High-viscoelasticity non-crosslinking carboxymethyl chitin hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medical biopolymer materials, in particular to high-viscoelasticity non-crosslinked carboxymethyl chitin hydrogel and a preparation method and application thereof, the hydrogel is prepared from high-viscoelasticity non-crosslinked carboxymethyl chitin, and the content of carboxymethyl chitin in the hydrogel is 5-30 mg / mL. The preparation method of the high-viscoelasticity carboxymethyl chitin comprises the following steps: 1) alkalization: suspending chitin in a sodium hydroxide aqueous solution, and stirring and alkalizing at 5-30 DEG C; 2) etherification: dissolving substituted acetic acid or salt thereof, dropwise adding the dissolved substituted acetic acid or salt thereof into the alkalized system, and carrying out etherification reaction at 5-30 DEG C; and 3) separating and refining a crude product. The high-viscoelasticity carboxymethyl chitin hydrogel prepared by the preparation method disclosed by the invention not only keeps the original characteristics of biocompatibility and the like of chitin, but also has relatively high viscoelasticity and shear viscosity, and also solves the problem that a chitin derivative is difficult to tolerate terminal moist heat sterilization.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biopolymer materials, and specifically relates to a high-viscoelasticity non-crosslinked carboxymethyl chitin hydrogel and a preparation method and application thereof. Background Art

[0002] Chitin is widely distributed in nature, present in the cell walls of certain fungi and algae, as well as in the skeletons and shells of shrimp, crabs, and insects. Its annual production is second only to cellulose on Earth, making it the second largest natural polysaccharide resource. Structurally, chitin is composed of N-acetyl-D-glucosamine polymerized through β-1,4-glycosidic bonds. Chitosan, also known as chitosan, is a polysaccharide compound derived from chitin after deacetylation (generally believed to have a deacetylation degree greater than 50%). It is composed of glucosamine and N-acetyl-D-glucosamine polymerized through β-1,4-glycosidic bonds.

[0003] Chitin is a natural biopolymer that is biocompatible, biodegradable, and sustainably produced, with potential applications in many clinical fields. However, due to strong intra- and inter-molecular hydrogen bonding, chitin is almost insoluble in all solvents (including acidic and alkaline aqueous solutions, as well as various organic solvents). Chitosan, obtained by deacetylation of chitin, also has good solubility only in acidic solutions, hindering its widespread application.

[0004] Carboxymethyl chitin and carboxymethyl chitosan are chitin derivatives obtained by introducing a large number of carboxymethyl groups onto the hydroxyl and / or amino groups of chitin via a substitution reaction. The introduction of carboxymethyl groups disrupts the inherent hydrogen bonding within chitin, while the introduced carboxyl groups or carboxylates themselves exhibit good water solubility. This strategy addresses the solubility issue of chitin and has been widely recognized by the industry. For example, Shuchongning, a carboxymethyl chitosan irrigant, and Shuyining, a carboxymethyl chitosan hydrogel, produced by Shijiazhuang Yishengtang Medical Products Co., Ltd., are carboxymethyl chitosan products; and Qitejie, produced by Shanghai Qisheng Biological Products Co., Ltd., is a carboxymethyl chitin product.

[0005] The introduction of carboxymethyl groups solves the solubility problem of chitin, but some issues remain unresolved. Current carboxymethyl chitin or carboxymethyl chitosan derivatives often use large amounts of carboxymethylating agents for over-substitution (reaction sites are hydroxyl groups and deacetylated amino groups), making it difficult to control the degree of carboxymethyl substitution. Excessively high carboxymethyl substitution can significantly improve the solubility of the product, but it also creates other problems, such as: 1) The product has poor viscoelastic properties, and its physical and chemical properties (especially elastic modulus and viscous modulus) are not ideal when used as a viscoelastic supplement for intra-articular injection. Its viscoelasticity is much lower than that of sodium hyaluronate or cross-linked sodium hyaluronate products (such as Xinweike) used as viscoelastic supplements; 2) The product has good fluidity and low shear viscosity. When used for postoperative adhesion prevention, it has insufficient adhesion to body tissues and is easily lost from target tissue areas (such as wound sutures), failing to achieve the optimal state of postoperative adhesion prevention; 3) The thermal stability is poor and it cannot withstand terminal moist heat sterilization. Only process sterilization control technology can be used. Compared with terminal moist heat sterilization, products prepared by sterilization filtration and process sterilization production technology have greater biosafety risks during clinical use.

[0006] In summary, there is an urgent need in this field to develop a chitin derivative that matches the technical characteristics of the product required for clinical applications, so as to fully develop chitin, the world's second largest natural polysaccharide resource. Summary of the Invention

[0007] In view of the shortcomings of existing chitin research and development technology, the present invention provides a high-viscoelastic non-cross-linked carboxymethyl chitin hydrogel and its preparation method and application.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin comprises the following steps:

[0010] (1) Alkalization: suspend chitin in sodium hydroxide aqueous solution and stir at 5-30°C to alkalize;

[0011] The mass percentage of sodium hydroxide in the sodium hydroxide aqueous solution is 40% to 55%, preferably 45% to 55%; the mass ratio of chitin to sodium hydroxide aqueous solution is 1.0:5.0 to 1.0:25.0, preferably 1.0:7.0 to 1.0:15.0. Further preferably, the alkalization temperature is 10 to 25°C.

[0012] (2) Etherification: dissolving a substituted acetic acid or a salt thereof and then adding it dropwise to the alkalized system to carry out an etherification reaction at 5-30° C.; the substituted acetic acid comprises at least one of iodoacetic acid, bromoacetic acid, and chloroacetic acid; and the molar amount thereof is 3.0-10.0 equivalents, preferably 3.5-7.0 equivalents, relative to the chitin repeating unit N-acetyl-D-glucosamine.

[0013] Preferably, substituted chloroacetic acid or its salt is dissolved in an alcohol solution, and the alcohol solution is isopropanol and / or 2-butanol; preferably, the etherification temperature is 10-25°C;

[0014] Based on the principle of halogen exchange, potassium iodide, sodium iodide, etc. can be added to promote the substitution reaction; since the reaction system is heterogeneous, phase transfer catalysts such as sodium dodecyl sulfonate and sodium dodecyl sulfate can be added.

[0015] (3) Crude product separation and purification: The reactants can be precipitated with aqueous ethanol to obtain a crude product, which can then be redissolved and filtered to obtain a crude product filtrate. The crude product filtrate can be precipitated with aqueous ethanol to obtain a refined product. The aqueous ethanol is 75% to 99% ethanol, preferably 80% to 95% ethanol. Aqueous ethanol can remove inorganic salts and precipitate the target product, thereby improving product quality. Similarly, the use of organic solvents such as isopropyl alcohol and acetone can achieve the same effect.

[0016] Alternatively, the crude filtrate may be subjected to ultrafiltration to remove small molecule impurities and then lyophilized.

[0017] Whether in the alkalization step or the etherification step, extending the reaction time objectively promotes deacetylation, thereby increasing the amino group content. Rheological test results show that the elastic modulus and viscous modulus also decrease. Therefore, reaction time is also an important process parameter. Simply controlling the material ratio and reaction temperature cannot ensure that the product has high viscoelasticity. The present invention, through extensive experimental exploration and adjustment of the preparation process parameters, ultimately obtains a highly viscoelastic non-crosslinked carboxymethyl chitin. The high viscoelastic properties of the product are unique. In contrast, the prior art generally uses a far excess of chloroacetic acid and a wide range of reaction temperatures to highly carboxymethylate chitin to improve the water solubility of the product, but the resulting product also does not have (or is not clearly reported to have) good viscoelasticity.

[0018] Based on the above method, the present invention produces non-cross-linked carboxymethyl chitin with high viscoelasticity. Cross-linked carboxymethyl chitin / carboxymethyl chitosan often exhibits high viscoelastic properties. However, based on existing literature reports and test data from the inventors' research team on commercially available carboxymethyl chitin / carboxymethyl chitosan products, no non-cross-linked carboxymethyl chitin / carboxymethyl chitosan with high viscoelasticity has been found.

[0019] The present invention also provides a method for preparing a high-viscoelastic non-cross-linked carboxymethyl chitin hydrogel, which is prepared using the above-mentioned high-viscoelastic carboxymethyl chitin of the present invention. The carboxymethyl chitin content in the hydrogel is 5 to 30 mg / mL; the content can be adjusted according to the physical and chemical performance parameters required for clinical use scenarios. If high viscoelasticity is required (such as a viscoelastic supplement), the carboxymethyl chitin content is increased, and if appropriate fluidity is required (such as postoperative adhesion prevention), the carboxymethyl chitin content is reduced.

[0020] Taking the prepared hydrogel with 2.0% carboxymethyl chitin content as an example, the elastic modulus (2.5Hz) is not less than 132Pa, the viscous modulus (2.5Hz) is not less than 45Pa, and the shear viscosity (10S -1 ) is not less than 20Pa.S. In comparison, the elastic modulus (2.5Hz) of commercially available carboxymethyl chitin derivatives is 4-34Pa, the viscous modulus (2.5Hz) is 4-49Pa, and the shear viscosity (10S -1 ) 0.1~7Pa.S. It can be seen that compared with commercially available carboxymethyl chitin derivative products, the carboxymethyl chitin hydrogel provided by the present invention has higher elastic modulus, viscous modulus and shear viscosity, and can be adapted to different clinical application fields.

[0021] The high-viscoelastic non-cross-linked carboxymethyl chitin hydrogel of the present invention can be used to prepare internal injection preparations, internal filling preparations or external drug carrier preparations.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The high-viscoelastic carboxymethyl chitin hydrogel prepared by the present invention not only maintains the original biocompatibility and other properties of chitin, but also has higher viscoelastic properties and shear viscosity, which are much higher than the existing chitosan products on the market; it can match the performance parameters of sodium hyaluronate anti-adhesion products and intra-articular injection products, has unique application value, and may occupy a place in the application field of sodium hyaluronate in the future, with good application prospects.

[0024] (2) Compared to sodium hyaluronate products with high market development maturity, the highly viscoelastic carboxymethyl chitin hydrogel prepared by the present invention has better metabolic stability (the body contains hyaluronidase, but no carboxymethyl chitin-specific metabolic enzyme). Because the human body does not have a specific metabolic enzyme for carboxymethyl chitin, the carboxymethyl chitin product prepared by the present invention is more stable than hyaluronic acid in the body, which has a potential advantage in terms of long-term product effect.

[0025] (3) The present invention solves the problem that chitin derivatives are difficult to withstand terminal moist heat sterilization. Existing carboxymethyl chitin, carboxymethyl chitosan, and non-cross-linked hyaluronic acid products have poor thermal stability. The corresponding preparations are difficult to withstand terminal moist heat sterilization and can only be controlled by process sterilization. This increases the difficulty of sterilization control of the final product and poses a biosafety risk in clinical use. However, the carboxymethyl chitin hydrogel prepared by the present invention can withstand moist heat sterilization and is highly safe for use.

[0026] (4) The highly viscoelastic carboxymethyl chitin hydrogel prepared by the present invention has good safety. The safety evaluation results conducted at the animal level show that the product of the present invention has good safety.

[0027] (5) The raw material chitin used in the present invention is the second largest natural polysaccharide in nature. Compared with sodium hyaluronate, which has limited resources, the carboxymethyl chitin product provided by the present invention has greater economic advantages and industrialization potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the TGA data of the raw material prepared in Example 3.

[0029] Figure 2 This is a graph showing the elastic modulus (G') and viscous modulus (G") data of a 2.0% hydrogel sample of the raw material prepared in Example 5.

[0030] Figure 3 This is a shear viscosity (η) graph of a 2.0% hydrogel sample of the raw material prepared in Example 5.

[0031] Figure 4 This is the animal intradermal irritation experiment of Example 13. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. Technology and methods known to those of ordinary skill in the relevant art may not be discussed in detail, but in appropriate cases, the technology and methods should be considered as a part of the specification. In all examples shown and discussed here, any specific value should be interpreted as merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments may have different values.

[0034] In the following examples, the experimental methods without specific conditions are generally measured according to national standards; if there is no corresponding national standard, the general standard requirements or general methods are used.

[0035] Example 1

[0036] A method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin, comprising the following steps:

[0037] (1) Alkalization: Weigh 8.00 g of chitin, add it to 80 g of 50% sodium hydroxide aqueous solution, and stir continuously at 15-25° C. to alkalize for 18 h.

[0038] (2) Etherification: Add 80 mL of isopropanol to the alkalized system and stir for about 30 min. Weigh 13.00 g (3.5 equivalents) of chloroacetic acid and dissolve it in 32 mL of isopropanol. Adjust the reaction system temperature below 25°C and control the temperature at 10-25°C. Add the chloroacetic acid solution in isopropanol dropwise for 1.2 h. After the addition is complete, stir and react at 19-25°C for 46 h.

[0039] (3) Crude product separation and purification: 800 mL of 80% ethanol was added to the reactant, stirred for 10 min, allowed to stand, and the supernatant was decanted. Then, 95% ethanol was added, stirred for 10 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0040] The crude carboxymethyl chitin was redissolved in 5 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The product was filtered and the filter cake was dried under reduced pressure to obtain 3.26 g of white powdery solid carboxymethyl chitin.

[0041] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0042] Example 2

[0043] A method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin, comprising the following steps:

[0044] (1) Alkalization: Weigh 8.01 g of chitin and add it to 80 g of a 50% sodium hydroxide aqueous solution. Stir continuously at 15-25° C. to alkalinize for 17.5 h.

[0045] (2) Etherification: Add 80 mL of isopropanol to the alkalized system and stir for about 30 min. Weigh 18.58 g (5.0 equivalents) of chloroacetic acid and dissolve it in 32 mL of isopropanol. Adjust the reaction system temperature below 25°C and control the temperature at 10-25°C. Add the chloroacetic acid solution in isopropanol dropwise for 0.6 h. After the addition is complete, stir and react at 19-25°C for 46 h.

[0046] (3) Crude product separation and purification: 800 mL of 80% ethanol was added to the reactant, stirred for 10 min, allowed to stand, and the supernatant was decanted. Then, 95% ethanol was added, stirred for 10 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0047] The crude carboxymethyl chitin was redissolved in 5 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The filter cake was filtered and dried under reduced pressure to obtain 6.37 g of white powdery solid carboxymethyl chitin.

[0048] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0049] Example 3

[0050] A method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin, comprising the following steps:

[0051] (1) Alkalization: Weigh 16.00 g of chitin, add it to 160 g of a 50% sodium hydroxide aqueous solution, and continue stirring at 20° C. to alkalize for 23.2 h.

[0052] (2) Etherification: Add 0.48 g of sodium lauryl sulfate and 160 mL of isopropanol to the alkalized system and stir for about 30 min. Weigh 37.19 g (5.0 equivalents) of chloroacetic acid and dissolve it in 64 mL of isopropanol. Adjust the reaction system temperature below 25°C and control the temperature at 10-25°C. Add the chloroacetic acid solution in isopropanol dropwise for 1.0 h. After the addition is complete, stir and react at 20°C for 18 h.

[0053] (3) Crude product separation and purification: 1.6 L of 80% ethanol was added to the reactant, stirred for 10 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 10 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0054] The crude carboxymethyl chitin was re-dissolved in 10 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The filter cake was then filtered and dried under reduced pressure to obtain 10.25 g of white powdery solid carboxymethyl chitin. The TGA data of the product is shown in the figure below. Figure 1 shown.

[0055] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0056] Example 4

[0057] A method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin, comprising the following steps:

[0058] (1) Alkalization: Weigh 16.00 g of chitin, add it to 160 g of a 50% sodium hydroxide aqueous solution, and continue stirring at 20° C. to alkalize for 18.5 h.

[0059] (2) Etherification: Add 0.62 g of potassium iodide and 160 mL of isopropanol to the alkalized system and stir for about 30 min. Weigh 37.17 g (5.0 equivalents) of chloroacetic acid and dissolve it in 64 mL of isopropanol. Adjust the reaction system temperature to below 25°C and control the temperature at 10-25°C. Add the chloroacetic acid solution in isopropanol dropwise for 6.0 h. After the addition is complete, stir and react at 20°C for 41 h.

[0060] (3) Crude product separation and purification: 1.6 L of 80% ethanol was added to the reactant, stirred for 10 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 10 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0061] The crude carboxymethyl chitin was redissolved in 10 L of purified water and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The product was filtered and the filter cake was dried under reduced pressure to obtain 12.00 g of white powdery solid carboxymethyl chitin.

[0062] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0063] Example 5

[0064] A method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin, comprising the following steps:

[0065] (1) Alkalization: Weigh 16.00 g of chitin, add it to 160 g of a 50% sodium hydroxide aqueous solution, and continue stirring at 10° C. to alkalize for 18.5 h.

[0066] (2) Etherification: Add 0.48 g of sodium lauryl sulfate and 160 mL of isopropanol to the alkalized system and stir for about 30 min. Weigh 37.18 g (5.0 equivalents) of chloroacetic acid and dissolve it in 64 mL of isopropanol. Adjust the reaction system temperature below 25°C and control the temperature at 10-25°C. Add the chloroacetic acid solution in isopropanol dropwise for 6.0 h. After the addition is complete, stir and react at 20°C for 41 h.

[0067] (3) Crude product separation and purification: 1.6 L of 80% ethanol was added to the reactant, stirred for 10 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 10 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0068] The crude carboxymethyl chitin was redissolved in 10 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The product was filtered and the filter cake was dried under reduced pressure to obtain 11.93 g of white powdery solid carboxymethyl chitin.

[0069] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0070] Example 6

[0071] A method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin, comprising the following steps:

[0072] (1) Alkalization: Weigh 16.00 g of chitin, add it to 160 g of a 50% sodium hydroxide aqueous solution, and stir continuously at 10° C. to alkalinize for 18 h.

[0073] (2) Etherification: Add 160 mL of isopropanol to the alkalized system and stir for about 30 min. Weigh 37.18 g (5.0 equivalents) of chloroacetic acid and dissolve it in 64 mL of isopropanol. Adjust the reaction system temperature below 25°C and control the temperature at 10-25°C. Add the chloroacetic acid solution in isopropanol dropwise for 6.1 h. After the addition is complete, stir and react at 20°C for 65 h.

[0074] (3) Crude product separation and purification: 1.6 L of 80% ethanol was added to the reactant, stirred for 10 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 10 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0075] The crude carboxymethyl chitin was redissolved in 10 L of purified water and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then freeze-dried to obtain 10.49 g of white spongy solid carboxymethyl chitin.

[0076] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0077] Example 7

[0078] A method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin, comprising the following steps:

[0079] (1) Alkalization: Weigh 16.00 g of chitin, add it to 160 g of a 50% sodium hydroxide aqueous solution, and continue stirring at 10° C. to alkalize for 17.7 h.

[0080] (2) Etherification: Add 160 mL of isopropanol to the alkalized system and stir for approximately 30 min. Weigh 37.18 g (5.0 equivalents) of chloroacetic acid and dissolve it in 64 mL of isopropanol. Adjust the reaction system temperature below 25°C and control the temperature at 10-25°C. Add the chloroacetic acid solution in isopropanol dropwise for 5.2 h. After the addition is complete, stir and react at 10°C for 40.7 h.

[0081] (3) Crude product separation and purification: 1.6 L of 80% ethanol was added to the reactant, stirred for 10 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 10 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0082] The crude carboxymethyl chitin was redissolved in 10 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The product was filtered and the filter cake was dried under reduced pressure to obtain 10.66 g of white powdery solid carboxymethyl chitin.

[0083] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0084] Example 8

[0085] A method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin, comprising the following steps:

[0086] (1) Alkalization: Weigh 12.01 g of chitin, add it to 120 g of a 40% sodium hydroxide aqueous solution, and continue stirring at 20° C. to alkalize for 16.5 h.

[0087] (2) Etherification: Add 80 mL of isopropanol to the alkalized system and stir for about 30 minutes. Weigh 27.88 g (5.0 equivalents) of chloroacetic acid and dissolve it in 48 mL of isopropanol. Adjust the reaction system temperature to below 20°C and control the temperature at 5-20°C. Add the chloroacetic acid solution in isopropanol dropwise for 6 hours. After the addition is complete, stir and react at 20°C for 41 hours.

[0088] (3) Crude product separation and purification: 600 mL of 80% ethanol was added to the reactant, stirred for 10 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 10 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0089] The crude carboxymethyl chitin was redissolved in 7.5 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The product was filtered and the filter cake was dried under reduced pressure to obtain 7.77 g of white powdery solid carboxymethyl chitin.

[0090] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0091] Example 9

[0092] A method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin, comprising the following steps:

[0093] (1) Alkalization: Weigh 12.01 g of chitin, add it to 120 g of a 50% sodium hydroxide aqueous solution, and stir continuously at 20° C. to alkalinize for 17 h.

[0094] (2) Etherification: Add 0.36 g of sodium dodecylsulfonate and 120 mL of isopropanol to the alkalized system and stir for about 30 min. Weigh 27.90 g (5.0 equivalents) of chloroacetic acid and dissolve it in 48 mL of isopropanol. Adjust the reaction system temperature below 20°C and control the temperature at 8-20°C. Add the chloroacetic acid solution in isopropanol dropwise for 6 h. After the addition is complete, stir and react at 20°C for 41 h.

[0095] (3) Crude product separation and purification: 1.2 L of 80% ethanol was added to the reactant, stirred for 20 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 20 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0096] The crude carboxymethyl chitin was redissolved in 7.5 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The product was filtered and the filter cake was dried under reduced pressure to obtain 8.79 g of white powdery solid carboxymethyl chitin.

[0097] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0098] Example 10

[0099] A method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin, comprising the following steps:

[0100] (1) Alkalization: Weigh 11.99 g of chitin, add it to 120 g of a 50% sodium hydroxide aqueous solution, and stir continuously at 20° C. to alkalinize for 17 h.

[0101] (2) Etherification: Add 120 mL of isopropanol to the alkalized system and stir for about 30 minutes. Weigh 39.03 g (7.0 equivalents) of chloroacetic acid and dissolve it in 48 mL of isopropanol. Adjust the reaction system temperature below 20°C and control the temperature at 8-20°C. Add the chloroacetic acid solution in isopropanol dropwise for 6.3 hours. After the addition is complete, stir and react at 20°C for 40.5 hours.

[0102] (3) Crude product separation and purification: 1.2 L of 80% ethanol was added to the reactant, stirred for 20 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 20 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0103] The crude carboxymethyl chitin was redissolved in 7.5 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The product was filtered and the filter cake was dried under reduced pressure to obtain 7.46 g of white powdery solid carboxymethyl chitin.

[0104] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G"), and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0105] In order to highlight the beneficial effects of the present invention, the following comparative example experiments are given.

[0106] Comparative Example 1

[0107] A method for preparing carboxymethyl chitin, comprising the following steps:

[0108] (1) Alkalization: Weigh 8.00 g of chitin, add it to 80 g of a 50% sodium hydroxide aqueous solution, and continue stirring at 20° C. to alkalize for 18.0 h.

[0109] (2) Etherification: Add 80 mL of isopropanol to the alkalized system and stir for about 30 minutes. Weigh 52.00 g (14.0 equivalents) of chloroacetic acid and dissolve it in 32 mL of isopropanol. Adjust the reaction system temperature to below 20°C and control the temperature at 10-20°C. Add the chloroacetic acid solution in isopropanol dropwise for 1.0 hour. After the addition is complete, stir and react at 20°C for 70 hours.

[0110] (3) Crude product separation and purification: 800 mL of 80% ethanol was added to the reactant, stirred for 10 min, allowed to stand, and the supernatant was decanted. Then, 95% ethanol was added, stirred for 10 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0111] The crude carboxymethyl chitin was redissolved in 5 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The filter cake was filtered and dried under reduced pressure to obtain 8.92 g of white powdery solid carboxymethyl chitin.

[0112] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G") and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0113] Comparative Example 2

[0114] A method for preparing carboxymethyl chitin, comprising the following steps:

[0115] (1) Alkalization: Weigh 12.01 g of chitin, add it to 120 g of a 50% sodium hydroxide aqueous solution, and continue stirring at 20° C. to alkalize for 18.0 h.

[0116] (2) Etherification: Add 120 mL of purified water to the alkalized system and stir for about 30 min. Weigh 27.92 g (5 equivalents) of chloroacetic acid and dissolve it in 48 mL of purified water. Adjust the reaction system temperature to below 20°C and add the isopropanol solution of chloroacetic acid dropwise at a temperature of 5-20°C over 6 h. After the addition is complete, stir and react at 20°C for 40 h.

[0117] (3) Crude product separation and purification: 1200 mL of 80% ethanol was added to the reactant, stirred for 15 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 15 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0118] The crude carboxymethyl chitin was redissolved in 7.5 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The product was filtered and the filter cake was dried under reduced pressure to obtain 5.05 g of white powdery solid carboxymethyl chitin.

[0119] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G") and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0120] Comparative Example 3

[0121] A method for preparing carboxymethyl chitin, comprising the following steps:

[0122] (1) Alkalization: Weigh 16.04 g of chitin and 80.47 g of sodium hydroxide, add them to 240 mL of isopropanol, and continue stirring at 20°C to alkalize for 20.0 h.

[0123] (2) Etherification: Add 80 mL of isopropanol to the alkalized system and stir for about 30 min. Weigh 37.16 g (5.0 equivalents) of chloroacetic acid and dissolve it in 64 mL of isopropanol. Adjust the reaction system temperature to below 20°C and control the temperature at 10-20°C. Add the chloroacetic acid solution in isopropanol dropwise for 0.9 h. After the addition is complete, stir and react at 20°C for 92 h.

[0124] (3) Crude product separation and purification: Filter by suction, and dry the filter cake under reduced pressure to obtain crude carboxymethyl chitin.

[0125] The crude carboxymethyl chitin was redissolved in 3.6 L of phosphate buffer, but a large amount of solid remained insoluble. The solution was filtered through a 0.22 μm PP filter membrane, and the filtrate was concentrated by ultrafiltration. 95% ethanol was added dropwise to precipitate the product. The product was filtered and the filter cake was dried under reduced pressure to obtain 3.13 g of white powdery solid carboxymethyl chitin.

[0126] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G") and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0127] Comparative Example 4

[0128] A method for preparing carboxymethyl chitin, comprising the following steps:

[0129] (1) Alkalization: Weigh 12.00 g of chitin, add it to 102 g of a 35% sodium hydroxide aqueous solution, and continue stirring at 10° C. to alkalize for 17 h.

[0130] (2) Etherification: Add 120 mL of isopropanol to the alkalized system and stir for about 30 minutes. Weigh 27.89 g (5.0 equivalents) of chloroacetic acid and dissolve it in 48 mL of isopropanol. Adjust the reaction system temperature to below 20°C and control the temperature at 8-20°C. Add the chloroacetic acid solution in isopropanol dropwise for 6 hours. After the addition is complete, stir and react at 20°C for 41 hours.

[0131] (3) Crude product separation and purification: 1.2 L of 80% ethanol was added to the reactant, stirred for 20 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 20 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0132] The crude carboxymethyl chitin was redissolved in 7.5 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The product was filtered and the filter cake was dried under reduced pressure to obtain 12.77 g of white powdery solid carboxymethyl chitin.

[0133] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G") and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0134] Comparative Example 5

[0135] A method for preparing carboxymethyl chitin, comprising the following steps:

[0136] (1) Alkalization: Weigh 12.06 g of chitin, add it to 120 g of a 50% sodium hydroxide aqueous solution, and stir continuously at 35° C. to alkalinize for 18 h.

[0137] (2) Etherification: Add 120 mL of isopropanol to the alkalized system and stir for about 30 minutes. Weigh 27.90 g (5.0 equivalents) of chloroacetic acid and dissolve it in 48 mL of isopropanol. Adjust the reaction system temperature to below 20°C and control the temperature at 5-20°C. Add the chloroacetic acid solution in isopropanol dropwise for 6 hours. After the addition is complete, stir and react at 35°C for 40 hours.

[0138] (3) Crude product separation and purification: 1.2 L of 80% ethanol was added to the reactant, stirred for 10 min, allowed to stand, and the supernatant was decanted. Then 95% ethanol was added, stirred for 10 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0139] The crude carboxymethyl chitin was redissolved in 7.5 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The product was filtered and the filter cake was dried under reduced pressure to obtain 7.07 g of white powdery solid carboxymethyl chitin.

[0140] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G") and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0141] Comparative Example 6

[0142] A method for preparing carboxymethyl chitin, comprising the following steps:

[0143] (1) Alkalization: Weigh 12.00 g of chitin, add it to 120 g of a 50% potassium hydroxide aqueous solution, and continue stirring at 20° C. to alkalize for 16.5 h.

[0144] (2) Etherification: Add 120 mL of isopropanol to the alkalized system and stir for about 30 minutes. Weigh 27.90 g (5.0 equivalents) of chloroacetic acid and dissolve it in 48 mL of isopropanol. Adjust the reaction system temperature to below 20°C and control the temperature at 5-20°C. Add the chloroacetic acid solution in isopropanol dropwise for 6 hours. After the addition is complete, stir and react at 20°C for 41 hours.

[0145] (3) Crude product separation and purification: 600 mL of 80% ethanol was added to the reactant, stirred for 15 min, allowed to stand, and the supernatant was decanted. Then, 95% ethanol was added, stirred for 15 min, allowed to stand, filtered, and dried under reduced pressure to obtain crude carboxymethyl chitin.

[0146] The crude carboxymethyl chitin was redissolved in 7.5 L of phosphate buffer and filtered through a 0.22 μm PP filter membrane. The filtrate was concentrated by ultrafiltration and then 95% ethanol was added dropwise to precipitate the product. The product was filtered and the filter cake was dried under reduced pressure to obtain 8.25 g of white powdery solid carboxymethyl chitin.

[0147] A 2.0% carboxymethyl chitin solution was prepared using phosphate buffer solution. The elastic modulus (G'), viscous modulus (G") and shear viscosity (η) of the solution were measured using a rheometer. The data are shown in Table 1.

[0148] Example 11

[0149] The carboxymethyl chitin prepared in Examples 1-10 and Comparative Examples 1-6 was prepared into hydrogels with a carboxymethyl chitin content of 2% (without drying, the water content of the carboxymethyl chitin was 12% to 15%) using a PBS solution. The elastic modulus G' and viscous modulus G" at a frequency of 2.5 Hz and the viscosity modulus G" at a frequency of 10 s were measured using a rheometer (rotational rheometer, model HAAKE RS1, manufacturer Thermo Scientific). -1 The shear viscosity η under 0.05 % is shown in Table 1.

[0150] Table 1 Rheological test results of carboxymethyl chitin hydrogel and commercial products

[0151]

[0152]

[0153] (Note: Commercially available products 1 to 3 are carboxymethyl chitin derivatives, and commercial products 4 to 9 are cross-linked sodium hyaluronate products. Commercial product 1 is Shuyining, manufactured by Shijiazhuang Yishengtang Medical Products Co., Ltd., batch number 20240505; commercial product 2 is Qitejie, manufactured by Shanghai Qisheng Biological Products Co., Ltd., batch number M2311CH71; commercial product 3 is KioMedine VS One, manufactured by KiOmed Pharma SA, batch number 14KA231025; marketed product 4 is Synvisc, manufactured by Genzyme Corporation, batch number CRSP006B; marketed product 5 is HyruanONE, manufactured by LG Chem, Ltd., batch number SIH22068; marketed product 6 is Haimei, manufactured by Shanghai Qisheng Biological Products Co., Ltd., batch number Q2305QS11; marketed product 7 is Gong Ankang, manufactured by Changzhou Bairuiji Biopharmaceutical Co., Ltd., batch number 2311010; marketed product 8 is Hai Ruiji, manufactured by Changzhou Bairuiji Biopharmaceutical Co., Ltd., batch number 2302008; marketed product 9 is Gong Weijia, manufactured by Zhejiang Jingjia Medical Technology Co., Ltd., batch number JG240408.

[0154] As can be seen from Table 1, the modulus data and shear viscosity data of the 2.0% hydrogel prepared with carboxymethyl chitin obtained under different reaction conditions before sterilization are in a wide range. In the embodiment: the elastic modulus is 132-286 Pa, the viscous modulus is 45-76 Pa, and the shear viscosity is 20-42 Pa.S; while in the comparative example, the elastic modulus is 6-23 Pa, the viscous modulus is 2-36 Pa, and the shear viscosity is 0.3-4 Pa.S. The parameter range of commercially available products varies greatly depending on their clinical uses: the elastic modulus is 4-266 Pa, the viscous modulus is 4-58 Pa, and the shear viscosity is 0.1-19 Pa.S; among them, the elastic modulus of the carboxymethyl chitin derivative product is 4-34 Pa, the viscous modulus is 4-49 Pa, and the shear viscosity is 0.1-7 Pa.S. Among them, the elastic modulus (G') and the viscous modulus (G") of the 2.0% hydrogel sample of the raw material prepared in Example 5 are as follows: Figure 2 The shear viscosity (η) of the 2.0% hydrogel sample of the raw material prepared in Example 5 is shown as Figure 3 shown.

[0155] It can be seen that compared with commercially available products, especially compared with commercially available carboxymethyl chitin derivative products, the carboxymethyl chitin hydrogel provided by the present invention has higher elastic modulus, viscous modulus and shear viscosity, and can be adapted to different clinical application fields.

[0156] Example 12

[0157] In this case, the carboxymethyl chitin obtained in Example 5 was formulated into a prescription preparation, and its stability under moist heat sterilization was investigated.

[0158] Carboxymethyl chitin was prepared into a hydrogel according to the formulation in Table 2, and the hydrogel was dispensed into multiple vials, stoppered, and capped. The vials were sterilized with moist heat at 121°C (sanitary sterilizer, model YXQ.WG-203, Zhangjiagang Valin Medical Equipment Co., Ltd.) for 8 to 14 min. The elastic modulus G' and viscous modulus G" of the sterilized products were tested at a frequency of 2.5 Hz, as well as the viscosity modulus G" at a frequency of 10 s. -1 The shear viscosity η under 0.05 % is shown in Table 3.

[0159] Table 2 Carboxymethyl chitin hydrogel formulations

[0160]

[0161] Table 3 Rheological test results of carboxymethyl chitin hydrogel

[0162]

[0163] The rheological test results of the carboxymethyl chitin hydrogel in Table 3 are as follows:

[0164] (1) The carboxymethyl chitin hydrogel provided by the present invention has excellent wet heat sterilization stability and can withstand wet heat sterilization at 121°C for 14 minutes, which can meet the wet heat sterilization requirements of the product terminal.

[0165] (2) The corresponding formulations with different carboxymethyl chitin contents exhibit different viscoelasticity and shear viscosity. Compared with the rheological data of the commercially available products in Table 1, the products obtained after moist heat sterilization of formulations with different carboxymethyl chitin contents can be compared with the commercially available cross-linked sodium hyaluronate products for viscoelastic and anti-adhesion applications, respectively, and have good development and application prospects.

[0166] Example 13 Animal skin test of prescription preparation

[0167] Intradermal irritation tests were conducted using New Zealand male rabbits, with reference to GB / T 16886.10-2017, "Requirements for Biological Irritation Evaluation." The dorsal skin of the New Zealand male rabbits was depilated, and the sample was injected intradermally with a syringe at a rate of 200 μL per injection site, five injections per sample. After injection, the samples were observed and scored 24, 48, and 72 hours later. The results are shown in Table 4. Conclusion: The carboxymethyl chitin hydrogel samples provided by this invention all achieved intradermal irritation scores of less than 1.0, meeting the requirements of GB / T 16886.10-2017, "Biological Irritation Evaluation."

[0168] The test product information is as follows:

[0169] Test sample No. 1: Figure 4 Upper left panel: Sample of Formulation 1 (1.5% carboxymethyl chitin hydrogel) of Example 12 sterilized for 14 minutes.

[0170] Test sample No. 2: Figure 4 Lower left panel: Sample of Formulation 2 (2.0% carboxymethyl chitin hydrogel) of Example 12 sterilized for 14 minutes.

[0171] Normal saline: Figure 4 Upper right panel, negative control group, 0.9% sodium chloride injection.

[0172] Test product No. 3: Figure 4 Lower right panel, 1.5% sorbitol in PBS solution.

[0173] Table 4 Animal skin test 72h scoring data

[0174] Test sample Test sample No. 1 Test sample No. 2 Normal saline Test product No. 3 score 0.11 0.12 0 0

[0175] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin, characterized in that: The steps include: (1) Alkalization: suspend chitin in sodium hydroxide aqueous solution and stir at 5-30°C to alkalize; (2) Etherification: dissolve substituted acetic acid or its salt and add dropwise to the alkalized system at 5-30°C for etherification reaction; (3) Crude product separation and purification.

2. The method for preparing highly viscoelastic carboxymethyl chitin according to claim 1, wherein: The mass percentage of sodium hydroxide in the sodium hydroxide aqueous solution is 40% to 55%, preferably 45% to 55%; the mass ratio of chitin to the sodium hydroxide aqueous solution is 1.0:5.0 to 1.0:25.0, preferably 1.0:7.0 to 1.0:15.

0.

3. The method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin according to claim 1, wherein: In the step (1), the alkalization temperature is preferably 10-25°C.

4. The method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin according to claim 1, wherein: The substituted acetic acid includes at least one of iodoacetic acid, bromoacetic acid and chloroacetic acid; and its molar amount is 3.0 to 10.0 equivalents, preferably 3.5 to 7.0 equivalents, relative to the chitin repeating unit N-acetyl-D-glucosamine.

5. The method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin according to claim 4, wherein: Substituted chloroacetic acid or its salt is dissolved in an alcohol solution, wherein the alcohol solution is isopropanol and / or 2-butanol; the etherification temperature is preferably 10-25°C; In the step (2), based on the halogen exchange principle, potassium iodide or sodium iodide can be added to promote the substitution reaction; since the reaction system is heterogeneous, a phase transfer catalyst can be added, and the phase transfer catalyst includes sodium dodecyl sulfonate or sodium dodecyl sulfate.

6. The method for preparing highly viscoelastic non-cross-linked carboxymethyl chitin according to claim 1, wherein: The step (3) specifically comprises: precipitating the reactants with an organic solvent to obtain a crude product, then re-dissolving the crude product and filtering it to obtain a crude product filtrate; precipitating the crude product filtrate with an organic solvent to obtain a refined product; the organic solvent is at least one of hydrous ethanol, isopropanol, and acetone; the hydrous ethanol is 75% to 99% ethanol, preferably 80% to 95% ethanol; Alternatively, the crude filtrate is ultrafiltered to remove small molecule impurities and then lyophilized.

7. Highly viscoelastic non-cross-linked carboxymethyl chitin prepared according to any one of the preparation methods of claims 1 to 6.

8. A method for preparing a highly viscoelastic non-crosslinked carboxymethyl chitin hydrogel, characterized by: The hydrogel is prepared using the highly viscoelastic non-cross-linked carboxymethyl chitin according to claim 7, wherein the content of carboxymethyl chitin in the hydrogel is 5 to 30 mg / mL; The content can be adjusted according to the physical and chemical performance parameters required for clinical use scenarios. If high viscoelasticity is required, the content of carboxymethyl chitin can be increased, and if appropriate fluidity is required, the content of carboxymethyl chitin can be reduced.

9. The highly viscoelastic non-cross-linked carboxymethyl chitin hydrogel prepared by the preparation method of claim 8, characterized in that: The elastic modulus (2.5Hz) of the hydrogel with 2.0% carboxymethyl chitin content is not less than 132Pa, the viscous modulus (2.5Hz) is not less than 45Pa, and the shear viscosity (10S -1 ) is not less than 20Pa.S.

10. Use of the highly viscoelastic carboxymethyl chitin hydrogel according to claim 9 in the preparation of an internal injection preparation, an internal filling preparation or an external drug carrier preparation.

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