CN114948913A

CN114948913A - Gel film with biological adhesion and application thereof

Info

Publication number: CN114948913A
Application number: CN202210504976.7A
Authority: CN
Inventors: 殷黎晨; 刘寻; 王梦如
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2022-05-10
Filing date: 2022-05-10
Publication date: 2022-08-30

Abstract

The invention belongs to the field of medical materials, and particularly relates to a gel film with biological adhesion and application thereof. The gel film has ROS removing function, can be attached to biological tissues such as endothelium, intestinal mucosa, bile duct and the like, and can entrap functional small molecules and biological macromolecules; the gel film is formed by mixing liquid A and liquid B, wherein the liquid A comprises a substance containing a phenylboronic acid structure, and the liquid B comprises plant polyphenol and a pharmaceutical adjuvant. The solution A and the solution B are sprayed on the surface of biological tissues simultaneously to form a gel film immediately, and functional small molecules or biological macromolecules can be controllably released under the condition of ROS or acidity, so that the gel film has a good anti-inflammatory effect.

Description

Gel film with biological adhesion and application thereof

Technical Field

The invention belongs to the field of medical materials, and particularly relates to a gel film with biological adhesion and application thereof.

Background

Inflammatory Bowel Disease (IBD) is a chronic, recurrent disease of the gastrointestinal tract, including Crohn's Disease (CD) and Ulcerative Colitis (UC). The symptoms of the patients with the inflammatory bowel disease are mainly repeated abdominal pain, diarrhea, mucous bloody stool and the like, and the health of the patients is seriously harmed. The current IBD treatment drugs mainly comprise glucocorticoids and immunosuppressants, but the treatment effect is not ideal, and the side effect of systemic administration is large. The inflammation targeted drug delivery system can improve the concentration of the drug at the inflammation part and reduce the toxic and side effects on normal tissues. Among them, enema is the main form of targeted drug delivery, however, it is difficult to retain the enema at the site of intestinal inflammation because IBD patients are often accompanied by diarrhea and intestinal irritation. Multiple administrations can seriously affect patient compliance. Therefore, the development of the high-efficiency safe, drug-controlled-release and bioadhesive gel stent has important research significance and application value.

Conventional hydrogel patches are generally prepared in vitro and injected into the site of inflammation by means of an enema. The preparation process of the hydrogel is complex, high temperature and organic solvent are possibly involved, and the adhesion, the controllable release of the drug and the biodegradation of the hydrogel at an inflammation part are not ideal. There is a need in the art for a new bioadhesive gel material that overcomes the above problems and allows for targeted treatment of inflammation.

Gel drug-loading systems are reported in a few cases, but few gels are used for enema targeted drug delivery, the conventional gel drug system is not applicable due to the special application mode and the special application positions, and the existing enema gel system is not ideal in adhesion, drug controlled release and biodegradation of inflammation positions.

Disclosure of Invention

In view of the above prior art, the present invention discloses novel bioadhesive gel films and controlled release drug systems that can be used to treat inflammation, particularly inflammatory bowel disease.

The invention provides a gel film with biological adhesiveness, which consists of a solution A and a solution B, wherein the solution A comprises a substance containing a phenylboronic acid structure; the solution B comprises plant polyphenol and pharmaceutic adjuvant.

Preferably, the substance containing a phenylboronic acid structure is a polymer containing a phenylboronic acid structure or a small molecule compound containing a phenylboronic acid structure.

Preferably, the polymer containing a phenylboronic acid structure has a structure represented by the following formula:

wherein n represents that the polymer has a rereading unit and has no specific numerical value meaning.

Further, the polymer containing a phenylboronic acid structure has a structure represented by the following formula:

wherein R is ₁ 、R ₂ Each independently selected from linear aliphatic chain hydrocarbons or branched aliphatic chain hydrocarbons;

n indicates that the polymer has repeating units and has no specific numerical meaning.

The polymer containing a phenylboronic acid structure can be obtained by covalent coupling of a water-soluble polymer (such as polyethylene glycol, polypeptide and the like) obtained by radical self-polymerization (including RAFT polymerization, ATRP polymerization and the like) or a water-soluble natural macromolecule and a monomer containing the phenylboronic acid structure.

Preferably, the small molecule compound containing a phenylboronic acid structure has a structure shown in the following formula:

wherein R is a structure comprising a secondary, tertiary or quaternary amine.

Further, the small molecule compound containing the phenylboronic acid structure has a structure shown in the following formula:

preferably, the plant polyphenol is a natural polyphenol compound or a synthetic polyphenol compound, and has a structure shown in the following formula:

wherein R' is an aliphatic chain, aromatic chain or heterocyclic chain structure.

Further, the plant polyphenol has a structure represented by the following formula:

preferably, the concentration of the substances with the phenylboronic acid structure in the solution A is 1-30 wt%; the concentration of the plant polyphenol in the solution B is 1-30 wt%.

Preferably, the medicinal auxiliary material in the solution B is a water-soluble biomacromolecule medicinal auxiliary material, such as sodium alginate, chitosan or hyaluronic acid.

The invention also provides an application of the gel film with biological adhesion in preparing an anti-inflammatory and reactive oxygen Radical (ROS) scavenging drug or a drug slow-release system, which comprises the following steps: the solution A and the solution B are sprayed on the surface of the tissue through the catheter at the same time, and the gel film carrying the medicine can be formed.

The system has simple application method, strong practicability and good adhesiveness.

The invention also provides an anti-inflammatory, ROS-scavenging drug or drug delivery system comprising the bioadhesive gel film described above.

In the bioadhesive gel film or the bioadhesive controllable release drug system disclosed by the invention, the gel film has good bioadhesive performance and ROS scavenging function, plant polyphenol and phenylboronic acid quickly react, and if drugs exist, the plant polyphenol and the phenylboronic acid are wrapped by gel; under the high-concentration ROS environment of inflammation parts, ROS is eliminated, the gel film is degraded to slowly release active drugs, and the gel film has an anti-inflammatory effect and plays a synergistic effect with the drugs.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the invention has the advantages that the preparation process of the gel bracket (gel film) is simple, and the gel bracket (gel film) is formed immediately by simply mixing the solution A and the solution B, thereby being beneficial to clinical transformation; the gel scaffold (gel film) is self-degradable at the inflammation site; the gel stent (gel film) has good drug-encapsulating effect and good drug slow-release effect.

The invention provides a gel film (gel stent) with bioadhesion and controllable drug release. The invention changes the defects of the traditional gel preparation, instantly forms a gel film by mixing the solution A and the solution B, and instantly forms the gel film by spraying the solution A (containing the medicine) and the solution B on the inner side surface of the intestinal cavity of the organism at the same time, which can be regarded as a gel bracket structure. Relevant experiments show that the gel film disclosed by the invention is good in bioadhesion, has ROS degradability and an anti-inflammatory effect, and has an obvious anti-inflammatory effect when an intestinal barrier protection drug Dervitin is encapsulated on a rat IBD model, so that the effects of promoting intestinal mucosa repair and regulating intestinal flora are achieved.

Drawings

FIG. 1 is a nuclear magnetic spectrum of Polymer PB of example 1.

FIG. 2 is a nuclear magnetic spectrum of monomer TSPBA in example 1.

FIG. 3 is a scanning electron micrograph of a gel film of example 1.

FIG. 4 is a fluorescence image of Cy 5-gel film attached to rat colon in vitro in example 3.

FIG. 5 is a graph of the adhesion of E133-gel film to rat colon in flowing water in example 3.

FIG. 6 is a fluorescence image of HD-gel film in vivo adhesion to rat colon in example 4.

FIG. 7 is a 3D reconstructed CT image of the colon of rat adhered to the gel film in vivo in example 4.

FIG. 8 is a graph of in vitro degradation of the gel film of example 5.

FIG. 9 is a graph of the quantification of the in vitro release of averin entrapped in the gel film of example 5.

FIG. 10 is a graph evaluating the effect of gel film treatment on rat gastrointestinal kinetics in example 6.

FIG. 11 is a graph for evaluating the safety of the gel film in rats in example 7.

FIG. 12 is a graph of the in vitro anti-inflammatory and anti-ROS evaluation of gel films in example 8.

FIG. 13 is a graph of the evaluation of gel films against neutrophil and macrophage migration in vitro in example 9.

Figure 14 is a graph of the efficacy of Di @ Gel on rat acute colitis evaluated in example 10.

FIG. 15 is a graph of the efficacy of Di @ Gel on rat chronic colitis evaluated in example 11.

Figure 16 is a graph of the efficacy of Di @ Gel in evaluating acute colitis in dogs in example 12.

Figure 17 is a graph of the assessment of Di @ Gel in example 13 to modulate macrophage polarization in vivo.

FIG. 18 is a graph of the assessment of Di @ Gel modulating intestinal flora in rats with acute colitis as in example 14.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The raw materials of the invention are the existing materials, the specific preparation operation and the experimental test are the conventional technologies, the animal experiment meets the animal experiment requirements of Suzhou university, and the colitis rat is obtained by conventional modeling.

Preparation example 1

Dimethylamino ethyl methacrylate (5g,31.8mmol) and azobisisobutyronitrile (52.2mg,0.318mmol) were charged into a 25mL polymerization flask, vacuum-pulled, purged with nitrogen for 15 minutes to remove oxygen, sealed, and the reaction solution was reacted at 65 ℃ for 24 hours. Dissolving the reaction product in dichloromethane, precipitating with n-hexane, washing for three times, and vacuum-drying to obtain a polymer 1, and performing nuclear magnetism on deuterated chloroform;

polymer 1(0.3g) was dissolved in dimethylformamide (20mL), and 4- (bromomethyl) phenylboronic acid (0.6g) was added and stirred at room temperature for 24 h. After the reaction is finished, the reaction solution is added into a 3500Da dialysis bag, and is dialyzed for 2 days by deionized water, and the dialysate is changed every 12 hours. The solution was freeze dried to give product PB which was nuclear magnetized with heavy water as shown in FIG. 1.

Dissolving tetramethylpropanediamine (0.1g) in dimethylformamide (10mL), adding 4- (bromomethyl) phenylboronic acid (0.5g), and stirring at 65 ℃ for 24 h; the reaction solution was added to 100mL tetrahydrofuran, filtered, washed three times with tetrahydrofuran, vacuum dried to obtain the product TSPBA, and nuclear magnetic resonance was performed with deuterated dimethyl sulfoxide, see FIG. 2.

The drug divartin was synthesized as follows:

an aqueous formaldehyde solution (0.2mol, 37% w/v) was added dropwise to a hydrazine hydrate solution (0.15mol) under ice-bath conditions. After stirring at room temperature for 20 minutes, the reaction mixture was left at room temperature for 3 days. The crude product was washed with hot isopropanol and filtered to give the divaritin as a white powder (18% yield) which was then dried under vacuum for elemental analysis as shown in table 1.

TABLE 1 elemental analysis of drug Divertin example 1

Chemical elements	C	H	N
				Ratio (%)	39.81	7.154	47.93

Preparation example 2

And (2) preparing the rest polymers containing the phenylboronic acid structure through amidation reaction, dissolving hyaluronic acid in aqueous solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), stirring for 6 hours at room temperature (25 +/-5 ℃), then adding 4-Dimethylaminopyridine (DMAP) and 4-aminomethylphenylboronic acid, stirring for 48 hours at room temperature, after the reaction is finished, adding the reaction solution into a 3500Da dialysis bag, dialyzing for 2 days with deionized water, changing dialysate every 12 hours, and freeze-drying the solution to obtain the polymer containing the phenylboronic acid structure.

The preparation method of the rest of the small molecular compounds containing the phenylboronic acid structure is the same as that of the small molecular compounds containing the phenylboronic acid structure in preparation example 1, except that the tetramethylpropanediamine is replaced by a corresponding poly-tertiary amine structure, so that the small molecular compounds containing the phenylboronic acid structure are obtained; wherein the molar weight of the 4- (bromomethyl) phenylboronic acid is 1 to 3 times of the molar content of the tertiary amine.

Example 1

Preparation of gel film (gel scaffold)

10mg of polymer PB was dissolved in 100. mu.L of deionized water to prepare solution A at a concentration of 10% (w/v); dissolving natural polyphenol Tannin (TA) in 100 μ L deionized water, and preparing solution B at concentration of 10% (w/v); and mixing the solution A and the solution B in equal volume to obtain the gel film.

Example 2

Dissolving 20mg of small molecule TSPBA in 100 mu L of deionized water to prepare solution A with the concentration of 20% (w/v), and dissolving TA and Sodium Alginate (SA) in 100 mu L of deionized water to prepare solution B, wherein the concentration of TA is 10% (w/v), and the concentration of SA is 1% (w/v); and mixing the solution A and the solution B in equal volume to obtain the gel film.

Example 3

10mg of polymer PB and drug Divertin (the dosage of rat (100mg/kg) and the dosage of dog (30 mg/kg)) were dissolved in 100. mu.L of deionized water to prepare solution A; dissolving 10mg of TA in 100 mu L of deionized water, and preparing solution B at the concentration of 10% (w/v); and mixing the solution A and the solution B in equal volume to obtain the drug-loaded gel film.

Example 4

Dissolving 20mg of small molecule TSPBA and drug diverin (the dosage of a rat (100mg/kg) and the dosage of a dog (30 mg/kg)) in 100 mu L of deionized water to prepare solution A, and dissolving TA and SA in 100 mu L of deionized water to prepare solution B, wherein the TA concentration is 10% (w/v) and the SA concentration is 1% (w/v); and mixing the solution A and the solution B in equal volume to obtain the drug-loaded gel film.

The gel scaffold (gel film) structure was characterized by scanning electron microscopy.

The solution A and the solution B of the second example are used in the following examples; for testing purposes, test agents, such as fluorescent agents, contrast agents, and the like, may be routinely added.

Example 5

In vitro adhesion of gel films

Normal SD rats (6-8 weeks, 180-. 3 sections of colon slices with the length of 2cm and the width of 1cm are cut, and 50 mu L of TSPBA solution and TA/SA solution loaded with hematoporphyrin dihydrochloride (HD, 0.15mg/mL) are sprayed on the mucous membrane of the colon cavity surface at the same time to form a gel film immediately. The colon plates were placed in a petri dish containing PBS (5mL, 10mM, pH 7.4) and shaken on a shaker at 37 ℃ and 200 rpm. Pieces of intestine were removed at 0, 12, 24, 48, 72 hours, respectively, imaged on an in vivo imaging system fluorescence imager (IVIS), and analyzed for fluorescence intensity.

The mucoadhesive strength of the gel film under water flow was tested. Taking 3 segments of colon pieces with length of 2.5cm and width of 1cm, spraying 70 μ L of TSPA solution and TA/SA solution loaded with brilliant blue pigment (E133, 0.5%, w/v) on the mucosa of the colon cavity surface to form gel film immediately. The gel was placed in a stream of water at a flow rate of 120mL/min, the colon was photographed at 2, 6, 12 and 24 hours, respectively, and the area of the remaining gel film on the colonic mucosa was quantitatively analyzed using ImageJ.

Example 6

In vivo adhesion of gel films

Colitis rats were separately anesthetized and 250 μ L of the HD-loaded TSPBA solution and TA/SA solution were sprayed simultaneously from anus to colon using a long tube syringe to form a gel film immediately. Intestinal fluorescence intensity was analyzed by IVIS at 0, 12, 24, 48 and 72 hours (n-3 at each time point) after enema, respectively. Also in parallel experiments, rats were sacrificed at various time points, the colon removed, and intestinal fluorescence intensity analyzed using IVIS.

CT imaging to assess gel film retention in rats

TSPBA and TA/SA were added to 1mL iohexol contrast agent (30 g iodine per 100mL solution), and vortexed to dissolve completely, resulting in a TSPBA contrast agent solution with a concentration of 20% (w/v) and a TA/SA contrast agent solution with a concentration of 10%/1% (w/v). Colitis rats were randomly divided into two groups: gel film (Gel) and Control (Control) groups of 3 pieces each. Gel group 250. mu.L of TSPBA contrast agent solution and TA/SA contrast agent solution were sprayed into the colon simultaneously, immediately forming a Gel film. The same method as in Control group was used to administer an equal volume of contrast agent. At 0, 12, 24, 48 and 72 hours after the enema, the rats were subjected to continuous scanning imaging by placing them on CT under isoflurane (1%) anesthesia. The CT imaging parameters are: current 615 uA; the voltage is 60 kV; the number of projections 499; total scan time 7 minutes. The original image is processed and 3D images of different axial directions are reconstructed.

Example 7

H ₂ O ₂ Degradation of gel films and Divertin Release under conditions

A50. mu.L TSPA solution and TA/SA solution were sprayed simultaneously into both vials, immediately forming a gel film. 5mL of PBS buffer (pH 7.4) and 5mL of H-containing buffer were added ₂ O ₂ (1mM) in PBS (pH 7.4), placed on a shaker at 37 ℃ and 200rpm for 0, 2, 5 and 7 days, respectively, the gel film was observed for degradation and recorded by photographing.

50 mu L of TSPBA solution and TA/SA solution loaded with Cy5(300mg/mL) are sprayed on the mucous membranes of the two segments of the colonic cavity surfaces simultaneously to form gel films immediately, and the gel films are respectively put into a dialysis bag of 3500 Da. Placing into a container containing 100mL of PBS buffer (pH 7.4) and 100mL of H ₂ O ₂ (1mM) in a PBS-buffered (pH 7.4) beaker, placed on a shaker at 37 ℃ at 200 rpm. At 0, 2, 4, 6, 8, 12, 24, 48 and 72 hours, respectively, 500. mu.L of fresh PBS was removed from the dialysis bag and supplemented with 500. mu.L. The fluorescence signal of Cy5 was detected using a SynergyNEO HTS multimode microplate reader with 633nm excitation and 670nm emission.

Example 8

Rat gastrointestinal kinetics assay

Healthy rats were randomly divided into two groups: control (Control) and Gel film (Gel), 6 of each. After fasting but not water deprivation for 12 hours, 250 μ L of physiological saline and gel film enema were administered, respectively. After the enema, the rats were housed in individual cages and the following experiments were performed, respectively:

effect of gel film on gastrointestinal transport in rats. 12 hours after gel film enema, each rat received 1mL of gastric lavage fluid containing phenol red (60mg) and methylcellulose (5mg) and placed in a separate cage. The time for the first red stool to be excreted after gavage was taken as the gastrointestinal transit time.

The colonic bead exclusion experiment evaluated the effect of the gel film on colonic motility. 12 hours after the gel film enema, glass beads (diameter 5mm) were inserted into the colon from the anus of the rat by 2cm using a glass rod under light anesthesia with ether, and the rat was placed in a separate cage and the time from insertion to expulsion of the beads was recorded.

The rats were observed to excrete feces within 72 hours after administration of the gel film. Feces excreted by each rat between 9-12 am were collected daily, counted, weighed (wet weight), dried (105 ℃, 24 hours) and weighed (dry weight).

Example 9

Safety evaluation of gel films in rats

Male SD rats were randomly divided into two groups: control group (Control) and Gel film group (Gel) 6 of them were administered 250. mu.L of physiological saline and Gel film enema, respectively, 12 hours after fasting but not water-feeding. The body weight of the rats was measured daily during the experiment and the rats were observed for drinking and eating water. On day 7, rats were sacrificed and major organs (heart, liver, spleen, lung, kidney, stomach, small intestine and colon) were collected for histopathological analysis.

Example 10

In vitro anti-inflammatory effect

Anti-inflammatory effects in neutrophils. Intraperitoneal injection of thioglycolate (4mL, 3% wt) into SD rats induced neutrophil production. After 4 hours, the rats were euthanized, the peritoneal cavity was perfused with sterile Hank's balanced salt solution (HBSS, 8mL), the peritoneal lavage fluid was collected, and neutrophils were collected by centrifugation at 400g, 4 ℃ for 10 minutes. Extracting neutrophils at a rate of 1 × 10 per well ⁶ Each of the cells was inoculated into a 12-well plate, and Divertin (250. mu.M), 2. mu.L of a gel film, and 2. mu.L of a gel film coated with Divertin were added to each well, respectively, and then the cells were stimulated with lipopolysaccharide (LPS, 100ng/mL) and cultured in DMEM medium containing 10% FBS for 12 hours. Supernatants from wells were collected and TNF-. alpha.and IL-6 concentrations were determined using an ELISA kit.

Anti-inflammatory effects in macrophages. RAW264.7 cells were plated at 2X 10 per well ⁵ Each was inoculated into a 12-well plate and cultured overnight in DMEM medium containing 10% FBS. Discard old cultureDivertin (250. mu.M), 2. mu.L of gel film loaded with Divertin were added to each well, and then LPS (100ng/mL) was used to stimulate the cells for 12 hours. Supernatants from wells were collected and TNF-. alpha.and IL-6 concentrations were determined using an ELISA kit.

In vitro anti-ROS therapeutic effect. RAW264.7 cells were plated at 1X 10 per well ⁵ One was inoculated into 12-well plates and incubated overnight in DMEM medium containing 10% FBS. Old medium was discarded and cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 100ng/mL) for 2 hours. Divertin (250. mu.M), 2. mu.L of gel film loaded with Divertin were added to each well and incubated for 8 hours. The cells were incubated for 30 minutes with 2 ', 7' -dichlorofluorescein diacetate (DCFH-DA, 10. mu.M) and washed 3 times with ice PBS. And (3) observing the fluorescence distribution in the cells under a laser confocal scanning microscope.

To quantify the intracellular fluorescence intensity, RAW264.7 cells were treated at 1X 10 ⁶ One was inoculated into 12-well plates and incubated overnight in DMEM medium containing 10% FBS. The intracellular fluorescence intensity was analyzed by flow cytometry through the same procedure as described above.

Example 11

Therapeutic effect against macrophage and neutrophil migration in vitro

The gel films were evaluated for their ability to resist neutrophil migration using the Transwell migration assay. Neutrophils in the abdominal cavity of rats were extracted at 2X 10 as described previously ⁵ One was inoculated into the upper chamber of a Transwell, and Divertin (250. mu.M), 2. mu.L of a gel film, and 2. mu.L of a gel film loaded with Divertin were added to the upper chamber, respectively, and incubated for 8 hours. Cells migrating to the lower chamber were observed and counted under an optical microscope.

In a similar manner, the hydrogel was tested for activity against macrophage migration. RAW264.7 cells at 2X 10 ⁵ One was inoculated into the upper chamber of a Transwell, and Divertin (250. mu.M), 2. mu.L of a gel film, and 2. mu.L of a gel film coated with Divertin were added to the upper chamber, respectively, and incubated in a medium containing 20ng of monocyte chemotactic protein (MCP-1) for 12 hours. The old medium was discarded, the cells in the upper chamber were wiped off with a cotton swab, and the cells 10 on the bottom surface of the lower chamber were fixed with 4% Paraformaldehyde (PFA)15 min, 0.1% crystal violet staining of cells for 10 min. Finally, observing under an optical microscope, photographing and counting.

Example 12

Construction and treatment of acute colitis rat model

30 healthy male SD rats (6-8 weeks, 180-. After the rats were fasted but not deprived of water for 12-24 hours, light hemp ether, paraffin oil lubricated polyethylene tube was inserted 8 cm from the anal orifice and 250. mu.L of a mixed solution of ethanol (50%, v/v) and trinitrobenzenesulfonic acid (TNBS, 5%, w/v, 100mg/kg) or an equal volume of 0.9% physiological saline was slowly injected. After 12 hours, the rats in the collitis group with successful induction were randomly divided into 4 groups of 6 rats each, which were: saline control group (PBS), divaretin treatment group (Di, 100mg/kg), Gel film treatment group (Gel), and divaretin-loaded Gel film treatment group (Di @ Gel), 250. mu.L of the above enema was administered intracolonically under light anesthesia with diethyl ether. The body weight of the rats was measured daily, and the fecal status, and the drinking water and diet were observed. After 72 hours, each rat was gavaged with fluorescein isothiocyanate labeled dextran (FD4) with a molecular weight of 4KDa at a dose of 600mg/kg, bled from the orbit after 4 hours, and the rats were sacrificed, fresh feces were collected from the colon, and the colon and major organs (heart, liver, spleen, lung, kidney) were harvested for further testing of anti-inflammatory effects and for pathological analysis.

Example 13

Construction and treatment of chronic colitis rat model

30 healthy male SD rats (6-8 weeks, 180-. The Colitis group received TNBS-50% ethanol enemas weekly (on

days

0, 7, 14, 21, 28, and 35) at TNBS doses of 75, 100, 115, 120, 125, and 125 mg/kg. The Normal group received an equal volume of saline enema weekly. After the fourth enema induction, the rats in the collitis group were randomly divided into 4 groups of 6 rats, a saline control group (PBS), a divardin treatment group (Di, 100mg/kg), a Gel film treatment group (Gel), and a divardin-entrapped Gel film treatment group (Di @ Gel). On

days

30, 36 and 42, 250 μ L of the enema was administered intracolonically under light anesthesia with diethyl ether. During the experiment, the body weight of the rats was measured every other day, and the fecal status and the drinking water status were observed. On day 48, FD4 was gavaged at a dose of 600mg/kg, after 4 hours orbital bleeding was collected, the rats were sacrificed, the colon and major organs (heart, liver, spleen, lung, kidney) were collected and further examined for anti-inflammatory effects and pathological analysis.

Example 14

Construction and treatment of canine acute colitis model

Gastrointestinal endoscopy the in vivo adhesion of the gel films on the colonic mucosa of dogs was evaluated. Healthy females (1 year old, 10-12kg) beagle dogs were fasted without water deprivation for 12 hours and then orally administered 500mL of mannitol solution (containing mannitol 50mg) for 12 hours of bowel preparation. Using a xylazine hydrochloride injection (0.01mg/kg, intramuscular injection) and

[ (teletamine, zolazepam), intramuscular injection]The dogs were anesthetized. 2mL of TSPBA solution mixed with E133 (0.5%, w/v) and TA/SA solution were simultaneously sprayed onto the colonic mucosa of a dog under a gastrointestinal endoscope (FUJINON/EG-590WR) using a two-channel spray tube, followed immediately by repeatedly rinsing and aspirating the inside of the colon lumen to which the gel film was adhered using an endoscope water injection tube. Endoscopy was performed at 0, 6, 12 and 24 hours to record the adhesion of the hydrogel to the colonic mucosal tissue.

14 healthy beagle dogs were subjected to the above-mentioned intestinal preparation in a xylazine hydrochloride injection (0.01mg/kg, intramuscular injection) and

[ (teletamine, zolazepam), intramuscular injection]Under anesthesia, the intestinal lumen is cleaned with warm normal saline. Slowly pushing an endoscope into colon, observing normal colon mucosa, inserting polyethylene hose coated with paraffin oil into colon for 20cm, slowly injecting (5mL/min) ethanol (95% v/v, 20mL) through the hose, and keeping dog at head-tail high position for 5 min; then will beAcetic acid (12% v/v, 8mL) was injected slowly; after 3 minutes, the colon was rinsed with physiological saline (100 mL). 24 hours after induction, endoscopic observations of the inflamed colonic mucosa were again made. Colitis dogs were then divided into saline control (PBS, n-4) and divaritin-loaded Gel film treatment (Di @ Gel, n-6). On

days

1 and 4, 2mL of the enema described above was administered, and the dose of Divertin was 30 mg/kg. During the experiment, the dogs were observed for defecation and diet water on the same day. On day 7, 5mL jugular venous blood was collected, dogs were sacrificed, and the colon and major organs (heart, liver, spleen, lung, kidney) were removed for further testing for anti-inflammatory effects and for pathological analysis.

Example 15

Rat colon Lamina Propria Mononuclear Cells (LPMC) were isolated and flow analyzed

After sacrifice, the colon was removed to remove mesenteric adipose tissue, the colon was opened longitudinally, the feces were cleared, rinsed with ice PBS and cut into small pieces. The intestinal pieces were incubated in digestive juice 1 (HBSS containing 5mM EDTA and 1mM DTT) for 20 minutes at 37 ℃. After filtration through a 100mm cell filter, washed with ice PBS, 10mL of digest 2 (HBSS containing 1.5% FBS, 200U/mL collagenase type 3 and 0.01mg/mL DNase I) was added, and incubated at 37 ℃ for 1 hour. Centrifugation was performed in 40/80Percol gradient at 1,000g for 20 min at 4 ℃, washed 3 times with ice PBS, collected for LPMC extraction and resuspended in PBS containing 10% FBS. The isolated LPMC was labeled with APC-CD11b/c antibody, PE-CD80 antibody and FITC-CD163 antibody to label M1 and M2 macrophages. After PBS washing, cells were analyzed by flow cytometry.

Inflammatory factors, inflammatory mediators and oxidative mediators are detected in colon tissue. Rat and dog colon tissues were homogenized, centrifuged at 10,000 g at 4 ℃ for 15 minutes, and the supernatants were assayed for inflammatory cytokines and oxidative mediator content, including tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10) and Myeloperoxidase (MPO), by ELISA. Expression levels of Malondialdehyde (MDA) in colon tissue were detected using the MDA kit.

And (4) histological analysis. A section of rat and dog colon was fixed in 4% PFA, paraffin embedded, cut into 4mm tissue sections, and stained with hematoxylin and eosin (H & E) and Masson's dye. And finally observing by using an optical microscope.

Example 16

Intestinal flora analysis

Total DNA was extracted from feces using Qiagen MagAttract Power Microbiome DNA kit. The extracted DNA was used as a template to amplify the V4 region of the 16S rRNA gene, and the amplification product was purified and subjected to high-throughput sequencing. These sequences were analyzed by Usearch and QIIME, clustered into Operational Taxa (OTUs) using the UPARSE algorithm, and sorted using the Silva database and the unclust classifier in QIIME. The α -diversity was analyzed by Shannon index (Shannon) and Inverse Simpson (Inverse-Simpson), and the β -diversity was evaluated by principal component analysis (PCoA).

The above examples conclude that the method of the gel scaffold (gel film) of the present invention is simple, and the gel scaffold (gel film) is formed immediately by simply mixing the solution A and the solution B, which is beneficial to clinical transformation; the gel scaffold (gel film) has good biological adhesiveness; the gel scaffold (gel film) has an ROS removing function and has a good anti-inflammatory effect; the gel scaffold (gel film) is self-degradable at the inflammation site; the gel stent (gel film) has good drug-encapsulating effect and good drug slow-release effect.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A gel film with biological adhesiveness is composed of solution A and solution B, and is characterized in that the solution A comprises a substance containing a phenylboronic acid structure; the solution B comprises plant polyphenol and pharmaceutic adjuvant.

2. The gel film according to claim 1, wherein the substance having a phenylboronic acid structure is a polymer having a phenylboronic acid structure or a small molecule compound having a phenylboronic acid structure.

3. The gel film according to claim 2, wherein the polymer having a phenylboronic acid structure has a structure represented by the following formula:

wherein n represents that the polymer has an rereaded unit, and no specific numerical value is provided.

4. The gel film according to claim 3, wherein the polymer having a phenylboronic acid structure has a structure represented by the following formula:

n represents that the polymer has repeating units and has no specific numerical meaning.

5. The gel film of claim 2, wherein the small molecule compound containing a phenylboronic acid structure has a structure represented by the following formula:

wherein R is a structure comprising a secondary, tertiary or quaternary amine.

6. The gel film of claim 5, wherein the small molecule compound containing a phenylboronic acid structure has a structure represented by the following formula:

7. the gel film of claim 1, wherein the plant polyphenol is a natural polyphenol compound or a synthetic polyphenol compound having a structure represented by the following formula:

8. the gel film according to claim 1, wherein the concentration of the substance having a phenylboronic acid structure in the solution A is 1-30 wt%; the concentration of the plant polyphenol in the solution B is 1-30 wt%.

9. Use of a bioadhesive gel film according to any one of claims 1-8 for the preparation of an anti-inflammatory, ROS-scavenging drug or a drug delivery system.

10. An anti-inflammatory, ROS scavenging drug or drug delivery system comprising a bioadhesive gel film according to any one of claims 1 to 8.