Near-infrared light response shape memory antibacterial stent for preventing postoperative pancreatic fistula

Abstract

The invention discloses a near-infrared light response shape memory antibacterial stent for preventing postoperative pancreatic fistula, and relates to the technical field of biomedical organic polymer materials. The preparation method of the shape memory antibacterial scaffold comprises the following steps: purifying caprolactone; pretreating six-arm polyethylene glycol; the purified caprolactone, the pretreated six-arm polyethylene glycol and stannous octoate are used for preparing 6aPEG-PCL; the preparation method comprises the following steps: preparing 6aPEG-PCL-AC from 6aPEG-PCL and potassium carbonate; preparing a polymer film from the 6aPEG-PCL-AC and TPO (Thermoplastic Polyolefin); a polymer film is placed in hot water to be rolled into a pipe shape, fixed in ice water and then placed in a Tris-HCl buffer solution containing dopamine to prepare the shape memory antibacterial stent. The stent disclosed by the invention has the advantages of good biocompatibility, timely degradability, excellent antibacterial property, Young modulus matching and corresponding anti-slip function.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical organic polymer materials, and in particular to a near-infrared light-responsive shape-memory antibacterial stent for preventing postoperative pancreatic fistula. Background Art

[0002] Periampullary tumors refer to benign or malignant tumors that occur in the area around the ampulla of Vater. Tumors in this area can originate from the pancreas, duodenum, lower common bile duct, or ampulla complex structure, usually within 2 cm of the major duodenal papilla. Surgery is the only chance for cure. Pancreaticoduodenectomy is the surgical treatment of choice. Its standard surgical resection includes the removal of the head of the pancreas, the first part of the small intestine, the gallbladder, and the bile duct. The regional anatomy of this procedure is complex and there are considerable risks in the postoperative recovery process. It is one of the most difficult and complex surgical procedures.

[0003] Due to the improvement of surgical instrument performance, the maturity of surgical techniques, and the progress of perioperative management, the postoperative complication rate has been significantly reduced, and the survival rate has been improved. However, the incidence of the most common complication, wound infection (23.5%), and the most dangerous complication, pancreatic leakage (21.4%), remains high. The main causes of postoperative pancreatic fistula are: ① anastomotic exudation and poor anastomotic healing; ② functional or mechanical "unsmoothness" of the anastomosis causes increased pressure in the intestinal cavity; ③ pancreatic enzyme activation corrodes the anastomosis; ④ anastomotic infection. Severe pancreatic fistula can cause abdominal infection, bleeding, gastric emptying disorder, and sepsis. Clinical studies have found that catheter drainage, the preferred interventional treatment for severe pancreatic fistula, has better clinical results and the placement of a support tube with the most appropriate pancreatic duct diameter is the best. The implantation of pancreatic tubular stents can drain pancreatic juice into the distal intestinal cavity or outside the body, avoiding the erosion of the anastomosis by pancreatic juice. At the same time, it also plays an effective supporting role for the pancreaticojejunal anastomosis, avoiding stenosis and obstruction of the pancreatic duct, ensuring the patency and continuity of the pancreatic duct mucosal anastomosis during the anastomosis process, and preventing bile or intestinal fluid from flowing back to the anastomosis to activate pancreatic enzymes, thereby effectively reducing the incidence of pancreatic fistula after pancreaticoduodenal surgery. In addition, pancreatic duct stents also play a positive role in protecting the long-term function of the residual pancreas. The problems of drainage catheters currently used in clinical practice are as follows: ① Most of the pancreatic duct catheters designed for anastomosis of the pancreatic duct and the digestive tract are replaced by silicone tubes used in other surgeries; ② The current tube diameter specifications are single, and the surgical operator needs to temporarily trim them during the operation; ③ The silicone tubes currently used in hospitals have a smooth surface and do not have an anti-dislocation function; ④ They are non-degradable and difficult to be excreted from the body, with potential immunogenicity risks and increased risks of infection and obstruction. Therefore, developing a degradable polymer stent with good antibacterial activity, appropriate degradation time and Young's modulus, good biocompatibility and anti-slip function is of great clinical significance for preventing postoperative infection and pancreatic fistula.

[0004] Polycaprolactone (PCL) is a polymer widely used in biomaterials and sustainable packaging applications, with biocompatibility, biodegradability and higher toughness. PCL is widely used due to its advantages such as wide source of raw materials, biodegradability, sufficient mechanical strength, excellent shape memory properties, and good thermal stability, but its high hydrophobicity and low in vivo degradability make it less suitable for widespread transportation purposes. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula, which has good biocompatibility, timely degradation, excellent antibacterial properties, matching Young's modulus and corresponding anti-slip function.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: a method for preparing a near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula is provided, comprising the following steps: (1) The caprolactone is allowed to stand and dehydrated by calcium hydride, and then distilled under reduced pressure to obtain purified caprolactone; the six-arm polyethylene glycol is dissolved in deionized water, chloroform is added, and the mixture is shaken and extracted, and then allowed to stand and decomposed, and the lower clear liquid is collected, and the extraction is repeated three times, and the mixture is dried over anhydrous sodium sulfate overnight, filtered, and rotary evaporated to a viscous colorless transparent liquid, and then the product is precipitated using frozen anhydrous ether, and the precipitate is collected after suction filtration, and vacuum dried to obtain the pretreated six-arm polyethylene glycol; (2) placing the purified caprolactone obtained in step (1), the pretreated six-arm polyethylene glycol and stannous octoate in a nitrogen environment, evacuating at 40°C for 4 h, then heating to 140°C for reaction for 6 h, adding dichloromethane to dissolve, concentrating by rotary evaporation, and precipitating with ice-cold anhydrous ethanol, filtering, and vacuum drying to obtain 6aPEG-PCL; (3) placing the 6aPEG-PCL and potassium carbonate obtained in step (2) in an ice-water bath, adding dichloromethane to dissolve, then adding a dichloromethane solution of acryloyl chloride dropwise, reacting in an ice-water bath for 1 h, then adjusting the temperature to 25°C to react for 24 h, concentrating by rotary evaporation, precipitating in a frozen ethanol / water mixed solution, filtering, and vacuum drying to obtain 6aPEG-PCL-AC; (4) The 6aPEG-PCL-AC and 2,4,6-trimethylbenzoyl-diphenylphosphine oxide obtained in step (3) were placed in a polytetrafluoroethylene crucible, dichloromethane was added and stirred for 6 h until completely dissolved, and the mixture was placed at room temperature for 12 h to naturally evaporate the solvent, and then placed on a polydimethylsiloxane template, heated to 70°C and evacuated for 30 min, and then covered with another polydimethylsiloxane template to form a film, and irradiated with a 130 W ultraviolet lamp at 365 nm for 1 h to obtain a polymer film; (5) The polymer film obtained in step (4) is placed in 60°C hot water and rolled into a tube, then placed in 0°C ice water for fixation, and then placed in a Tris-HCl buffer solution containing dopamine, stirred for reaction at room temperature for 48 h, washed with deionized water, and dried to obtain a near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula.

[0007] Furthermore, the ratio of six-arm polyethylene glycol to caprolactone was 1:2, and the shape memory transition temperature of the shape memory antibacterial stent was reduced to near the normal physiological temperature of the human body.

[0008] Furthermore, in step (1), the mass ratio of caprolactone to calcium hydride is 10:2; and the mass volume ratio of six-arm polyethylene glycol, deionized water and chloroform is 10 g:20 mL:20 mL.

[0009] Furthermore, in step (1), the mass volume ratio of purified caprolactone, pretreated six-arm polyethylene glycol, stannous octoate and dichloromethane is 10 g:5 g:0.15 g:20 mL.

[0010] Furthermore, in step (3), the mass volume ratio of 6aPEG-PCL, potassium carbonate and dichloromethane is 9 g:1.25 g:20 mL.

[0011] Furthermore, in step (3), in the dichloromethane solution of acryloyl chloride, the mass volume ratio of acryloyl chloride to dichloromethane is 0.815 g:10 mL.

[0012] Furthermore, in step (3), in the ethanol / water mixed solution, the volume ratio of ethanol to water is 9:1.

[0013] Furthermore, in step (4), the mass volume ratio of 6aPEG-PCL-AC, 2,4,6-trimethylbenzoyl-diphenylphosphine oxide and dichloromethane is 1 g:0.04 g:2 mL.

[0014] Furthermore, in step (4), the thickness of the polymer film is 800±50 μm.

[0015] Furthermore, in step (5), the shape memory antibacterial stent has a length of 4-6 cm, an inner diameter of 2.2-3.2 mm, and an outer diameter of 3-4 mm.

[0016] Furthermore, in step (5), the concentration of the Tris-HCl buffer solution is 20 mM, and the concentration of dopamine is 2 mg / mL.

[0017] The present invention also provides a near-infrared light responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula, which is prepared by the preparation method of the near-infrared light responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula.

[0018] The present invention has the following beneficial effects: 1. The near-infrared light-responsive shape-memory antibacterial stent for preventing postoperative pancreatic fistula of the present invention has good biocompatibility, timely degradation, excellent antibacterial performance, matching Young's modulus and corresponding anti-slip function.

[0019] 2. The polymer 6aPEG-PCL is obtained by ring-opening polymerization of caprolactone and six-arm polyethylene glycol (6aPEG) of the present invention, which has biodegradable properties and shape memory effect, and realizes shape memory in vitro, and also realizes shape memory in vivo when implanted in the body.

[0020] 3. The special properties of polydopamine give the stent good antibacterial activity. Its excellent photothermal conversion ability enables the stent to be placed in the main pancreatic duct with the most suitable diameter after pancreaticoduodenectomy, and slowly rebounds and fits the inner wall of the pancreatic duct in response to near-infrared light, thereby preventing slippage and further realizing shape memory in the body. In addition, the rich catechol and secondary amine structures of polydopamine, as well as the hydrogen peroxide formed by PDA, can denature the protein of the cell membrane, destroy the structure of the bacterial cell membrane, and cause bacterial death, thereby achieving the purpose of antibacterial. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the H NMR spectrum of 6aPEG-PCL-AC; Figure 2 is the Fourier transform infrared spectrum of 6aPEG-PCL-AC; Figure 3 The results of cell compatibility experiments. DETAILED DESCRIPTION

[0022] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0023] Example 1 A near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula, the preparation method of which comprises the following steps: (1) 10 g of caprolactone was allowed to stand for 12 h to remove water by 2 g of calcium hydride, and then distilled under reduced pressure to obtain purified caprolactone; 10 g of six-arm polyethylene glycol was dissolved in 20 mL of deionized water, and 20 mL of chloroform was added. After shaking extraction, the mixture was allowed to stand for stratification, and the lower clear liquid was collected. The extraction was repeated three times, and the mixture was dried overnight with 5 g of anhydrous sodium sulfate, filtered, and evaporated to a viscous colorless transparent liquid. The product was then precipitated with frozen anhydrous ether, and the precipitate was collected after suction filtration and vacuum dried to obtain pretreated six-arm polyethylene glycol; (2) 10 g of the purified caprolactone obtained in step (1), 5 g of pretreated six-arm polyethylene glycol and 0.15 g of stannous octoate were placed in a nitrogen environment, vacuumed at 40 °C for 4 h, then heated to 140 °C for 6 h, and then 20 mL of dichloromethane was added to dissolve, concentrated by rotary evaporation, and precipitated with ice-cold anhydrous ethanol, filtered, and vacuum dried to obtain 6aPEG-PCL; (3) 9 g of 6aPEG-PCL obtained in step (2) and 1.25 g of potassium carbonate were placed in an ice-water bath, 20 mL of dichloromethane was added to dissolve, and then a dichloromethane solution of acryloyl chloride (0.815 g: 10 mL) was added dropwise. After the addition was completed, the mixture was reacted in an ice-water bath for 1 h, and then the temperature was adjusted to 25 °C and reacted for 24 h. The mixture was concentrated by rotary evaporation, precipitated in a frozen ethanol / water (volume ratio 9:1) mixed solution, filtered, and vacuum dried to obtain 6aPEG-PCL-AC; (4) 1 g of 6aPEG-PCL-AC obtained in step (3) and 0.04 g of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide were placed in a polytetrafluoroethylene crucible, 2 mL of dichloromethane was added and stirred for 6 h until completely dissolved, and the mixture was placed at room temperature for 12 h to evaporate the solvent naturally, and then placed on a polydimethylsiloxane template, heated to 70 °C and evacuated for 30 min, and then covered with another polydimethylsiloxane template to form a film, and irradiated with a 130 W ultraviolet lamp at 365 nm for 1 h to obtain a polymer film (800 ± 50 μm); (5) The polymer film obtained in step (4) is placed in 60°C hot water and rolled into a tube, then placed in 0°C ice water for fixation, and then placed in a 20 mM Tris-HCl buffer solution containing dopamine (2 mg / mL), stirred for reaction at room temperature for 48 h, washed with deionized water, and dried to obtain a near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula.

[0024] Test Example 1 The nuclear magnetic resonance hydrogen spectrum (1H-NMR) and Fourier transform infrared spectrum (FT-IR) of 6aPEG-PCL-AC in Example 1 were obtained as follows: Dissolve 5 mg of 6a PEG-PCL-AC powder in deuterated chloroform (CDCl3), mix the liquid and inject it into the NMR tube. 1H-NMR nuclear magnetic resonance was used to detect the molecular structure of the test samples to verify whether 6a PEG-PCL-AC was successfully ring-opened and whether the double bonds were successfully connected, and the molecular weight of 6a PEG-PCL-AC was calculated by analyzing the characteristic peak area of ​​the nuclear magnetic spectrum. Because water will interfere with the sample spectrum, the 6a PEG sample, 6a PEG-PCL sample, and 6a PEG-PCL-AC sample were thoroughly dried before the experiment. Take an appropriate amount of the test sample and put it into a clean agate mortar, add KBr particles to the three test samples at a ratio of 1:100, mix them evenly and grind them into fine powder (particle size <2 μm) to avoid the influence of scattered light. Use a hydraulic press to press the ground powder into thin sheets, and then perform infrared spectrum measurement. The results are as follows: Figure 1-2 shown.

[0025] Depend on Figure 1 It can be seen that the chemical shift δ = 4.05 ppm (1) is attributed to the proton peak of -CH2-O- (1) of 6a PEG, and δ = 2.29 ppm (3), 1.63 ppm (4), 1.37 ppm (5), 4.05 ppm (6) are attributed to the proton peaks of -CH2-CH2-CH2-CH2-CH2- (3, 4, 5, 6) in the PCL repeating unit, which indicates that the caprolactone has been successfully ring-opened. The characteristic peak areas of δ = 4.22 ppm (2) and δ = 4.05 ppm (6) are integrated to determine the number of monomer units in each block, and the average number of caprolactone repeating units on each PEG single chain is calculated to be 52.6, and the molecular weight of the PCL segment is 35978. The peaks at δ = 6.12 ppm (7), 5.80 ppm (8), and 6.36 ppm (9) belong to the -CH=CH-C- (7, 8, 9) proton peaks in the propylene group, which verified that acryloyl chloride was successfully grafted onto 6aPEG-PCL.

[0026] Depend on Figure 2 It can be seen that the 1106 cm -1 The -COC- stretching vibration peak at 1732 cm -1 The peak at 1639 cm-1 belongs to the C=O stretching vibration peak in the PCL chain segment, proving that PCL is successfully generated from polycaprolactone in the ring-opening reaction. -1 The peak at 6a belongs to the C=C stretching vibration peak of the propionyl group, indicating that the double bond was successfully connected with acryloyl chloride. The characteristic peaks of the FT-IR images of the three samples indicate that 6a PEG-PCL-AC was successfully synthesized.

[0027] Test Example 2 The cell compatibility of polymer films and polydopamine-coated films with L929 cells was investigated by CCK8 method, and the process was as follows: The film was cut into 6×6×0.1 mm 3 The size of L929 cells was soaked in 75% ethanol and irradiated with UV light for 24 h; they were washed with PBS three times in a sterile operating table, and then soaked in PBS and irradiated with UV light for 24 h. 4 The cells were inoculated into a 12-well plate at a density of 100 cells. After 12 hours of complete attachment of the cells, the prepared film was added for culture at 37°C and 5% CO2. On the 1st, 3rd and 5th days after incubation, the extract was discarded and rinsed with sterile PBS. 200 µL of CCK8 solution diluted with DMEM to 10% was added to each well, and then incubated in the dark at 37°C for 2-4 hours until the solution turned orange-red. After incubation, 100 µL of the supernatant was transferred to a 96-well plate, and its absorbance was measured at 450 nm with an enzyme reader to determine the compatibility of the scaffold. The results are shown in Figure 2. Figure 3 shown.

[0028] Depend on Figure 3 It can be seen that after 1, 3, 5, and 7 days of co-incubation of cells with the material, the cells grew well and the survival rate was above 95%, indicating that the 6a PEG-PCL cross-linked material is safe and non-toxic and has good cell compatibility with L929.

[0029] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula, characterized in that: The following steps are involved: (1) The caprolactone is allowed to stand and dehydrated by calcium hydride, and then distilled under reduced pressure to obtain purified caprolactone; the six-arm polyethylene glycol is dissolved in deionized water, chloroform is added, and the mixture is shaken and extracted, and then allowed to stand and decompose, and the lower clear liquid is collected, and the extraction is repeated three times, and the mixture is dried over anhydrous sodium sulfate overnight, filtered, and rotary evaporated to a viscous colorless transparent liquid, and then the product is precipitated using frozen anhydrous ether, and the precipitate is collected after suction filtration, and vacuum dried to obtain the pretreated six-arm polyethylene glycol; (2) placing the purified caprolactone obtained in step (1), the pretreated six-arm polyethylene glycol and stannous octoate in a nitrogen environment, evacuating at 40°C for 4 h, then heating to 140°C for reaction for 6 h, adding dichloromethane to dissolve, concentrating by rotary evaporation, and precipitating with ice-cold anhydrous ethanol, filtering, and vacuum drying to obtain 6aPEG-PCL; (3) placing the 6aPEG-PCL and potassium carbonate obtained in step (2) in an ice-water bath, adding dichloromethane to dissolve, then adding a dichloromethane solution of acryloyl chloride dropwise, reacting in an ice-water bath for 1 h, then adjusting the temperature to 25°C to react for 24 h, concentrating by rotary evaporation, precipitating in a frozen ethanol / water mixed solution, filtering, and vacuum drying to obtain 6aPEG-PCL-AC; (4) The 6aPEG-PCL-AC and 2,4,6-trimethylbenzoyl-diphenylphosphine oxide obtained in step (3) were placed in a polytetrafluoroethylene crucible, dichloromethane was added and stirred for 6 h until completely dissolved, and the mixture was placed at room temperature for 12 h to naturally evaporate the solvent, and then placed on a polydimethylsiloxane template, heated to 70°C and evacuated for 30 min, and then covered with another polydimethylsiloxane template to form a film, and irradiated with a 130 W ultraviolet lamp at 365 nm for 1 h to obtain a polymer film; (5) The polymer film obtained in step (4) is placed in 60°C hot water and rolled into a tube, then placed in 0°C ice water for fixation, and then placed in a Tris-HCl buffer solution containing dopamine, stirred for reaction at room temperature for 48 h, washed with deionized water, and dried to obtain a near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula.

2. The method for preparing the near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula according to claim 1, characterized in that: In step (1), the mass ratio of caprolactone to calcium hydride is 10:2; the mass volume ratio of six-arm polyethylene glycol, deionized water and chloroform is 10 g:20 mL:20 mL.

3. The method for preparing the near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula according to claim 1, characterized in that: In step (1), the mass volume ratio of purified caprolactone, pretreated six-arm polyethylene glycol, stannous octoate and dichloromethane is 10 g: 5 g: 0.15 g: 20 mL.

4. The method for preparing the near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula according to claim 1, characterized in that: In step (3), the mass volume ratio of 6aPEG-PCL, potassium carbonate and dichloromethane is 9 g:1.25 g:20 mL.

5. The method for preparing the near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula according to claim 1, characterized in that: In step (3), in the dichloromethane solution of acryloyl chloride, the mass volume ratio of acryloyl chloride to dichloromethane is 0.815 g:10 mL.

6. The method for preparing the near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula according to claim 1, characterized in that: In step (3), in the ethanol / water mixed solution, the volume ratio of ethanol to water is 9:

1.

7. The method for preparing the near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula according to claim 1, characterized in that: In step (4), the mass volume ratio of 6aPEG-PCL-AC, 2,4,6-trimethylbenzoyl-diphenylphosphine oxide and dichloromethane is 1 g:0.04 g:2 mL.

8. The method for preparing the near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula according to claim 1, characterized in that: In step (4), the thickness of the polymer film is 800±50 μm.

9. The method for preparing the near-infrared light-responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula according to claim 1, characterized in that: In step (5), the shape memory antibacterial stent has a length of 4-6 cm, an inner diameter of 2.2-3.2 mm, and an outer diameter of 3-4 mm.

10. A near-infrared light responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula, prepared by the method for preparing the near-infrared light responsive shape memory antibacterial stent for preventing postoperative pancreatic fistula according to any one of claims 1 to 9.

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