Preparation method of temperature-sensitive sustained-release gel preparation of intestinal flora transplantation liquid and preparation

Abstract

The invention discloses a preparation method of an intestinal flora transplantation liquid temperature-sensitive sustained-release gel preparation and the preparation. The invention belongs to the technical field of biological medicine, and relates to a preparation method of an intestinal flora transplantation liquid temperature-sensitive sustained-release gel preparation and the preparation. According to the method, standard intestinal flora suspension is used as a functional raw material, poloxamer 407 and poloxamer 188 are used as matrixes, and some cryopreservation protective agents and other formulas are used as auxiliary materials, and the preparation method comprises the following steps: (1) preparing standard intestinal flora liquid; (2) gradually adding the following components according to the preparation concentration: 15%-22% of poloxamer 407, 0.5%-4% of poloxamer 188, 5%-20% of glycerol, 0.5%-1% of sodium carboxymethyl cellulose and the balance of 0.9% sodium chloride aqueous solution; (3) mixing and stirring the suspension intestinal bacterial liquid at room temperature, stirring the suspension intestinal bacterial liquid by a method of uniformly stirring the suspension intestinal bacterial liquid by an electric stirring blade until the suspension intestinal bacterial liquid does not have blocks and is uniformly dispersed, continuously stirring the suspension intestinal bacterial liquid until the liquid is viscous and uniform, and converting a viscous solution into a gel state at a corresponding temperature; and (4) sequentially putting the prepared intestinal flora gel preparation into freezing refrigerators at-20 DEG C and-80 DEG C for freezing and storing for later use. The preparation process is simple, the preparation is rapid, the auxiliary materials are medicinal and have good biocompatibility, and the activity of bacteria is not damaged.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a preparation method and preparation of a thermosensitive sustained-release gel preparation of intestinal flora transplantation solution. Background Art

[0002] Fecal microbiota transplantation (FMT) is a therapeutic approach that involves transplanting functional intestinal microbiota from the feces of healthy individuals into the intestines of patients or those with suboptimal health. The goal is to reestablish a new intestinal microbiota, restore its diversity and biological functions, and treat both intestinal and extraintestinal diseases. The earliest documented use of this method for treatment was by Ge Hong during the Eastern Jin Dynasty in China. In his book, "Emergency Prescriptions for the Elbow," he documented the use of fecal solution to treat food poisoning and severe diarrhea. Over the past decade, FMT has achieved breakthroughs in the treatment of various intestinal and extraintestinal diseases, with efficacy rates exceeding 90% in the treatment of Clostridium difficile infection. Currently, FMT is performed in several main ways: FMT involves oral administration of frozen-dried fecal microbiota powder or pellets encapsulated in acid-resistant enteric-coated capsules. Alternatively, FMT involves transplanting fecal microbiota isolated from the feces of healthy individuals into the intestines via nasogastric tube insertion, gastrointestinal tube insertion, or endoscopic spraying. This approach aims to restore the homeostasis of the intestinal microbiota and treat various intestinal diseases caused by intestinal dysbiosis. Each method has its own advantages and disadvantages in terms of clinical research and feasibility. Furthermore, the conclusions on the efficacy of different microbiota transplantation methods vary.

[0003] The most commonly used method is to prepare enteric-coated capsules of freeze-dried powder derived from isolated fecal microbiota. The capsule transplantation method has the advantages of being minimally invasive and convenient, and can avoid endoscopic operation, thereby eliminating the risk of perforation. However, there are some disadvantages in preparing freeze-dried powder: 1: Inherent disadvantages of current freeze-drying technology: (1) High cost: The price of large freeze-dryers is relatively high, and the cost of operation and maintenance is also high. A laboratory-grade freeze-dryer is not expensive, but a commercial freeze-dryer suitable for large-scale production can cost hundreds of thousands to millions. (2) Long processing time: The freeze-drying process takes a long time to complete, especially for large-scale freeze-drying, which takes several days from the beginning of freezing to the actual drying. (3) High energy consumption: Due to the need to maintain a low temperature and vacuum environment, the energy consumption of freeze-drying is relatively high. 2: Activity problem: The activity of the intestinal flora freeze-dried powder capsules may be low, which directly affects the efficacy. Studies have shown that the tedious and long freeze-drying process, the low temperature effect, freezing effect and dehydration effect during the freeze-drying process will greatly reduce the activity of the flora, with a reduction ratio of about 40%.

[0004] With the advancement of FMT technology and its proven clinical efficacy against various intestinal and extraintestinal diseases, research on formulations based on intestinal flora fluid has become a current research hotspot and focus. Studies have shown that the activity of liquid enteric-coated capsules containing live bacteria can reach 95%. Liquid capsules can effectively protect the active ingredients of the bacteria, preventing them from being affected during freeze-drying, storage, and transportation. However, the liquid bacterial solution alone has high fluidity, and the intestinal bacterial solution itself is alkaline, which can easily damage the shell of the enteric-coated capsule. In addition, sudden release also directly affects the efficacy. Studies have shown that slow release is more conducive to achieving the maximum therapeutic effect of FMT technology. Therefore, research and development of new formulations based on intestinal flora fluid, and improving parameters such as fluidity, viscosity, and sustained-release rate, are crucial for the research and product development of liquid enteric-coated capsules containing live bacteria. Liquid gels are currently a major research direction for liquid flora formulations.

[0005] Thermosensitive hydrogels are temperature-sensitive hydrogels that undergo volume phase transitions within a specific temperature range. For example, some thermosensitive hydrogels transition from a swollen state to a contracted state when the temperature rises. This is because temperature changes alter the intermolecular forces within the hydrogel (such as hydrogen bonds). Thermosensitive hydrogels are widely used in a variety of fields, including drug sustained release and tissue engineering. Their temperature-responsive properties can be exploited to control the rate and amount of drug release. The most widely used are gel formulations at physiological temperature (37°C), which can achieve a sustained release effect within the human body. Poloxamer, a nonionic triblock copolymer composed of polyethylene oxide (PEO) and polypropylene oxide (PPO), possesses unique properties. Its hydrophilic polyethylene oxide segments and hydrophobic polypropylene oxide segments interact to form a hydrogel under certain conditions, such as when the concentration reaches a certain level or when the temperature changes. Active ingredients are encapsulated in the gel, slowing the release rate and prolonging the duration of drug efficacy. Therefore, the most widely used and safe thermosensitive hydrogels currently disclosed in literature and patents are various types of poloxamer complexes and formulations containing them. Existing research primarily focuses on their application in pharmaceutical formulations, such as eye drops, suppositories, and gels, with fewer reports on their application in intestinal flora transplantation solutions. Summary of the Invention

[0006] With the progress and clinical efficacy of FMT in treating intestinal and extraintestinal diseases, the research and development of liquid enteric-coated capsules with live bacteria has become a major focus, with the main purpose being to avoid freeze-drying and maintain a high level of active bacteria. Improving parameters such as the fluidity, viscosity, and sustained-release rate of the bacterial solution is currently the primary condition for the development of liquid enteric-coated capsules with live bacteria, which is also more conducive to calibration and achieves better results after transplantation. In order to solve the problems caused by the inherent physical and chemical properties of the direct intestinal flora liquid mentioned above, the present invention provides a method for preparing a thermosensitive sustained-release gel formulation of intestinal flora transplant solution. Compared with the prior art, the preparation method provided by the present invention does not require a complex preparation process and can quickly prepare a thermosensitive sustained-release gel formulation based on intestinal flora solution, which has good commercial prospects. During the experimental process, the research and development team of this application found that thermosensitive hydrogels formed based on poloxamer generally need to be placed or stirred for a long time (>12 hours) under low temperature conditions of 4 degrees Celsius. Therefore, the solution must be stored in a refrigerator for use to allow the poloxamer to continuously swell and form a gel. This makes the overall preparation and use inconvenient, especially large-scale production. However, further analysis and experimentation by the R&D team revealed that continuous stirring could achieve gelation in approximately 10 minutes. This short process is crucial for maintaining the activity of the intestinal flora. Furthermore, gelation at body temperature prolongs the retention time of intestinal bacteria in the intestine. As the gel polymer continuously degrades and the gel contents are continuously diluted and dissolved by intestinal fluid, a long-term sustained-release function of the bacteria is gradually achieved. Overall, the intestinal flora gel preparation of the present invention has a simple preparation process, is suitable for industrial production, and exhibits sustained-release properties.

[0007] In order to achieve the above-mentioned object, the technical solution adopted by the present invention is as follows: a preparation method and preparation of a thermosensitive sustained-release gel preparation of intestinal flora transplantation solution, the preparation method comprising the following steps:

[0008] (1) Preparation of standard intestinal flora transplantation solution or slurry: aseptically collect fresh healthy donor feces, weigh it, add sterile saline at a mass ratio of 1:5, homogenize it, filter it through gauze of different mesh sizes to remove residues, and then centrifuge it at 4 degrees Celsius. Discard the supernatant to obtain the centrifugal precipitate, wash it with 0.9% saline, repeat the washing and centrifugation several times, and the obtained intestinal flora solution is set aside;

[0009] (2) gradually adding the following components to the bacterial solution prepared in step (1), including poloxamer 407, poloxamer 188, glycerol, sodium carboxymethyl cellulose, and 0.9% sodium chloride solution;

[0010] (3) The suspension is continuously mixed and stirred at room temperature, and the suspension is evenly broken up by an electric stirring blade until there is no lump and the dispersion is uniform, and the stirring is continued until the liquid becomes uniformly viscous. At the corresponding temperature, the viscous solution can be transformed into a gel state;

[0011] (4) The prepared intestinal flora gel preparation was first frozen at -20 degrees and then placed in a -80 degrees freezer for storage.

[0012] Furthermore, the mass ratio of poloxamer 407 in step (2) is 15% to 22%.

[0013] Preferably, the mass ratio of poloxamer 407 in step (2) is 18% to 20%.

[0014] Furthermore, in the step (2), the poloxamer 407 is a poloxamer 407 having a polymer weight ratio of 100-300:10-70.

[0015] Furthermore, the mass ratio of Poloxamer 188 in step (2) is 0.5% to 4%.

[0016] Preferably, the mass ratio of Poloxamer 188 in step (2) is 1% to 2%.

[0017] Furthermore, the mass ratio of glycerol in step (2) is 5% to 20%.

[0018] Preferably, the mass ratio of glycerol in step (2) is 10%.

[0019] Furthermore, the mass ratio of sodium carboxymethyl cellulose in step (2) is 0.5% to 1%.

[0020] Furthermore, the sodium chloride solution in step (2) is a sterile sodium chloride aqueous solution.

[0021] The beneficial effects achieved by the present invention using the above structure are as follows: (1) The preparation method provided by the present invention does not require a complicated preparation process, and can quickly prepare a thermosensitive sustained-release gel preparation based on intestinal bacterial fluid, which has good commercial prospects; (2) Through research and development, the present invention can make poloxamer gel in about 10 minutes, thereby maintaining the activity of bacteria in the intestinal bacterial fluid and prolonging the retention time of intestinal bacteria in the intestine. As the gel polymer is continuously degraded and the gel contents are continuously diluted and dissolved by the intestinal fluid, the long-term sustained-release function of the bacteria is gradually realized; (3) The intestinal flora gel preparation of the present invention has a simple preparation process, is suitable for industrial production, and has a sustained-release function. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The status of intestinal bacterial fluid and intestinal bacterial fluid gel preparation at different temperatures;

[0023] Figure 2 In vitro release profile of intestinal flora liquid gel simulating intestinal environment.

[0024] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0027] Materials, reagents, or instruments used in this invention, unless the manufacturer is indicated, are all commercially available. Among them: Poloxamer 407 and Poloxamer 188 were purchased from BASF (China) Co., Ltd.; glycerol and carboxymethyl cellulose were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the screening of healthy donors was strictly carried out in accordance with the relevant guidelines of the Chinese Expert Consensus on Standardized Technical Specifications for Enterobacteriaceae Transplantation Preparation and Quality Control Laboratories (2023 Edition). The donor screening process included four stages: initial questionnaire screening, on-site interview, comprehensive physical examination, and medical verification.

[0028] Example 1

[0029] Preparation of a thermosensitive sustained-release intestinal flora liquid gel preparation containing 18% poloxamer 407 + 1% poloxamer 188

[0030] (1) Preparation of standard intestinal flora transplantation solution or slurry: Aseptically collect fresh healthy donor feces, weigh them, add sterile saline at a mass ratio of 1:5, homogenize them, filter them through gauze of different mesh sizes to remove residues, and then centrifuge them at 4 degrees Celsius. Discard the supernatant to obtain the centrifugal precipitate, wash it with 0.9% saline, repeat the washing and centrifugation several times, and the obtained intestinal flora solution is set aside;

[0031] (2) The bacterial solution prepared in S1 was gradually added with the following components: 18% poloxamer 407, 1% poloxamer 188, 10% glycerol, 1% sodium carboxymethyl cellulose, and 0.9% sodium chloride solution;

[0032] (3) The suspension is continuously mixed and stirred at room temperature, and the suspension is evenly broken up by an electric stirring blade until there is no lump and the dispersion is uniform, and the stirring is continued until the liquid becomes uniformly viscous. At the corresponding temperature, the viscous solution can be transformed into a gel state;

[0033] (4) Determine the phase transition temperature of the bacterial liquid gel preparation by the test tube inversion method. Take 2 ml of the liquid preparation at 4°C and pipette it into a 5 mL test tube. Place the test tube in a gradually heated water bath and repeatedly tilt it. Record the temperature at which complete gelation occurs.

[0034] (5) The prepared intestinal flora gel preparation was first frozen at -20 degrees and then placed in a -80 degrees freezer for storage.

[0035] Example 2

[0036] Preparation of a thermosensitive sustained-release intestinal flora liquid gel preparation containing 18% poloxamer 407 + 2% poloxamer 188

[0037] (1) Preparation of standard intestinal flora transplantation solution or slurry: aseptically collect fresh healthy donor feces, weigh it, add sterile saline at a mass ratio of 1:5, homogenize it, filter it through gauze of different mesh sizes to remove residues, and then centrifuge it at 4 degrees Celsius. Discard the supernatant to obtain the centrifugal precipitate, wash it with 0.9% saline, repeat the washing and centrifugation several times, and the obtained intestinal flora solution is set aside;

[0038] (2) The bacterial solution prepared in S1 was gradually added with the following components: 18% poloxamer 407, 2% poloxamer 188, 10% glycerol, 1% sodium carboxymethyl cellulose, and 0.9% sodium chloride solution;

[0039] (3) The suspension is continuously mixed and stirred at room temperature, and the suspension is evenly broken up by an electric stirring blade until there is no lump and the dispersion is uniform, and the stirring is continued until the liquid becomes uniformly viscous. At the corresponding temperature, the viscous solution can be transformed into a gel state;

[0040] (4) Determine the phase transition temperature of the bacterial liquid gel preparation by the test tube inversion method. Take 2 ml of the liquid preparation at 4°C and pipette it into a 5 mL test tube. Place the test tube in a gradually heated water bath and repeatedly tilt it. Record the temperature at which complete gelation occurs.

[0041] (5) The prepared intestinal flora gel preparation was first frozen at -20 degrees and then placed in a -80 degrees freezer for storage.

[0042] Example 3

[0043] Preparation of a thermosensitive sustained-release intestinal flora liquid gel preparation containing 18% poloxamer 407 + 3% poloxamer 188

[0044] Preparation of standard intestinal flora transplantation solution or slurry: Aseptically collect fresh healthy donor feces, weigh it, add sterile saline at a mass ratio of 1:5, homogenize it, filter it through gauze of different mesh sizes to remove residue, and then centrifuge it at 4 degrees Celsius. Discard the supernatant to obtain the centrifugal precipitate, wash it with 0.9% saline, repeat the washing and centrifugation several times, and the obtained intestinal flora solution is set aside.

[0045] The bacterial solution prepared in S1 was gradually added with the following components: 18% poloxamer 407, 2% poloxamer 188, 10% glycerol, 1% sodium carboxymethyl cellulose, and 0.9% sodium chloride solution;

[0046] The suspension is continuously mixed and stirred at room temperature, and the mixture is evenly broken up by an electric stirring blade until there is no lump and the mixture is evenly dispersed. The mixture is continuously stirred until the liquid becomes viscous and uniform. The viscous solution can be transformed into a gel state at a corresponding temperature.

[0047] The phase transition temperature of the bacterial liquid gel preparation was determined by the test tube inversion method. 2 ml of the liquid preparation was taken at 4°C and pipetted into a 5 mL test tube. The test tube was placed in a gradually heated water bath and tilted repeatedly. The temperature at which complete gelation occurred was recorded.

[0048] The prepared intestinal flora gel preparations were first frozen at -20 degrees and then placed in a -80 degrees freezer for storage.

[0049] Example 4

[0050] Preparation of a thermosensitive sustained-release intestinal flora liquid gel preparation containing 18% poloxamer 407 + 4% poloxamer 188

[0051] (1) Preparation of standard intestinal flora transplantation solution or slurry: aseptically collect fresh healthy donor feces, weigh it, add sterile saline at a mass ratio of 1:5, homogenize it, filter it through gauze of different mesh sizes to remove residues, and then centrifuge it at 4 degrees Celsius. Discard the supernatant to obtain the centrifugal precipitate, wash it with 0.9% saline, repeat the washing and centrifugation several times, and the obtained intestinal flora solution is set aside;

[0052] (2) The bacterial solution prepared in S1 was gradually added with the following components: 18% poloxamer 407, 4% poloxamer 188, 10% glycerol, 1% sodium carboxymethyl cellulose, and 0.9% sodium chloride solution;

[0053] (3) The suspension is continuously mixed and stirred at room temperature, and the suspension is evenly broken up by an electric stirring blade until there is no lump and the dispersion is uniform, and the stirring is continued until the liquid becomes uniformly viscous. At the corresponding temperature, the viscous solution can be transformed into a gel state;

[0054] (4) Determine the phase transition temperature of the bacterial liquid gel preparation by the test tube inversion method: take 2 ml of the liquid preparation at 4°C, pipette it into a 5 mL test tube, place the test tube in a gradually heated water bath, and repeatedly tilt it, and record the temperature of complete gelation;

[0055] (5) The prepared intestinal flora gel preparation was first frozen at -20 degrees and then placed in a -80 degrees freezer for storage.

[0056] Example 5

[0057] Preparation of a thermosensitive sustained-release intestinal flora liquid gel preparation containing 20% poloxamer 407 + 1% poloxamer 188

[0058] (1) Preparation of standard intestinal flora transplantation solution or slurry: aseptically collect fresh healthy donor feces, weigh it, add sterile saline at a mass ratio of 1:5, homogenize it, filter it through gauze of different mesh sizes to remove residues, and then centrifuge it at 4 degrees Celsius. Discard the supernatant to obtain the centrifugal precipitate, wash it with 0.9% saline, repeat the washing and centrifugation several times, and the obtained intestinal flora solution is set aside;

[0059] (2) The bacterial solution prepared in S1 was gradually added with the following components: 20% poloxamer 407, 1% poloxamer 188, 10% glycerol, 1% sodium carboxymethyl cellulose, and 0.9% sodium chloride solution;

[0060] (3) The suspension is continuously mixed and stirred at room temperature, and the suspension is evenly broken up by an electric stirring blade until there is no lump and the dispersion is uniform, and the stirring is continued until the liquid becomes uniformly viscous. At the corresponding temperature, the viscous solution can be transformed into a gel state;

[0061] (4) Determine the phase transition temperature of the bacterial liquid gel preparation by the test tube inversion method: take 2 ml of the liquid preparation at 4°C, pipette it into a 5 mL test tube, place the test tube in a gradually heated water bath, and repeatedly tilt it, and record the temperature of complete gelation;

[0062] (5) The prepared intestinal flora gel preparation was first frozen at -20 degrees and then placed in a -80 degrees freezer for storage.

[0063] Example 6

[0064] Preparation of a thermosensitive sustained-release intestinal flora liquid gel preparation containing 20% poloxamer 407 and 2% poloxamer 188

[0065] (1) Preparation of standard intestinal flora transplantation solution or slurry: aseptically collect fresh healthy donor feces, weigh it, add sterile saline at a mass ratio of 1:5, homogenize it, filter it through gauze of different mesh sizes to remove residues, and then centrifuge it at 4 degrees Celsius. Discard the supernatant to obtain the centrifugal precipitate, wash it with 0.9% saline, repeat the washing and centrifugation several times, and the obtained intestinal flora solution is set aside;

[0066] (2) The bacterial solution prepared in S1 was gradually added with the following components: 20% poloxamer 407, 2% poloxamer 188, 10% glycerol, 1% sodium carboxymethyl cellulose, and 0.9% sodium chloride solution;

[0067] (3) The suspension is continuously mixed and stirred at room temperature, and the suspension is evenly broken up by an electric stirring blade until there is no lump and the dispersion is uniform, and the stirring is continued until the liquid becomes uniformly viscous. At the corresponding temperature, the viscous solution can be transformed into a gel state;

[0068] (4) Determine the phase transition temperature of the bacterial liquid gel preparation by the test tube inversion method: take 2 ml of the liquid preparation at 4°C, pipette it into a 5 mL test tube, place the test tube in a gradually heated water bath, and repeatedly tilt it, and record the temperature of complete gelation;

[0069] (5) The prepared intestinal flora gel preparation was first frozen at -20 degrees and then placed in a -80 degrees freezer for storage.

[0070] Example 7

[0071] Preparation of a thermosensitive sustained-release intestinal flora liquid gel preparation containing 20% poloxamer 407 and 3% poloxamer 188

[0072] (1) Preparation of standard intestinal flora transplantation solution or slurry: aseptically collect fresh healthy donor feces, weigh it, add sterile saline at a mass ratio of 1:5, homogenize it, filter it through gauze of different mesh sizes to remove residues, and then centrifuge it at 4 degrees Celsius. Discard the supernatant to obtain the centrifugal precipitate, wash it with 0.9% saline, repeat the washing and centrifugation several times, and the obtained intestinal flora solution is set aside;

[0073] (2) The bacterial solution prepared in S1 was gradually added with the following components: 20% poloxamer 407, 3% poloxamer 188, 10% glycerol, 1% sodium carboxymethyl cellulose, and 0.9% sodium chloride solution;

[0074] (3) The suspension is continuously mixed and stirred at room temperature, and the suspension is evenly broken up by an electric stirring blade until there is no lump and the dispersion is uniform, and the stirring is continued until the liquid becomes uniformly viscous. At the corresponding temperature, the viscous solution can be transformed into a gel state;

[0075] (4) Determine the phase transition temperature of the bacterial liquid gel preparation by the test tube inversion method. Take 2 ml of the liquid preparation at 4°C and pipette it into a 5 mL test tube. Place the test tube in a gradually heated water bath and repeatedly tilt it. Record the temperature at which complete gelation occurs.

[0076] (5) The prepared intestinal flora gel preparation was first frozen at -20 degrees and then placed in a -80 degrees freezer for storage.

[0077] Example 8

[0078] Preparation of a thermosensitive sustained-release intestinal flora liquid gel preparation containing 20% poloxamer 407 + 4% poloxamer 188

[0079] (1) Preparation of standard intestinal flora transplantation solution or slurry: aseptically collect fresh healthy donor feces, weigh it, add sterile saline at a mass ratio of 1:5, homogenize it, filter it through gauze of different mesh sizes to remove residues, and then centrifuge it at 4 degrees Celsius. Discard the supernatant to obtain the centrifugal precipitate, wash it with 0.9% saline, repeat the washing and centrifugation several times, and the obtained intestinal flora solution is set aside;

[0080] (2) The bacterial solution prepared in S1 was gradually added with the following components: 120% poloxamer 407, 4% poloxamer 188, 10% glycerol, 1% sodium carboxymethyl cellulose, and 0.9% sodium chloride solution;

[0081] (3) The suspension is continuously mixed and stirred at room temperature, and the suspension is evenly broken up by an electric stirring blade until there is no lump and the dispersion is uniform, and the stirring is continued until the liquid becomes uniformly viscous. At the corresponding temperature, the viscous solution can be transformed into a gel state;

[0082] (4) Determine the phase transition temperature of the bacterial liquid gel preparation by the test tube inversion method. Take 2 ml of the liquid preparation at 4°C and pipette it into a 5 mL test tube. Place the test tube in a gradually heated water bath and repeatedly tilt it. Record the temperature at which complete gelation occurs.

[0083] (5) The prepared intestinal flora gel preparation was first frozen at -20 degrees and then placed in a -80 degrees freezer for storage.

[0084] Experimental Example 1

[0085] Determination of the critical gelling temperature of intestinal flora hydrogel preparations by test tube inversion method

[0086] Place 2 mL of the thermosensitive gel solution in a test tube and insert a precision thermometer with an accuracy of 0.1°C in a constant temperature water bath. The mercury bulb of the thermometer is completely submerged below the water surface, and the gel liquid level is 2 cm below the water bath liquid level. Set the initial temperature of the water bath to 18°C and the heating rate to 0.2°C per minute. Take out the test tube and turn it over every 0.1°C rise in temperature, and observe the gelation of the liquid. When the solution solidifies, that is, when no flow is detected within 30 seconds after inverting the test tube, read the thermometer scale value. This temperature is the gelation temperature. Each sample is measured in parallel 3 times, and the results are averaged.

[0087] Table 1 Phase transition temperatures of intestinal flora hydrogel preparations prepared under different mass ratios

[0088] Poloxamer 407 mass ratio Poloxamer 188 mass ratio Phase transition temperature 18％ 1％ 26.9℃ 18％ 2％ 28.8℃ 18％ 3％ 30.4℃ 18％ 4％ 33.9℃ 20％ 1％ 22.7℃ 20％ 2％ 23.1℃ 20％ 3％ 24.9℃ 20％ 4％ 26.9℃

[0089] Experimental results: The present invention sets up gel preparations formed under different mass ratios of poloxamer 407 and poloxamer 188, and performs ratio screening to determine the optimal formation ratio. The results are shown in Table 1. Existing studies have shown that adding a certain concentration of poloxamer 188 to poloxamer 407 can increase the strength and stability of the gel, but as the concentration of 188 increases, the gelation temperature will also decrease. Configurations with different ratios can form intestinal flora liquid gel preparations. We selected a preparation formed under the conditions of a mass ratio of 20% poloxamer 407 and 2% poloxamer 188 to study its gel state. The results are shown in Table 1. Figure 1 As shown, the thermosensitive hydrogel is in a gel-like, semi-solid state at 37°C. When the glass bottle is inverted, the gel does not flow (B). At 4°C, it is in a liquid state and flows to the bottom when inverted (C). This demonstrates that the preparation has good gel properties at 37°C.

[0090] Experimental Example 2

[0091] Evaluation of the in vitro sustained-release effect of a thermosensitive sustained-release intestinal flora hydrogel preparation containing 20% poloxamer 407 and 2% poloxamer 188

[0092] 1 mL of hydrogel was pipetted onto the bottom of a beaker (liquid preparation at 4°C). The beaker was then placed at 37°C. After gelation, 9 mL of deionized water and a solution simulating intestinal pH (pH 6.8, a mixture of 250 mL of 0.1 mol / L KH2PO4 and 118 mL of 0.2 mol / L NaOH) were added, respectively. The solution was shaken in a 37°C shaking water bath to simulate in vitro release. Every hour, 1 mL of the release medium was collected (for 24 hours) and replaced with the same volume of solution. The quality of the released enteric bacteria was indirectly determined by measuring the protein concentration using changes in water-soluble protein concentration using a UV-visible spectrophotometer. In this experiment, lysozyme was used to dissolve the bacterial cell wall, combined with ultrasonic cell disruption to dissolve the protein. The protein concentration was then measured to indirectly determine the content of the released bacteria. The specific method was as follows: the release medium collected at different time points was centrifuged at 6000 rpm for 20 minutes, and the supernatant was discarded to obtain bacterial slurry. Add 10 mL of distilled water to wash the sludge, and repeat the above steps to obtain the washed sludge to remove the residual buffer. Accurately measure 10 mL of PBS (pH 7.2) into the sludge, add a certain amount of lysozyme, and hydrolyze at 40°C for 120 minutes. Then, use a cell disruptor to sonicate for 10 minutes to dissolve the protein and obtain an enzymatic solution. Stain with Coomassie Brilliant Blue G-250 reagent, and then measure the absorbance at a wavelength of 595 nm using a UV-visible spectrophotometer. Calculate the protein concentration of the measured intestinal bacteria according to the regression equation of the protein standard curve.

[0093] Experimental results: Figure 2 As shown, when the hydrogel is under simulated intestinal relaxation, the liquid will gradually erode the hydrogel, increasing the pore size of the three-dimensional network structure, and water molecules will gradually enter and exit the hydrogel. On the one hand, the concentration of the matrix material in the hydrogel will gradually decrease, leading to the disintegration of the hydrogel. On the other hand, under the action of osmotic pressure, the bacteria can be gradually released. The whole process is a slow-release process.

[0094] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

[0095] The present invention and its embodiments are described above. This description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by this and, without departing from the purpose of the present invention, designs structures and embodiments similar to this technical solution without inventiveness, they shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing a thermosensitive sustained-release gel preparation for intestinal flora transplantation, characterized by: The preparation method comprises the following steps: (1) Preparation of standard intestinal flora transplantation solution or slurry: aseptically collect fresh healthy donor feces, weigh it, add sterile saline at a mass ratio of 1:5, homogenize it, filter it through gauze of different mesh sizes to remove residues, and then centrifuge it at 4 degrees Celsius. Discard the supernatant to obtain the centrifugal precipitate, wash it with 0.9% saline, repeat the washing and centrifugation several times, and the obtained intestinal flora solution is set aside; (2) gradually adding the following components to the bacterial solution prepared in step (1), including poloxamer 407, poloxamer 188, glycerol, sodium carboxymethyl cellulose, and 0.9% sodium chloride solution; (3) The suspension is continuously mixed and stirred at room temperature, and the suspension is evenly broken up by an electric stirring blade until there is no lump and the dispersion is uniform, and the stirring is continued until the liquid becomes uniformly viscous. At the corresponding temperature, the viscous solution can be transformed into a gel state; (4) The prepared intestinal flora gel preparation was first frozen at -20 degrees and then placed in a -80 degrees freezer for storage.

2. The method for preparing a thermosensitive sustained-release gel preparation of intestinal flora transplantation solution according to claim 1, characterized in that: The mass ratio of Poloxamer 407 in step (2) is 15% to 22%.

3. The method for preparing a thermosensitive sustained-release gel preparation of intestinal flora transplantation solution according to claim 2, characterized in that: The mass ratio of Poloxamer 407 in step (2) is 18% to 20%.

4. The method for preparing a thermosensitive sustained-release gel preparation for intestinal flora transplantation according to claim 3, characterized in that: In the step (2), the poloxamer 407 is a poloxamer 407 having a polymer weight ratio of 100-300:10-70.

5. The method for preparing a thermosensitive sustained-release gel preparation for intestinal flora transplantation according to claim 4, characterized in that: The mass ratio of Poloxamer 188 in the step (2) is 0.5% to 4%.

6. The method for preparing a thermosensitive sustained-release gel preparation for intestinal flora transplantation according to claim 5, characterized in that: The mass ratio of Poloxamer 188 in step (2) is 1% to 2%.

7. The method for preparing a thermosensitive sustained-release gel preparation for intestinal flora transplantation according to claim 6, characterized in that: The mass ratio of glycerol in step (2) is 10%.

8. The method for preparing a thermosensitive sustained-release gel preparation for intestinal flora transplantation according to claim 7, characterized in that: The mass ratio of sodium carboxymethyl cellulose in step (2) is 0.5% to 1%.

9. The method for preparing a thermosensitive sustained-release gel preparation for intestinal flora transplantation according to claim 8, characterized in that: The sodium chloride solution in step (2) is a sterile sodium chloride aqueous solution.

10. The thermosensitive sustained-release gel preparation for intestinal flora transplantation according to claim 9, characterized in that: The preparation is prepared according to the preparation method according to claim 1.

Images