CN115737887A - Skin wound dressing and preparation method thereof - Google Patents
Skin wound dressing and preparation method thereof Download PDFInfo
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- CN115737887A CN115737887A CN202211382329.XA CN202211382329A CN115737887A CN 115737887 A CN115737887 A CN 115737887A CN 202211382329 A CN202211382329 A CN 202211382329A CN 115737887 A CN115737887 A CN 115737887A
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Abstract
The invention belongs to the technical field of biomedical materials, and provides a skin wound dressing and a preparation method thereof. The method comprises the steps of mixing and freeze-drying Sulfated Chitosan (SCS) and aqueous solution of mono-succinylated pullulan (st-Pu) according to different proportions, crosslinking 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine (EDC) and N-hydroxysuccinimide (NHS) to obtain a crosslinked membrane material, and spraying resveratrol ethanol solution on the crosslinked membrane to obtain the SCS/st-Pu composite membrane loaded with resveratrol, namely the skin wound dressing. The skin wound dressing prepared by the method has the characteristics of better antibiosis, antioxidation, wound exudate absorption, chronic wound healing promotion and the like, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a skin wound dressing and a preparation method thereof.
Background
Wound healing is a complex process involving a variety of cells, as well as cytokines, growth factors, proteases, and extracellular matrix components, and includes processes of hemostasis, inflammation, cell migration and proliferation, wound closure, and the like. Wound dressings are applied to the injured site, providing a physical barrier to protect the wound and promote repair. Natural, inexpensive, biocompatible and biodegradable polysaccharides are gaining increasing attention as wound dressings.
Pullulan (Pu) is a water-soluble exopolysaccharide produced by fermentation of aureobasidium pullulans, which has good water solubility and is easily filmed. Pullulan gel can improve wound regeneration, angiogenesis and collagen synthesis, and accelerate wound contraction (Thangavel P, vildanathan S P, kuttalam I, et al. Clinical administration of pullulan gel sheets tissue regeneration by enhancing collagen synthesis and wind communication in rates. International j ournal of biological macrogels, 2020, 149. Easily soluble sulfonated chitosan derivatives (SCS) can interact with cytokines, enzymes and membrane receptors. Such as: sulfated chitosan-collagen hydrogels stimulate macrophage secretion, up-regulate interleukin-4 and TGF- β 1 while inhibiting interleukin-6, promote collagen deposition and neovascularization, and accelerate diabetic wound healing (Shen T, dai K, yuY, et al. Sulfated chicken wounds dynamic mechanical wounds and diabetic wounds in diabetes mellitus. Acta biomateriala, 2020, 117. Resveratrol (RES) is used as a natural polyphenol compound, and has antioxidant, antiinflammatory, angiogenesis promoting and antibacterial effects. For example: the resveratrol-loaded peptide hydrogel can inhibit inflammation, promote collagen deposition, accelerate wound healing and inhibit scar formation when being coated on rat wounds (Zhao C, zhu L, wu Z, et al. Resveratrol-loaded peptide-hydrogel inhibition diagnosis formation in wind and health promotion in regeneration biological materials,2020,7 (1): 99-107.). At present, researches on the aspect of preparing a wound dressing film for chronic wound healing by combining sulfated chitosan, succinylated pullulan and natural polyphenol resveratrol are not reported.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides the skin wound dressing and the preparation method thereof, wherein the composite membrane material of sulfated chitosan and succinylated pullulan is crosslinked, and resveratrol with a proper proportion is loaded.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a skin wound dressing, which comprises the following steps:
(1) Mixing pullulan, succinic anhydride and dimethyl sulfoxide, mixing with 4-dimethylaminopyridine after complete dissolution, and sequentially stirring, precipitating with ethanol and filtering to obtain a precipitate 1;
(2) Mixing the precipitate 1 with water to enable the precipitate 1 to be just dissolved, and sequentially dialyzing and freeze-drying to obtain mono-succinylated pullulan;
(3) Mixing chitosan, dichloroacetic acid and dimethylformamide, and stirring overnight to obtain a mixed solution 1;
(4) Mixing and stirring chlorosulfonic acid and dimethylformamide to obtain a mixed solution 2;
(5) Mixing the mixed solution 1 and the mixed solution 2 under the conditions of stirring and nitrogen, then reacting for 1.5-2.5 h at 55-65 ℃, adding deionized water after the reaction is finished, adjusting the pH value to be neutral, and then carrying out alcohol precipitation and suction filtration to obtain a precipitate 2;
(6) Mixing the precipitate 2 with water to enable the precipitate 2 to be just dissolved, and sequentially dialyzing and freeze-drying to obtain sulfated chitosan;
(7) Respectively mixing the mono-succinylated pullulan and the sulfated chitosan with water to obtain a mono-succinylated pullulan aqueous solution and a sulfated chitosan aqueous solution;
(8) Mixing the mono-succinylated pullulan aqueous solution with a sulfated chitosan aqueous solution, and freeze-drying to obtain a freeze-dried film;
(9) Immersing the freeze-dried membrane in a crosslinking solution for crosslinking reaction, washing with deionized water, and freeze-drying to obtain a mono-succinylated pullulan-sulfated chitosan crosslinked membrane;
(10) Loading the resveratrol ethanol solution on the mono-succinylated pullulan polysaccharide-sulfated chitosan cross-linked film, and drying to obtain the skin wound dressing.
Preferably, in the step (1), the molar ratio of the pullulan to the succinic anhydride to the 4-dimethylaminopyridine is 0.8-1.2 and is within a range from 0.8 to 1.2 to 0.1-0.3, the molecular weight of the pullulan is 20-2000 KDa, the dosage ratio of the pullulan to the dimethyl sulfoxide is 1g.
Preferably, in the step (2), the dialysis is carried out for 45 to 50 hours at room temperature by using a 8000 to 14000Da Mw dialysis bag, the freeze-drying temperature is-60 to-70 ℃, the vacuum degree of the freeze-drying is less than 5pa, and the freeze-drying time is 20 to 25 hours.
Preferably, in the step (3), the mixing ratio of the chitosan, the dichloroacetic acid and the dimethylformamide is 0.8-1.2g, and the molecular weight of the chitosan is 5-2000 KDa;
the mixing volume ratio of the chlorosulfonic acid to the dimethylformamide in the step (4) is 3-4, the stirring temperature in the step (4) is 0-4 ℃, and the stirring time in the step (4) is 25-35 min.
Preferably, the mixing ratio in the step (5) is calculated according to the molar ratio of chitosan in the mixed solution 1 to chlorosulfonic acid in the mixed solution 2, the molar ratio of chitosan in the mixed solution 1 to chlorosulfonic acid in the mixed solution 2 is 1;
and (6) dialyzing for 70-75 h at room temperature by using a 3000-4000 Da Mw dialysis bag, wherein the freeze-drying temperature is-60-70 ℃, the vacuum degree of freeze-drying is less than 5pa, and the freeze-drying time is 20-25 h.
Preferably, the concentration of the aqueous solution of the mono-succinylated pullulan and the aqueous solution of sulfated chitosan in step (7) are independently 0.8 to 1.2wt%.
Preferably, the mixing weight ratio of the aqueous solution of the mono-succinylated pullulan to the aqueous solution of the sulfated chitosan in the step (8) is 30-70, the freeze-drying temperature is-60 to-70 ℃, the vacuum degree of the freeze-drying is less than 5pa, and the freeze-drying time is 20-25 h.
Preferably, the crosslinking solution in step (9) uses an ethanol solution as a solvent, and comprises the following components in concentration: 80-120 mmol/L of 1-ethyl- (3-dimethylaminopropyl) carbodiimide, 20-30 mmol/L of N-hydroxysuccinimide, 70-80 vt% of ethanol solution, 0-4 ℃ of crosslinking reaction, 20-25 h of crosslinking reaction time, 2-4 times of deionized water washing, 0.8-1.2 h of washing time, 60-70 ℃ of freeze-drying temperature, 5pa of vacuum degree of freeze-drying and 20-25 h of freeze-drying time.
Preferably, the concentration of the resveratrol ethanol solution in the step (10) is 8-12 mg/mL, the loading amount of the resveratrol ethanol solution is 2.5-15% of the weight of the mono-succinylated pullulan-sulfated chitosan cross-linked membrane, the drying temperature is 45-55 ℃, and the drying time is 20-25 h.
The invention also provides the skin wound dressing prepared by the preparation method.
Compared with the prior art, the invention has the following beneficial effects:
the pullulan in the invention is used as natural polysaccharide, has excellent water solubility, high water absorption, flexibility and film forming property, has certain elasticity and stretchability, and has excellent mechanical property. Chitosan (CS) as a common wound dressing has the characteristics of good biocompatibility, hemostasis, antibiosis, wound healing promotion and the like, but the water solubility of the CS is poor, so that the chitosan needs to be modified. Sulfated Chitosan (SCS) belongs to a chitosan derivative, and has good water solubility, angiogenesis promotion and growth factor binding capacity. Resveratrol (RES) is used as a natural polyphenol compound, and has antioxidant, antiinflammatory, angiogenesis promoting and antibacterial effects. The invention mixes and freezes Sulfated Chitosan (SCS) and aqueous solution of mono-succinylated pullulan (st-Pu) according to different proportions, cross-links through 1-ethyl- (3-dimethyl aminopropyl) carbonyl diimine (EDC) and N-hydroxyl succinimide (NHS) to obtain cross-linked membrane material, and sprays resveratrol ethanol solution on the cross-linked membrane to obtain SCS/st-Pu composite membrane loaded with resveratrol, namely the skin wound dressing. The skin wound dressing prepared by the method has the characteristics of better antibiosis, antioxidation, absorption of wound exudates, promotion of chronic wound healing and the like, and has good application prospect.
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 an infrared spectrum of a product prepared in example 1 of the present invention and a reference (note: a represents pullulan; b represents succinylated pullulan; c represents chitosan; d represents sulfated chitosan; e represents pullulan and sulfated chitosan after crosslinking);
FIG. 2 is a nuclear magnetic spectrum of the product prepared in example 1 of the present invention and a reference (note: a represents pullulan, b represents succinic anhydride, c represents succinylated pullulan, d represents chitosan, e represents sulfated chitosan);
FIG. 3 shows the results of in vitro antibacterial tests on the product prepared in example 1 of the present invention and a control (note: a represents SCS-st-Pu II; b represents RES-2.5%/SCS-st-Pu; c represents RES-5%/SCS-st-Pu; and d represents RES-15%/SCS-st-Pu);
FIG. 4 shows the results of an antioxidant assay of the product prepared in example 1 of the present invention and a control;
fig. 5 is a cytotoxicity evaluation (n = 3) of the product prepared in example 1 of the present invention;
FIG. 6 shows the results of a test of the product prepared according to example 1 of the present invention on the wound repair of diabetic rats (note: A represents a graph of wound repair in diabetic rats; B represents a graph of the relative percentage of the area of diabetic wounds (n = 3); a represents a blank control; B represents a 3Tegaderm dressing; c represents RES-2.5%/SCS-st-Pu; d represents RES-5%/SCS-st-Pu);
FIG. 7 shows the results of the wound healing experiments on normal rats using the product prepared in example 1 of the present invention (note: A represents a wound healing map in normal rats; B represents a relative percentage of wound area in rats (n = 3); a represents a blank; B represents Band-aid; c represents SCS-st-Pu; d represents RES-2.5%/SCS-st-Pu; and e represents RES-5%/SCS-st-Pu).
Detailed Description
The invention provides a preparation method of a skin wound dressing, which comprises the following steps:
(1) Mixing pullulan, succinic anhydride and dimethyl sulfoxide, mixing with 4-dimethylaminopyridine after complete dissolution, and sequentially stirring, precipitating with ethanol and filtering to obtain a precipitate 1;
(2) Mixing the precipitate 1 with water to enable the precipitate 1 to be just dissolved, and sequentially dialyzing and freeze-drying to obtain mono-succinylated pullulan;
(3) Mixing chitosan, dichloroacetic acid and dimethylformamide, and stirring overnight to obtain a mixed solution 1;
(4) Mixing chlorosulfonic acid and dimethylformamide and stirring to obtain a mixed solution 2;
(5) Mixing the mixed solution 1 and the mixed solution 2 under the conditions of stirring and nitrogen, then reacting for 1.5-2.5 h at 55-65 ℃, adding deionized water after the reaction is finished, adjusting the pH value to be neutral, and then carrying out alcohol precipitation and suction filtration to obtain a precipitate 2;
(6) Mixing the precipitate 2 with water to enable the precipitate 2 to be just dissolved, and sequentially dialyzing and freeze-drying to obtain sulfated chitosan;
(7) Respectively mixing the mono-succinylated pullulan and the sulfated chitosan with water to obtain a mono-succinylated pullulan aqueous solution and a sulfated chitosan aqueous solution;
(8) Mixing the mono-succinylated pullulan aqueous solution with a sulfated chitosan aqueous solution, and freeze-drying to obtain a freeze-dried film;
(9) Immersing the freeze-dried membrane in a cross-linking solution for cross-linking reaction, washing with deionized water, and freeze-drying to obtain a mono-succinylated pullulan-sulfated chitosan cross-linked membrane;
(10) Loading the resveratrol ethanol solution on the mono-succinylated pullulan polysaccharide-sulfated chitosan cross-linked film, and drying to obtain the skin wound dressing.
In the present invention, the molar ratio of pullulan, succinic anhydride and 4-dimethylaminopyridine in step (1) is preferably 0.8 to 1.2; the molecular weight of the pullulan is preferably 20-2000 KDa, more preferably 500-1500 KDa, and even more preferably 1000KDa; the dosage ratio of the pullulan to the dimethyl sulfoxide is preferably 1g; the temperature in the stirring process is preferably 45-55 ℃, and more preferably 50 ℃; the stirring speed is preferably 80-120 r/min, more preferably 90-110 r/min, and even more preferably 100r/min; the stirring time is preferably 20 to 25 hours, and more preferably 24 hours; the ethanol precipitation is preferably performed by ethanol precipitation, and the dosage ratio of the ethanol to the pullulan is preferably 180-220mL (total volume) 3g, and more preferably 200mL.
In the present invention, in the step (2), the dialysis is preferably performed for 45 to 50 hours at room temperature by using a 8000 to 14000Da Mw dialysis bag, and more preferably for 48 hours at room temperature by using a 12000Da Mw dialysis bag; the freeze-drying temperature is preferably-60 to-70 ℃, and more preferably-65 ℃; the vacuum degree of the freeze-drying is preferably less than 5pa, and more preferably 3pa; the freeze-drying time is preferably 20 to 25 hours, and more preferably 24 hours.
In the present invention, in step (3), the mixing ratio of the chitosan, dichloroacetic acid and dimethylformamide is preferably 0.8 to 1.2g, and more preferably 1 g; the molecular weight of the chitosan is preferably 5-2000 KDa, more preferably 500-1500 KDa, and further preferably 1000KDa;
the mixing volume ratio of chlorosulfonic acid to dimethylformamide in step (4) is preferably 3 to 4, and more preferably 3.3; the stirring temperature in the step (4) is preferably 0-4 ℃, and more preferably 3 ℃; the stirring time in the step (4) is preferably 25 to 35min, more preferably 28 to 32min, and still more preferably 30min.
In the present invention, the mixing ratio in step (5) is preferably calculated by a molar ratio of chitosan in mixed solution 1 to chlorosulfonic acid in mixed solution 2, and the molar ratio of chitosan in mixed solution 1 to chlorosulfonic acid in mixed solution 2 is preferably 1; the component for adjusting the pH value is preferably a sodium hydroxide solution, and the molar concentration of the sodium hydroxide solution is preferably 1-3 mol/L, and more preferably 2mol/L;
in the step (6), the dialysis is preferably performed for 70 to 75 hours at room temperature by using a 3000 to 4000Da Mw dialysis bag, and is further preferably performed for 72 hours at room temperature by using a 3500Da Mw dialysis bag; the freeze-drying temperature is preferably-60 to-70 ℃, and more preferably-65 ℃; the vacuum degree of the freeze-drying is preferably less than 5pa, and more preferably 3pa; the time for freeze-drying is preferably 20 to 25 hours, and more preferably 24 hours.
In the present invention, the concentration of the aqueous solution of the mono-succinylated pullulan and the aqueous solution of sulfated chitosan in step (7) is preferably 0.8 to 1.2wt%, and more preferably 1wt%.
In the present invention, the mixing weight ratio of the aqueous solution of the mono-succinylated pullulan to the aqueous solution of sulfated chitosan in step (8) is preferably 30 to 70, more preferably 30 or 50; the freeze-drying temperature is preferably-60 to-70 ℃, and more preferably-65 ℃; the vacuum degree of the freeze-drying is preferably less than 5pa, and more preferably 3pa; the freeze-drying time is preferably 20 to 25 hours, and more preferably 24 hours.
In the present invention, the crosslinking solution in step (9) preferably uses an ethanol solution as a solvent, and contains the following components in concentration: 80-120 mmol/L of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 20-30 mmol/L of N-hydroxysuccinimide, and further preferably ethanol solution is used as a solvent, and the ethanol solution comprises the following components in concentration: 100mmol/L of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 25mmol/L of N-hydroxysuccinimide; the concentration of the ethanol solution is preferably 70-80 vt%, and more preferably 75vt%; the temperature of the crosslinking reaction is preferably 0-4 ℃, and more preferably 3 ℃; the time of the crosslinking reaction is preferably 20 to 25 hours, and more preferably 24 hours; the number of washing with deionized water is preferably 2 to 4, and more preferably 3; the time of each washing is preferably 0.8-1.2 h, and more preferably 1h; the freeze-drying temperature is preferably-60 to-70 ℃, and more preferably-65 ℃; the vacuum degree of the freeze-drying is preferably less than 5pa, and more preferably 3pa; the time for freeze-drying is preferably 20 to 25 hours, and more preferably 24 hours.
In the invention, the concentration of the resveratrol ethanol solution in the step (10) is preferably 8-12 mg/mL, and more preferably 10mg/mL; the load capacity of the resveratrol ethanol solution is preferably 2.5-15% of the weight of the mono-succinylated pullulan-sulfated chitosan crosslinked membrane, and is further preferably 5% of the weight of the mono-succinylated pullulan-sulfated chitosan crosslinked membrane; the drying temperature is preferably 45-55 ℃, more preferably 48-52 ℃, and even more preferably 50 ℃; the drying time is preferably 20 to 25 hours, and more preferably 24 hours.
The invention also provides the skin wound dressing prepared by the preparation method.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Pullulan in the following examples was purchased from Shandong kana Biotech limited; succinic anhydride was purchased from national pharmaceutical group chemical agents, ltd; 4-dimethylaminopyridine was purchased from national pharmaceutical group chemical agents, inc.; dimethyl sulfoxide was purchased from chemical reagents ltd of miuiou, department of tianjin; chitosan was purchased from zhejiang gold chitosan pharmaceutical ltd; dichloroacetic acid was purchased from Kemi Euro Chemicals, inc., tianjin; dimethylformamide was purchased from chemical reagents ltd, miuiou, department of tianjin; chlorosulfonic acid (HClSO) 3 ) Purchased from Shanghai Xiandong Biotechnology GmbH; resveratrol was purchased from mcelin reagent.
Example 1
A preparation method of a skin wound dressing comprises the following steps:
(1) Dissolving 3.0g of pullulan in 20mL of DMSO for later use; dissolving 1.8g of succinic anhydride (succinic anhydride/Pu sugar unit =1/1,mol/mol) in 4mLDMSO, then dripping into pullulan sugar solution, adding 0.45g of DMAP (DMAP: succinic anhydride =1:5,mol/mol) after complete dissolution, stirring in a constant-temperature water bath at 50 ℃ for 24h for reaction, stopping the reaction, dripping the reaction solution into 200mL of absolute ethyl alcohol for precipitation, carrying out suction filtration, washing with an appropriate amount of absolute ethyl alcohol, dissolving the precipitate in an appropriate amount of water, dialyzing for 48h, and freeze-drying (the freeze-drying temperature is-65 ℃, the vacuum degree of freeze-drying is 3pa, the freeze-drying time is 24h, the same below) to obtain white spongy solid, namely the mono-succinylated pullulan (st-Pu).
(2) In a three-necked flask, 1g of chitosan was added, and stirred overnight using 40mL of formamide and 0.8mL of dichloroacetic acid as solvents. 3.4mL of HClSO was added at 4 deg.C 3 Slowly dropwise adding the mixture into 13.4mL of Dimethylformamide (DMF), stirring for 30min, adding the mixture into a chitosan solution under magnetic stirring and nitrogen protection, reacting at 60 ℃ for 2h, adding 10mL of deionized water, adjusting the pH value to be neutral by using 2M NaOH, precipitating with 4-fold ethanol, washing a precipitate with ethanol, dissolving the precipitate in water, dialyzing the water for 72h by using a 3500Da Mw cutoff dialysis membrane, and freeze-drying to obtain sulfated chitosan (2, 6-SCS).
(3) st-Pu and SCS are dissolved in deionized water to obtain mixed solutions with the concentration of 1% (w/v) respectively. st-Pu and SCS were mixed in different weight ratios (30.
(4) 10mg/mL of resveratrol absolute ethanol solution is prepared, and the solution is sprayed on 20mg of membrane material according to the weight of 2.5 percent, 5 percent and 15 percent of the cross-linked membrane material respectively to obtain different resveratrol-loaded membranes (RES-2.5 percent/SCS-st-Pu I, RES-5 percent/SCS-st-Pu I, RES-15 percent/SCS-st-Pu I and RES-2.5 percent/SCS-st-Pu II, RES-5 percent/SCS-st-Pu II and RES-15 percent/SCS-st-Pu II series resveratrol-loaded membrane materials). Placing in ventilated dark place, after the anhydrous alcohol is volatilized, wetting the membrane material with a small amount of water to restore the original state, and drying at 50 deg.C for 24h to obtain the skin wound dressing. The obtained skin wound dressing is stored under the dark and dry condition at room temperature.
Experimental example 1
Infrared spectrum detection:
taking the sample prepared in example 1 as an example, a proper amount of sample is taken for infrared detection, and KBr tabletting method is adopted, and the thickness is 40-4000cm -1 Wave number scanning. And (3) measuring the infrared spectra of the mixed sample of pullulan, succinylated pullulan, chitosan, sulfated chitosan and SCS/st-Pu. The results of the detection are shown in FIG. 1. As can be seen from FIG. 1, the succinylated pullulan was found to be 1734cm -1 The successful synthesis of succinylated pullulan can be proved by the appearance of a new ester peak. Sulfated chitosan is in the range of 1245cm -1 Asymmetric vibration absorption peak at O = S = O, 807cm -1 Is a symmetric vibration absorption peak of C-O-C. The existence of a sulfate structure in the product and the successful synthesis of the sulfonic acid group are shown. Furthermore, in the mapping analysis of the crosslinked composite (e.g. in FIG. 1), a characteristic absorption peak of Sulfated Chitosan (SCS) was found at 1734cm -1 The characteristic peak of succinylated pullulan (st-Pu) is shown, and the SCS is successfully crosslinked with the st-Pu.
Experimental example 2
And (3) detecting nuclear magnetic resonance spectrum:
of the samples prepared in example 1 of the invention 1 The H-NMR spectrum is shown in FIG. 2, wherein a in FIG. 2 is of pullulan 1 H-NMR spectrum,. Delta. =4.84ppm, H 1,α-1,6 Proton peak, δ =5.25-5.29ppm is H 1,α-1,4 Proton peak, delta =3.0-4.0ppm is H in sugar ring 2,3,4,5,6,6’ Proton peak. B in FIG. 2 is succinic anhydride 1 H-NMR spectrum showed the appearance of-OCCH at 2.44-2.66ppm 2 CH 2 Proton peak on CO-group. C in FIG. 2 is of succinyl pullulan 1 H-NMR spectrum showed that methylene protons of succinic anhydride appeared at delta =2.55-2.67ppm with slight shift in chemical shift. Meanwhile, all peaks of pullulan appeared in succinyl pullulan, demonstrating that succinyl pullulanAnd (4) successfully synthesizing. In FIG. 2 d and e are chitosan and sulfated chitosan, respectively 1 H-NMR chart, there were some significant differences between the two. Chitosan 1 H-NMR spectrum analysis: delta =1.85ppm is the methyl proton peak in N-acetylglucosamine, delta =2.85ppm is the H-2 proton peak of the glucosamine ring, delta =3.6-4.0ppm is the H-3,4,5 and 6 proton peaks of the glucosamine ring, and delta =4.60ppm is the H-1 proton peak of the glucosamine ring. The H-2 proton absorption peak of the glucosamine ring in sulfated chitosan (e in FIG. 2) is significantly reduced, a new H-2S peak appears at 3.27ppm, and it is slightly shifted to a low field due to the negative charge of the sulfonic acid group. Due to-SO 3 - On the influence of the C6 proton, a new peak, an absorption peak of H-6S, was found at 3.58 ppm. 4.25ppm A small, blunt peak of H-3S, i.e., very little reaction of-OH at the 3-position, was observed, indicating that sulfonation predominantly occurred at C 6 -OH and C 2 -NH 2 。
Experimental example 3
In vitro antibacterial experiments:
the dressing of example 1 of the present invention was evaluated for antibacterial activity using colony counting method, and common escherichia coli (e.coli, gram negative bacteria) and staphylococcus aureus (s.aureus, gram positive bacteria) were used as bacterial models. The specific steps of the antibacterial experiment are as follows: prior to the experiment, the film material was sterilized by UV light for 30 minutes, followed by preparation of 10 5 CFU/mL of Staphylococcus aureus and Escherichia coli liquid. Under aseptic conditions, 20mL of the suspension containing 10 5 The culture solution of CFU/mL bacteria is put into a centrifuge tube together with the sample, and put into a constant temperature oscillator at 37 ℃ so that the sample is fully contacted with the bacteria solution. After 24h incubation, the bacterial suspension was serially diluted in decimal form, and 100. Mu.L of each dilution was dropped onto pre-sterilized and cooled agar medium. The bacterial solution was then spread evenly on agar medium with a sterile cotton swab. Each dish was placed upside down in the incubator and after 24h, the plate medium was removed and the colonies were counted. Percent bacteria reduction = (N-M)/N × 100%, where M is the number of colonies in the experimental group and N is the number of colonies in the blank group.
Bacterial infections can delay and prevent normal wound repair, especially in diabetic wounds, and are more susceptible to bacterial attack. Thus, effective antimicrobial capacity is an important assessment factor for wound dressings. FIG. 3 is a photograph of colonies after 24 hours of incubation of Escherichia coli and Staphylococcus aureus. Through formula calculation, the bacteriostatic rates of the resveratrol-loaded (RES, 2.5-15% of load respectively prepared and w/w) membrane dressing group to staphylococcus aureus are respectively 64% (RES-2.5%/SCS-st-Pu II), 91% (RES-5%/SCS-st-Pu II) and 99% (RES-15%/SCS-st-Pu II) and the bacteriostatic rates to escherichia coli are respectively 27% (RES-2.5%/SCS-st-Pu II), 40% (RES-5%/SCS-st-Pu II) and 82% (RES-15%/SCS-st-Pu II) by taking sulfated chitosan cross-succinylated pullulan II (SCS-st-Pu II) group as a control. The data result shows that the RES/SCS-st-Pu film dressing prepared by the invention has better bacteriostatic activity on escherichia coli and staphylococcus aureus, and particularly has obvious inhibition on staphylococcus aureus.
Experimental example 4
In vitro antioxidant assay:
0.03g of DPPH powder is precisely weighed and prepared into 0.03mg/mL solution by absolute ethyl alcohol for later use. Different proportions of drug-containing material (20 mg) were immersed in 20mL of absolute ethanol solution at 37 ℃ in the dark. Subsequently 2mL of sample solution was collected at (2, 6, 12, 24, 48 and 72 h) and replaced with the same volume of absolute ethanol to maintain the same volume of solution. Adding equal volume of DPPH solution into all the sample solutions, mixing, reacting at room temperature in the dark for 30min, and measuring the decrease of DPPH at 517nm with an ultraviolet spectrophotometer. The calculation formula is as follows:
where Abs is the absorbance of the solution of the composite material containing RES, absc is the corresponding solution without RES, and Abs0 is the absorbance of the DPPH solution.
The antioxidant activity is beneficial to controlling the generation of excessive active oxygen on the wound surface, the active oxygen is closely related to wound inflammation and chronic wounds, and RES is a free radical scavenger. The compound has three phenol groups, can transfer protons from the phenol groups to free radicals, and plays a role in scavenging the free radicals; meanwhile, it can inhibit lipid peroxidation and induce various intracellular antioxidases, and therefore, RES has strong antioxidant activity. The antioxidant activity of each film dressing group at different time points is shown in FIG. 4, the SCS-st-Pu II and SCS-st-Pu I groups have no substantial antioxidant activity, the antioxidant activity of the SCS-st-Pu II and SCS-st-Pu I with the same drug loading amount has no obvious difference, and the clearance of the RES/SCS-st-Pu film dressing to DPPH is in a dose-dependent relationship. After 2h, the DPPH clearance rates of different drug-loaded film dressings were, in order from high to low, 93% (RES-15%/SCS-st-Pu II), 91% (RES-15%/SCS-st-Pu I), 60% (RES-5%/SCS-st-Pu I), 59% (RES-5%/SCS-st-Pu II), 52% (RES-2.5%/SCS-st-Pu II) and 50% (RES-2.5%/SCS-st-Pu I). The clearance rate of DPPH is increased along with the increase of the soaking time of the film dressing in the ethanol solution, and the clearance rate of all groups is basically unchanged after 24 hours.
Experimental example 5
Evaluation of membrane material cytotoxicity:
and performing cytotoxicity evaluation on the film dressing by adopting an MTT (diphenyl tetrazolium bromide) method. The experimental selection of NIH/3T3 mouse embryonic fibroblasts was based on CO at 37 ℃ and 5% 2 Under the condition, the culture was carried out in a DMEM medium containing 10% fetal bovine serum. The film dressing is treated by an extraction method. 0.02g of SCS-st-Pu I, RES-2.5%/SCS-st-Pu I, RES-5%/SCS-st-Pu I, RES-15%/SCS-st-Pu I, SCS-st-Pu II, RES-2.5%/SCS-st-Pu II, RES-5%/SCS-st-Pu II and RES-15 SCS-st-Pu II were weighed respectively, sterilized by ultraviolet rays for 30min, the membrane dressing was immersed in 10mL of DMEM culture solution, placed in a 37 ℃ incubator for 24h, and the medium was filter-sterilized with a 0.22 μm sterile filter.
NIH/3T3 cells at 1X 10 4 One well was inoculated to a 96-well plate at 200. Mu.L/well and incubated overnight in an incubator. The stock culture was discarded, the cells were washed twice with PBS buffer and 200. Mu.L of the sample extract was added. Under the same condition, DMEM culture solution is added as negative control; only DMEM culture solution is added to non-inoculated cells to serve as a blank control 1, only sample leaching liquor is added to non-inoculated cells to serve as a blank control 2, and each group is provided with three multiple wells. Cultivation of24. After 48 and 72h, the 96-well plate was removed and incubated for an additional 4h with 20 μ LMTT solution. The culture medium was discarded, 150. Mu.L of DMSO was added to each well, shaken for 10min, and the absorbance was measured at 490nm with a microplate reader. The Cell viability (percent) was calculated according to the following formula.
In the formula, OD sample Absorbance for the experimental group well; OD control Absorbance in negative control wells; OD 1 Blank control 1 group Absorbance, OD 2 Blank 2 absorbance.
To evaluate the biological safety of the film dressing, the membrane dressing was examined for NIH/3T3 cytotoxicity using the MTT method. Cell viability of each group after 24, 48 and 72h is shown in fig. 5, the cell activity of each dressing at 24h is from high to low in order of 96% (SCS-st-Pu ii), 91% (SCS-st-Pu i), 90% (RES-2.5%/SCS-st-Pu ii), 87% (RES-2.5%/SCS-st-Pu i), 84% (RES-5% SCS-st-Pu ii), 84% (RES-5%/SCS-st-Pu i), 64% (RES-15%/SCS-st-Pu ii) and 61% (RES-15%/SCS-st-Pu i), the membrane dressing cell viability is reduced after loading, and when the drug loading reaches 15%, the cell viability is significantly reduced, indicating that a high content of RES is toxic to the cells, i.e. a low concentration of RES can enhance NIH/3T3 cells, a high concentration of RES can reduce cell viability, and cell toxicity is generated. At 72h, the RES-15%/SCS-st-Pu II and RES-15%/SCS-st-Pu I groups showed cell viability as low as 48% and 46%, while the other groups showed an increasing trend with time.
Experimental example 6
Diabetic rat wound healing experiment:
establishing and repairing a full-thickness incision model of a diabetic rat: 12 male 12-week Wistar diabetic rats were selected and randomized into 4 groups, the placebo, 3Tegaderm dressing, RES-2.5%/SCS-st-Pu ii and RES-5%/SCS-st-Pu ii groups. The food and water were freely available to the environment in ventilated cages at a temperature of (21. + -. 2 ℃) and a relative humidity of (55. + -. 10%). Before the experiment, ether is used for anesthesiaRats, shaved back hair, surgical area sterilized with 75% ethanol, and under sterile conditions, a full-thickness circular wound of diameter (15 mm x 15 mm) was created on the back of each rat using a sterile biopsy punch. The sterilized film dressing is applied to wounds of diabetic rats and fixed by medical gauze and adhesive tapes, and the rats after molding are fed with single cages. Blood glucose and body weight levels were monitored. Wound healing status was photographed at 0, 3, 7 and 14d with a digital camera and the area of each photographed wound was calculated with ImageJ software. Extent of wound healing by percent area contraction (W) of skin wound A ) To calculate:
in the formula, A 0 Is the initial wound area; a. The t Is the wound area on day t.
Diabetic wounds are difficult to heal due to excessive inflammation and blocked new blood vessels. The experimental study establishes a full-thickness skin excision model of a diabetic rat, selects RES-2.5%/SCS-st-Pu II and RES-5%/SCS-st-Pu II film dressing, and applies the dressing on the wound surface of the diabetic rat for evaluation. As shown in fig. 6, the blank group of 3d diabetic rat wounds had severe infection with a large wound area; the rat wound surface applied with 3Tegaderm dressing (3M dressing for short) group is slightly infected; the RES-2.5%/SCS-st-Pu II and RES-5%/SCS-st-Pu II film dressing the wound healing rate of the rat wound treatment group was significantly better than that of the 3M dressing. 7d, the wound healing rate of each group was 78% (RES-2.5%/SCS-st-Pu II group), 63% (RES-5%/SCS-st-Pu II group), 35% (3M dressing group) and 28% (blank group) in order from high to low. The healing rates of the 14d, RES-2.5%/SCS-st-Pu II and RES-5%/SCS-st-Pu II membrane dressings were 99% and 96%, respectively. While the healing rates of the blank and 3M dressing groups were 55% and 81%, respectively. In conclusion, the RES-2.5%/SCS-st-Pu II and RES-5%/SCS-st-Pu II film dressings prepared by the invention have obviously better effect on treating the wound surface of the diabetic rat than that of the 3M dressing group sold on the market. Therefore, the RES/SCS-st-Pu film dressing with good antibacterial, anti-inflammatory and antioxidant effects can effectively promote the healing of diabetic wounds.
Experimental example 7
Normal rat wound healing experiment:
a rat full-thickness incision model is established to evaluate the wound healing promotion capability of the film dressing, healthy male Wistar rats are selected and randomly divided into 5 groups, and each group comprises 3 rats. The food and water can be freely obtained by separately feeding the food and water in ventilated cages with the temperature of (21 +/-2) DEG C and the relative humidity of (55 +/-10)%. Rats were anesthetized prior to the experiment, the back hair shaved, the surgical area sterilized with 75% ethanol, and a full-thickness circular wound of diameter (15 mm x 15 mm) was created on the back of each rat using a sterile biopsy punch under sterile conditions. The experiment is divided into SCS-st-Pu, RES-2.5%/SCS-st-Pu, RES-5%/SCS-st-Pu, commercially available band-aid and blank groups. The film dressing was applied to the wound after sterilization, fixed with medical gauze and tape, and the wound was photographed at 0, 3, 7 and 14d with a camera. And calculate the area of each wound with ImageJ software. Extent of wound healing by percent wound area shrinkage (W) A ) To calculate:
in the formula, A 0 Is the initial wound area; a. The t Is the wound area on day t.
A full-layer incision model is established on the back of a Wistar rat to study the wound repair capacity of the film dressing, and the wound repair conditions and the wound healing rates of different dressings to the rat are shown in figure 7. Compared with a blank control group, the self-made film dressing promotes wound healing, SCS-st-Pu II is superior to SCS-st-Pu I, and the drug-loaded film dressing shows a faster healing rate, wherein the RES-2.5%/SCS-st-Pu film dressing has the best effect. The wound healing rate is shown in fig. 7B, the wound healing rate of each group 7d is 71% (RES-2.5%/SCS-st-Pu), 65% (RES-5%/SCS-st-Pu), 58% (SCS-st-Pu), 45% (woundplast group) and 39% (blank group) from high to low, and the RES/SCS-st-Pu film dressing shows better wound healing promoting effect compared with the woundplast group. No. 14d, RES/SCS-st-Pu film dressing group rats all grow new skin, almost no scab is generated, and the wound healing rate reaches 97 percent; the wound of the rats in the blank group is scabbed, the wound is not completely closed, and the healing rate is 64%.
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)
1. A preparation method of a skin wound dressing is characterized by comprising the following steps:
(1) Mixing pullulan, succinic anhydride and dimethyl sulfoxide, mixing with 4-dimethylaminopyridine after complete dissolution, and sequentially stirring, precipitating with ethanol and filtering to obtain a precipitate 1;
(2) Mixing the precipitate 1 with water to ensure that the precipitate 1 is just dissolved, and sequentially dialyzing and freeze-drying to obtain mono-succinylated pullulan;
(3) Mixing chitosan, dichloroacetic acid and dimethylformamide, and stirring overnight to obtain a mixed solution 1;
(4) Mixing and stirring chlorosulfonic acid and dimethylformamide to obtain a mixed solution 2;
(5) Mixing the mixed solution 1 and the mixed solution 2 under the conditions of stirring and nitrogen, then reacting for 1.5-2.5 h at 55-65 ℃, adding deionized water after the reaction is finished, adjusting the pH value to be neutral, and then carrying out alcohol precipitation and suction filtration to obtain a precipitate 2;
(6) Mixing the precipitate 2 with water to enable the precipitate 2 to be just dissolved, and sequentially dialyzing and freeze-drying to obtain sulfated chitosan;
(7) Respectively mixing the mono-succinylated pullulan and the sulfated chitosan with water to obtain a mono-succinylated pullulan aqueous solution and a sulfated chitosan aqueous solution;
(8) Mixing the aqueous solution of the mono-succinylated pullulan polysaccharide with an aqueous solution of sulfated chitosan, and freeze-drying to obtain a freeze-dried film;
(9) Immersing the freeze-dried membrane in a cross-linking solution for cross-linking reaction, washing with deionized water, and freeze-drying to obtain a mono-succinylated pullulan-sulfated chitosan cross-linked membrane;
(10) Loading the resveratrol ethanol solution on the mono-succinylated pullulan polysaccharide-sulfated chitosan cross-linked film, and drying to obtain the skin wound dressing.
2. The preparation method of a skin wound dressing according to claim 1, wherein in step (1), the mole ratio of pullulan, succinic anhydride and 4-dimethylaminopyridine is 0.8-1.2, 0.8-1.2.
3. A method for preparing a skin wound dressing according to claim 1 or 2, wherein the dialysis in step (2) is carried out at room temperature for 45-50 h by using 8000-14000 DaMw dialysis bags, the freeze-drying temperature is-60-70 ℃, the vacuum degree of the freeze-drying is less than 5pa, and the freeze-drying time is 20-25 h.
4. A method for preparing a skin wound dressing according to claim 3, wherein in the step (3), the mixing ratio of the chitosan, the dichloroacetic acid and the dimethylformamide is 0.8-1.2 g, and is 35-45 mL, and the molecular weight of the chitosan is 5-2000 KDa;
the mixing volume ratio of the chlorosulfonic acid to the dimethylformamide in the step (4) is 3-4, the stirring temperature in the step (4) is 0-4 ℃, and the stirring time in the step (4) is 25-35 min.
5. A method for preparing a skin wound dressing according to claim 4, wherein the mixing ratio in step (5) is calculated according to the molar ratio of chitosan in the mixed solution 1 to chlorosulfonic acid in the mixed solution 2, the molar ratio of chitosan in the mixed solution 1 to chlorosulfonic acid in the mixed solution 2 is 1 to 6 to 8, the component for adjusting the pH value is a sodium hydroxide solution, and the molar concentration of the sodium hydroxide solution is 1 to 3mol/L;
and (3) dialyzing for 70-75 h at room temperature by using a 3000-4000 DaMw dialysis bag in the dialysis step (6), wherein the freeze-drying temperature is-60 to-70 ℃, the vacuum degree of freeze-drying is less than 5pa, and the freeze-drying time is 20-25 h.
6. A method for preparing a skin wound dressing according to claim 5, wherein the concentrations of the aqueous solution of the mono-succinylated pullulan and the aqueous solution of sulfated chitosan in step (7) are independently 0.8 to 1.2wt%.
7. A method for preparing a skin wound dressing according to claim 6, wherein the mixing weight ratio of the aqueous solution of the mono-succinylated pullulan to the aqueous solution of sulfated chitosan in step (8) is 30-70, the freeze-drying temperature is-60 to-70 ℃, the vacuum degree of the freeze-drying is less than 5pa, and the freeze-drying time is 20-25 h.
8. A method of preparing a skin wound dressing according to claim 7, wherein the cross-linking solution in step (9) is an ethanol solution, and comprises the following components in concentration: 80-120 mmol/L of 1-ethyl- (3-dimethylaminopropyl) carbodiimide, 20-30 mmol/L of N-hydroxysuccinimide, 70-80 vt% of ethanol solution, 0-4 ℃ of crosslinking reaction, 20-25 h of crosslinking reaction time, 2-4 times of deionized water washing, 0.8-1.2 h of washing time, 60-70 ℃ of freeze-drying temperature, 5pa of vacuum degree of freeze-drying and 20-25 h of freeze-drying time.
9. The method for preparing a skin wound dressing according to claim 8, wherein the concentration of the resveratrol ethanol solution in step (10) is 8-12 mg/mL, the loading capacity of the resveratrol ethanol solution is 2.5-15% of the weight of the mono-succinylated pullulan-sulfated chitosan cross-linked film, the drying temperature is 45-55 ℃, and the drying time is 20-25 h.
10. A wound dressing of skin prepared by the process of any one of claims 1 to 9.
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