Preparation method of medical antibacterial hydrogel dressing
By preparing medical antibacterial hydrogel dressings with high crosslinking three-dimensional network structures, the problems of insufficient strength of hydrogel dressings and poor biosafety of antibacterial agents are solved, and higher strength and antibacterial effects are achieved.
Patent Information
- Application Number
- CN202510591529.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-08
- Publication Date
- 2025-08-08
AI Technical Summary
The tensile strength of existing medical hydrogel dressings is poor and prone to breakage. Commonly used antibacterial agents have biosafety problems, which are prone to bacterial infections.
MA-CTS is prepared by reacting chitosan with methacrylic anhydride, and MAS-ABI is prepared by reacting chitosan with 4-methacrylamidosaicylic acid and 2-aminobenzimidazole, and then crosslinking with hydroxyethyl vinyl ether to form a medical antibacterial hydrogel dressing with a high crosslinking degree of three-dimensional network structure.
The strength of the hydrogel dressing is increased by about 30%-40%, which enhances antibacterial properties, ensures that it is not easy to break during use, and significantly improves the ability to inhibit bacteria.
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Figure CN120441785A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical dressings, and in particular to a method for preparing a medical antibacterial hydrogel dressing. Background Art
[0002] Wound repair has always been a major issue facing humanity. To accelerate wound repair, people have long hoped to find methods or drugs to promote or accelerate this process. Early on, dry wounds, especially large burn wounds, were treated primarily to prevent infection. Later, it was discovered that wounds healed better if they maintained a moist environment. Guided by the theory of "moist wound healing" and driven by advances in materials science and industry, wound dressings have undergone revolutionary changes, from traditional gauze dressings to synthetic and biological dressings, and finally to new medical biosynthetic dressings. These new dressings, with a variety of raw materials and dosage forms, are suitable for different wounds and at different healing stages. Hydrogel dressings are a representative example.
[0003] Hydrogel dressings have a unique three-dimensional network structure that effectively locks moisture within them. When applied to a wound, these dressings slowly release moisture, maintaining a slightly moist environment and promoting wound healing. Existing medical hydrogels offer excellent breathability and biocompatibility; they have no side effects in medical use and exhibit a high swelling rate. However, these hydrogels suffer from poor tensile and gel strength, making them prone to breakage and fragmentation during transportation and use, making their application inconvenient. Furthermore, if wet dressings are used improperly, they can easily breed bacteria and cause infection. Therefore, antimicrobial agents are often added to hydrogel dressings. Most commonly used antimicrobial agents on the market are organic, which have relatively poor biosafety and can easily cause hemolysis and pain. Natural antimicrobial agents, such as chitosan and honey, also have relatively weak antimicrobial properties. Summary of the Invention
[0004] In view of the problems existing in the prior art, the object of the present invention is to provide a method for preparing a medical antibacterial hydrogel dressing.
[0005] The purpose of the present invention is achieved by adopting the following technical solutions:
[0006] A method for preparing a medical antibacterial hydrogel dressing comprises the following steps:
[0007] Step 1: chitosan (CTS) is added to an acetic acid solution, stirred until fully dissolved, and then methacrylic anhydride (MAAH) is added and stirred for 10-20 hours, followed by dialysis, filtration and drying to obtain MA-CTS;
[0008] Step 2: Weigh 4-methylacrylamidosalicylic acid (MAS) and add it to dichloromethane. Under nitrogen protection, fully dissolve it. First, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), stir evenly in an ice-water bath, then warm to room temperature, add N-hydroxysuccinimide (NHS), stir for 0.5-1.5h, and then add 2-aminobenzimidazole (2-ABI). Stir at room temperature for 12-24h. After the reaction is completed, remove the solvent under reduced pressure, dialyze and dry to obtain MAS-ABI;
[0009] Step 3: Add MAS-ABI and MA-CTS to deionized water, then add hydroxyethyl vinyl ether (HEVE), stir thoroughly, add initiator, heat to 60-80°C, stir and react for 4-12 hours. After the reaction is completed, dialyze, filter and dry to obtain a medical antibacterial hydrogel dressing material.
[0010] Preferably, in step 1, the deacetylation degree of chitosan (CTS) is ≥85%, and the molecular weight is 10-50 kDa.
[0011] Preferably, in step 1, the mass fraction of the acetic acid solution is 1%-5%.
[0012] Preferably, in step 1, the mass volume ratio of chitosan (CTS), methacrylic anhydride (MAAH) and acetic acid solution is 1 g: (8-12) g: (60-100) mL.
[0013] Preferably, in step 1, the dialysis is performed using a 5-10 kDa dialysis bag in pure water for 3-5 days, with the dialysate replaced every day; and the drying is performed by vacuum drying.
[0014] Preferably, in step 2, the mass volume ratio of 4-methylacrylamidosalicylic acid (MAS), EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide), NHS (N-hydroxysuccinimide), 2-aminobenzimidazole (2-ABI) and dichloromethane is (1.8-2.6) g: (1.9-2.7) g: (0.2-0.4) g: (1.1-1.5) g: (20-40) mL.
[0015] Preferably, in step 2, the dialysis is performed using a 12 kDa dialysis bag in a 1% NaCl solution for 2-5 days, with the dialysis solution being replaced every day.
[0016] Preferably, in step 3, the mass volume ratio of MAS-ABI, MA-CTS, hydroxyethyl vinyl ether (HEVE) and deionized water is (2-4.5) g: (1.2-2) g: (0.2-0.6) g: (20-40) mL.
[0017] Preferably, in step 3, the initiator is ammonium persulfate (APS), and the amount added is 1.2%-2.6% of the mass of MA-CTS.
[0018] Preferably, in step 3, the dialysis is performed using a 5-10 kDa dialysis bag in pure water for 3-5 days, with the dialysate replaced every day; and the drying is performed by vacuum drying.
[0019] The beneficial effects of the present invention are:
[0020] 1. The present invention prepares a medical antibacterial hydrogel dressing, which is prepared by cross-linking polymerization using MA-CTS, MAS-ABI, and hydroxyethyl vinyl ether (HEVE). The hydrogel dressing prepared by the present invention has a highly cross-linked three-dimensional network structure. It is rich in chitosan, salicylamide groups, benzimidazole groups, and other structures, which not only give the hydrogel dressing super strong water absorption and moisture retention, but also extremely excellent antibacterial performance. In addition, compared with conventional chitosan hydrogel dressings, the strength is increased by about 30%-40%, which can ensure that it will not easily break during use.
[0021] 2. MA-CTS is prepared by acrylate treatment of chitosan (CTS) with methacrylic anhydride (MAAH), which grafts a large number of acrylic acid groups onto the chitosan. MAS-ABI is prepared by the EDC / NHS condensation reaction of 4-methacrylamidosalicylic acid (MAS) and 2-aminobenzimidazole (2-ABI), converting the salicylic acid in MAS to salicylamide and introducing benzimidazole groups.
[0022] 3. Hydroxyethyl vinyl ether (HEVE) is a water-soluble ether compound containing a vinyl group. The present invention utilizes it as a cosolvent for MA-CTS and MAS-ABI on the one hand, and as a cross-linking agent in the polymerization reaction of MA-CTS and MAS-ABI on the other hand, thereby enhancing the hydrophilicity of the final product while also increasing a certain degree of stability. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The present invention is further described with reference to the accompanying drawings. However, the embodiments in the accompanying drawings do not constitute any limitation to the present invention. A person skilled in the art can obtain other drawings based on the following drawings without creative effort.
[0024] Figure 1 Schematic diagram of the SEM of the medical antibacterial hydrogel dressing prepared in Example 1 of the present invention. DETAILED DESCRIPTION
[0025] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the present invention. Changes or adjustments in their relative relationships, without substantially changing the technical content, should also be regarded as the scope of the present invention.
[0026] In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.
[0027] The present invention will be further described below with reference to the following examples.
[0028] Example 1
[0029] A method for preparing a medical antibacterial hydrogel dressing comprises the following steps:
[0030] Step 1, preparation of MA-CTS:
[0031] 1 g of chitosan (CTS, 30 kDa, degree of deacetylation ≥ 85%) was added to 80 mL of 3% acetic acid solution and stirred until fully dissolved. 10 g of methacrylic anhydride (MAAH) was then added and stirred for 15 h. The product was then dialyzed in pure water using an 8 kDa dialysis bag for 4 days, with the dialysate replaced every day. The product was filtered and vacuum dried to obtain MA-CTS.
[0032] Step 2, preparation of MAS-ABI:
[0033] 2.2 g of 4-methylacrylamidosalicylic acid (MAS) was weighed and added to 30 mL of dichloromethane. Under the protection of nitrogen, it was fully dissolved. 2.3 g of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) was first added, and the mixture was stirred evenly in an ice-water bath. The mixture was then warmed to room temperature, and 0.3 g of NHS (N-hydroxysuccinimide) was added. The mixture was stirred for 1 h, and then 1.3 g of 2-aminobenzimidazole (2-ABI) was added. The mixture was stirred at room temperature for 18 h. After the reaction was completed, the solvent was removed under reduced pressure, and the mixture was dialyzed in a 1% NaCl solution using a 12 kDa dialysis bag for 3 days. The dialysate was replaced every day, and then vacuum dried to obtain MAS-ABI.
[0034] Step 3, cross-linking polymerization:
[0035] Add 3.5g MAS-ABI and 1.6g MA-CTS to 30mL deionized water, then add 0.4g hydroxyethyl vinyl ether (HEVE), stir thoroughly, add initiator ammonium persulfate (APS), the addition amount is 1.9% of the mass of MA-CTS, heat to 70℃, stir and react for 8h, cool to room temperature after the reaction, dialyze in pure water using an 8kDa dialysis bag for 4 days, change the dialysate every day, filter and vacuum dry to obtain a medical antibacterial hydrogel dressing material.
[0036] Example 2
[0037] A method for preparing a medical antibacterial hydrogel dressing comprises the following steps:
[0038] Step 1, preparation of MA-CTS:
[0039] 1 g of chitosan (CTS, 10 kDa, degree of deacetylation ≥ 85%) was added to 60 mL of 1% acetic acid solution and stirred until fully dissolved. 8 g of methacrylic anhydride (MAAH) was then added and stirred for 10 h. The product was then dialyzed in pure water using a 5 kDa dialysis bag for 3 days, with the dialysate replaced daily. The product was filtered and vacuum dried to obtain MA-CTS.
[0040] Step 2, preparation of MAS-ABI:
[0041] 1.8 g of 4-methylacrylamidosalicylic acid (MAS) was weighed and added to 20 mL of dichloromethane. Under nitrogen, it was fully dissolved. 1.9 g of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) was added first, and the mixture was stirred evenly in an ice-water bath. The mixture was then warmed to room temperature, and 0.2 g of NHS (N-hydroxysuccinimide) was added. The mixture was stirred for 0.5 h, and then 1.1 g of 2-aminobenzimidazole (2-ABI) was added. The mixture was stirred at room temperature for 12 h. After the reaction, the solvent was removed under reduced pressure, and the mixture was dialyzed in a 1% NaCl solution using a 12 kDa dialysis bag for 2 days. The dialysate was replaced every day, and then dried in vacuo to obtain MAS-ABI.
[0042] Step 3, cross-linking polymerization:
[0043] Add 2g MAS-ABI and 1.2g MA-CTS to 20mL deionized water, then add 0.2g hydroxyethyl vinyl ether (HEVE), stir thoroughly, add initiator ammonium persulfate (APS), the addition amount is 1.2% of the mass of MA-CTS, heat to 60℃, stir and react for 4h. After the reaction, use 5kDa dialysis bag to dialyze in pure water for 3 days, change the dialysate every day, filter and vacuum dry to obtain medical antibacterial hydrogel dressing material.
[0044] Example 3
[0045] A method for preparing a medical antibacterial hydrogel dressing comprises the following steps:
[0046] Step 1, preparation of MA-CTS:
[0047] 1 g of chitosan (CTS, 40 kDa, degree of deacetylation ≥ 85%) was added to 70 mL of 2% acetic acid solution and stirred until fully dissolved. 10 g of methacrylic anhydride (MAAH) was then added and stirred for 15 h. The product was then dialyzed in pure water using an 8 kDa dialysis bag for 5 days, with the dialysate replaced daily. The product was filtered and vacuum dried to obtain MA-CTS.
[0048] Step 2, preparation of MAS-ABI:
[0049] 2 g of 4-methylacrylamidosalicylic acid (MAS) was weighed and added to 40 mL of dichloromethane. Under nitrogen, the mixture was fully dissolved. 2.3 g of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) was added first, and the mixture was stirred evenly in an ice-water bath. The mixture was then warmed to room temperature, and 0.3 g of NHS (N-hydroxysuccinimide) was added. The mixture was stirred for 1 h, and then 1.4 g of 2-aminobenzimidazole (2-ABI) was added. The mixture was stirred at room temperature for 16 h. After the reaction, the solvent was removed under reduced pressure, and the mixture was dialyzed in a 1% NaCl solution using a 12 kDa dialysis bag for 2-5 days. The dialysate was replaced every day, and then dried in vacuo to obtain MAS-ABI.
[0050] Step 3, cross-linking polymerization:
[0051] Add 3g MAS-ABI and 1.8g MA-CTS to 30mL deionized water, then add 0.4g hydroxyethyl vinyl ether (HEVE), stir thoroughly, add initiator ammonium persulfate (APS), the addition amount is 1.8% of the mass of MA-CTS, heat to 70℃, stir and react for 10h. After the reaction, use 8kDa dialysis bag to dialyze in pure water for 4 days, change the dialysate every day, filter and vacuum dry to obtain a medical antibacterial hydrogel dressing material.
[0052] Example 4
[0053] A method for preparing a medical antibacterial hydrogel dressing comprises the following steps:
[0054] Step 1, preparation of MA-CTS:
[0055] 1 g of chitosan (CTS, 50 kDa, degree of deacetylation ≥ 85%) was added to 100 mL of 5% acetic acid solution and stirred until fully dissolved. 12 g of methacrylic anhydride (MAAH) was then added and stirred for 10-20 h. The mixture was then dialyzed in pure water using a 10 kDa dialysis bag for 5 days, with the dialysate replaced daily. The mixture was filtered and vacuum dried to obtain MA-CTS.
[0056] Step 2, preparation of MAS-ABI:
[0057] 2.6 g of 4-methylacrylamidosalicylic acid (MAS) was weighed and added to 40 mL of dichloromethane. Under nitrogen, it was fully dissolved. 2.7 g of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) was first added and stirred evenly in an ice-water bath. The mixture was then warmed to room temperature and 0.4 g of NHS (N-hydroxysuccinimide) was added. The mixture was stirred for 1.5 h, and then 1.5 g of 2-aminobenzimidazole (2-ABI) was added. The mixture was stirred at room temperature for 24 h. After the reaction, the solvent was removed under reduced pressure and dialyzed in a 1% NaCl solution using a 12 kDa dialysis bag for 5 days. The dialysate was replaced every day, and then dried in vacuo to obtain MAS-ABI.
[0058] Step 3, cross-linking polymerization:
[0059] Add 4.5g MAS-ABI and 2g MA-CTS to 40mL deionized water, then add 0.6g hydroxyethyl vinyl ether (HEVE), stir thoroughly, add initiator ammonium persulfate (APS), the addition amount is 2.6% of the mass of MA-CTS, heat to 80℃, stir and react for 12h. After the reaction is completed, use a 10kDa dialysis bag to dialyze in pure water for 5 days, change the dialysate every day, filter and vacuum dry to obtain a medical antibacterial hydrogel dressing material.
[0060] Comparative Example 1
[0061] A method for preparing a medical antibacterial hydrogel dressing, which differs from Example 1 in that MAS-ABI is not added, comprises the following steps:
[0062] Step 1, preparation of MA-CTS:
[0063] 1 g of chitosan (CTS, 30 kDa, degree of deacetylation ≥ 85%) was added to 80 mL of 3% acetic acid solution and stirred until fully dissolved. Then, 10 g of methacrylic anhydride (MAAH) was added and stirred for 15 h. The product was then dialyzed in pure water using an 8 kDa dialysis bag for 4 days, with the dialysate replaced every day. After filtration, the product was vacuum dried to obtain MA-CTS.
[0064] Step 2, cross-linking polymerization:
[0065] Add 1.6g MA-CTS to 30mL deionized water, then add 0.4g hydroxyethyl vinyl ether (HEVE), stir thoroughly, add initiator ammonium persulfate (APS), the addition amount is 1.9% of the mass of MA-CTS, heat to 70℃, stir and react for 8h. After the reaction, use 8kDa dialysis bag to dialyze in pure water for 4 days, change the dialysate every day, filter and vacuum dry to obtain a medical antibacterial hydrogel dressing material.
[0066] Comparative Example 2
[0067] A method for preparing a medical antibacterial hydrogel dressing, which differs from Example 1 in that MAS-ABI is replaced with 4-methacrylamidosalicylic acid (MAS), comprises the following steps:
[0068] Step 1, preparation of MA-CTS:
[0069] 1 g of chitosan (CTS, 30 kDa, degree of deacetylation ≥ 85%) was added to 80 mL of 3% acetic acid solution and stirred until fully dissolved. Then, 10 g of methacrylic anhydride (MAAH) was added and stirred for 15 h. The product was then dialyzed in pure water using an 8 kDa dialysis bag for 4 days, with the dialysate replaced every day. After filtration, the product was vacuum dried to obtain MA-CTS.
[0070] Step 2, cross-linking polymerization:
[0071] 2.2g 4-methylacrylamidosalicylic acid (MAS) and 1.6g MA-CTS were added to 30mL deionized water, and then 0.4g hydroxyethyl vinyl ether (HEVE) was added. After thorough stirring, the initiator ammonium persulfate (APS) was added in an amount of 1.9% of the mass of MA-CTS. The temperature was raised to 70°C and stirred for 8h. After the reaction was completed, the material was dialyzed in pure water using an 8kDa dialysis bag for 4 days. The dialysate was replaced every day. After filtration, the material was vacuum dried to obtain a medical antibacterial hydrogel dressing material.
[0072] Experimental example
[0073] For the optimal embodiment of the present invention, Example 1, and its corresponding control examples 1-2, the prepared medical antibacterial hydrogel dressing material was tested for its tensile strength, antibacterial rate, and moisture permeability.
[0074] Tensile strength: With reference to ISO 527-2-2012, the tensile strength of the hydrogel dressing material was tested using a universal material testing machine.
[0075] Shear adhesion strength: Referring to ASTM F2258, the adhesion of the hydrogel was tested by tensile pull-off test.
[0076] Antibacterial properties: Referring to ASTM E2149, the antibacterial properties of the hydrogel were tested using the dynamic contact method, including Escherichia coli, Staphylococcus aureus and Candida albicans.
[0077] The results are as follows:
[0078]
[0079] As can be seen from the table above, Example 1 of the present invention maintains high water absorbency while also exhibiting higher strength. Its tensile strength is approximately 30%-40% higher than that of the control, and its adhesion strength is also somewhat improved. Furthermore, the chitosan dressing introduced with salicylamide and benzimidazole groups in Example 1 significantly enhances its antibacterial capacity, exhibiting superior antibacterial activity against Candida albicans and effectively inhibiting bacterial growth and reproduction.
[0080] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification.
[0081] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.
Claims
1. A method for preparing a medical antibacterial hydrogel dressing, characterized in that: The following steps are involved: Step 1: chitosan is added to an acetic acid solution, stirred until fully dissolved, and then methacrylic anhydride is added and stirred for 10-20 hours, followed by dialysis, filtration and drying to obtain MA-CTS; Step 2: Weigh 4-methylacrylamidosalicylic acid and add it to dichloromethane. Under the protection of nitrogen, fully dissolve it. First, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, stir evenly in an ice-water bath, then warm to room temperature, add N-hydroxysuccinimide, stir for 0.5-1.5 hours, then add 2-aminobenzimidazole, stir at room temperature for 12-24 hours. After the reaction, remove the solvent under reduced pressure, dialyze and dry to obtain MAS-ABI; Step 3: Add MAS-ABI and MA-CTS to deionized water, then add hydroxyethyl vinyl ether, stir thoroughly, add initiator, heat to 60-80°C, stir and react for 4-12 hours. After the reaction is completed, dialyze, filter and dry to obtain a medical antibacterial hydrogel dressing material.
2. The method for preparing a medical antibacterial hydrogel dressing according to claim 1, characterized in that: In the step 1, the deacetylation degree of chitosan is ≥85%, and the molecular weight is 10-50 kDa.
3. The method for preparing a medical antibacterial hydrogel dressing according to claim 1, characterized in that: In the step 1, the mass fraction of the acetic acid solution is 1%-5%.
4. The method for preparing a medical antibacterial hydrogel dressing according to claim 1, characterized in that: In the step 1, the mass volume ratio of chitosan, methacrylic anhydride and acetic acid solution is 1 g: (8-12) g: (60-100) mL.
5. The method for preparing a medical antibacterial hydrogel dressing according to claim 1, characterized in that: In step 1, the dialysis is performed using a 5-10 kDa dialysis bag in pure water for 3-5 days, and the dialysate is replaced every day; and the drying is performed by vacuum drying.
6. The method for preparing a medical antibacterial hydrogel dressing according to claim 1, characterized in that: In the step 2, the mass volume ratio of 4-methylacrylamidosalicylic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, 2-aminobenzimidazole and dichloromethane is (1.8-2.6) g: (1.9-2.7) g: (0.2-0.4) g: (1.1-1.5) g: (20-40) mL.
7. The method for preparing a medical antibacterial hydrogel dressing according to claim 1, characterized in that: In step 2, the dialysis is performed using a 12 kDa dialysis bag in a 1% NaCl solution for 2-5 days, with the dialysate being replaced every day.
8. The method for preparing a medical antibacterial hydrogel dressing according to claim 1, characterized in that: In step 3, the mass volume ratio of MAS-ABI, MA-CTS, hydroxyethyl vinyl ether and deionized water is (2-4.5) g: (1.2-2) g: (0.2-0.6) g: (20-40) mL.
9. The method for preparing a medical antibacterial hydrogel dressing according to claim 1, characterized in that: In step 3, the initiator is ammonium persulfate, and the amount added is 1.2%-2.6% of the mass of MA-CTS.
10. The method for preparing a medical antibacterial hydrogel dressing according to claim 1, characterized in that: In step 3, the dialysis is performed using a 5-10 kDa dialysis bag in pure water for 3-5 days, with the dialysate replaced every day; and the drying is performed by vacuum drying.