Medical cannula jacket, medical cannula and preparation method thereof

Abstract

The present application provides a medical cannula jacket, a medical cannula and a preparation method, which belong to the field of medical device technology. The medical cannula jacket is prepared with biocompatible silk protein, so that it maintains a stable structure during the cannula operation, is not easy to deform or damage, and provides good support for the tissue. The use of silk protein can also accelerate the healing of damaged tissues by promoting angiogenesis. The microporous structure on the medical cannula jacket can efficiently store bioactive substances such as viscous protein, realize the slow release of viscous protein, and provide long-term protection and tissue repair functions. Viscous protein has excellent biocompatibility and biological activity, can promote cell adhesion, migration and tissue repair, and enhance the therapeutic effect. The medical cannula jacket is designed to be disposable to avoid cross infection and improve the safety of examination and the therapeutic effect. The medical cannula jacket is used for tissue protection, repair and anti-inflammatory treatment, and is suitable for the inspection of medical cannulas such as bronchoscopes and gastroscopes.

Description

Technical Field

[0001] The invention belongs to the technical field of medical devices and relates to a medical cannula jacket, a medical cannula and a preparation method thereof. Background Art

[0002] Medical cannulas are tubes inserted into the human body through holes on the surface and inside the body to input and withdraw gases or liquids. Common medical cannulas include bronchoscope cannulas and gastroscope cannulas, which are widely used in the examination and treatment of diseases of the respiratory and digestive systems, and are indispensable diagnostic and treatment tools in modern medicine.

[0003] During the use of medical cannulas, mechanical friction between the cannulas and the airway or digestive tract mucosa may cause mucosal damage, inflammatory response, or even bleeding, especially in the case of long-term operation or when the patient's airway or digestive tract is relatively fragile. This mechanical damage not only increases the patient's pain, but may also cause postoperative complications such as infection or scar formation, affecting the treatment effect and patient recovery.

[0004] At present, lubricants or local anesthetics are commonly used clinically to reduce the irritation of the mucosa during intubation examinations, but these methods have limited effects and cannot provide long-term protection or promote tissue repair. In addition, traditional lubricants have a short duration of action and are difficult to cover the entire examination process, and have poor protection effects in complex lesion areas such as tumors or stenosis. Therefore, it is of great significance to develop a medical intubation jacket that can continuously protect the mucosa, reduce damage and promote tissue repair during the examination process. Summary of the invention

[0005] The purpose of the present invention is to provide a medical cannula jacket, a medical cannula and a preparation method to solve the problem that the existing medical cannula is prone to mechanical damage.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present application provides a medical cannula jacket, which is made of biocompatible silk protein, and the surface of the jacket has micropores for efficient storage and sustained release of viscous protein.

[0008] Silk protein is a high-molecular biomaterial extracted from natural silk, which has the characteristics of soft texture and lightness. After purification, the immunogenicity of silk protein is extremely low, and it is not easy to induce immune rejection reaction in the human body, showing good biocompatibility. In addition, silk protein is mainly composed of glycine and alanine, and forms stable fibers through β-folding structure, which gives it high tensile strength and elasticity. These characteristics enable the coat made of silk protein to maintain a stable structure during the intubation operation, not easy to deform or damage, and also provide good support for the tissue. In addition, the coat made of silk protein can also accelerate the healing of damaged tissue by promoting angiogenesis.

[0009] The micropores on the surface of the outer coat have a high specific surface area, controllable pore size and porosity, making them ideal carriers for loading and sustained release of bioactive substances. In the present application, by loading the viscous protein in the microporous structure, the stable storage and slow release of the viscous protein can be achieved, thereby continuously protecting the mucosa during the intubation inspection, reducing mechanical damage, and promoting tissue repair. In the present application, the pore size of the micropores is 10 nm to 200 nm, and the porosity is 40-80%. Preferably, the porosity of the micropores is 60%.

[0010] In the present application, the adhesive protein is selected from one or more of fibronectin, collagen, laminin and elastin. Among them, fibronectin is an important component of the extracellular matrix, has excellent biocompatibility and biological activity, and can promote cell adhesion, migration and tissue repair.

[0011] In addition, the medical cannula jacket in the present application is a disposable item, and the disposable design can avoid cross infection and improve the safety and convenience of clinical operations.

[0012] In a second aspect, the present application provides a method for preparing a medical cannula jacket, the method comprising:

[0013] S01: The soluble silk protein solution and the glutaraldehyde solution are evenly mixed in a volume ratio of 5:1-20:1, poured into a mold, and after freezing, vacuum drying, washing, and drying, a silk protein scaffold is formed.

[0014] Cut the silk cocoons into pieces and boil them in a 0.5% sodium carbonate solution for 30 minutes to remove the sericin in the silk. After boiling, wash with deionized water and dry to obtain purified silk fibers. Dissolve the purified silk fibers in a lithium bromide solution with a molar concentration of 9.3M and stir at 60°C until completely dissolved. Put the dissolved purified silk fibers into a dialysis bag for dialyzed purification to obtain a soluble silk protein solution. In the present application, the concentration of the soluble silk protein solution is 3% w / v-10% w / v; preferably 6% w / v.

[0015] Mix a soluble silk protein solution with a concentration of 3% w / v-10% w / v and a glutaraldehyde solution with a concentration of 0.1% w / v-1% w / v in a volume ratio of 5:1-20:1, and pour it into the outer mold. After freezing at -80°C for 24 hours, use a freeze dryer to vacuum freeze dry for 24 hours at a vacuum degree of 0.1-0.01mbar. After vacuum freeze drying, use deionized water to wash and remove the residual glutaraldehyde solution. Finally, dry at room temperature for 6-8 hours to form a silk protein scaffold with a microporous structure.

[0016] The addition of glutaraldehyde solution with cross-linking effect makes the silk protein in the soluble silk protein solution undergo cross-linking reaction, preventing it from over-shrinking or collapsing during vacuum freeze-drying, and forming silk protein fibers with stable structure. When the cross-linked silk protein fibers are frozen to low temperature, the water molecules in the solution form ice crystals. The growth of ice crystals will squeeze out the silk protein molecules, forming a network structure around the ice crystals. Under vacuum conditions, the ice crystals directly sublimate into water vapor, leaving behind a microporous structure composed of a silk protein network.

[0017] In the present application, the preferred concentration of the glutaraldehyde solution is 0.5% w / v, and the volume ratio of the soluble silk protein solution to the glutaraldehyde solution is preferably 10:1.

[0018] S02: Dissolve the viscous protein in PBS (phosphate buffered saline) to prepare a protein solution with a concentration of 30-70µg / mL. Preferably, the concentration of the protein solution is 50µg / mL.

[0019] S03: After the protein solution is loaded on the surface of the silk fibroin scaffold, the surface is modified with a polydopamine solution to obtain a medical cannula jacket.

[0020] The protein solution is loaded on the surface of the silk fibroin scaffold by vacuum adsorption or spraying, so that the silk fibroin is stored in the micropores. Specifically, when the vacuum adsorption method is adopted, the silk fibroin scaffold is placed in a vacuum container, the protein solution is added and the silk fibroin scaffold is completely immersed. The vacuum pump is started, the vacuum degree is maintained at 0.1-0.01mbar, and the adsorption is 1-2h. The silk fibroin scaffold is taken out, and the excess protein solution on the surface is gently sucked off to form a silk fibroin scaffold with a surface-loaded protein solution. When the spraying method is adopted, the silk fibroin scaffold is fixed on a sterile platform, and the protein solution is evenly sprayed on the surface of the silk fibroin scaffold using a spray gun or a micro-sprayer or other spraying device. After each spraying, it is left to stand for 1-2min to allow the protein solution to penetrate into the interior of the silk fibroin scaffold. Repeat the spraying 3-5 times to ensure that the surface of the silk fibroin scaffold is evenly covered with the protein solution to form a silk fibroin scaffold with a surface-loaded protein solution.

[0021] Dopamine hydrochloride is dissolved in a Tris-HCl buffer solution with a pH of 8.5 and a concentration of 10 mM to prepare a polydopamine solution with a concentration of 1-2 mg / mL. Preferably, the concentration of the polydopamine solution is 1.5 mg / mL.

[0022] After the surface of the silk fibroin scaffold is loaded with protein solution, a polydopamine solution with a concentration of 1-2 mg / mL is modified on the surface of the silk fibroin scaffold by immersion method, oscillation method or vacuum adsorption method to improve the adhesion and stability of the medical cannula jacket to obtain the medical cannula jacket.

[0023] Specifically, when the immersion method is used, the silk fibroin scaffold with the protein solution loaded on the surface is completely immersed in the polydopamine solution and allowed to stand at room temperature or 37°C for 30 minutes to 24 hours. After the standing time is over, the silk fibroin scaffold is taken out with tweezers and gently rinsed with deionized water to remove the unreacted polydopamine solution. The silk fibroin scaffold is placed in an oven at room temperature or 37°C to dry to obtain a medical cannula jacket.

[0024] When the oscillation method is used, the silk fibroin scaffold with the protein solution loaded on the surface is completely immersed in the polydopamine solution and placed in an oscillator. The oscillation is performed at an oscillation speed of 50-150 rpm for 30 min-24 h. After the oscillation is completed, the silk fibroin scaffold is taken out with tweezers and gently rinsed with deionized water to remove the unreacted polydopamine solution. The silk fibroin scaffold is placed in an oven at room temperature or 37°C to dry to obtain a medical cannula jacket.

[0025] When the vacuum adsorption method is used, the silk fibroin scaffold with the protein solution on the surface is completely immersed in the polydopamine solution and placed in a vacuum container. The vacuum pump is started, the vacuum degree is maintained at 0.1-0.01 mbar, and the adsorption is performed for 1-2 hours. The silk fibroin scaffold is taken out with tweezers and gently rinsed with deionized water to remove the unreacted polydopamine solution. The silk fibroin scaffold is placed in an oven at room temperature or 37°C to dry to obtain a medical cannula jacket.

[0026] In a third aspect, the present application provides a medical cannula, comprising a cannula body and a medical cannula jacket attached to the surface of the cannula body.

[0027] In a fourth aspect, the present application provides a method for preparing a medical cannula, the method comprising:

[0028] S01: Apply biocompatible glue on the surface of the medical cannula body, wherein the biocompatible glue can be medical grade epoxy resin glue, medical grade silicone glue or medical grade cyanoacrylate glue.

[0029] S02: attaching the medical cannula jacket to the surface of the medical cannula body and fixing it with biocompatible glue.

[0030] S03: The fixed medical cannula is packaged and sterilized by low-temperature plasma or ethylene oxide to ensure its sterility, thereby obtaining the medical cannula.

[0031] The present invention has the following beneficial effects:

[0032] (1) The medical cannula jacket is made of biocompatible silk protein, which has good biocompatibility, high tensile strength and elasticity, excellent flexibility and adhesion, so that the jacket can maintain a stable structure during the cannulation operation, not easily deformed or damaged, and can also provide good support for the tissue. In addition, the jacket made of silk protein can accelerate the healing of damaged tissue by promoting angiogenesis.

[0033] (2) The medical cannula jacket has a microporous structure that can efficiently store bioactive substances such as adhesive proteins, achieve the slow release of adhesive proteins, and provide long-term protection and tissue repair functions. At the same time, adhesive proteins have excellent biocompatibility and bioactivity, can promote cell adhesion, migration and tissue repair, and enhance the therapeutic effect.

[0034] (3) The disposable design of the medical intubation jacket can avoid cross infection and significantly improve the safety and therapeutic effect of medical intubation examinations such as bronchoscopy and gastroscopy.

[0035] (4) The medical cannula sheath has the functions of mucosal protection, tissue repair and anti-inflammatory treatment, meeting diverse clinical needs.

[0036] (5) The medical cannula cover is used for tissue protection, repair and anti-inflammatory treatment, and is suitable for the examination of medical cannulas such as bronchoscopes and gastroscopy. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a scanning electron microscope image of the medical cannula jacket in the embodiment of the present application;

[0038] Figure 2 This is a graph showing the comparison of the cytotoxicity of the medical cannula jacket and the polyurethane jacket extract on epithelial cells in the embodiments of the present application;

[0039] Figure 3 This is a comparison result of the adsorption effect of the medical cannula jacket and the polyurethane jacket on sticky proteins in the embodiment of the present application;

[0040] Figure 4 This is a graph showing the hemolysis rate comparison between the medical cannula jacket and the polyurethane jacket in the embodiment of the present application;

[0041] Figure 5 This is a graph showing the effects of the medical cannula jacket and the polyurethane jacket on the inflammatory factor IL-1β in rat oral mucosal injury in the examples of this application;

[0042] Figure 6 This is a graph showing the effects of the medical cannula jacket and the polyurethane jacket on the inflammatory factor IL-6 in rat oral mucosal injury in the examples of this application. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0044] Example 1

[0045] The present application embodiment provides a medical cannula jacket, and the preparation method of the medical cannula jacket comprises:

[0046] S101: Cut the silk cocoons into pieces and put them into a 0.5% sodium carbonate solution and boil them for 30 minutes. Wash them with deionized water and dry them to obtain purified silk fibers. Dissolve the purified silk fibers in a lithium bromide solution with a molar concentration of 9.3M and stir them at 60°C until they are completely dissolved. Put the dissolved purified silk fibers into a dialysis bag for dialysis purification to obtain a soluble silk protein solution. Mix the soluble silk protein solution with a concentration of 6% w / v and the glutaraldehyde solution with a concentration of 0.5% w / v in a volume ratio of 10:1 and pour them into the outer mold. After freezing at -80°C for 24 hours, use a freeze dryer to vacuum freeze-dry for 24 hours at a vacuum degree of 0.1mbar. After vacuum freeze-drying, use deionized water to wash and remove the residual glutaraldehyde solution. Finally, dry at room temperature for 6 hours to form a silk protein scaffold with a microporous structure.

[0047] S102: Dissolve fibronectin in PBS to prepare a protein solution with a concentration of 50 µg / mL.

[0048] S103: Place the silk fibroin scaffold in a vacuum container, add the protein solution and completely immerse the silk fibroin scaffold. Start the vacuum pump, maintain the vacuum degree at 0.1 mbar, and adsorb for 1 hour. Take out the silk fibroin scaffold, gently suck off the excess protein solution on the surface, and form a silk fibroin scaffold with a surface-loaded protein solution. Dissolve dopamine hydrochloride in a Tris-HCl buffer with a pH of 8.5 and a concentration of 10 mM to prepare a polydopamine solution with a concentration of 1.5 mg / mL. Completely immerse the silk fibroin scaffold with a surface-loaded protein solution in the polydopamine solution and let it stand at room temperature or 37°C for 24 hours. After standing, take out the silk fibroin scaffold with tweezers, gently rinse with deionized water to remove the unreacted polydopamine solution. Dry the silk fibroin scaffold in an oven at room temperature or 37°C to obtain a medical cannula jacket.

[0049] Example 2

[0050] The present application embodiment provides a medical cannula jacket, and the preparation method of the medical cannula jacket includes:

[0051] S201: Cut the silk cocoons into pieces and put them into a 0.5% sodium carbonate solution and boil them for 30 minutes. Wash them with deionized water and dry them to obtain purified silk fibers. Dissolve the purified silk fibers in a lithium bromide solution with a molar concentration of 9.3M and stir them at 60°C until they are completely dissolved. Put the dissolved purified silk fibers into a dialysis bag for dialysis purification to obtain a soluble silk protein solution. Mix the soluble silk protein solution with a concentration of 3% w / v and the glutaraldehyde solution with a concentration of 0.1% w / v in a volume ratio of 5:1 and pour them into the outer mold. After freezing at -80°C for 24 hours, use a freeze dryer to vacuum freeze dry for 24 hours at a vacuum degree of 0.01mbar. After vacuum freeze drying, use deionized water to wash and remove the residual glutaraldehyde solution. Finally, dry at room temperature for 8 hours to form a silk protein scaffold with a microporous structure.

[0052] S202: Dissolve collagen in PBS to prepare a protein solution with a concentration of 30 µg / mL.

[0053] S203: The silk fibroin scaffold is fixed on a sterile platform, and the protein solution is evenly sprayed on the surface of the silk fibroin scaffold using a spray gun or a micro-sprayer or other spraying device. After each spraying, the scaffold is left to stand for 2 minutes to allow the protein solution to penetrate into the interior of the silk fibroin scaffold. The spraying is repeated 3 times to ensure that the surface of the silk fibroin scaffold is evenly covered with the protein solution to form a silk fibroin scaffold with a surface loaded with the protein solution. Dopamine hydrochloride is dissolved in a Tris-HCl buffer with a pH of 8.5 and a concentration of 10 mM to prepare a polydopamine solution with a concentration of 2 mg / mL. The silk fibroin scaffold with the surface loaded with the protein solution is completely immersed in the polydopamine solution and placed in an oscillator. Oscillate at an oscillation speed of 100 rpm for 30 minutes. After the oscillation is completed, the silk fibroin scaffold is removed with tweezers and gently rinsed with deionized water to remove the unreacted polydopamine solution. The silk fibroin scaffold is placed in an oven at room temperature or 37°C to dry to obtain a medical cannula jacket.

[0054] Example 3

[0055] The present application embodiment provides a medical cannula jacket, and the preparation method of the medical cannula jacket includes:

[0056] S301: Cut the silk cocoons into pieces and put them into a 0.5% sodium carbonate solution and boil them for 30 minutes. Wash them with deionized water and dry them to obtain purified silk fibers. Dissolve the purified silk fibers in a lithium bromide solution with a molar concentration of 9.3M and stir them at 60°C until they are completely dissolved. Put the dissolved purified silk fibers into a dialysis bag for dialysis purification to obtain a soluble silk protein solution. Mix the soluble silk protein solution with a concentration of 10% w / v and the glutaraldehyde solution with a concentration of 1% w / v in a volume ratio of 20:1 and pour them into the outer mold. After freezing at -80°C for 24 hours, use a freeze dryer to vacuum freeze dry for 24 hours at a vacuum degree of 0.05mbar. After vacuum freeze drying, use deionized water to wash and remove the residual glutaraldehyde solution. Finally, dry at room temperature for 7 hours to form a silk protein scaffold with a microporous structure.

[0057] S302: Dissolve laminin in PBS to prepare a protein solution with a concentration of 70 µg / mL.

[0058] S303: Place the silk fibroin scaffold in a vacuum container, add the protein solution and completely immerse the silk fibroin scaffold. Start the vacuum pump, maintain the vacuum degree at 0.01 mbar, and adsorb for 2 hours. Take out the silk fibroin scaffold, gently remove the excess protein solution on the surface, and form a silk fibroin scaffold with a surface loaded with protein solution. Dopamine hydrochloride is dissolved in a Tris-HCl buffer with a pH of 8.5 and a concentration of 10 mM to prepare a polydopamine solution with a concentration of 1 mg / mL. The silk fibroin scaffold with a surface loaded with protein solution is completely immersed in the polydopamine solution and placed in a vacuum container. Start the vacuum pump, maintain the vacuum degree at 0.01 mbar, and adsorb for 2 hours. Take out the silk fibroin scaffold with tweezers, gently rinse with deionized water to remove the unreacted polydopamine solution. Dry the silk fibroin scaffold in an oven at room temperature or 37°C to obtain a medical cannula jacket.

[0059] Example 4

[0060] The present application embodiment provides a medical cannula jacket, and the preparation method of the medical cannula jacket comprises:

[0061] S401: Cut the silk cocoons into pieces and put them into a 0.5% sodium carbonate solution and boil for 30 minutes. Wash them with deionized water and dry them to obtain purified silk fibers. Dissolve the purified silk fibers in a lithium bromide solution with a molar concentration of 9.3M and stir them at 60°C until they are completely dissolved. Put the dissolved purified silk fibers into a dialysis bag for dialysis purification to obtain a soluble silk protein solution. Mix the soluble silk protein solution with a concentration of 7% w / v and the glutaraldehyde solution with a concentration of 0.6% w / v in a volume ratio of 15:1 and pour them into the outer mold. After freezing at -80°C for 24 hours, use a freeze dryer to vacuum freeze dry for 24 hours at a vacuum degree of 0.03mbar. After vacuum freeze drying, use deionized water to wash and remove the residual glutaraldehyde solution. Finally, dry at room temperature for 6 hours to form a silk protein scaffold with a microporous structure.

[0062] S402: Dissolve elastin in PBS to prepare a protein solution with a concentration of 40 µg / mL.

[0063] S403: The silk fibroin scaffold is fixed on a sterile platform, and the protein solution is evenly sprayed on the surface of the silk fibroin scaffold using a spray gun or a micro-sprayer or other spraying device. After each spraying, the scaffold is left to stand for 2 minutes to allow the protein solution to penetrate into the interior of the silk fibroin scaffold. The spraying is repeated 4 times to ensure that the surface of the silk fibroin scaffold is evenly covered with the protein solution to form a silk fibroin scaffold with a surface loaded with the protein solution. Dopamine hydrochloride is dissolved in a Tris-HCl buffer with a pH of 8.5 and a concentration of 10 mM to prepare a polydopamine solution with a concentration of 1.8 mg / mL. The silk fibroin scaffold with the surface loaded with the protein solution is completely immersed in the polydopamine solution and left to stand at room temperature or 37°C for 12 hours. After the standing period, the silk fibroin scaffold is removed with tweezers and gently rinsed with deionized water to remove the unreacted polydopamine solution. The silk fibroin scaffold is placed in an oven at room temperature or 37°C to dry to obtain a medical cannula jacket.

[0064] Example 5

[0065] The present application embodiment provides a medical cannula, and the preparation method of the medical cannula includes:

[0066] S501: Apply medical-grade epoxy resin glue on the surface of the medical cannula body prepared in Example 1.

[0067] S502: attaching the medical cannula jacket to the surface of the medical cannula body and fixing it with medical-grade epoxy resin glue.

[0068] S503: The fixed medical cannula is packaged and sterilized by low-temperature plasma or ethylene oxide to ensure its sterility, thereby obtaining the medical cannula.

[0069] Comparative Example 1

[0070] The comparative example of the present application provides a medical cannula jacket, and the preparation method of the medical cannula jacket comprises:

[0071] D101: Dissolve polyurethane in a mixed solvent of dichloromethane and acetone to prepare a polyurethane solution with a mass concentration of 15%. Use the spin coating method to evenly coat the polyurethane solution on the glass plate, and control the coating thickness to 0.1mm. Place the coated glass plate in a ventilated place to naturally evaporate the solvent to form a uniform polyurethane film.

[0072] D102: Dissolve fibronectin in PBS to prepare a protein solution with a concentration of 40 µg / mL.

[0073] D103: Soak the polyurethane film in the protein solution for 2 hours, take it out and air dry it. Then immerse it in a PEG (English name: Polyethylene glycol; Chinese name: Polyethylene glycol) solution, and use the immersion and pulling method to evenly coat the PEG on the film surface, and dry it naturally at room temperature to form a polyurethane coat.

[0074] Comparative Example 2

[0075] The comparative example of the present application provides a bronchoscope cannula, and the preparation method of the bronchoscope cannula comprises:

[0076] D201: The polyurethane jacket prepared in Comparative Example 1 was cut into a shape suitable for the size of the bronchoscope and fitted onto the surface of the bronchoscope, ensuring that there were no bubbles or wrinkles.

[0077] D202: The bronchoscope is sterilized with ethylene oxide and the bronchoscope cannula is obtained.

[0078] The present invention uses scanning electron microscopy to detect the medical cannula jacket prepared in Example 1, and obtains the attached Figure 1 . Figure 1 It can be seen that the medical cannula jacket prepared in Example 1 of the present application is a fiber structure, and a microporous structure with a pore size of about 10-200 nm exists between the microstructures.

[0079] In the examples of the present application, the medical cannula jacket prepared in Example 1 and the polyurethane jacket prepared in Comparative Example 1 were tested for epithelial cell cytotoxicity, viscous protein adsorption efficiency, hemolysis, and the effects on inflammatory factors IL-1β and IL-6 of rat oral mucosa, and the like, which are described in detail below. Among them, the medical cannula jacket prepared in Example 1 is marked as A, and the polyurethane jacket prepared in Comparative Example 1 is marked as B.

[0080] 1. Cytotoxicity assay for epithelial cells

[0081] Epithelial cell cytotoxicity assays include:

[0082] S01: Epithelial cells cultured to P3-P5 were added to the culture medium and suspended to prepare a cell suspension. 4 The cells were inoculated at a density of 1 / well in a 96-well plate, with 100 μL of cell suspension inoculated in each well, and each sample was repeated 3 times. The inoculated culture plate was placed in an incubator and pre-cultured at 37°C and 5% CO2 for 4 hours.

[0083] S02: After the pre-culture, the culture medium was discarded, and the experimental group was added with culture medium containing different concentrations of the extract of medical cannula jacket A, the control group was added with culture medium containing different concentrations of the extract of polyurethane jacket B, and the blank group was added with culture medium without extract. The experimental group, control group and blank group were placed in an incubator and cultured at 37°C and 5% CO2 for 48 hours.

[0084] S03: After the incubation, 10ul CCK-8 reagent was added to each well and the incubation was continued for 2h. The absorbance value at 450nm wavelength was detected using an ELISA instrument to determine the cell proliferation rate and obtain the attached Figure 2 .

[0085] By the attached Figure 2 It can be seen that during the epithelial cell culture process, the proliferation rate of epithelial cells in the experimental group reached more than 95%, and the apoptosis rate was about 5%. The proliferation rate of epithelial cells in the control group was 60%, and the apoptosis rate was as high as 40%. This shows that the medical cannula jacket prepared in Example 1 of the present application has less cytotoxicity to epithelial cells and can maintain a high cell activity. This shows that the microporous structure on the surface of the medical cannula jacket can efficiently store sticky proteins, protect and repair cells, reduce cell apoptosis, and improve cell survival rate.

[0086] 2. Detection of adhesive protein adsorption efficiency

[0087] The BCA method was used to detect the adsorption rate of sticky protein. The specific process is as follows:

[0088] S01: Prepare BSA (English name: Bovine albumin; Chinese name: bovine serum albumin) standard solutions with concentrations of 0, 10, 20, 50, and 100 µg / mL, and draw a standard curve. Take 50 µL of each concentration of BSA standard solution and add it to a 96-well plate.

[0089] S02: The medical cannula jacket A prepared in Example 1 and the polyurethane jacket B prepared in Comparative Example 1 are immersed in PBS respectively, the adsorbed protein is eluted by shaking, the eluate is collected, and the insoluble impurities are removed by centrifugation.

[0090] S03: Add 50 µL of the eluate to a 96-well plate, add 200 µL of the BSA standard solution to each well, mix well, and incubate at 37°C for 30 min. After incubation, use a microplate reader to measure the absorbance at a wavelength of 562 nm.

[0091] S04: Calculate the protein concentration in the eluent according to the standard curve; calculate the protein adsorption per unit area based on the eluent volume and the surface area of ​​the medical cannula jacket and the traditional protective film, and obtain the protein adsorption capacity per unit area. Figure 3 .

[0092] By the attached Figure 3 It can be seen that the protein adsorption rate of the medical cannula jacket A prepared in Example 1 is as high as over 90%, and the protein adsorption rate of the polyurethane jacket B prepared in Comparative Example 1 is less than 50%. This shows that compared with the polyurethane jacket B prepared in Comparative Example 1, the medical cannula jacket A prepared in Example 1 has the highest adsorption efficiency for sticky proteins. This shows that the microporous structure on the surface of the medical cannula jacket can efficiently store sticky proteins, and more sticky proteins can be loaded by vacuum adsorption or spraying than by short-time immersion.

[0093] 3. Hemolytic test

[0094] Specific methods include:

[0095] S01: Place the collected fresh human blood in a low-speed centrifuge and centrifuge at 2000r / min for 10 minutes. Discard the supernatant and add physiological saline to the precipitate in the centrifuge tube to dilute the precipitate 10 times to prepare a red blood cell suspension.

[0096] S02: Add 1200 μl of normal saline and 300 μl of diluted human blood to the medical cannula jacket A and polyurethane jacket B, respectively, mix gently, and incubate at 37°C for 2 h.

[0097] S03: After the incubation, each mixture was centrifuged at 3000r / min for 10min, and the hemolysis phenomenon was photographed after centrifugation. The supernatant was aspirated and placed on an ELISA analyzer for OD value detection, and the hemolysis rate was calculated to obtain the attached Figure 4 Among them, the positive control was added with distilled water, the negative control was added with normal saline, and the hemolysis rate = (absorbance of the sample - negative control) / (positive control - negative control).

[0098] By the attached Figure 4It can be seen that during the hemolytic experiment, the hemolytic rate of the medical cannula jacket A provided in Example 1 of the present application is about 5%, and the hemolytic rate of the polyurethane jacket B provided in Comparative Example 1 is about 60%. It can be seen that compared with the polyurethane jacket B provided in Comparative Example 1, the hemolytic rate of the medical cannula jacket A provided in Example 1 is lower, and accordingly, the tissue compatibility is better. This shows that the medical cannula jacket prepared using the biopolymer material silk protein has good degradability and tissue compatibility, and at the same time, the microporous structure and the load of sticky protein enhance its tissue adhesion and effectively reduce the hemolytic rate.

[0099] 4. Detection of the effects on inflammatory factors IL-1β and IL-6 in rat oral mucosa

[0100] Twenty-four healthy male SD rats, aged 8-10 weeks and weighing 200-250 g, were selected and kept in a standard laboratory environment with a temperature of 22±2°C and a humidity of 50±10% under a 12-hour light / dark cycle, with free access to water and food. The 24 SD rats were randomly divided into 4 groups, with 6 rats in each group. They were named as follows: normal group A, model group B, medical cannula jacket group C, and polyurethane jacket group D.

[0101] 40 mg / kg sodium pentobarbital was injected into the peritoneal cavity of 24 SD rats to ensure that the rats were fully anesthetized. The anesthetized rats were fixed on the experimental table and the mouth was exposed using an opener. For rats in model group B, medical cannula jacket group C, and polyurethane jacket group D, 50% glacial acetic acid was evenly applied to the oral mucosa of the rats, such as the buccal mucosa or tongue mucosa, with a cotton swab for 30 seconds. After the application time, the residual glacial acetic acid was rinsed with saline to remove the residual glacial acetic acid. For normal group A, an equal amount of saline was applied. For medical cannula jacket group C, the medical cannula jacket was cut to a suitable size and applied to the burned mucosa. For polyurethane jacket group D, the polyurethane jacket was cut to a suitable size and applied to the burned mucosa. All rats were killed 48 hours later, and the oral mucosal tissue of the rats was taken, 1 mL of pre-cooled PBS homogenization buffer containing protease inhibitors was added, and homogenized using an ultrasonic disruptor. The homogenate was centrifuged at 12000 rpm for 15 minutes at 4°C, and the supernatant was collected. The supernatant was used for ELISA-IL-6 and ELISA-IL-1β detection.

[0102] The detection process of ELISA-IL-6 and ELISA-IL-1β is the same. The detection process of ELISA-IL-1β is described in detail below:

[0103] According to the kit instructions, dilute the standard into a series of concentrations, such as 0, 15.6, 31.2, 62.5, 125, 250, 500, 1000 pg / mL. Prepare the washing buffer, chromogenic substrate, and stop solution. In the 96-well plate pre-coated with antibodies, add 100µL of standard and sample to each well, set up 3 replicates for each sample and standard, and add only diluent to the blank control well. After adding the liquid, cover the plate with a sealing film and incubate at 37°C for 1.5 hours. After the incubation, discard the liquid in the well, add 300µL of washing buffer to each well, let it stand for 30 seconds, and then discard it. Repeat 3-5 times. Discard the washing buffer, add 100µL of enzyme-labeled secondary antibody (HRP labeled) to each well, and incubate at 37°C for 1 hour. After the incubation, discard the liquid in the well and wash 3-5 times. Discard the washing buffer and add 100 µL of TMB colorimetric substrate to each well. Incubate at room temperature for 15-30 minutes in the dark. After the incubation, add 50 µL of 2 M H2SO4 to each well and mix gently to terminate the reaction. Use a microplate reader to read the absorbance of each well at a wavelength of 450 nm to obtain the attached Figure 5 , 6 .

[0104] By the attached Figure 5 It can be seen that during the inflammatory factor detection process, the inflammatory factor concentration of IL-1β in the control group A was about 22pg / mL, and the inflammatory factor concentration of IL-1β in the model group B was about 50pg / mL. After the medical cannula jacket group C treated the oral mucosal injury of rats, the inflammatory factor concentration of IL-1β was about 25pg / mL, close to the control group A. After the polyurethane jacket group D treated the oral mucosal injury of rats, the inflammatory factor concentration of IL-1β was about 45pg / mL.

[0105] By the attached Figure 6 It can be seen that during the inflammatory factor detection process, the inflammatory factor concentration of IL-6 in the control group A was about 40pg / mL, and the inflammatory factor concentration of IL-6 in the model group B was about 90pg / mL. After the medical cannula jacket group C treated the oral mucosal injury of rats, the inflammatory factor concentration of IL-6 was about 45pg / mL, close to the control group A. After the polyurethane jacket group D treated the oral mucosal injury of rats, the inflammatory factor concentration of IL-6 was about 80pg / mL.

[0106] It can be seen that compared with the polyurethane jacket provided in Comparative Example 1, the medical cannula jacket provided in Example 1 can effectively improve the increased levels of IL-1β and IL-6 inflammatory factors caused by oral mucosal damage in rats, reduce inflammation, repair damaged mucosa, and provide long-term protective effect on tissues.

[0107] In summary, the medical cannula jacket provided in the embodiments of the present application adopts biocompatible materials, constructs a microporous structure, loads sticky proteins and performs surface modification, which can not only provide long-term protection, but also promote tissue repair and anti-inflammatory treatment, enhance cell activity, reduce hemolysis rate, and significantly improve the safety and therapeutic effect of intubation examinations such as bronchoscopy and gastroscopy.

[0108] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A medical cannula jacket, characterized in that: The outer coat is made of silk protein, and the surface of the outer coat has micropores for storing and slowly releasing sticky protein; The preparation method of the medical cannula jacket comprises: The soluble silk protein solution and the glutaraldehyde solution are mixed evenly in a volume ratio of 5:1-20:1, poured into a mold, and after freezing, vacuum drying, washing, and drying, a silk protein scaffold is formed; Dissolve the sticky protein in PBS to prepare a protein solution with a concentration of 30-70µg / mL; After the protein solution is loaded on the surface of the silk fibroin scaffold, the surface is modified with a polydopamine solution to obtain a medical cannula jacket.

2. The medical cannula jacket according to claim 1, characterized in that: The micropores have a pore size of 10-200 nm and a porosity of 40-80%.

3. The medical cannula jacket according to claim 1, characterized in that: The preparation method of the soluble silk protein solution comprises: The chopped silk cocoons were placed in a 0.5% sodium carbonate solution and boiled for 30 minutes, washed and dried to obtain purified silk fibers; The purified silk fibers are dissolved in a lithium bromide solution, stirred at 60° C. until completely dissolved, and purified by dialysis to obtain a soluble silk protein solution.

4. The medical cannula jacket according to claim 1, characterized in that: The concentration of the soluble silk protein solution is 3% w / v-10% w / v, and the concentration of the glutaraldehyde solution is 0.1% w / v-1% w / v.

5. The medical cannula cover according to claim 1, characterized in that: The adhesive protein includes one or more of fibronectin, collagen, laminin and elastin.

6. The medical cannula cover according to claim 1, characterized in that: The protein solution loading method includes vacuum adsorption method or spray method.

7. The medical cannula cover according to claim 1, characterized in that: The surface modification method of the polydopamine solution includes immersion method, oscillation method or vacuum adsorption method.

8. A medical cannula, characterized in that: The invention comprises a cannula body and the medical cannula cover according to any one of claims 1 to 7 which is attached to the surface of the cannula body.

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