Preparation method of anti-adhesion, pro-repair and antibacterial composite patch

Abstract

The application discloses a preparation method of an anti-adhesion, repair-promoting and antibacterial composite patch. L-lactide and glycolide are used as monomers to synthesize four-arm polylactic acid-glycolic acid 4arm-PLGA through ring-opening polymerization; then, L-lysine and 1,2-propylene glycol are used to prepare hydrophilic BLPD through esterification reaction, and the 4arm-PLGA is copolymerized with the 4arm-PLGA to obtain a block copolymer 4arm-PLGA-BLPD; subsequently, the proportion of PCL, the 4arm-PLGA-BLPD, the 4arm-PLGA and an antibacterial drug is regulated, and the composite patch is prepared through continuous electrospinning technology. The composite patch has good biosafety, antibacterial property, anti-adhesion between the patch and human organs and repair-promoting property, and has a wide application prospect in surgical operations, especially in hernia repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a method for preparing an anti-adhesion, repair-promoting, and antibacterial composite patch. Background Technology

[0002] Abdominal wall hernias are common clinical conditions caused by the protrusion of internal organs through a weak abdominal wall or missing fascia. Currently, surgical treatment for abdominal wall hernias typically involves using synthetic, non-degradable mesh made of polypropylene. However, due to the inherent properties of polypropylene, over time after implantation, the mesh can severely adhere to the organs, causing not only severe foreign body sensation but also chronic pain, intestinal obstruction, and other complications, necessitating a second surgery.

[0003] Based on the tissue engineering regeneration therapy strategy, this invention designs and develops a patch material with a clearly defined composition using fully biodegradable polymer materials and amino acid molecules. Through a simple and easy-to-implement electrospinning technique, a composite patch with good biocompatibility, anti-adhesion properties, and the ability to promote autologous tissue regeneration and repair is obtained, thereby improving the postoperative life of patients and having broad application value. Summary of the Invention

[0004] The purpose of this invention is to provide a composite patch that is biocompatible, anti-adhesive, and can promote autologous tissue regeneration and repair, in order to solve the problems of existing polypropylene patches that are non-degradable, have a hard feel, and are prone to excessive adhesion.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: An anti-adhesion, repair-promoting, and antibacterial composite patch includes a repair-promoting layer, a support layer, and an antibacterial layer, wherein the support layer is disposed between the repair-promoting layer and the antibacterial layer; The composite patch is prepared using the following steps: Step 1: Mix L-lactide and glycolide, then add stannous isooctanoate catalyst and pentaerythritol initiator, and react under vacuum at 150~180 ℃ for 8~12 h. After cooling, dissolving and precipitating, the reaction product is dried to obtain four-armed polylactic acid-glycolic acid 4arm-PLGA. Step 2: Dissolve p-toluenesulfonic acid and 1,2-propanediol by heating, then add lysine and toluene and mix. Separate the mixture using a Dianstar apparatus with water reflux at a temperature of 100-120 °C for 6-8 h. Cool and let stand at room temperature, then remove toluene by rotary evaporation to obtain sulfonated lysine. Step 3: Add isopropanol to the product from Step 2, reflux at 110°C until the product dissolves, cool the product to room temperature and recrystallize it in a -20°C refrigerator. Repeat the recrystallization step three times, and then dry the product in a vacuum drying oven at 30°C for 8-12 hours to obtain polylysine p-toluenesulfonate BLPD. Step 4: Dissolve 4arm-PLGA in dichloromethane, then add triethylamine and seal in an ice-water bath. Dissolve triphosgene in dichloromethane to prepare a triphosgene solution with a concentration of 0.02 g / mL, and add it dropwise to 4arm-PLGA at a rate of 1 mL / h to obtain a 4arm-PLGA-Cl solution. Dissolve BLPD in pyridine solution, then add it dropwise to the 4arm-PLGA-Cl solution. Reflux at 60 °C under a nitrogen atmosphere for 12 h. After cooling and precipitation, the reaction product is dried to obtain 4arm-PLGA-BLPD. Step 5: Dissolve 4arm-PLGA-BLPD and polycaprolactone in hexafluoroisopropanol to prepare a polymer mixture stock solution A with a mass fraction of 10-20%. Add polymer mixture stock solution A to an electrospinning machine for electrospinning. The spinning voltage is 10-20 kV, the spinning distance is 10-15 cm, and the spinning solution propulsion speed is 0.2-2 mL / h to obtain a functional layer that promotes repair. Step 6: Dissolve 4arm-PLGA and polycaprolactone in hexafluoroisopropanol to prepare a polymer mixture stock solution B with a mass fraction of 10-20%. Add polymer mixture stock solution B to an electrospinning machine for electrospinning. The spinning voltage is 10-20 kV, the spinning distance is 10-15 cm, and the spinning solution feed speed is 0.2-2 mL / h. Based on step 5, a support layer is obtained. Step 7: Dissolve the antibacterial drug, 4arm-PLGA, and polycaprolactone in hexafluoroisopropanol to prepare a polymer mixture stock solution C with a mass fraction of 10-20%. Add the polymer mixture stock solution C to an electrospinning machine for electrospinning. The spinning voltage is 10-20 kV, the spinning distance is 10-15 cm, and the spinning solution feed speed is 0.2-2 mL / h. Based on Step 6, an antibacterial layer is obtained. Step 8: Vacuum dry the multilayer scaffold at room temperature to remove residual organic solvents from the scaffold, and sterilize it with ethylene oxide or irradiation to obtain a composite patch with antibacterial, anti-adhesion and repair-promoting properties.

[0006] In step one, the molar ratio of L-lactide to glycolide is 1:0.1~9, the molar ratio of L-lactide to catalyst stannous isooctanoate is 400~3000:1, and the molar ratio of L-lactide to initiator pentaerythritol is 50~600:1.

[0007] In step two, the mass ratio of toluenesulfonic acid to 1,2-propanediol is 1.1~1.5:1; the molar ratio of lysine to p-toluenesulfonic acid is 1:1.1~1.5; and the volume ratio of 1,2-propanediol to toluene is 1:5.

[0008] In step four, the mass ratio of 4arm-PLGA to dichloromethane is 1:5, the mass ratio of 4arm-PLGA to triethylamine is 6000~6500:1, the mass ratio of 4arm-PLGA to BLPD is 20:7, the mass ratio of BLPD to pyridine is 7:20, and the volume ratio of triphosgene solution to pyridine is 1:2.

[0009] In step five, the mass ratio of 4arm-PLGA-BLPD to polycaprolactone is 1:1.

[0010] In step six, the mass ratio of 4arm-PLGA to polycaprolactone is 1:1.

[0011] In step seven, the antibacterial drug is emodin, with a purity of HPLC grade and above 90%. The ratio of the mass of the antibacterial drug to the sum of the masses of 4arm-PLGA and polycaprolactone is 1:10~20. Beneficial effects

[0012] 1. Lightweight: A high specific area nanofiber membrane is obtained through electrospinning, and this membrane does not cause a change in the weight of the patch; 2. Flexibility: Compared with polypropylene, the electrospun membrane formed by mixing PLGA and PCL in a four-arm structure has high strength and high flexibility, which is beneficial for wound coverage; 3. Biocompatibility: PLGA and PCL are FDA-approved biodegradable materials that can be absorbed by the human body; 4. Anti-adhesion: The high specific surface area and high porosity of the nanofiber membrane enable it to adhere moderately to the tissue, which avoids tissue damage caused by adhesion and helps to ensure the support of the patch for the gap. 5. Promotes repair: By adding L-lysine, it accelerates tissue repair. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the composite patch of the present invention.

[0014] Figure 2 This is a SEM image of the repair-promoting layer of the composite patch in Example 1.

[0015] Figure 3 This is a SEM image of the antibacterial layer of the composite patch in Example 1.

[0016] Figure 4The results are the cell viability test results after co-incubating the 4arm-PLGA-BLPD material extract with L929 cells (mouse fibroblasts) for 24 h and 48 h in Example 1.

[0017] Figure 5 The results of the composite patch in Example 1 against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) are shown.

[0018] Figure 6 The results of the composite patch in Example 1 were used in a rat model of abdominal wall defects. Implementation

[0019] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0022] 1. 7.2 g L-lactide (LA) and 1.3 g glycolide (GA) were added to a two-necked flask, along with 0.0027 g of stannous isooctanoate catalyst (Sn(Oct)2) and 0.18 g of pentaerythritol (PET) initiator. The mixture was evacuated for 5 min. The flask was sealed and placed in an oven at 160 °C for 8 h, shaking once every 2 h. After the reaction was complete, the vacuum was maintained and the mixture was cooled to room temperature. The product was dissolved in dichloromethane, and the solution was slowly added to a large amount of methanol to precipitate the product. The collected product was dried in a vacuum drying oven at room temperature for 24 h to obtain four-armed polylactic-glycolic acid (4arm-PLGA).

[0023] 2. Dissolve 6.276 g of p-toluenesulfonic acid and 6 mL of 1,2-propanediol by ultrasonic heating. Then add 4.3857 g of L-lysine and 20 mL of toluene. After mixing thoroughly, reflux the solution using a Dianstar apparatus at 100–120 °C for 8 hours. After cooling to room temperature, remove the toluene solution using a rotary evaporator to obtain sulfonated lysine.

[0024] 3. Add an appropriate amount of isopropanol to the sulfonated lysine and reflux at 110 °C until the product dissolves. After cooling the product to room temperature, recrystallize it in a -20 °C refrigerator. Repeat this step three times, and then dry the product in a vacuum drying oven at 30 °C for 8-12 h to obtain polylysine p-toluenesulfonate (BLPD).

[0025] 4. Dissolve 10 g of 4arm-PLGA in 20 mL of dichloromethane, then add 165 μL of triethylamine, and seal the apparatus in an ice-water bath. Dissolve 0.2 g of triphosgene in dichloromethane to prepare a triphosgene solution with a concentration of 0.02 g / mL, and add 5 mL dropwise to the flask at a rate of 1 mL / h to obtain a 4arm-PLGA-Cl solution. Dissolve 3.5 g of BLPD in 10 mL of pyridine solution, then add it dropwise to the obtained 4arm-PLGA-Cl solution and mix. Then transfer the solution to a nitrogen atmosphere at 60 °C and reflux for 12 h. After the reaction cools to room temperature, add the product to a large amount of methanol solution to obtain a white flocculent product. Dry the product in a vacuum drying oven at 50 °C for 12 h to obtain 4arm-PLGA-BLPD.

[0026] 5. Dissolve 1g of 4arm-PLGA-BLPD and 1g of polycaprolactone (PCL) in hexafluoroisopropanol to prepare a 15% (w / w) 4arm-PLGA-BLPD / PCL mixture. Add the mixture to an electrospinning machine for electrospinning. The spinning voltage is 15kV, the spinning distance is 15cm, and the propulsion speed of the spinning solution is 0.2mL / h to obtain a functional layer that promotes repair.

[0027] 6. Dissolve 1g of 4arm-PLGA and 1g of PCL in hexafluoroisopropanol to prepare a 15% (w / w) 4arm-PLGA-BLPD / PCL mixture. Add the mixture to an electrospinning machine for electrospinning. The spinning voltage is 15 kV, the spinning distance is 15 cm, and the propulsion speed of the spinning solution is 0.2 mL / h. The support layer is obtained based on step 5.

[0028] 7. Dissolve 200 mg of emodin, 1 g of 4arm-PLGA and 1 g of PCL in hexafluoroisopropanol to prepare a 15% (w / w) 4arm-PLGA-BLPD / PCL mixture. Add the mixture to an electrospinning machine for electrospinning. The spinning voltage is 15 kV, the spinning distance is 15 cm, and the spinning solution advance speed is 0.1 mL / h. Based on step 6, an antibacterial layer is obtained.

[0029] 8. Vacuum drying of multilayer scaffolds at room temperature to remove residual organic solvents in the scaffolds, followed by sterilization by ultraviolet irradiation; resulting in a composite patch with antibacterial, anti-adhesion, and repair-promoting properties.

[0030] like Figure 3 As shown in the SEM image of the antibacterial layer, the rhomboid-shaped substance is the antibacterial drug emodin.

[0031] L929 cells (mouse fibroblasts) were seeded into 96-well plates (1 × 10⁶ cells per well). 4 Cells were incubated in DMEM medium for 24 hours. 4arm-PLGA and 4arm-PLGA-BLPD were co-incubated for 24 hours to obtain extracts. Cells were then co-incubated with the 4arm-PLGA and 4arm-PLGA-BLPD extracts at 37 ± 0.5 ºC for 24 and 48 hours, respectively. Cell absorbance at 450 nm was measured using a microplate reader. Figure 4 As shown, 4arm-PLGA-BLPD can promote the proliferation of L929 cells.

[0032] In vitro antibacterial assays were performed using the colony forming unit (CFU) method. Specifically, samples were sliced ​​into 1-1 cm sections. 2 (N=5) size, exposed to a concentration of 10 5 The ATCC bacterial strain was administered in 5 mL of culture medium containing CFU / mL. Subsequently, each bacterial suspension was diluted 100 µL in a sterile petri dish and mixed with 15 mL of LB agar medium (containing 2% agar). The petri dishes were then incubated at 37 ± 2 °C for 24 hours. Colony counting was performed in triplicate, and colony area was measured using Image J. The antibacterial efficacy of the composite patch against *Escherichia coli* (Gram-negative) and *Staphylococcus aureus* (Gram-positive) was tested. Figure 5 As shown, the composite patch has a wide range of antibacterial effects, especially against Staphylococcus aureus.

[0033] Using rats as an experimental model, a rat model of abdominal wall defect was created. Commercially available polypropylene (PP) and composite patches were placed and fixed to the defect site, and the repair process was observed at fixed time points. Figure 6 As shown, the large intestine of untreated rats flowed out from the incision and formed severe adhesions with the abdominal wall; commercially available polypropylene (PP) also showed severe adhesions with the abdominal wall; while the composite patch prepared above showed good repair of the abdominal wall defect and no adhesions were observed.

[0034] As shown in the figure, this material can be prepared into patch material through electrospinning technology, which has broad application prospects in abdominal wall repair and other fields.

Claims

1. A composite patch for preventing adhesion, promoting repair, and inhibiting bacteria, characterized in that: It includes a repair-promoting layer, a support layer, and an antibacterial layer, wherein the support layer is disposed between the repair-promoting layer and the antibacterial layer; The composite patch is prepared using the following steps: Step 1: Mix L-lactide and glycolide, then add stannous isooctanoate catalyst and pentaerythritol initiator, and react under vacuum at 150~180 ℃ for 8~12 h. After cooling, dissolving and precipitating, the reaction product is dried to obtain four-armed polylactic acid-glycolic acid 4arm-PLGA. The molar ratio of L-lactide to glycolide is 1:0.1~9, the molar ratio of L-lactide to catalyst stannous isooctanoate is 400~3000:1, and the molar ratio of L-lactide to initiator pentaerythritol is 50~600:

1. Step 2: Dissolve p-toluenesulfonic acid and 1,2-propanediol by heating, then add lysine and toluene and mix. Separate the mixture using a Dianstar apparatus with water reflux at a temperature of 100-120 °C for 6-8 h. Cool and let stand at room temperature, then remove toluene by rotary evaporation to obtain sulfonated lysine. Step 3: Add isopropanol to the product from Step 2, reflux at 110°C until the product dissolves, cool the product to room temperature and recrystallize it in a -20°C refrigerator. Repeat the recrystallization step three times, and then dry the product in a vacuum drying oven at 30°C for 8-12 hours to obtain polylysine p-toluenesulfonate BLPD. Step 4: Dissolve 4arm-PLGA in dichloromethane, then add triethylamine and seal in an ice-water bath. Dissolve triphosgene in dichloromethane to prepare a triphosgene solution with a concentration of 0.02 g / mL, and add it dropwise to 4arm-PLGA at a rate of 1 mL / h to obtain a 4arm-PLGA-Cl solution. Dissolve BLPD in pyridine solution, then add it dropwise to the 4arm-PLGA-Cl solution. Reflux at 60 °C under a nitrogen atmosphere for 12 h. After cooling and precipitation, the reaction product is dried to obtain 4arm-PLGA-BLPD. Step 5: Dissolve 4arm-PLGA-BLPD and polycaprolactone in hexafluoroisopropanol to prepare a polymer mixture stock solution A with a mass fraction of 10-20%. Add polymer mixture stock solution A to an electrospinning machine for electrospinning. The spinning voltage is 10-20 kV, the spinning distance is 10-15 cm, and the spinning solution propulsion speed is 0.2-2 mL / h to obtain a functional layer that promotes repair. The mass ratio of 4arm-PLGA-BLPD to polycaprolactone is 1:1; Step 6: Dissolve 4arm-PLGA and polycaprolactone in hexafluoroisopropanol to prepare a polymer mixture stock solution B with a mass fraction of 10-20%. Add polymer mixture stock solution B to an electrospinning machine for electrospinning. The spinning voltage is 10-20 kV, the spinning distance is 10-15 cm, and the spinning solution feed speed is 0.2-2 mL / h. A support layer is formed on the functional layer in step 5. The mass ratio of 4arm-PLGA to polycaprolactone is 1:1; Step 7: Dissolve the antibacterial drug, 4arm-PLGA, and polycaprolactone in hexafluoroisopropanol to prepare a polymer mixture stock solution C with a mass fraction of 10-20%. Add the polymer mixture stock solution C to an electrospinning machine for electrospinning. The spinning voltage is 10-20 kV, the spinning distance is 10-15 cm, and the spinning solution feed speed is 0.2-2 mL / h. An antibacterial layer is formed on the support layer of Step 6. The antibacterial drug is emodin, and the ratio of the mass of the antibacterial drug to the sum of the masses of 4arm-PLGA and polycaprolactone is 1:10~20. Step 8: Vacuum dry the multilayer scaffold at room temperature, and sterilize it to obtain a composite patch.

2. The anti-adhesion, repair-promoting, and antibacterial composite patch according to claim 1, characterized in that, In step two, the mass ratio of toluenesulfonic acid to 1,2-propanediol is 1.1~1.5:1; the molar ratio of lysine to p-toluenesulfonic acid is 1:1.1~1.5; and the volume ratio of 1,2-propanediol to toluene is 1:

5.

3. The anti-adhesion, repair-promoting, and antibacterial composite patch according to claim 1, characterized in that, In step four, the mass ratio of 4arm-PLGA to dichloromethane is 1:5, the mass ratio of 4arm-PLGA to triethylamine is 6000~6500:1, the mass ratio of 4arm-PLGA to BLPD is 20:7, the mass ratio of BLPD to pyridine is 7:20, and the volume ratio of triphosgene solution to pyridine is 1:2.

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