Preparation method of temperature-sensitive submucosal injection based on freeze-dried particles for endoscopic submucosal dissection

Abstract

The invention discloses a preparation method of a temperature-sensitive submucosal injection based on freeze-dried particles for endoscopic submucosal dissection, and belongs to the technical field of regenerative medicine. The injection comprises chitosan-polyvinylpyrrolidone freeze-dried particles and beta-sodium glycerophosphate freeze-dried particles, can be stored for a long time, is convenient to transport and is simple and convenient to use. When in use, the two freeze-dried particles can be respectively and quickly dissolved in low-temperature injection water, and the low-temperature submucosal injection is formed after the two freeze-dried particles are mixed. The low-temperature injection is very low in viscosity, does not need to use a special injection device, can be used for submucosal injection by using a conventional endoscope injection needle which is clinically used at present, and is high in injection speed. Meanwhile, after the low-temperature injection is injected under the mucous membrane layer, high-strength hydrogel can be rapidly formed under the action of body temperature, the upheaval state of the mucous membrane layer is more stably supported, ESD operation is facilitated, intraoperative and postoperative bleeding can be reduced, postoperative wound healing is promoted, and the low-temperature injection has important clinical practical application significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of regenerative medicine, and particularly relates to a preparation method and application of a lyophilized particle-based temperature-sensitive submucosal injection for endoscopic submucosal dissection. BACKGROUND

[0002] Early lesions of the digestive tract are mainly located in the mucosa and submucosa, and endoscopic submucosal dissection (ESD) has become the main diagnosis and treatment method for early lesions of the digestive tract. Perforation and hemorrhage are common complications of ESD, which limits the promotion of ESD. In order to reduce complications, a submucosal injection is usually injected under the mucosa of the lesion to make the mucosa rise, so as to lift the lesion mucosa and separate it from the muscle layer, which provides the operator with a better view and operating space to reduce damage to blood vessels and avoid injury to the muscle layer.

[0003] At present, the commonly used submucosal injection in clinical practice is a solution type injection containing medical dyes such as methylene blue, hydroxyethyl starch and hyaluronic acid. They quickly spread to the surrounding after injection and are absorbed by the tissue, which significantly reduces the height of the mucosa, and repeated injection is required during the operation, which increases the difficulty of operation.

[0004] The chitosan-beta-glycerophosphate sodium temperature-sensitive gel can be injected into the mucosa under the endoscopic injection needle due to its liquid state at low temperature, and can quickly change to a gel state under the action of body temperature, thereby better lifting the mucosa, and repeated injection is not required during the operation, which is expected to be used as a new type of endoscopic submucosal injection.

[0005] Since the chitosan-beta-glycerophosphate sodium low-temperature mixed solution cannot be stored for a long time, it needs to be prepared using a chitosan solution and a beta-glycerophosphate sodium solution before use. However, long-term storage of the chitosan solution may cause degradation of chitosan, thereby affecting the gelation performance of the injection. In addition, long-term storage of the beta-glycerophosphate sodium solution may cause precipitation, affecting its use concentration. Therefore, it can be seen that the current chitosan-beta-glycerophosphate sodium temperature-sensitive gel submucosal injection cannot meet the use requirements of ESD, and it is urgent to develop a new submucosal injection to overcome the defects of the prior art, improve the storage stability of the injection components, and facilitate clinical preparation and use, while maintaining its mucosa lifting effect. SUMMARY

[0006] In view of the problems existing in the prior art, the present application provides a preparation method and application of a lyophilized particle-based temperature-sensitive submucosal injection for endoscopic submucosal dissection, which aims to improve the storage stability of the components of the injection, and facilitate clinical preparation and use, while maintaining its mucosa lifting effect.

[0007] To achieve the above-mentioned purposes, the present application provides the following technical solutions.

[0008] The present application provides a preparation method of a lyophilized particle-based temperature-sensitive submucosal injection solution for endoscopic submucosal dissection, characterized in that the method comprises the following steps: S1. Under stirring, a polyvinylpyrrolidone solution is added to a chitosan solution with a pH value of 5.5-6.2 to prepare a mixed solution A, and the mass concentration ratio of polyvinylpyrrolidone to chitosan in the mixed solution A is 3-7; S2. The mixed solution A is finally frozen at-80℃ to constant temperature through gradient cooling, and then freeze-dried to obtain a chitosan-polyvinylpyrrolidone lyophilized product B; S3. The chitosan-polyvinylpyrrolidone lyophilized product B is ground, and then larger particles are removed through sieving to obtain chitosan-polyvinylpyrrolidone lyophilized particles C that can pass through the sieve; S4. Under stirring, β-glycerophosphate sodium is dissolved in water for injection to obtain a β-glycerophosphate sodium solution D; S5. The β-glycerophosphate sodium solution D is finally frozen at-80℃ to constant temperature through gradient cooling, and then freeze-dried to obtain a β-glycerophosphate sodium lyophilized product E; S6. The β-glycerophosphate sodium lyophilized product E is ground, and then larger particles are removed using a sieve to obtain β-glycerophosphate sodium lyophilized particles F that can pass through the sieve; S7. The chitosan-polyvinylpyrrolidone lyophilized particles C are fully dissolved using water for injection containing a medical coloring indicator, and placed in an ice bath to obtain a redissolved low-temperature chitosan-polyvinylpyrrolidone solution G; S8. The β-glycerophosphate sodium lyophilized particles F are fully dissolved using water for injection containing a medical coloring indicator, and placed in an ice bath to obtain a redissolved low-temperature β-glycerophosphate sodium solution H; S9. The low-temperature β-glycerophosphate sodium solution H is added to the low-temperature chitosan-polyvinylpyrrolidone solution G, and mixed uniformly to obtain a low-temperature temperature-sensitive submucosal injection solution.

[0009] Further, the degree of deacetylation of chitosan in step S1 is 95% or higher, and the molecular weight is 5000-400000 Da; the molecular weight of polyvinylpyrrolidone is 10000-400000 Da.

[0010] Further, the temperature of stirring in step S1 is room temperature-37℃, the stirring rate is 500-2000 rpm, and the stirring time is 20-40 minutes.

[0011] Further, the gradient cooling in step S2 is that the mixed solution A is placed in -20℃ and -40℃ in sequence to constant temperature, and finally frozen in -80℃ to constant temperature.

[0012] Further, the freeze-drying condition in step S2 is -50℃ to -30℃, and the vacuum degree is 10 to 40 Pa.

[0013] Further, the grinding medium in the grinding condition in step S3 is zirconium oxide, and the grinding time is 10 to 30 minutes.

[0014] Further, the freeze-dried particles C of chitosan-polyvinylpyrrolidone that can pass through the screen in step S3 is obtained by screening the ground particles with a 65 to 220 mesh screen to obtain smaller particles that can pass through the screen.

[0015] Further, the stirring rate in step S4 is 1000 to 2000 rpm, and the stirring time is 30 to 60 minutes.

[0016] Further, the gradient cooling in step S5 is that the solution D is placed in -20℃ and -40℃ in sequence to constant temperature, and finally frozen in -80℃ to constant temperature.

[0017] Further, the freeze-drying condition in step S5 is -50℃ to -30℃, and the vacuum degree is 10 to 40 Pa.

[0018] Further, the grinding medium in the grinding condition in step S6 is zirconium oxide, and the grinding time is 10 to 30 minutes.

[0019] Further, the freeze-dried particles F of β-glycerophosphate sodium that can pass through the screen in step S6 is obtained by screening the ground particles with a 35 to 150 mesh screen to obtain smaller particles that can pass through the screen.

[0020] Further, the medical coloring indicator in steps S7 and S8 is one or a combination of methylene blue and indigo carmine.

[0021] Further, the concentration of chitosan in the low-temperature temperature-sensitive submucosal injection solution in step S9 is 0.3 to 1% (w / v, g / mL), the concentration of polyvinylpyrrolidone is 0.9 to 7% (w / v, g / mL), the concentration of β-glycerophosphate sodium is 5 to 10% (w / v, g / mL), and the concentration of the medical coloring indicator is 0.001 to 0.04% (w / v, g / mL).

[0022] The application also provides a use of the freeze-dried particle-based temperature-sensitive submucosal injection solution prepared by any of the above methods in the preparation of a submucosal injection for endoscopic submucosal dissection.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0024] 1. The temperature-sensitive submucosal injection solution based on lyophilized particles proposed in this invention consists of two types of lyophilized particles: chitosan-polyvinylpyrrolidone lyophilized particles and β-glycerophosphate sodium lyophilized particles. These are reconstituted and mixed before use to obtain a low-temperature endoscopic submucosal injection solution. The lyophilized granulated injection components avoid the instability phenomena such as degradation and precipitation that may occur in chitosan solution and β-glycerophosphate sodium solution during long-term storage, thus improving their storage stability. Furthermore, the lyophilized granulated formulation significantly reduces volume, which is also beneficial for transportation and storage.

[0025] 2. The two types of lyophilized particles in the thermosensitive submucosal injection solution based on lyophilized particles proposed in this invention can be rapidly dissolved in water for injection, forming two-component solutions. In particular, the chitosan-polyvinylpyrrolidone lyophilized particles exhibit excellent water solubility. However, chitosan solutions alone, even after lyophilization, still require relatively strong acidic conditions to dissolve. Furthermore, the dissolved chitosan solution is excessively acidic, resulting in a high degree of protonation of the chitosan, which prevents thermosensitive gelation when mixed with sodium β-glycerophosphate solution, significantly worse than un-lyophilized chitosan solutions of the same concentration. This invention addresses this issue by adding polyvinylpyrrolidone to induce hydrogen bonding between the chitosan and polyvinylpyrrolidone, thereby disrupting the intermolecular hydrogen bonds of chitosan to a certain extent. This significantly increases the water solubility of the composite lyophilized particles, allowing for rapid dissolution in water for injection while maintaining their thermosensitive gelation properties when mixed with sodium β-glycerophosphate. Therefore, this invention solves the key problems of difficult resolution and inability to form thermosensitive gels from individual chitosan lyophilized particles.

[0026] 3. In addition to enabling chitosan-polyvinylpyrrolidone lyophilized particles to dissolve in water for injection, the polyvinylpyrrolidone used in this invention can also significantly improve the integrity and mechanical strength of the gel, thereby improving the maintenance effect of mucosal elevation after submucosal injection.

[0027] 4. The two types of lyophilized granules of this invention can be separately packaged in two sterile vials. In use, the granules can be dissolved and mixed evenly by injecting low-temperature water for injection containing a medical color indicator into the vial using a standard clinical syringe, thus obtaining the injection solution, which can then be injected. This avoids the problem of requiring sterile stirring and mixing conditions for conventional liquid injection solutions.

[0028] 5. The temperature-sensitive submucosal injection solution based on lyophilized particles proposed in this invention has a very low viscosity, does not require the use of special injection devices, and can be injected using conventional endoscopic injection needles currently used in clinical practice. Moreover, it has a high injection rate, rapid mucosal elevation, fast gelation, and slow mucosal collapse, eliminating the need for repeated injections. It can meet the specific needs of ESD surgery, greatly reduce the difficulty of the surgery, significantly shorten the operation time, and reduce complications.

[0029] 6. The thermosensitive submucosal injection solution based on lyophilized particles proposed in this invention enhances the hydrogen bond interaction between chitosan and polyvinylpyrrolidone during the lyophilization process. Therefore, the elastic modulus (G') of the low-temperature injection solution prepared using the solution after reconstitution of chitosan-polyvinylpyrrolidone lyophilized particles and the gel formed by it at body temperature are both greater than those of the injection solution and its gel prepared using the solution of chitosan-polyvinylpyrrolidone without lyophilization. This makes the thermosensitive submucosal injection solution based on lyophilized particles have a better effect on maintaining mucosal elevation. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0031] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0033] The thermosensitive submucosal injection solution based on lyophilized particles proposed in this invention comprises two parts: chitosan-polyvinylpyrrolidone lyophilized particles and β-glycerophosphate sodium lyophilized particles. It can be stored for a long time, is convenient to transport, and is easy to use. In use, these two lyophilized particles can be rapidly dissolved separately in cryogenic water for injection, and after mixing, they form a cryogenic submucosal injection solution. This cryogenic injection solution has a very low viscosity, requiring no special injection device; it can be injected submucosally using conventional endoscopic injection needles currently used in clinical practice, and its injection rate is high. Simultaneously, after injection into the submucosal layer, this cryogenic injection solution rapidly forms a high-strength hydrogel under body temperature, providing more stable support for the mucosal elevation, facilitating ESD procedures, reducing intraoperative and postoperative bleeding, and promoting postoperative wound healing.

[0034] Example 1.

[0035] Under stirring conditions (room temperature, stirring speed 500 rpm, stirring time 20 minutes), a 10000 Da polyvinylpyrrolidone solution was added to a chitosan solution with a pH of 6.2. The chitosan had a degree of deacetylation of 95% and a molecular weight of 5000 Da. The mixture was stirred until homogeneous to obtain mixture A, with a mass concentration ratio of 3:1 for polyvinylpyrrolidone to chitosan. Mixture A was then subjected to a gradient cooling process: sequentially placed at -20℃, then -40℃ until constant temperature was reached, and finally frozen at -80℃ until constant temperature was reached. The constant temperature was determined by maintaining the temperature for at least 1 hour. Afterward, freeze-drying was performed at -50℃ under a vacuum of 10 Pa to obtain chitosan-polyvinylpyrrolidone freeze-dried product B. Then, chitosan-polyvinylpyrrolidone freeze-dried product B was ground using zirconium oxide as the grinding medium for 10 minutes. Next, the ground particles were sieved through a 65-mesh sieve to remove larger particles, obtaining chitosan-polyvinylpyrrolidone lyophilized particles C that could pass through the sieve. Sodium β-glycerophosphate was dissolved in water for injection under stirring conditions (room temperature, stirring speed 1000 rpm, stirring time 30 minutes) to obtain sodium β-glycerophosphate solution D. Sodium β-glycerophosphate solution D was subjected to a gradient cooling process: solution D was successively placed at -20℃, -40℃ to constant temperature, and finally frozen at -80℃ to constant temperature, followed by freeze-drying at -50℃ and a vacuum of 10 Pa to obtain sodium β-glycerophosphate lyophilized product E. Sodium β-glycerophosphate lyophilized product E was then ground using zirconium oxide as the grinding medium for 10 minutes. Larger particles were then removed using a 35-mesh sieve, obtaining sodium β-glycerophosphate lyophilized particles F that could pass through the sieve. Both types of lyophilized particles C and F were sealed and stored in a drying oven at 25℃, and the storage time was recorded. The morphological changes of the two freeze-dried particles were observed once a month, and they were reconstituted to prepare low-temperature injection solution 1#, and the changes in gelation time in a 37℃ water bath were tested.

[0036] When reconstituted the above-mentioned lyophilized particles, chitosan-polyvinylpyrrolidone lyophilized particles C were fully dissolved in water for injection containing methylene blue, a medical coloring indicator, and placed in an ice bath to obtain a reconstituted low-temperature chitosan-polyvinylpyrrolidone solution G. Simultaneously, sodium β-glycerophosphate lyophilized particles F were fully dissolved in water for injection containing methylene blue, and placed in an ice bath to obtain a reconstituted low-temperature sodium β-glycerophosphate solution H. Then, the low-temperature sodium β-glycerophosphate solution H was added to the low-temperature chitosan-polyvinylpyrrolidone solution G and mixed thoroughly to obtain a low-temperature thermosensitive submucosal injection solution. The concentration of chitosan in this low-temperature thermosensitive submucosal injection solution was 1% (w / v, g / mL), the concentration of polyvinylpyrrolidone was 7% (w / v, g / mL), the concentration of sodium β-glycerophosphate was 5% (w / v, g / mL), and the concentration of methylene blue was 0.001% (w / v, g / mL).

[0037] In addition, 2% (w / v, g / mL) chitosan solution, 20% (w / v, g / mL) polyvinylpyrrolidone solution, and 56% (w / v, g / mL) β-glycerophosphate sodium solution were prepared, sealed, and stored at 25°C, and the storage time was recorded. The state of each solution was observed monthly, and a low-temperature injection solution 2# with the same component concentrations as the above-mentioned injection solution 1# was prepared using these solutions. The change in gelation time of this solution in a 37°C water bath was then tested.

[0038] Experiments showed that the morphology of the two types of freeze-dried particles did not change significantly during long-term storage. Initially, the gelation time of both low-temperature injection solutions 1# and 2# at 37℃ was 2.5 min. After 13 months, the gelation time of low-temperature injection solution 2# increased to 3.5 min, while the gelation time of low-temperature injection solution 1# remained at 2.5 min. This indicates that freeze-drying granulation can effectively improve the stability of the components of the injection solution during long-term storage and maintain the gelation time of the prepared low-temperature injection solution. In addition, at 15 months, a small amount of precipitation was observed in the 56% (w / v, g / mL) β-glycerophosphate sodium solution, indicating that the long-term stability of high-concentration solution-state β-glycerophosphate sodium is lower than that of freeze-dried granulated solution.

[0039] Example 2.

[0040] Under stirring conditions (stirring temperature 37℃, stirring speed 2000rpm, stirring time 40 minutes), a polyvinylpyrrolidone (PVP) solution with a molecular weight of 400,000 Da was added to a chitosan solution with a pH of 5.5. The degree of deacetylation of the chitosan was 98%, and the molecular weight of the chitosan was 400,000 Da. The mixture was stirred until homogeneous to obtain mixture A, with a mass concentration ratio of PPVP to chitosan of 7:1. Mixture A was then subjected to a gradient cooling process, successively placed at -20℃, -40℃ until isothermal, and finally frozen at -80℃ until isothermal. The isothermal condition was defined as maintaining a constant temperature for more than 1 hour. Afterward, it was freeze-dried at -30℃ under a vacuum of 40 Pa to obtain chitosan-PVP freeze-dried product B. Then, chitosan-PVP freeze-dried product B was ground using zirconium oxide as the grinding medium for 30 minutes. Next, the ground particles were sieved through a 220-mesh sieve to remove larger particles, obtaining chitosan-polyvinylpyrrolidone lyophilized particles C that could pass through the sieve. Sodium β-glycerophosphate was dissolved in water for injection under stirring conditions (room temperature, stirring speed 2000 rpm, stirring time 60 minutes) to obtain sodium β-glycerophosphate solution D. Sodium β-glycerophosphate solution D was subjected to a gradient cooling process: solution D was successively placed at -20℃, -40℃ to constant temperature, and finally frozen at -80℃ to constant temperature, followed by freeze-drying at -30℃ and a vacuum of 40 Pa to obtain sodium β-glycerophosphate lyophilized product E. Sodium β-glycerophosphate lyophilized product E was then ground using zirconium oxide as the grinding medium for 30 minutes. Larger particles were then removed using a 150-mesh sieve, obtaining sodium β-glycerophosphate lyophilized particles F that could pass through the sieve. Both types of lyophilized particles C and F were sealed and stored in a drying oven at 25℃.

[0041] When reconstituted the above-mentioned lyophilized particles, chitosan-polyvinylpyrrolidone lyophilized particles C were fully dissolved in water for injection containing the medical coloring indicator indigo carmine, and placed in an ice bath to obtain a reconstituted low-temperature chitosan-polyvinylpyrrolidone solution G. Simultaneously, β-glycerophosphate sodium lyophilized particles F were fully dissolved in water for injection containing the medical coloring indicator indigo carmine, and placed in an ice bath to obtain a reconstituted low-temperature β-glycerophosphate sodium solution H. Then, the low-temperature β-glycerophosphate sodium solution H was added to the low-temperature chitosan-polyvinylpyrrolidone solution G and mixed thoroughly to obtain a low-temperature thermosensitive submucosal injection solution 1#. The concentration of chitosan in this No. 1 low-temperature thermosensitive submucosal injection is 0.3% (w / v, g / mL), the concentration of polyvinylpyrrolidone is 0.9% (w / v, g / mL), the concentration of sodium β-glycerophosphate is 10% (w / v, g / mL), and the concentration of indigo carmine, a medical staining indicator, is 0.04% (w / v, g / mL).

[0042] In addition, three mixtures A with mass concentration ratios of polyvinylpyrrolidone (PVP) to chitosan of 2, 3, and 8 were prepared respectively to obtain corresponding chitosan-PVP lyophilized particles C (control groups 2-4), while other conditions remained unchanged. The experiment revealed that in control group 2, the PPV concentration was too low, preventing the chitosan-PVP lyophilized particles C from dissolving in water for injection, thus making it impossible to prepare an injection solution. Furthermore, in control group 4, the PPV concentration was too high, significantly reducing the hydrogen bonding between chitosan molecules, which prevented the subsequent low-temperature thermosensitive gel injection solution from forming at 37°C. In contrast, the chitosan-PVP lyophilized particles C in control group 3 dissolved in water for injection, and the subsequent injection solution formed thermosensitively. This experiment demonstrates that a mass concentration ratio of 3-7 for PPVP to chitosan in mixture A proposed in this invention is the most suitable range for use.

[0043] Example 3.

[0044] Under stirring conditions (stirring temperature 30℃, stirring speed 1250 rpm, stirring time 30 minutes), a polyvinylpyrrolidone (PVP) solution with a molecular weight of 200,000 Da was added to a chitosan solution with a pH of 5.9. The degree of deacetylation of the chitosan was 96%, and the molecular weight of the chitosan was 200,000 Da. The mixture was stirred until homogeneous to obtain mixture A, with a mass concentration ratio of PPVP to chitosan of 5:1. Mixture A was then subjected to a gradient cooling process, successively placed at -20℃, -40℃ until constant temperature, and finally frozen at -80℃ until constant temperature. The constant temperature was determined by maintaining the temperature unchanged for more than 1 hour. Afterward, freeze-drying was performed at -40℃ under a vacuum of 25 Pa to obtain chitosan-PVP freeze-dried product B. Then, chitosan-PVP freeze-dried product B was ground using zirconium oxide as the grinding medium for 20 minutes. Next, the ground particles were sieved through a 140-mesh sieve to remove larger particles, obtaining chitosan-polyvinylpyrrolidone lyophilized particles C that could pass through the sieve. Sodium β-glycerophosphate was dissolved in water for injection under stirring conditions (room temperature, stirring speed 1500 rpm, stirring time 45 minutes) to obtain sodium β-glycerophosphate solution D. Sodium β-glycerophosphate solution D was subjected to a gradient cooling process: solution D was successively placed at -20℃, -40℃ to constant temperature, and finally frozen at -80℃ to constant temperature, followed by freeze-drying at -40℃ and a vacuum of 25 Pa to obtain sodium β-glycerophosphate lyophilized product E. Sodium β-glycerophosphate lyophilized product E was then ground using zirconium oxide as the grinding medium for 20 minutes. Larger particles were then removed using a 90-mesh sieve, obtaining sodium β-glycerophosphate lyophilized particles F that could pass through the sieve. Both types of lyophilized particles C and F were sealed and stored in a drying oven at 25℃.

[0045] When reconstituted the above-mentioned lyophilized particles, chitosan-polyvinylpyrrolidone lyophilized particles C were fully dissolved in water for injection containing the medical coloring indicator indigo carmine, and placed in an ice bath to obtain a reconstituted low-temperature chitosan-polyvinylpyrrolidone solution G. Simultaneously, β-glycerophosphate sodium lyophilized particles F were fully dissolved in water for injection containing the medical coloring indicator indigo carmine, and placed in an ice bath to obtain a reconstituted low-temperature β-glycerophosphate sodium solution H. Then, the low-temperature β-glycerophosphate sodium solution H was added to the low-temperature chitosan-polyvinylpyrrolidone solution G and mixed thoroughly to obtain a low-temperature thermosensitive submucosal injection solution 1#. The concentration of chitosan in this No. 1 low-temperature thermosensitive submucosal injection is 0.65% (w / v, g / mL), the concentration of polyvinylpyrrolidone is 3.95% (w / v, g / mL), the concentration of sodium β-glycerophosphate is 7.5% (w / v, g / mL), and the concentration of indigo carmine, a medical staining indicator, is 0.02% (w / v, g / mL).

[0046] In addition, a control group of low-temperature thermosensitive submucosal injection solution #2 was prepared by mixing freshly prepared 2% (w / v, g / mL) chitosan solution, 20% (w / v, g / mL) polyvinylpyrrolidone solution, and 56% (w / v, g / mL) sodium β-glycerophosphate solution. The concentrations of each component in this control group injection solution #2 were the same as those in injection solution #1. The elastic modulus of the gels formed by the two injection solutions at 37°C was measured. It was found that the elastic modulus of the gel of injection solution #1 was 14 Pa, which was higher than that of the gel of injection solution #2 (10 Pa). Furthermore, 1 mL of each of the low-temperature injection solutions #1 and #2 was injected into the submucosal layer of the esophagus of Bama miniature pigs using an endoscopic injection needle. It was found that at 0 min after injection, the mucosal elevation height of injection solution #1 was 12 mm, which was higher than that of injection solution #2 (10 mm). At 30 min after injection, the mucosal elevation height of injection solution #1 was 10.5 mm, which was higher than that of injection solution #2 (9.2 mm). At 60 minutes post-injection, the mucosal elevation height of injection solution #1 was 9.9 mm, higher than that of injection solution #2 (8.2 mm). These experimental results indicate that, under the same component concentration, the lyophilization granulation process significantly improved the elastic modulus of the gel formed by the injection solution, resulting in a marked improvement in its ability to maintain mucosal elevation compared to the control group of conventionally prepared fresh injection solutions.

[0047] Example 4.

[0048] Under stirring conditions (stirring temperature 32℃, stirring speed 900rpm, stirring time 35 minutes), a polyvinylpyrrolidone (PVP) solution with a molecular weight of 300,000 Da was added to a chitosan solution with a pH of 6.2. The degree of deacetylation of the chitosan was 97%, and the molecular weight of the chitosan was 300,000 Da. The mixture was stirred until homogeneous to obtain mixture A, with a mass concentration ratio of PPVP to chitosan of 4. Mixture A was then subjected to a gradient cooling process, successively placed at -20℃, -40℃ until constant temperature, and finally frozen at -80℃ until constant temperature. The constant temperature was determined by maintaining the temperature unchanged for more than 1 hour. Afterwards, freeze-drying was performed at -35℃ under a vacuum of 15 Pa to obtain chitosan-PVP freeze-dried product B. Chitosan-PVP freeze-dried product B was then ground using zirconium oxide as the grinding medium for 18 minutes. Next, the ground particles were sieved through a 100-mesh sieve to remove larger particles, obtaining chitosan-polyvinylpyrrolidone lyophilized particles C that could pass through the sieve. Sodium β-glycerophosphate was dissolved in water for injection under stirring conditions (room temperature, stirring speed 1100 rpm, stirring time 35 minutes) to obtain sodium β-glycerophosphate solution D. Sodium β-glycerophosphate solution D was subjected to a gradient cooling process: solution D was successively placed at -20℃, -40℃ to constant temperature, and finally frozen at -80℃ to constant temperature, followed by freeze-drying at -45℃ and a vacuum of 35 Pa to obtain sodium β-glycerophosphate lyophilized product E. Sodium β-glycerophosphate lyophilized product E was then ground using zirconium oxide as the grinding medium for 12 minutes. Larger particles were then removed using a 120-mesh sieve, obtaining sodium β-glycerophosphate lyophilized particles F that could pass through the sieve. Both types of lyophilized particles C and F were sealed and stored in a drying oven at 25℃.

[0049] When reconstituted the above-mentioned lyophilized particles, chitosan-polyvinylpyrrolidone lyophilized particles C were fully dissolved in water for injection containing methylene blue, a medical coloring indicator, and placed in an ice bath to obtain a reconstituted low-temperature chitosan-polyvinylpyrrolidone solution G. Simultaneously, β-glycerophosphate sodium lyophilized particles F were fully dissolved in water for injection containing methylene blue, and placed in an ice bath to obtain a reconstituted low-temperature β-glycerophosphate sodium solution H. Then, the low-temperature β-glycerophosphate sodium solution H was added to the low-temperature chitosan-polyvinylpyrrolidone solution G and mixed thoroughly to obtain a low-temperature thermosensitive submucosal injection solution 1#. The concentration of chitosan in this No. 1 low-temperature thermosensitive submucosal injection is 0.85% (w / v, g / mL), the concentration of polyvinylpyrrolidone is 3.4% (w / v, g / mL), the concentration of sodium β-glycerophosphate is 9% (w / v, g / mL), and the concentration of methylene blue, a medical staining indicator, is 0.008% (w / v, g / mL).

[0050] In addition, low-temperature thermosensitive submucosal injection solutions 2-4# were prepared using chitosan solutions with pH values ​​of 5.0, 5.5, and 6.5, respectively, while other conditions remained unchanged, serving as a control group. The experiment revealed that the chitosan solution with pH value 6.5, due to its pH exceeding the isoelectric point of chitosan (6.2), resulted in chitosan precipitation and could not be used to prepare thermosensitive gels. Furthermore, the low-temperature thermosensitive submucosal injection solution 2# prepared using a chitosan solution with pH value 5.0 exhibited very slow gelation at 37°C, taking approximately 20 minutes, and was therefore unsuitable for use as an endoscopic submucosal injection solution. However, the low-temperature thermosensitive submucosal injection solutions 3# and 1# prepared using chitosan solutions with pH values ​​of 5.5 and 6.2 respectively, gelled within 5 minutes and could be used for injection to lift the mucosa. Therefore, these experimental results indicate that 5.5-6.2 is the optimal pH range for chitosan solutions.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a lyophilized particle-based thermosensitive submucosal injection solution for endoscopic submucosal dissection, characterized in that... The method includes the following steps: S1. Under stirring conditions, polyvinylpyrrolidone solution is added to chitosan solution with pH value of 5.5-6.2, and the mixture is stirred and mixed evenly to obtain mixture A. The mass concentration ratio of polyvinylpyrrolidone to chitosan in mixture A is 3-7. S2. Mixture A is subjected to gradient cooling and finally frozen at -80℃ to a constant temperature, and then freeze-dried to obtain chitosan-polyvinylpyrrolidone freeze-dried product B; S3. Grind the chitosan-polyvinylpyrrolidone lyophilized product B, then sieve to remove larger particles to obtain chitosan-polyvinylpyrrolidone lyophilized particles C that can pass through the sieve. S4. Dissolve sodium β-glycerophosphate in water for injection under stirring conditions to obtain sodium β-glycerophosphate solution D; S5. The sodium β-glycerophosphate solution D was subjected to gradient cooling and finally frozen at -80℃ to a constant temperature, and then freeze-dried to obtain the freeze-dried sodium β-glycerophosphate product E. S6. Grind the lyophilized sodium β-glycerophosphate product E, and then use a sieve to remove larger particles to obtain lyophilized sodium β-glycerophosphate particles F that can pass through the sieve; S7. When using, use water for injection containing medical coloring indicator to fully dissolve chitosan-polyvinylpyrrolidone lyophilized particles C, place in an ice bath, and obtain a reconstituted low-temperature chitosan-polyvinylpyrrolidone solution G. S8. Use water for injection containing a medical coloring indicator to fully dissolve the lyophilized sodium β-glycerophosphate particles F, place them in an ice bath, and obtain a reconstituted low-temperature sodium β-glycerophosphate solution H; S9. Add the low-temperature β-glycerophosphate sodium solution H to the low-temperature chitosan-polyvinylpyrrolidone solution G, mix well, and obtain a low-temperature thermosensitive submucosal injection solution.

2. The method for preparing the lyophilized particle-based thermosensitive submucosal injection solution for endoscopic submucosal dissection as described in claim 1, characterized in that, In step S1, the degree of deacetylation of chitosan is 95% or higher, and the molecular weight is 5000-400000 Da; the molecular weight of polyvinylpyrrolidone is 10000-400000 Da.

3. The method for preparing the lyophilized particle-based thermosensitive submucosal injection solution for endoscopic submucosal dissection as described in claim 1, characterized in that, In step S1, the stirring temperature is room temperature to 37°C, the stirring speed is 500 to 2000 rpm, and the stirring time is 20 to 40 minutes.

4. The method for preparing the lyophilized particle-based thermosensitive submucosal injection solution for endoscopic submucosal dissection as described in claim 1, characterized in that, In steps S2 and S5, the gradient cooling involves placing the mixed solution A and the β-glycerophosphate sodium solution D at -20℃ and -40℃ respectively until they reach a constant temperature, and finally freezing them at -80℃ until they reach a constant temperature. In steps S2 and S5, the freeze-drying conditions are -50℃ to -30℃ and a vacuum degree of 10 to 40 Pa.

5. The method for preparing the lyophilized particle-based thermosensitive submucosal injection solution for endoscopic submucosal dissection as described in claim 1, characterized in that, In steps S3 and S6, the grinding medium is zirconium oxide, and the grinding time is 10 to 30 minutes.

6. The method for preparing the lyophilized particle-based thermosensitive submucosal injection solution for endoscopic submucosal dissection as described in claim 1, characterized in that, In step S3, obtaining chitosan-polyvinylpyrrolidone freeze-dried particles C that can pass through a sieve involves sieving the grinding particles using a 65-220 mesh sieve to obtain smaller particles that can pass through the sieve. In step S6, obtaining β-glycerophosphate sodium freeze-dried particles F that can pass through a sieve involves sieving the grinding particles using a 35-150 mesh sieve to obtain smaller particles that can pass through the sieve.

7. The method for preparing the lyophilized particle-based thermosensitive submucosal injection solution for endoscopic submucosal dissection as described in claim 1, characterized in that, In step S4, the stirring speed is 1000-2000 rpm and the stirring time is 30-60 minutes.

8. The method for preparing the lyophilized particle-based thermosensitive submucosal injection solution for endoscopic submucosal dissection as described in claim 1, characterized in that, In steps S7 and S8, the coloring indicator used in traditional Chinese medicine is one of methylene blue and indigo carmine, or a combination thereof.

9. The method for preparing the lyophilized particle-based thermosensitive submucosal injection solution for endoscopic submucosal dissection as described in claim 1, characterized in that, In step S9, the concentration of chitosan in the low-temperature thermosensitive submucosal injection solution is 0.3–1% (w / v, g / mL), the concentration of polyvinylpyrrolidone is 0.9–7% (w / v, g / mL), the concentration of sodium β-glycerophosphate is 5–10% (w / v, g / mL), and the concentration of medical staining indicator is 0.001–0.04% (w / v, g / mL).

10. The use of a temperature-sensitive submucosal injection solution based on lyophilized particles prepared by the preparation method according to any one of claims 1 to 9 in the preparation of a submucosal injection for endoscopic submucosal dissection.