Chitosan-based temperature-sensitive endoscopic submucosal injection, and preparation method therefor and use thereof

Abstract

Disclosed in the present disclosure are a chitosan-based temperature-sensitive endoscopic submucosal injection, and a preparation method therefor and use thereof. The injection comprises chitosan suitable for conventional pH value, polyvinylpyrrolidone, sodium β-glycerophosphate, and sodium bicarbonate. The injection has good low-temperature stability and high injectability, and can be injected by means of utilizing a conventional endoscopic injection needle currently in clinical use. Meanwhile, after being injected into the submucosa, the injection can rapidly form a hydrogel with relatively high mechanical strength higher-strength hydrogel under the action of body temperature, and exhibits higher tissue adhesion, which enables more stable support for the elevated state of the mucosal layer, thereby delaying the height decrease, better facilitating ESD operation, and reducing the occurrence probability of complications. Therefore, the injection holds significant practical value in clinical applications.

Description

A chitosan-based thermosensitive submucosal injection solution for endoscopy, its preparation method and application

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202410504444.2, filed on April 25, 2024, with the China National Intellectual Property Administration, entitled “A method for preparing a chitosan-based thermosensitive endoscopic submucosal injection solution”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure belongs to the field of regenerative medicine technology, and in particular relates to a chitosan-based thermosensitive endoscopic submucosal injection solution, its preparation method and application. Background Technology

[0004] Endoscopic submucosal dissection (ESD) is a minimally invasive technique that involves completely removing diseased mucosa from the submucosa under endoscopic guidance. Because it can completely remove early-stage gastrointestinal tumors in a single endoscopic procedure, ESD has broad clinical application prospects. However, the cutting and dissection of the diseased mucosa during ESD is very difficult, requires a high level of skill, and carries a high risk of complications such as perforation and bleeding, limiting the widespread adoption of ESD.

[0005] To facilitate mucosal cutting and dissection, it is routinely practiced in clinical practice to inject methylene blue-containing saline solution around the lesion mucosa as a submucosal injection solution. This causes the lesion mucosal layer to bulge, separating it from the underlying muscle layer, improving the operative field of vision and space, avoiding damage to the muscle layer, reducing vascular damage, and lowering the chance of complications. However, physiological saline has almost no viscosity and quickly diffuses into the surrounding tissue after injection and is rapidly absorbed. Therefore, within minutes of injection, the bulging mucosal layer collapses significantly, greatly reducing the cutting space. Furthermore, physiological saline continuously flows out during the cutting process, exacerbating the reduction in mucosal height. Therefore, it is necessary to repeatedly inject physiological saline during the operation, which greatly increases the complexity of the surgery, prolongs the operation time, and the complication rate remains high.

[0006] To address the shortcomings of saline solution, solutions such as sodium hyaluronate, glycerol fructose, and hydroxyethyl starch have been attempted as alternative submucosal injection solutions. These solutions have a higher viscosity than saline, which slows their diffusion to surrounding tissues after injection, thus reducing tissue absorption and maintaining the height of the edema better than saline. However, because they are essentially still flowable liquids, they still leak during incision and have poor resistance to mucosal pressure, resulting in a rapid decrease in edema height, failing to fundamentally improve the problem.

[0007] Chitosan-based thermosensitive gels have the potential to overcome the shortcomings of the aforementioned solution-type injectable solutions. A mixture of chitosan and sodium β-glycerophosphate maintains a solution state at low temperatures and transforms into a hydrogel state at body temperature; this injectable system has already been applied in areas such as drug delivery and sustained release. Considering its ability to undergo a liquid-hydrogel phase transition in response to temperature, it also has the potential to be a novel submucosal injection solution for ESD. However, conventional chitosan-sodium β-glycerophosphate thermosensitive gels still have some significant drawbacks when applied to ESD procedures.

[0008] 1. Poor injectability: To enhance gel-forming ability, the final chitosan concentration of conventional chitosan-β-glycerophosphate sodium thermosensitive gel is generally 2% (w / v or g / mL), which is considered the saturation concentration of chitosan. At this concentration, the chitosan solution has a very high viscosity, and the addition of β-glycerophosphate sodium has no significant effect on the viscosity. However, the inner diameter of the endoscopic injection needle is about 2 mm, and its length is close to 2 m, resulting in very high injection resistance. Therefore, under conventional chitosan concentration conditions, in vivo endoscopic injection cannot be achieved. It is necessary to significantly reduce the final chitosan concentration and its viscosity to reduce injection resistance and improve injectability.

[0009] 2. Poor gelling ability under low chitosan concentrations: Since the backbone of the thermosensitive gel is chitosan, its gelling ability at body temperature is significantly reduced, or even non-gelling, when the chitosan concentration is significantly decreased. This leads to rapid diffusion into surrounding tissues after injection, resulting in a limited height of the raised mucosal layer and difficulty in successful cutting. Although using high concentrations of sodium β-glycerophosphate can promote gelling to some extent, high concentrations of sodium β-glycerophosphate have significant cytotoxicity. Therefore, the concentration of sodium β-glycerophosphate must be kept at a low level to ensure biocompatibility. The isoelectric point of chitosan is approximately 6.2. Chitosan will precipitate when the pH exceeds 6.2. pH has a certain influence on gelling ability. The pH of conventional chitosan solutions is generally less than or equal to 5.4. High-pH chitosan solutions (pH>5.4) have improved gelling ability due to increased deprotonation. However, the preparation conditions for high-pH chitosan solutions are demanding, requiring the use of excessive powder for dissolution, additional standing and centrifugation steps, or prolonged dialysis. Precisely controlling the pH is difficult. Therefore, it is necessary to find other ways to increase the overall pH, improve the gelling ability of chitosan solutions with conventional pH values ​​at low concentrations, shorten the gelation time after injection, reduce its diffusion to surrounding tissues, and improve the elevation height and effect of the mucosa.

[0010] 3. Poor low-temperature stability under low-concentration chitosan conditions: Under low chitosan concentration conditions, the pH of the low-temperature mixture is relatively high, which enhances the hydrogen bonding and hydrophobic interactions between chitosan molecules, easily causing chitosan precipitation and resulting in poor stability. New modification techniques need to be developed to appropriately reduce the interactions between chitosan molecules without affecting the temperature-sensitive properties, thereby improving stability and enabling the stable preparation of low-temperature injection solutions.

[0011] 4. Low gel strength under low chitosan concentration: The framework of the thermosensitive gel prepared from chitosan and sodium β-glycerophosphate is a three-dimensional network formed between chitosan molecules. Reducing the chitosan concentration weakens the network density, leading to a decrease in gel strength and its ability to resist mucosal pressure, resulting in a more significant decrease in the height of the bulge. Increasing the gel strength will help improve the gel's resistance to mucosal pressure and slow down the rate of decrease in bulge height.

[0012] 5. Poor tissue adhesion of chitosan thermosensitive gel: Besides gelation time and gel strength, the reduction in mucosal elevation height is also significantly related to the affinity of the gel-tissue interface. Insufficient adhesion between chitosan and tissue makes it prone to slippage under mucosal pressure or cutting, affecting the elevation effect. Therefore, improving the tissue adhesion of thermosensitive gel is beneficial for better maintaining the elevation state of the mucosal layer and further slowing down the height loss.

[0013] The above analysis shows that currently used solution-based submucosal injection solutions are insufficient to meet the needs of ESD. Conventional chitosan-β-glycerophosphate sodium thermosensitive gel has significant defects in injectability, low-temperature stability, gelling ability, gel strength, and tissue adhesion, which do not meet the application requirements of ESD. Therefore, there is an urgent need to develop new chitosan-based thermosensitive endoscopic submucosal injection solutions to overcome the shortcomings of existing technologies and further improve the safety and effectiveness of ESD procedures.

[0014] Public content

[0015] To address the aforementioned issues, this disclosure provides a method for preparing a chitosan-based thermosensitive submucosal injection solution for endoscopic submucosal dissection (ESD). A novel chitosan-based thermosensitive gel injection solution is constructed using chitosan at a conventional pH, polyvinylpyrrolidone, sodium β-glycerophosphate, sodium bicarbonate, and dopamine. The aim is to improve the injectability, low-temperature stability, gel strength, and tissue adhesion of the chitosan thermosensitive gel, providing more stable support for the mucosal elevation, facilitating ESD procedures, and reducing the incidence of complications.

[0016] To achieve the above-mentioned objectives, this disclosure provides the following technical solutions.

[0017] This disclosure provides a chitosan-based thermosensitive endoscopic submucosal injection solution, the injection solution comprising chitosan with conventional pH, polyvinylpyrrolidone, sodium β-glycerophosphate, and sodium bicarbonate.

[0018] Furthermore, the injection solution also includes dopamine.

[0019] This disclosure also provides the application of the chitosan-based thermosensitive endoscopic submucosal injection solution in the preparation of thermosensitive submucosal injections for endoscopic submucosal dissection.

[0020] This disclosure also provides the application of the chitosan-based thermosensitive endoscopic submucosal injection solution in the preparation of a thermosensitive submucosal injection for postoperative procedures following endoscopic submucosal dissection.

[0021] This disclosure also provides a method for preparing the chitosan-based thermosensitive endoscopic submucosal injection solution, characterized in that the method includes the following steps:

[0022] S1. Chitosan powder with a molecular weight of 4000 Da to 800000 Da is dissolved in a dilute acid solution under stirring to obtain a chitosan solution A with a pH less than or equal to 5.4.

[0023] S2. Dissolve polyvinylpyrrolidone powder and methylene blue powder in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue, wherein the molecular weight of the polyvinylpyrrolidone powder is 10,000 Da to 500,000 Da.

[0024] S3. Dissolve sodium β-glycerophosphate powder in water under stirring to obtain sodium β-glycerophosphate solution C;

[0025] S4. Dissolve sodium bicarbonate powder in water under stirring to obtain sodium bicarbonate solution D;

[0026] S5. Place the solutions A, B, C, and D in an ice bath to pre-cool them thoroughly;

[0027] S6. The solutions B, C, and D, which are pre-cooled in an ice bath, are added dropwise to the solution A, which is pre-cooled in an ice bath, in strict order of B, C, and D under vortex oscillation conditions. Each solution is thoroughly mixed after being added dropwise to obtain a chitosan-based thermosensitive submucosal injection solution, which is placed in an ice bath for in vivo injection.

[0028] The chitosan-based thermosensitive submucosal injection solution contains chitosan at a concentration of 0.2%–1%, polyvinylpyrrolidone at a concentration of 0.2%–1.9%, sodium β-glycerophosphate at a concentration of 4%–10%, methylene blue at a concentration of 0.001%–0.02%, and sodium bicarbonate at a concentration of 0.005M–0.03M. The concentrations of chitosan, polyvinylpyrrolidone, sodium β-glycerophosphate, and methylene blue are all expressed as w / v or g / mL.

[0029] Further, in step S1, the degree of deacetylation of the chitosan is 85% or higher, and the dilute acid solution is a 0.05M to 0.2M hydrochloric acid solution or a 0.5% to 3% acetic acid solution on a v / v basis.

[0030] This disclosure also provides the application of the thermosensitive submucosal injection solution prepared by the above preparation method in the preparation of thermosensitive submucosal injections for endoscopic submucosal dissection.

[0031] This disclosure also provides a method for preparing the chitosan-based thermosensitive endoscopic submucosal injection solution, the method comprising the following steps:

[0032] S1. Chitosan powder with a molecular weight of 4000 Da to 800000 Da is dissolved in a dilute acid solution under stirring to obtain a chitosan solution A with a pH less than or equal to 5.4.

[0033] S2. Dissolve polyvinylpyrrolidone powder and methylene blue powder in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue, wherein the molecular weight of the polyvinylpyrrolidone powder is 10,000 Da to 500,000 Da.

[0034] S3. Dissolve sodium β-glycerophosphate powder in water under stirring to obtain sodium β-glycerophosphate solution C;

[0035] S4. Dissolve sodium bicarbonate powder in water under stirring to obtain sodium bicarbonate solution D;

[0036] S5. Dissolve dopamine powder in sodium bicarbonate solution D under stirring to obtain a mixed solution E of sodium bicarbonate and dopamine, and polymerize at room temperature for 20 min.

[0037] S6. Place the solutions A, B, C, and E in an ice bath to pre-cool them thoroughly;

[0038] S7. The solutions B, C, and E, which are pre-cooled in an ice bath, are added dropwise to the solution A, which is pre-cooled in an ice bath, in strict order of B, C, and E under vortex oscillation conditions. Each solution is thoroughly mixed after being added dropwise to obtain a chitosan-based thermosensitive submucosal injection solution, which is placed in an ice bath for in vivo injection.

[0039] The chitosan-based thermosensitive submucosal injection solution contains chitosan at a concentration of 0.2%–1%, polyvinylpyrrolidone at a concentration of 0.2%–1.9%, sodium β-glycerophosphate at a concentration of 4%–10%, methylene blue at a concentration of 0.001%–0.02%, sodium bicarbonate at a concentration of 0.005M–0.03M, and dopamine at a concentration of 0%–2%. The concentrations of chitosan, polyvinylpyrrolidone, sodium β-glycerophosphate, methylene blue, and dopamine are all expressed as w / v or g / mL.

[0040] Further, in step S1, the degree of deacetylation of the chitosan is 85% or higher, and the dilute acid solution is a 0.05M to 0.2M hydrochloric acid solution or a 0.5% to 3% acetic acid solution on a v / v basis.

[0041] This disclosure also provides the application of the thermosensitive submucosal injection solution prepared by the above preparation method in the preparation of thermosensitive submucosal injections for postoperative procedures after endoscopic submucosal dissection.

[0042] Compared with the prior art, the beneficial effects of this disclosure are as follows.

[0043] 1. This disclosure discloses a novel chitosan-based thermosensitive gel injection solution constructed using chitosan with a conventional pH value (pH ≤ 5.4), polyvinylpyrrolidone, sodium β-glycerophosphate, and sodium bicarbonate. Specifically, based on the traditional chitosan / sodium β-glycerophosphate system, the chitosan concentration is reduced to improve injectability; polyvinylpyrrolidone is used to maintain stability at low chitosan concentrations; sodium bicarbonate is used to enhance the thermosensitive gelling ability under low-concentration, conventional pH chitosan conditions; and the synergistic effect of polyvinylpyrrolidone and sodium bicarbonate improves gel strength. Furthermore, dopamine can be added to the above-mentioned new system. Dopamine can polymerize into polydopamine, which further enhances gel strength through interactions with chitosan molecules. In addition, the introduction of polydopamine enhances the adhesion between the gel and the tissue interface, resulting in a tighter bond between the gel and surrounding tissue, reducing gel movement under mucosal pressure and during cutting, and improving the stability of the supporting mucosa. Therefore, the chitosan-based thermosensitive gel proposed in this disclosure can meet all the requirements of ESD for submucosal injection solutions, overcome the defects of the prior art, and better maintain the bulging state of the mucosa, which is beneficial to ESD operation.

[0044] 2. The temperature-sensitive submucosal injection solution for endoscopy proposed in this disclosure uses a conventional pH chitosan solution (pH less than or equal to 5.4), instead of a high pH chitosan solution (pH > 5.4). This is because the conventional preparation conditions for chitosan solution involve directly dissolving chitosan powder in dilute acid, resulting in a pH value generally less than or equal to 5.4, low viscosity, and a simple and controllable preparation process. Although higher pH chitosan solutions exhibit stronger temperature-sensitive gelling ability, high pH chitosan solutions (pH > 5.4) require special processes for preparation. These include adding excessive chitosan to consume hydrogen ions, followed by sedimentation and centrifugation, or prolonged dialysis to control the pH inside and outside the dialysis bag. The preparation process is complex, requires high precision, and exhibits poor stability and controllability, making it unsuitable for mass production of injection solutions. Therefore, the technical method disclosed herein, by establishing a new composite system and regulating the synergistic effect between components, enables the preparation of a temperature-sensitive submucosal injection solution suitable for ESD using conventional pH chitosan solution (pH less than or equal to 5.4) as the raw material. This breaks through the bottleneck of the prior art and greatly simplifies the preparation process of the injection solution.

[0045] 3. Submucosal injection solutions used for ESD must first possess good injectability to endoscopic needles. Endoscopic injection needles have an inner diameter of approximately 2 mm and a length approaching 2 m, resulting in very high injection resistance. Therefore, it is essential to reduce the viscosity of the cryogenic injection solution. The viscosity of the chitosan solution plays a decisive role in the overall viscosity of the cryogenic injection solution; reducing the final concentration of chitosan improves its injectability. The conventional concentration of chitosan solution used to prepare temperature-sensitive gels is 2% (w / v or g / mL). Under these conditions, the viscosity of the chitosan solution is very high, leading to a high viscosity in the cryogenic injection solution, making it unsuitable for injection through an endoscopic needle. This disclosure reduces the final concentration of chitosan to 0.2–1% (w / v or g / mL), significantly lowering the viscosity. Furthermore, this disclosure uses a chitosan solution with a conventional pH value (pH less than or equal to 5.4), whose viscosity is significantly lower than that of high-pH chitosan solutions (pH > 5.4). This further reduces the viscosity of the cryogenic injection solution, further improving its injectability and meeting the requirements for endoscopic injection.

[0046] 4. Thermosensitive submucosal injection solutions for ESD should possess good low-temperature stability. Chitosan has an isoelectric point of approximately 6.2, meaning it precipitates when the pH exceeds 6.2. Under normal chitosan concentration conditions, sodium β-glycerophosphate can prevent chitosan precipitation at low temperatures, allowing the pH of the low-temperature injection solution to rise to the physiological pH range. However, under low chitosan concentration conditions, even in the presence of sodium β-glycerophosphate, chitosan molecules cannot tolerate high pH and precipitate, causing the low-temperature injection solution to become turbid and significantly reducing its gelling ability. To prevent chitosan molecule precipitation, this disclosure incorporates polyvinylpyrrolidone (PVP). Utilizing its hydrogen bonding with chitosan, PPVP reduces the interaction between chitosan molecules, inhibiting precipitation and significantly improving the stability of the low-temperature injection solution under low chitosan concentration conditions. Furthermore, the addition of PPVP does not affect the thermosensitivity of the injection solution and can also improve the integrity and strength of the gel.

[0047] 5. Thermosensitive submucosal injection solutions used for ESD should have the ability to rapidly gel at body temperature. The most important difference between thermosensitive submucosal injection solutions and conventional solution-based injection solutions is that they can transform into a hydrogel state at body temperature. This hydrogel is non-flowing and possesses a certain strength, thus better supporting the bulge of the mucosal layer. Because thermosensitive submucosal injection solutions are in solution form at injection, accelerated gelation at body temperature reduces the extent to which the solution diffuses into surrounding tissues, resulting in a higher initial bulge height, which is more conducive to ESD procedures. Since the backbone of thermosensitive gels is chitosan, reducing the chitosan concentration weakens the gel backbone, leading to a decrease in gelation ability at body temperature, or even no gelation at all. In particular, conventional pH chitosan solutions (pH ≤ 5.4) have a relatively low pH, and their gelation ability at low concentrations at body temperature is worse than that of high pH chitosan solutions (pH > 5.4). Although increasing the concentration of sodium β-glycerophosphate can accelerate gelation to some extent, high concentrations of sodium β-glycerophosphate exhibit significant cytotoxicity, and its final concentration should not exceed 10% (w / v or g / mL) to ensure biosafety. To accelerate the thermosensitive gelation ability of chitosan solutions with conventional pH values ​​(pH ≤ 5.4) at low concentrations, this disclosure adds sodium bicarbonate to the chitosan solution at lower concentrations of polyvinylpyrrolidone and sodium β-glycerophosphate. Sodium bicarbonate can slightly increase the pH of the injection solution, enhance the interaction between chitosan molecules, effectively promote gelation, and improve gel strength. Furthermore, the technical method of this disclosure is specifically designed to add polyvinylpyrrolidone solution, sodium β-glycerophosphate solution, and sodium bicarbonate solution to the chitosan solution in a strictly sequential order. This is because adding polyvinylpyrrolidone first weakens the interaction between chitosan molecules, thus preventing chitosan molecules from precipitating when the other two weak bases are added subsequently. Then, sodium β-glycerophosphate is added, which protects chitosan from precipitation at higher pH levels and also possesses thermosensitive properties. Then, sodium bicarbonate is added to further adjust the pH. Under the condition of sufficient reaction with sodium β-glycerophosphate, chitosan molecules remain stable and do not precipitate. Their intermolecular interactions are strengthened, and their temperature-sensitive gelling ability is significantly improved. Experiments showed that if a mixed solution of sodium β-glycerophosphate and sodium bicarbonate is prepared first and then added to a low-concentration mixture of chitosan and polyvinylpyrrolidone, chitosan precipitates, causing the solution to become turbid and its stability to decrease significantly. This is because chitosan molecules are more sensitive to alkaline environments under low-concentration chitosan conditions. Without sufficient reaction with sodium β-glycerophosphate, the addition of sodium bicarbonate causes chitosan molecules to lack sufficient steric hindrance, resulting in aggregation and precipitation. The disclosed technical method uses a low concentration of sodium bicarbonate to enable rapid gelation of the low-temperature injection solution at body temperature, exhibiting very high temperature responsiveness.

[0048] 6. Thermosensitive submucosal injection solutions used for ESD should possess good gel strength. Chitosan is the main component of the thermosensitive gel; at low chitosan concentrations, the gel skeleton has low density and low gel strength. Furthermore, chitosan solutions with conventional pH values ​​(pH ≤ 5.4) have relatively low pH, resulting in lower intermolecular interactions and also weaker strength. Reduced gel strength weakens the gel's resistance to mucosal pressure and the squeezing forces of gastrointestinal motility, leading to damage to the gel skeleton and a decrease in the height of the raised mucosal layer. This reduces the operating space for ESD electroresection, thus necessitating improved gel strength. The method disclosed in this paper utilizes hydrogen bonding between polyvinylpyrrolidone and chitosan to link the two polymers together, causing them to intertwine and increasing the density of the gel skeleton, thereby improving gel strength. In addition, the addition of sodium bicarbonate adjusts the pH, enhancing the interaction between chitosan molecules, making the skeleton more robust and synergistically strengthening the gel strength. In addition, dopamine can be added to the chitosan-polyvinylpyrrolidone-sodium β-glycerophosphate-sodium bicarbonate composite system. Dopamine polymerizes into polydopamine, which can interact with chitosan and polyvinylpyrrolidone to further enhance gel strength and better slow down the decrease in bulge height.

[0049] 7. Thermosensitive submucosal injection solutions used for ESD should have good gel-tissue affinity. Chitosan-based thermosensitive gels have low adhesion to the surface of digestive tract tissues and may slip under mucosal pressure or cutting, affecting the maintenance of mucosal elevation. To enhance the tissue adhesion of the gel, improve the integrity of the gel and tissue, and further slow down the reduction of mucosal height, this disclosure may optionally add dopamine. Dopamine can be polymerized into polydopamine, which has strong tissue adhesion, thereby allowing the gel surface to better adhere to the tissue surface, limiting gel displacement, and also improving the integrity and strength of the gel. In the later stages of injection, its improvement in gel strength and tissue adhesion can further slow down the collapse of the elevated mucosal height, making it more suitable for ESD operations in the later stages of injection.

[0050] 8. The chitosan-based thermosensitive submucosal injection solution proposed in this disclosure does not require the use of special injection devices. It can be injected submucosally using a routine clinical endoscopic injection needle. It has high stability, smooth in vivo injection, rapid mucosal elevation, rapid gelation, and slow mucosal collapse. The cutting process does not require repeated injections, which can meet the specific requirements of ESD procedure, greatly reduce the difficulty of the operation, and improve the safety and effectiveness of ESD procedure. Detailed Implementation

[0051] The present disclosure will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the subject matter of the present disclosure to the following embodiments; all technologies implemented based on the content of the present disclosure fall within its scope.

[0052] Unless otherwise specified, all reagents and materials used in this disclosure are commercially available.

[0053] This disclosure utilizes chitosan with a conventional pH value (pH less than or equal to 5.4), polyvinylpyrrolidone, sodium β-glycerophosphate, and sodium bicarbonate (with the option to add dopamine) to construct a novel chitosan-based thermosensitive gel injection solution. After being injected into the submucosal layer, this injection solution can rapidly form a hydrogel under body temperature, providing more stable support for the bulging state of the mucosal layer, which is beneficial for ESD operations.

[0054] Example 1

[0055] Chitosan powder (molecular weight 800,000 Da, degree of deacetylation 95%) was dissolved in 0.1 M dilute hydrochloric acid solution under stirring to obtain chitosan solution A (pH 5.4). Polyvinylpyrrolidone powder (molecular weight 500,000 Da) and methylene blue powder were dissolved in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue. Sodium β-glycerophosphate powder was dissolved in water under stirring to obtain sodium β-glycerophosphate solution C. Sodium bicarbonate powder was dissolved in water under stirring to obtain sodium bicarbonate solution D. Solutions A, B, C, and D were pre-cooled in an ice bath. The pre-cooled solutions B, C, and D were added dropwise to pre-cooled solution A in the order of B, C, and D under vortex oscillation, and each solution was thoroughly mixed after addition to obtain a chitosan-based thermosensitive submucosal injection solution, which was then placed in an ice bath.

[0056] This chitosan-based thermosensitive submucosal injection contains 0.2% (w / v or g / mL) chitosan, 0.2% (w / v or g / mL) polyvinylpyrrolidone, 10% (w / v or g / mL) sodium β-glycerophosphate, 0.02% (w / v or g / mL) methylene blue, and 0.03M sodium bicarbonate.

[0057] In addition, three injection solutions with polyvinylpyrrolidone (PVP) concentrations of 0.19% (w / v or g / mL), 1.9% (w / v or g / mL), and 2% (w / v or g / mL) were prepared, with other conditions remaining unchanged, thus obtaining three control group solutions. The experiment showed that the two injection solutions with PPVP concentrations of 0.2% (w / v or g / mL) and 1.9% (w / v or g / mL) maintained good stability at low temperatures, were clear and transparent, and had low viscosity, allowing for injection through an ESD injection needle. However, the control group with a PPVP concentration of 0.19% (w / v or g / mL) became turbid, with chitosan precipitation, indicating poor low-temperature stability. Furthermore, the control group with a PPVP concentration of 2% (w / v or g / mL) had very high viscosity, making injection through an ESD injection needle extremely difficult, and thus impossible due to the strong hydrogen bonding between chitosan and PPVP. The above experiments demonstrate that the polyvinylpyrrolidone concentration proposed in this disclosure is 0.2% to 1.9% (w / v or g / mL) to achieve excellent low-temperature stability and low viscosity for injection.

[0058] Example 2

[0059] Chitosan powder (molecular weight 4000 Da, degree of deacetylation 85%) was dissolved in 0.5% (v / v) acetic acid solution under stirring to obtain chitosan solution A (pH 5.0). Polyvinylpyrrolidone powder (molecular weight 10000 Da) and methylene blue powder were dissolved in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue. Sodium β-glycerophosphate powder was dissolved in water under stirring to obtain sodium β-glycerophosphate solution C. Sodium bicarbonate powder was dissolved in water under stirring to obtain sodium bicarbonate solution D. Dopamine powder was dissolved in sodium bicarbonate solution D under stirring to obtain a mixed solution E of sodium bicarbonate and dopamine, which was polymerized at room temperature for 20 min. Solutions A, B, C, and E were then pre-cooled in an ice bath. Solutions B, C, and E, pre-cooled in an ice bath, were added dropwise to solution A, pre-cooled in an ice bath, in the order of B, C, and E, under vortex oscillation. Each solution was thoroughly mixed after addition to obtain a chitosan-based thermosensitive submucosal injection solution, which was then placed in an ice bath.

[0060] This chitosan-based thermosensitive submucosal injection contains 1% chitosan (w / v or g / mL), 1.9% polyvinylpyrrolidone (w / v or g / mL), 4% sodium β-glycerophosphate (w / v or g / mL), 0.001% methylene blue (w / v or g / mL), 0.005 M sodium bicarbonate, and 2% dopamine (w / v or g / mL).

[0061] In addition, three injection solutions with polyvinylpyrrolidone (PVP) concentrations of 0.19% (w / v or g / mL), 0.2% (w / v or g / mL), and 2% (w / v or g / mL) were prepared respectively, while other conditions remained unchanged, thus obtaining three control group solutions. The experiment showed that the two injection solutions with PPVP concentrations of 0.2% (w / v or g / mL) and 1.9% (w / v or g / mL) maintained good stability at low temperatures, were clear and transparent, and had low viscosity, allowing for injection through an ESD injection needle. However, the control group with a PPVP concentration of 0.19% (w / v or g / mL) showed turbidity and chitosan precipitation, indicating poor low-temperature stability. Furthermore, the control group with a PPVP concentration of 2% (w / v or g / mL) had very high viscosity, making it very difficult to inject through an ESD injection needle and thus impossible to administer. The above experiments also demonstrate that the polyvinylpyrrolidone concentration proposed in this disclosure is 0.2% to 1.9% (w / v or g / mL) to achieve excellent low-temperature stability and low viscosity for injection.

[0062] Example 3

[0063] Chitosan powder (molecular weight 400,000 Da, degree of deacetylation 90%) was dissolved in 0.2 M dilute hydrochloric acid solution under stirring to obtain chitosan solution A (pH 4.8). Polyvinylpyrrolidone powder (molecular weight 250,000 Da) and methylene blue powder were dissolved in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue. Sodium β-glycerophosphate powder was dissolved in water under stirring to obtain sodium β-glycerophosphate solution C. Sodium bicarbonate powder was dissolved in water under stirring to obtain sodium bicarbonate solution D. Solutions A, B, C, and D were pre-cooled in an ice bath. Solutions B, C, and D, pre-cooled in the ice bath, were added dropwise to solution A in the order of B, C, and D under vortex oscillation, and thoroughly mixed after each addition to obtain a chitosan-based thermosensitive submucosal injection solution. This solution was placed in an ice bath, and then 5 mL was used for an ESD injection needle injectability test.

[0064] This chitosan-based thermosensitive submucosal injection contains 1% chitosan (w / v or g / mL), 1% polyvinylpyrrolidone (w / v or g / mL), 7% sodium β-glycerophosphate (w / v or g / mL), 0.01% methylene blue (w / v or g / mL), and 0.018 M sodium bicarbonate.

[0065] In addition, three injection solutions with chitosan concentrations of 1.1% (w / v or g / mL), 0.2% (w / v or g / mL), and 0.19% (w / v or g / mL) were prepared respectively, while other conditions remained unchanged, thus obtaining three control group solutions. The experiment showed that the injection solutions with chitosan concentrations of 0.2% (w / v or g / mL) and 1% (w / v or g / mL) could be smoothly injected through the ESD injection needle, while the 1.1% (w / v or g / mL) injection solution became viscous, exhibiting high injection resistance and failing to be injected. The 0.19% (w / v or g / mL) injection solution became turbid and unstable at low temperatures. Therefore, the above experiments demonstrate that the chitosan concentration of 0.2%–1% (w / v or g / mL) proposed in this disclosure is necessary to achieve excellent ESD needle injectability of the injection solution. Furthermore, two control group solutions were obtained by replacing the aforementioned chitosan solution with a pH of 4.8 using a chitosan solution with a pH of 5.4 and a high pH chitosan solution (pH 5.5), while keeping other conditions unchanged. The experiment revealed that the injection solution prepared with the high pH chitosan solution (pH 5.5) had significantly increased viscosity and poor flowability, making it unsuitable for ESD injection. In contrast, the injection solution prepared with the chitosan solution at pH 5.4 maintained a low viscosity and was injectable. Therefore, the method proposed in this disclosure, using a chitosan solution with a conventional pH (pH less than or equal to 5.4), is necessary to achieve excellent ESD needle injectability in the injection solution.

[0066] Example 4

[0067] Chitosan powder (molecular weight 200,000 Da, degree of deacetylation 92%) was dissolved in 0.18 M hydrochloric acid solution under stirring to obtain chitosan solution A (pH 4.5). Polyvinylpyrrolidone powder (molecular weight 100,000 Da) and methylene blue powder were dissolved in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue. Sodium β-glycerophosphate powder was dissolved in water under stirring to obtain sodium β-glycerophosphate solution C. Sodium bicarbonate powder was dissolved in water under stirring to obtain sodium bicarbonate solution D. Dopamine powder was dissolved in sodium bicarbonate solution D under stirring to obtain a mixed solution E of sodium bicarbonate and dopamine, which was polymerized at room temperature for 20 min. Solutions A, B, C, and E were then pre-cooled in an ice bath. Solutions B, C, and E, pre-cooled in an ice bath, were added dropwise to solution A, pre-cooled in an ice bath, in the order of B, C, and E, under vortex oscillation. Each solution was thoroughly mixed after addition to obtain a chitosan-based thermosensitive submucosal injection solution. The solution was placed in an ice bath, and then 5 mL was taken for an ESD injection needle injectability test.

[0068] This chitosan-based thermosensitive submucosal injection contains 0.6% chitosan (w / v or g / mL), 0.7% polyvinylpyrrolidone (w / v or g / mL), 8% sodium β-glycerophosphate (w / v or g / mL), 0.005% methylene blue (w / v or g / mL), 0.01M sodium bicarbonate, and 1% dopamine (w / v or g / mL).

[0069] In addition, four injection solutions with chitosan concentrations of 1% (w / v or g / mL), 0.2% (w / v or g / mL), 0.19% (w / v or g / mL), and 1.1% (w / v or g / mL) were prepared respectively, while other conditions remained unchanged, thus obtaining four control group solutions. The experiment showed that the injection solutions with chitosan concentrations of 0.2% (w / v or g / mL), 0.6% (w / v or g / mL), and 1% (w / v or g / mL) could be smoothly injected through the ESD injection needle. The 1.1% (w / v or g / mL) injection solution became viscous, exhibiting significant injection resistance and failing to achieve injection. The 0.19% (w / v or g / mL) injection solution became turbid and unstable at low temperatures. Therefore, the above experiments demonstrate that the chitosan concentration of 0.2%–1% (w / v or g / mL) proposed in this disclosure is necessary to achieve excellent ESD needle injectability of the injection solution. Furthermore, two control group solutions were obtained by replacing the aforementioned chitosan solution with a pH of 4.5 using a chitosan solution with a pH of 5.4 and a high pH chitosan solution (pH 6.1), while keeping other conditions unchanged. The experiment revealed that the injection solution prepared with the high pH chitosan solution (pH 6.1) had significantly increased viscosity and poor flowability, making it unsuitable for ESD injection. In contrast, the injection solution prepared with the chitosan solution at pH 5.4 maintained a low viscosity and was injectable. Therefore, the method proposed in this disclosure, using a chitosan solution with a conventional pH (pH less than or equal to 5.4), is necessary to achieve excellent ESD needle injectability in the injection solution.

[0070] Example 5

[0071] Chitosan powder (molecular weight 800,000 Da, degree of deacetylation 94%) was dissolved in 0.15 M dilute hydrochloric acid solution under stirring to obtain chitosan solution A (pH 5.0). Polyvinylpyrrolidone powder (molecular weight 500,000 Da) and methylene blue powder were dissolved in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue. Sodium β-glycerophosphate powder was dissolved in water under stirring to obtain sodium β-glycerophosphate solution C. Sodium bicarbonate powder was dissolved in water under stirring to obtain sodium bicarbonate solution D. Solutions A, B, C, and D were placed in an ice bath for thorough pre-cooling. Solutions B, C, and D, pre-cooled in an ice bath, were added dropwise to solution A, pre-cooled in an ice bath, in the strict order of B, C, and D under vortex oscillation. Each solution was thoroughly mixed after addition to obtain a chitosan-based thermosensitive submucosal injection solution. The solution was placed in an ice bath, and then 1 mL was placed in a test tube and immersed in a 37°C water bath. The shortest time for the solution to remain stably inverted and the gelation time were determined by the inversion method.

[0072] This chitosan-based thermosensitive submucosal injection contains 0.2% chitosan (w / v or g / mL), 0.2% polyvinylpyrrolidone (w / v or g / mL), 9% sodium β-glycerophosphate (w / v or g / mL), 0.006% methylene blue (w / v or g / mL), and 0.005 M sodium bicarbonate.

[0073] In addition, three injection solutions with sodium bicarbonate concentrations of 0.004M, 0.03M, and 0.035M were prepared respectively, while keeping other conditions unchanged, thus obtaining three control group solutions. The experiment showed that the gelation time of the 0.004M sodium bicarbonate injection solution was 10 minutes, which was too long, resulting in the solution remaining in a liquid state for an extended period after injection and easily spreading. In contrast, the gelation times of the 0.005M and 0.03M sodium bicarbonate injection solutions were 5 minutes and 2.5 minutes respectively, significantly shorter and suitable for ESD use. Furthermore, it was found that the gelation speed increased with increasing sodium bicarbonate concentration. The gelation time of the 0.035M sodium bicarbonate injection solution was only 1 minute, which was too short, causing needle clogging and making it unsuitable for ESD operations. Therefore, the method proposed in this disclosure, using sodium bicarbonate concentrations of 0.005M to 0.03M, is necessary to achieve a shorter gelation time suitable for ESD operations. Furthermore, two control group solutions were obtained by replacing the aforementioned chitosan solution with a pH of 5.0 using a chitosan solution of pH 5.0 and a high-pH chitosan solution (pH 5.9), while keeping other conditions unchanged. The experiment revealed that the injection solution prepared with the high-pH chitosan solution (pH 5.9) had a gelation time of 1 minute, which was too short and caused needle blockage, making it unsuitable for ESD injection. In contrast, the injection solution prepared with the pH 5.4 chitosan solution had a gelation time of 2 minutes, which was suitable for ESD use. Therefore, it can be concluded that the use of a conventional pH chitosan solution (pH less than or equal to 5.4) as proposed in this disclosure is necessary to achieve a gelation time suitable for ESD use in the injection solution, and it was found that increasing the pH of the chitosan accelerates gelation.

[0074] Example 6

[0075] Chitosan powder (molecular weight 4000 Da, degree of deacetylation 85%) was dissolved in 0.5% (v / v) acetic acid solution under stirring to obtain chitosan solution A (pH 5.2). Polyvinylpyrrolidone powder (molecular weight 10000 Da) and methylene blue powder were dissolved in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue. Sodium β-glycerophosphate powder was dissolved in water under stirring to obtain sodium β-glycerophosphate solution C. Sodium bicarbonate powder was dissolved in water under stirring to obtain sodium bicarbonate solution D. Dopamine powder was dissolved in sodium bicarbonate solution D under stirring to obtain a mixed solution E of sodium bicarbonate and dopamine, which was polymerized at room temperature for 20 min. Solutions A, B, C, and E were then pre-cooled in an ice bath. Solutions B, C, and E, pre-cooled in an ice bath, were added dropwise to solution A, pre-cooled in an ice bath, in the strict order of B, C, and E, under vortex oscillation. Each solution was thoroughly mixed after addition to obtain a chitosan-based thermosensitive submucosal injection solution. The solution was placed in an ice bath, and then 1 ml was placed in a test tube and immersed in a 37°C water bath. The shortest time for the solution to remain stably inverted and the gelation time were determined by the inversion method.

[0076] This chitosan-based thermosensitive submucosal injection contains 1% chitosan (w / v or g / mL), 1.9% polyvinylpyrrolidone (w / v or g / mL), 7.5% sodium β-glycerophosphate (w / v or g / mL), 0.013% methylene blue (w / v or g / mL), and 0.03 M sodium bicarbonate.

[0077] In addition, three injection solutions with sodium bicarbonate concentrations of 0.004M, 0.005M, and 0.035M were prepared respectively, while keeping other conditions unchanged, thus obtaining three control group solutions. The experiment showed that the gelation time of the 0.004M sodium bicarbonate injection solution was 9 minutes, which was too long, resulting in the solution remaining in a liquid state for an extended period after injection and easily spreading. The gelation times of the 0.005M and 0.03M sodium bicarbonate injection solutions were 5 minutes and 3 minutes respectively, significantly shorter and suitable for ESD use. The gelation time of the 0.035M sodium bicarbonate injection solution was only 1 minute, too short, causing needle clogging and unsuitable for ESD operations. Therefore, the method proposed in this disclosure, using sodium bicarbonate concentrations of 0.005M to 0.03M, is necessary to achieve a shorter gelation time suitable for ESD operations. Furthermore, two control group solutions were obtained by replacing the aforementioned chitosan solution with a pH of 5.2 using a chitosan solution with a pH of 5.4 and a high pH chitosan solution (pH 6.0), while keeping other conditions unchanged. The experiment revealed that the injection solution prepared with the high pH chitosan solution (pH 6.0) had a gelation time of 1 minute, which was too short, causing needle blockage and making it unsuitable for ESD injection. In contrast, the injection solution prepared with the pH 5.4 chitosan solution had a gelation time of 2 minutes, which was suitable for ESD use. Therefore, it can be concluded that only by using a chitosan solution with a conventional pH (pH less than or equal to 5.4) as proposed in this disclosure can the injection solution achieve a gelation time suitable for ESD use.

[0078] Example 7

[0079] Chitosan powder (molecular weight 500,000 Da, degree of deacetylation 90%) was dissolved in 0.08 M dilute hydrochloric acid solution under stirring to obtain chitosan solution A (pH 5.3). Polyvinylpyrrolidone powder (molecular weight 200,000 Da) and methylene blue powder were dissolved in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue. Sodium β-glycerophosphate powder was dissolved in water under stirring to obtain sodium β-glycerophosphate solution C. Sodium bicarbonate powder was dissolved in water under stirring to obtain sodium bicarbonate solution D. Solutions A, B, C, and D were pre-cooled in an ice bath. The pre-cooled solutions B, C, and D were added dropwise to pre-cooled solution A in the order of B, C, and D under vortex oscillation, and each solution was thoroughly mixed after addition to obtain a chitosan-based thermosensitive submucosal injection solution, which was then placed in an ice bath. Subsequently, the storage modulus, i.e., gel strength, of the gel formed at 37°C was measured using a rheometer. In addition, Bama miniature pigs were anesthetized, and the injection solution was injected at specific points into the submucosa of the esophagus using an endoscopic needle, with 1 mL injected at each point. The height of the mucosal elevation after injection and subsequent changes in height over time were then detected using endoscopic ultrasound.

[0080] This chitosan-based thermosensitive submucosal injection contains 0.8% chitosan (w / v or g / mL), 1.9% polyvinylpyrrolidone (w / v or g / mL), 6.5% sodium β-glycerophosphate (w / v or g / mL), 0.009% methylene blue (w / v or g / mL), and 0.02 M sodium bicarbonate.

[0081] In addition, two more injection solutions with polyvinylpyrrolidone (PVP) concentrations of 0.2% (w / v or g / mL) and 1% (w / v or g / mL) were prepared, with all other conditions remaining unchanged, thus obtaining two control group solutions. The experiment showed that the injection solution with a PPVP concentration of 1.9% (w / v or g / mL) had the highest gel strength at 15 Pa, while the injection solutions with PPVP concentrations of 0.2% (w / v or g / mL) and 1% (w / v or g / mL) had gel strengths of 8 Pa and 12 Pa, respectively. Furthermore, immediately after injection, the initial mucosal elevation height was 12 mm in all three groups, with no significant difference. At 40 minutes post-injection, the mucosal elevation height of the 1.9% (w / v or g / mL) polyvinylpyrrolidone (PVP) solution was 8 mm, showing the slowest decline. In contrast, the elevation mucosal heights of the 0.2% (w / v or g / mL) and 1% (w / v or g / mL) PPVP solutions were 4.5 mm and 6.5 mm, respectively. Therefore, this experiment demonstrates that increasing the PPVP concentration can improve gel strength, thereby slowing down the collapse of the elevated mucosal height and facilitating ESD procedures.

[0082] Example 8

[0083] Chitosan powder (molecular weight 50,000 Da, degree of deacetylation 95%) was dissolved in 0.12 M dilute hydrochloric acid solution under stirring to obtain chitosan solution A (pH 5.1). Polyvinylpyrrolidone powder (molecular weight 50,000 Da) and methylene blue powder were dissolved in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue. Sodium β-glycerophosphate powder was dissolved in water under stirring to obtain sodium β-glycerophosphate solution C. Sodium bicarbonate powder was dissolved in water under stirring to obtain sodium bicarbonate solution D. Solutions A, B, C, and D were pre-cooled in an ice bath. The pre-cooled solutions B, C, and D were added dropwise to pre-cooled solution A in the order of B, C, and D under vortex oscillation, and each solution was thoroughly mixed after addition to obtain a chitosan-based thermosensitive submucosal injection solution, which was then placed in an ice bath. Subsequently, the storage modulus, i.e., gel strength, of the gel formed at 37°C was measured using a rheometer. In addition, Bama miniature pigs were anesthetized, and the injection solution was injected at specific points into the submucosa of the esophagus using an endoscopic needle, with 1 mL injected at each point. The height of the mucosal elevation after injection and subsequent changes in height over time were then detected using endoscopic ultrasound.

[0084] This chitosan-based thermosensitive submucosal injection contains 1% chitosan (w / v or g / mL), 1.7% polyvinylpyrrolidone (w / v or g / mL), 10% sodium β-glycerophosphate (w / v or g / mL), 0.011% methylene blue (w / v or g / mL), and 0.03 M sodium bicarbonate.

[0085] In addition, two more injection solutions with sodium bicarbonate concentrations of 0.02M and 0.01M were prepared, with all other conditions remaining unchanged, thus obtaining two control group solutions. The experiment showed that the injection solution with a sodium bicarbonate concentration of 0.03M had the highest gel strength at 16 Pa, while the gel strengths of the 0.02M and 0.01M solutions were 13 Pa and 11 Pa, respectively. Furthermore, immediately after injection, the initial mucosal elevation height was 12 mm in all three groups, with no difference. However, at 40 minutes after injection, the mucosal elevation height of the 0.03M solution was 9 mm, showing the slowest decline, while the elevation heights of the 0.02M and 0.01M solutions were 7 mm and 5 mm, respectively. Therefore, this experiment demonstrates that increasing the sodium bicarbonate concentration can improve gel strength, thereby slowing down the collapse of the mucosal elevation and facilitating ESD procedures.

[0086] Example 9

[0087] Chitosan powder (molecular weight 370,000 Da, degree of deacetylation 95%) was dissolved in 1% (v / v) acetic acid solution under stirring to obtain chitosan solution A (pH 4.9). Polyvinylpyrrolidone powder (molecular weight 330,000 Da) and methylene blue powder were dissolved in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue. Sodium β-glycerophosphate powder was dissolved in water under stirring to obtain sodium β-glycerophosphate solution C. Sodium bicarbonate powder was dissolved in water under stirring to obtain sodium bicarbonate solution D. Dopamine powder was dissolved in sodium bicarbonate solution D under stirring to obtain a mixed solution E of sodium bicarbonate and dopamine, which was polymerized at room temperature for 20 min. Solutions A, B, C, and E were then pre-cooled in an ice bath. Solutions B, C, and E, pre-cooled in an ice bath, were added dropwise to solution A, pre-cooled in an ice bath, in the strict order of B, C, and E, under vortex oscillation. Each solution was thoroughly mixed after addition to obtain a chitosan-based thermosensitive submucosal injection solution, which was then placed in an ice bath. The storage modulus, i.e., gel strength, of the gel formed at 37°C was then measured using a rheometer. Furthermore, Bama miniature pigs were anesthetized, and the injection solution was injected 1 mL at each point into the submucosal layer of the esophagus using an endoscopic needle. The height of the mucosal elevation after injection and its subsequent change over time were then observed using endoscopic ultrasound.

[0088] This chitosan-based thermosensitive submucosal injection contains 0.65% chitosan (w / v or g / mL), 1.1% polyvinylpyrrolidone (w / v or g / mL), 7.5% sodium β-glycerophosphate (w / v or g / mL), 0.015% methylene blue (w / v or g / mL), 0.02M sodium bicarbonate, and 2% dopamine (w / v or g / mL).

[0089] In addition, two more injection solutions with dopamine concentrations of 0% (w / v or g / mL) and 1% (w / v or g / mL) were prepared, with all other conditions remaining unchanged, thus obtaining two control group solutions. The experiment showed that the injection solution with a dopamine concentration of 2% (w / v or g / mL) had the highest gel strength at 15 Pa, while the gel strengths of the injection solutions with dopamine concentrations of 0% (w / v or g / mL) and 1% (w / v or g / mL) were 11 Pa and 13 Pa, respectively. Furthermore, immediately after injection, the initial mucosal elevation height was 12.5 mm in all three groups, with no difference. At 20 minutes after injection, the initial mucosal elevation height was 10.5 mm in all three groups, with no difference. At 40 minutes post-injection, the mucosal elevation height of the 2% (w / v or g / mL) dopamine injection was 9 mm, showing the slowest decline. In contrast, the elevation heights of the 0% (w / v or g / mL) and 1% (w / v or g / mL) dopamine injections were 5 mm and 7.5 mm, respectively. Therefore, this experiment demonstrates that polydopamine polymerized with dopamine can improve gel strength, and this gel strength increases with increasing dopamine concentration. Furthermore, while dopamine-containing injections initially slow the decline in mucosal elevation height after in vivo injection, their improved gel strength and tissue adhesion in the later stages of injection further slow down mucosal collapse, making them more suitable for ESD procedures in the later stages of injection.

[0090] Example 10

[0091] Chitosan powder (molecular weight 370,000 Da, degree of deacetylation 95%) was dissolved in 1% (v / v) acetic acid solution under stirring to obtain chitosan solution A (pH 4.9). Polyvinylpyrrolidone powder (molecular weight 330,000 Da) and methylene blue powder were dissolved in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue. Sodium β-glycerophosphate powder was dissolved in water under stirring to obtain sodium β-glycerophosphate solution C. Sodium bicarbonate powder was dissolved in water under stirring to obtain sodium bicarbonate solution D. Dopamine powder was dissolved in sodium bicarbonate solution D under stirring to obtain a mixed solution E of sodium bicarbonate and dopamine, which was polymerized at room temperature for 20 min. Solutions A, B, C, and E were then pre-cooled in an ice bath. Solutions B, C, and E, pre-cooled in an ice bath, were added dropwise to solution A, pre-cooled in an ice bath, in the strict order of B, C, and E, under vortex oscillation. Each solution was thoroughly mixed after addition to obtain a chitosan-based thermosensitive submucosal injection solution, which was then placed in an ice bath. Subsequently, the porcine esophagus was dissected and removed, laid flat on a plate and fixed with needles. The plate was then tilted at a 45° angle, and immediately 0.2 mL of the cryogenic injection solution was dropped onto a fixed position on the tilted esophageal surface. The furthest length of flow was measured to evaluate its adhesion to the tissue.

[0092] This chitosan-based thermosensitive submucosal injection contains 0.65% chitosan (w / v or g / mL), 1.1% polyvinylpyrrolidone (w / v or g / mL), 7.5% sodium β-glycerophosphate (w / v or g / mL), 0.015% methylene blue (w / v or g / mL), 0.02M sodium bicarbonate, and 2% dopamine (w / v or g / mL).

[0093] In addition, two more injection solutions with dopamine concentrations of 0% (w / v or g / mL) and 1% (w / v or g / mL) were prepared, with all other conditions remaining unchanged, thus obtaining two control group solutions. The experiment showed that the injection solution with a dopamine concentration of 2% (w / v or g / mL) had the shortest flow distance of 5 cm, while the flow distances for the dopamine injection solutions with concentrations of 0% (w / v or g / mL) and 1% (w / v or g / mL) were 8.2 cm and 7 cm, respectively. Therefore, this experiment demonstrates that dopamine can improve the adhesion between the gel and tissue, and that the adhesion is enhanced with increasing dopamine concentration.

[0094] Example 11

[0095] Chitosan powder (molecular weight 8000 Da, degree of deacetylation 90%) was dissolved in 1.1% (v / v) acetic acid solution under stirring to obtain chitosan solution A (pH 4.8). Polyvinylpyrrolidone powder (molecular weight 30000 Da) and methylene blue powder were dissolved in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue. Sodium β-glycerophosphate powder was dissolved in water under stirring to obtain sodium β-glycerophosphate solution C. Sodium bicarbonate powder was dissolved in water under stirring to obtain sodium bicarbonate solution D. Dopamine powder was dissolved in sodium bicarbonate solution D under stirring to obtain a mixed solution E of sodium bicarbonate and dopamine, which was polymerized at room temperature for 20 min. Solutions A, B, C, and E were then pre-cooled in an ice bath. Solutions B, C, and E, pre-cooled in an ice bath, were added dropwise to solution A, pre-cooled in an ice bath, in the order of B, C, and E, under vortex oscillation. Each solution was thoroughly mixed after addition to obtain injection solution 1, which was then placed in an ice bath.

[0096] This chitosan-based thermosensitive submucosal injection contains 0.8% chitosan (w / v or g / mL), 1.3% polyvinylpyrrolidone (w / v or g / mL), 7% sodium β-glycerophosphate (w / v or g / mL), 0.001% methylene blue (w / v or g / mL), 0.01M sodium bicarbonate, and 1% dopamine (w / v or g / mL).

[0097] In addition, solutions B, C, and E, which were pre-cooled in an ice bath, were added dropwise to solution A, which was pre-cooled in an ice bath, in the order of B, E, and C, under vortex oscillation conditions. Each solution was thoroughly mixed after being added, while other conditions remained unchanged, to obtain injection solution 2, which was then placed in an ice bath.

[0098] In addition, C and E were first mixed thoroughly in an ice bath to obtain a mixture F. Then, B and F were added dropwise to solution A, which was pre-cooled in an ice bath, with each solution being thoroughly mixed after addition, while keeping other conditions unchanged, to obtain injection solution 3, which was then placed in an ice bath.

[0099] In addition, dopamine was removed from injection solutions 1, 2, and 3, while other conditions remained unchanged, to obtain injection solutions 4, 5, and 6, which were then placed in an ice bath. Experiments showed that only injection solutions 1 and 4 were clear and could form complete gels at 37°C, while injection solutions 2, 3, 5, and 6 were turbid and could only form tiny fragments of gel at 37°C.

[0100] This experiment demonstrates that only by strictly adding the chitosan molecules B, C, and D, or B, C, and E sequentially to the pre-cooled solution A in an ice bath, as proposed in this disclosure, can a clear and homogeneous injection solution be obtained, which also possesses the ability to form a complete hydrogel at body temperature. This is because chitosan molecules are more sensitive to alkaline environments under low-concentration chitosan conditions. If D or E is added to A before C, or if C, D, or C and E are mixed before being added to A, the interaction between sodium β-glycerophosphate and chitosan will be insufficient, failing to form adequate steric hindrance. This leads to a rapid increase in alkalinity, causing a large amount of chitosan to precipitate, resulting in turbidity of the injection solution and a significant reduction in its gel-forming ability.

[0101] The above description is merely a preferred embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of this disclosure.

Claims

1. A chitosan-based thermosensitive submucosal injection solution for endoscopy, characterized in that, The injection solution includes chitosan with a standard pH value, polyvinylpyrrolidone, sodium β-glycerophosphate, and sodium bicarbonate.

2. The injection solution according to claim 1, characterized in that, The injection solution also includes dopamine.

3. The use of the chitosan-based thermosensitive endoscopic submucosal injection solution according to claim 1 or 2 in the preparation of a thermosensitive submucosal injection for endoscopic submucosal dissection.

4. The use of the chitosan-based thermosensitive endoscopic submucosal injection solution according to claim 1 or 2 in the preparation of a thermosensitive submucosal injection for postoperative procedures following endoscopic submucosal dissection.

5. A method for preparing a chitosan-based thermosensitive endoscopic submucosal injection solution according to claim 1 or 2, characterized in that, The method includes the following steps: S1. Chitosan powder with a molecular weight of 4000 Da to 800000 Da is dissolved in a dilute acid solution under stirring to obtain a chitosan solution A with a pH less than or equal to 5.

4. S2. Dissolve polyvinylpyrrolidone powder and methylene blue powder in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue, wherein the molecular weight of the polyvinylpyrrolidone powder is 10,000 Da to 500,000 Da. S3. Dissolve sodium β-glycerophosphate powder in water under stirring to obtain sodium β-glycerophosphate solution C; S4. Dissolve sodium bicarbonate powder in water under stirring to obtain sodium bicarbonate solution D; S5. Place the solutions A, B, C, and D in an ice bath to pre-cool them thoroughly; S6. The solutions B, C, and D, which are pre-cooled in an ice bath, are added dropwise to the solution A, which is pre-cooled in an ice bath, in strict order of B, C, and D under vortex oscillation conditions. Each solution is thoroughly mixed after being added dropwise to obtain a chitosan-based thermosensitive submucosal injection solution, which is placed in an ice bath for in vivo injection. The chitosan-based thermosensitive submucosal injection solution contains chitosan at a concentration of 0.2%–1%, polyvinylpyrrolidone at a concentration of 0.2%–1.9%, sodium β-glycerophosphate at a concentration of 4%–10%, methylene blue at a concentration of 0.001%–0.02%, and sodium bicarbonate at a concentration of 0.005M–0.03M. The concentrations of chitosan, polyvinylpyrrolidone, sodium β-glycerophosphate, and methylene blue are all expressed as w / v or g / mL.

6. The method for preparing the chitosan-based thermosensitive endoscopic submucosal injection solution according to claim 5, characterized in that, In step S1, the degree of deacetylation of the chitosan is 85% or higher, and the dilute acid solution is a 0.05M to 0.2M hydrochloric acid solution or a 0.5% to 3% acetic acid solution on a v / v basis.

7. The application of a thermosensitive submucosal injection solution prepared by the preparation method according to claim 5 or 6 in the preparation of a thermosensitive submucosal injection for endoscopic submucosal dissection.

8. A method for preparing a chitosan-based thermosensitive endoscopic submucosal injection solution according to claim 1 or 2, characterized in that, The method includes the following steps: S1. Chitosan powder with a molecular weight of 4000 Da to 800000 Da is dissolved in a dilute acid solution under stirring to obtain a chitosan solution A with a pH less than or equal to 5.

4. S2. Dissolve polyvinylpyrrolidone powder and methylene blue powder in water under stirring to obtain a mixed solution B of polyvinylpyrrolidone and methylene blue, wherein the molecular weight of the polyvinylpyrrolidone powder is 10,000 Da to 500,000 Da. S3. Dissolve sodium β-glycerophosphate powder in water under stirring to obtain sodium β-glycerophosphate solution C; S4. Dissolve sodium bicarbonate powder in water under stirring to obtain sodium bicarbonate solution D; S5. Dissolve dopamine powder in sodium bicarbonate solution D under stirring to obtain a mixed solution E of sodium bicarbonate and dopamine, and polymerize at room temperature for 20 min. S6. Place the solutions A, B, C, and E in an ice bath to pre-cool them thoroughly; S7. The solutions B, C, and E, which are pre-cooled in an ice bath, are added dropwise to the solution A, which is pre-cooled in an ice bath, in strict order of B, C, and E under vortex oscillation conditions. Each solution is thoroughly mixed after being added dropwise to obtain a chitosan-based thermosensitive submucosal injection solution, which is placed in an ice bath for in vivo injection. The chitosan-based thermosensitive submucosal injection solution contains chitosan at a concentration of 0.2%–1%, polyvinylpyrrolidone at a concentration of 0.2%–1.9%, sodium β-glycerophosphate at a concentration of 4%–10%, methylene blue at a concentration of 0.001%–0.02%, sodium bicarbonate at a concentration of 0.005M–0.03M, and dopamine at a concentration of 0%–2%. The concentrations of chitosan, polyvinylpyrrolidone, sodium β-glycerophosphate, methylene blue, and dopamine are all expressed as w / v or g / mL.

9. The method for preparing the chitosan-based thermosensitive endoscopic submucosal injection solution according to claim 8, characterized in that, In step S1, the degree of deacetylation of the chitosan is 85% or higher, and the dilute acid solution is a 0.05M to 0.2M hydrochloric acid solution or a 0.5% to 3% acetic acid solution on a v / v basis.

10. The application of a thermosensitive submucosal injection solution prepared by the preparation method according to claim 8 or 9 in the preparation of a thermosensitive submucosal injection for postoperative procedures after endoscopic submucosal dissection.