at a particle / mL concentration, bioactive substance II is loaded into chitosan-based fiber aggregate II via electrostatic adsorption.

[0156] Chitosan-based fiber aggregate III was immersed in a solution containing bioactive substance III at a concentration of 15 mg / mL (PBS with pH = 5.5 as solvent). Bioactive substance III was loaded into chitosan-based fiber aggregate III through electrostatic adsorption.

[0157] Chitosan-based fiber aggregates IV were immersed in a solution containing bioactive substance IV at a concentration of 8 mg / mL (PBS with pH = 5.5 as solvent). Bioactive substance IV was loaded into chitosan-based fiber aggregates III through electrostatic adsorption.

[0158] (3) The four-layer chitosan-based fiber assembly constituting the internal porous functional matrix is ​​laid layer by layer in the order of chitosan-based fiber assembly I, chitosan-based fiber assembly ⅠⅠ, chitosan-based fiber assembly III, and chitosan-based fiber assembly IV. During the laying process, after each layer is laid, a liquid treatment agent is evenly sprayed on the outer surface of the layer using a spraying device. Then, the next layer is laid until the entire layer is laid, resulting in a stacked chitosan-based fiber assembly. The amount of liquid treatment agent sprayed each time is 0.07 g / cm³. 2 ;

[0159] (4) The stacked chitosan-based fiber aggregates are needle-punched using a needle punching machine; the needle punching process parameters are: needle punching density of 200 needles / cm. 2 The depth is 3cm and the frequency is 0.9Hz (55 times / min);

[0160] (5) Evenly spray the liquid treatment agent onto the outer surface of the product treated in step (4), then align and overlap it with the pre-prepared outer protective layer and apply a pressure of 0.01 MPa. After maintaining this pressure for 5 minutes, allow it to dry at room temperature to obtain a multi-layered gradient structure chitosan-based dressing; the spraying amount of the liquid treatment agent is 0.03 g / cm³. 2 .

[0161] The final multi-layered gradient chitosan-based dressing is composed of an inner porous functional matrix and an outer protective layer. Chitosan-based fiber assembly I serves as a supporting reservoir layer, chitosan-based fiber assembly II as a proliferation-promoting layer, chitosan-based fiber assembly III as an anti-inflammatory regulating layer, and chitosan-based fiber assembly IV as a contact layer. The peel strength between chitosan-based fiber assembly I and chitosan-based fiber assembly II is 1.4 N / cm, between chitosan-based fiber assembly II and chitosan-based fiber assembly III is 1.6 N / cm, between chitosan-based fiber assembly III and chitosan-based fiber assembly IV is 1.8 N / cm, and between chitosan-based fiber assembly IV and the outer protective layer is 1.1 N / cm. The contact angle of the outer layer of the multi-layered gradient chitosan-based dressing is 68°, and the moisture permeability is 2950 g / (m²). 2 • 24h), absorption capacity is 22g / g; the multi-layer gradient structure chitosan-based dressing has a 48-hour inhibition rate of 97% against Staphylococcus aureus and a 48-hour inhibition rate of 96% against Escherichia coli; blood absorption is 4.5g, and hemostasis time is 42s.

[0162] Example 4

[0163] A method for preparing a multi-layered gradient chitosan-based dressing, the specific steps of which are as follows:

[0164] (1) Preparation of raw materials:

[0165] Organic acid: acetic acid;

[0166] Solvent: A mixture of chloroform and tetrahydrofuran with organic acid in a mass ratio of 2:3, wherein the organic acid content in the solvent is 45 wt%.

[0167] Liquid treatment agent: a mixture of solvent and aqueous ethanol solution; the aqueous ethanol solution contains 90 wt% anhydrous ethanol; the mass ratio of solvent to aqueous ethanol solution is 5:100;

[0168] Chitosan-based fiber aggregate I: Carboxyethyl chitosan needle-punched nonwoven fabric, weight 150 g / m² 2 Thickness 1.5mm, pore size 50μm;

[0169] Chitosan-based fiber aggregate II: Pure chitosan fiber needle-punched nonwoven fabric, weight 250 g / m² 2 Thickness 2.5mm;

[0170] Bioactive Substance I: Sodium Heparin: Sigma-Aldrich;

[0171] Bioactive substance II: Human basic fibroblast growth factor bFGF (manufacturer: StemRD, brand name: bFGF-250);

[0172] External protective layer material: pure chitosan fiber spunlace nonwoven fabric, weight 80g / m² 2 Thickness 0.3mm;

[0173] (2) Preparation of chitosan-based fiber assemblies loaded with bioactive substances:

[0174] Chitosan-based fiber aggregate I was immersed in a solution containing bioactive substance I at a concentration of 8 mg / mL (PBS with pH = 5.5 as solvent). Bioactive substance I was loaded into chitosan-based fiber aggregate I through electrostatic adsorption.

[0175] Chitosan-based fiber aggregate II was immersed in a solution containing bioactive substance II at a concentration of 100 μg / mL (the solvent was PBS solution containing 0.1 wt% BSA, pH = 7.2, and the BSA was from Sigma Aldrich B2064). Bioactive substance II was loaded into chitosan-based fiber aggregate II through electrostatic adsorption.

[0176] (3) The two layers of chitosan-based fiber assemblies constituting the internal porous functional matrix are laid layer by layer in the order of chitosan-based fiber assembly I and chitosan-based fiber assembly ⅠⅠ. During the laying process, after each layer is laid, a liquid treatment agent is evenly sprayed on the outer surface of the layer using a spraying device. Then, the next layer is laid until the laying is completed, resulting in a stacked chitosan-based fiber assembly. The amount of liquid treatment agent sprayed each time is 0.05 g / cm³. 2 ;

[0177] (4) The stacked chitosan-based fiber aggregates are needle-punched using a needle punching machine; the needle punching process parameters are: needle punching density of 210 needles / cm. 2 The depth is 3.3 cm and the frequency is 1.2 Hz (70 times / min);

[0178] (5) Evenly spray the liquid treatment agent onto the outer surface of the product treated in step (4), then align and overlap it with the pre-prepared outer protective layer and apply a pressure of 0.01 MPa. After maintaining this pressure for 5 minutes, allow it to dry at room temperature to obtain a multi-layered gradient structure chitosan-based dressing; the spraying amount of the liquid treatment agent is 0.02 g / cm³. 2 .

[0179] The final multi-layered gradient chitosan-based dressing is composed of an inner porous functional matrix and an outer protective layer. Chitosan-based fiber assembly I serves as a hydrophilic guiding layer, and chitosan-based fiber assembly II serves as a contact layer. The peel strength between chitosan-based fiber assembly I and chitosan-based fiber assembly II is 1.8 N / cm, and the peel strength between chitosan-based fiber assembly III and the outer protective layer is 1.2 N / cm. The contact angle of the outer layer of the multi-layered gradient chitosan-based dressing is 70°, and the moisture permeability is 2550 g / (m²). 2 • 24h), absorption capacity is 20g / g; the multi-layer gradient structure chitosan-based dressing has a 48-hour inhibition rate of 95% against Staphylococcus aureus and a 48-hour inhibition rate of 94% against Escherichia coli; blood absorption is 4g, and hemostasis time is 48s.

[0180] Example 5

[0181] A method for preparing a multi-layered gradient chitosan-based dressing, the specific steps of which are as follows:

[0182] (1) Preparation of raw materials:

[0183] Organic acid: formic acid;

[0184] Solvent: A mixture of toluene and acetone in a mass ratio of 3:7 with organic acids, wherein the organic acid content in the solvent is 50 wt%;

[0185] Liquid treatment agent: a mixture of solvent and aqueous ethanol solution; the aqueous ethanol solution contains 75 wt% anhydrous ethanol; the mass ratio of solvent to aqueous ethanol solution is 9:100;

[0186] Chitosan-based fiber aggregate I: Quaternized chitosan needle-punched nonwoven fabric, basis weight 250 g / m² 2 Thickness 2.5mm;

[0187] Chitosan-based fiber aggregate II: Pure chitosan fiber needle-punched nonwoven fabric, weight 120 g / m² 2 Thickness 1.2mm;

[0188] Chitosan-based fiber aggregate III: Thiohydrin-modified chitosan fibers, 120 g / m² 2 Thickness 1.2mm;

[0189] Bioactive Substance I: Human Basic Fibroblast Growth Factor bFGF (Manufacturer: StemRD, Brand: bFGF-250);

[0190] Bioactive substance II: VEGF, manufacturer: Sino Biological, brand name: VEGF165;

[0191] Bioactive Substance III: Chondroitin Sulfate: Sigma, extracted from shark cartilage;

[0192] External protective layer material: silanized chitosan electrospun nanofiber membrane, basis weight 30 g / m 2 Thickness 50μm;

[0193] (2) Preparation of chitosan-based fiber assemblies loaded with bioactive substances:

[0194] Chitosan-based fiber aggregate I was immersed in a solution containing bioactive substance I at a concentration of 100 μg / mL (the solvent was PBS solution containing 0.1 wt% BSA, pH = 7.2, and the BSA was from Sigma Aldrich B2064). Bioactive substance I was loaded into chitosan-based fiber aggregate I through electrostatic adsorption.

[0195] Chitosan-based fiber aggregate II was immersed in a solution containing bioactive substance II at a concentration of 100 μg / mL (PBS with pH = 5.5 as solvent). Bioactive substance II was loaded into chitosan-based fiber aggregate II through electrostatic adsorption.

[0196] Chitosan-based fiber aggregate III was immersed in a solution containing bioactive substance III at a concentration of 15 mg / mL (PBS with pH = 5.5 as solvent). Bioactive substance III was loaded into chitosan-based fiber aggregate III through electrostatic adsorption.

[0197] (3) The three-layer chitosan-based fiber assembly constituting the internal porous functional matrix is ​​laid layer by layer in the order of chitosan-based fiber assembly I, chitosan-based fiber assembly ⅠⅠ, and chitosan-based fiber assembly III. During the laying process, after each layer is laid, a liquid treatment agent is evenly sprayed on the outer surface of the layer using a spraying device. Then, the next layer is laid until the laying is completed, resulting in a stacked chitosan-based fiber assembly. The amount of liquid treatment agent sprayed each time is 0.09 g / cm³. 2 ;

[0198] (4) The stacked chitosan-based fiber aggregates are needle-punched using a needle punching machine; the needle punching process parameters are: needle punching density of 205 needles / cm². 2 The depth is 3.5cm and the frequency is 1.2Hz (70 times / min);

[0199] (5) Evenly spray the liquid treatment agent onto the outer surface of the product treated in step (4), then align and overlap it with the pre-prepared outer protective layer and apply a pressure of 0.01 MPa. After maintaining this pressure for 5 minutes, allow it to dry at room temperature to obtain a multi-layered gradient structure chitosan-based dressing; the spraying amount of the liquid treatment agent is 0.04 g / cm³. 2 .

[0200] The final multi-layered gradient chitosan-based dressing is composed of an inner porous functional matrix and an outer protective layer. Chitosan-based fiber aggregate I serves as the tissue remodeling layer, chitosan-based fiber aggregate II as the proliferation-promoting layer, and chitosan-based fiber aggregate III as the anti-inflammatory regulatory layer. The peel strength between chitosan-based fiber aggregate I and chitosan-based fiber aggregate II is 1.7 N / cm, the peel strength between chitosan-based fiber aggregate II and chitosan-based fiber aggregate III is 1.6 N / cm, and the peel strength between chitosan-based fiber aggregate III and the outer protective layer is 1.2 N / cm. The contact angle of the outer layer of the multi-layered gradient chitosan-based dressing is 65°, and the moisture permeability is 2650 g / (m²). 2 • 24h), absorption capacity is 23g / g; the multi-layer gradient structure chitosan-based dressing has a 48-hour inhibition rate of 98% against Staphylococcus aureus and a 48-hour inhibition rate of 97% against Escherichia coli; blood absorption is 4.8g, and hemostasis time is 40s.

[0201] Example 6

[0202] A method for preparing a multi-layered gradient chitosan-based dressing, the specific steps of which are as follows:

[0203] (1) Preparation of raw materials:

[0204] Organic acids: acetic acid and formic acid in a 1:1 mass ratio;

[0205] Solvent: A mixture of tetrahydrofuran and dichloromethane with organic acid in a mass ratio of 1:4, wherein the organic acid content in the solvent is 35wt%;

[0206] Liquid treatment agent: a mixture of solvent and aqueous ethanol solution; the aqueous ethanol solution contains 60 wt% anhydrous ethanol; the mass ratio of solvent to aqueous ethanol solution is 7:100;

[0207] Chitosan-based fiber aggregate I: Pure chitosan fiber needle-punched nonwoven fabric, weight 200 g / m² 2 Thickness 2mm;

[0208] Chitosan-based fiber aggregate II: Pure chitosan fiber needle-punched nonwoven fabric, weight 120 g / m² 2 Thickness 1.2mm;

[0209] Chitosan-based fiber aggregate III: Sulfonated polysaccharide needle-punched nonwoven fabric, 200 g / m² 2 Thickness 1.5mm;

[0210] Chitosan-based fiber aggregate IV: Carboxymethyl chitosan fiber needle-punched nonwoven fabric, 1.5 mm thick, 150 g / m² weight. 2The chitosan-based fibers have a diameter of 1.5 dtex and a length of 38 mm.

[0211] Bioactive substance I: Sodium alginate: DuPont, MANUCOL DH, G / M = 40 / 60;

[0212] Bioactive substance II: Exosomes derived from mesenchymal stem cells (extraction method refers to patent CN 110252440 A an exosome filtration extraction device and exosome extraction method);

[0213] Bioactive Substance III: Astragalus Polysaccharide: Shaanxi Jiahe Biotechnology, AGP-01;

[0214] Bioactive substance IV: Collagen: Sigma-Aldrich, C9879;

[0215] External protective layer material: silanized chitosan electrospun nanofiber membrane, basis weight 30 g / m 2 Thickness 50μm;

[0216] (2) Preparation of chitosan-based fiber assemblies loaded with bioactive substances:

[0217] Chitosan-based fiber aggregate I was immersed in a solution containing bioactive substance I at a concentration of 15 mg / mL (PBS with pH = 5.5 as solvent). Bioactive substance I was loaded into chitosan-based fiber aggregate I through electrostatic adsorption.

[0218] Chitosan-based fiber aggregate II was immersed in a solution with a concentration of 1×10⁻⁶. 7 In a solution containing bioactive substance II (solvent: PBS pH = 5.5) at a particle / mL concentration, bioactive substance II is loaded into chitosan-based fiber aggregate II via electrostatic adsorption.

[0219] Chitosan-based fiber aggregate III was immersed in a solution containing bioactive substance III at a concentration of 15 mg / mL (PBS with pH = 5.5 as solvent). Bioactive substance III was loaded into chitosan-based fiber aggregate III through electrostatic adsorption.

[0220] Chitosan-based fiber aggregate IV was immersed in a solution containing bioactive substance IV at a concentration of 5 mg / mL (PBS with pH = 5.5 as solvent). Bioactive substance IV was loaded into chitosan-based fiber aggregate IV through electrostatic adsorption.

[0221] (3) The four-layer chitosan-based fiber assembly constituting the internal porous functional matrix is ​​laid layer by layer in the order of chitosan-based fiber assembly I, chitosan-based fiber assembly ⅠⅠ, chitosan-based fiber assembly III, and chitosan-based fiber assembly IV. During the laying process, after each layer is laid, a liquid treatment agent is evenly sprayed on the outer surface of the layer using a spraying device. Then, the next layer is laid until the entire layer is laid, resulting in a stacked chitosan-based fiber assembly. The amount of liquid treatment agent sprayed each time is 0.06 g / cm³. 2 ;

[0222] (4) The stacked chitosan-based fiber aggregates are needle-punched using a needle punching machine; the needle punching process parameters are: needle punching density of 220 needles / cm². 2 The depth is 2.5cm, and the frequency is 0.8Hz (50 times / min);

[0223] (5) Evenly spray the liquid treatment agent onto the outer surface of the product treated in step (4), then align and overlap it with the pre-prepared outer protective layer and apply a pressure of 0.01 MPa. After maintaining this pressure for 5 minutes, allow it to dry at room temperature to obtain a multi-layered gradient structure chitosan-based dressing; the spraying amount of the liquid treatment agent is 0.03 g / cm³. 2 .

[0224] The final multi-layered gradient chitosan-based dressing is composed of an inner porous functional matrix and an outer protective layer. Chitosan-based fiber assembly I serves as a supporting reservoir layer, chitosan-based fiber assembly II as a proliferation-promoting layer, chitosan-based fiber assembly III as an anti-inflammatory regulating layer, and chitosan-based fiber assembly IV as a contact layer. The peel strength between chitosan-based fiber assembly I and chitosan-based fiber assembly II is 1.6 N / cm, between chitosan-based fiber assembly II and chitosan-based fiber assembly III is 1.7 N / cm, between chitosan-based fiber assembly III and chitosan-based fiber assembly IV is 1.6 N / cm, and between chitosan-based fiber assembly IV and the outer protective layer is 1.1 N / cm. The contact angle of the outer layer of the multi-layered gradient chitosan-based dressing is 68°, and the moisture permeability is 2750 g / (m²). 2 • 24h), absorption capacity is 19g / g; the multi-layer gradient structure chitosan-based dressing has a 48-hour inhibition rate of 96% against Staphylococcus aureus and a 48-hour inhibition rate of 95% against Escherichia coli; blood absorption is 4.3g, and hemostasis time is 44s.

Claims

1. A multi-layered gradient chitosan-based dressing, characterized in that: It is composed of an internal porous functional matrix and an external protective layer; The internal porous functional matrix comprises at least two layers of chitosan-based fiber assemblies, and the adjacent two layers of chitosan-based fiber assemblies are composited by spraying a liquid treatment agent followed by needle punching and drying. The composite of the internal porous functional matrix and the external protective layer is achieved by spraying a liquid treatment agent onto the outer surface of the internal porous functional matrix and then drying it. The liquid treatment agent is a mixture of a solvent and an aqueous ethanol solution. The solvent is a mixture of one or more of trifluoroethanol, hexafluoroisopropanol, dichloromethane, chloroform, toluene, tetrahydrofuran, and acetone with an organic acid, wherein the organic acid is one or more of acetic acid and formic acid. The aqueous ethanol solution contains 60–90 wt% anhydrous ethanol; The mass ratio of solvent to aqueous ethanol solution is 5–10:100; The outer protective layer is made of hydrophobically modified chitosan-based material.

2. The multi-layered gradient structure chitosan-based dressing according to claim 1, characterized in that, Chitosan-based fiber aggregates refer to chitosan-based needle-punched nonwoven fabrics obtained by needle-punching nonwoven fabrics of one or two chitosan-based fibers; chitosan-based fibers refer to pure chitosan fibers, silanized chitosan fibers, carboxymethyl chitosan fibers, carboxyethyl chitosan fibers, quaternized chitosan fibers, acylated modified chitosan fibers, thiolated chitosan fibers, or sulfonated chitosan fibers.

3. The multi-layered gradient structure chitosan-based dressing according to claim 2, characterized in that, The thickness of the chitosan-based fiber aggregate is 1–10 mm, and the basis weight is 100–300 g / m². 2 The chitosan-based fibers have a diameter of 1.2–3.6 dtex and a length of 30–60 mm.

4. The multi-layered gradient structure chitosan-based dressing according to claim 1, characterized in that, At least one layer of chitosan-based fiber assembly is a chitosan-based fiber assembly loaded with bioactive substances. The preparation process of the chitosan-based fiber assembly loaded with bioactive substances is as follows: the chitosan-based fiber assembly is immersed in a solution containing bioactive substances, and the bioactive substances are loaded in the chitosan-based fiber assembly through electrostatic adsorption; the bioactive substances are negatively charged bioactive substances.

5. The multi-layered gradient structure chitosan-based dressing according to claim 1, characterized in that, The hydrophobically modified chitosan-based material is a chitosan-based nanofiber membrane or a chitosan-based spunlace nonwoven fabric; the thickness of the chitosan-based nanofiber membrane is 20–100 μm; the thickness of the chitosan-based spunlace nonwoven fabric is 0.2–0.5 mm, and the basis weight is 40–150 g / m². 2 .

6. The multi-layered gradient structure chitosan-based dressing according to claim 1, characterized in that, The solvent contains 30–50 wt% organic acids.

7. The multi-layered gradient structure chitosan-based dressing according to claim 1, characterized in that, In multi-layered gradient chitosan-based dressings, the peel strength between any two adjacent layers is ≥1.0 N / cm.

8. A method for preparing a multi-layered gradient chitosan-based dressing according to any one of claims 1 to 7, characterized in that... Includes the following steps: (1) At least two layers of chitosan-based fiber assemblies constituting the internal porous functional matrix are laid layer by layer in a preset order. During the laying process, after each layer is laid, a liquid treatment agent is evenly sprayed on the outer surface of the layer, and then the next layer is laid until the laying is completed, and the stacked chitosan-based fiber assemblies are obtained. (2) The stacked chitosan-based fiber aggregates are needle-punched using a needle punching machine; (3) Spray the liquid treatment agent evenly on the outer surface of the product after the needle punching treatment in step (2), then align and overlap it with the pre-prepared outer protective layer and apply pressure. After drying at room temperature, a multi-layer gradient structure chitosan-based dressing is obtained.

9. The method for preparing a multi-layered gradient structure chitosan-based dressing according to claim 8, characterized in that, In step (1), the amount of liquid treatment agent sprayed each time is ≤0.1g / cm³. 2 .

10. The method for preparing a multi-layered gradient structure chitosan-based dressing according to claim 8, characterized in that, In step (3), the spraying rate of the liquid treatment agent is ≤0.05 g / cm³. 2 .

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