CN114276528A - Tissue adhesive and preparation method and application thereof - Google Patents
Tissue adhesive and preparation method and application thereof Download PDFInfo
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- CN114276528A CN114276528A CN202210030960.7A CN202210030960A CN114276528A CN 114276528 A CN114276528 A CN 114276528A CN 202210030960 A CN202210030960 A CN 202210030960A CN 114276528 A CN114276528 A CN 114276528A
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- 238000010438 heat treatment Methods 0.000 claims description 30
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- 239000002202 Polyethylene glycol Substances 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 23
- 239000003054 catalyst Substances 0.000 claims description 18
- 239000000178 monomer Substances 0.000 claims description 16
- RKDVKSZUMVYZHH-UHFFFAOYSA-N 1,4-dioxane-2,5-dione Chemical compound O=C1COC(=O)CO1 RKDVKSZUMVYZHH-UHFFFAOYSA-N 0.000 claims description 15
- 238000006116 polymerization reaction Methods 0.000 claims description 14
- KSBAEPSJVUENNK-UHFFFAOYSA-L tin(ii) 2-ethylhexanoate Chemical compound [Sn+2].CCCCC(CC)C([O-])=O.CCCCC(CC)C([O-])=O KSBAEPSJVUENNK-UHFFFAOYSA-L 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 7
- 239000012043 crude product Substances 0.000 claims description 6
- 239000002184 metal Substances 0.000 claims description 6
- 229910052751 metal Inorganic materials 0.000 claims description 6
- CANRESZKMUPMAE-UHFFFAOYSA-L Zinc lactate Chemical compound [Zn+2].CC(O)C([O-])=O.CC(O)C([O-])=O CANRESZKMUPMAE-UHFFFAOYSA-L 0.000 claims description 3
- 125000003545 alkoxy group Chemical group 0.000 claims description 3
- 125000000217 alkyl group Chemical group 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- 229940050168 zinc lactate Drugs 0.000 claims description 3
- 235000000193 zinc lactate Nutrition 0.000 claims description 3
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- 238000004519 manufacturing process Methods 0.000 claims 2
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- 239000000047 product Substances 0.000 description 51
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 50
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 36
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- 238000000113 differential scanning calorimetry Methods 0.000 description 8
- 238000001228 spectrum Methods 0.000 description 8
- 229920000436 Poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) Polymers 0.000 description 7
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- 210000004027 cell Anatomy 0.000 description 5
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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Images
Abstract
The invention belongs to the technical field of medical materials, and particularly relates to a tissue adhesive as well as a preparation method and application thereof. The tissue adhesives provided herein comprise a structural repeat unit of formula (I), a structural repeat unit of formula (II), and a structural repeat unit of formula (III). The tissue adhesive provided by the invention is a block copolymer containing a hydrophilic block and a hydrophobic block, has good biocompatibility and adhesiveness, can be fixed to an adhesive interface only by applying pressure during clinical use, does not need other curing processes, greatly reduces the operation difficulty during clinical use, and has wide application prospects in the biomedical field, such as wound adhesion and other fields.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a tissue adhesive as well as a preparation method and application thereof.
Background
Tissue adhesives can reduce tissue damage by non-invasively immobilizing skin tissue or organs, potentially eliminating the need for sutures or staples during certain surgical procedures. Currently, adhesion of most tissue adhesives relies on a curing step, with the crosslinkable components of the adhesive crosslinking upon mixing, thereby creating adhesion. Tissue adhesives of this type, in order to complete the curing step, must be deposited on the surgical site as a liquid at the time of clinical use, which presents difficulties to the surgeon's operation, which may lead to longer operation times and unforeseen complications, such as thromboembolism.
Disclosure of Invention
In view of the above, the present invention aims to provide a tissue adhesive, and a preparation method and an application thereof, wherein the tissue adhesive provided by the present invention has good biocompatibility and adhesiveness, and can be fixed to an adhesive interface only by applying pressure during use without other curing processes, thereby greatly reducing the operation difficulty during clinical use.
The invention provides a tissue adhesive, which comprises a structural repeating unit of a formula (I), a structural repeating unit of a formula (II) and a structural repeating unit of a formula (III);
preferably, the tissue adhesive has the structure of formula (a), formula (B), formula (C), formula (D), or formula (E):
in formulas (A) to (E), R has a structure of formula (IV):
a to f represent polymerization degrees.
Preferably, 9. ltoreq. a. ltoreq.182; b is more than or equal to 9 and less than or equal to 182; c is more than or equal to 9 and less than or equal to 182; d is more than or equal to 9 and less than or equal to 182; e is more than or equal to 9 and less than or equal to 182; f is more than 0 and less than or equal to 80; g is more than 0 and less than or equal to 120.
The invention provides a preparation method of a tissue adhesive, which comprises the following steps:
a) in the presence of a catalyst, carrying out polymerization reaction on a macroinitiator, lactide and glycolide to obtain a tissue adhesive;
the macroinitiator is polyethylene glycol and/or multi-arm polyethylene glycol.
Preferably, the lactide is L-type lactide or L/D mixed lactide.
Preferably, the molar ratio of the macroinitiator, lactide and glycolide is 1: (1-120): (1-80).
Preferably, the catalyst is one or more of zinc lactate, stannous octoate, alkyl metal and alkoxy metal.
Preferably, the temperature of the polymerization reaction is 80-130 ℃; the polymerization reaction time is 24-72 h.
Preferably, step a) specifically comprises:
a1) mixing a macroinitiator, lactide, glycolide, a catalyst and a solvent, and heating for reaction to obtain a crude product;
a2) removing the solvent and unreacted monomers from the crude product to obtain the tissue adhesive.
The invention provides a preparation for tissue adhesion, which comprises a tissue adhesive and a solvent, wherein the tissue adhesive is the tissue adhesive in the technical scheme or the tissue adhesive prepared by the preparation method in the technical scheme.
Compared with the prior art, the invention provides a tissue adhesive, and a preparation method and application thereof. The tissue adhesives provided herein comprise a structural repeat unit of formula (I), a structural repeat unit of formula (II), and a structural repeat unit of formula (III). The tissue adhesive provided by the invention is a block copolymer containing a hydrophilic block and a hydrophobic block, has good biocompatibility and adhesiveness, can be fixed to an adhesive interface only by applying pressure during clinical use, does not need other curing processes, greatly reduces the operation difficulty during clinical use, and has wide application prospects in the biomedical field, such as wound adhesion and other fields.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a nuclear magnetic spectrum of a tissue adhesive product of example 1 provided by the present invention;
FIG. 2 is a nuclear magnetic spectrum of a tissue adhesive product of example 2 provided by the present invention;
FIG. 3 is a nuclear magnetic spectrum of a tissue adhesive product of example 3 provided by the present invention;
FIG. 4 is a nuclear magnetic spectrum of a tissue adhesive product of example 4 provided by the present invention;
FIG. 5 is a nuclear magnetic spectrum of a tissue adhesive product of example 5 provided by the present invention;
FIG. 6 is a nuclear magnetic spectrum of a tissue adhesive product of example 6 provided by the present invention;
FIG. 7 is a nuclear magnetic spectrum of a tissue adhesive product of example 7 provided by the present invention;
FIG. 8 is a nuclear magnetic spectrum of a tissue adhesive product of example 8 provided by the present invention;
FIG. 9 is a GPC chart of a tissue adhesive product of example 1 provided by the present invention;
FIG. 10 is a GPC chart of a tissue adhesive product of example 2 provided by the present invention;
FIG. 11 is a GPC chart of a tissue adhesive product of example 3 provided by the present invention;
FIG. 12 is a GPC chart of a tissue adhesive product of example 4 provided by the present invention;
FIG. 13 is a GPC chart of a tissue adhesive product of example 5 provided by the present invention;
FIG. 14 is a GPC chart of a tissue adhesive product of example 6 provided by the present invention;
FIG. 15 is a GPC chart of a tissue adhesive product of example 7 provided by the present invention;
FIG. 16 is a DSC of a tissue adhesive product of example 1 provided by the present invention;
FIG. 17 is a DSC of a tissue adhesive product of example 2 provided by the present invention;
FIG. 18 is a DSC of a tissue adhesive product of example 3 provided by the present invention;
FIG. 19 is a DSC of a tissue adhesive product of example 4 provided by the present invention;
FIG. 20 is a DSC of a tissue adhesive product of example 5 provided by the present invention;
FIG. 21 is a DSC of a tissue adhesive product of example 6 provided by the present invention;
FIG. 22 is a DSC of a tissue adhesive product of example 7 provided by the present invention;
FIG. 23 is a graph of lap shear test results for tissue adhesive products of examples 1-8 provided herein;
FIG. 24 is a graph showing the results of a cell compatibility test for the tissue adhesive product of example 1 provided by the present invention;
fig. 25 is a graph of animal wound repair experimental results for the tissue adhesive product of example 1 provided by the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a tissue adhesive, which comprises a structural repeating unit of a formula (I), a structural repeating unit of a formula (II) and a structural repeating unit of a formula (III);
the tissue adhesive provided by the invention is a block copolymer containing structural repeating units of formula (I), formula (II) and formula (III), and has a hydrophilic block and a hydrophobic block. Wherein the hydrophilic block is a polyethylene glycol fragment structure or a multi-arm polyethylene glycol fragment structure consisting of the structural repeating units of the formula (I); the hydrophobic block is a lactic acid-co-glycolic acid fragment structure consisting of structural repeating units of a formula (II) and a formula (III).
In the present invention, the tissue adhesive preferably has a structure of formula (a), formula (B), formula (C), formula (D), or formula (E):
in formulas (A) to (E), R has a structure of formula (IV):
in the tissue adhesive having a polymeric chemical structure according to the present invention, a to f represent a degree of polymerization; preferably, 9. ltoreq. a. ltoreq.182; b is more than or equal to 9 and less than or equal to 182; c is more than or equal to 9 and less than or equal to 182; d is more than or equal to 9 and less than or equal to 182; e is more than or equal to 9 and less than or equal to 182; f is more than 0 and less than or equal to 80; g is more than 0 and less than or equal to 120; more preferably, 9. ltoreq. a.ltoreq.100; b is more than or equal to 9 and less than or equal to 100; c is more than or equal to 9 and less than or equal to 100; d is more than or equal to 9 and less than or equal to 100; e is more than or equal to 9 and less than or equal to 100; f is more than 0 and less than or equal to 40; g is more than 0 and less than or equal to 80; most preferably, a is 9-50; b is more than or equal to 9 and less than or equal to 50; c is more than or equal to 9 and less than or equal to 50; d is more than or equal to 9 and less than or equal to 50; e is more than or equal to 9 and less than or equal to 50; f is more than 0 and less than or equal to 20; g is more than 0 and less than or equal to 40. In one embodiment provided by the present invention, a may be specifically 9, 23, 45; c may be specifically 11; f can be 6, 8, 9, 12, 17; g may be 10, 11, 13, 14, 15, 18, 30.
In the present invention, the number average molecular weight of the tissue adhesive is preferably 2 × 103~1×104Specifically, it may be 2 × 103、2.3×103、2.5×103、2.7×103、3×103、3.2×103、3.4×103、3.6×103、3.8×103、4×103、4.2×103、4.4×103、4.6×103、4.8×103、5×103、5.2×103、5.5×103、5.7×103、6×103、6.2×103、6.5×103、6.7×103、7×103、7.2×103、7.5×103、8×103、8.5×103、9×103、9.5×103Or 1X 104。
The tissue adhesive provided by the invention is a block copolymer containing a hydrophilic block and a hydrophobic block, has good biocompatibility and adhesiveness, can be fixed to an adhesive interface only by applying pressure during clinical use, does not need other curing processes, greatly reduces the operation difficulty during clinical use, and has wide application prospects in the biomedical field, such as wound adhesion and other fields. More specifically, the present invention provides tissue adhesives that include the following advantages:
1) the transparent and colloidal state is presented, the fixed part of the object is basically invisible when the transparent base material is used, and the wound condition can be observed in real time when the transparent base material is used for adhering skin tissues;
2) has high pressure-sensitive adhesiveness and can be repeatedly peeled from the substrate after a long time without damaging the substrate;
3) the paint can be dissolved in solvents such as water, alcohol, dichloromethane and the like, and can be used in a mode of dissolving in the solvent firstly and then volatilizing the solvent after spraying;
4) has biodegradability, and can be gradually degraded with time after the biodegradable material acts.
The invention also provides a preparation method of the tissue adhesive, which comprises the following steps:
a) and (3) carrying out polymerization reaction on the macroinitiator, Lactide (LA) and Glycolide (GA) in the presence of a catalyst to obtain the tissue adhesive.
In the preparation method provided by the invention, the macroinitiator is polyethylene glycol and/or multi-arm polyethylene glycol, and the multi-arm polyethylene glycol can be specifically selected from one or more of three-arm polyethylene glycol, four-arm polyethylene glycol, six-arm polyethylene glycol and eight-arm polyethylene glycol; the number average molecular weight of the macroinitiator is preferably 400-8000, and specifically can be 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500 or 8000.
In the preparation method provided by the invention, the lactide is preferably L-type lactide or L/D mixed type lactide, and the molar ratio of L-type lactide to D-type lactide in the L/D mixed type lactide is preferably 1: (0-1); the preferable molar ratio of the lactide to the macroinitiator is (1-120): 1, more preferably (1 to 100): 1, most preferably (1-80): 1, specifically 1:1, 2:1, 5:1, 7:1, 9:1, 10:1, 11:1, 11.3:1, 12:1, 15:1, 17:1, 20:1, 20.5:1, 23:1, 25:1, 27:1, 30:1, 32:1, 34:1, 35:1, 37:1, 40:1, 45:1 or 50: 1.
In the preparation method provided by the invention, the mole ratio of the glycolide to the macroinitiator is preferably (1-80): 1, more preferably (1 to 60): 1, most preferably (1-40): 1, specifically 1:1, 2:1, 5:1, 5.5:1, 6:1, 7:1, 9:1, 10:1, 12:1, 13:1, 13.5:1, 15:1, 18:1, 20:1, 23:1, 25:1 or 30: 1.
In the preparation method provided by the invention, the catalyst is preferably one or more of zinc lactate, stannous octoate, alkyl metal and alkoxy metal; the amount of the catalyst is preferably 0.1 to 0.5% of the total molar amount of lactide and glycolide, and specifically may be 0.1%, 0.12%, 0.15%, 0.17%, 0.2%, 0.23%, 0.25%, 0.27%, 0.3%, 0.32%, 0.35%, 0.37%, 0.4%, 0.42%, 0.45%, 0.47%, or 0.5%.
In the preparation method provided by the invention, the polymerization reaction is carried out in an oxygen-free and water-free environment; the polymerization reaction temperature is preferably 80-130 ℃, and specifically can be 80 ℃, 82 ℃, 85 ℃, 87 ℃, 90 ℃, 92 ℃, 95 ℃, 97 ℃, 100 ℃, 102 ℃, 105 ℃, 107 ℃, 110 ℃, 112 ℃, 115 ℃, 117 ℃, 120 ℃, 123 ℃, 125 ℃, 127 ℃ or 130 ℃; the polymerization reaction time is preferably 24-72 h, and specifically can be 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h, 48h, 50h, 52h, 54h, 56h, 58h, 60h, 62h, 64h, 66h, 68h, 70h or 72 h.
In the preparation method provided by the invention, more specific preparation steps preferably comprise:
a1) mixing a macroinitiator, lactide, glycolide, a catalyst and a solvent, and heating for reaction to obtain a crude product;
a2) removing the solvent and unreacted monomers from the crude product to obtain the tissue adhesive.
In the above preparation steps provided by the present invention, in step a1), the specific kinds and the dosage ratios of the macroinitiator, the lactide, the glycolide and the catalyst have been described above, and are not described herein again; the solvent is preferably toluene; the specific process of mixing is preferably as follows: firstly, mixing and dissolving a macromolecular initiator, lactide and glycolide in a solvent, and then mixing the mixture with the initiator diluted by the solvent; the temperature and time of the heating reaction are the same as those of the polymerization reaction described above and will not be described in detail.
In the above preparation step provided by the present invention, the specific method for removing the solvent and the unreacted monomer in step a2) is preferably: firstly, removing the solvent by adopting a method of vacuumizing and liquid nitrogen freezing; then adding chloroform for dissolving; pouring into the ice ether, stirring, standing and settling to obtain a polymer; then removing the supernatant and dissolving with chloroform; then pouring into the ice ether for stirring, standing and settling to obtain a polymer; finally, the supernatant is discarded to obtain a pure tissue adhesive product.
The preparation method provided by the invention takes polyethylene glycol and branched chain derivatives thereof as macroinitiators, and obtains the block polymer containing the hydrophilic block and the hydrophobic block by initiating the ring-opening polymerization reaction of lactide monomers and glycolide monomers, and the block polymer has good biocompatibility and adhesiveness and has wide application prospects in the biomedical field, such as the field of wound adhesion and the like. In addition, the preparation method provided by the invention can adjust the molecular weight and the repeating unit ratio of the block polymer by controlling the molecular weight of the initiator, the monomer dosage ratio, the reaction conditions and the like, so that the adhesion and the strength range of the prepared tissue adhesive can better meet the use requirements of different scenes, and the application field of the tissue adhesive is widened.
The invention also provides a preparation for tissue adhesion, which comprises a tissue adhesive and a solvent, wherein the tissue adhesive is the tissue adhesive in the technical scheme or the tissue adhesive prepared by the preparation method in the technical scheme. The preparation provided by the invention takes the tissue adhesive provided by the invention as an adhesive, has good biocompatibility and adhesiveness, and has wide application prospect in the biomedical field, such as the field of wound adhesion and the like.
For the sake of clarity, the following examples are given in detail.
Example 1
Vacuumizing and baking a reaction bottle, introducing nitrogen, standing and cooling to room temperature, repeatedly carrying out three times to remove water, adding 2g (0.002mol) of PEG (with the number average molecular weight of 1000), 2.1g (0.018mol) of Glycolide (GA) and 4.88g (0.034mol) of L-type lactide (LLA), vacuumizing and introducing nitrogen to keep an oxygen-free and water-free environment in the reaction bottle, adding about 40ml of toluene serving as a solvent through a syringe, heating to 100 ℃, stirring to dissolve the toluene, adding 0.8ml of stannous octoate serving as a catalyst diluted by the anhydrous toluene after half an hour, heating to 110 ℃, and reacting for 48 hours.
Stopping heating, vacuumizing, removing the solvent toluene by a liquid nitrogen freezing method, adding 10ml of chloroform for dissolving, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, dissolving by 10ml of chloroform, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, and removing the diethyl ether to obtain the tissue adhesive product.
The chemical name of the tissue adhesive product prepared in this example is poly (lactic-co-glycolic acid) polyethylene glycol copolymer, abbreviated as PLGA-PEG-PLGA, and the chemical structure is shown in formula (a), wherein a is 23, f is 8, and g is 13.
The tissue adhesive product prepared in this example was named form x-y-z according to molecular weight and monomer molar ratio; wherein x represents the number average molecular weight of PLGA (in kDa), y represents the number average molecular weight of PEG (in kDa), and z represents the monomer molar ratio LA/GA; the tissue adhesive products prepared in this example were named 1.3-1-1.7.
Example 2
Vacuumizing and baking the reaction bottle, introducing nitrogen, standing and cooling to room temperature, repeatedly carrying out three times to remove water, adding 0.8g (0.002mol) of PEG (with the number average molecular weight of 400), 2.1g (0.018mol) of GA and 4.88g (0.034mol) of LLA (polyethylene glycol), vacuumizing and introducing nitrogen to keep an oxygen-free and water-free environment in the reaction bottle, adding about 40ml of solvent toluene through an injector, heating to 100 ℃, stirring to dissolve, adding 0.8ml of catalyst stannous octoate diluted by the anhydrous toluene after half an hour, heating to 110 ℃, and reacting for 48 hours.
Stopping heating, vacuumizing, removing the solvent toluene by a liquid nitrogen freezing method, adding 10ml of chloroform for dissolving, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, dissolving by 10ml of chloroform, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, and removing the diethyl ether to obtain the tissue adhesive product.
The chemical name of the tissue adhesive product prepared in this example is poly (lactic-co-glycolic acid) polyethylene glycol copolymer, abbreviated as PLGA-PEG-PLGA, and the chemical structure is shown in formula (a), wherein a is 9, f is 9, and g is 15.
The tissue adhesive product prepared in this example was named 1.6-0.4-1.6 according to molecular weight and monomer molar ratio.
Example 3
Vacuumizing and baking the reaction bottle, introducing nitrogen, standing and cooling to room temperature, repeatedly carrying out three times to remove water, adding 4g (0.002mol) of PEG (with the number average molecular weight of 2000), 2.1g (0.018mol) of GA and 4.88g (0.034mol) of LLA, vacuumizing and introducing nitrogen to keep an oxygen-free and water-free environment in the reaction bottle, adding about 40ml of toluene serving as a solvent through an injector, heating to 100 ℃, stirring for dissolving, adding 0.8ml of stannous octoate serving as a catalyst diluted by the anhydrous toluene after half an hour, heating to 110 ℃, and reacting for 48 hours.
Stopping heating, vacuumizing, removing the solvent toluene by a liquid nitrogen freezing method, adding 10ml of chloroform for dissolving, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, dissolving by 10ml of chloroform, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, and removing the diethyl ether to obtain the tissue adhesive product.
The chemical name of the tissue adhesive product prepared in this example is poly (lactic-co-glycolic acid) polyethylene glycol copolymer, abbreviated as PLGA-PEG-PLGA, and the chemical structure is shown in formula (a), wherein a is 45, f is 8, and g is 14.
The tissue adhesive product prepared in this example was named 1.4-2-1.7 according to molecular weight and monomer molar ratio.
Example 4
Vacuumizing and baking the reaction bottle, introducing nitrogen, standing and cooling to room temperature, repeatedly carrying out three times to remove water, adding 2g (0.002mol) of PEG (with the number average molecular weight of 1000), 1.28g (0.011mol) of GA and 5.93g (0.041mol) of LLA, vacuumizing and introducing nitrogen to keep an oxygen-free and water-free environment in the reaction bottle, adding about 40ml of toluene serving as a solvent through a syringe, heating to 100 ℃, stirring for dissolving, adding 0.8ml of stannous octoate serving as a catalyst diluted by the anhydrous toluene after half an hour, heating to 110 ℃, and reacting for 48 hours.
Stopping heating, vacuumizing, removing the solvent toluene by a liquid nitrogen freezing method, adding 10ml of chloroform for dissolving, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, dissolving by 10ml of chloroform, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, and removing the diethyl ether to obtain the tissue adhesive product.
The chemical name of the tissue adhesive product prepared in this example is poly (lactic-co-glycolic acid) polyethylene glycol copolymer, abbreviated as PLGA-PEG-PLGA, and the chemical structure is shown in formula (a), wherein a is 23, f is 6, and g is 18.
The tissue adhesive product prepared in this example was named 1.6-1-3.2 according to molecular weight and monomer molar ratio.
Example 5
Vacuumizing and baking the reaction bottle, introducing nitrogen, standing and cooling to room temperature, repeatedly carrying out three times to remove water, adding 1g (0.001mol) of PEG (with the number average molecular weight of 1000), 2.1g (0.018mol) of GA and 4.88g (0.034mol) of LLA (polyethylene glycol), vacuumizing and introducing nitrogen to keep an oxygen-free and water-free environment in the reaction bottle, adding about 40ml of toluene serving as a solvent through an injector, heating to 100 ℃, stirring for dissolving, adding 0.8ml of stannous octoate serving as a catalyst diluted by the anhydrous toluene after half an hour, heating to 110 ℃, and reacting for 48 hours.
Stopping heating, vacuumizing, removing the solvent toluene by a liquid nitrogen freezing method, adding 10ml of chloroform for dissolving, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, dissolving by 10ml of chloroform, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, and removing the diethyl ether to obtain the tissue adhesive product.
The chemical name of the tissue adhesive product prepared in this example is poly (lactic-co-glycolic acid) polyethylene glycol copolymer, abbreviated as PLGA-PEG-PLGA, and the chemical structure is shown in formula (a), wherein a is 23, f is 17, and g is 30.
The tissue adhesive product prepared in this example was named 3.1-1-1.8 according to molecular weight and monomer molar ratio.
Example 6
Vacuumizing and baking the reaction bottle, introducing nitrogen, standing and cooling to room temperature, repeatedly carrying out three times to remove water, adding 3g (0.003mol) of PEG (with the number average molecular weight of 1000), 2.1g (0.018mol) of GA and 4.89g (0.034mol) of LLA, vacuumizing and introducing nitrogen to keep an oxygen-free and water-free environment in the reaction bottle, adding about 40ml of toluene serving as a solvent through an injector, heating to 100 ℃, stirring for dissolving, adding 0.8ml of stannous octoate serving as a catalyst diluted by the anhydrous toluene after half an hour, and heating to 110 ℃ for reaction for 48 hours.
Stopping heating, vacuumizing, removing the solvent toluene by a liquid nitrogen freezing method, adding 10ml of chloroform for dissolving, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, dissolving by 10ml of chloroform, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, and removing the diethyl ether to obtain the tissue adhesive product.
The chemical name of the tissue adhesive product prepared in this example is poly (lactic-co-glycolic acid) polyethylene glycol copolymer, abbreviated as PLGA-PEG-PLGA, and the chemical structure is shown in formula (a), wherein a is 23, f is 6, and g is 10.
The tissue adhesive product prepared in this example was named 1-1-1.7 according to the molecular weight and the monomer molar ratio.
Example 7
Vacuumizing and baking the reaction bottle, introducing nitrogen, standing and cooling to room temperature, repeatedly carrying out three times to remove water, adding 2g (0.002mol) of PEG (with the number average molecular weight of 1000), 3.1g (0.027mol) of GA and 3.6g (0.018mol) of LLA, vacuumizing and introducing nitrogen to keep an oxygen-free and water-free environment in the reaction bottle, adding about 40ml of toluene serving as a solvent through a syringe, heating to 100 ℃, stirring to dissolve the toluene, adding 0.8ml of stannous octoate serving as a catalyst diluted by the anhydrous toluene after half an hour, heating to 110 ℃, and reacting for 48 hours.
Stopping heating, vacuumizing, removing the solvent toluene by a liquid nitrogen freezing method, adding 10ml of chloroform for dissolving, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, dissolving by 10ml of chloroform, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, and removing the diethyl ether to obtain the tissue adhesive product.
The chemical name of the tissue adhesive product prepared in this example is poly (lactic-co-glycolic acid) polyethylene glycol copolymer, abbreviated as PLGA-PEG-PLGA, and the chemical structure is shown in formula (a), wherein a is 23, f is 12, and g is 11.
The tissue adhesive product prepared in this example was named 1.5-1-1 according to the molecular weight and the monomer molar ratio.
Example 8
Vacuumizing and baking the reaction bottle, introducing nitrogen, standing and cooling to room temperature, repeating the steps for three times to remove water, adding 2g (0.001mol) of four-arm PEG (with the number average molecular weight of 2000), 1.4g (0.012mol) of GA and 3.25g (0.023mol) of LLA3, vacuumizing and introducing nitrogen to keep an oxygen-free and water-free environment in the reaction bottle, adding about 40ml of toluene serving as a solvent through an injector, heating to 100 ℃, stirring to dissolve, adding 0.8ml of stannous octoate serving as a catalyst diluted by the anhydrous toluene after half an hour, and heating to 110 ℃ to react for 48 hours.
Stopping heating, vacuumizing, removing the solvent toluene by a liquid nitrogen freezing method, adding 10ml of chloroform for dissolving, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, dissolving by 10ml of chloroform, pouring into 500ml of diethyl ether for stirring, standing and settling to obtain a polymer, removing the supernatant, and removing the diethyl ether to obtain the tissue adhesive product.
The chemical name of the tissue adhesive product prepared in this example is poly (lactic-co-glycolic acid) polyethylene glycol copolymer, abbreviated as 4arm-PEG-PLGA, and the chemical structure is shown in formula (C), wherein C is 11, f is 6, and g is 10.
The tissue adhesive product prepared in this example was named 4a-1.1 based on PLGA molecular weight; wherein 1.1 represents the number average molecular weight (in kDa) of PLGA.
Example 9
8mg of the polymer prepared in examples 1 to 8 was used for nuclear magnetic testing, and the results are shown in FIGS. 1 to 8.
As can be seen from FIGS. 1 to 8, the number average molecular weights of the polymers prepared in examples 1 to 8 were 3.6X 103Da、3.6×103Da、4.8×103Da、4.2×103Da、7.2×103Da、3×103Da、4×103Da、6.2×103Da。
Example 10
4mg of the polymer prepared in examples 1 to 7 was used for Gel Permeation Chromatography (GPC) measurement, and the results are shown in FIGS. 9 to 15.
As can be seen from FIGS. 9 to 15, the molecular weight distributions of the polymers prepared in examples 1 to 7 are all between 1.2 and 1.35.
Example 11
The results of Differential Scanning Calorimetry (DSC) measurements of 4mg of the polymers prepared in examples 1 to 7 are shown in FIGS. 16 to 22.
As can be seen from FIGS. 16 to 22, the melting points of the polymers prepared in examples 1 to 7 were 50.96 ℃, 52.09 ℃, 47 ℃, 52.82 ℃, 52.23 ℃, 48.52 ℃ and 48.69 ℃, respectively, and the polymers were applied by heating to about 50 ℃ to a fluid state.
Example 12
0.04g of the polymer prepared in examples 1-8 was coated on PMMA plates with casing adhered on the surface, and each plate was coated with a length of 1cm, a width of 1cm and an area of 1cm2And the two plates are overlapped and pressed by a weight for lap joint shearing test.
The test result is shown in FIG. 23, and it can be seen from FIG. 23 that the adhesive strength of the polymer is between 0 and 200kpa, which can be controlled according to the molecular weight and the monomer molar ratio.
Example 13
The polymer prepared in example 1 was used for cell compatibility experiments, and mouse fibroblast cells 3T3 were seeded in 96-well plates at a density of 5000 cells per well, 200 μ L of complete medium (90% DMEM medium + 10% newborn bovine serum) per well, and incubated in an incubator for 24 h; diluting the leaching liquor to four dilutions of 100%, 50%, 25% and 12% by using a culture medium; taking out the culture plate after 24h, respectively adding 20 mu L of PBS buffer solution with pH of 7.4 and leaching liquor diluents with different concentrations into the culture plate, and placing the culture plate in an incubator for incubation for 24 h; and taking out the culture plate after 24h, sucking the culture medium, washing the culture plate for 2-3 times by using PBS (phosphate buffer solution), adding 10% CCK-8 solution in a dark place, placing the culture plate in an incubator for incubation for 1h, and testing the absorbance of the culture plate at 450nm by using an enzyme-labeling instrument.
The cytotoxicity results of the materials at different concentrations on 3T3 cells are shown in fig. 24, in which the ordinate represents the cell viability and the calculation formula is: cell viability (%) (sample OD/control OD) × 100%, where OD value represents absorbance at 490 nm. As can be seen from FIG. 24, the cell viability of the leaching solution group is greater than 80%, which indicates that the polymer has no cytotoxicity and better cell compatibility.
Example 14
The polymer prepared in example 1 was used in animal wound repair experiments to evaluate the wound closure performance of the polymer tissue adhesive, creating a full thickness skin wound of 2cm in length on the back of rats; applying the polymeric tissue adhesive to the wound surface to close the wound, after heating the polymeric tissue adhesive to about 50 ℃; the blank control group only performs gauze pressing hemostasis; wound sites were photographed on days 0, 7, and 14; on day 14 post-surgery, animals were euthanized.
The test results are shown in fig. 25. As can be seen from fig. 25, the wound repair effect of the experimental group rats was better than that of the blank group.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
3. The tissue adhesive of claim 2, wherein 9 ≦ a ≦ 182; b is more than or equal to 9 and less than or equal to 182; c is more than or equal to 9 and less than or equal to 182; d is more than or equal to 9 and less than or equal to 182; e is more than or equal to 9 and less than or equal to 182; f is more than 0 and less than or equal to 80; g is more than 0 and less than or equal to 120.
4. A method of preparing a tissue adhesive, comprising the steps of:
a) in the presence of a catalyst, carrying out polymerization reaction on a macroinitiator, lactide and glycolide to obtain a tissue adhesive;
the macroinitiator is polyethylene glycol and/or multi-arm polyethylene glycol.
5. The production method according to claim 4, wherein the lactide is L-type lactide or L/D mixed type lactide.
6. The method according to claim 4, wherein the molar ratio of the macroinitiator, lactide and glycolide is 1: (1-120): (1-80).
7. The preparation method according to claim 4, wherein the catalyst is one or more of zinc lactate, stannous octoate, alkyl metal and alkoxy metal.
8. The preparation method according to claim 4, wherein the temperature of the polymerization reaction is 80-130 ℃; the polymerization reaction time is 24-72 h.
9. The method according to claim 4, wherein step a) comprises in particular:
a1) mixing a macroinitiator, lactide, glycolide, a catalyst and a solvent, and heating for reaction to obtain a crude product;
a2) removing the solvent and unreacted monomers from the crude product to obtain the tissue adhesive.
10. A tissue adhesive preparation comprising a tissue adhesive and a solvent, wherein the tissue adhesive is the tissue adhesive according to any one of claims 1 to 3 or the tissue adhesive obtained by the production method according to any one of claims 4 to 9.
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110070320A1 (en) * | 2009-09-23 | 2011-03-24 | Soonkap Hahn | Biodegradable thermoresponsive 3-arm polyethylene glycol poly(lactide-co-glycolide) copolymer for ginseng administration |
CN105131264A (en) * | 2015-10-13 | 2015-12-09 | 山东师范大学 | Biodegradable medical temperature-sensitive hydrogel and preparing method thereof |
EP3085397A1 (en) * | 2015-04-23 | 2016-10-26 | Covidien LP | Adhesive compositions |
CN106543454A (en) * | 2016-11-25 | 2017-03-29 | 山东师范大学 | Medical photo-polymerization type hydrogel of a kind of low swelling biodegradable and preparation method thereof |
CN113444232A (en) * | 2021-07-12 | 2021-09-28 | 东北师范大学 | Degradable polymer for wound dressing, preparation method thereof and wound dressing |
-
2022
- 2022-01-12 CN CN202210030960.7A patent/CN114276528A/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110070320A1 (en) * | 2009-09-23 | 2011-03-24 | Soonkap Hahn | Biodegradable thermoresponsive 3-arm polyethylene glycol poly(lactide-co-glycolide) copolymer for ginseng administration |
EP3085397A1 (en) * | 2015-04-23 | 2016-10-26 | Covidien LP | Adhesive compositions |
CN105131264A (en) * | 2015-10-13 | 2015-12-09 | 山东师范大学 | Biodegradable medical temperature-sensitive hydrogel and preparing method thereof |
CN106543454A (en) * | 2016-11-25 | 2017-03-29 | 山东师范大学 | Medical photo-polymerization type hydrogel of a kind of low swelling biodegradable and preparation method thereof |
CN113444232A (en) * | 2021-07-12 | 2021-09-28 | 东北师范大学 | Degradable polymer for wound dressing, preparation method thereof and wound dressing |
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