WO2022196946A1 - Barrière d'adhérence de type poudre comprenant un polymère biocompatible et son procédé de préparation - Google Patents

Barrière d'adhérence de type poudre comprenant un polymère biocompatible et son procédé de préparation Download PDF

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WO2022196946A1
WO2022196946A1 PCT/KR2022/002055 KR2022002055W WO2022196946A1 WO 2022196946 A1 WO2022196946 A1 WO 2022196946A1 KR 2022002055 W KR2022002055 W KR 2022002055W WO 2022196946 A1 WO2022196946 A1 WO 2022196946A1
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powder
adhesion
type anti
pvp
acid
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PCT/KR2022/002055
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Korean (ko)
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권오형
이혜영
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금오공과대학교 산학협력단
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Publication of WO2022196946A1 publication Critical patent/WO2022196946A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/042—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/08—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/043—Proteins; Polypeptides; Degradation products thereof
    • A61L31/045—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/23—Carbohydrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/23—Carbohydrates
    • A61L2300/236—Glycosaminoglycans, e.g. heparin, hyaluronic acid, chondroitin

Definitions

  • the present invention relates to a powder-type anti-adhesion agent comprising a biocompatible polymer and a method for producing the same, and more particularly, to a powder-type anti-adhesion agent prepared by cross-linking a natural polymer using a cross-linking agent. It has excellent biocompatibility. . In addition, it is possible to improve tissue adhesion by introducing an adhesive, and provides an excellent effect of anti-adhesion ability.
  • Adhesion formation in surgery is the most common problem that occurs after almost all abdominal surgeries. Abnormal adhesion due to excessive formation of fibrous tissue between organs and surrounding tissues or between organs is called adhesions. Adhesion in the abdominal cavity after surgery occurs frequently in 67 ⁇ 93% of patients, which causes serious problems such as female infertility, chronic pelvic pain, and small bowel obstruction. .
  • Methods of effectively preventing tissue adhesion include minimizing wounds during surgery, taking pharmaceutical agents, or using an adhesion prevention barrier that physically separates the damaged area from adjacent tissues or organs. There is this.
  • Anti-adhesion membranes in the form of solutions, gels, and films that are currently commercialized can separate damaged tissues from surrounding tissues, and are therefore more widely used in clinical practice than drug types.
  • it is being commercialized because it has been clinically proven to be the most effective, but there is a problem in that it cannot be continuously accurately positioned on the wound site due to the movement of the internal organs when applied to internal organs.
  • it since it is recognized as a foreign substance in the tissue, it is reported that the effect of preventing organ adhesion is insufficient due to agglomeration of each other.
  • laparoscopic surgery is a non-invasive approach. It has been reported that it has an anti-adhesion effect of 40% compared to open surgery, but it is reported that commercially available anti-adhesion membranes focus on open surgery.
  • Korean Patent Laid-Open No. 10-2005-0111419 discloses an anti-adhesion agent using a beta-glucan derivative.
  • the present invention has been devised to solve the problems of the prior art, and an object of the present invention is to provide a powder-type anti-adhesion agent excellent in biocompatibility and a manufacturing method thereof by cross-linking a natural polymer using a cross-linking agent.
  • one aspect of the present invention is,
  • biocompatible polymers comprising biocompatible polymers; and a crosslinking agent, wherein the biocompatible polymer is crosslinked by the crosslinking agent.
  • a powder-type anti-adhesion agent is provided.
  • the biocompatible polymer is chitosan, starch, cellulose, alginate, gelatin, hyaluronic acid, sodium hyaluronate, collagen, polyvinyl alcohol (PVA), alginic acid, pectin, carrageenan, chondroitin (sulfate), dextran (sulfate), polylysine (polylysine), carboxymethyl titin, fibrin, agarose, pullulan, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), hydroxypropyl Methylcellulose (HPMC), sodium carboxymethylcellulose, polyalcohol, gum arabic, cyclodextrin, dextrin, glucose, fructose, trehalose, glucose, maltose, lactose, lactulose, fructose, turanose, melitose, melezitose , from the
  • the biocompatible polymer may include sodium hyaluronate.
  • the crosslinking agent is glyoxal, glutaraldehye, citric acid, 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), adipic dihydrazide (ADH), N,N-methylene bis at least one selected from the group consisting of acrylamide (N,N-methylene bisacrylamide), dialdehyde, oxalic acid, and genipin may include
  • the powder-type anti-adhesion agent may further include an adhesive.
  • the adhesive may include at least one selected from the group consisting of poly(vinyl pyrrolidone, PVP), pullulan, sodium carboxymethylcellulose, and dopamine. .
  • Another aspect of the present invention (a) preparing a solution containing a biocompatible polymer; and (b) adding a crosslinking agent to the solution and freeze-drying, and then preparing a powder-type anti-adhesion agent through a grinding and washing process; is provided.
  • step (b) Before the introduction of the crosslinking agent in step (b), the step of adding a catalyst to the solution may be further included.
  • the catalyst may be an acid catalyst.
  • the catalyst may include at least one selected from the group consisting of hydrochloric acid (HCl), nitric acid (HNO 3 ), sulfuric acid (H 2 SO 4 ), phosphoric acid (H 3 PO 4 ), and hydrofluoric acid (HF).
  • HCl hydrochloric acid
  • HNO 3 nitric acid
  • sulfuric acid H 2 SO 4
  • phosphoric acid H 3 PO 4
  • HF hydrofluoric acid
  • the solution of step (a) may further include an adhesive.
  • the weight ratio of the biocompatible polymer and the pressure-sensitive adhesive in the solution of step (a) may be 3:7 to 7:3.
  • the concentration of the biocompatible polymer and the pressure-sensitive adhesive in the solution may be 1 to 10 wt%.
  • the powder-type anti-adhesion agent of the present invention is produced by cross-linking a natural polymer using a cross-linking agent, and has excellent biocompatibility.
  • the powder-type anti-adhesion agent of the present invention has excellent tissue adhesiveness and excellent anti-adhesion ability by introducing an adhesive.
  • UV-vis standard curve of glyoxal at 214 nm is a UV-vis standard curve of glyoxal at 214 nm.
  • FIG. 3 is a graph showing the absorption rate according to particle size of the powder-type anti-adhesion agent according to Examples 1 and 2, (a) is the absorption rate of x-HA, (b) is the absorption rate of x-HA-PVP (5:5).
  • Figure 4 is a graph showing the adhesive force of HA, pullulan, CMC and PVP.
  • Example 5 is a graph showing the adhesive strength according to the type of adhesive of the powder-type anti-adhesion agent according to Example 2;
  • Example 6 is a graph showing the adhesive strength according to the polymer concentration (HA concentration) of the powder-type anti-adhesion agent according to Example 1.
  • Example 7 is a graph showing the adhesive strength according to the weight ratio of HA and PVP of the powder-type anti-adhesion agent according to Example 2;
  • FIG. 11 is a graph showing the absorption rate of the powder-type anti-adhesion agent according to Examples 1 and 2.
  • Example 12 is an analysis result of the biodegradation behavior of the powder-type anti-adhesion agent according to Example 1.
  • Example 13 is a biodegradation behavior analysis result of the powder-type anti-adhesion agent according to Example 2.
  • 16 is an in vitro anti-adhesion test result of the powder-type anti-adhesion agent according to Examples 1 and 2.
  • 17 is an anti-adhesion ability evaluation result of a control group to which an anti-adhesion agent is not applied.
  • FIG. 20 is an image of a powder-type anti-adhesion agent according to Example 2 attached to a tissue, (a) is a liver tissue, (b) is a cecum tissue, (c) is an abdominal (abdomial) ) is an organization.
  • Figure 21 is the result of confirming the anti-adhesion ability through tissue staining
  • (a) and (b) are the results of H & E staining and masson's trichrome staining of the control group to which the anti-adhesion agent is not applied, respectively
  • (c) and (d) are performed, respectively
  • These are the results of H&E staining and masson's trichrome staining of the powder-type anti-adhesion agent according to Example 1.
  • Example 22 is a result of confirming the anti-adhesion ability through tissue staining of the powder-type anti-adhesion agent according to Example 2, (a) and (b) are the healed H&E staining and masson's trichrome staining results, respectively, (c) and (d) ) is the result of H&E staining and masson's trichrome staining of the healed .
  • the present invention is a biocompatible polymer; and a cross-linking agent, wherein the biocompatible polymer is cross-linked by the cross-linking agent.
  • the biocompatible polymer is chitosan, starch, cellulose, alginate, gelatin, hyaluronic acid, sodium hyaluronate, collagen, polyvinyl alcohol (PVA), alginic acid, pectin, carrageenan, chondroitin (sulfate), dextran (sulfate), polylysine (polylysine), carboxymethyl titin, fibrin, agarose, pullulan, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), hydroxypropyl Methylcellulose (HPMC), sodium carboxymethylcellulose, polyalcohol, gum arabic, cyclodextrin, dextrin, glucose, fructose, trehalose, glucose, maltose, lactose, lactulose, fructose, turanose, melitose, melezitose , from the
  • HA is a linear polysaccharide having a disaccharide repeating unit combined with ⁇ -1,4-D-glucuronic acid and ⁇ -1,3-N-acetyl-D-glucosamine. It exists as hyaluronate.
  • the biological function of HA is to maintain the elasticity of the connective tissue in liquid form, such as joint synovial fluid and ocular vitreous fluid, supramolecular assembly of proteoglycan in the extracellular matrix, cell migration, differentiation, and signal transduction between cells. play a role Specifically, it is a biopolymer material that is widely distributed throughout connective tissue, epithelial tissue, and nervous tissue, and has excellent non-immunity, biocompatibility, and biodegradability.
  • the functional groups mainly responsible for crosslinking in HA molecules are hydroxyl group and carboxyl group. Hydroxyl groups can be crosslinked by ether bonds and acetal bonds, and carboxyl groups can be crosslinked by ester bonds.
  • the crosslinking agent is glyoxal, glutaraldehye, citric acid, 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone , DVS), adipic dihydrazide (ADH), At least one selected from the group consisting of N,N-methylene bisacrylamide, dialdehyde, oxalic acid, and genipin It may include, preferably glyoxal.
  • Glyoxal has the smallest dialdehyde group in existence, and is a crosslinking agent that forms acetal or hemiacetal bonds under acid catalysis with hydroxyl groups present in polysaccharides, and forms primary amine and shiff base bonds. Glyoxal is less toxic than glutaraldehyde with the same dialdehyde group, and if the concentration is maintained below 100 ppm, cells are not damaged and high biocompatibility can be maintained.
  • the powder-type anti-adhesion agent may further include an adhesive.
  • the pressure-sensitive adhesive may include at least one selected from the group consisting of poly(vinyl pyrrolidone), PVP), pullulan, sodium carboxymethylcellulose, and dopamine. , preferably PVP.
  • PVP is an amorphous synthetic polymer with FDA-approved representative bioabsorption and biocompatibility characteristics, and is used in various medicines and pharmaceutical fields.
  • PVP can form a complex with various compounds by forming hydrogen bonds with HA and a polymer having a hydroxyl group such as poly(vinyl alcohol).
  • the powder-type anti-adhesion agent of the present invention has excellent biocompatibility without generating inflammatory or immune responses that can cause adhesions in the body, and has an effect that does not require secondary surgery to be naturally decomposed and removed during tissue healing.
  • the powder-type anti-adhesion agent of the present invention with handling convenience can also be applied to laparoscopic surgery and can effectively cover defective areas easily by air spraying.
  • step a a solution containing a biocompatible polymer is prepared (step a).
  • the solution of step (a) may further include an adhesive.
  • the weight ratio of the biocompatible polymer and the pressure-sensitive adhesive in the solution of step (a) may be 3:7 to 7:3, preferably 4:6 to 6:4.
  • the concentration of the biocompatible polymer and the pressure-sensitive adhesive in the solution may be 1 to 10 wt%, preferably 2 to 4 wt%.
  • the concentration of the biocompatible polymer in the solution may be 1 to 10 wt%, preferably 2 to 4 wt%.
  • a crosslinking agent is added to the solution and freeze-dried, followed by grinding and washing to prepare a powder-type anti-adhesion agent (step b).
  • the powder-type anti-adhesion agent is the same as the description of the powder-type anti-adhesion agent of the present invention described above, and therefore, specific details thereof will be referred to.
  • step (b) Before the introduction of the crosslinking agent in step (b), the step of adding a catalyst to the solution may be further included.
  • the catalyst may be an acid catalyst.
  • the catalyst may include at least one selected from the group consisting of hydrochloric acid (HCl), nitric acid (HNO 3 ), sulfuric acid (H 2 SO 4 ), phosphoric acid (H 3 PO 4 ) and hydrofluoric acid (HF), preferably may include hydrochloric acid.
  • HCl hydrochloric acid
  • HNO 3 nitric acid
  • sulfuric acid H 2 SO 4
  • phosphoric acid H 3 PO 4
  • HF hydrofluoric acid
  • Example 1 Preparation of a powder-type anti-adhesion agent (x-HA) containing HA cross-linked with a Glyoxal cross-linking agent
  • HA hyaluronate
  • tertiary distilled water pH adjusted to 4-5 using 1 N HCl as an acid catalyst
  • 0.13 ml of a 40 wt% glyoxal solution in H 2 O, Sigma-Aldrich
  • 100 rpm using a mechanical stirrer at room temperature for 2 hours.
  • post-crosslinking was performed at room temperature for 48 hours.
  • the reaction was terminated by adjusting the pH to 7 using 1 N NaOH, and the reaction solution was pre-frozen and freeze-dried for 3 days. After freeze-drying, it was ground into a powder using a pestle, and the unreacted crosslinking agent was washed with acetone. At this time, it was replaced with new acetone once every 3 hours, and washing was performed for a total of 6 hours. After washing, acetone was removed to prepare a powder-type anti-adhesion agent (x-HA).
  • x-HA powder-type anti-adhesion agent
  • Example 2 Preparation of powder-type anti-adhesion agent (x-HA-PVP) comprising HA and PVP cross-linked with a Glyoxal cross-linking agent
  • HA-PVP polyvinylpyrrolidone
  • a powder-type anti-adhesion agent (x-HA-PVP) was prepared in the same manner as in Example 1, except that the HA-PVP solution was used instead of the HA solution in Example 1.
  • Comparative Example 1 Preparation of a powder-type anti-adhesion agent (x-HA-BDDE) containing HA cross-linked with a BDDE cross-linking agent
  • the solution crosslinked with BDDE is placed in an ice mold, pre-freeze in a deep freezer (-80°C), freeze-dried for 2 days using a freeze dryer, and pulverized using a mortar to obtain a powder-type anti-adhesion agent (x- HA-BDDE) was prepared.
  • Comparative Example 2 Preparation of a powder-type anti-adhesion agent (x-HA-PVP-BDDE) comprising HA and PVP cross-linked with a BDDE cross-linking agent
  • Comparative Example 1 except for dissolving 3% (w/v) HA and PVP instead of dissolving 3% (w/v) HA in a 0.25 M NaOH solution containing 1% (v/v) BDDE in Comparative Example 1
  • a powder-type anti-adhesion agent (x-HA-PVP-BDDE) was prepared in the same manner as described above.
  • FTIR Fourier transform infrared spectroscopy
  • FIG. 1 The FTIR analysis results are shown in FIG. 1, in which (a) is an FTIR spectrum of x-HA-PVP (5:5), (b) is x-HA, (c) is PVP, (d) is HA to be.
  • the hydroxyl group of HA reacts with the aldehyde group of glyoxal to form an acetal bond.
  • -COC- groups which show the formation of acetal bonds, were observed at 1140 and 1050 cm -1 .
  • -COC-peak was observed in x-HA-PVP (5:5) at 1290 cm -1 , the CN stretching peak of PVP and 1050 cm -1 in HA.
  • x-HA-PVP (5:5) has the characteristic functional groups of HA and PVP at the same time. It can be seen that the PVP remains even after the washing process. The hydroxyl group of HA covalently bonds with the aldehyde group of glyoxal to form an acetal bond. At this time, PVP does not participate in the reaction and forms a weak hydrogen bond with the OH group of HA.
  • glyoxal crosslinking agent 40 wt% of glyoxal crosslinking agent was determined as a standard solution, diluted to a certain concentration to draw a standard curve, and UV absorbance was measured in the range of 100 to 2000 ppm. 2 is a UV-vis standard curve of glyoxal at 214 nm. In Examples 1 and 2, 3 ml of the solution after washing with acetone for 6 hours was removed, the solvent was evaporated by vacuum distillation, and then 3 ml of distilled water was added.
  • the x-HA and x-HA-PVP (5:5) powder washing solutions showed absorbances of 0.009 and 0.012, respectively, which were 41 and 56 ppm, indicating that they were less than 100 ppm. there was. Since there is a study result that Glyoxal cross-linking agent is biocompatible when it is less than 100 ppm, it was found that the unreacted cross-linking agent was washed up to a concentration harmless to the human body with x-HA and x-HA-PVP (5:5) powder.
  • the crosslinking reaction was carried out with a polymer concentration of 3 wt%, an amount of a crosslinking agent of 0.26 ml, and a reaction time of 48 h. Above, it was separated into sizes of 250-500 ⁇ m, 125-250 ⁇ m, and 125 ⁇ m or less.
  • the optimum particle size was determined by calculating the absorption rate for each particle size as shown in Equation 1 below. Water absorption rate was evaluated by measuring the weight when 50 ⁇ l of distilled water was added to 0.1 g of powder for each particle size and no longer absorbed, and the absorption rate (W A ) was calculated.
  • Equation 1 W initial is the initial weight of the powder, and W absop is the weight of the powder when maximally absorbed.
  • the absorption rate according to the particle size is shown in FIG. 3 .
  • (a) is the absorption rate of x-HA and (b) is the absorption rate of x-HA-PVP (5:5).
  • x-HA and x-HA-PVP powders showed the maximum absorption rates at a particle size of 250 to 500 ⁇ m.
  • the crosslinked x-HA powder showed an absorption rate of 640% at a particle size of 250-500 ⁇ m, and the x-HA-PVP powder showed an absorption rate of 769% at a particle size of 250-500 ⁇ m.
  • a certain amount of distilled water was added to the cross-linked x-HA and x-HA-PVP powder to form a gel, and then a rheometer was used to measure the polymer concentration, the cross-linking agent concentration, and the adhesive force according to the reaction time.
  • the tack adhesion mode was used with the Discovery Hybrid Rheometer (DHR-II TA instruments, USA).
  • a 20 mm parallel plate (peltier plate steel, 110258) was mounted on the upper part of the rheometer, and pig skin with a thickness of 20 to 30 mm was attached, and the sample was placed on the lower plate.
  • the control group was HA
  • the experimental group was carboxymethylcellulose (CMC, Na salt), pullulan and PVP
  • the adhesive strength test was carried out. 4
  • the average negative force of HA was -0.89 N
  • pullulan was -0.3 N
  • CMC was -1.0 N
  • PVP was -2.34 N.
  • PVP was the material with the greatest adhesive force as it produced a large negative force.
  • Example 2 When HA reacts with glyoxal, it is assumed that the reaction is 1:1 by calculating the number of moles of reactive functional groups of HA.
  • 0.26 ml of glyoxal was added, and the final solution was carried out at a concentration of 3 wt%.
  • the reaction time was fixed at 48 hours, and HA, pullulan, CMC, and PVP were added as adhesives and cross-linked with HA in a ratio of 5:5 to compare the adhesive strength, which was 3 wt% x-HA, 3 wt% x, respectively.
  • -HA-pullulan, 3 wt% x-HA-CMC, 3 wt% x-HA-PVP The adhesive force according to the type of adhesive is shown in FIG. 5 .
  • Example 1 adhesiveness was measured by varying the cross-linked HA polymer concentration to 1, 2, 3, 4, 5, and 6 wt % under the conditions where the amount of cross-linking agent was 0.26 ml and the reaction time was fixed at 48 hours. 6 shows.
  • the adhesiveness is -0.75, -0.75, -0.74, -0.55, -0.30, and -0.14 N. It can be seen that this is lowered, which proves that the adhesiveness of HA is lowered during the crosslinking reaction. From 1 wt% to 3 wt%, there was almost no difference in adhesiveness, and at a polymer concentration of 3 wt% or more, it was observed that the adhesiveness was reduced during crosslinking.
  • the concentration of the polymer increases, the number of reactive functional groups increases, and even if the same amount of the crosslinking agent is added, the network becomes denser and the bond between the polymer chains becomes stronger. Accordingly, it was judged that the adhesive force of the surface was reduced.
  • Example 2 the amount of cross-linking agent was fixed at 0.26 ml, the reaction time was fixed at 48 hours, the ratios of HA and PVP were 7:3, 5:5, and 3:7, respectively, and the concentration of the final cross-linked polymer solution was 1, 3 , 6 wt%, the ratio showing the highest adhesiveness was measured, and the results are shown in FIG. 7 and Tables 1 to 3.
  • BDDE which is known as a crosslinking agent for crosslinking a typical hydroxyl group
  • PVP was introduced under the same conditions to observe adhesion, and the results are shown in FIG. 10 .
  • Inflammatory exudate is produced during the tissue self-healing process after surgery.
  • the absorption rates of the crosslinked x-HA and x-HA-PVP powders according to Examples 1 and 2 were calculated in order to evaluate whether they adhere well to the correct target site while absorbing the exudate, and the results are shown in FIG. 11 .
  • the absorption rate of the non-crosslinked HA powder 1061% was obtained, and the absorption rate of the non-crosslinked PVP was 254%.
  • the absorption rate was measured by varying the weight ratio of HA and PVP. Referring to FIG. 11 , it was found that the cross-linked HA powder had a reduced absorption rate. The absorption rate according to the PVP ratio was the highest at 7:3, and it was found that it decreased as the PVP content increased. However, it was found that the difference between the absorption rates of 7:3 and 5:5 was small with a difference of approximately 5%, and the adhesion was found to be excellent with a difference of 34%, showing that the ratio of 5:5 was excellent (see (b) of FIG.
  • the powder-type anti-adhesion agent has a predominantly adhesive property by spraying the powder on the peritoneum and tissue and applying distilled water. Therefore, although the absorption rate is slightly different from the ratio of 7:3, the ratio of 5:5 has excellent adhesion, so the optimal condition with PVP is 5:5.
  • the x-HA and x-HA-PVP powders according to Examples 1 and 2 were incubated with HAase at 37° C., and enzyme stability was evaluated using a carbazole assay using the Bitter-Muir method. Standard materials and HA, x-HA, x-HA-PVP powder (20 mg) were put together with 10 ml HAase solution (10 U/ml, in PBS) and incubated at 37°C.
  • 0.5 ml of the supernatant is taken out, and 0.5 ml of fresh HAase solution (10 U/ml ) was replaced with 0.5 ml of the supernatant was carefully put into 3 ml of 0.025 M sodium tetraborate ⁇ 10H 2 O (in sulfuric acid) solution prepared in a vial so that the layers were separated.
  • the vial was closed, shaken slowly, and cooled at 4°C.
  • the cooled vial was heated in a boiling water bath for 10 minutes to inactivate the enzyme, and then cooled at room temperature.
  • D-glucuronic acid lactone was used as a standard material, and the amount of uronic acid in the sample was quantified by drawing a standard curve at a concentration of 4-60 ⁇ g/ml.
  • the concentration of glucuronic acid in the supernatant is C 1
  • the final concentration of glucuronic acid in the supernatant is C 2 to calculate the decomposition rate (W D ) as shown in Equation 2 below.
  • the control group was non-crosslinked HA, and the experimental group was subjected to biodegradation experiments by varying the concentration of x-HA crosslinked in Example 1 to 1, 3, and 6 wt%, 3, 6, 12, 24, 48, 72, 96 , 120 and 144 h time intervals were taken out and the amount of uronic acid produced by decomposition by hyaluronic acid enzyme was quantified, and the results are shown in FIG. 12 . Referring to FIG. 12 , it was confirmed that all decomposition of HA without crosslinking was achieved to 98% in 3 h. As the concentration of cross-linked x-HA increases, the number of functional groups increases and the degree of cross-linking increases.
  • FIG. 13 The results of analyzing the biodegradation behavior by varying the content ratio of HA and PVP in Example 2 are shown in FIG. 13 .
  • the ratio of 3:7 with the highest PVP content was decomposed in 48 h, and 5:5, the optimal ratio of absorption and adhesion, was decomposed after 120 h, that is, 4 days. It can be seen that the difference from 7:3, which has a higher HA content, is small, which is judged to have resistance to HAase enzymes by infiltrating PVP through the network structure of cross-linked HA to form hydrogen bonds.
  • 3 wt% of the prepared x-HA and x-HA-PVP powders were applied as anti-adhesion powders, it was found that they had resistance to enzymes for 3 to 4 days, which is the tissue healing period.
  • the cytotoxicity grade 1 shows that both x-HA and x-HA-PVP (5:5) powder cross-linked with a value corresponding to >80% correspond to the corresponding grade, and shows low cytotoxicity. Furthermore, since it is biocompatible to apply as an anti-adhesion powder, it induces a low inflammatory response and shows that it is harmless in the human body.
  • x-HA and x-HA-PVP 5:5) according to Examples 1 and 2, the growth behavior of cells was visually evaluated using Live/Dead staining.
  • Each of 5.0 ⁇ 10 4 cells was seeded in a 35 mm petri dish and cultured for 4 hours to adhere the cells, and then the sample eluate was allowed to contact the cells directly. Thereafter, cell growth behavior was observed by incubation for 1, 3, and 5 days at 37° C., 5% CO 2 conditions.
  • Live/Dead staining was performed using 2 ⁇ M calcein AM (Live staining) and 4 ⁇ M ethidium homodimer-1 (EthD-1, Dead staining) for 30 minutes. Live cells were stained green and dead cells were stained red, and under a fluorescence microscope. Stained living and dead cells were observed.
  • the experimental group checked the cell growth behavior by placing the cross-linked x-HA and x-HA-PVP (5:5) powders according to Examples 1 and 2 on the already adhered cells.
  • the control group observed cells cultured for the same time as the experimental group without any treatment, and the results are shown in FIG. 15 .
  • the sample used in the experiment used the same NIH3T3 (mouse fibroblast) cells as in the previous cell experiment, and was cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin.
  • the eluate (2 ml) was applied to the bottom of a 35 mm petri dish, and NIH3T3 fibroblasts (5.0 ⁇ 10 4 cells) were inoculated. After culturing for 24 hours, a 35 mm petri dish was observed under a microscope, and the results are shown in FIG. 16 .
  • Example 1 8-week-old male Sprague Dawley rats (Hyochang Science, 250-300 g) ) was used to conduct animal experiments.
  • the experimental control group did not receive any treatment, and the experimental group used x-HA and x-HA-PVP (5:5) powder as an anti-adhesion agent.
  • the experiment was carried out with 5 animals in both the experimental control group and the experimental group. The experiment proceeded as follows.
  • the control group did not receive any additional treatment after the procedure of (4), and the experimental group sprayed 0.1 g of powder on the wounded peritoneal membrane and cecum, and then sprayed 300 ⁇ l of physiological saline.
  • potassium chloride (KCl) was injected and euthanized, and the degree of adhesion was evaluated.
  • FIG. 17 is the anti-adhesion ability evaluation result of the control group to which the anti-adhesion agent is not applied
  • FIG. 18 is the anti-adhesion ability evaluation result of the powder-type anti-adhesion agent according to Example 1
  • FIG. 19 is the powder-type anti-adhesion agent according to Example 2 of the anti-adhesion ability evaluation result.
  • the control group showed a total of 100% adhesion with 2 heads of grade 3 and 3 heads of grade 4.
  • grade 2 1 heads and grade 4 2 heads were evaluated, and an average of grade 2 adhesions was achieved in a total of 60%.
  • FIG. 19 when cross-linked x-HA-PVP (5:5) powder was applied, it was evaluated as 0 grade 5 heads, resulting in a total adhesion of 0%. In contrast to the control group showing severe adhesion grade, overall x-HA and x-HA-PVP (5:5) powders showed low adhesion scores. Through this, it was confirmed that x-HA and x-HA-PVP (5:5) powders had excellent anti-adhesion effects as anti-adhesion agents.
  • FIG. 20 is an image of a powder-type anti-adhesion agent according to Example 2 attached to a tissue, (a) is a liver tissue, (b) is a cecum tissue, (c) is an abdominal (abdomial) ) is an organization.
  • a tissue is a liver tissue
  • b is a cecum tissue
  • c is an abdominal (abdomial) ) is an organization.
  • HA as a bio-derived polymer was used as a main material, and cross-linking was performed with glyoxal due to its low mechanical properties.
  • PVP was introduced to improve adhesion.
  • In vitro HAase enzyme digestion test showed that x-HA-PVP(5:5) powder was degraded 24 h earlier than powder without PVP.
  • SD rats fed with x-HA powder developed adhesions by 60%, whereas all SD rats to which x-HA-PVP(5:5) powder was applied developed adhesions. knew it wasn't.
  • PVP PVP
  • H&E staining is a technique for staining with two dyes, hematoxylin and eosin.
  • the principle of H&E staining is a chemical interaction between tissue and dye, and hematoxylin, a basic dye, is oxidized by mercuric oxide, an oxidizing agent, to form hematein.
  • Hematain combines with alum, a mordant, to form an alum-hematein lake in a salted state. These hematein lakes are cations and combine with the phosphate groups of negatively charged DNA to give them a purple color.
  • eosin an acid dye, has an anion and is dyed pink by ionic bonding with the cytoplasm, extracellular matrix, and collagen that have a positive cation.
  • Masson's trichrome staining is a method that looks at the difference in the size of dye molecules, the pores in the tissue structure, and the difference in dye permeability. This is a method for selectively staining glue, fibrin, muscle and red blood cells with three dyes. Depending on the permeability of the tissue, tissues with low permeability are colored by small dye molecules, and large dye molecules permeate into tissues with high permeability. According to the principle of dye permeability according to the size of the dye molecule, the glue fiber is dyed blue by anilin blue, the largest dye molecule, and acid fuchsin, a medium-sized dye molecule, and biebrich scarlet, the smallest dye molecule, are mixed. The muscle fibers and cytoplasm are stained red with the staining solution. In addition, weigret's hematoxylin, which has acid resistance, stains the cell nucleus in dark brown.
  • Example 22 is a result of confirming the anti-adhesion ability through tissue staining of the powder-type anti-adhesion agent (x-HA-PVP (5:5)) according to Example 2, (a) and (b) are H&E of the healed peritoneal membrane, respectively; Staining and masson's trichrome staining results, (c) and (d) are H&E staining and masson's trichrome staining results of the healed appendix.
  • x-HA-PVP (5:5) powder-type anti-adhesion agent
  • the experimental group to which x-HA powder was applied was connected with a complex fibrous layer between the pink-colored peritoneal membrane and the purple-colored muscle layer of the appendix, and it was confirmed that the new tissue was stained with a light pink color.
  • the cross-linked x-HA-PVP (5:5) powder was applied, it was confirmed that no new tissue was formed between the peritoneal membrane and the cecum membrane. New mesothelial cell layers were identified in blue and pink colors, respectively, and it was confirmed that they were completely recovered without adhesions.
  • the x-HA-PVP (5:5) powder prepared through two types of tissue staining did not completely cause adhesions, and it was confirmed that the damaged peritoneal membrane and the cecum were completely recovered, and histologically demonstrated excellent anti-adhesion ability. .
  • the powder-type anti-adhesion agent of the present invention is produced by cross-linking a natural polymer using a cross-linking agent, and has excellent biocompatibility.
  • the powder-type anti-adhesion agent of the present invention has excellent tissue adhesiveness and excellent anti-adhesion ability by introducing an adhesive.

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Abstract

La présente invention se rapporte à une barrière d'adhérence de type poudre comprenant un polymère biocompatible et à son procédé de préparation, et plus précisément, à une barrière d'adhérence de type poudre préparée par réticulation d'un polymère naturel à l'aide d'un agent de réticulation, la barrière d'adhérence de type poudre présentant une excellente biocompatibilité. De plus, par l'introduction d'un adhésif, les propriétés d'adhérence tissulaire peuvent être améliorées, et l'effet d'une excellente capacité anti-adhérence est fourni.
PCT/KR2022/002055 2021-03-15 2022-02-10 Barrière d'adhérence de type poudre comprenant un polymère biocompatible et son procédé de préparation WO2022196946A1 (fr)

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