CN110755695A

CN110755695A - Chitosan gel with physiological responsiveness and application thereof in uterine cavity anti-adhesion

Info

Publication number: CN110755695A
Application number: CN201911130179.1A
Authority: CN
Inventors: 张继业; 杨显伟; 李建; 莫菲; 马佳; 张沛沛
Original assignee: Shaanxi Wufang Medical Technology Co Ltd; Xian Jiaotong University
Current assignee: Shaanxi Wufang Medical Technology Co Ltd; Xian Jiaotong University
Priority date: 2019-11-18
Filing date: 2019-11-18
Publication date: 2020-02-07

Abstract

The invention discloses a chitosan gel with physiological responsiveness and application thereof in uterine cavity anti-adhesion, belonging to the technical field of pharmacy. The active ingredients with the functions of stopping bleeding, inhibiting bacteria, promoting healing and the like are added into the non-chemical cross-linked temperature-sensitive sterile hydrogel matrix, are in a sol state with good fluidity at room temperature, are convenient for administration in uterine cavities, are rapidly gelled and cured at the physiological temperature of uterus, complete physical isolation on wound surfaces after uterine surgery, prevent adhesion, simultaneously play the function of physiological active materials and promote the wound surfaces to heal quickly. After the uterus is recovered, the gel is dissolved and eroded under the physiological action of self proliferation and secretion and is discharged out of the body along with the secretion without manual intervention. Has the advantages of simple application, predictable effect, no need of secondary intervention and the like. Can be used for preventing adhesion after various uterine operations.

Description

Chitosan gel with physiological responsiveness and application thereof in uterine cavity anti-adhesion

Technical Field

The invention belongs to the technical field of pharmacy, and relates to chitosan gel with physiological responsiveness and application thereof in uterine cavity anti-adhesion.

Background

At present, after the uterine operation, estrogen is mostly used clinically to promote the repair of endometrium, so as to achieve the purpose of preventing uterine Adhesion (IUA), in addition, other positive interventions are basically not available, and reports show that after the gynecological operation, the incidence rate of postoperative IUA of different levels is up to more than 50%. The clinical manifestations of IUA are mainly hypomenorrhea and even amenorrhea, dysmenorrhea, secondary infertility, and poor pregnancy outcome, including recurrent abortion, premature birth, pre-placer placenta, etc.

Hysteroscopic hysteromyomectomy (TCRA) is the primary treatment of IUA at present. In order to prevent recurrence of IUA after TCRA treatment, a combined artificial cycle treatment scheme of placing Intrauterine Devices (IUDs) in a uterine cavity is mostly adopted to promote endometrial hyperplasia and cover a wound surface, but the clinical treatment effect is still not ideal. Since the disposable saccule uterine pear-shaped stent J-BUS-253000 developed by Kuke (COOK) company in the United states in 2013 enters the clinic, the disposable saccule uterine pear-shaped stent J-BUS-253000 becomes a main intervention means for preventing IUA recurrence after TCRA, and the total effective rate is good. However, the overall treatment cost is high, and after the IUA is corrected, the patient needs to go back to the hospital again, and the balloon is taken out from the cervical orifice with the help of the doctor, so that the process is complicated, and the compliance of the patient is not high. Therefore, finding new effective strategies for preventing IUA is of particular importance.

After the uterus operation, the temperature-sensitive gel in a sol state is poured into the uterine cavity, and the gel is rapidly formed under the action of body temperature to isolate the damaged uterine basal layer. According to the characteristics of the physiological cycle of a human body, the uterus can be quickly healed in the proliferation stage, the secretion of the endometrium is less in the proliferation stage, the gel material can be slowly degraded under the condition of body temperature, the rigidity of the gel is reduced, and the original space reticular structure is gradually lost. After the proliferation period, the uterus enters the secretion period, the intima is thickened, the pressure of the uterine cavity is increased, and meanwhile, the secretion is increased. Under the combined action of secretion erosion and uterine cavity pressure, the temperature-sensitive gel is gradually discharged out of the uterus through the cervical orifice, the physiological response process is completed, clinical intervention is not needed, and the adaptability of a patient is good.

Temperature Sensitive Hydrogel (Thermal Sensitive Hydrogel) is a temperature Sensitive gel system. Different from the traditional common hydrogel, the TSH is a liquid with good fluidity in a low-temperature environment, and after the temperature is increased to a certain level, the state of the system is changed into a gel state, and the critical phase transition temperature is called as the 'gelation temperature'. The temperature-sensitive hydrogel is convenient to administer and easy to control dosage, can be uniformly distributed and adhered to an administration part after being converted into a gel state, and can provide physical support with certain strength or drug sustained release capability. The currently widely used temperature-sensitive material is poloxamer (poloxamer), which is a copolymer composed of Polyoxyethylene (PEO) and polyoxypropylene (PPO), spherical micelles with PPO as the inner core and PEO as the outer shell can be formed in water, and as the temperature rises, when the gelation temperature is reached, the micelles are intertwined and stacked to form gel.

At present, poloxamer 407(p407) which is already recorded in the united states pharmacopoeia is studied most deeply, but poloxamer 188 which can adjust the gelling temperature is often added into the formula to obtain the ideal gelling temperature because the phase transition temperature of p407 is not sharp at a low concentration and the phase transition temperature of p407 is low at a high concentration.

At present, poloxamer and chitosan are taken as main materials for the temperature-sensitive gel material in the field of life science, but the chitosan is taken as a main substrate and needs to be used together with glycerophosphate to realize the temperature-sensitive characteristic, and the glycerophosphate has certain toxicity, is easy to generate inflammatory reaction in vivo and can not meet the safety requirement.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a chitosan gel with physiological responsiveness and application thereof in uterine cavity anti-adhesion, and the physiological responsiveness temperature-sensitive chitosan gel is non-toxic and has good safety.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses a chitosan gel with physiological responsiveness, which comprises the following components:

a) anti-blocking materials, chitosan and/or chitosan derivatives;

b) a temperature sensitive matrix material consisting of poloxamer 407 and poloxamer 188;

c) solvent, water;

in the chitosan gel with physiological responsiveness, the mass concentration (w/v) of chitosan is less than or equal to 1%, the mass concentration (w/v) of poloxamer 407 is 17% -21%, and the mass concentration (w/v) of poloxamer 188 is 1% -3%.

Preferably, pharmaceutically acceptable pharmaceutical excipients are further added into the chitosan gel with physiological responsiveness.

Further preferably, the pharmaceutical excipients comprise one or more of a viscosity regulator, a preservative, a surfactant, a suspending agent, an antioxidant, a stabilizer and a humectant.

Preferably, the water is water for injection, physiological saline or purified water.

Preferably, the chitosan derivative is carboxymethyl chitosan, carboxylated chitosan, hydroxypropyl chitosan, or chitosan hydrochloride.

Preferably, the gel temperature of the chitosan gel with physiological responsiveness is between 28 and 33 ℃.

The preparation method of the chitosan gel with physiological responsiveness comprises the following steps:

and (2) putting poloxamer 407 and poloxamer 188 into normal saline, water for injection or purified water at a temperature of lower than 28 ℃ in a clean environment, stirring until the mixture is infinitely swollen, adding chitosan and/or derivatives thereof, fully and uniformly mixing, and performing sterilization treatment to obtain the chitosan gel with physiological responsiveness.

The invention also discloses a temperature-sensitive gel system for preventing uterine cavity adhesion, which consists of a temperature-sensitive gel administration device and the chitosan gel with physiological responsiveness;

the temperature-sensitive gel administration device comprises a sol pusher for containing chitosan gel with physiological responsiveness, wherein the front end of the sol pusher is provided with a sol conveying pipe, and the end part of the sol conveying pipe is provided with a plurality of sol outflow holes; a cervical orifice positioner is also arranged on the sol delivery pipe;

when the temperature-sensitive gel administration device is used, chitosan gel with physiological responsiveness is filled in the sol pusher at the temperature lower than 28 ℃, the chitosan gel with physiological responsiveness is injected into a target position through a sol outflow hole at the end part of the sol conveying pipe, and the temperature-sensitive gel administration device is removed after 30-60 seconds.

Preferably, an auxiliary fixing rod is further arranged at the end part of the cervical orifice positioner close to the sol pusher, and an auxiliary fixing rod handle is arranged at the end part of the auxiliary fixing rod.

The temperature-sensitive gel system for preventing the uterine cavity adhesion is used for uterine perfusion in a sol state through a cervical orifice by an administration device at room temperature (lower than 28 ℃), and the sol rapidly finishes phase transformation to form gel under the action of intrauterine temperature (37 ℃), so that a first physiological response process is finished. The gel is distributed on the inner wall of the whole uterus in a gel state, the damaged uterine basal layer is physically isolated, adhesion in the uterine healing process is prevented, along with the healing of endometrium, the uterus enters a proliferation period under the regulation of estrogen and progestogen, the gel material is slowly degraded under the action of body temperature, after the uterus enters a secretion period, the gel is rapidly degraded under the action of a plurality of degrading enzymes of secretion, the rigidity of the gel is reduced, the pressure generated by the proliferation of the endometrium and the corrosion action of the secretion are responded, the gel recovers the liquid state again and is discharged out of a body from a cervical orifice through a vagina, and the second physiological response process is completed.

Compared with the prior art, the invention has the following beneficial effects:

the chitosan gel with physiological responsiveness disclosed by the invention is characterized in that active ingredients with the functions of stopping bleeding, inhibiting bacteria, promoting healing and the like are added into a non-chemically crosslinked temperature-sensitive sterile hydrogel matrix, the hydrogel is a sol with good fluidity at room temperature, so that the administration of the chitosan gel in a uterine cavity is convenient, the chitosan gel is rapidly gelled and solidified at physiological temperature of a uterus, the physical isolation of a wound surface after a uterine operation is finished, the adhesion is prevented, and the chitosan function is exerted to promote the rapid healing of the wound surface. The matrix material does not use a cross-linking agent, so that the damage of the cross-linking agent to a human body can be effectively avoided, the preparation method of the physiological response temperature-sensitive chitosan gel is simple, the experimental conditions are mild, special equipment is not needed, the investment cost is low, and the used reagents are conventional reagents, so that the method is convenient for industrial implementation and is environment-friendly.

Compared with Cuk J-BUS-253000, the invention has the advantages of low cost, simple operation, no foreign body sensation to patients, no need of removing the implanted device after the intervention, and the loaded active ingredients can promote the healing of endometrium, prevent inflammation and infection to a certain extent.

The invention discloses a chitosan gel with physiological responsiveness, which can be applied to IUA prevention after uterine operation, and based on the temperature-sensitive gel system for preventing uterine cavity adhesion, under the assistance of a vaginal dilator, the temperature-sensitive chitosan gel with physiological responsiveness is injected into uterus through a cervical orifice by means of a temperature-sensitive gel administration device in the system, and the administration device is removed after about 30-60 seconds.

Drawings

FIG. 1 is a schematic diagram of a physiologically responsive chitosan gel for preventing uterine cavity adhesion according to the present invention using a syringe; 1 is a sol outflow hole; 2 is a sol conveying pipe; 3 is sol; 4 is a sol pusher; 5 is a cervical orifice positioner; 6 is an auxiliary fixing rod; 7 is an auxiliary fixing rod handle;

FIG. 2 is a graph of temperature as a function of mass concentration (w/v) for P407 and P188 over the experimental range of the present invention;

FIG. 3 is a three-dimensional graph of a gelation temperature change model constructed by the present invention with P407 and P188 mass concentration (w/v);

FIG. 4 is a vaginal secretion smear at various physiological periods in female rats; wherein A is the prophase of estrus; b is estrus; c is the anaphase of estrus; d is estrus interval;

FIG. 5 is a drawing showing rat model making and sampling; wherein, A is the uterus treated by LPS in the molding; b is post-mortem uterine tissue; c is isolated uterus;

FIG. 6 is MASSON staining pattern (X100) of rat uterine sections; wherein, A is a normal group; b is a molding set; c is intervention group; d is a positive control group;

FIG. 7 is a view of the mouse peritoneal adhesion model; wherein A is the treatment of the abdominal mucosa; b, treating the cecum;

FIG. 8 is a adhesion level classification chart; wherein A is grade III viscosity; b is II-grade adhesion; c is I-grade adhesion; d is no adhesion.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

example 1 preparation of physiologically responsive Chitosan gel for prevention of uterine adhesions 1.1 screening of prescription

Locking the range of P407 mass concentration (w/v) at 17% -21%, locking the range of P188 mass concentration (w/v) at 1% -3%, establishing a star point design by taking P407 and P188 as independent variables and the gelling temperature as an effective amount, and showing in table 1:

TABLE 1 Star design-Effect surface optimization method factor horizon

The preparation method comprises the following steps: weighed calculated amounts of P407 and P188 were added to the vessel, calculated amounts of purified water were added, and the mixture was stirred until the solid swelled indefinitely. Adding 0.5% chitosan hydrochloride, and stirring until complete dissolution.

And (4) analyzing results: the coding factor equation of the model is obtained by design expert:

T＝26.5-6.79*A+2.59*B-0.85*A*B+2.07A²+1.04B²

f is 93.00 and P <0.0001<0.01, which shows that the regression equation is significant and has research significance.

The gelation temperature is shown in figure 2 as a function of P407 and P188 mass concentration (w/v) within the experimental range.

1.2 prescription prediction

From the above formula, a recipe with a desired gelling temperature of 28-33 ℃ can be deduced.

TABLE 2 predicted temperature recipe and actual temperature of recipe

Experiments prove that the actual gelling temperature is not greatly different from the predicted gelling temperature, and the P40717.55% and the P1881.15% fall within the expected range, so that the group is drawn as an optimal prescription. The gelation temperature is shown in the figure 3 along with the changes of P407 and P188 mass concentration (w/v).

1.3 gel stability Studies

1.3.1 Chitosan content determination method verification:

standard curve: precisely sucking 0.4g/L chitosan standard solution 0.0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mL, respectively placing into 10mL glass test tubes with covers and scales, respectively sequentially adding 2mol/L sodium acetate buffer solution with pH 5.5 and 1% ninhydrin reagent 1.0mL, and adding ultrapure water to 3.0mL, mixing well, heating in boiling water bath for 10min, taking out, rapidly cooling to room temperature, adding 60% ethanol to dilute to 10mL, shaking well, standing for 20min, and measuring absorbance at 570 nm. A sample containing 0.0ml of the chitosan standard solution was used as a blank.

Precision: samples to which 0.4 chitosan hydrochloride standard solution was added were taken for 6 consecutive determinations.

Stability: absorbance was measured at 10, 15, 30, 45, and 60 min.

Repeatability: 1g of chitosan gel was precisely weighed, diluted with 9.0mL of ultrapure water, and 1m was measured according to the above method. Six groups were repeated.

And (3) sample recovery rate: the diluted gel was taken at 1.0mL and added with 0.1, 0.2, 0.3, 0.4mL of standard solution as determined above.

1.3.2 investigation indexes:

viscosity: measured using a rotational viscometer.

And (3) placing the prepared gel in a liquid containing cylinder of the viscometer, selecting a proper rotor and a proper rotating speed, and measuring the viscosity value.

And (3) gelling temperature: an inversion method is adopted.

Respectively weighing 10g of CTS gel, putting the CTS gel into 3 small-size penicillin bottles, covering plugs with temperature sensors, putting the bottles into a constant-temperature electric water bath kettle, taking 20 ℃ as an initial temperature, raising the temperature by 1 ℃ every time, keeping the temperature for 10min after the temperature is stable, then inverting the penicillin bottles, observing the flowing state of the gel, recording the temperature indication of the sensors as the gelation temperature within 30s, and otherwise, repeating the operation until the gelation temperature of all samples is measured.

1.3.3 results:

the preferred prescription is 17.55% P4071.115% P1880.5% CTS, the indices are as follows

The content of chitosan is as follows: taking chitosan hydrochloride amount (g) as an abscissa, absorbance A as an ordinate, and a linear regression equation of which y is 587.73x-0.0086, R²0.9921, good linearity, good methodological parameters,% calculated as 98.28%.

Viscosity: greater than 100000cp at 37 ℃

And (3) gelling temperature: 30.50 deg.C

The product can be used for quality control of industrial production by determining chitosan content by mature method. The product has high viscosity at 37 deg.C, and can be effectively adhered to uterine cavity to prevent adhesion. In addition, the gel temperature of the product is 30.5 ℃, and is within the optimal temperature range of 28-33 ℃, so the product is in a liquid state at room temperature, can be rapidly heated up under the influence of the temperature of the uterine cavity after entering the uterine cavity, is in a semisolid gel state, is very convenient to administer, and is easier to coat uniformly on the inner wall of the uterine cavity.

Example 2 intervention of physiologically responsive Chitosan gels on the uterine adhesion model in rats

2.1 Molding method

The 10% chloral hydrate solution 30ml/kg is injected into the abdominal cavity of the oestrous rat, and the anaesthetized rat is fixed on an operation plate with a constant temperature pad in a backward position. Removing hair from rat abdomen, sterilizing rat abdomen with iodophor, opening a wound of about 3cm along the lower part of abdominal midline, exposing uterus, making a 2mm incision on the upper part of uterus, scraping with curette to the far end (about 3cm in length) until roughness and smoothness are reached (the uterus can be held by finger abdomen), and suturing uterus. Clamping uterine artery, ovarian artery-uterine artery traffic branch, bilateral uterine proximal end and bilateral uterine distal end with non-invasive vascular clamps, puncturing the lower part of uterus with a 1ml sterile syringe, injecting about 0.4ml of LPS solution of 10mg/L in parallel with the uterus, withdrawing the LPS solution after 30min, and loosening the vascular clamps. Injecting about 0.3ml of each group of medicine into the needle hole along the injection of LPS, flushing the abdominal cavity with normal saline, suturing the wound with non-absorbable cotton thread in a layered manner, and sterilizing with iodophor. The surgery is performed under sterile conditions. And 7d, killing, taking the uterus on the treatment side and the uterus on the opposite side allele, and placing the uterus in a cell fixing solution for fixation. Paraffin embedding was then performed, and sections (0.4 μm) were cut, and one piece of Masson was stained per uterus.

The estrus screening method comprises the following steps: and (3) sucking 20 mu L of physiological saline by a pipette gun, injecting the physiological saline into a rat vagina, washing the physiological saline, coating the physiological saline on an adhesive glass slide, dripping about 10 mu L of the Dacron staining solution A on the smear part, dripping 10 mu L of the Dacron staining solution B after 30-60s, and observing the smear by a 400X optical microscope after the smear part is dried.

As a result, as shown in fig. 4, it can be seen from fig. 4 that the non-keratinized epithelial cells at the prophase of estrus are more, and the cells are round or oval nucleated cells; the keratinized epithelial cells in estrus are more and are anucleate cells with irregular shapes; the keratinized epithelial cells, the non-keratinized epithelial cells, the white blood cells in the anaphase of estrus each account for about 1/3; the most of the estrus is white blood cells, and the round shape is small.

2.2 grouping and intervention method

Grouping: dividing 30 female SD rats (200-250 g in 6-8 weeks) into six cages, each cage is provided with 5 rats, screening the rats in estrus every day, and randomly performing each group to perform an operation experiment, wherein the rat uterus is distributed bilaterally in a Y shape, and the other side of the rat uterus is used as a blank control without intervention after the operation. The photographs of the mold and sample taken during the experiment are shown in FIG. 5.

The intervention method comprises the following steps: a1 mL syringe was used to inject the gel through the needle during LPS treatment.

Building a module: no other intervention was performed after LPS treatment.

Intervention group: 0.5% CTS gel was injected.

Positive control group: gongkang gel.

2.3 evaluation index

Including HE staining, Masson staining, degree of adhesion.

Observation indexes and detection methods:

① number of glands 4 fields were randomly selected under HE staining, and the number of glands (. times.200) in each field was counted and averaged, the number of glands in the control group was smaller than the number of glands in the control group, and the degree of adhesion was higher.

② area ratio of endometrial fibrosis 4 fields were randomly selected under a high power microscope under Masson staining, and the ratio of the area of endometrial fibrosis per field (x 200) was calculated as the percentage of the total area of endometrial interstitial fibrosis to the sum of the area of endometrial stroma and glands, and the average was calculated.

Scoring of gland number and fibrosis ratio:

the semi-quantitative score of endometrial fibrosis comprises the sum of 2 parameter scores of endometrial gland number and collagen fiber number change, and the semi-quantitative score of endometrial fibrosis is obtained.

HE staining standard:

the number of endometrial glands per high power of endoscopic field (x 200) in normal endometrium of rat was taken as the standard (score 0).

Randomly selecting 4 fields of view of an endometrial slice at the rat model side, reading the number of endometrial glands under each high power of visual field of the endoscope, and calculating the average number, if the number of the endometrial glands is reduced by 1-10% (1 minute); reducing by 11-25% (2 min); reducing by 26-50% (3 min); reducing by 51-75% (4 min); the decrease is 76-100% (5 points), and the increase is negative.

Masson staining reading standard:

the ratio of collagen fiber components contained in normal endometrium of rat per high power of visual field (x 200) was 0.

Randomly selecting 4 visual fields of an endometrial slice at the rat model side, reading the ratio of endometrial fiber components under each high-power endoscopic visual field, and calculating the average number, wherein if the ratio is increased by 1-10% (1 min); increasing by 11-25% (2 min); increase 26% -50% (3 min); increase 51% -75% (4 minutes); the increase is 76-100% (5 min), and the decrease is negative.

2.4 results of the experiment

As shown in FIG. 6, it can be seen that the collagen fibers of the model group are significantly increased and the number of glands is also decreased compared with the normal group; the fibrosis level of the intervention group is similar to that of the normal group, and the number of glands is less than that of the normal group and the number of the glands is more than that of the modeling groups; the positive control group has a fibrosis level higher than that of the intervention group and smaller than that of the modeling group, and the number of glands is close to that of the intervention group.

Analysis of the semi-quantitative scores resulted in the following table 3:

TABLE 3 rat uterus adhesion score table

Performing normality test on the three groups, wherein the intervention group W is 0.911, and the intervention group P is 0.442; positive control group W ═ 0.972, P ═ 0.554; the die set W is 0.496 and P is 0.005. Wherein the intervention group and the positive control group obey normal distribution, and the modeling group does not obey normal distribution. Therefore, the comparison between the three groups should adopt the rank-sum test, the comparison between the intervention group and the positive control group adopts the t test, and the intervention group and the positive control group adopt the rank-sum test when being compared with the modeling group in pairs.

The module building group, the intervention group and the positive control group have the following rank sum test results: chi shape²13.122, P0.001 < 0.05, the degree of uterine adhesion is not identical in three groups of rats; the rank and test results of the molding group and the intervention group are as follows: z is-2.994, P is 0.003 < 0.05, and the difference of uterine adhesion degree between the model group and the intervention group has statistical significance; the rank sum test results of the modeling group and the positive control group were: z is-3.000, P is 0.003 < 0.05, and the difference of uterine adhesion degree between the model group and the positive control group has statistical significance; the t test results of the intervention group and the positive control group are as follows: t-2.314, P-0.043 < 0.05, and the difference of uterine adhesion degree between the dried group and the positive control group has statistical significance.

The uterus adhesion degree of the intervention group and the positive control group is different from that of the artificial model group, and the scores of the intervention group and the positive control group are lower than that of the artificial model group, so that the intervention group and the positive control group can effectively prevent the uterus adhesion. Although the difference between the intervention group and the positive control group is close to 0.05, the difference is not as high as the difference between the P value and 0.003, but the intervention group score is obviously lower than the positive control group score, which indicates that the prescription has better prevention effect than the positive control drug. According to data analysis, the intervention group and the positive control group have little difference in the gland number score, but the positive control group has deeper fibrosis degree and more collagen. The main component of the positive control drug is hyaluronic acid, and the positive control drug is reported in related documents: hyaluronic acid can bind with hyaluronic acid binding protein on fibroblasts, so that the fibroblasts show motility, migrate to tendon injury, participate in healing, and regulate collagen synthesis. It is expected that more collagen fibers will be expressed during recovery of damaged mucosa relative to the intervention group, and it is therefore normal to obtain this result.

Example 3 intervention of physiologically responsive Chitosan gel on mouse Abdominal junction model

3.1 Molding

The mice were weighed and anesthetized with a 3.5% chloral hydrate intraperitoneal injection of 0.01 ml/g. The electric hair remover shaves off the abdominal hair. Spraying 75% ethanol to the whole body for sterilization, and sterilizing the abdomen with iodophor. The mouse is fixed on the foam board in the upward position, and the abdomen is opened by the surgical scissors in a layered mode. The right muscle layer was turned over with both hemostats to bring the peritoneal mucosa outward, a 1 x 0.5cm rectangular area was gently stroked with a small blade, and the peritoneal mucosa in the area was removed with forceps, as shown in fig. 7, and the hemostats were removed. The cecum was removed, worn around the cecum with gauze about 20 times to a bleeding state as shown in fig. 7, and then returned to the abdominal cavity. Each group of drugs was evenly spread on the peritoneal and cecal wound surfaces at approximately 0.5 ml. Mice were sutured separately on the ventral skin. The surgery is performed under sterile conditions.

3.2 grouping

60 Kunming female mice (about 30g in 6-8 weeks) were randomly divided into six groups of 10 mice each, each consisting of a model group, a 0% CTS gel group, a 0.5% CTS gel group, a 1% CTS gel group, and a 1.5% CTS gel, and were fed adaptively for one week.

3.3 intervention methods

Uniformly coating the medicine on the surfaces of peritoneal wound and cecum;

building a module: no administration of the drug;

0% CTS gel group: coating 0% CTS gel;

0.5% CTS gel group: coating with 0.5% CTS gel;

1% CTS gel group: coating 1% CTS gel;

1.5% CTS gel group: coating with 1.5% CTS gel;

positive control group: coating the Gongkang gel.

3.4 evaluation index

The evaluation index is the degree of adhesion: after 7d, the patient was sacrificed, the abdomen was cut open, and the right muscle layer was lifted with forceps. Completely no adhesion, marking as no adhesion, and giving a score of 0; slight pulling can mark the separation of the bonds as grade I bonds, giving 1 point; slightly tearing the adhesive to separate the adhesive into II-grade adhesive and giving 2 points; no separation by hard tearing was scored as grade III adhesion, giving a score of 3. Four adhesion level differences are shown in fig. 8.

3.5 results of the experiment

As shown in fig. 7 and tables 4 and 5 below:

TABLE 4 degree of peritoneal adhesions after CTS gel Dry

TABLE 5 Effect of CTS gel on degree of peritoneal adhesions Scoring

The normality analysis of each group is not compliant with normal distribution, so the rank sum test is adopted. Rank sum test, χ, was performed on each group²The value is 34.026, P is 0.000 < 0.05, and therefore the degree of abdominal adhesions in each group cannot be considered the same. Pairwise rank sum test results were as follows: the difference in uterine adhesion between the model and the other groups was statistically significant, while the differences between the other groups were not significant, and the P value was closer to 0.05 in the 0% CTS gel group than in the other groups (except the model), so the likelihood of the 0% CTS gel adhering to the uterus of the other groups was not as great as the likelihood of the adhesion between the other groups. It can be seen from the data that: the 0% CTS gel was also less anti-blocking than the other groups, and the gel effect was better with the addition of CTS, thus it was seen that the expected anti-blocking effect could not be obtained only by the action of the gel skeleton itself, and the gel had better anti-blocking effect with the addition of CTS. The difference between the 0.5 percent of the CTS gel and the CTS gel prevention effect of other concentration groups has no statistical significance, the CTS content is increased, the anti-adhesion effect basically has no significant change, and the high-efficiency low-quantity product is selected as much as possible from the safety aspect. Therefore, the final CTS content of the product is determined to be 0.5%.

In conclusion, the temperature-sensitive gel with physiological response characteristics, which can prevent uterine adhesion after an operation, is disclosed by the invention. The active ingredients with the functions of stopping bleeding, inhibiting bacteria, promoting healing and the like are added into the non-chemical cross-linked temperature-sensitive sterile hydrogel matrix, are in a sol state with good fluidity at room temperature, are convenient for administration in uterine cavities, are rapidly gelled and cured at the physiological temperature of uterus, complete physical isolation on wound surfaces after uterine surgery, prevent adhesion, simultaneously play the function of physiological active materials and promote the wound surfaces to heal quickly. After the uterus is recovered, the gel is dissolved and eroded under the physiological action of self proliferation and secretion and is discharged out of the body along with the secretion without manual intervention

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A physiologically responsive chitosan gel, comprising:

a) anti-blocking materials, chitosan and/or chitosan derivatives;

c) solvent, water;

in the chitosan gel with physiological responsiveness, the mass concentration (w/v) of chitosan is less than or equal to 1%, the mass concentration (w/v) of poloxamer 407 is 17-21%, and the mass concentration (w/v) of poloxamer 188 is 1-3%.

2. The physiologically responsive chitosan gel according to claim 1, wherein a pharmaceutically acceptable pharmaceutical excipient is further added to said physiologically responsive chitosan gel.

3. The physiologically responsive chitosan gel according to claim 2, wherein said pharmaceutical excipients comprise one or more of viscosity modifiers, preservatives, surfactants, suspending agents, antioxidants, stabilizers and humectants.

4. The physiologically responsive chitosan gel according to claim 1, wherein said water is water for injection, physiological saline or purified water.

5. The physiologically responsive chitosan gel according to claim 1, wherein said chitosan derivative is carboxymethyl chitosan, carboxylated chitosan, hydroxypropyl chitosan, chitosan hydrochloride, or chitosan lactate.

6. The physiologically responsive chitosan gel according to claim 1, wherein said physiologically responsive chitosan gel has a gelling temperature between 28-33 ℃.

7. A temperature-sensitive gel system for preventing uterine cavity adhesion, which is characterized by comprising a temperature-sensitive gel administration device and the chitosan gel with physiological responsiveness of any one of claims 1 to 6;

the temperature-sensitive gel administration device comprises a sol pusher (4) for containing chitosan gel with physiological responsiveness, wherein the front end of the sol pusher (4) is provided with a sol conveying pipe (2), and the end part of the sol conveying pipe (2) is provided with a plurality of sol outflow holes (1); a cervical orifice positioner (5) is also arranged on the sol delivery pipe (2);

when the temperature-sensitive gel administration device is used, chitosan gel with physiological responsiveness is filled in the sol pusher (4) at the temperature lower than 30 ℃, the chitosan gel with physiological responsiveness is injected into a target position through the sol outflow hole (1) at the end part of the sol delivery pipe (2), and the temperature-sensitive gel administration device is removed after 30-60 seconds.

8. The temperature-sensitive gel system for preventing the adhesion of the uterine cavity according to claim 7, wherein an auxiliary fixing rod (6) is further arranged at one end of the cervical orifice locator (5) close to the sol pusher (4), and an auxiliary fixing rod handle (7) is arranged at the end of the auxiliary fixing rod (6).

9. Use of the physiologically responsive chitosan gel of claim 1 in the preparation of a clinical agent for uterine cavity anti-adhesion.