Submucosal injection and preparation method thereof

Abstract

The invention discloses a submucosal injection and a preparation method thereof.The submucosal injection is prepared from poloxamer and sodium hyaluronate, the mass fraction of the poloxamer in the raw materials is 12%-20%, and the mass fraction of hyaluronic acid in the raw materials is 0.05%-0.2%; or the hyaluronic acid-poloxamer grafted copolymer is prepared from the following components in percentage by mass: 12 to 18 percent of hyaluronic acid-poloxamer grafted copolymer. The submucous membrane injection is easy to inject and can form gel at the body temperature (37 DEG C), the height of the submucous membrane cushion layer meets the surgical requirement, the submucous membrane cushion layer can maintain enough surgical operation time, separation of a lesion mucous membrane and a muscular layer is facilitated, resection operation of a doctor on the mucous membrane is facilitated, loss caused to submucous membrane tissue is reduced, and the mucous membrane submucous membrane injection is safe and reliable. Complications such as perforation are prevented, bleeding caused by mucous membrane tissue excision is reduced, and the method has special advantages compared with other non-gel materials.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, in particular to a submucosal injection solution and a preparation method thereof. Background Art

[0002] Endoscopic mucosal resection (EMR) and endoscopic mucosal dissection (ESD) are mature treatments for early gastrointestinal cancer and precancerous lesions. Submucosal injection refers to the injection of liquid into the submucosal layer to form a submucosal fluid cushion (SFC) between the diseased mucosa and the smooth muscle layer, thereby lifting the diseased mucosa and forming a submucosal isolation layer to separate the mucosal layer from the muscle layer. Submucosal space can be established through submucosal injection, which can reduce the risk of complications and save surgical time. The longer the intraoperative SFC is maintained, the less repeated injections are needed to maintain the height of the liquid cushion, which can simplify the operation, shorten the operation time, and reduce complications. Therefore, SFC has become one of the decisive factors in ensuring the safety of minimally invasive endoscopic surgery.

[0003] At present, the submucosal injection solutions commonly used in clinical practice include normal saline, glucose, glycerol fructose, and sodium hyaluronate. Among them, normal saline is the most commonly used submucosal injection solution, but because it is quickly absorbed by the surrounding tissues in the mucosal layer, it is insufficient to maintain the height of the mucosal bulge and the time is short; the main problem with glucose for submucosal injection is that it damages tissues as a hypertonic solution; the liquid pad formed after submucosal injection of glycerol fructose lasts relatively long, but more smoke will appear during ESD electrosurgical resection, interfering with the surgical field and affecting endoscopic operation; sodium hyaluronate (0.4% HA) is currently considered to be a submucosal injection solution with better effect, and its products include Blue Beacon TM 、Blue Eye TM and Multiple studies have confirmed its effectiveness as an injection solution, with significant advantages in mucosal ridge height, boundaries, and maintenance time. However, when the ridge height and time of sodium hyaluronate meet the requirements of submucosal injection, the viscosity of the liquid is too high, which is not conducive to injection, limiting its wide application.

[0004] In view of this, a submucosal injection solution that meets the requirements for mucosal elevation height and maintenance time and is easy to inject is particularly necessary. Summary of the invention

[0005] The purpose of the present invention is to provide a submucosal injection solution and a preparation method thereof, and to provide a submucosal injection solution which has a mucosal protrusion height and a maintenance time that meet the requirements and is easy to inject.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a submucosal injection solution, wherein the raw materials include poloxamer and sodium hyaluronate, wherein the mass fraction of the poloxamer in the raw material is 12%-20%, and the mass fraction of the hyaluronic acid in the raw material is 0.05%-0.2%;

[0008] Or, comprising 12%-18% by mass of hyaluronic acid-poloxamer graft copolymer.

[0009] In an optional embodiment, the molecular weight of the hyaluronic acid is 100 kDa-1000 kDa or 1000 Da-5000 Da.

[0010] In an optional embodiment, a dye is further included. Preferably, the dye is one of indigo carmine, methylene blue, phenol red, Congo red, toluidine blue and crystal violet.

[0011] In a second aspect, the present invention provides a method for preparing the submucosal injection solution described in the aforementioned embodiment, comprising:

[0012] The poloxamer and the hyaluronic acid aqueous solution are mixed to dissolve the poloxamer to obtain a mixed solution, and the mixed solution is subjected to high-pressure homogenization to obtain the submucosal injection solution.

[0013] In an optional embodiment, the mass fraction of hyaluronic acid in the submucosal injection solution is 0.05wt%-0.2wt%;

[0014] And / or, the mass fraction of the poloxamer is 12wt%-20wt%;

[0015] And / or, the molecular weight of the hyaluronic acid is 400 kDa-1000 kDa;

[0016] And / or, high pressure homogenization is carried out in a high-speed dispersing homogenizer, the homogenization pressure is 100-500 bar, and the homogenization number is 1 time;

[0017] and / or, the poloxamer is poloxamer 407;

[0018] And / or, the dissolution temperature is 3-5°C and the dissolution time is 12-24h.

[0019] In a third aspect, the present invention provides a method for preparing the submucosal injection solution described in the aforementioned embodiment, comprising:

[0020] Preparation of hyaluronic acid-poloxamer graft copolymer: under ice bath condition, poloxamer, 4-(N,N-dimethylamino)pyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are sequentially added to a hyaluronic acid solution, and reacted under nitrogen protection condition, and the reaction liquid is dialyzed and freeze-dried in turn to obtain a hyaluronic acid-poloxamer graft copolymer;

[0021] Preparation of submucosal injection solution: the hyaluronic acid-poloxamer graft copolymer is dissolved in water and then subjected to high-pressure homogenization to obtain the submucosal injection solution.

[0022] In an optional embodiment, the solvent of the hyaluronic acid solution is N,N-dimethylformamide;

[0023] And / or, the molecular weight of the hyaluronic acid is in the range of 1000-5000Da;

[0024] and / or, in the pre-reaction solution, the mass ratio of hyaluronic acid, poloxamer, 4-(N,N-dimethylamino)pyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is (0.16-0.7):2:(0.1-0.4):(0.1-0.4);

[0025] and / or, the reaction time is 18-36h;

[0026] and / or, the mass fraction of the hyaluronic acid-poloxamer graft copolymer in the submucosal injection solution is 12%-18%;

[0027] And / or, high pressure homogenization is carried out in a high-speed dispersing homogenizer, the homogenization pressure is 100-500 bar, and the homogenization number is 1 time;

[0028] And / or, the poloxamer is poloxamer 407.

[0029] In a fourth aspect, the present invention provides a method for preparing the submucosal injection solution described in the aforementioned embodiment, comprising:

[0030] Preparation of hyaluronic acid-poloxamer graft copolymer: under ice bath condition, poloxamer, 4-(N,N-dimethylamino)pyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are sequentially added to a hyaluronic acid solution, and reacted under nitrogen protection condition, and the reaction liquid is dialyzed and freeze-dried in turn to obtain a hyaluronic acid-poloxamer graft copolymer;

[0031] Preparation of submucosal injection solution: The hyaluronic acid-poloxamer graft copolymer and poloxamer are dissolved in water to obtain a hyaluronic acid-poloxamer graft copolymer aqueous solution and a poloxamer aqueous solution, and then mixed and subjected to high-pressure homogenization to obtain the submucosal injection solution.

[0032] In an optional embodiment, the solvent of the hyaluronic acid solution is N,N-dimethylformamide;

[0033] And / or, the hyaluronic acid has a molecular weight range of 100 kDa-400 kDa;

[0034] and / or, in the pre-reaction solution, the mass ratio of hyaluronic acid, poloxamer, 4-(N,N-dimethylamino)pyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 0.25:(5-10):(0.16-0.26):(0.19-0.3);

[0035] and / or, the reaction time is 18-36h;

[0036] And / or, the mass fraction of the hyaluronic acid-poloxamer graft copolymer aqueous solution is 14%-18%;

[0037] And / or, the mass fraction of the poloxamer aqueous solution is 14%-18%;

[0038] and / or, the hyaluronic acid-poloxamer graft copolymer aqueous solution and the poloxamer aqueous solution are mixed at a volume ratio of 1:5-10;

[0039] And / or, high pressure homogenization is carried out in a high-speed dispersing homogenizer, the homogenization pressure is 100-500 bar, and the homogenization number is 1 time;

[0040] And / or, the poloxamer is poloxamer 407.

[0041] The present invention has the following beneficial effects:

[0042] The submucosal injection gel of the present invention is easy to inject and can gel at body temperature (37°C) to maintain the height of the submucosal cushion layer. The submucosal cushion layer can be maintained for a sufficient time for surgical operations, which is beneficial to the separation of the diseased mucosa and the muscular layer, facilitates the doctor's mucosal resection operation, reduces the loss of submucosal tissue, prevents complications such as perforation, and reduces bleeding caused by the resection of mucosal tissue. This has special advantages over other non-gel materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0044] Figure 1 A preparation method of submucosal injection solution and a schematic diagram of its gelling mechanism;

[0045] Figure 2 This is the gel formation diagram of sample 4 and reference substance 1 after homogenization (reference substance 1 on the left and sample 4 on the right);

[0046] Figure 3This is the elevation height diagram of sample 4 injected into the pig stomach submucosal layer;

[0047] Figure 4 This is a photo of the gel obtained by injecting sample 4 into the submucosal layer of pig stomach. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0049] The embodiment of the present application provides a submucosal injection solution, the raw materials include poloxamer and sodium hyaluronate, the mass fraction of the poloxamer in the raw material is 12%-20%, and the mass fraction of the hyaluronic acid in the raw material is 0.05%-0.2%;

[0050] Or, comprising 12%-18% by mass of hyaluronic acid-poloxamer graft copolymer.

[0051] Poloxamer is a synthetic polyester material, a block copolymer composed of polyoxyethylene and polyoxypropylene. It has a thermosensitive sol-gel transition property. It is liquid at room temperature, and the molecular chains are in a self-assembled micelle state. It can undergo a phase transition at human body temperature, the micelle state disintegrates, and the molecular chains cross-link into a network to form a semisolid gel.

[0052] Sodium hyaluronate is a polysaccharide material with very strong water storage capacity. It has a higher viscosity at a lower concentration, has certain anti-inflammatory and pro-repair properties, and has good biocompatibility.

[0053] Thermosensitive hydrogel is liquid at room temperature, and can undergo phase transition when close to body temperature to form a non-chemically cross-linked semisolid gel, which can meet the requirements for submucosal injection and may become an ideal submucosal injection substance. The thermosensitive submucosal injection proposed in this embodiment has simple raw materials, is easy to prepare, and has a low viscosity at a relatively low temperature (4-25°C). At a relatively high temperature (30-40°C), hyaluronic acid molecules and poloxamer molecules form a hydrogel in a three-dimensional network state through hydrogen bonds. For submucosal injection, it can provide a high SFC that meets the surgical requirements and can maintain a sufficiently long uplift time. The material has a low viscosity and can be easily injected through a general endoscopic syringe (low injection pressure). It has good biocompatibility with tissues, does not damage tissues, is easy to prepare and manage, has a low production cost, and meets the basic requirements of submucosal injection.

[0054] In an optional embodiment, the molecular weight of the hyaluronic acid is 100 kDa-1000 kDa or 1000 Da-5000 Da, and hyaluronic acid with different molecular weights can be selected according to different preparation methods.

[0055] In an optional embodiment, a dye is further included, preferably, the dye is one of indigo carmine, methylene blue, phenol red, Congo red, toluidine blue and crystal violet. Specifically, the type and amount of the dye can be added according to the type and depth of the desired color, and the amount added is small, which has little effect on the viscosity, injection performance, bulge height, maintenance time, etc. of the submucosal injection solution.

[0056] Another embodiment of the present invention provides a method for preparing the submucosal injection solution described in the above embodiment, comprising:

[0057] The poloxamer and the hyaluronic acid aqueous solution are mixed to dissolve the poloxamer to obtain a mixed solution, and the mixed solution is subjected to high-pressure homogenization to obtain the submucosal injection solution.

[0058] This embodiment adopts physical method to prepare the submucosal injection solution, the mechanism is as follows Figure 1 As shown, poloxamer and hyaluronic acid are mixed and then subjected to high-pressure homogenization to form composite micelles to reduce viscosity. Due to the intermolecular force between poloxamer and hyaluronic acid, the addition of hyaluronic acid can significantly increase the gel viscosity of poloxamer. After passing through the high-pressure homogenizer, the viscosity of the solution is further reduced because the poloxamer micelles wrap the hyaluronic acid molecules, which can reduce the injection pressure.

[0059] In this embodiment, the hyaluronic acid aqueous solution can be obtained by dissolving hyaluronic acid in deionized water and stirring for 12-24 hours, and a dye can also be added as needed.

[0060] In an optional embodiment, the mass fraction of hyaluronic acid in the submucosal injection solution is 0.05wt%-0.2wt%;

[0061] And / or, the mass fraction of the poloxamer is 12wt%-20wt%;

[0062] And / or, the molecular weight of the hyaluronic acid is 400 kDa-1000 kDa;

[0063] And / or, high-pressure homogenization is carried out in a high-speed dispersing homogenizer, the homogenization pressure is 100-500 bar, the homogenization number is 1 time, and the viscosity of the obtained sample is further reduced as the homogenization number increases, resulting in failure to form gel;

[0064] and / or, the poloxamer is poloxamer 407;

[0065] And / or, the dissolution temperature is 3-5°C and the dissolution time is 12-24h.

[0066] In this embodiment, too little hyaluronic acid content, too little hyaluronic acid molecular weight, too little poloxamer content and too much homogenization pressure may lead to failure of gelation, or conversely may lead to excessive viscosity, increasing the difficulty of injection.

[0067] Another embodiment of the present invention provides a method for preparing the submucosal injection solution described in the above embodiment, comprising:

[0068] Preparation of hyaluronic acid-poloxamer graft copolymer: under ice bath condition, poloxamer, 4-(N,N-dimethylamino)pyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are sequentially added to a hyaluronic acid solution, and reacted under nitrogen protection condition, and the reaction liquid is dialyzed and freeze-dried in turn to obtain a hyaluronic acid-poloxamer graft copolymer;

[0069] Preparation of submucosal injection solution: the hyaluronic acid-poloxamer graft copolymer is dissolved in water and then subjected to high-pressure homogenization to obtain the submucosal injection solution.

[0070] In this embodiment, hyaluronic acid and poloxamer are reacted through Steglich esterification to form a hyaluronic acid-poloxamer graft copolymer conjugate, and ultra-low molecular weight hyaluronic acid is grafted onto the poloxamer main chain to increase the viscosity of the poloxamer. The solution viscosity is then reduced by high-pressure homogenization to achieve the purpose of reducing the injection pressure, making it easier to inject through a common gastrointestinal endoscope while maintaining its temperature sensitivity and being able to provide sufficient ridge height and maintenance time.

[0071] In an optional embodiment, the solvent of the hyaluronic acid solution is N,N-dimethylformamide, and the amount of the solvent used is sufficient to fully dissolve the reactants and the catalyst, so as to facilitate their removal in the subsequent dialysis freeze-drying step;

[0072] And / or, the molecular weight of the hyaluronic acid is in the range of 1000-5000Da;

[0073] and / or, in the pre-reaction solution, the mass ratio of hyaluronic acid, poloxamer, 4-(N,N-dimethylamino)pyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is (0.16-0.7):2:(0.1-0.4):(0.1-0.4);

[0074] and / or, the reaction time is 18-36h;

[0075] and / or, the mass fraction of the hyaluronic acid-poloxamer graft copolymer in the submucosal injection solution is 12%-18%;

[0076] And / or, high-pressure homogenization is carried out in a high-speed dispersing homogenizer, the homogenization pressure is 100-500 bar, the homogenization number is 1 time, and the viscosity of the obtained sample is further reduced as the homogenization number increases, resulting in failure to form gel;

[0077] And / or, the poloxamer is poloxamer 407.

[0078] In this embodiment, 4-(N,N-dimethylamino)pyridine (DMAP) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) are used as catalysts to graft hyaluronic acid onto poloxamer, which can reduce the amount of poloxamer required for submucosal injection to a certain extent.

[0079] Another embodiment of the present invention provides a method for preparing the submucosal injection solution described in the above embodiment, comprising:

[0080] Preparation of hyaluronic acid-poloxamer graft copolymer: under ice bath condition, poloxamer, 4-(N,N-dimethylamino)pyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are sequentially added to a hyaluronic acid solution, and reacted under nitrogen protection condition, and the reaction liquid is dialyzed and freeze-dried in turn to obtain a hyaluronic acid-poloxamer graft copolymer;

[0081] Preparation of submucosal injection solution: The hyaluronic acid-poloxamer graft copolymer and poloxamer are dissolved in water to obtain a hyaluronic acid-poloxamer graft copolymer aqueous solution and a poloxamer aqueous solution, and then mixed and subjected to high-pressure homogenization to obtain the submucosal injection solution.

[0082] Poloxamer is grafted onto the main chain of low molecular weight hyaluronic acid, and the grafted product poloxamer is then mixed to reduce the concentration of hyaluronic acid. After high-pressure homogenization, the viscosity of the solution and the injection pressure are reduced, making it easier to inject through a common gastrointestinal endoscope while maintaining its temperature sensitivity, providing sufficient ridge height and maintenance time.

[0083] In an optional embodiment, the solvent of the hyaluronic acid solution is N,N-dimethylformamide, and the amount of the solvent used is sufficient to fully dissolve the reactants and the catalyst, so as to facilitate their removal in the subsequent dialysis freeze-drying step;

[0084] And / or, the hyaluronic acid has a molecular weight range of 100 kDa-400 kDa;

[0085] and / or, in the pre-reaction solution, the mass ratio of hyaluronic acid, poloxamer, 4-(N,N-dimethylamino)pyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 0.25:(5-10):(0.16-0.26):(0.19-0.3);

[0086] and / or, the reaction time is 18-36h;

[0087] And / or, the mass fraction of the hyaluronic acid-poloxamer graft copolymer aqueous solution is 14%-18%;

[0088] And / or, the mass fraction of the poloxamer aqueous solution is 14%-18%;

[0089] and / or, the hyaluronic acid-poloxamer graft copolymer aqueous solution and the poloxamer aqueous solution are mixed at a volume ratio of 1:5-10;

[0090] And / or, high-pressure homogenization is carried out in a high-speed dispersing homogenizer, the homogenization pressure is 100-500 bar, the homogenization number is 1 time, and the viscosity of the obtained sample is further reduced as the homogenization number increases, resulting in failure to form gel;

[0091] And / or, the poloxamer is poloxamer 407.

[0092] In this embodiment, 4-(N,N-dimethylamino)pyridine (DMAP) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) are used as catalysts to graft hyaluronic acid onto poloxamer, which can reduce the amount of poloxamer required for submucosal injection to a certain extent.

[0093] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0094] Example 1

[0095] 0.2 g of hyaluronic acid (molecular weight 800-1000 kDa) was weighed and added to 319.8 g of deionized water. Indigo carmine was added and stirred for 20 h. Then, 80 g of poloxamer 407 was added and dissolved at 4 °C for 24 h. A high-speed dispersing homogenizer was used to perform a homogenization cycle at a pressure of 200 bar to obtain sample 1.

[0096] Example 2

[0097] Weigh 0.8 g of hyaluronic acid (molecular weight 800-1000 kDa), add 351.2 g of deionized water, then add methyl blue and stir for 24 hours, then add 48 g of poloxamer 407, dissolve at 4°C for 30 hours, and use a high-speed dispersing homogenizer to perform a homogenization cycle at a pressure of 300 bar to obtain sample 2.

[0098] Example 3

[0099] 0.8 g of hyaluronic acid (molecular weight 400-600 kDa) was weighed and added to 319.2 g of deionized water. Methyl blue was then added and stirred for 24 h. Then, 80 g of poloxamer 407 was added and dissolved at 4 °C for 36 h. A high-speed dispersing homogenizer was used to perform a homogenization cycle at a pressure of 500 bar to obtain sample 3.

[0100] Example 4

[0101] Weigh 0.2 g of hyaluronic acid (molecular weight 400-600 kDa), add 351.8 g of deionized water, then add methyl blue and stir for 12 hours, then add 48 g of poloxamer 407, dissolve at 4°C for 24 hours, and use a high-speed dispersing homogenizer at a pressure of 100 bar to perform a homogenization cycle to obtain sample 4.

[0102] Example 5

[0103] Weigh 0.4 g of hyaluronic acid (molecular weight 600-800 kDa), add 339.6 g of deionized water, then add methyl blue and stir for 18 hours, then add 60 g of poloxamer 407, dissolve at 4°C for 24 hours, and use a high-speed dispersing homogenizer at a pressure of 400 bar to perform a homogenization cycle to obtain sample 5.

[0104] Example 6

[0105] Weigh 0.4 g of hyaluronic acid (molecular weight 600-800 kDa), add 327.6 g of deionized water, then add methyl blue and stir for 18 hours, then add 72 g of poloxamer 407, dissolve at 4°C for 30 hours, and use a high-speed disperser homogenizer at a pressure of 400 bar to perform a homogenization cycle to obtain sample 6.

[0106] Example 7

[0107] Weigh 0.6 g of hyaluronic acid (molecular weight 600-800 kDa), add 339.4 g of deionized water, then add methyl blue and stir for 18 hours, then add 60 g of poloxamer 407, dissolve at 4°C for 30 hours, and use a high-speed dispersing homogenizer at a pressure of 400 bar to perform a homogenization cycle to obtain sample 7.

[0108] Example 8

[0109] 0.4 g of hyaluronic acid (molecular weight 800-1000 kDa) was weighed and added to 339.6 g of deionized water. Methyl blue was then added and stirred for 18 h. Then, 60 g of poloxamer 407 was added and dissolved at 4°C for 30 h. A high-speed dispersing homogenizer was used to perform a homogenization cycle at a pressure of 500 bar to obtain sample 8.

[0110] Example 9

[0111] 0.16 g of hyaluronic acid (molecular weight of 1000) was weighed and dissolved in N,N-dimethylformamide, and 2 g of poloxamer 407, 0.1 g of 4-(N,N-dimethylamino)pyridine (DMAP), and 0.1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were added in sequence. The mixture was reacted under nitrogen protection for 18 h in an ice bath, and then dialyzed and freeze-dried to obtain a hyaluronic acid-poloxamer graft copolymer. The copolymer was mixed with methyl blue to prepare a solution of 18 wt% of the hyaluronic acid-poloxamer graft copolymer. The solution was homogenized once at a pressure of 100 bar using a high-speed disperser homogenizer to obtain sample 9.

[0112] Example 10

[0113] 0.7 g of hyaluronic acid (molecular weight of 5000) was weighed and dissolved in N, N-dimethylformamide, and 2 g of poloxamer 407, 0.4 g of 4-(N, N-dimethylamino)pyridine (DMAP), and 0.4 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were added in sequence. The mixture was reacted under nitrogen protection for 36 h in an ice bath, and then dialyzed and freeze-dried to obtain a hyaluronic acid-poloxamer graft copolymer. A 14 wt% hyaluronic acid-poloxamer graft copolymer solution was prepared by mixing the copolymer with methyl blue, and the mixture was homogenized once at a pressure of 500 bar using a high-speed dispersing homogenizer to obtain sample 10.

[0114] Embodiment 11

[0115] 0.5 g of hyaluronic acid (molecular weight of 2000) was weighed and dissolved in N, N-dimethylformamide, and 2 g of poloxamer 407, 0.24 g of 4-(N, N-dimethylamino)pyridine (DMAP), and 0.28 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were added in sequence. The mixture was reacted under nitrogen protection for 24 h in an ice bath, and then dialyzed and freeze-dried to obtain a hyaluronic acid-poloxamer graft copolymer. A 15 wt% hyaluronic acid-poloxamer graft copolymer solution was prepared by mixing the copolymer with methyl blue, and the mixture was homogenized once at a pressure of 400 bar using a high-speed disperser homogenizer to obtain sample 11.

[0116] Example 12

[0117] 0.25 g of hyaluronic acid (molecular weight of 100-200 kDa) was weighed and dissolved in N, N-dimethylformamide, and 5 g of poloxamer 407, 0.16 g of 4-(N, N-dimethylamino)pyridine (DMAP), and 0.19 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were added in sequence. The mixture was reacted under nitrogen protection for 18 h in an ice bath, and then dialyzed and freeze-dried to obtain a hyaluronic acid-poloxamer graft copolymer. A 14 wt % hyaluronic acid-poloxamer graft copolymer solution and a poloxamer 407 solution were prepared respectively. The two solutions were mixed in a volume ratio of 1:10 and then mixed with methyl blue. The mixture was homogenized once at a pressure of 400 bar using a high-speed disperser homogenizer to obtain sample 12.

[0118] Embodiment 13

[0119] 0.25 g of hyaluronic acid (molecular weight of 300-400 kDa) was weighed and dissolved in N,N-dimethylformamide, and 10 g of poloxamer 407, 0.26 g of 4-(N,N-dimethylamino)pyridine (DMAP), and 0.3 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were added in sequence. The mixture was reacted under nitrogen protection for 36 h in an ice bath, dialyzed and freeze-dried to obtain a hyaluronic acid-poloxamer graft copolymer. 18% hyaluronic acid-poloxamer graft copolymer solution and poloxamer 407 solution were prepared respectively. The two solutions were mixed in a volume ratio of 1:5 and then mixed with methyl blue. The mixture was homogenized once at a pressure of 500 bar using a high-speed disperser homogenizer to obtain sample 13.

[0120] Embodiment 14

[0121] 0.25 g of hyaluronic acid (molecular weight 200-300 kDa) was weighed and dissolved in N, N-dimethylformamide, and 8 g of poloxamer 407, 0.2 g of 4-(N, N-dimethylamino)pyridine (DMAP), and 0.3 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were added in sequence. The mixture was reacted under nitrogen protection for 24 h in an ice bath, dialyzed and freeze-dried to obtain a hyaluronic acid-poloxamer graft copolymer. A 15% hyaluronic acid-poloxamer graft copolymer solution and a poloxamer 407 solution were prepared respectively. The two were mixed in a volume ratio of 1:8 and then mixed with methyl blue. The mixture was homogenized once at a pressure of 100 bar using a high-speed disperser homogenizer to obtain sample 14.

[0122] Comparative Example 1

[0123] Weigh 0.16 g of hyaluronic acid (molecular weight 400-600 kDa), add 351.84 g of deionized water, add methyl blue and stir for 18 hours, then add 48 g of poloxamer 407, dissolve at 4 ° C for 30 hours, use a high-speed dispersing homogenizer at a pressure of 100 bar to homogenize once, and obtain reference substance 1.

[0124] Comparative Example 2

[0125] Weigh 0.2 g of hyaluronic acid (molecular weight 400-600 kDa), add 355.8 g of deionized water, add methyl blue and stir for 18 hours, then add 44 g of poloxamer 407, dissolve at 4 ° C for 30 hours, use a high-speed dispersing homogenizer at a pressure of 100 bar to homogenize once, and obtain reference substance 2.

[0126] Comparative Example 3

[0127] Weigh 0.2 g of hyaluronic acid (molecular weight 200-400 kDa), add 351.8 g of deionized water, add methyl blue and stir for 18 hours, then add 48 g of poloxamer 407, dissolve at 4°C for 30 hours, and use a high-speed dispersing homogenizer at a pressure of 100 bar to perform a homogenization cycle to obtain reference substance 3.

[0128] Comparative Example 4

[0129] Weigh 0.8 g of hyaluronic acid (molecular weight 800-1000 kDa), add 319.2 g of deionized water, add methyl blue and stir for 24 hours, then add 80 g of poloxamer 407, dissolve at 4 ° C for 36 hours, and use a high-speed dispersing homogenizer at a pressure of 600 bar to perform a homogenization cycle to obtain reference substance 4.

[0130] Test Example 1

[0131] 3 mL of the solution before and after homogenization was taken into a centrifuge tube, and the gelation time was tested in a 37°C water bath. The centrifuge tube was tilted every 15 seconds. If the hydrogel did not flow, it was judged to be gelled. The gelation time of the samples in the embodiment and the comparative example was recorded. The test results are shown in Table 1.

[0132] Test Example 2

[0133] The viscosity of the sample before and after homogenization was tested using a viscometer at 25° C., rotor No. 1, and a rotation speed of 12 rpm. The test results are shown in Table 1.

[0134] Test Example 3

[0135] Fresh ex vivo porcine stomach was used as an in vitro model to evaluate the injection resistance of the submucosal injection solution of the present invention for submucosal injection in a constant temperature water bath at 37° C. The injection resistance was measured using a precision digital pressure gauge, with one side of the three-way tube connected to a 23G endoscopic injection needle, one side connected to a digital pressure gauge, and one side connected to a syringe. The test results are shown in Table 1.

[0136] Test Example 4

[0137] Use a syringe with a 23G needle to inject 3 mL into the submucosal layer of fresh isolated porcine stomach, repeat 3 times for each group, use a ruler to record the submucosal bulge height at 0, 30, 60, and 90 minutes after submucosal injection, and calculate the maintenance rate of the mucosal bulge height at 90 minutes, maintenance rate = measured height / initial bulge height × 100%. The test results are shown in Tables 1 and 2.

[0138] Table 1

[0139]

[0140]

[0141] Table 2

[0142]

[0143] As can be seen from Table 1, the gelation time of the examples and comparative examples before and after homogenization was tested in a 37°C water bath. The results show that examples 1-8 gelled in 60 seconds before high-pressure homogenization and also gelled within 90 seconds after homogenization. The gelation time was not significantly prolonged, and rapid gelation was achieved. However, from reference products 1-4, it was shown that the solution with a hyaluronic acid content of less than 0.05wt%, a molecular weight of less than 400-600kDa, a poloxamer 407 content of less than 12wt%, and a homogenization pressure of more than 500bar did not gel after high-pressure homogenization. The gelation diagram of sample 4 and reference product 1 after homogenization is shown in FIG. Figure 2 . In addition, samples 5-8 show that reducing the hyaluronic acid content to 0.1%-0.15% and the molecular weight to 600-1000, the poloxamer content has little effect on the gelation time. Samples 9-11 and 12-14 have increased gelation time due to hyaluronic acid grafted with poloxamer or poloxamer grafted with hyaluronic acid, respectively, but can quickly gel in 90s, and can reduce the dosage of poloxamer and hyaluronic acid, making the submucosal injection safer and non-toxic, especially the samples obtained in Examples 12-14, the dosage of hyaluronic acid is significantly reduced.

[0144] According to the viscosity data before and after high-pressure homogenization of each sample in Table 1, it can be seen that the viscosity of the submucosal injection obtained in Examples 1-14 of the present invention is reduced after high-pressure homogenization. In Example 1-8, poloxamer 407 and hyaluronic acid form a more dispersed and uniform solution after passing through a high-pressure homogenizer, and poloxamer wraps hyaluronic acid, and the viscosity of the solution is further reduced. And Examples 9-14 form more dispersed and uniform micelles after high-pressure homogenization, which reduces viscosity. In addition, the viscosity of the submucosal injection is closely related to the injection resistance. As shown in Table 1, the injection resistance of the submucosal injection of the present invention is slightly larger than that of normal saline, but it does not significantly affect the operation process of the entire submucosal injection, and is significantly smaller than the material currently on the market on the bed, which is attributed to the reduction in viscosity after high-pressure homogenization, indicating that the submucosal injection of the present invention has good injectability and low injection pressure.

[0145] According to the results in Tables 1 and 2, the mucosal elevation height of the submucosal injection of the present invention is 11-13 mm, and the specific morphology is as follows: Figure 3-4 As shown, after submucosal injection, the mucosa at the injection site was significantly elevated, and the liquid pad was well formed, reaching the height of the bulge of the commercially available product. The submucosal injection of the present invention can maintain the height of the submucosal pad at 83%-100% within 90 minutes, and the liquid pad descending speed is significantly slower than that of physiological saline and other materials. Other materials can significantly reduce the height of the submucosal pad within 90 minutes after injection, and the maintenance rate is only 61-76%.

[0146] The method can prepare a thermosensitive submucosal injection solution, which can quickly gel, and the viscosity and injection pressure are reduced after high-pressure homogenization.

[0147] The present invention combines poloxamer and hyaluronic acid by physical and chemical methods to achieve the purpose of reducing viscosity and enhancing the duration of bulge maintenance. One of the physical methods is to mix poloxamer and hyaluronic acid and then form composite micelles through high-pressure homogenization to reduce viscosity. Since poloxamer 407 and hyaluronic acid can form intermolecular forces, the addition of hyaluronic acid can significantly increase the gel viscosity of poloxamer 407. After passing through a high-pressure homogenizer, the viscosity of the solution is further reduced, and the injection pressure can be reduced. This is because the poloxamer micelles wrap the hyaluronic acid molecules. The chemical method is to form a hyaluronic acid-poloxamer conjugate by Steglich esterification reaction of hyaluronic acid and poloxamer. One method is to graft ultra-low molecular weight hyaluronic acid onto the poloxamer main chain to increase the viscosity of the poloxamer, and then reduce the viscosity of the solution through high-pressure homogenization to achieve the purpose of reducing injection pressure; the other method is to graft poloxamer onto the low molecular weight hyaluronic acid main chain, and then reduce the poloxamer concentration by mixing with poloxamer, and then reduce the solution viscosity and injection pressure after high-pressure homogenization. These low-viscosity poloxamer and hyaluronic acid mixed solutions are still temperature-sensitive at human body temperature. The viscosity of the solution at room temperature can meet the injection requirements, has good injectability, and is easier to inject through a general gastrointestinal endoscope.

[0148] The method can prepare a thermosensitive submucosal injection solution, and the submucosal bulge height maintenance rate can reach 100%.

[0149] The present invention uses a thermosensitive submucosal injection solution obtained by high-pressure homogenization of poloxamer and hyaluronic acid. The intermolecular force between poloxamer and hyaluronic acid enhances the mechanical strength and adhesion of the gel. The in vitro animal model shows that the mucosal layer of the present invention is significantly raised after submucosal injection, meeting the height required for surgery, and no significant decrease is observed over time, and the elevation time can be maintained for a sufficiently long time, which is conducive to the lesion removal operation, so that no submucosal injection is required again during the operation.

[0150] The method can prepare a thermosensitive submucosal injection solution, which can reduce the dosage of poloxamer and hyaluronic acid, making the product safer, non-toxic and more biocompatible.

[0151] The present invention adopts a thermosensitive submucosal injection obtained by combining poloxamer and hyaluronic acid through high-pressure homogenization, which is safe and non-toxic, has good biocompatibility, and reduces production costs. Since poloxamer and hyaluronic acid synergistically enhance gel strength, the concentrations of poloxamer and hyaluronic acid are less than the commercially available amounts, and the low concentration of poloxamer makes the product safe and non-toxic, and the reduction of hyaluronic acid also reduces costs.

[0152] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A submucosal injection, characterized in that: Comprising poloxamer and sodium hyaluronate, wherein the mass fraction of the poloxamer in the raw material is 12%-20%, and the mass fraction of the hyaluronic acid in the raw material is 0.05%-0.2%; Or, comprising 12%-18% by mass of hyaluronic acid-poloxamer graft copolymer.

2. The submucosal injection according to claim 1, characterized in that: The molecular weight of the hyaluronic acid is 100 kDa-1000 kDa or 1000 Da-5000 Da.

3. The submucosal injection according to claim 1, characterized in that: The method further comprises a dye, and preferably, the dye is one of indigo carmine, methylene blue, phenol red, Congo red, toluidine blue and crystal violet.

4. A method for preparing the submucosal injection solution according to any one of claims 1 to 3, characterized in that: include: The poloxamer and the hyaluronic acid aqueous solution are mixed to dissolve the poloxamer to obtain a mixed solution, and the mixed solution is subjected to high-pressure homogenization to obtain the submucosal injection solution.

5. The method for preparing the submucosal injection according to claim 4, characterized in that: The mass fraction of hyaluronic acid in the submucosal injection solution is 0.05wt%-0.2wt%; And / or, the mass fraction of the poloxamer is 12wt%-20wt%; And / or, the molecular weight of the hyaluronic acid is 400 kDa-1000 kDa; And / or, high pressure homogenization is carried out in a high speed dispersing homogenizer, the homogenization pressure is 100-500 bar, and the homogenization number is 1 time; and / or, the poloxamer is poloxamer 407; And / or, the dissolution temperature is 3-5°C and the dissolution time is 12-24h.

6. A method for preparing the submucosal injection solution according to claim 4, characterized in that: include: Preparation of hyaluronic acid-poloxamer graft copolymer: under ice bath condition, poloxamer, 4-(N,N-dimethylamino)pyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are sequentially added to a hyaluronic acid solution, and reacted under nitrogen protection condition, and the reaction liquid is dialyzed and freeze-dried in turn to obtain a hyaluronic acid-poloxamer graft copolymer; Preparation of submucosal injection solution: the hyaluronic acid-poloxamer graft copolymer is dissolved in water and then subjected to high-pressure homogenization to obtain the submucosal injection solution.

7. The method for preparing a submucosal injection according to claim 6, characterized in that: The solvent of the hyaluronic acid solution is N,N-dimethylformamide; And / or, the molecular weight of the hyaluronic acid is in the range of 1000-5000Da; and / or, in the pre-reaction solution, the mass ratio of hyaluronic acid, poloxamer, 4-(N,N-dimethylamino)pyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is (0.16-0.7):2:(0.1-0.4):(0.1-0.4); and / or, the reaction time is 18-36h; and / or, the mass fraction of the hyaluronic acid-poloxamer graft copolymer in the submucosal injection solution is 12%-18%; And / or, high pressure homogenization is carried out in a high-speed dispersing homogenizer, the homogenization pressure is 100-500 bar, and the homogenization number is 1 time; And / or, the poloxamer is poloxamer 407.

8. A method for preparing the submucosal injection solution according to claim 4, characterized in that: include: Preparation of hyaluronic acid-poloxamer graft copolymer: under ice bath condition, poloxamer, 4-(N,N-dimethylamino)pyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are sequentially added to a hyaluronic acid solution, and reacted under nitrogen protection condition, and the reaction liquid is dialyzed and freeze-dried in turn to obtain a hyaluronic acid-poloxamer graft copolymer; Preparation of submucosal injection solution: The hyaluronic acid-poloxamer graft copolymer and poloxamer are dissolved in water to obtain a hyaluronic acid-poloxamer graft copolymer aqueous solution and a poloxamer aqueous solution, and then mixed and subjected to high-pressure homogenization to obtain the submucosal injection solution.

9. The method for preparing a submucosal injection according to claim 8, characterized in that: The solvent of the hyaluronic acid solution is N,N-dimethylformamide; And / or, the hyaluronic acid has a molecular weight range of 100 kDa-400 kDa; and / or, in the pre-reaction solution, the mass ratio of hyaluronic acid, poloxamer, 4-(N,N-dimethylamino)pyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 0.25:(5-10):(0.16-0.26):(0.19-0.3); and / or, the reaction time is 18-36h; And / or, the mass fraction of the hyaluronic acid-poloxamer graft copolymer aqueous solution is 14%-18%; And / or, the mass fraction of the poloxamer aqueous solution is 14%-18%; and / or, the hyaluronic acid-poloxamer graft copolymer aqueous solution and the poloxamer aqueous solution are mixed at a volume ratio of 1:5-10; And / or, high pressure homogenization is carried out in a high-speed dispersing homogenizer, the homogenization pressure is 100-500 bar, and the homogenization number is 1 time; And / or, the poloxamer is poloxamer 407.

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