WO2015096170A1 - 一种ε-聚赖氨酸水凝胶及其制备方法和应用 - Google Patents
一种ε-聚赖氨酸水凝胶及其制备方法和应用 Download PDFInfo
- Publication number
- WO2015096170A1 WO2015096170A1 PCT/CN2013/090814 CN2013090814W WO2015096170A1 WO 2015096170 A1 WO2015096170 A1 WO 2015096170A1 CN 2013090814 W CN2013090814 W CN 2013090814W WO 2015096170 A1 WO2015096170 A1 WO 2015096170A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- polylysine
- nitrophenyl chloroformate
- polyethylene glycol
- preparation
- tyramine
- Prior art date
Links
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- 238000006243 chemical reaction Methods 0.000 claims description 58
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 57
- 229920001223 polyethylene glycol Polymers 0.000 claims description 56
- 239000002202 Polyethylene glycol Substances 0.000 claims description 55
- 229960003732 tyramine Drugs 0.000 claims description 55
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 51
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 45
- DZGWFCGJZKJUFP-UHFFFAOYSA-N tyramine Chemical compound NCCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-N 0.000 claims description 43
- 239000000243 solution Substances 0.000 claims description 40
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 36
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 34
- 229920001577 copolymer Polymers 0.000 claims description 32
- NXLNNXIXOYSCMB-UHFFFAOYSA-N (4-nitrophenyl) carbonochloridate Chemical compound [O-][N+](=O)C1=CC=C(OC(Cl)=O)C=C1 NXLNNXIXOYSCMB-UHFFFAOYSA-N 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 26
- 239000008363 phosphate buffer Substances 0.000 claims description 19
- -1 p-nitrophenyl chloroformate-polyethylene Chemical group 0.000 claims description 18
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- 239000007864 aqueous solution Substances 0.000 claims description 7
- 229920000573 polyethylene Polymers 0.000 claims description 7
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 6
- ZSZYCGVNBKEVPH-UHFFFAOYSA-N tyramine phosphate Chemical compound NCCC1=CC=C(OP(O)(O)=O)C=C1 ZSZYCGVNBKEVPH-UHFFFAOYSA-N 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 2
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 claims description 2
- 102000003992 Peroxidases Human genes 0.000 claims 1
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Classifications
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- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
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- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0019—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
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- A61L15/42—Use of materials characterised by their function or physical properties
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- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
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- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/009—Materials resorbable by the body
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
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- C08G69/10—Alpha-amino-carboxylic acids
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/40—Polyamides containing oxygen in the form of ether groups
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/48—Polymers modified by chemical after-treatment
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
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- 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
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/80—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special chemical form
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2377/00—Characterised by the use of polyamides obtained by reactions forming a carboxylic amide link in the main chain; Derivatives of such polymers
- C08J2377/04—Polyamides derived from alpha-amino carboxylic acids
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
Definitions
- the invention relates to an ⁇ -polylysine hydrogel, a preparation method and application thereof, and belongs to the field of medical technology application. Background technique
- Hydrogel medical wound excipients are a new type of wound excipient developed in recent years. Compared with traditional excipients, hydrogels can promote wound healing and reduce pain in patients. It can improve the microenvironment of the wound and inhibit the growth of bacteria. Hydrogels are particularly suitable for common skin injuries such as abrasions, scratches, and acne. For these wounds, doctors have traditionally used sterile gauze and topical antibiotics. The gauze is easy to adhere to the skin wound tissue. When changing the dressing, it often destroys the epithelium and granulation tissue of the novice, causing bleeding, which is not only bad for the healing of the wound, but also makes the patient unbearable.
- Hydrogel excipient When applied to the wound with a hydrogel adjuvant, it not only does not stick to the wound, does not damage the new tissue, but also kills various bacteria and prevents wound infection.
- Hydrogel excipient is a good wound excipient. It is a polymer swelling body with a three-dimensional network structure with certain moisture. It has good water absorption and biocompatibility, and can closely conform to uneven wounds without Adhesion can occur, reducing the chance of bacterial growth and easy replacement, and can penetrate various pharmaceutical ingredients and growth factors to promote wound healing.
- the hydrogel itself has a large heat capacity and a gentle cooling effect on the wound, which can alleviate the pain of the wound. Therefore, domestic and foreign scholars have conducted extensive research on hydrogel excipients and achieved some results. Currently, hydrogel excipients account for the majority of the global excipients and bandage markets.
- the materials currently used in wound excipients and commercially produced mainly include fibrin glue, cyanoacrylate, chitosan, etc., but they have their defects, which limit their wide application in clinical surgical medicine, such as fibrin.
- Gum is mainly derived from animal blood tissue, so there is a risk of blood virus infection.
- cyanoacrylate has good hemostasis effect, it is not the best choice for wound tissue healing because of its cytotoxicity.
- Chitosan has the function of rapid hemostasis. Can be processed into a variety of medical accessories products, but due to its poor mechanical properties, brittleness, poor water solubility and other shortcomings become its main obstacles for the application of surgical wounds.
- Polylysine is a natural polymer material that forms a hydrogel and is produced by microbial fermentation.
- the molecular weight of ⁇ -polylysine is between 3,000 and 5,000 Daltons, similar to the protein composition and function in the natural extracellular matrix (ECM).
- ECM extracellular matrix
- the significant advantage of polylysine in distinguishing it from other wound excipient materials is that The following aspects: First, the molecular chain has a large number of amino reactive groups, which combine with hydrogen ions in an aqueous solution or acidic environment to carry a positive charge to form a cationic polymer, which can be well bound to the cell surface.
- polylysine is a natural polymer material, it has good biocompatibility and its degradation products.
- Lysine is an essential amino acid in human body.
- polylysine is an amino acid polymer, which has good water solubility and overcomes the difficulty of many tissue healing materials being difficult to dissolve in water.
- polylysine carries a large amount of active amino groups. The positive charge associated with hydrogen ion binding in aqueous solution or acidic environment will destroy the structure of tissue cells to some extent, and has certain cytotoxicity. To be safely applied to wound tissue adhesion, it must be modified to reduce Cytotoxicity.
- PEG is a water-soluble and biocompatible polymer that is widely used for surface modification of biological materials, modification of peptides and protein drugs.
- PEG modification has a significant inhibitory effect on some of the induced apoptosis, significantly reducing the intracellular ROS (reactive oxygen species) content and protecting the mitochondrial membrane potential stability.
- PEG modification mainly reduces the cytotoxicity of polylysine by reducing the production of intracellular ROS (reactive oxygen species), and the high degree of modification has a significant effect of reducing toxicity.
- PEGylated polylysine can significantly reduce the cytotoxicity of polylysine, and the ability to reduce toxicity is significantly enhanced with the increase in molecular weight and degree of modification of PEG.
- hydrogel medical materials applied to wound excipients are mainly divided into chemical synthesis and biopolymer materials.
- there are many defects in the formation process of hydrogel materials due to poor mechanical strength, poor biocompatibility, and cytotoxicity. . Summary of the invention
- the technical problem to be solved by the present invention is to provide a medical wound adjuvant polylysine hydrogel.
- the technical problem to be solved by the present invention is to provide a method for preparing the above hydrogel.
- the final technical problem to be solved by the present invention is to provide the use of the above hydrogel.
- ⁇ is any natural number in 20 30, and m is any natural number in 50 70.
- the preparation method of the above polylysine hydrogel comprises the following steps:
- step (3) obtained grafted polyethylene glycol (PEG) and tyramine £ - polylysine dissolved in water or phosphate buffer, horseradish peroxidase with hydrogen peroxide
- the aqueous solution was mixed and stirred at room temperature for 5 to 60 s to form a polylysine hydrogel.
- the number average molecular weight of the polyethylene glycol ranges from 4,000 to 6,000, preferably 4,000.
- the polyethylene glycol is dissolved in dichloromethane so that the concentration of the solute polyethylene glycol is 50 to 2 G0 g/L, preferably 50 to 150 g/L; p-nitrophenyl chloroformate
- the concentration of the solute p-nitrophenyl chloroformate is 20 to 40 g/L, preferably 25 to 35 g/L.
- step (1) 4-dimethylaminopyridine and triethylamine are further added to react at room temperature for 30 minutes.
- the reaction molar ratio of polyethylene glycol, 4-dimethylaminopyridine, triethylamine and p-nitrophenyl chloroformate is 1:2 ⁇ 5:2 ⁇ 5:2 ⁇ 5 'Optimized 1:3:3:3.
- the cold diethyl ether temperature is -41; -20 ° C, preferably -4 ° C, and the amount of cold ether is 20 to 40 times, preferably 30 times, the volume of the reaction liquid after the rotary distillation.
- the vacuum freeze-drying temperature is -40'C ⁇ -80 °C, preferably -60'C, and the pressure is 10 30 Pa, preferably
- the rotary steaming temperature is 50 ° C, and it is steamed to 5 to 10%, preferably 5%, of the volume of the raw liquid.
- step (1) all operations are carried out under anhydrous and anaerobic conditions at room temperature, and the anaerobic conditions may be protected by means of an inert gas, preferably by nitrogen.
- the phosphate buffer solution is a phosphate buffer solution having a pH of 7.4 and 0.01 to 0.2 mol/L.
- the specification is preferably a phosphate buffer solution having a pH of 7.4 and 0.01 mol/L.
- the p-nitrophenyl chloroformate-polyethylene glycol-p-nitrophenyl chloroformate copolymer is dissolved in a phosphate buffer, and the solute is p-nitrophenyl chloroformate-
- concentration of the polyethylene glycol-p-nitrophenyl chloroformate copolymer is 50 to 100 g/L, preferably 80 g/L; in the phosphate buffer solution containing tyramine, the concentration of the solute tyramine is 3 to 10 g L Preferably, it is 2 to 6 g/L.
- the molar ratio of the p-nitrophenyl chloroformate-polyethylene glycol-p-nitrophenyl chloroformate copolymer to tyramine is 2: 0.5 to 1.5, preferably 2:1.
- step (2) it is preferred to carry out a reaction at room temperature for 10 hours to obtain a p-nitrophenyl chloroformate-polyethylene glycol-tyramine copolymer solution.
- step (2) all operations are carried out under room temperature and anaerobic conditions, and the anaerobic conditions may be protected by means of an inert gas, preferably by nitrogen gas.
- the polylysine is dissolved in the phosphate buffer, and the concentration of the solute polylysine is 0.434.09 g/L, preferably 0.4 to 0.8 g/L.
- the molar ratio of the polylysine to the p-nitrophenyl chloroformate-polyethylene glycol-p-nitrophenyl chloroformate copolymer is 0.04 to 0.07:2, preferably 0.05: 2.
- step (3) it is preferred to react at room temperature for 24 hours.
- dialysis is preferably carried out for 5 days.
- the lyophilization temperature is -60 °C.
- step (3) all operations are carried out under room temperature and anaerobic conditions, and the anaerobic conditions may be protected by means of an inert gas, preferably by nitrogen gas.
- the grafted polyethylene glycol obtained in the step (3) and the tyrosine & polylysine are dissolved in water or phosphate buffer, and the solute grafted polyethylene glycol and tyramine ⁇ -poly
- concentration of the acid is 5 to 10% by weight, preferably 10% by weight.
- step (4) all the reaction operations are carried out under room temperature and anaerobic conditions, and the anaerobic conditions may be protected by means of an inert gas, preferably by nitrogen gas.
- the polylysine hydrogel prepared by the above method is also within the scope of the present invention.
- the use of the above s-polylysine hydrogel in the preparation of wound excipients can be applied to the medical or cosmetic field.
- Advantageous Effects of the Invention uses a natural polymer material polylysine as a scaffold adjuvant scaffold to modify a polylysine molecular skeleton by grafting polyethylene glycol and tyramine, and then in horseradish peroxidase and Hydrogel medical material forming a polylysine-polyethylene glycol-tyramine three-dimensional network structure under the action of hydrogen peroxide, the wound auxiliary material has high mechanical strength, fast gel time and adjustable gel strength and reaction It has many advantages such as mild conditions and low tissue toxicity, and overcomes many defects such as poor mechanical strength, poor biocompatibility and cytotoxicity of existing wound excipients, and has great application prospects in the medical field.
- Figure 1 is a schematic diagram and molecular structure of the prepared polylysine-polyethylene glycol-tyramine
- Figure 2 is a nuclear magnetic resonance spectroscopy H-NMR spectrum of polylysine, polylysine-polyethylene glycol-tyramine, the solvent is D 2 0;
- Figure 3 is an electron micrograph (SEM) of a polylysine-polyethylene glycol-tyramine hydrogel
- Polylysine purchased from Nanjing Xuankai Biotechnology Co., Ltd.;
- PNC PNC (p-nitrophenyl chloroformate)
- DMAP 4-dimethylaminopyridine
- TA tyramine
- TEA triethylamine
- PEG Polyethylene glycol
- Magnetic stirrer Model 85-2C, Shanghai Twisting Instrument Equipment Co., Ltd.
- Freeze dryer Model FD-1C-50, Beijing Bo Yikang Experimental Instrument Co., Ltd.
- Infrared spectrometer Model Nicolet 380, Thermo Company, USA.
- Example 1 Preparation of PNC-PEG-PNC.
- Example 1 The procedure of Example 1 was different except that the molar ratio of PEG, DMAP, TEA, and PNC was controlled to be 1:2:2:3, and the obtained PNC-PEG-PNC yield was 43%.
- EXAMPLES Preparation of PNC-PEG-PNC.
- Example 8 Preparation of PNC-PEG-PNC.
- Example 9 Preparation of PNC-PEG-PNC.
- the specification differs from the method of Example 1 in that the molar ratio of PEG, DMAP, TEA, and PNC is controlled to be 1:5:5:3, and the obtained PNC-PEG-PNC yield is 69%.
- Example 10 Preparation of PNC-PEG-PNC.
- Example 11 Preparation of PNC-PEG-PNC.
- Example 12 Preparation of grafted PEG and tyramine-polylysine.
- PNC-PEG-tyramine copolymer solution 0.14g (0.04mmol) of ⁇ -polylysine was weighed and dissolved in 280ml, pH 7.4, 0.01M phosphate buffer solution under room temperature and anaerobic conditions. After the lysine is completely dissolved, it is added to the PNC-PEG-tyramine copolymer solution, and the reaction is stirred at room temperature for 24 hours.
- the PNC salt is removed by filtration, and the mixture is placed in a dialysis bag having a molecular weight cut off of 7000 D & Dialysis in pure water to remove unreacted materials and by-products, 5-6 times of ultrapure water per day, dialysis for 5 days, and finally freeze-drying at -60 ° C to obtain polylysine of grafted PEG and tyramine, yield 65 %.
- Example 13 Preparation of e-polylysine grafted with PEG and tyramine.
- Example 15 Preparation of e-polylysine grafted with PEG and tyramine.
- Example 16 Preparation of £-polylysine grafted with PEG and tyramine.
- Example 17 Preparation of £-polylysine grafted with PEG and tyramine.
- Example 18 Preparation of e-polylysine grafted with PEG and tyramine.
- Example 19 Preparation of ⁇ -polylysine grafted with PEG and tyramine.
- Example 12 The same procedure as in Example 12 was carried out except that the poly-lysine was completely dissolved and then added to the PNC-PEG-tyramine copolymer solution, and the reaction was stirred at room temperature for 12 hours, and the yield of the obtained e- pL-PEG-TA was 29. %.
- Example 20 Preparation of £-polylysine grafted with PEG and tyramine.
- Example 21 Preparation of a hydrogel.
- the grafted PEG prepared from Example 13 and the polylysine copolymer of tyramine were dissolved in a pH 7.4, 0.01 M phosphate buffer solution to prepare a solution having a concentration of 10% by weight, and then horseradish oxide enzyme was added. (Enzyme activity >250U, Sigma) and hydrogen peroxide, the final concentration of horseradish peroxidase and hydrogen peroxide were 0.03mg/ml, 0.06wt%, magnetically stirred at 25 °C to form polylysine hydrogel Glue with a gel time of 30 s.
- Example 22 Preparation of a hydrogel.
- the grafted PEG prepared from Example 13 and the tyramine & polylysine copolymer were dissolved in a pH 7.4, 0.01 M phosphate buffer solution to prepare a solution having a concentration of 10% by weight, followed by the addition of horseradish oxide.
- the final concentration of enzyme (enzyme activity >250U, Sigma) and hydrogen peroxide, horseradish peroxidase and hydrogen peroxide was 0.05mg/ml, 0.06wt%, magnetically stirred at 25 °C to form polylysine water. Gel with a gel time of 25 s.
- Example 23 Preparation of a hydrogel.
- the grafted PEG prepared from Example 13 and the tyramine & polylysine copolymer were dissolved in a pH 7.4, 0.01 M phosphate buffer solution to prepare a solution having a concentration of 10% by weight, followed by the addition of horseradish oxide.
- the final concentration of enzyme (enzyme activity >250U, Sigma) and hydrogen peroxide, horseradish peroxidase and hydrogen peroxide was 0.08mg/ml, 0.06wt%, and magnetic stirring at 25 °C to form polylysine water. Gel with a gel time of 22 s.
- Example 24 Preparation of a hydrogel.
- the grafted PEG prepared from Example 13 and the polylysine copolymer of tyramine were dissolved in a pH 7.4, 0.01 M phosphate buffer solution to prepare a solution having a concentration of 10% by weight, and then horseradish oxide enzyme was added.
- horseradish oxide enzyme was added.
- hydrogen peroxide, horseradish peroxidase and hydrogen peroxide were respectively 0.12mg/L, 0.06wt%, magnetically stirred at 25 °C to form polylysine hydrogel Glue with a gel time of 5 s.
- Example 25 Preparation of a hydrogel.
- Example 26 Preparation of a hydrogel.
- Example 21 The procedure of Example 21 was different except that the concentration of hydrogen peroxide was controlled to be 0.02% by weight, and the polylysine hydrogel was formed by magnetic stirring at 25 ° C with a gel time of 39 s.
- Example 27 Preparation of a hydrogel.
- Example 28 Preparation of a hydrogel.
- Example 29 The difference from the method of Example 21 is that the concentration of the hydrogel material s-pL-PEG-TA is controlled to be 5 wt%, and magnetic stirring is carried out at 25 ° C to form a polylysine hydrogel having a gel time of 54 s. .
- Example 29 The difference from the method of Example 21 is that the concentration of the hydrogel material s-pL-PEG-TA is controlled to be 5 wt%, and magnetic stirring is carried out at 25 ° C to form a polylysine hydrogel having a gel time of 54 s. .
- Example 30 Wound healing experiment.
- Example 1 The wound dressing of Example 1 was applied to the wound with 7 (% % alcohol disinfection, the control group was sutured by conventional surgical needles, and the control group 2 was coated with commercially available medical wound excipient fibrin glue, and the control group was used.
- the hydrogel dressing treatment group was superior to the suture-only treatment group; using the ⁇ -polylysine cross-linked polymer hydrogel dressing treatment group, the wound area was significantly smaller, and the healing speed was fast, and the surface was smooth and smooth. , showing excellent biocompatibility and ability to promote wound healing.
- the polylysine hydrogel wound dressing of the invention has good biocompatibility, is favorable for cell attachment growth, has a promoting effect on wound healing, and effectively reduces exudation of tissue fluid, and has a broad application prospect in the field of medical wound dressing.
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Abstract
本发明公开了一种ε-聚赖氨酸水凝胶,其具有如下结构单元(I),其中,n取值为20~30 中的任意自然数,m取值为50~70中的任意自然数。本发明还公开了上述ε-聚赖氨酸水凝胶的制备方法和应用。本发明的聚赖氨酸水凝胶对受体无毒害,且具有生物降解性和生物相容性。本发明制得的伤口组织愈合材料可以高效、稳定、安全的应用于伤口组织粘合。
Description
说 明 书 一种 聚赖氨酸水凝胶及其制备方法和应用 技术领域
本发明涉及一种 ε-聚赖氨酸水凝胶及其制备方法和应用, 属于医疗技术应用领域。 背景技术
水凝胶医用创伤辅料是近年来发展起来的一种新型的创伤辅料。 与传统的辅料相 比, 水凝胶能促进伤口更好地愈合、 减轻患者的疼痛, 它能改善创面的微环境、 抑制细 菌的生长。 水凝胶特别适用于常见的体表创伤, 如擦伤、 划伤、 褥疮等各种皮肤损伤。 对于这些伤口, 传统上医生一般用无菌纱布及外用抗生素处理。伹是纱布易于皮肤伤口 组织黏连, 换药时常常破坏新手的上皮和肉芽组织, 引起出血, 这不但不利于伤口的愈 合, 而且使病人疼痛难忍。 用水凝胶辅料敷贴在伤口上时, 它不但不粘连伤口、 不破坏 新生组织, 而且能杀死各种细菌、 避免伤口感染。 水凝胶辅料是一种良好的创面辅料, 它是含有一定水分的三维网状结构的高分子溶胀体, 具有良好的吸水性和生物相容性, 能与不平整的创面密切贴合而不会发生粘连, 减少了细菌滋生的机会且易于更换, 并且 可以渗入各种药物成分和生长因子, 可以促进伤口的愈合。 水凝胶本身的热容量大, 对 伤口有温和的冷却作用, 能减轻伤口的疼痛。 因此, 国内外学者对水凝胶辅料进行了广 泛的硏究并取得了一些成果。 目前, 水凝胶辅料占全球辅料和绷带市场的大部分。
目前应用于创伤辅料并已商业化生产的材料主要有纤维蛋白胶、氰基丙烯酸酯、 壳 聚糖等, 但由于都分别有其缺陷而限制了在临床外科医学中的广泛应用, 如纤维蛋白胶 主要来源于动物血液组织, 所以存在血液病毒感染的风险, 氰基丙烯酸酯虽然止血效果 良好但由于细胞毒性较大也不是伤口组织愈合的最佳选择材料,壳聚糖具有快速止血的 功能, 可以加工成各种医用辅料产品, 但由于其力学性能差、 脆性较大、 水溶性差等缺 点成为其应用于外科创伤辅料的主要阻碍因素。
聚赖氨酸是一种形成水凝胶的天然高分子材料, 由微生物发酵法制得。 ε-聚赖氨 酸的分子量在三千到五千道尔顿之间, 与天然细胞外基质(ECM)中的蛋白成分和功能 类似, 聚赖氨酸区别于其他创伤辅料材料的显著优势在于以下几个方面: 首先, 分子链 上拥有大量的氨基活性基团,其在水溶液或者酸性环境下结合氢离子带上正电荷形成阳 离子聚合物,这种阳离子聚合物可以很好的结合在细胞表面从而达到创伤组织粘合的目 的, 其次, 由于聚赖氨酸是天然高分子材料, 具有良好的生物相容性, 并且其降解产物
赖氨酸为人体必需氨基酸, 最后, 聚赖氨酸为氨基酸聚合物, 其水溶性良好, 克服了众 多组织愈合材料难溶于水的困难, 但由于聚赖氨酸携带大量的活性氨基基团, 其在水溶 液或者酸性环境下结合氢离子带上正电荷会一定程度上破坏组织细胞结构,具有一定的 细胞毒性, 要使其安全的应用于伤口组织粘合, 必须对其进行改性以降低细胞毒性。
PEG是一种水溶性和生物相容性良好的高分子, 被广泛用于生物材料的表面改性、 多肽和蛋白药物的修饰保护。 PEG修饰对一些引发的细胞凋亡有明显的抑制作用,显著 降低胞内 ROS (活性氧簇) 含量, 并保护线粒体膜电位的稳定。 PEG修饰主要通过减少 胞内 ROS (活性氧簇) 的产生来降低聚赖氨酸的细胞毒性,高修饰度有明显的降低毒性的 作用。 PEG化聚赖氨酸可以显著降低聚赖氨酸的细胞毒性, 降低毒性的能力随 PEG的 分子量和修饰度的增大而明显加强。
目前应用于创伤辅料的水凝胶医用材料主要分为化学合成和生物高分子材料两种, 但水凝胶材料形成过程存在材料机械强度差、生物相容性不好、细胞毒性大等诸多缺陷。 发明内容
本发明所要解决的技术问题是提供一种医用创伤辅料聚赖氨酸水凝胶。
本发明还要解决的技术问题是提供上述水凝胶的制备方法。
本发明最后要解决的技术问题是提供上述水凝胶的应用。
为解决上述技术问题, 本发明采用的技术方案如下:
一种 聚
其中, η取值为 20 30中的任意自然数, m取值为 50 70中的任意自然数。
上述 聚赖氨酸水凝胶的制备方法, 它包括如下步骤:
(1)将聚乙二醇 (PEG)溶解在二氯甲烷中, 再加入 4-二甲氨基吡啶 (DMAP)和三乙 胺 (TEA)室温下反应 20~150min, 反应结束后在室温、惰性气体保护条件下将反应液滴 加到对硝基苯基氯甲酸酯 (PNC)的二氯甲垸溶液中, 滴加完毕后在室温、惰性气体保护
说 明 书 条件下反应 12〜72h, 反应结束后,先将反应液旋蒸再滴加到冷乙醚中, 过滤后取沉淀再 真空冷冻干燥得到对硝基苯基氯甲酸酯-聚乙二醇-酪胺共聚物溶液共聚物 (PNC-PEG-PNC);
(2) 将歩骤 (1)得到的对硝基苯基氯甲酸酯-聚乙二醇-酪胺共聚物溶液共聚物 (PNC-PEG-PNC)溶解在磷酸缓冲液中, 然后加入溶有酪胺 (TA)的磷酸缓冲液, 室温下反 应 5~15h得到对硝基苯基氯甲酸酯-聚乙二醇 -酪胺共聚物 (PNC-PEG-酪胺)溶液:
(3)将 s-聚赖氨酸溶解在磷酸缓冲液中, 再加入歩骤 (2)得到的对硝基苯基氯甲酸酯 -聚乙二醇 -酪胺共聚物 (PNC-PEG-酪胺)溶液, 室温下反应 12~72h, 反应结束后过滤除去 对硝基苯基氯甲酸酯 (PNC)盐沉淀, 过滤后得到的滤液再在纯水中透析 4~8天, 冻干得 到接枝聚乙二醇 (PEG)和酪胺的 -聚赖氨酸;
(4)将步骤 (3)得到的接枝聚乙二醇(PEG)和酪胺的£ -聚赖氨酸溶解在水或者磷酸缓 冲液中, 加入辣根过氧化物酶与过氧化氢的混合水溶液, 室温下搅拌 5〜60s形成聚赖氨 酸水凝胶。
步骤 (1)中, 聚乙二醇的数均分子量范围为 4000~6000 , 优选 4000。
歩骤 (1)中, 聚乙二醇溶解在二氯甲烷中, 使得溶质聚乙二醇的浓度为 50〜2G0g/L, 优选 50~150g/L; 对硝基苯基氯甲酸酯的二氯甲垸溶液中, 溶质对硝基苯基氯甲酸酯的 浓度为 20~40g/L, 优选 25~35g/L。
步骤 (1)中, 再加入 4-二甲氨基吡啶和三乙胺室温下优选反应 30min。
歩骤 (1)中, 滴加完毕后在室温、 惰性气体保护条件下优选反应 24h。
歩骤 (1)中, 聚乙二醇、 4-二甲氨基吡啶、 三乙胺、 对硝基苯基氯甲酸酯的反应摩尔 比为 1 :2~5 :2~5 :2~5 ' 优选 1 :3 :3 :3。
歩骤 (1)中, 所述的冷乙醚温度为 -41;〜 -20°C, 优选 -4°C, 冷乙醚用量为旋蒸后反应 液体积的 20~40倍, 优选 30倍。
步骤 (1)中, 真空冷冻干燥温度为 -40'C~ -80°C, 优选 -60'C, 压力为 10 30 Pa, 优选
10Pa。
步骤 (1)中, 旋蒸温度为 50°C, 旋蒸到原液体积的 5〜10%, 优选 5%。
步骤 (1)中,所有操作都在室温无水无氧条件下进行,所述的无氧条件可采用通入惰 性气体的方式保护, 优选通入氮气保护。
步骤 (2)、 (3)和 (4)中, 所述的磷酸缓冲液为 pH7.4、 0.01~0.2mol/L的磷酸缓冲液,
说 明 书 优选 pH7.4、 0.01mol/L的磷酸缓冲液。
步骤 (2)中, 对硝基苯基氯甲酸酯-聚乙二醇 -对硝基苯基氯甲酸酯共聚物溶解在磷酸 缓冲液中, 溶质对硝基苯基氯甲酸酯-聚乙二醇-对硝基苯基氯甲酸酯共聚物的浓度为 50〜100g/L, 优选 80g/L; 溶有酪胺的磷酸缓冲液中, 溶质酪胺的浓度为 3〜10g L, 优选 2〜6g/L。
步骤 (2)中, 对硝基苯基氯甲酸酯-聚乙二醇 -对硝基苯基氯甲酸酯共聚物与酪胺的摩 尔比为 2:0.5〜1.5, 优选 2: 1。
步骤 (2)中, 优选室温下反应 10h得到对硝基苯基氯甲酸酯 -聚乙二醇-酪胺共聚物溶 液。
歩骤 (2)中,所有操作都在室温无氧条件下进行,所述的无氧条件可采用通入惰性气 体的方式保护, 优选通入氮气保护。
歩骤 (3)中, 聚赖氨酸溶解在磷酸缓冲液中, 溶质聚赖氨酸浓度为 0.434.09g/L, 优 选 0.4〜0.8g/L。
步骤 (3)中, 聚赖氨酸与对硝基苯基氯甲酸酯-聚乙二醇-对硝基苯基氯甲酸酯共聚 物的摩尔比为 0.04〜0.07: 2, 优选 0.05:2。
歩骤 (3)中, 优选室温下反应 24h。
步骤 (3)中, 优选透析 5天。
步骤 (3)中, 冻干温度为 -60°C。
歩骤 (3)中,所有操作都在室温无氧条件下进行,所述的无氧条件可采用通入惰性气 体的方式保护, 优选通入氮气保护。
步骤 (4)中, 将步骤 (3)得到的接枝聚乙二醇和酪胺的 &聚赖氨酸溶解在水或者磷酸 缓冲液中, 溶质接枝聚乙二醇和酪胺的 ε-聚赖氨酸浓为 5〜10wt%, 优选 10wt%。
步骤 (4)中,辣根过氧化物酶与过氧化氢的混合水溶液中,溶质辣根过氧化物酶和过 氧化氢在水溶液中的浓度分别为 0.003~0.12mg/mL和 0.02~0.07wt%, 优选 0.12mg/mL 和 0.06wt%。
步骤 (4)中,所有反应操作都在室温无氧条件下进行,所述的无氧条件可采用通入惰 性气体的方式保护, 优选通入氮气保护。
上述方法制备得到的 聚赖氨酸水凝胶也在本发明的保护范围之内。
上述 s-聚赖氨酸水凝胶在制备创伤辅料中的应用, 可以应用于医疗或者美容领域。
说 明 书 有益效果: 本发明通过使用天然高分子材料聚赖氨酸作为创伤辅料支架, 通过接枝 聚乙二醇和酪胺对聚赖氨酸分子骨架进行修饰,再在辣根过氧化物酶和过氧化氢的作用 下形成聚赖氨酸-聚乙二醇 -酪胺三维网状结构的水凝胶医用材料, 该创伤辅料材料具有 机械强度大、 凝胶时间快且凝胶强度可调控、 反应条件温和、 组织毒性低等诸多优势, 克服了现有创伤辅料存在的材料机械强度差、生物相容性不好、细胞毒性大等诸多缺陷, 在医疗领域具有很大应用前景。
附图说明
图 1 是制备的聚赖氨酸 -聚乙二醇-酪胺的示意图及分子结构;
图 2是聚赖氨酸、 聚赖氨酸 -聚乙二醇-酪胺的核磁共振氢谱 H-NMR)谱图, 溶剂为 D20;
图 3 是聚赖氨酸-聚乙二醇-酪胺水凝胶的电镜图片 (SEM);
图 4 是老鼠背部伤口愈合实验, a)对照组为人工缝线愈合, b)为纤维蛋白胶敷料处理组, c)为氰基丙烯酸酯敷料处理组, d)聚赖氨酸-聚乙二醇水凝胶辅料, e)为本发明聚赖氨 酸水凝胶敷料处理组。
具体实施方式
根据下述实施例, 可以更好地理解本发明。 然而, 本领域的技术人员容易理解, 实 施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的 本发明。
以下实施例所用试剂来源如下:
聚赖氨酸: 购于南京轩凯生物科技有限公司;
PNC(PNC (对硝基苯基氯甲酸酯 ))、 DMAP(4-二甲氨基吡啶)购自 Sigma-Aldrich 公 司; TA (酪胺) 购自 Energy chemical; TEA (三乙胺)、 PEG (聚乙二醇)购自国药集团。
以下实施例所用设备来源如下:
磁力搅拌器: 型号 85-2C, 上海扭航仪器设备有限公司。
冷冻干燥机: 型号 FD-1C-50, 北京博医康实验仪器有限公司。
真空干燥箱: 型号 YZG-600, 南京焱泰电热设备有限公司。
红外光谱仪: 型号 Nicolet 380, 美国 Thermo公司。
核磁共振仪: 型号 AVANCE AV-500, 美国 Bruker Daltonics 公司。
说 明 书
实施例 1 : PNC-PEG-PNC的制备。
在室温并在无水无氧条件下, 称取 PEG4000 20g(5mmol)置于 500ml三口烧瓶中, 加入 400ml无水二氯甲烷, 在磁力搅拌下待 PEG完全溶解后加 1.832g(15mmol)DMAP 和 1.518g(15mmol)TEA, 室温下搅拌反应 30 分钟, 反应结束后将其滴加到 3.022g(15mmol)PNC溶液 (以 100ml无水二氯甲烷为溶剂)中, 递交完毕后在室温并在氮 气保护下搅拌反应 24h, 反应结束后, 先将反应液用旋转蒸发仪浓縮至 25ml, 再滴加到 -4Ό冷乙醚中, 冷乙醚用量为 750ml, 形成沉淀后进行过滤取沉淀再 -60°C, 10Pa真空冷 冻干燥得到 PNC-PEG-PNC共聚物, 收率为 79%。 实施例 2: PNC-PEG-PNC的制备。
在室温并在无水无氧条件下, 称取 PEG4000 20g(5mmol)置于 500ml三口烧瓶中, 加入 200ml无水二氯甲烷, 在磁力搅拌下待 PEG完全溶解后加 1.832g(15mmoDDMAP 和 ] .518g(15mm0l)TEA , 室温下搅拌反应 30 分钟, 反应结束后将其滴加到 3.022g(15mmol)PNC溶液 (以 100ml无水二氯甲烷为溶剂)中, 递交完毕后在室温并在氮 气保护下搅拌反应 24h, 反应结束后, 先将反应液用旋转蒸发仪浓縮至 15ml, 再滴加到 -4Ό冷乙醚中, 冷乙醚用量为 450ml, 形成沉淀后进行过滤取沉淀再 -60°C, 10Pa真空冷 冻干燥得到 PNC-PEG-PNC共聚物, 收率为 87%。 实施例 3: PNC-PEG-PNC的制备。
在室温并在无水无氧条件下, 称取 PEG6000 20g(5mmol)置于 500ml三口烧瓶中, 加入 100ml无水二氯甲垸, 在磁力搅拌下待 PEG完全溶解后加 1.832g(15mmol)DMAP 和 ] .518g(15mmol)TEA, 室温下搅拌反应 30 分钟, 反应结束后将其滴加到 3.022g(15mmol)PNC溶液 (以 100ml无水二氯甲烷为溶剂)中, 递交完毕后在室温并在氮 气保护下搅拌反应 24h, 反应结束后, 先将反应液用旋转蒸发仪浓縮至 10ml, 再滴加到 -4°C冷乙醚中, 冷乙醚用量为 300ml, 形成沉淀后进行过滤取沉淀再 -60°C, 10Pa真空冷 冻干燥得到 PNC-PEG-PNC共聚物, 收率为 62%。 实施例 4: PNC-PEG-PNC的制备。
说 明 书 在室温并在无水无氧条件下, 称取 PEG4000 20g(5mmol)置于 500ml三口烧瓶中, 加入 200ml无水二氯甲烷, 在磁力搅拌下待 PEG完全溶解后加 1.832g(15mmol)DMAP 和 1.518g(15mmol)TEA, 室温下搅拌反应 30 分钟, 反应结束后将其滴加到 3.022g(15mmol)PNC溶液 (以 120ml无水二氯甲垸为溶剂)中, 递交完毕后在室温并在氮 气保护下搅拌反应 24h, 反应结束后, 先将反应液用旋转蒸发仪浓缩至 16ml, 再滴加到 -4°C冷乙醚中, 冷乙醚用量为 480ml, 形成沉淀后进行过滤取沉淀再 -60°C, 10Pa真空冷 冻干燥得到 PNC-PEG-PNC共聚物, 收率为 77%。 实施例 5: PNC-PEG-PNC的制备。
在室温并在无水无氧条件下, 称取 PEG4000 20g 5mmol)置于 500ml三口烧瓶中, 加入 200ml无水二氯甲烷, 在磁力搅抨下待 PEG完全溶解后加 1.832g(15mmol)DMAP 和 ] .518g 15mmol)TEA , 室温下搅拌反应 30 分钟, 反应结束后将其滴加到 3.022g(15mmol)PNC溶液 (;以 86ml无水二氯甲烷为溶剂)中,递交完毕后在室温并在氮气 保护下搅拌反应 24h, 反应结束后, 先将反应液用旋转蒸发仪浓缩至 14.3ml, 再滴加到 -4°C冷乙醚中, 冷乙醚用量为 429ml, 形成沉淀后进行过滤取沉淀再 -60°C , 10Pa真空冷 冻干燥得到 PNC-PEG-PNC共聚物, 收率为 53%。 实施例 6: PNC-PEG-PNC的制备。
同实施例 1的方法,所不同的是控制 PEG、DMAP、TEA、PNC投料摩尔比为 :1 :2:2:3 , 所得 PNC-PEG-PNC收率为 43%。 实施例 Ί·· PNC-PEG-PNC的制备。
同实施例 1的方法,所不同的是控制 PEG、DMAP、TEA、PNC投料摩尔比为 :1 :2:5:3, 所得 PNC-PEG-PNC收率为 51%。 实施例 8: PNC-PEG-PNC的制备。
同实施例 1的方法,所不同的是控制 PEG、DMAP、TEA、PNC投料摩尔比为 :1 :5:2:3 , 所得 PNC-PEG-PNC收率为 38%。 实施例 9: PNC-PEG-PNC的制备。
说 明 书 同实施例 1的方法,所不同的是控制 PEG、DMAP、TEA、PNC投料摩尔比为 :1 :5:5:3, 所得 PNC- PEG-PNC收率为 69%。 实施例 10: PNC-PEG-PNC的制备。
同实施例 2的方法, 所不同的是加入 DMAP和 TEA后, 室温下搅拌反应 20分钟, 所得 PNC-PEG-PNC收率为 64%。 实施例 11 : PNC-PEG-PNC的制备。
同实施例 2的方法,所不同的是加入 DMAP和 TEA后,室温下搅拌反应 150分钟, 所得 PNC-PEG-PNC收率为 81%。 实施例 12: 接枝 PEG和酪胺的 -聚赖氨酸的制备。
在室温无氧条件下, 称取 TA 0.069g(0.5mmol)溶解在 14ml、 pH7.4、 0.01M磷酸盐 缓冲液中, 同时将实施例 2制备得到的 PNC-PEG-PNC共聚物 4g(2mmol)溶解在 50ml、 pH7.4、 0.01M磷酸盐缓冲液中, 待 PNC-PEG-PNC完全溶解后, 将酪胺磷酸盐溶液在 无氧条件下加入其中, 室温下搅拌反应 10h, 反应结束得到 PNC-PEG-酪胺共聚物溶液; 在室温无氧条件下, 称取 ε-聚赖氨酸 0.14g(0.04mmol)溶解在 280ml、 pH7.4、 0.01M磷 酸盐缓冲液中, 待&聚赖氨酸完全溶解后加入到 PNC-PEG-酪胺共聚物溶液, 室温下搅 拌反应 24h, 反应结束后过滤除去 PNC盐, 将混合液置于截止分子量为 7000D&的透析 袋中, 再在大量超纯水中透析除去未反应物质及副产物, 每天换 5-6次超纯水, 透析 5 天, 最后 -60°C冷冻干燥得到接枝 PEG和酪胺的 聚赖氨酸, 收率为 65%。 实施例 13 : 接枝 PEG和酪胺的 e-聚赖氨酸的制备。
在室温无氧条件下, 称取 TA0.137g(lmmol)溶解在 28ml、 pH7.4、 0.01M磷酸盐缓 冲液中, 同时将实施例 2制备得到的 PNC-PEG-PNC共聚物 4g(2mmol)溶解在 50ml、 pH7.4、 0.01M磷酸盐缓冲液中, 待 PNC-PEG-PNC完全溶解后, 将酪胺磷酸盐溶液在 无氧条件下加入其中, 室温下搅拌反应 10h, 反应结束得到 PNC-PEG-酪胺共聚物溶液; 在室温无氧条件下, 称取 聚赖氨酸 0.14g (0.04mmol)溶解在 280ml、 pH7.4、 0.01M磷 酸盐缓冲液中, 待 s-聚赖氨酸完全溶解后加入到 PNC-PEG-酪胺共聚物溶液, 室温下搅
说 明 书 拌反应 24h, 反应结束后过滤除去 PNC盐, 将混合液置于截止分子量为 7000Da的透析 袋中, 再在大量超纯水中透析除去未反应物质及副产物, 每天换 5-6次超纯水, 透析 5 天, 最后 -60°C冷冻干燥得到接枝 PEG和酪胺的 &聚赖氨酸, 收率为 77%。 由图 2, 下 面的图是聚赖氨酸氢谱图谱, 上面的图谱为合成的 s-pL-PEG-TA核磁氢谱图可以看到, 在原有聚赖氨酸的基础峰上出现化学位移为 3.5和 7.0的峰分别是接枝上的 PEG和酪胺, 表明合成的 ε-pL-PEG-TA合成正确。 实施例 14: 接枝 PEG和酪胺的 £ -聚赖氨酸的制备。
同实施例 12的方法, 所不同的是控制 PNC-PEG-PNC、 TA、 聚赖氨酸 ?-pL)的投 料摩尔比控制为 2: 1.5:0.04, 所得 £-pL-PEG-TA收率为 69%。 实施例 15 : 接枝 PEG和酪胺的 e-聚赖氨酸的制备。
同实施例 12的方法, 所不同的是控制 PNC-PEG-PNC、 ΤΑ、 聚赖氨酸 ?-pL)的投 料摩尔比控制为 2: 1 :0.06, 所得 s-pL-PEG-TA收率为 58%。 实施例 16: 接枝 PEG和酪胺的 £-聚赖氨酸的制备。
同实施例 12的方法, 所不同的是控制 PNC-PEG-PNC、 ΤΑ、 聚赖氨酸 (ε-pL)的投 料摩尔比控制为 2: 1.5:0.07, 所得 £-pL-PEG-TA收率为 63%。 实施例 17: 接枝 PEG和酪胺的 £ -聚赖氨酸的制备。
同实施例 12的方法, 所不同的是, 待 PNC-PEG-PNC完全溶解后, 将酪胺磷酸盐 溶液在无氧条件下加入其中, 室温下搅拌反应 5h, 所得 £-pL-PEG-TA收率为 45%。 实施例 18: 接枝 PEG和酪胺的 e-聚赖氨酸的制备。
同实施例 12的方法, 所不同的是, 待 PNC-PEG-PNC完全溶解后, 将酪胺磷酸盐 溶液在无氧条件下加入其中, 室温下搅拌反应 15h, 所得 e-pL-PEG-TA收率为 72%。 实施例 19: 接枝 PEG和酪胺的 ε -聚赖氨酸的制备。
同实施例 12的方法,所不同的是,待 聚赖氨酸完全溶解后加入到 PNC-PEG-酪胺 共聚物溶液, 室温下搅拌反应 12h, 所得 e-pL-PEG-TA收率为 29%。
说 明 书
实施例 20: 接枝 PEG和酪胺的 £ -聚赖氨酸的制备。
同实施例 12的方法,所不同的是,待 聚赖氨酸完全溶解后加入到 PNC-PEG-酪胺 共聚物溶液, 室温下搅拌反应 72h, 所得 ε-pL-PEG-TA收率为 67%。 实施例 21 : 水凝胶的制备。
将实施例 13制备得到的接枝 PEG和酪胺的 聚赖氨酸共聚物溶解在 pH7.4、 0.01M 磷酸盐缓冲液中, 配制成浓度为 10wt%的溶液, 再加入辣根氧化物酶 (酶活 >250U, Sigma)和过氧化氢,辣根过氧化物酶和过氧化氢的终浓度分别为 0.03mg/ml, 0.06wt%, 25 °C下磁力搅拌形成聚赖氨酸水凝胶, 其凝胶时间为 30s。 实施例 22: 水凝胶的制备。
将实施例 13制备得到的接枝 PEG和酪胺的&聚赖氨酸共聚物溶解在 pH7.4、 0.01M 磷酸盐缓冲液中, 配制成浓度为 10wt%的溶液, 再加入辣根氧化物酶 (酶活 >250U, Sigma)和过氧化氢,辣根过氧化物酶和过氧化氢的终浓度分别为 0.05mg/ml, 0.06wt%, 25 °C下磁力搅拌形成聚赖氨酸水凝胶, 其凝胶时间为 25s。 实施例 23 : 水凝胶的制备。
将实施例 13制备得到的接枝 PEG和酪胺的&聚赖氨酸共聚物溶解在 pH7.4、 0.01M 磷酸盐缓冲液中, 配制成浓度为 10wt%的溶液, 再加入辣根氧化物酶 (酶活 >250U, Sigma)和过氧化氢,辣根过氧化物酶和过氧化氢的终浓度分别为 0.08mg/ml, 0.06wt%, 25 °C下磁力搅拌形成聚赖氨酸水凝胶, 其凝胶时间为 22s。 实施例 24: 水凝胶的制备。
将实施例 13制备得到的接枝 PEG和酪胺的 聚赖氨酸共聚物溶解在 pH7.4、 0.01M 磷酸盐缓冲液中, 配制成浓度为 10wt%的溶液, 再加入辣根氧化物酶 (酶活 >250U, Sigma) 和过氧化氢, 辣根过氧化物酶和过氧化氢的终浓度分别为 0.12mg/L, 0.06wt%, 25 °C下磁力搅拌形成聚赖氨酸水凝胶, 其凝胶时间为 5s。 实施例 25 : 水凝胶的制备。
说 明 书 同实施例 21 的方法, 所不同的是控制过氧化氢的浓度为 0.04wt%, 25 °C下磁力搅 拌形成聚赖氨酸水凝胶, 其凝胶时间为 17s。 实施例 26: 水凝胶的制备。
同实施例 21 的方法, 所不同的是控制过氧化氢的浓度为 0.02wt%, 25 °C下磁力搅 拌形成聚赖氨酸水凝胶, 其凝胶时间为 39s。 实施例 27: 水凝胶的制备。
同实施例 21的方法 '所不同的是控制水凝胶材料 s-pL-PEG-TA的浓度为 7wt%, 25 °C 下磁力搅拌形成聚赖氨酸水凝胶, 其凝胶时间为 25s。 实施例 28 : 水凝胶的制备。
同实施例 21的方法 '所不同的是控制水凝胶材料 s-pL-PEG-TA的浓度为 5wt%, 25 °C 下磁力搅拌形成聚赖氨酸水凝胶, 其凝胶时间为 54s。 实施例 29:
将实施例 24制得的聚赖氨酸 -PEG-酪胺水凝胶样品进行冻干后,少量固定于硅片后 过夜风干, 在扫描电镜 100倍下观察其表面交联形态, 如图 3。 实施例 30: 伤口愈合实验。
在老鼠背部剪毛后, 再用硫化钠溶液脱毛 48h, 每只老鼠皮下注射 0.5%利多卡因注 射液 0.5 mL局麻, 供 3处, 在背部用手术刀划出直径约 2.0 cm的切口两处, 用 7(^ %酒 精消毒, 将实施例 1中的创伤敷料涂布在伤口处, 对照组一用常规的手术针线缝合, 对 照组二采用市售医用创伤辅料纤维蛋白胶涂敷,对照组三采用市售医用创伤辅料多抹棒 涂敷, 对照组四采用聚赖氨酸 -PEG水凝胶 [Polylysine-Modified PEG-Based Hydrogels to Enhance the Neuro-Electrode Interface, Journal of Biomaterials Science, Polymer Edition Volume 22, Issue 4-6, 2011]涂敷。 实验过程中各组均未发生细菌感染现象, 分别于 0, 7, 14夭后观察伤口愈合愔况。参见图 5, a)对照组为人工缝线愈合, b)为纤维蛋白胶敷料处 理组, c)为氰基丙烯酸酡敷料处理组, d) 聚赖氨酸 -PEG (聚乙二醇)水凝胶辅料, e ) 为 本发明聚赖氨酸水凝胶敷料处理组。 随着治疗时间的延长, 四组老鼠伤口均有愈合的现
说 明 书 象, 水凝胶敷料处理组均优于仅用缝合处理组; 使用 ε-聚赖氨酸交联聚合物水凝胶敷料 处理组, 伤口面积明显变小, 且愈合速度快, 表面光滑平整, 显示出优良的生物相容性 及促进伤口愈合的能力。
综合评价: 本发明的 聚赖氨酸水凝胶创伤敷料, 生物相容性良好, 有利于细胞附 着生长, 对伤口愈合具有促进作用, 有效减少组织液渗出, 在医用创伤敷料领域应用前 景广阔。
Claims
(1) 将聚乙二醇溶解在二氯甲烷中, 再加入 4-二甲氨基吡啶和三乙胺室温下反应 20~150min, 反应结束后在室温、 惰性气体保护条件下将反应液滴加到对硝基苯基氯甲 酸酯的二氯甲烷溶液中, 滴加完毕后在室温、惰性气体保护条件下反应 12~72h, 反应结 束后, 先将反应液旋蒸再滴加到冷乙醚中, 过滤后取沉淀再真空冷冻干燥得到对硝基苯 基氯甲酸酯-聚乙二醇-对硝基苯基氯甲酸酯共聚物;
(2) 将步骤 (1)得到的对硝基苯基氯甲酸酯-聚乙二醇-对硝基苯基氯甲酸酯共聚物溶 解在磷酸缓冲液中, 然后加入溶有酪胺的磷酸缓冲液, 室温下反应 5~15h得到对硝基苯 基氯甲酸酯-聚乙二醇 -酪胺共聚物溶液;
(3) 将 £-聚赖氨酸溶解在磷酸缓冲液中, 再加入歩骤 (2)得到的对硝基苯基氯甲酸酯 -聚乙二醇 -酪胺共聚物溶液, 室温下反应 12~72h, 反应结束后过滤除去对硝基苯基氯甲 酸酯盐沉淀, 过滤后得到的滤液再在纯水中透析 4~8天, 冻干得到接枝聚乙二醇和酪胺 的 ε-聚赖氨酸;
(4) 将步骤 (3)得到的接枝聚乙二醇和酪胺的 £-聚赖氨酸溶解在水或者磷酸缓冲液 中, 加入辣根过氧化物酶与过氧化氢的混合水溶液, 室温下搅拌 5~60s形成聚赖氨酸水 凝胶。
3、根据权利要求 2所述的 ε-聚赖氨酸水凝胶的制备方法, 其特征在于, 步骤 (1)中, 聚乙二醇溶解在二氯甲垸中, 使得溶质聚乙二醇的浓度为 50~200g/L; 对硝基苯基氯甲 酸酯的二氯甲烷溶液中, 溶质对硝基苯基氯甲酸酯的浓度为 20~40g/L。
4、根据权利要求 2所述的 ε-聚赖氨酸水凝胶的制备方法, 其特征在于, 步骤 (1)中,
权 利 要 求 书 聚乙二醇、 4-二甲氨基吡啶、 三乙胺、 对硝基苯基氯甲酸酯的反应摩尔比为 1:2-5 :2-5:2-5 o
5、根据权利要求 2所述的 ε-聚赖氨酸水凝胶的制备方法, 其特征在于, 步骤 (1)中, 所述的冷乙醚温度为 -4 'C ~ -20 °C , 冷乙醚用量为旋蒸后反应液体积的 20~40倍。
6、根据权利要求 2所述的 ε-聚赖氨酸水凝胶的制备方法, 其特征在于, 步骤 (1)中, 真空冷冻干燥温度为 -40°C~ -80 °C , 压力为 10~30 Pa。
7、根据权利要求 2所述的 s-聚赖氨酸水凝胶的制备方法, 其特征在于, 步骤 (1)中, 所有操作都在室温无水无氧条件下进行。
8、 根据权利要求 2所述的 £-聚赖氨酸水凝胶的制备方法, 其特征在于, 步骤 (2)、 (3)和 (4)中, 所述的磷酸缓冲液为 pH7.4、 G.01~0.2mOl/L的磷酸缓冲液。
9、根据权利要求 2所述的 £ -聚赖氨酸水凝胶的制备方法, 其特征在于, 步骤 (2)中, 对硝基苯基氯甲酸酯-聚乙二醇-对硝基苯基氯甲酸酯共聚物溶解在磷酸缓冲液中, 溶质 硝基苯基氯甲酸酯-聚乙二醇-对硝基苯基氯甲酸酯共聚物的浓度为 50~100g/L; 溶有酪 胺的磷酸缓冲液中, 溶质酪胺的浓度为 3~10g L。
10、 根据权利要求 2所述的 e-聚赖氨酸水凝胶的制备方法, 其特征在于, 歩骤 (2) 中, 对硝基苯基氯甲酸酯 -聚乙二醇-对硝基苯基氯甲酸酯共聚物与酪胺的摩尔比为 2:0·5~·1 ·5。
11、 根据权利要求 2所述的 e-聚赖氨酸水凝胶的制备方法, 其特征在于, 步骤 (2) 中, 所有操作都在室温无氧条件下进行。
12、 根据权利要求 2所述的 ε-聚赖氨酸水凝胶的制备方法, 其特征在于, 歩骤 (3) 中, 聚赖氨酸溶解在磷酸缓冲液中, 溶质聚赖氨酸浓度为 0.43~1.09g/L。
13、 根据权利要求 2所述的 ε-聚赖氨酸水凝胶的制备方法, 其特征在于, 歩骤 (3) 中, 5-聚赖氨酸与对硝基苯基氯甲酸酯-聚乙二醇-对硝基苯基氯甲酸酯共聚物的摩尔比 为 0.04~0.07: 2。
14、 根据权利要求 2所述的 ε-聚赖氨酸水凝胶的制备方法, 其特征在于, 步骤 (3) 中, 所有操作都在室温无氧条件下进行。
15、 根据权利要求 2所述的 ε-聚赖氨酸水凝胶的制备方法, 其特征在于, 步骤 (4) 中, 将步骤 (3)得到的接枝 PEG (聚乙二醇)和酪胺的 ε-聚赖氨酸溶解在氷或者磷酸缓冲液 中, 溶质接枝 PEG (聚乙二醇)和酪胺的 聚赖氨酸浓度为 10~15wt%。
权 利 要 求 书
16、 根据权利要求 2所述的 ε-聚赖氨酸水凝胶的制备方法, 其特征在于, 歩骤 (4) 中, 辣根过氧化物酶与过氧化氢的混合水溶液中, 溶质辣根过氧化物酶和过氧化氢在水 溶液中的浓度分别为 0.003~0.12mg/mL和 0.02~0.07wt%。
Π、 根据权利要求 2所述的 ε-聚赖氨酸水凝胶的制备方法, 其特征在于, 步骤 (4) 中, 所有反应操作都在室温无氧条件下进行。
18、 权利要求 2~17任意一项方法制备得到的 聚赖氨酸水凝胶。
19,权利要求 1或权利要求 18所述的 £-聚赖氨酸水凝胶在制备创伤辅料中的应用。
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