Glucose responsive fluorescent molecule modified intestinal serosa patch and application thereof in early warning of intestinal fistula

Abstract

The invention discloses an intestinal serous membrane patch modified with glucose responsive fluorescent molecules and application of the intestinal serous membrane patch to early warning of intestinal fistula, the intestinal serous membrane patch comprises an electrospinning fiber membrane and the fluorescent molecules modified on the electrospinning fiber membrane, the fiber membrane is obtained by processing a polyvinyl alcohol solution through an electrostatic spinning technology, and the fluorescent molecules are fluorescein isothiocyanate modified with aminophenylboronic acid. The fluorescent molecules and the polyvinyl alcohol fiber membrane are connected through boric acid ester bonds, and the boric acid ester bonds have glucose response characteristics. The patch is used for covering the serous membrane side of the intestinal tract, when intestinal fistula occurs, intestinal contents containing glucose are in contact with the patch, and along with breakage of boric acid ester bonds, fluorescent molecules are released into an enteric cavity and detected in excrement, so that early warning of the intestinal fistula is realized. The intestinal serosa patch provided by the invention realizes early warning of intestinal fistula based on glucose response characteristics of boric acid ester bonds and a fluorescence detection technology, and has the advantages of strong specificity, sensitive response and convenient operation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical material engineering, and more specifically, relates to an intestinal serosal patch modified with glucose-responsive fluorescent molecules and its application in intestinal fistula early warning. Background Art

[0002] Intestinal fistula is a common and extremely dangerous postoperative complication after intestinal anastomosis surgery. It occurs when the anastomosis fails to heal or reopens due to factors such as ischemia, infection, high tension, and digestive fluid stimulation, leading to leakage of intestinal contents into the abdominal cavity and causing infection or systemic sepsis. Due to the rapid progression and extremely serious consequences of intestinal fistulas, early diagnosis is particularly important. Patent application number CN201980065647.3 provides a pharmaceutical composition containing a therapeutically effective amount of a self-assembling peptide for the treatment or prevention of intestinal ulcers or fistulas. Patent application number CN201210400991.3 provides methods and compositions for treating fistulas using adipose tissue-derived stromal stem cells. However, the aforementioned patented technologies can only be applied to treat intestinal fistulas after they have occurred, and currently, there is a lack of effective strategies for early diagnosis of intestinal fistulas in clinical practice. Although some biomarkers, such as elevated procalcitonin, are considered to indicate the development of intestinal fistulas, elevated procalcitonin can occur in a variety of diseases, including infection, inflammation, and tumors, and is therefore not specific for the diagnosis of intestinal fistulas. Furthermore, intestinal fistulas are insidious in their early stages, with atypical clinical manifestations, making them difficult to detect. Once patients experience symptoms or signs such as abdominal pain and muscle tension, it often indicates that intestinal contents have leaked into the abdominal cavity, making treatment difficult and associated with a high mortality rate. Therefore, the development of a highly specific, sensitive, and operable early detection system for intestinal fistulas is urgent. Summary of the Invention

[0003] (1) Technical problems solved

[0004] Against the above background, the present invention provides an intestinal serosal patch modified with glucose-responsive fluorescent molecules for achieving early warning of intestinal fistula. The patch comprises a fibrous membrane base and a fluorescent molecule modified thereon. The fluorescent molecule and the fibrous membrane are connected by a boronate bond. The boronate bond has glucose-responsive properties. The patch is covered on the intestinal serosal side and fits perfectly. When intestinal fistula occurs, intestinal contents containing glucose will come into contact with the patch. As the boronate bond breaks, the fluorescent molecule will be released into the intestinal cavity and detected in the excreta, thereby achieving intestinal fistula warning. This solves the current problem of the lack of effective intestinal fistula warning or early detection methods.

[0005] (2) Technical solution

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A intestinal serosal patch comprises a fiber membrane substrate and fluorescent molecules modified thereon; the fiber membrane is obtained by processing a polyvinyl alcohol solution using an electrospinning process; the fluorescent molecule is isothiocyanate fluorescein modified with aminophenylboronic acid; the fluorescent molecule and the polyvinyl alcohol fiber membrane are connected via a borate ester bond.

[0008] Preferably, the fiber membrane has a fiber diameter of 0.2 μm-5 μm.

[0009] Preferably, the fiber membrane is in the shape of a rectangle.

[0010] Preferably, the length of the fiber membrane is 4-8 cm.

[0011] Preferably, the width of the fiber membrane is 1-3 cm.

[0012] Preferably, the fiber membrane has a thickness of 50 μm-500 μm.

[0013] Preferably, the material of the fiber membrane includes but is not limited to polyvinyl alcohol.

[0014] Preferably, the main body of the fluorescent molecule includes but is not limited to fluorescein isothiocyanate.

[0015] In another aspect, the present invention provides a method for preparing the above-mentioned intestinal serosal patch, comprising the following steps:

[0016] S1: Synthesis of fluorescent molecules: 4-aminophenylboronic acid and fluorescein isothiocyanate were dissolved in methanol solution and reacted in the dark. The product was separated and purified by silica gel chromatography column and finally subjected to reduced pressure distillation to obtain fluorescein isothiocyanate modified with 4-aminophenylboronic acid.

[0017] S2: Modification of fluorescent molecules in polyvinyl alcohol: Add the fluorescein isothiocyanate modified with 4-aminophenylboronic acid obtained in step S1 to a polyvinyl alcohol aqueous solution, adjust the pH to alkaline using sodium hydroxide solution, heat in a water bath and react while stirring to obtain a polyvinyl alcohol solution modified with a fluorescent molecule.

[0018] S3: The solution obtained in step S2 is allowed to stand at room temperature to defoam, and is spun using electrospinning technology to obtain an intestinal serosal patch.

[0019] Preferably, in step S1, the 4-aminophenylboronic acid and fluorescein isothiocyanate are dissolved in a methanol solution, the temperature of the light-proof reaction is 25-50°C, the reaction time is 12-48 h, the concentration of the 4-aminophenylboronic acid in the methanol solution is 1-10 mmol / L, the concentration of the fluorescein isothiocyanate in the methanol solution is 1-10 mmol / L, and the developing solvent used for separation and purification by silica gel chromatography comprises dichloromethane and ethanol.

[0020] Preferably, in step S2, the pH of the fluorescent molecule modified in polyvinyl alcohol is adjusted to 9-12 using sodium hydroxide, and the concentration of the fluorescein isothiocyanate modified with 4-aminophenylboronic acid is 1-10 mg / mL.

[0021] Preferably, in step S3, the voltage of the electrospinning is 12 kV-25 kV, the flow rate of the electrospinning is 0.5 mL / h-2 mL / h, and the diameter of the electrospinning nozzle is 15 G-23 G.

[0022] Another aspect of the present invention provides the use of the intestinal serosa patch in early warning or early diagnosis of intestinal fistula.

[0023] The present invention also provides a method for using the intestinal serosal patch in early warning or early diagnosis of intestinal fistula, which specifically comprises the following steps:

[0024] S1: Place the patch on the outside (serosa) of the intestinal anastomosis, with the side modified with fluorescent molecules facing the intestinal tissue.

[0025] S2: Collect postoperative fecal matter and perform fluorescence detection at the excitation wavelength. If fluorescence is detected in the fecal matter, it indicates the occurrence of intestinal fistula.

[0026] (3) Beneficial effects

[0027] The beneficial effects produced by the present invention are:

[0028] (1) The intestinal serosa patch prepared by the present invention is used for the detection of intestinal fistula and has a sensitive early warning. The intestinal serosa patch designed by the present invention has a base of a polyvinyl alcohol fiber membrane, and the fluorescent molecule modified thereon is aminophenylboronic acid functionalized isothiocyanate fluorescein. The fiber membrane and the fluorescent molecule are connected by a boronate ester bond. The boronate ester bond formed between aminophenylboronic acid and polyvinyl alcohol is essentially a reversible covalent bond. In solution, glucose mainly exists in a cyclic structure, and its C1 and C2 cis-vicinal diol configuration has a much stronger binding ability with borate than the random vicinal diol in polyvinyl alcohol. Under the influence of thermodynamic drive and steric hindrance, glucose tends to replace borate and bind to polyethylene glycol, thereby releasing aminophenylboronic acid functionalized isothiocyanate fluorescein. After the fluorescent molecule is released into the intestinal cavity, it will be discharged from the stool with the peristalsis of the intestine, thereby being used for early warning of intestinal fistula.

[0029] (2) The intestinal serosal patch prepared by the present invention is used for the detection of intestinal fistula, and the method is convenient. In order to detect whether there is fluorescence in the excreta, it is only necessary to use excitation light of a specific wavelength for irradiation and read it after passing through a filter of a specific wavelength, without the need for expensive or bulky equipment. It is suitable for bedside detection and implementation in primary hospitals. In addition, the patient specimen used in this detection method is excreta, which is convenient and non-invasive, and does not cause secondary harm to the patient.

[0030] (3) The intestinal serosa patch prepared by the present invention is used for the detection of intestinal fistula and has a leak-proof function. The intestinal serosa patch designed by the present invention has a polyvinyl alcohol fiber membrane as its base, which has excellent mechanical properties. The fiber membrane is made of many nanometer-scale diameter fibers interwoven together. On the one hand, it is soft and can fit perfectly with the intestinal tissue. On the other hand, it has strong tensile strength and can withstand large tissue tension. When intestinal fistula occurs, the fiber membrane can prevent intestinal contents from leaking into the intestinal cavity and causing fatal peritonitis or shock, which is of great significance for blocking the progression of the disease and saving the patient's life.

[0031] (4) The intestinal serosa patch prepared by the present invention is used for the detection of intestinal fistulas and is biodegradable, safe and absorbable. The intestinal serosa patch designed by the present invention uses materials including polyvinyl alcohol, fluorescein isothiocyanate, and aminophenylboronic acid, all of which are biosafe and biodegradable materials. The intestinal serosa patch designed by the present invention has good biocompatibility and controllable degradation properties, avoiding the long-term foreign body reaction that may be caused by traditional materials, and does not require secondary surgery for removal. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0033] Figure 1 : Schematic diagram of the structure of the intestinal serosal patch, where: 1-fiber membrane, 2-fluorescent molecules.

[0034] Figure 2 : H NMR spectrum of fluorescein isothiocyanate functionalized with aminophenylboronic acid.

[0035] Figure 3Preparation and characterization of the intestinal serosal patch. A: Reaction equation for the formation of a boronate bond between aminophenylboronic acid-functionalized fluorescein isothiocyanate and polyvinyl alcohol; B: Image of the intestinal serosal patch modified with a glucose-responsive fluorescent molecule; Scale: 2 cm; C: Thickness of the intestinal serosal patch measured using a micrometer; D: Scanning electron micrograph of the intestinal serosal patch; E: Fluorescence micrograph of the intestinal serosal patch.

[0036] Figure 4 Schematic diagram of the application of the intestinal serosa patch in early warning of intestinal fistula. A: Establishment of an intestinal fistula animal model and application of the intestinal serosa patch; B: Detection of fluorescence signals in feces. Left: Intestinal fistula model group; Right: Sham operation group. DETAILED DESCRIPTION

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0038] Example 1: Preparation of an intestinal serosa patch modified with a glucose-responsive fluorescent molecule for intestinal fistula detection

[0039] The present invention designs an intestinal serosa patch modified with glucose-responsive fluorescent molecules for intestinal fistula early warning. The intestinal serosa patch is used for intestinal fistula early warning, has the characteristics of sensitive detection and convenient operation, and also has a leak-proof function. The intestinal serosa patch is biodegradable, safe and absorbable. Figure 1 As shown, the intestinal serosa patch includes an electrospun fiber membrane 1 and a fluorescent molecule 2 modified thereon. The fiber membrane 1 is processed using an electrospinning process from a polyvinyl alcohol solution. The fluorescent molecule 2 is isothiocyanate fluorescein modified with aminophenylboronic acid. The fiber membrane 1 and the fluorescent molecule 2 are connected by a boronate ester bond. The boronate ester bond has glucose-responsive properties. When an intestinal fistula occurs, intestinal contents containing glucose will come into contact with the patch. As the boronate ester bond breaks, the fluorescent molecule will be released into the intestinal cavity and detected in the feces, thus providing an early warning of intestinal fistula.

[0040] A method for preparing an intestinal serosa patch modified with a glucose-responsive fluorescent molecule comprises the following steps:

[0041] S1. Synthesis of fluorescent molecules: 4-aminophenylboronic acid and fluorescein isothiocyanate were dissolved in methanol solution and reacted in the dark. The product was separated and purified by silica gel chromatography column and finally subjected to reduced pressure distillation to obtain fluorescein isothiocyanate modified with 4-aminophenylboronic acid.

[0042] In some embodiments, the concentration of the 4-aminophenylboronic acid in the methanol solution is 1-10 mmol / L;

[0043] In some embodiments, the concentration of the fluorescein isothiocyanate in the methanol solution is 1-10 mmol / L;

[0044] In some embodiments, the 4-aminophenylboronic acid and fluorescein isothiocyanate are dissolved in a methanol solution, and the reaction temperature is 25-50° C. in the dark.

[0045] In some embodiments, the 4-aminophenylboronic acid and fluorescein isothiocyanate are dissolved in a methanol solution, and the reaction time in the dark is 12-48 h.

[0046] In some embodiments, the developing solvent used for separation and purification on a silica gel chromatography column comprises dichloromethane and ethanol.

[0047] S2, modification of fluorescent molecules in polyvinyl alcohol: polyvinyl alcohol is dissolved in a mixed solvent of deionized water and N,N-dimethylformamide, the isothiocyanate fluorescein modified with 4-aminophenylboronic acid obtained in step S1 is added to the polyvinyl alcohol aqueous solution, the pH is adjusted to alkaline using sodium hydroxide solution, and the reaction is carried out to obtain a polyvinyl alcohol solution modified with a fluorescent molecule.

[0048] In some embodiments, the concentration of fluorescein isothiocyanate modified with 4-aminophenylboronic acid is 1-10 mg / mL;

[0049] In some embodiments, the mass fraction of the polyvinyl alcohol solution is 6-15 wt%;

[0050] In some embodiments, the volume fraction of N,N-dimethylformamide in the mixed solvent of deionized water and N,N-dimethylformamide is 30%-70%;

[0051] In some embodiments, sodium hydroxide solution is used to adjust the pH to 9-12;

[0052] In some embodiments, the water bath is heated at a temperature of 30-50°C;

[0053] In some embodiments, the reaction time of heating in a water bath and stirring is 1-8 h.

[0054] S3, the solution obtained in step S2 is allowed to stand at room temperature to defoam, and is spun using electrospinning technology to obtain an intestinal serosal patch.

[0055] In some embodiments, the defoaming time is 2-14 hours;

[0056] In some embodiments, the electrospinning voltage is 12 kV-25 kV;

[0057] In some embodiments, the electrospinning flow rate is 0.5 mL / h-2 mL / h;

[0058] In some embodiments, the diameter of the electrospinning nozzle is 15 G-23 G;

[0059] In some embodiments, the distance between the electrospinning nozzle and the collecting roller is 8 cm to 15 cm;

[0060] In some embodiments, the rotation speed of the electrospinning collection roller is 100 r / min- 300 r / min;

[0061] In some embodiments, the thickness of the polyvinyl alcohol fiber membrane is 0.08 mm-0.12 mm;

[0062] In some embodiments, the fiber diameter of the polyvinyl alcohol fiber membrane is 0.2 m-2 μm.

[0063] The intestinal serosa patch is used to early-warning intestinal fistulas. The patch is applied to the outer serosa of the intestinal anastomosis, with the side modified with fluorescent molecules facing the intestinal tissue. Postoperative fecal matter is collected and excited for fluorescence at an excitation wavelength, then detected through a filter with a specific wavelength. Detection of fluorescence in the fecal matter indicates the development of an intestinal fistula.

[0064] In some embodiments, the excitation wavelength is 475-500 nm;

[0065] In some embodiments, the filter wavelength is 520-550 nm.

[0066] The following is the specific implementation process:

[0067] 1. Synthesis of fluorescent molecules

[0068] 4-Aminophenylboronic acid (29 mg, 0.21 mmol) and fluorescein isothiocyanate (80 mg, 0.21 mmol) were dissolved in 50 mL of methanol and reacted in the dark at 30 °C for 24 h. The reaction mixture was then concentrated and distilled under reduced pressure. The product was then separated and purified by silica gel column chromatography (dichloromethane / methanol 6:1) and finally distilled under reduced pressure to obtain aminophenylboronic acid-functionalized fluorescein isothiocyanate powder.

[0069] The powder was dissolved in dimethyl sulfoxide and the H NMR spectrum of the product was obtained by nuclear magnetic resonance. Figure 2 As shown in Figure 5, a broad peak appeared near 10.0 ppm, corresponding to the protons of the thiourea group (-NH-CS-NH-), proving the occurrence of the reaction.

[0070] 2. Modification of fluorescent molecules in polyvinyl alcohol

[0071] Dissolve 10 g of polyvinyl alcohol in a mixture of 90 g of deionized water and N,N-dimethylformamide (40% by volume). Add 10 mg of fluorescein isothiocyanate modified with 4-aminophenylboronic acid (from Step 1) to 10 mL of the polyvinyl alcohol solution. Adjust the pH to 10 with sodium hydroxide solution. Heat in a 40°C water bath with stirring for 6 h to obtain a fluorescent molecule-modified polyvinyl alcohol solution.

[0072] 3. Electrospinning to prepare intestinal serosa patches

[0073] The solution obtained in step 2 was allowed to stand at room temperature for 6 h to defoam. 6 mL of spinning solution was extracted and electrospun under a high voltage electric field of 24 kV. The spinning flow rate was 0.5 mL / h, the diameter of the spinning nozzle was 23 G, the distance between the nozzle and the collecting roller was 10 cm, and the speed of the collecting roller was 300 r / min. Figure 3 As shown, the prepared polyvinyl alcohol fiber membrane modified with aminophenylboronic acid-fluorescein isothiocyanate exhibits uniform texture. The thickness of the fiber membrane was measured using a vernier caliper and was found to be approximately 0.7 mm. Scanning electron microscopy (SEM) observations of the fiber membrane's micromorphology revealed that it was composed of nanofibers of uniform diameter, arranged in a crisscross pattern. Fluorescence microscopy observations of the fluorescence distribution of the fiber membrane revealed a strong, uniformly distributed fluorescence signal across the fibers.

[0074] Example 2: Application of intestinal serosa patch in early warning of intestinal fistula

[0075] like Figure 4 As shown, an intestinal serosa patch is used for early warning of intestinal fistulas. An intestinal fistula model was first established through laparotomy: 10-12 week-old male Sprague-Dawley rats were anesthetized with 20% urethane, followed by routine hair removal, disinfection, and sterile surgical draping. A 4-5 cm midline incision was made, and the abdominal cavity was opened layer by layer. The small intestine was carefully retracted and protected with gauze moistened with sterile saline. A circular incision approximately 5 mm in diameter was made along the contralateral mesenteric edge of the intestine to establish an intestinal fistula model. An intestinal serosa patch modified with a glucose-responsive fluorescent molecule was placed over the fistula surface and secured with a horizontal mattress suture. The intestine was carefully reinserted, and the peritoneal cavity was flushed with sterile saline. After ensuring hemostasis, the abdominal cavity was closed layer by layer. In the sham surgery group, no circular incision was made, and all other procedures were the same. On the third postoperative day, feces were collected, and fluorescence was observed using a small animal imaging device. The experiment confirmed that fluorescent signals were detected in the excreta of the intestinal fistula model group, while no fluorescent signals were detected in the excreta of the sham operation group, proving that the intestinal serosal patch modified with glucose-responsive fluorescent molecules can achieve early warning of intestinal fistula.

[0076] In summary, the present invention provides an intestinal serosal patch modified with a glucose-responsive fluorescent molecule for realizing early warning of intestinal fistula. The patch comprises a fiber membrane base and a fluorescent molecule modified thereon. The fluorescent molecule and the fiber membrane are connected by a boronate bond. The boronate bond has glucose-responsive properties. The patch is covered on the intestinal serosal side and fits perfectly. When intestinal fistula occurs, intestinal contents containing glucose will come into contact with the patch. As the boronate bond breaks, the fluorescent molecule will be released into the intestinal cavity and detected in the excreta, thereby realizing intestinal fistula warning. Solve the problem of the current lack of effective intestinal fistula warning or early detection means.

[0077] Finally, it should be noted that the above embodiments are intended only to illustrate the present invention and are not intended to limit the scope of protection of the present invention. In addition, after reading the technical contents of the present invention, those skilled in the art may make various changes, modifications, or variations to the present invention, and all such equivalent forms also fall within the scope of protection defined by the present application.

Claims

1. An intestinal serosal patch, characterized in that: The invention comprises a fiber membrane (1) and a fluorescent molecule (2) modified on the fiber membrane (1); the fiber membrane (1) comprises a polyvinyl alcohol fiber membrane, and the polyvinyl alcohol fiber membrane is processed by an electrospinning process; the fluorescent molecule (2) comprises isothiocyanate fluorescein modified with aminophenylboronic acid; the fiber membrane (1) and the fluorescent molecule (2) are connected via a boronate bond, and the boronate bond has glucose response characteristics.

2. The intestinal serosal patch according to claim 1, characterized in that The fiber diameter of the fiber membrane (1) is 0.2 μm-5 μm.

3. The intestinal serosa patch according to claim 1, wherein The thickness of the fiber membrane (1) is 50 μm-500 μm.

4. The intestinal serosal patch according to claim 1, wherein The material of the fiber membrane (1) also includes a spinnable high molecular polymer.

5. The intestinal serosa patch according to claim 1, wherein The main body of the fluorescent molecule (2) also includes a fluorescent molecule with biological safety.

6. The method for preparing an intestinal serosa patch according to claim 1, characterized in that: The following steps are involved: S1: Synthesis of fluorescent molecules: 4-aminophenylboronic acid and fluorescein isothiocyanate were dissolved in methanol solution and reacted in the dark. The product was separated and purified by silica gel chromatography and finally distilled under reduced pressure to obtain fluorescein isothiocyanate modified with 4-aminophenylboronic acid. S2: Modification of fluorescent molecules in polyvinyl alcohol: adding the fluorescein isothiocyanate modified with 4-aminophenylboronic acid obtained in step S1 to a polyvinyl alcohol aqueous solution, adjusting the pH to alkaline using sodium hydroxide solution, heating in a water bath and reacting while stirring to obtain a polyvinyl alcohol solution modified with a fluorescent molecule; S3: The solution obtained in step S2 is allowed to stand at room temperature to defoam, and is spun using electrospinning technology to obtain an intestinal serosal patch.

7. The method for preparing an intestinal serosa patch according to claim 6, wherein: In step S1, the 4-aminophenylboronic acid and fluorescein isothiocyanate are dissolved in a methanol solution, the light-proof reaction temperature is 25-50° C., the reaction time is 12-48 h, the concentration of the 4-aminophenylboronic acid in the methanol solution is 1-10 mmol / L, the concentration of the fluorescein isothiocyanate in the methanol solution is 1-10 mmol / L, and the developing solvent used for separation and purification by silica gel chromatography contains dichloromethane and ethanol.

8. The method for preparing an intestinal serosa patch according to claim 6, wherein: In step S2, the pH of the fluorescent molecule modified in polyvinyl alcohol is adjusted to 9-12 using sodium hydroxide, and the concentration of the fluorescein isothiocyanate modified with 4-aminophenylboronic acid is 1-10 mg / mL.

9. The method for preparing an intestinal serosa patch according to claim 6, wherein: In the step S3, the voltage of the electrospinning is 12 kV-25 kV, the flow rate of the electrospinning is 0.5 mL / h-2 mL / h, and the diameter of the electrospinning nozzle is 15 G-23 G.

10. Use of the intestinal serosal patch according to any one of claims 1 to 5 in early warning or early diagnosis of intestinal fistula.

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