An ico-dextrin-containing wound dressing and a method for preparing the same

Abstract

The application discloses an ectoine-containing wound dressing and a preparation method thereof, and belongs to the technical field of medical dressing preparation.The ectoine-containing wound dressing comprises the following components in proportion by weight: HNTs@IPA, sodium alginate, oligomeric proanthocyanidin, N-isopropyl acrylamide, acrylic acid, ectoine and deionized water; wherein the HNTs@IPA is obtained by loading isopropyl alcohol on halloysite nanotubes.The ectoine in-situ crystallization and long-acting slow release are realized by loading isopropyl alcohol on halloysite nanotubes, the traditional glutaraldehyde is replaced by oligomeric proanthocyanidin as a crosslinking agent, the wound dressing with excellent mechanical properties is prepared, and the tensile strength, toughness and biological safety of the dressing are significantly improved, so that the wound dressing can be used for repairing complex wounds.

Description

Technical Field

[0001] This invention relates to the field of medical dressing preparation technology, specifically to a wound dressing containing ectoine and its preparation method. Background Technology

[0002] Modern wound repair theory has long since moved beyond the traditional concept of dry healing, shifting towards the theory of moist healing. This theory states that in an ideal environment that is moist, slightly acidic, low-oxygen, and sterile, wound healing speed can be increased by approximately 40%. This environment effectively prevents scab formation and tissue dehydration and necrosis, while promoting the migration, proliferation, and differentiation of epidermal cells, accelerating granulation tissue growth, and significantly reducing patient pain. Based on this theory, the research and development of functional wound dressings has become a cutting-edge hot topic in the field of biomedical materials. Ideal modern dressings not only need to possess excellent biocompatibility, water absorption / retention capacity, and gas permeability, but should also be able to actively participate in and regulate the healing process.

[0003] Ectoine, a potent and biocompatible solute derived from extremophiles, has shown great potential in skin repair in recent years. Its unique molecular structure allows it to form a stable hydration protective shell with water molecules, providing not only extreme hydration to wounds and stabilizing cell membranes and biomolecular structures, but also effectively inhibiting the release of inflammatory factors and resisting secondary damage to damaged cells from the external environment. Therefore, developing a wound dressing with ectoine as its core active ingredient is an important direction in current wound repair research.

[0004] However, existing technologies still face many challenges and shortcomings in developing wound dressings containing active ingredients such as ectoine. Firstly, regarding the loading and release control of the active ingredient, many preparation methods simply incorporate ectoine into the dressing matrix through physical blending. This approach results in weak binding between ectoine and the matrix material. Once in contact with wound exudate, the active ingredient is released rapidly in large quantities within a short period, producing a burst release effect. This not only wastes the active ingredient and shortens the effective action time of the dressing, but may also lead to excessively high local concentrations, failing to achieve optimal therapeutic effects and failing to achieve sustained or controlled release that matches the wound healing cycle. Secondly, the performance of the dressing substrate itself needs improvement. Many hydrogel dressings, while possessing good moisturizing properties, generally have poor mechanical strength. After absorbing exudate, they are prone to swelling and rupture, making it difficult to maintain their integrity and causing inconvenience for clinical use and replacement.

[0005] Existing technology CN118045217B discloses a wound repair dressing containing ectoine and its preparation method. This invention combines ectoine with natural polymer materials such as chitosan and sodium alginate to prepare hydrogels or freeze-dried sponges. While this preparation method utilizes the biocompatibility of natural materials, the controlled-release mechanism of ectoine is relatively simple, mainly relying on physical embedding, making it difficult to avoid burst release effects, thus limiting the utilization rate and long-term effectiveness of ectoine. Furthermore, it does not adequately consider the mechanical properties, fatigue resistance, and fit and stability of the dressing in complex wounds, limiting its application potential and effectiveness in broader clinical scenarios. Therefore, developing a wound dressing that can achieve long-term, controllable release of ectoine while possessing excellent mechanical properties remains a pressing technical challenge in this field. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention discloses a wound dressing containing ectoine and its preparation method, the details of which are as follows.

[0007] This invention protects a wound dressing containing ectoine, comprising the following components by weight: 5-15 parts HNTs@IPA, 15-25 parts sodium alginate, 1-3 parts oligomeric proanthocyanidins, 80-120 parts N-isopropylacrylamide, 3-7 parts acrylic acid, 40-60 parts ectoine, and 800-1000 parts deionized water; The HNTs@IPA is obtained by loading isopropanol onto halloysite nanotubes.

[0008] Preferably, the ectoine-containing wound dressing comprises the following components by weight: 8-12 parts HNTs@IPA, 18-22 parts sodium alginate, 2 parts oligomeric proanthocyanidins, 90-110 parts N-isopropylacrylamide, 4-6 parts acrylic acid, 45-55 parts ectoine, and 850-950 parts deionized water.

[0009] Preferably, the ectoine-containing wound dressing comprises the following components by weight: 10 parts HNTs@IPA, 20 parts sodium alginate, 2 parts oligomeric proanthocyanidins, 100 parts N-isopropylacrylamide, 5 parts acrylic acid, 50 parts ectoine, and 900 parts deionized water.

[0010] Preferably, the HNTs@IPA is prepared by the following method: halloysite nanotubes are uniformly dispersed in isopropanol, centrifuged at 8000 rpm for 10 min, and the precipitate is collected to obtain HNTs@IPA.

[0011] This invention also protects the preparation method of the above-mentioned ectoine-containing wound dressing, which specifically includes the following steps: Step 1: Dissolve sodium alginate in deionized water, add oligomeric proanthocyanidins powder, stir and mix evenly at 40°C in the dark, add N-isopropylacrylamide, acrylic acid, ectoine and HNTs@IPA, stir and mix evenly, transfer to a mold, add ammonium persulfate and TEMED under nitrogen protection, seal and let stand at 25°C for 6 hours to obtain ectoine-containing gel; Step 2: Immerse the ectoine-containing wound gel in a calcium chloride aqueous solution for 2 hours, then remove it and place it in deionized water. Change the water every 4 hours and continue washing for 48 hours. After removing it, freeze it at -20℃ for 12 hours, then freeze-dry it, and then perform vacuum swing adsorption drying to obtain the ectoine-containing wound dressing.

[0012] Preferably, the weight of the ammonium persulfate is 1% to 2% of N-isopropylacrylamide.

[0013] Preferably, the weight of TEMED is 10% to 15% of ammonium persulfate.

[0014] Preferably, the concentration of the calcium chloride aqueous solution is 0.2M.

[0015] Preferably, the specific conditions for freeze-drying are: temperature -50℃, pressure less than 10Pa, and freeze-drying time of 48h.

[0016] Preferably, the specific operation of the vacuum oscillation adsorption drying is as follows: evacuate to 10 Pa and maintain for 5 min, then fill with high-purity argon gas to 80 kPa and maintain for 5 min, and repeat this cycle 100 times.

[0017] The present invention has the following beneficial effects: (1) In the process of preparing ectoine-containing wound dressings, during the formation of P(NIPAM-co-AA), the strong hydrophilicity of the P(NIPAM-co-AA) molecular chain changes the solvent environment and induces in-situ nucleation and crystallization of ectoine. During the process, these ectoine crystals no longer simply diffuse, but dissolve first and then diffuse. The crystals limit the release rate of ectoine through slow dissolution, thus achieving long-term release of ectoine. At the same time, as a reinforcing filler, the ectoine crystals are uniformly dispersed in the dressing matrix, which significantly improves the tensile strength and toughness of the dressing.

[0018] (2) In this invention, isopropanol is loaded onto halloysite nanotubes. In the early stage of P(NIPAM-co-AA) polymerization, the slow release of isopropanol maintains the local high solubility of ectoine around HNTs@IPA, preventing premature crystallization. As polymerization proceeds and isopropanol volatilizes, the supersaturation of the system increases gradually, and ectoine slowly nucleates and crystallizes on the surface of HNTs@IPA, ensuring the uniformity of ectoine crystal size.

[0019] (3) This invention uses oligomeric proanthocyanidins instead of traditional glutaraldehyde as a crosslinking agent, thus avoiding the cytotoxicity of glutaraldehyde. The oligomeric proanthocyanidin molecular chain has a large number of phenolic hydroxyl groups, which can form a large number of hydrogen bonds with the carboxyl and hydroxyl groups on the alginate molecular chain and the carboxyl group on P (NIPAM-co-AA), thereby crosslinking the two molecular chains through hydrogen bonds, improving the energy dissipation capacity of the two molecular chains, and improving the tear resistance of the dressing. At the same time, as a powerful antioxidant, oligomeric proanthocyanidins are slowly released during the dressing process, which can protect the wound tissue and ectoine from oxidative stress damage, thereby accelerating wound healing.

[0020] (4) The ectoine-containing wound dressing of the present invention can be used for the healing of complex wounds, such as joint wounds, postoperative incisions and burn wounds, which can effectively shorten the healing period and reduce the risk of infection. In the preparation of the dressing, non-toxic cross-linking agent oligomeric proanthocyanidins are used instead of traditional chemical cross-linking agent glutaraldehyde, which avoids damage to new cells and will not irritate the wound during use, making it suitable for sensitive people. The ectoine crystals uniformly distributed in the dressing can continuously and stably release ectoine during the use of the dressing, reducing the frequency of dressing replacement. This not only improves the comfort and convenience of patients, but also ensures the reliability of its application in active sites such as joints, making it extremely promising in the market. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the preparation method of the ectoine-containing wound dressing of the present invention. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] The halloysite nanotubes used in the embodiments of this application are from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd., with CAS number 1332-58-7, diameter of 50-300nm, and length of 1-10um; the oligomeric proanthocyanidins used are from Shanghai Aladdin Biochemical Technology Co., Ltd., with CAS number 222838-60-0, purity of 95%, and catalog number G493214.

[0024] This invention prepares a wound dressing containing ectoine, comprising the following components by weight: 5-15 parts HNTs@IPA, 15-25 parts sodium alginate, 1-3 parts oligomeric proanthocyanidins, 80-120 parts N-isopropylacrylamide, 3-7 parts acrylic acid, 40-60 parts ectoine, and 800-1000 parts deionized water; The HNTs@IPA is obtained by loading isopropanol onto halloysite nanotubes.

[0025] In one embodiment of this application, the ectoine-containing wound dressing comprises the following components by weight: 8-12 parts HNTs@IPA, 18-22 parts sodium alginate, 2 parts oligomeric proanthocyanidins, 90-110 parts N-isopropylacrylamide, 4-6 parts acrylic acid, 45-55 parts ectoine, and 850-950 parts deionized water.

[0026] In one embodiment of this application, the ectoine-containing wound dressing comprises the following components by weight: 10 parts HNTs@IPA, 20 parts sodium alginate, 2 parts oligomeric proanthocyanidins, 100 parts N-isopropylacrylamide, 5 parts acrylic acid, 50 parts ectoine, and 900 parts deionized water.

[0027] In one embodiment of this application, the HNTs@IPA is prepared by the following method: halloysite nanotubes are vacuum dried at 60°C and -0.1 MPa for 12 h, cooled to room temperature, added to isopropanol and dispersed evenly, centrifuged at 8000 rpm for 10 min, and the precipitate is collected to obtain HNTs@IPA.

[0028] It is understood that in this embodiment, by pre-loading isopropanol, a good solvent for ectoine, into the cavity of halloysite nanotubes, the slow and continuous release of isopropanol during subsequent mixing is ensured, avoiding drastic changes in system polarity caused by direct addition of isopropanol. Halloysite nanotubes are natural nanomaterials with a hollow tubular structure. The air and moisture in the cavity are first removed by vacuum drying, and then the tubes are immersed in isopropanol. Under the action of capillary action and pressure difference, isopropanol molecules fill the cavity and adhere to the inner wall of the cavity through van der Waals forces.

[0029] One embodiment of this application also discloses a method for preparing the above-mentioned ectoine-containing wound dressing, which specifically includes the following steps: Step 1: Dissolve sodium alginate in deionized water, add oligomeric proanthocyanidins powder, stir and mix evenly at 40°C in the dark, add N-isopropylacrylamide, acrylic acid, ectoine and HNTs@IPA, stir and mix evenly, transfer to a mold, add ammonium persulfate and TEMED under nitrogen protection, seal and let stand at 25°C for 6 hours to obtain ectoine-containing gel; It is understood that in the embodiments of this application, the alginate molecular chain is rich in carboxyl and hydroxyl groups, and is bonded to the oligomeric proanthocyanidin molecules by hydrogen bonds with a large number of phenolic hydroxyl groups. Under heating conditions of 40°C, the molecular thermal motion is intensified, and the oligomeric proanthocyanidin molecules are uniformly distributed on the alginate molecular chain. The added HNTs@IPA will slowly release isopropanol, which is a good solvent for ectoine. Ectoine has higher solubility and concentration around HNTs@IPA. After the addition of ammonium persulfate and TEMED, the ammonium persulfate will decompose at room temperature under the catalysis of TEMED to generate sulfate free radicals, which attack the carbon-carbon double bonds of N-isopropylacrylamide and acrylic acid, initiating a chain polymerization reaction to generate P(NIPAM-co) rich in amide bonds and carboxyl groups. -AA), which has strong hydrophilicity, these P (NIPAM-co-AA) form a polymer network. The amide bonds and carboxyl groups on it will combine with a large number of water molecules through hydrogen bonds, resulting in a decrease in the proportion of free water in the system and a decrease in the solubility of ectoine. As the polymer network continues to form, the dissolved ectoine gradually precipitates out. The dispersed HNTs@IPA in the system provides a surface for ectoine to attach and arrange in an orderly manner, which greatly reduces the nucleation energy barrier and becomes an ideal precipitation site for ectoine. During the precipitation process, ectoine molecules first form tiny crystal nuclei on the surface of HNTs@IPA. As ectoine molecules in the surrounding supersaturated solution continue to precipitate out, they are deposited in an orderly manner on the crystal nuclei. The crystal nuclei eventually grow into ectoine crystals.

[0030] Step 2: Immerse the ectoine-containing wound gel in a calcium chloride aqueous solution for 2 hours, then remove it and place it in deionized water. Change the water every 4 hours and continue washing for 48 hours. After removing it, freeze it at -20℃ for 12 hours, then freeze-dry it, and then perform vacuum swing adsorption drying to obtain the ectoine-containing wound dressing.

[0031] It is understood that in the embodiments of this application, alginate is a block copolymer composed of mannuronic acid and guluronic acid, wherein the carboxyl and hydroxyl groups on guluronic acid form a uniform groove structure in space, and calcium ions in calcium chloride aqueous solution chelate with the carboxyl groups of multiple different alginate long chains through ionic bonds to form an "egg box model" cross-linked structure, generating a stable hydrogel structure, which improves the stability and anti-dissolution of the alginate network, and ensures that the dressing can still maintain its intact shape after absorbing wound exudate and will not easily disintegrate.

[0032] In one embodiment of this application, the weight of the ammonium persulfate is 1% to 2% of N-isopropylacrylamide.

[0033] In one embodiment of this application, the weight of TEMED is 10% to 15% of ammonium persulfate.

[0034] In one embodiment of this application, the concentration of the calcium chloride aqueous solution is 0.2M.

[0035] In one embodiment of this application, the specific conditions for freeze-drying are: temperature -50°C, pressure less than 10 Pa, and freeze-drying time of 48 h.

[0036] In one embodiment of this application, the specific operation of the vacuum oscillation adsorption drying is as follows: evacuate to 10 Pa and maintain for 5 min, then fill with high-purity argon gas to 80 kPa and maintain for 5 min, and repeat this cycle 100 times.

[0037] It is understood that in the embodiments of this application, vacuum oscillation adsorption drying is used to first promote the desorption of residual isopropanol adsorbed on the surface of the dressing through low vacuum, while high-pressure argon can purge and remove the desorbed isopropanol from the system, thereby improving the biosafety of the dressing.

[0038] Example 1 This embodiment prepares a wound dressing containing ectoine, which comprises the following components by weight: 10 parts HNTs@IPA, 20 parts sodium alginate, 2 parts oligomeric proanthocyanidins, 100 parts N-isopropylacrylamide, 5 parts acrylic acid, 50 parts ectoine and 900 parts deionized water.

[0039] The ectoine-containing wound dressing of this embodiment is prepared through the following steps: Step 1: Preparation of HNTs@IPA: The HNTs@IPA was prepared by the following method: Halloysite nanotubes were vacuum dried at 60℃ and -0.1MPa for 12h, cooled to room temperature, and dispersed evenly in isopropanol. The mixture was centrifuged at 8000rpm for 10min, and the precipitate was collected to obtain HNTs@IPA.

[0040] Step 2, Alginate Complex Solution: Dissolve sodium alginate in deionized water, add oligomeric proanthocyanidins powder, and stir and mix evenly at 40°C in the dark to obtain an alginate complex solution. Add N-isopropylacrylamide, acrylic acid, ectoine and HNTs@IPA to the alginate complex solution, stir and mix evenly, transfer to a mold, add ammonium persulfate and TEMED under nitrogen protection, seal and let stand at 25°C for 6 hours to obtain ectoine-containing gel; The ammonium persulfate is 1% of N-isopropylacrylamide by weight, and the TEMED is 10% of ammonium persulfate by weight.

[0041] Step 3: Immerse the ectoine-containing wound gel in a 0.2M calcium chloride aqueous solution for 2 hours, then remove it and place it in deionized water. Change the water every 4 hours and continue washing for 48 hours. Then freeze it in a -20℃ refrigerator for 12 hours, and freeze-dry it at -50℃ and a pressure of less than 10Pa for 48 hours. Finally, perform vacuum swing adsorption drying to obtain the ectoine-containing dressing. The specific operation of the vacuum oscillation adsorption drying is as follows: evacuate to 10 Pa and maintain for 5 min, then fill with high-purity argon gas to 80 kPa and maintain for 5 min, repeat this cycle 100 times.

[0042] Example 2 This embodiment prepares a wound dressing containing ectoine, which comprises the following components by weight: 8 parts HNTs@IPA, 22 parts sodium alginate, 2 parts oligomeric proanthocyanidins, 110 parts N-isopropylacrylamide, 6 parts acrylic acid, 45 parts ectoine, and 950 parts deionized water.

[0043] The ectoine-containing wound dressing of this embodiment is prepared through the following steps: Step 1: Preparation of HNTs@IPA: The HNTs@IPA was prepared by the following method: Halloysite nanotubes were vacuum dried at 60℃ and -0.1MPa for 12h, cooled to room temperature, and dispersed evenly in isopropanol. The mixture was centrifuged at 8000rpm for 10min, and the precipitate was collected to obtain HNTs@IPA.

[0044] Step 2, Alginate Complex Solution: Dissolve sodium alginate in deionized water, add oligomeric proanthocyanidins powder, and stir and mix evenly at 40°C in the dark to obtain an alginate complex solution. Add N-isopropylacrylamide, acrylic acid, ectoine and HNTs@IPA to the alginate complex solution, stir and mix evenly, transfer to a mold, add ammonium persulfate and TEMED under nitrogen protection, seal and let stand at 25°C for 6 hours to obtain ectoine-containing gel; The ammonium persulfate is 1% of N-isopropylacrylamide by weight, and the TEMED is 10% of ammonium persulfate by weight.

[0045] Step 3: Immerse the ectoine-containing wound gel in a 0.2M calcium chloride aqueous solution for 2 hours, then remove it and place it in deionized water. Change the water every 4 hours and continue washing for 48 hours. Then freeze it in a -20℃ refrigerator for 12 hours, and freeze-dry it at -50℃ and a pressure of less than 10Pa for 48 hours. Finally, perform vacuum swing adsorption drying to obtain the ectoine-containing dressing. The specific operation of the vacuum oscillation adsorption drying is as follows: evacuate to 10 Pa and maintain for 5 min, then fill with high-purity argon gas to 80 kPa and maintain for 5 min, repeat this cycle 100 times.

[0046] Example 3 This embodiment prepares an ectoine-containing wound dressing, which comprises the following components by weight: 12 parts HNTs@IPA, 18 parts sodium alginate, 2 parts oligomeric proanthocyanidins, 110 parts N-isopropylacrylamide, 6 parts acrylic acid, 55 parts ectoine, and 850 parts deionized water.

[0047] The ectoine-containing wound dressing of this embodiment is prepared through the following steps: Step 1: Preparation of HNTs@IPA: The HNTs@IPA was prepared by the following method: Halloysite nanotubes were vacuum dried at 60℃ and -0.1MPa for 12h, cooled to room temperature, and dispersed evenly in isopropanol. The mixture was centrifuged at 8000rpm for 10min, and the precipitate was collected to obtain HNTs@IPA.

[0048] Step 2, Alginate Complex Solution: Dissolve sodium alginate in deionized water, add oligomeric proanthocyanidins powder, and stir and mix evenly at 40°C in the dark to obtain an alginate complex solution. Add N-isopropylacrylamide, acrylic acid, ectoine and HNTs@IPA to the alginate complex solution, stir and mix evenly, transfer to a mold, add ammonium persulfate and TEMED under nitrogen protection, seal and let stand at 25°C for 6 hours to obtain ectoine-containing gel; The ammonium persulfate is 1% of N-isopropylacrylamide by weight, and the TEMED is 10% of ammonium persulfate by weight.

[0049] Step 3: Immerse the ectoine-containing wound gel in a 0.2M calcium chloride aqueous solution for 2 hours, then remove it and place it in deionized water. Change the water every 4 hours and continue washing for 48 hours. Then freeze it in a -20℃ refrigerator for 12 hours, and freeze-dry it at -50℃ and a pressure of less than 10Pa for 48 hours. Finally, perform vacuum swing adsorption drying to obtain the ectoine-containing dressing. The specific operation of the vacuum oscillation adsorption drying is as follows: evacuate to 10 Pa and maintain for 5 min, then fill with high-purity argon gas to 80 kPa and maintain for 5 min, repeat this cycle 100 times.

[0050] Comparative Example 1 Halloysite nanotubes were not loaded with isopropanol; instead, an equal amount of halloysite nanotubes were used to replace HNTs@IPA. The rest of the process was the same as in Example 1.

[0051] Comparative Example 2 The oligomeric proanthocyanidin powder was replaced with an equal amount of the traditional crosslinking agent glutaraldehyde, and the rest was the same as in Example 1.

[0052] Comparative Example 3 Vacuum oscillating adsorption drying was not performed in step three; the rest was the same as in Example 1.

[0053] Test Example 1: The mechanical properties of the wound dressings prepared in Examples 1-3 and Comparative Examples 1-3 were tested. The test results are shown in Table 1 below: Table 1. Results of Mechanical Performance Testing

[0054] As shown in Table 1, the concentration of the active ingredient in the wound dressing prepared in Example 1 is higher than that in Example 2, resulting in improved mechanical strength. In Example 3, the concentration of the active ingredient exceeds the optimal range, leading to a decrease in mechanical strength. In Comparative Example 1, the halloysite nanotubes were not loaded with isopropanol, resulting in uncontrolled nucleation and crystallization rate of ectoin and uneven crystal size, weakening the physical reinforcement effect on the dressing and reducing the tensile strength. In Comparative Example 2, glutaraldehyde was used instead of oligomeric proanthocyanidins as a crosslinking agent, increasing the crosslinking strength. However, the lack of hydrogen bonds between the alginate molecular chain and P(NIPAM-co-AA) and oligomeric proanthocyanidins resulted in a decrease in elongation at break.

[0055] Test Example 2: The in vitro cytotoxicity, skin sensitization, and intradermal irritation of the ectoine-containing dressings prepared in Examples 1, 2, and 3 were tested according to GB / T 16886.1-2011 Biological Evaluation of Medical Devices Part 1: Evaluation and Testing in Risk Management Processes, GB / T 16886.5-2017 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests, and GB / T 16886.10-2017 Biological Evaluation of Medical Devices Part 10: Irritation and Skin Sensitization Tests.

[0056] 1. In vitro cytotoxicity assay using the MTT assay: Step 1: According to GB / T 16886.12 standard, the ectoine-containing wound dressings prepared in Example 1, Comparative Example 2, and Comparative Example 3 were respectively prepared into 1cm diameters. 2 The sample fragments were added to MEM culture medium at a ratio of 0.2 g / ml and extracted in a 37℃, 5% CO2 incubator for 24 h. The sample extract was collected and filtered for sterilization.

[0057] Step 2: In the logarithmic growth phase, L929 cells were incubated at a rate of 1×10⁻⁶. 4 The cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 hours until the cells adhered. The original culture medium was then removed. Three experimental groups were set up, each with 100% concentration of sample extract added. A negative control group was added with MEM culture medium, and a positive control group was added with MEM culture medium containing 0.64% phenol. Each group had 6 replicates.

[0058] Step 3: Continue culturing in a 37℃, 5% CO2 incubator for 24 hours. Then, add 20 μL of 5 mg / mL MTT solution to each well and continue culturing for 4 hours. Terminate the culture, aspirate the liquid from the wells, add 150 μL of DMSO to each well, and shake for 10 minutes to fully dissolve the formazan crystals. Use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 490 nm and calculate the relative cell proliferation rate.

[0059] Relative cell proliferation rate (RGR) = (OD value of experimental group / OD value of negative control group) × 100%.

[0060] 2. Skin sensitization tests were conducted using the guinea pig maximum dose method: Step 1: The test subjects were the ectoine-containing wound dressings prepared in Examples 1, 2 and 3 above. The test animals were healthy adult white guinea pigs with an average weight of 300-350g.

[0061] Step 2: Setting up experimental and control groups: Guinea pigs in the experimental group were intradermally injected with a 1:1 mixture of the above-mentioned sample extract and Freund's complete adjuvant, while the control group was injected with an equal volume of physiological saline. One week later, the experimental group underwent enhanced induction by topical application of the sample extract to the original injection site, while the control group underwent enhanced induction by topical application of physiological saline to the original injection site.

[0062] Step 3: On day 14 after sensitization and induction, challenge the guinea pigs on the contralateral back with the sample extract. Observe the skin at the challenge site for allergic reactions such as erythema and edema at 24h, 48h, and 72h, and calculate the sensitization rate.

[0063] The detection results data for Test Example 2 are shown in Table 2 below: Table 2. Results of Biological Evaluation Tests

[0064] As shown in Table 2, the ectoine-containing dressing prepared in Example 1 exhibits negligible in vitro cytotoxicity and minimal intradermal irritation, indicating good biocompatibility. In Comparative Example 2, the ectoine-containing dressing, due to the use of the chemical cross-linking agent glutaraldehyde, remains in the dressing, exhibiting significant cytotoxicity and high sensitization. Comparative Example 3, which underwent vacuum oscillation adsorption drying, failed to effectively remove residual isopropanol, resulting in cell inhibition; however, its cytotoxicity was lower than that of Example 1. Therefore, the above data demonstrate that the present invention, by using oligomeric proanthocyanidins instead of traditional glutaraldehyde as a cross-linking agent in the preparation of the ectoine-containing dressing, significantly improves the biocompatibility of the dressing; the vacuum oscillation adsorption drying effectively removes residual isopropanol, further enhancing the biocompatibility of the dressing.

[0065] Test Example 3: Testing the wound healing effect of ectoine-containing wound dressings prepared in Examples 1-3 and Comparative Examples 1-3 on joint wounds: Step 1: Select healthy male SD rats with an average weight of 250-300g for the experiment. After anesthetizing the rats, prepare and disinfect the skin at the knee joint of the right hind limb to create a circular full-thickness skin defect with a diameter of 10mm as a wound model.

[0066] Step 2: The rats were randomly divided into 6 groups of 10 rats each, corresponding to Examples 1-3 and Comparative Examples 1-3 respectively. The corresponding group of ectoine-containing wound dressings were applied to the surface of the rat wounds and fixed with medical tape and elastic bandages. The dressings were changed every two days.

[0067] Step 3: Record the wound area of ​​rats on days 0, 3, 7, 10 and 14 after drug administration, and calculate the wound healing rate.

[0068] Wound healing rate = ((initial wound area - wound area at a specific time) / initial wound area) × 100%.

[0069] The detection results of test example 3 are shown in Table 3 below: Table 3. Wound Healing Rate Results

[0070] As shown in Table 3, the ectoine-containing wound dressing of Example 1 had the best wound healing effect on rats, with almost complete wound healing by day 14. In Comparative Example 1, because the halloysite nanotubes were not loaded with isopropanol, the ectoine was not evenly dispersed in the dressing, resulting in a lower healing rate compared to Example 1. In Comparative Example 2, because glutaraldehyde was used as a crosslinking agent, the residual glutaraldehyde in the dressing had certain cytotoxicity, limiting wound healing. In Comparative Example 3, because vacuum swing adsorption drying was not performed, a small amount of residual isopropanol caused slight irritation to the rat wounds, and the healing effect was not as good as the Example 1 group.

[0071] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A wound dressing containing ectoine, characterized in that, The product comprises the following components by weight: 5-15 parts HNTs@IPA, 15-25 parts sodium alginate, 1-3 parts oligomeric proanthocyanidins, 80-120 parts N-isopropylacrylamide, 3-7 parts acrylic acid, 40-60 parts ectoine, and 800-1000 parts deionized water. The HNTs@IPA is obtained by loading isopropanol onto halloysite nanotubes.

2. The ectoine-containing wound dressing according to claim 1, characterized in that, The product comprises the following components by weight: 8–12 parts HNTs@IPA, 18–22 parts sodium alginate, 2 parts oligomeric proanthocyanidins, 90–110 parts N-isopropylacrylamide, 4–6 parts acrylic acid, 45–55 parts ectoine, and 850–950 parts deionized water.

3. The ectoine-containing wound dressing according to claim 1, characterized in that, The product comprises the following components by weight: 10 parts HNTs@IPA, 20 parts sodium alginate, 2 parts oligomeric proanthocyanidins, 100 parts N-isopropylacrylamide, 5 parts acrylic acid, 50 parts ectoine, and 900 parts deionized water.

4. The ectoine-containing wound dressing according to claim 1, characterized in that, The HNTs@IPA was prepared by the following method: halloysite nanotubes were vacuum dried at 60℃ and -0.1MPa for 12h, cooled to room temperature, and dispersed evenly in isopropanol. The mixture was centrifuged at 8000rpm for 10min, and the precipitate was collected to obtain HNTs@IPA.

5. A method for preparing an ectoine-containing wound dressing as described in any one of claims 1 to 4, characterized in that, Specifically, the following steps are included: Step 1: Dissolve sodium alginate in deionized water, add oligomeric proanthocyanidins powder, stir and mix evenly at 40°C in the dark, add N-isopropylacrylamide, acrylic acid, ectoine and HNTs@IPA, stir and mix evenly, transfer to a mold, add ammonium persulfate and TEMED under nitrogen protection, seal and let stand at 25°C for 6 hours to obtain ectoine-containing gel; Step 2: Immerse the ectoine-containing wound gel in a calcium chloride aqueous solution for 2 hours, then remove it and place it in deionized water. Change the water every 4 hours and continue washing for 48 hours. After removing it, freeze it at -20℃ for 12 hours, then freeze-dry it, and then perform vacuum swing adsorption drying to obtain the ectoine-containing wound dressing. The specific operation of the vacuum oscillation adsorption drying is as follows: evacuate to 10 Pa and maintain for 5 min, then fill with high-purity argon gas to 80 kPa and maintain for 5 min, repeat this cycle 100 times.

6. The preparation method according to claim 5, characterized in that, The weight of the ammonium persulfate is 1% to 2% of N-isopropylacrylamide.

7. The preparation method according to claim 5, characterized in that, The weight of TEMED is 10% to 15% of ammonium persulfate.

8. The preparation method according to claim 5, characterized in that, The concentration of the calcium chloride aqueous solution is 0.2M.

9. The preparation method according to claim 5, characterized in that, The specific conditions for freeze-drying are: temperature -50℃, pressure less than 10Pa, and freeze-drying time of 48h.

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