CN117695447A

CN117695447A - Tissue repair patch with multilayer structure and preparation method and application thereof

Info

Publication number: CN117695447A
Application number: CN202311727416.9A
Authority: CN
Inventors: 高长有; 张丽丽; 董晓飞; 刘小伟; 聂兴成
Original assignee: Shaoxing Research Institute Of Zhejiang University; Zhejiang University ZJU
Current assignee: Shaoxing Research Institute Of Zhejiang University; Zhejiang University ZJU
Priority date: 2023-12-15
Filing date: 2023-12-15
Publication date: 2024-03-15

Abstract

The invention relates to the field of functional composite materials, in particular to a tissue repair patch with a multilayer structure, and a preparation method and application thereof. The patch is a polymer patch with self-adhesive property, antibacterial property, postoperative adhesion prevention and biocompatibility, and is a multilayer structure formed by alternately arranging a tissue adhesion layer and an anti-adhesion layer, wherein the multilayer structure comprises at least 4 layers, the anti-adhesion structure layer comprises a high molecular polymer with film forming property, and the tissue adhesion layer comprises a poly (N-vinyl pyrrolidone-acrylic acid-N-succinimidyl ester) copolymer, an antibacterial agent or a cross-linking agent and a plasticizer; the patch of the invention is particularly suitable for rapid adhesion and sealing of soft tissues in vivo and can be used as an auxiliary means for connecting, sealing, reinforcing and healing wounds in vivo.

Description

Tissue repair patch with multilayer structure and preparation method and application thereof

Technical Field

The invention belongs to the field of functional composite materials, and particularly relates to a tissue repair patch with a multilayer structure, and a preparation method and application thereof. The patch is a polymer patch with self-adhesive property, antibacterial property, postoperative adhesion prevention and biocompatibility, is particularly suitable for rapid adhesion and sealing of in-vivo soft tissues, and can be used as an auxiliary means for in-vivo wound connection, sealing, reinforcement and healing.

Background

Tens of millions of people each year suffer from different types of tissue damage including surgical wounds, burns, perforations, ulcers, and the like. Can effectively seal various wounds in time, can achieve the effects of stopping bleeding, preventing the leakage of contents, providing antibacterial barriers and the like, and finally promoting the healing of the wounds. Conventional wound closure typically uses sutures, staples, hemostatic clips, and the like. However, these methods are not applicable to densely distributed organs and tissues such as liver, lung, dura, etc. In addition, the use of sutures, staples, and like instruments can cause additional tissue damage, increasing the risk of bacterial infection. In addition, suturing is not ideal and does not control the tissue response during the healing process, resulting in closed leaks, such as cerebral spinal fluid leakage after cranium and spinal surgery, etc.

The tissue repair patch may be used as an adhesive tape to adhesively close a wound without penetrating the skin, forming a completely sealed protective barrier on the wound surface, reducing the risk of leakage and infection, helping to heal the wound and providing a better aesthetic effect. Compared with the stitching nails and the stitching lines, the tissue repair patch is quick and convenient to use, can be used for closing wounds in emergency situations, and can be easily removed and replaced when needed. In addition, the tissue repair patch can also carry antibacterial, anti-inflammatory or therapeutic drugs, and can be slowly released to a wound part to improve the curative effect, so that better wound closure effect is finally achieved, the time and difficulty of wound treatment are reduced, the effect of reducing surgical infection and complications is achieved, and obvious benefits are provided for patients and doctors.

However, to date, most commercial tissue repair patches have been limited to in vitro applications due to limitations in material properties and biocompatibility. For tissue patches used in vivo, few are clinically approved, and the performance is far from ideal, and the defects of weak adhesive capability, poor mechanical performance, easy rupture and debonding and the like exist, so that the tissue patches are usually used in combination with sutures and staples, and more of the tissue patches are still in a preclinical research stage. Mechanical properties of tissue, such as elasticity, rigidity, and hardness, vary from tissue type to tissue type. The conformability of an adhesive material to tissue is largely dependent on its ability to match the mechanical properties of the underlying layer of tissue over a period of time. The main challenge at present is to achieve a sufficient and stable adhesion to different soft tissues in a dynamic humid environment in the body.

In addition to strong and stable adhesion to soft tissues, it is also desirable to resist adhesion to other tissues to avoid post-operative organ adhesions. Postoperative organ adhesion is a serious surgical complication. Inflammation, tissue damage, or abnormal mechanical stimulation during healing, which may occur during surgery, may induce fibrosis of fibroblasts, resulting in post-operative tissue adhesions. Postoperative organ adhesions can occur at any site where surgery is performed, including the abdomen, pelvis, joints, heart, etc. Its symptoms and severity vary from patient to patient and from type of surgery, pain and organ dysfunction are often induced, as intestinal adhesions can lead to ileus, joint adhesions can lead to joint movement limitation, etc., and death can occur in severe cases. In order to reduce the risk of post-operative organ adhesions, measures are required to minimize tissue trauma during surgery or to use biological barriers or lubricants to reduce the risk of adhesions.

In addition, postoperative infection is also one of the common and serious complications after surgery. Postoperative infections may cause a series of symptoms such as redness, pain, fever, increased secretions, difficult wound healing, etc. The severity of the infection varies from individual to individual, type of surgery, and type of bacteria. In some cases, the postoperative infection may be severe, spread to surrounding tissues or enter the blood circulation, causing systemic infection, and life threatening. Thus, bacterial infections after surgery need to be treated and prevented seriously. In clinic, sterile procedures, prevention of use of antibiotics, and periodic monitoring of wounds are often employed to reduce the risk of infection. The tissue repair patch with antibacterial and anti-infection capabilities can further reduce the risk of postoperative infection and better promote wound healing.

Based on the above, the invention provides a brand new tissue repair patch, which is helpful for improving the treatment effect and widening the application scene.

Disclosure of Invention

Aiming at the defects of the prior art, the invention discloses an antibacterial and postoperative adhesion-preventing tissue repair patch with a multilayer structure. The patch is composed of alternating tissue adhering and anti-adhering faces. The tissue adhesive surface has stable and excellent tissue adhesive performance, can be effectively adhered to tissues to form a layer of airtight barrier, provides additional mechanical support and protection, enhances the stability of an operation area, supports the regeneration of damaged tissues and promotes the repair of tissue injury. The back surface is a smooth anti-adhesion surface, which can prevent adhesion of proteins, fibroblasts and the like and avoid postoperative tissue adhesion. The tissue patch is added with the hyperbranched polylysine which is safe and nontoxic and has good biocompatibility and broad-spectrum antibacterial agent. The antibacterial patch is mutually coupled with a tissue repair patch substrate through covalent bonds, so that exudation and loss of medicines are avoided, and the antibacterial effect is strong and stable. Structurally, the patch of the invention is composed of at least 4 layers of alternating tissue adhesion surface and anti-adhesion surface, and has excellent mechanical property and adhesion property, uniformity and stability.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention synthesizes a biological adhesive polymer: poly (N-vinylpyrrolidone-acrylic acid-N-succinimidyl acrylate). Which is prepared by free radical polymerization of the monomers N-vinylpyrrolidone, acrylic acid and N-succinimidyl acrylate. Wherein the pyrrolidone pendant groups contained therein can provide excellent structural mechanical properties to the substrate and adhesion properties to tissue surfaces through hydrogen bonding or van der waals forces. Wherein the carboxyl groups contained therein can form hydrogen bonds with the tissue surface to adhere to each other. At the same time, the carboxyl is also a reaction functional group for the cross-linking of the base material and the cross-linking agent and the cross-coupling of the antibacterial agent hyperbranched polylysine. The pendant succinimidyl ester groups contained therein can interact with amino groups on the tissue surface to form amide covalent bonds and thereby provide strong and stable adhesion.

The preparation method of the polymer comprises the following steps:

dimethyl sulfoxide (DMSO) was heated to 80 ℃ in an oil bath while oxygen-free nitrogen was bubbled through the solvent to remove dissolved oxygen. After adding N-vinylpyrrolidone (NVP), acrylic acid (AAc) and acrylic acid-N-succinimidyl ester (AAc-NHS) in a ratio to DMSO, the initiator Azoisobutyronitrile (AIBN) is added. The reaction was carried out under nitrogen atmosphere for 18h. Wherein the molar ratio of N-vinylpyrrolidone, acrylic acid and N-succinimidyl acrylate can be adjusted to obtain tissue patches of different mechanical and adhesive properties. Wherein, the molar ratio of the N-vinyl pyrrolidone monomer is preferably 20% -80%, more preferably 30% -70%, and most preferably 40% -60%; the molar ratio of the acrylic acid monomer is preferably 10% to 50%, more preferably 15% to 40%, most preferably 20% to 30%; the molar ratio of the acrylic acid-N-succinimidyl ester monomer is preferably 10% to 50%, more preferably 15% to 40%, and most preferably 20% to 30%. The amount of the initiator azoisobutyronitrile is generally not less than 0.2% by mass of the monomers.

After the reaction was completed, it was cooled to room temperature, and isopropyl alcohol (IPA) was added to precipitate a white polymer, which was isolated by filtration. The resulting polymer was washed several times in isopropanol to remove unpolymerized reaction monomers and initiator, followed by drying and purification of the polymer to obtain a tissue-adhesive polymer, i.e. poly (N-vinylpyrrolidone-acrylic acid-N-succinimidyl ester). The molecular weight is generally about 30000. The polymer may be self-crosslinking at elevated temperatures or crosslinked by a crosslinking agent, in the form of a tough gel.

The anti-adhesion layer of the tissue patches referred to in the present invention may also be used as a structural laminate for adjusting the mechanical properties of the patch, such as stiffness and toughness. In a preferred embodiment, the patch is comprised of alternating anti-adhesion and bioadhesive layers, which is better than a patch with a single structural layer.

The anti-adhesion layer according to the present invention is prepared from polylactic-co-glycolic acid (PLGA). Wherein the polymerization ratio of lactic acid and glycolic acid is preferably 0.2 to 0.8, more preferably 0.4 to 0.6, and most preferably 0.5. Which is generally biocompatible and degradable, the degradation rate being related to its molecular weight. In order to match the regeneration time of the tissue in the body, the molecular weight thereof is preferably 50000 to 400000, more preferably 100000 ～ 300000, most preferably 150000 ～ 250000. The complete degradation of PLGA of this molecular weight in vivo takes about 50 days.

The hyperbranched polylysine (HBPL) of the antibacterial agent contains a plurality of terminal amino groups, is positively charged after being partially protonated in the solution, can destroy the biological membranes of bacteria and fungi to inhibit the growth of the bacteria and fungi, and simultaneously, the unprotonated amino groups can be coupled with carboxyl groups on the tissue adhesive polymer to play a role of a base material crosslinking agent. The amount of HBPL added to the patch is preferably 0.1% to 1%, more preferably 0.2% to 0.8%, and most preferably 0.4% to 0.6%.

The properties of the tissue patches may be optimized by the addition of other cross-linking agents and plasticizers. Hydroxyl-containing substances such as glycerol, sucrose, hydroxypropyl cellulose, and low molecular weight polyethylene glycol (PEG) can be coupled by condensation of hydroxyl groups with carboxyl groups on the substrate to adjust the flexibility or elasticity of the patch. The preferred plasticizer in the present invention is a low molecular weight polyethylene glycol, preferably having a molecular weight of less than 1000, more preferably less than 600, and even more preferably less than 400. The addition amount thereof is preferably 0 to 50%, more preferably 10 to 40%, most preferably 20 to 30%. In addition, fibroin (silk fibroin) has good biocompatibility, flexibility, air and moisture permeability and tensile strength, and is an ideal plasticizer for tissue patches to improve the mechanical properties of the tissue patches. The amount of silk fibroin added in the present invention is preferably 0 to 40%, more preferably 10 to 30%, most preferably 15 to 25%.

The patch according to the present invention can be prepared by referring to the following modes:

first, two solutions were prepared as follows A, B:

solution a: PLGA is dissolved in methylene dichloride to prepare a solution with the mass fraction of 5 percent

Solution B: a solution of 2% by mass was prepared by dissolving poly (N-vinylpyrrolidone-acrylic acid-N-succinimidyl acrylate) in 50% w/w isopropanol. The plasticizer PEG is then added ₂₀₀ The final mass concentrations of the silk fibroin and the antibacterial agent HBPL are respectively 1%, 0.4% and 0.04%.

The solution was then cast onto silicone-backed release paper to prepare the layers:

casting layer 1: casting the solution A on release paper with the back of organic silicon, and drying for 30 minutes at room temperature;

casting layer 2: casting solution B on layer 1 and drying at 85℃for 4 hours;

casting layer 3: casting the solution A onto the layer 2 and drying at room temperature for 30 minutes;

casting layer 4: solution B was cast onto layer 3 and dried at 85℃for 4 hours.

And after the drying is finished, the patch of the invention is obtained.

The total thickness of the patch is between 0.02mm and 1mm, and the patch can be prepared or cut into different sizes.

Compared with the prior art, the invention has the beneficial effects that:

(1) The tissue repair patch provided by the invention is in a dry state, can be stored for a long time in a room temperature sealed dry state, and is convenient and concise to use.

(2) The tissue repair patch has excellent mechanical properties, the Young modulus is about 240MPa, and the tensile stress at break is about 7 MPa.

(3) The tissue repair patch is suitable for forming stable adhesion on the surface of moist tissues, and is used for sealing pulmonary air leakage, cerebrospinal fluid exudation, hemostasis and the like.

(4) The anti-adhesion surface of the tissue repair patch can reduce the adhesion of protein and fibroblasts, and is used for reducing postoperative tissue adhesion.

(5) The tissue repair patch has good antibacterial performance, and the average antibacterial rate of common bacteria including staphylococcus aureus, escherichia coli and candida albicans is more than 80 percent.

(6) The tissue repair patch provided by the invention is safe and nontoxic and has good biocompatibility.

Drawings

The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention.

FIG. 1 equation of the synthesis reaction of tissue adhesive polymers in example 1 of the present invention

FIG. 2 molecular weight distribution of the synthetic tissue adhesive polymer of example 2 of the present invention

FIG. 3 IR spectrum of synthetic tissue adhesive polymer in example 3 of the present invention

FIG. 4 is a schematic representation of the structure of a tissue repair patch according to embodiment 4 of the present invention

FIG. 5 measurement of tensile Strength of tissue repair patches described in example 5 of the present invention

FIG. 6 adhesion measurement of the tissue repair patch of example 6 of the present invention to fresh pork liver

FIG. 7 antimicrobial properties of the tissue repair patch of example 7 of the present invention

Detailed Description

Example 1: preparation of bioadhesive polymers

200mL of Dimethylsulfoxide (DMSO) was heated to 80℃in an oil bath while oxygen-free nitrogen was bubbled through the solvent to remove dissolved oxygen. After adding 12.3. 12.3g N-vinylpyrrolidone (NVP), 3.94g of acrylic acid (AAc) and 9.2g of acrylic acid-N-succinimidyl ester (AAc-NHS) to DMSO, 0.08g of Azoisobutyronitrile (AIBN) was added. The reaction was carried out for 18h under nitrogen (FIG. 1).

After the reaction was completed, the reaction mixture was cooled to room temperature, 2000mL of isopropyl alcohol (IPA) was added to precipitate a polymer, and the polymer was isolated by filtration. The resulting polymer was washed 3 times in isopropanol, and then the polymer was lyophilized to obtain a white powder, i.e., the tissue-adhesive polymer.

Example 2: molecular weight determination of tissue adhesive polymers

The molecular weight distribution of the synthesized tissue viscosity polymer was characterized by Gel Permeation Chromatography (GPC). The molecular weight is generally about 30000 (FIG. 2).

Example 3: infrared spectra of tissue adhesive polymers

The functional groups of the synthesized tissue adhesive polymer were characterized using fourier infrared transform spectroscopy (FTIR) (fig. 3).

Example 4: preparation of tissue repair patches

The tissue repair patch of the present invention includes four alternating layers of anti-adhesion structures and tissue adhesion layers (fig. 4).

The anti-adhesion structure layer is made of PLGA, and the tissue adhesion layer is prepared from a tissue adhesive polymer, a plasticizer and an antibacterial agent.

The preparation method of the tablet comprises the following steps:

first, two solutions were prepared as follows A, B:

solution a: PLGA was dissolved in methylene chloride to prepare a 5% mass fraction solution.

casting layer 1: 3mL of solution A was cast on release paper and dried at room temperature for 30 minutes;

casting layer 2: 5mL of solution B was cast onto layer 1 and dried at 65℃for 4 hours;

casting layer 3: 3mL of solution a was cast onto layer 2 and dried at room temperature for 30 minutes;

casting layer 5: 5mL of solution B was cast onto layer 3 and dried at 65℃for 4 hours.

And (3) peeling off the patch from the release paper after drying, and cutting the patch into a required shape to obtain the patch. The patch has a thickness of about 50-100 μm.

Example 5: physical characteristics and mechanical Properties

The tissue repair patches of the present invention are transparent or white translucent films in appearance. The tensile strength was measured by a universal material tester, and the Young's modulus was about 240MPa, and the tensile stress at break was about 7MPa (FIG. 5).

Example 6: adhesion properties

The in vitro adhesive properties of the tissue repair patches of the present invention were quantified by adhesion to fresh pork liver. The patch was cut into strips 15cm long and 1cm wide and its tissue-adhering surface was adhered to the surface of fresh pork liver slices. Lightly pressing to tightly attach the patch to the pork liver. After 5min, 180 ° peel strength was measured by a universal material tester. Wherein, the average adhesion work of the four-layer structure patch to fresh pork liver is better than that of the double-layer patch, which is about 14J/m ² Left and right.

Example 7: antibacterial property

The antibacterial performance of the tissue repair patch of the invention is quantified by a 24h antibacterial rate against E.coli and Staphylococcus aureus. The antibacterial rate is generally greater than 99% (fig. 6).

Claims

1. The tissue repair patch with the multilayer structure is characterized by being of a multilayer structure formed by alternately arranging a tissue adhesion layer and an anti-adhesion layer, wherein the multilayer structure comprises at least 4 layers; the anti-adhesion layer is a polymer with film forming property, and the tissue adhesion layer comprises a tissue adhesive polymer and an antibacterial agent.

2. The multi-layered tissue repair patch of claim 1 wherein the polymer having film-forming properties is natural collagen or a synthetic polymer comprising polyglycolic acid, polylactic acid, polycaprolactone, or a copolymer of a plurality thereof and the tissue-adhesive polymer is poly (N-vinylpyrrolidone-acrylic acid-N-succinimidyl acrylate).

3. The multi-layered construction tissue repair patch of claim 1 wherein plasticizers and cross-linking agents are also incorporated into the tissue adhesive layer.

4. The method of preparing a multilayered tissue repair patch according to claim 1, comprising the steps of:

1) Preparing an anti-adhesion structural layer: dissolving a film-forming polymer in a proper solvent to prepare a solution A, casting the solution A on release paper dip-coated with organic silicon, and drying the release paper at room temperature to obtain an anti-adhesion layer 1;

2) Preparation of bioadhesive layer: mixing the bioadhesive polymer, the plasticizer, the cross-linking agent and the antibacterial agent in a proper solvent to obtain a solution B, casting the solution B on the anti-adhesion layer 1, and drying the solution B at 65 ℃ or higher to obtain an adhesion layer 2;

3) Casting the solution A onto the adhesive layer 2, and drying at room temperature to obtain an anti-adhesive layer 3;

4) Casting the solution B onto the anti-adhesion layer 3, and drying at 65 ℃ or higher to obtain an adhesion layer 4;

5) And (3) completely drying to obtain the patch, or continuously preparing a plurality of adhesive layer-anti-adhesive layer structures on the adhesive layer 4, and completely drying to obtain the patch.

5. The method of producing a multilayered tissue repair patch according to claim 4, wherein the film-forming polymer is polylactic acid-glycolic acid copolymer (PLGA) having a polymerization ratio of 0.2 to 0.8 and a molecular weight of 50000 to 400000.

6. The method of claim 4, wherein the bioadhesive polymer is formed by copolymerizing N-vinyl pyrrolidone, acrylic acid, and acrylic acid-N-succinimidyl ester, wherein the molar content of the polymerized N-vinyl pyrrolidone is 20% -80%, preferably 30% -70%, and more preferably 40% -60%; the polymerization mole content of the acrylic acid is 10% -50%, preferably 15% -40%, more preferably 20% -30%; the molar content of the acrylic acid-N-succinimidyl ester polymerization is 10-50%, preferably 15-40%, more preferably 20-30%.

7. The method of claim 4, wherein the antimicrobial agent is hyperbranched polylysine and the amount of the antimicrobial agent added is 0.1% -1%, preferably 0.2% -0.8%, more preferably 0.4% -0.6% (relative to the total mass of the patch).

8. The method of claim 4, wherein the cross-linking agent is glycerol, sucrose, hydroxypropyl cellulose or low molecular weight polyethylene glycol (PEG), the molecular weight of which is less than 1000, and the amount of the cross-linking agent added is 0-50%, preferably 10-40%, more preferably 20-30% (relative to the total mass of the patch).

9. The method of claim 4, wherein the plasticizer is natural fibroin or silk fibroin, and the addition amount is 0-40%, preferably 10-30%, more preferably 15-25%.

10. The method of producing a multilayered tissue repair patch according to claim 4, wherein the film-forming polymer is dissolved in methylene chloride in a mass fraction of 0.5% to 20%, preferably 1% to 10%, more preferably 2% to 5%;

the bioadhesive polymer is dissolved in 30-70% w/w isopropyl alcohol, with a mass fraction of 0.5-20%, preferably 1-10%, more preferably 2-5%;

the plasticizer, the cross-linking agent and the antibacterial agent are dissolved in water.