CN103961738A - Chitosan-nano-silver wound dressing and preparation method thereof - Google Patents
Chitosan-nano-silver wound dressing and preparation method thereof Download PDFInfo
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Abstract
The invention provides a chitosan-nano-silver wound dressing which comprises a water absorption antibiosis layer and a hydrophobic layer for gluding a wound, wherein the water absorption antibiosis layer comprises chitosan and nano-silver; according to the water absorption antibiosis layer, spongy chitosan is soaked in sol contained nano-silver, and the nano-silver is uniformly compounded on the spongy chitosan through freeze drying; the other side adopts the hydrophobic layer. A preparation method of the chitosan-nano-silver wound dressing comprises the following steps: (1) preparing chitosan-nano-silver sponge; (2) uniformly spraying an ethanol solution on one surface of the chitosan-nano-silver sponge prepared in step (1), prefreezing under minus 18 DEG C, and then performing freeze drying to obtain the chitosan-nano-silver wound dressing. According to the invention, the chitosan, the nano-silver, fatty acid and other hydrophobic modifiers for preparing the chitosan-nano-silver wound dressing have high biosecurity and have no toxic or side effects on human cells; in addition, the chitosan is of a sponge structure, excellent in softness and high in breathability.
Description
Technical field
The invention belongs to medical dressing biomaterial preparing technical field, be specifically related to a kind of chitosan-nanometer silver wound dressing and preparation method thereof.
Background technology
Skin is the vitals of human body, is body and the extraneous natural cover for defense and the bridge of communication.Because burn, wound and skin ulcer etc. are former thereby cause the damaged of skin histology, may cause wound infection.At present, effective ways of reply skin wound are to use wound dressing.
Traditional wound dressing comprises gauze, absorbent cotton, binder etc.But from medical angle, these traditional wound dressings, owing to not possessing treatment and repairing the performances such as wound, do not have antibacterial activity yet, in clinical middle range of application, are limited to.Thereby can promote wound healing, high-efficiency antimicrobial, use Medical dressing more easily to grow up gradually.
Chitosan is biodegradable, good biocompatibility, have antibacterial activity and have no side effect, and can provide in wound non-albumen substrate to promote cell proliferation, tissue growth and blood clotting, and alleviate the pain of teleneuron.In addition, chitosan has good hydrophilic, and absorbing wound exudate, keeps the certain moistening degree of wound fast.According to wound " wet union " theory, moistening environment is conducive to slough and fibrinous dissolving, is conducive to cell proliferation and differentiation and movement, accelerates the formation of granulation tissue.And, thering is the Chitosan in Wound Dressing of excellent hydrophilic, can make it to be difficult for and new granulation tissue's adhesion, more can ease the pain during change dressings, the secondary mechanical damage causing while avoiding using.Therefore, chitosan-based wound dressing obtains people's extensive concern, and commercial chitosan dressing has Medical C AD(cellulose acetate at present) chitosan dressing, chitosan anti-bacteria wound-protecting film, nation of nation the biological external dressing of various chitosans etc. such as paste.
Simple chitosan dressing is unsatisfactory to the inhibition of pathogen, therefore in relevant research, generally in chitosan, adds antibacterial to improve its antibacterial activity.Inorganic antiseptic is due to the antibacterial activity of its broad-spectrum high efficacy and be difficult for producing drug resistance, thereby is widely used in wound dressing.Wherein the material of silver series receives much concern in inorganic antiseptic field in recent years, and as commercial Kang Lidi argentiferous chitosan dressing at present, this dressing has added silver ion on the basis of chitosan bleeding-stopping dressing, thereby improves its anti-infection ability.Yet the dressing of silver ion in use can cause wound concentration of silver ions too high, if life-time service, the silver ion of excessively assembling may have certain risk to the health of human body.In recent years along with the development of nanotechnology, it is found that nanometer silver to the antibacterial activity of encountered pathogenic bacteria far above silver salt, and compare with silver salt, can overcome the too fast and unmanageable shortcoming of silver ion release in silver salt with antibacterial activity, lower to the toxicity of human body.For this reason, preparation becomes study hotspot in recent years containing the chitosan dressing of nanometer silver.
The advantages such as the existing chitosan dressing containing nanometer silver generally has the wound exudate of absorption, promote wound blood coagulation and wound healing, biocompatibility is good.Yet these dressing are all exposed in air conventionally, with extraneous exchanging gas in, also may cause some unclean liquid infiltration to enter wound, thereby stimulate wound, affect wound healing.This is also to apply clinically at present the existing weak point of wound dressing.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, and chitosan-nanometer silver wound dressing that a kind of surface has water-proof function and preparation method thereof is provided.This wound dressing laminating wound face pliability is high, good permeability, biological safety are high, and preparation method is simple.
The present invention is for solving the problems of the technologies described above by the following technical solutions:
A kind of chitosan-nanometer silver wound dressing, this wound dressing comprises water suction antibiotic layer and the hydrophobic layer of the wound face of fitting, described water suction antibiotic layer chitosan-containing and nanometer silver, this layer is that spongy chitosan is immersed in the colloidal sol containing nanometer silver, by cryodesiccated method, nanometer silver is evenly compounded on spongy chitosan; Another side is hydrophobic layer.
Press such scheme, described nanometer silver particle diameter is 10-20nm; Described hydrophobic layer comprises one or more in hydrophobically modified agent palmitic acid, stearic acid, arachidic acid or behenic acid.Hydrophobic layer consists of straight chain fatty acid, is the structure of smooth planar, and a small amount of hole exists, and can prevent that extraneous liquid from infiltrating.Due to hydrophobic layer very thin (thickness is at 1-10 μ m), and there is a small amount of hole to exist, so do not affect the breathability of dressing.
Press such scheme, the preparation method of described spongy chitosan is: chitosan is dissolved in the acetum of 0.5-2wt%, preparing mass volume ratio is 1-3%(w/v) chitosan-acetic acid solution, and carry out below precooling 8-16h at-18 ℃, then carry out lyophilization processing, subsequently products therefrom is immersed in and in alkali liquor, removes residual acetic acid, more repeatedly rinse to neutrality with deionized water, obtain spongy chitosan.
Preferably, described deacetylating degree of chitosan is 70-100%, and viscosity is 100-200cPas.
Preferably, the condition that described lyophilization is processed is: freezer dryer condenser temperature is equal to or less than-40 ℃, vacuum≤10Pa, and sublimation drying is 16-24h.
The present invention also provides the preparation method of above-mentioned chitosan-nanometer silver wound dressing, and it comprises the following steps:
(1) spongy chitosan is immersed in nano silver colloidal sol at 20-30 ℃, in this nano silver colloidal sol, nanometer silver concentration is 10-100 μ g/mL, soaks after 16-24h at precooling 6-10h below-18 ℃, obtains chitosan-nanometer silver sponge;
(2) alcoholic solution of the hydrophobically modified agent that is 10-50mmol/L by concentration is evenly sprayed in the one side of step (1) gained chitosan-nanometer silver sponge, and carry out below precooling 30-60min at-18 ℃, then carry out lyophilization processing and obtain chitosan-nanometer silver wound dressing.
Press such scheme, the preparation method of the described nano silver colloidal sol of step (1) is: the silver nitrate aqueous solution that the polyvinylpyrrolidone aqueous solution that is 1-20g/L by concentration is 0.1-0.5mol/L with concentration mixes, polyvinylpyrrolidone aqueous solution and silver nitrate aqueous solution volume ratio are 10:1-20:1, after stirring, slowly drip the sodium borohydride aqueous solution that concentration is 0.01-0.1mol/L, drop rate is within every 20 seconds, to drip 10 μ L, extremely mixed solution colour is yellowish-brown and samples by till the generation of sodium chloride solution evaluation nothing precipitation, obtains nano silver colloidal sol.
Press such scheme, the described lyophilization treatment conditions of step (2) are: freezer dryer condenser temperature is equal to or less than-40 ℃, vacuum≤10Pa, and sublimation drying is 10-12h.
Press such scheme, the preparation method of described spongy chitosan is: chitosan is dissolved in the acetum of 0.5-2wt%, prepare the chitosan-acetic acid solution that mass volume ratio is 1-3%, and carry out below precooling 8-16h at-18 ℃, then carry out lyophilization processing, subsequently products therefrom is immersed in and in alkali liquor, removes residual acetic acid, more repeatedly rinse to neutrality with deionized water, obtain spongy chitosan.
Press such scheme, described deacetylating degree of chitosan is 70-100%, and viscosity is 100-200cPas.
The invention has the beneficial effects as follows: 1. the hydrophobically modified agent biological safeties such as chitosan, nanometer silver and fatty acid that the prepared chitosan-nanometer silver wound dressing of the present invention is used are high, human body cell is had no side effect, and chitosan is sponge structure, flexibility is good, breathability is high, is also difficult for and wound surface adhesion; 2. this wound dressing has one side water suction, and the characteristic of another side waterproof can when absorbing wound sepage, can prevent that again extraneous unholiness liquid and antibacterial from infiltering wound, and the detailed bacterium of test chart cannot grow on this wound dressing surface; 3. contained nano-Ag particles size uniform in the wound dressing that prepared by the present invention, antibacterial activity is high; This wound dressing to human normal cell as HEK293(HEKC) there is no toxicity, and the growth of cell is had to facilitation, there is excellent wound repair ability and promote healing ability; 5. wound dressing of the present invention has good bacteriostatic activity to clinical common pathogen (staphylococcus aureus, methicillin-resistant staphylococcus aureus, Pseudomonas aeruginosa, tolerant Pseudomonas aeruginosa, escherichia coli, drug resistance escherichia coli); 6. wound dressing provided by the invention has good tensile strength, ductility and water absorbing properties (hot strength reaches 0.022-0.072MPa; Extension rate reaches 20.663-40.764%; Water absorption rate reaches 1165-1309%); 7. compare with normal blood coagulation, wound dressing product provided by the invention can make clotting time in advance more than 29%, more than rate can reach the twice of conventional medical gauze (14.60%) in advance.
Accompanying drawing explanation
Fig. 1 is the prepared chitosan-nanometer silver wound dressing hydrophobic layer surface water droplet test photo of the embodiment of the present invention 1;
Fig. 2 is the prepared chitosan-nanometer silver wound dressing water suction antibiotic layer surface water droplet test photo of embodiment 1;
Fig. 3 is the SEM photo on the prepared chitosan-nanometer silver wound dressing hydrophobic layer of embodiment 1 and water suction antibiotic layer surface;
Fig. 4 is the prepared chitosan-nanometer silver wound dressing hydrophobic layer surface water droplet contact photo of embodiment 2;
Fig. 5 is the prepared chitosan-nanometer silver wound dressing water suction antibiotic layer surface water droplet contact photo of embodiment 2;
Fig. 6 is the TEM photo of the nano-Ag particles of the prepared chitosan-nanometer silver wound dressing stripping of embodiment 3;
Fig. 7 is the prepared Chitosan in Wound Dressing bacterial penetration test photo of embodiment 4 and comparative example 1, and wherein left figure is the situation of wound dressing superficial growth antibacterial after cultivation 24h, and right figure is situation about taking off after wound dressing;
Fig. 8 is the SEM photo that the wound dressing of embodiment 5 preparations carries out anti-bacterial attachment test rear hydrophobic layer surface and water suction antibiotic layer surface;
Fig. 9 is mice wound healing test photo, is wherein from left to right followed successively by the blank group that any processing is not done in use, and Ai Kexin
tMthe wound dressing of Nanometer dressing and embodiment 6 preparations is processed the healing state of rear wound;
Figure 10 is for doing the result of cytotoxicity test to embodiment 1 to 4, wherein the result after 1-4 days is cultivated in abscissa representative; Vertical coordinate is the absorbance that cell records at 490nm place after MTT dyeing.
The specific embodiment
For making those skilled in the art understand better technical scheme of the present invention, below in conjunction with accompanying drawing, the present invention is described in further detail.
Comparative example 1
This comparative example preparation is containing nanometer silver and without the Chitosan in Wound Dressing of asymmetric moistening modification.
The preparation of spongy chitosan: (deacetylation is 82% to take 1.2g chitosan, viscosity is 200cPas), be dissolved in the acetum that 50mL concentration is 1wt%, after dissolving completely, pour the chitosan-acetic acid solution obtaining into glass culture dish and put into refrigerator in-20 ℃ of precooling 12h.Then be positioned in vacuum freeze drier and carry out lyophilization processing, wherein the parameter of freezer dryer is: condenser temperature is-40 ℃, and vacuum is 10Pa, and cooling time is 20 hours.Use subsequently the ammonia spirit immersion treatment 2 hours of 2.0mol/L, more repeatedly with deionized water, wash, until obtain neutral spongy chitosan.
The preparation of Chitosan in Wound Dressing: it is 0.3cm that above-mentioned spongy chitosan is cut into 2cm * 2cm(thickness) small pieces, and through ultraviolet radiation disinfection, encapsulation, obtain Chitosan in Wound Dressing product.
Embodiment 1
The preparation of spongy chitosan: (deacetylation is 70% to take 0.8g chitosan, viscosity is 100cPas), be dissolved in the acetum that 50mL concentration is 0.5wt%, after dissolving completely, pour the chitosan-acetic acid solution obtaining into glass culture dish and put into refrigerator in-20 ℃ of precooling 12h.Then be positioned in vacuum freeze drier and carry out lyophilization processing, wherein the parameter of freezer dryer is: condenser temperature is-40 ℃, and vacuum is 10Pa, and cooling time is 18 hours.Use subsequently the sodium hydroxide solution immersion treatment 2 hours of 1.0mol/L, more repeatedly with deionized water, wash, until obtain neutral spongy chitosan.
The preparation of nano silver colloidal sol: take 1.2g polyvinylpyrrolidone (PVP, weight average molecular weight Mw=55,000), be dissolved in 60mL deionized water, drip the silver nitrate aqueous solution that 6mL concentration is 0.1mol/L, stir the sodium borohydride solution that slowly drips 0.04mol/L after 30 minutes, every 20 seconds, drip 10 μ L, until mixed solution colour is yellowish-brown and sampling is identified without precipitation and generated by sodium chloride solution, it is 10 μ g/mL that gained nano silver colloidal sol is diluted to nanometer silver concentration.
The preparation of chitosan-nanometer silver wound dressing:
(1) preparation of chitosan-nanometer silver sponge: at 25 ℃, above-mentioned spongy chitosan is immersed in the nano silver colloidal sol of above-mentioned preparation, pours out immersion after immersion 24h, and put into refrigerator in-20 ℃ of precooling 6h, obtain chitosan-nanometer silver sponge;
(2) the asymmetric wettability modification of chitosan-nanometer silver sponge: the alcoholic solution that preparation palmitic acid concentration is 20mmol/L, and be sprayed on equably on chitosan-nanometer silver sponge obtained above surface, then put into refrigerator and continue freezing 30min in-20 ℃, then be positioned over and in vacuum freeze drier, carry out lyophilization processing, wherein the parameter of freezer dryer is: condenser temperature-40 ℃, vacuum 10Pa, cooling time is chitosan-nanometer silver wound dressing that 10h obtains having asymmetric wettability.
It is 0.3cm that the chitosan-nanometer silver wound dressing obtaining is cut into 2cm * 2cm(thickness) small pieces, and through ultraviolet radiation disinfection, encapsulation, obtain chitosan-nanometer silver wound dressing product.
With syringe, get 1 * 10
-5the rhdamine B solution of mmol/L drips respectively the two sides at above-mentioned square small pieces, observes drop in the adhesional wetting situation on two sides, and photo as shown in Figure 1, 2.By photo as seen on palmitic acid modification face drop can slide off surface; On unmodified, drop infilters inside, and test result shows that this chitosan-nanometer silver wound dressing product has one side waterproof, the characteristic of another side water suction.
By SEM, test the prepared chitosan-nanometer silver wound dressing product of the present embodiment, its surface microstructure as shown in Figure 3.As seen from the figure, palmitic acid modification face is fine and close planar structure; And unmodified be fine and close loose structure, and aperture is between 100-200 μ m.The surface microstructure that two sides is totally different, makes product have one side waterproof (the easy landing of water droplet), the characteristic of another side water suction.
Embodiment 2
The preparation of spongy chitosan: (deacetylation is 95% to take 1.0g chitosan, viscosity is 100cPas), be dissolved in the acetum that 50mL concentration is 1wt%, after dissolving completely, pour the chitosan-acetic acid solution obtaining into glass culture dish and put into refrigerator in-18 ℃ of precooling 12h.Then be positioned in vacuum freeze drier and carry out lyophilization processing, wherein the parameter of freezer dryer is: condenser temperature-55 ℃, and vacuum 4Pa, cooling time is 24 hours.Use subsequently the sodium hydroxide solution immersion treatment 2 hours of 2.0mol/L, more repeatedly with deionized water, wash, until obtain neutral spongy chitosan.
The preparation of nano silver colloidal sol: take 0.05g polyvinylpyrrolidone (PVP, weight average molecular weight Mw=55,000), be dissolved in 50mL deionized water, drip the silver nitrate aqueous solution that 2.5mL concentration is 0.1mol/L, stir the sodium borohydride solution that slowly drips 0.01mol/L after 30 minutes, every 20 seconds, drip 10 μ L, until mixed solution colour is yellowish-brown and sampling is identified without precipitation and generated by sodium chloride solution, it is 20 μ g/mL that gained nano silver colloidal sol is diluted to nanometer silver concentration.
The preparation of chitosan-nanometer silver wound dressing:
(1) preparation of chitosan-nanometer silver sponge: at 30 ℃, above-mentioned spongy chitosan is immersed in the nano silver colloidal sol of above-mentioned preparation, pours out immersion after immersion 24h, and put into refrigerator in-18 ℃ of precooling 8h, obtain chitosan-nanometer silver sponge;
(2) the asymmetric wettability modification of chitosan-nanometer silver sponge: the alcoholic solution that preparation arachidic acid concentration is 10mmol/L, and be sprayed on equably on chitosan-nanometer silver sponge obtained above surface, then put into refrigerator and continue freezing 60min in-18 ℃, then be positioned over and in vacuum freeze drier, carry out lyophilization processing, wherein the parameter of freezer dryer is: condenser temperature-55 ℃, vacuum 4Pa, cooling time is chitosan-nanometer silver wound dressing that 12h obtains having asymmetric wettability.
It is 0.4cm that the chitosan-nanometer silver wound dressing obtaining is cut into 2cm * 2cm(thickness) small pieces, and through ultraviolet radiation disinfection, encapsulation, obtain chitosan-nanometer silver wound dressing product.
Chitosan-nanometer silver wound dressing product that the present embodiment is prepared carries out water contact angle test, and use contact angle measurement respectively test water drops in the arachidic acid modification face of product and the contact angle of unmodified.As shown in Figure 4 and Figure 5, the contact angle of water droplet on arachidic acid modification face is 138 °; Water droplet is absorbed into the inside of product soon at unmodified face, its contact angle is about 0 °.Test result shows, this product has the hydrophilic asymmetric wettability of the hydrophobic another side of one side, and this specific character can, when absorbing wound sepage, can prevent that again extraneous unholiness liquid from infiltering wound.
Embodiment 3
The preparation of spongy chitosan: (deacetylation is 100% to take 1.5g chitosan, viscosity is 200cPas), be dissolved in the acetum that 50mL concentration is 1.5wt%, after dissolving completely, pour the chitosan-acetic acid solution obtaining into glass culture dish and put into refrigerator in-22 ℃ of precooling 8h.Then be positioned in vacuum freeze drier and carry out lyophilization processing, wherein the parameter of freezer dryer is: condenser temperature-58 ℃, and vacuum 8Pa, cooling time is 16h.Use subsequently the ammonia spirit immersion treatment 2 hours of 1.2mol/L, more repeatedly with deionized water, wash, until obtain neutral spongy chitosan.
The preparation of nano silver colloidal sol: take 0.5g polyvinylpyrrolidone (PVP, weight average molecular weight Mw=55,000), be dissolved in 45mL deionized water, drip the silver nitrate aqueous solution that 2.5mL concentration is 0.2mol/L, stir the sodium borohydride solution that slowly drips 0.1mol/L after 30 minutes, every 20 seconds, drip 10 μ L, until mixed solution colour is yellowish-brown and sampling is identified without precipitation and generated by sodium chloride solution, it is 30 μ g/mL that gained nano silver colloidal sol is diluted to nanometer silver concentration.
The preparation of chitosan-nanometer silver wound dressing:
(1) preparation of chitosan-nanometer silver sponge: at 20 ℃, above-mentioned spongy chitosan is immersed in the nano silver colloidal sol of above-mentioned preparation, pours out immersion after immersion 20h, and put into refrigerator in-20 ℃ of precooling 6h, obtain chitosan-nanometer silver sponge;
(2) the asymmetric wettability modification of chitosan-nanometer silver sponge: the alcoholic solution that preparation stearic acid concentration is 50mmol/L, and be sprayed on equably on chitosan-nanometer silver sponge obtained above surface, then put into refrigerator and continue freezing 40min in-20 ℃, then be positioned over and in vacuum freeze drier, carry out lyophilization processing, wherein the parameter of freezer dryer is: condenser temperature-58 ℃, vacuum 8Pa, cooling time is the chitosan-nanometer silver wound dressing that obtains having asymmetric wettability for 12 hours.
Chitosan-nanometer silver wound dressing that the present embodiment is prepared cuts out the small pieces that quality is 20mg, is dissolved in the aqueous acetic acid that 5mL concentration is 1wt%, and the liquid obtaining is carried out to centrifugal treating, gets supernatant and obtains nano silver dispersion.By TEM, test pattern and the size of nano-Ag particles in dispersion liquid, as shown in Figure 6.TEM test result shows, contained nano-Ag particles size uniform in chitosan-nanometer silver wound dressing prepared by the present embodiment, and particle diameter is 10-20nm.
Embodiment 4
The preparation of spongy chitosan: (deacetylation is 82% to take 1.2g chitosan, viscosity is 200cPas), be dissolved in the acetum that 50mL concentration is 2wt%, after dissolving completely, pour the chitosan-acetic acid solution obtaining into glass culture dish and put into refrigerator in-20 ℃ of precooling 12h.Then be positioned in vacuum freeze drier and carry out lyophilization processing, wherein the parameter of freezer dryer is: condenser temperature-46 ℃, and vacuum 6Pa, cooling time is 20h.Use subsequently the ammonia spirit immersion treatment 2 hours of 2.0mol/L, more repeatedly with deionized water, wash, until obtain neutral spongy chitosan.
The preparation of nano silver colloidal sol: take 0.45g polyvinylpyrrolidone (PVP, weight average molecular weight Mw=55,000), be dissolved in 30mL deionized water, drip the silver nitrate aqueous solution that 2.0mL concentration is 0.3mol/L, stir the sodium borohydride solution that slowly drips 0.05mol/L after 30 minutes, every 20 seconds, drip 10 μ L, until mixed solution colour is yellowish-brown and sampling is identified without precipitation and generated by sodium chloride solution, it is 60 μ g/mL that gained nano silver colloidal sol is diluted to nanometer silver concentration.
The preparation of chitosan-nanometer silver wound dressing:
(1) preparation of chitosan-nanometer silver sponge: at 25 ℃, above-mentioned spongy chitosan is immersed in the nano silver colloidal sol of above-mentioned preparation, pours out immersion after immersion 16h, and put into refrigerator in-19 ℃ of precooling 7h, obtain chitosan-nanometer silver sponge;
(2) the asymmetric wettability modification of chitosan-nanometer silver sponge: the alcoholic solution that preparation behenic acid concentration is 50mmol/L, and be sprayed on equably on chitosan-nanometer silver sponge obtained above surface, then put into refrigerator and continue freezing 50min in-19 ℃, then be positioned over and in vacuum freeze drier, carry out lyophilization processing, wherein the parameter of freezer dryer is: condenser temperature-46 ℃, vacuum 6Pa, cooling time is chitosan-nanometer silver wound dressing that 10h obtains having asymmetric wettability.
It is 0.2cm that the chitosan-nanometer silver wound dressing obtaining is cut into 2cm * 2cm(thickness) small pieces, and through ultraviolet radiation disinfection, encapsulation, obtain chitosan-nanometer silver wound dressing product.
The Chitosan in Wound Dressing product of chitosan-nanometer silver wound dressing product prepared by the present embodiment and comparative example 1 preparation carries out contrast test: the hydrophobic one side of chitosan-nanometer silver wound dressing makes progress, be laid in side by side on agar culture medium with Chitosan in Wound Dressing, above wound dressing, (bacterial concentration is 10 at two kinds then respectively to drip 200 μ L Escherichia coli bacteria liquids
6cFU/mL, in every milliliter of bacterium liquid, the colony counts of antibacterial is 10
6individual), observe place 24 hours in 37 ℃ of incubators after.The hydrophobic table of chitosan-nanometer silver wound dressing bacterium liquid prepared by visible the present embodiment is still stayed surface and is not infiltered inside, and the bacterium liquid on matched group Chitosan in Wound Dressing surface has infiltered sample; Two kinds of wound dressings are opened, and it is clean that chitosan-nanometer silver wound dressing bottom still keeps, and there is no colony growth, and large stretch of bacterium colony (Fig. 7) has been observed in the bottom of matched group Chitosan in Wound Dressing.Test result shows: nanometer silver-chitosan dressing product prepared by the present embodiment can prevent that extraneous antibacterial from infiltering inside.
Embodiment 5
The preparation of spongy chitosan: (deacetylation is 87% to take 0.5g chitosan, viscosity is 150cPas), be dissolved in the acetum that 50mL concentration is 0.8wt%, after dissolving completely, pour the chitosan-acetic acid solution obtaining into glass culture dish and put into refrigerator in-19 ℃ of precooling 15h.Then be positioned in vacuum freeze drier and carry out lyophilization processing, wherein the parameter of freezer dryer is: condenser temperature-55 ℃, and vacuum 4Pa, cooling time is 18 hours.Use subsequently the sodium hydroxide solution immersion treatment 2 hours of 1.5mol/L, more repeatedly with deionized water, wash, until obtain neutral spongy chitosan.
The preparation of nano silver colloidal sol: take 0.25g polyvinylpyrrolidone (PVP, weight average molecular weight Mw=55,000), be dissolved in 50mL deionized water, drip the silver nitrate aqueous solution that 3mL concentration is 0.5mol/L, stir the sodium borohydride solution that slowly drips 0.08mol/L after 30 minutes, every 20 seconds, drip 10 μ L, until mixed solution colour is yellowish-brown and sampling is identified without precipitation and generated by sodium chloride solution, it is 100 μ g/mL that gained nano silver colloidal sol is diluted to nanometer silver concentration.
The preparation of chitosan-nanometer silver wound dressing:
(1) preparation of chitosan-nanometer silver sponge: at 20 ℃, above-mentioned spongy chitosan is immersed in the nano silver colloidal sol of above-mentioned preparation, pours out immersion after immersion 24h, and put into refrigerator in-19 ℃ of precooling 9h, obtain chitosan-nanometer silver sponge;
(2) the asymmetric wettability modification of chitosan-nanometer silver sponge: the alcoholic solution that preparation palmitic acid concentration is 30mmol/L, and be sprayed on equably on chitosan-nanometer silver sponge obtained above surface, then put into refrigerator and continue freezing 45min in-19 ℃, then be positioned over and in vacuum freeze drier, carry out lyophilization processing, wherein the parameter of freezer dryer is: condenser temperature-55 ℃, vacuum 6Pa, cooling time is 9h, obtains having chitosan-nanometer silver wound dressing of asymmetric wettability.
It is 0.3cm that the chitosan-nanometer silver wound dressing obtaining is cut into 2cm * 2cm(thickness) small pieces, and through ultraviolet radiation disinfection, encapsulation, obtain chitosan-nanometer silver wound dressing product.
Chitosan-nanometer silver wound dressing product prepared by the present embodiment carries out anti-bacterial attachment test: from escherichia coli (E.coli) culture plate, picking colony is made into suspension, and diluted concentration to 10
6cFU/mL.Wound dressing was immersed in rapidly in bacterium liquid after 3 seconds, again wound dressing is immersed in to LB(bacteriolyze meat soup) in culture fluid, after shaken cultivation 24 hours, from culture fluid, take out wound dressing, add the glutaraldehyde solution of 2.5wt% to fix, after 15 minutes, use successively more aseptic PBS(phosphate buffer) solution and sterilized water repeatedly rinse 3 times, and then rinse 3 dehydrations, natural air drying with absolute methanol.Wound dressing water suction after fixing with SEM observation and the situation of hydrophobic two sides bacterial adhesion.Glutaraldehyde solution can make rapidly protein denaturation, and antibacterial is fixed on above ground wound dressing, can be by scanning electron microscopic observation to the antibacterial shell by staying after methanol dehydration, dried if there is antibacterial to exist.SEM test picture is visible, and escherichia coli shell has not all been observed on wound dressing two sides prepared by the present embodiment, shows antibacterial cannot grow on this wound dressing (Fig. 8).
Embodiment 6
The preparation of spongy chitosan: (deacetylation is 97% to take 1.3g chitosan, viscosity is 200cPas), be dissolved in the acetum that 50mL concentration is 1.2wt%, after dissolving completely, pour the chitosan-acetic acid solution obtaining into glass culture dish and put into refrigerator in-18 ℃ of precooling 12h.Then be positioned in vacuum freeze drier and carry out lyophilization processing, wherein the parameter of freezer dryer is: condenser temperature-50 ℃, and vacuum 8Pa, cooling time is 18h.Use subsequently the ammonia spirit immersion treatment 2h of 1.25mol/L, more repeatedly with deionized water, wash, until obtain neutral spongy chitosan.
The preparation of nano silver colloidal sol: take 0.5g polyvinylpyrrolidone (PVP, weight average molecular weight Mw=55,000), be dissolved in 40mL deionized water, drip the silver nitrate aqueous solution that 3mL concentration is 0.4mol/L, stir the sodium borohydride solution that slowly drips 0.05mol/L after 30 minutes, every 20 seconds, drip 10 μ L, until mixed solution colour is yellowish-brown and sampling is identified without precipitation and generated by sodium chloride solution, it is 50 μ g/mL that gained nano silver colloidal sol is diluted to nanometer silver concentration.
The preparation of chitosan-nanometer silver wound dressing:
(1) preparation of chitosan-nanometer silver sponge: at 30 ℃, above-mentioned spongy chitosan is immersed in the nano silver colloidal sol of above-mentioned preparation, pours out immersion after immersion 24h, and put into refrigerator in-18 ℃ of precooling 10h, obtain chitosan-nanometer silver sponge;
(2) the asymmetric wettability modification of chitosan-nanometer silver sponge: the alcoholic solution that preparation stearic acid concentration is 50mmol/L, and be sprayed on equably on chitosan-nanometer silver sponge obtained above surface, then put into refrigerator and continue freezing 60min in-18 ℃, then be positioned over and in vacuum freeze drier, carry out lyophilization processing, wherein the parameter of freezer dryer is: condenser temperature-50 ℃, vacuum 8Pa, cooling time is 11h, obtains having chitosan-nanometer silver wound dressing of asymmetric wettability.
It is 0.3cm that the chitosan-nanometer silver wound dressing obtaining is cut into 2cm * 2cm(thickness) small pieces, and through ultraviolet radiation disinfection, encapsulation, obtain chitosan-nanometer silver wound dressing product.
Chitosan-nanometer silver wound dressing the product that uses the present embodiment to prepare is treated the experiment of mouse back incision, and wound healing effect of the present invention is described.Also select commercial " Ai Kexin (Acasin here
tM) Nanometer dressing " make positive controls, and do not make a group of any processing and make blank group.Method of testing is as follows: get 15 of healthy BALB/c mouse, entirely male, 6-8 week is large, and body weight is in 20g left and right.Be divided at random 3 groups, 5 every group, be respectively " blank " group, " Ai Kexin
tMnanometer dressing " group and " embodiment 6 " group.Successively mice is done after inhalation anesthesia with ether, with scalpel, at mouse back, mark the wound that is about 10mm, subcutaneous tissue is fully exposed.Then according to being grouped in mice wound, be correspondingly processed.Wherein blank group is not done any processing; Other two groups before applying ointment or plaster first with the abundant moistening Ai Kexin of physiological saline solution
tMwound dressing prepared by Nanometer dressing and the present embodiment, is then fixed on wound by infusion patch.After processing wound, put mice into little mouse cage continuation raising according to grouping, and within some days after surgery, observe the healing state of mice wound, experiment photo as shown in Figure 9.From mice wound healing situation, within postoperative the 2nd day, use the mice wound healing degree of the wound dressing of embodiment 6 preparations to be obviously better than not deal with use Ai Kexin
tMthe mice of Nanometer dressing; And the 8th day after surgery, use the mice wound of the wound dressing of embodiment 6 preparations substantially heal, and other two groups of visible obvious wounds still.As can be seen here, for the wound due to exterior trauma, wound dressing product provided by the invention has excellent wound repair ability and promotes healing ability.
Embodiment 7: the mechanical strength of product and water absorbing properties test
Below by hot strength, extension rate and the water absorption rate test of embodiment 1-4, mechanical strength and the water absorbing properties of wound dressing product of the present invention is described.Test result is as shown in table 1.
Table 1: the hot strength of product, extension rate and water absorption rate
Test result shows, product provided by the invention has good tensile strength, ductility and water absorbing properties.
Embodiment 8: the bacteriostatic activity test of product
Below by the Bactericidal test of embodiment 1-3, the bacteriostatic activity of wound dressing product of the present invention is described.The nano silver colloidal sol concentration that wherein embodiment 1-3 soaks is respectively 10 μ g/mL, 20 μ g/mL, 30 μ g/mL.Method of testing is as follows: the Chitosan in Wound Dressing product of the wound dressing product that embodiment 1-3 is prepared and matched group comparative example 1 preparation is cut into the circular shaped patches that size homogeneous, diameter are 1cm, respectively staphylococcus aureus, methicillin-resistant staphylococcus aureus, Pseudomonas aeruginosa, tolerant Pseudomonas aeruginosa, escherichia coli, these six kinds of pathogen of drug resistance escherichia coli is done to inhibition zone test.Method of testing: compound concentration 10
8the bacterium liquid of CFU/mL, drip 40 μ L and be coated with on LB solid medium and evenly, the one side that wound dressing is hydrophilic contacts with the solid medium that scribbles bacterium liquid, then puts under 37 ℃ of environment of biochemical cultivation case and cultivates, and observes the inhibition zone size of dressing periphery in culture medium after 48h.Test result is as shown in table 2.Test result is visible, and Chitosan in Wound Dressing does not have bacteriostatic activity, and has added three groups of samples of the embodiment 1-3 of nanometer silver, and silver content is different as far as possible, but all can be observed obvious inhibition zone.Experiment shows, chitosan-nanometer silver wound dressing of the present invention has good bacteriostatic activity to clinical common pathogen (comprising the pathogen that antibiosis is have to drug resistance).
Table 2 inhibition zone test result (unit: mm)
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Argentiferous matched group not | |
| Staphylococcus aureus | 10 | 15 | 13 | - |
| Methicillin-resistant staphylococcus aureus | 11 | 14 | 13 | - |
| Pseudomonas aeruginosa | 9 | 13 | 12 | - |
| Tolerant Pseudomonas aeruginosa | 9 | 13 | 14 | - |
| Escherichia coli | 10 | 18 | 14 | - |
| Drug resistance escherichia coli | 9 | 16 | 14 | - |
Note: the diameter that the numerical value in table 2 is inhibition zone, "-" represents not have inhibition zone
Embodiment 9: the cytotoxicity test of product
Cytotoxicity test below by embodiment 1-4, illustrates the safety of product of the present invention to human normal cell.Method of testing is as follows: the wound dressing product that embodiment 1-4 is prepared is cut into the circular shaped patches of size homogeneous, diameter is 1cm, to HEK293(HEKC) do cytotoxicity test, concrete steps are as follows: get in exponential phase and complete adherent HEK293 cell and be made into cell suspension, adjusting concentration extremely every 1mL contains 10
5the cell of the order of magnitude.In 24 orifice plates, the circular shaped patches of a built-in testing is put in every hole, and adds the cell suspension of 1mL to continue to cultivate; Prepare in addition one group and do not add the blank group that any sample only adds 1mL cell suspension.With mtt assay, successively the cell of having cultivated after 1-4 days is done to survival rate test, concrete method of testing: add the MTT(tetrazolium bromide that 100 μ L concentration are 5mg/mL toward having cultivated in 24 orifice plates of corresponding natural law) solution, place 6h at 37 ℃ after, suck the liquid in porous plate and add 750 μ L DMSO(dimethyl sulfoxide), velocity fluctuation 20min with 150rpm/min in shaking table fully dissolves the bluish violet crystallization generating, then adopt enzyme-linked immunosorbent assay instrument to measure its absorbance value under 490nm wavelength, indirectly reflect thus the survival volume of cell.As shown in figure 10, along with the increase of incubation time, with respect to blank group, the cell of embodiment 1-4 group all has obvious growth to result.Test result shows, wound dressing product provided by the invention does not have toxicity to human normal cell, and the growth of cell is had to facilitation.
Embodiment 10: the blood coagulation test of product
Blood coagulation test below by embodiment 1-4, illustrates the promotion coagulation result of wound dressing of the present invention to blood of human body.Method of testing is as follows: using commercially available hospital gauze as the matched group of testing.The wound dressing product that embodiment 1-4 is prepared and hospital gauze respectively cut out the small pieces that quality is 10mg, are placed in 5mL centrifuge tube, separately get the matched group that a centrifuge tube is made normal clotting time.Every centrifuge tube all adds the anticoagulation of the fresh collection of 1mL, and then adds the CaCl that 100 μ L concentration are 0.2mol/L
2solution coagulant blood, records the time that blood solidifies completely, the results are shown in Table 3.
Table 3: clotting time test result (unit: s)
| Blank | Gauze | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | |
| 1 | 720 | 612 | 499 | 505 | 504 | 507 |
| 2 | 717 | 598 | 502 | 506 | 515 | 510 |
| 3 | 727 | 638 | 510 | 503 | 512 | 515 |
| Meansigma methods | 721 | 616 | 504 | 505 | 510 | 511 |
| Rate in advance | - | 14.60% | 30.18% | 30.04% | 29.25% | 29.21% |
From this test result, to compare with normal blood coagulation, wound dressing product provided by the invention can make clotting time in advance more than 29%, more than rate can reach the twice of conventional medical gauze (14.60%) in advance.
Be understandable that, above embodiment is only used to principle of the present invention is described and the illustrative embodiments that adopts, yet the present invention is not limited thereto.For those skilled in the art, without departing from the spirit and substance in the present invention, can make various modification and improvement, these modification and improvement are also considered as protection scope of the present invention.
Claims (10)
1. chitosan-nanometer silver wound dressing, this wound dressing comprises water suction antibiotic layer and the hydrophobic layer of the wound face of fitting, described water suction antibiotic layer chitosan-containing and nanometer silver, this layer is that spongy chitosan is immersed in the colloidal sol containing nanometer silver, by cryodesiccated method, nanometer silver is evenly compounded on spongy chitosan; Another side is hydrophobic layer.
2. chitosan-nanometer silver wound dressing according to claim 1, is characterized in that: described nanometer silver particle diameter is 10-20nm; Described hydrophobic layer comprises one or more in hydrophobically modified agent palmitic acid, stearic acid, arachidic acid or behenic acid.
3. chitosan-nanometer silver wound dressing according to claim 1 and 2, the preparation method that it is characterized in that described spongy chitosan is: chitosan is dissolved in the acetum of 0.5-2wt%, prepare the chitosan-acetic acid solution that mass volume ratio is 1-3%, and carry out below precooling 8-16h at-18 ℃, then carry out lyophilization processing, subsequently products therefrom is immersed in and in alkali liquor, removes residual acetic acid, more repeatedly rinse to neutrality with deionized water, obtain spongy chitosan.
4. chitosan-nanometer silver wound dressing according to claim 3, is characterized in that described deacetylating degree of chitosan is 70-100%, and viscosity is 100-200cPas.
5. chitosan-nanometer silver wound dressing according to claim 3, is characterized in that the condition that described lyophilization is processed is: freezer dryer condenser temperature is equal to or less than-40 ℃, vacuum≤10Pa, and sublimation drying is 16-24h.
6. according to the preparation method of the arbitrary described chitosan-nanometer silver wound dressing of claim 1-5, it is characterized in that comprising the following steps:
(1) spongy chitosan is immersed in nano silver colloidal sol at 20-30 ℃, in this nano silver colloidal sol, nanometer silver concentration is 10-100 μ g/mL, soaks after 16-24h at precooling 6-10h below-18 ℃, obtains chitosan-nanometer silver sponge;
(2) alcoholic solution of the hydrophobically modified agent that is 10-50mmol/L by concentration is evenly sprayed in the one side of step (1) gained chitosan-nanometer silver sponge, and carry out below precooling 30-60min at-18 ℃, then carry out lyophilization processing and obtain chitosan-nanometer silver wound dressing.
7. the preparation method of chitosan-nanometer silver wound dressing according to claim 6, it is characterized in that: the preparation method of the described nano silver colloidal sol of step (1) is: the silver nitrate aqueous solution that the polyvinylpyrrolidone aqueous solution that is 1-20g/L by concentration is 0.1-0.5mol/L with concentration mixes, polyvinylpyrrolidone aqueous solution and silver nitrate aqueous solution volume ratio are 10:1-20:1, after stirring, slowly drip the sodium borohydride aqueous solution that concentration is 0.01-0.1mol/L, drop rate is within every 20 seconds, to drip 10 μ L, extremely mixed solution colour is yellowish-brown and samples by sodium chloride solution evaluation without till precipitating generation, obtain nano silver colloidal sol.
8. the preparation method of chitosan-nanometer silver wound dressing according to claim 6, it is characterized in that: the described lyophilization treatment conditions of step (2) are: freezer dryer condenser temperature is equal to or less than-40 ℃, vacuum≤10Pa, sublimation drying is 10-12h.
9. the preparation method of chitosan-nanometer silver wound dressing according to claim 6, it is characterized in that, the preparation method of described spongy chitosan is: chitosan is dissolved in the acetum of 0.5-2wt%, prepare the chitosan-acetic acid solution that mass volume ratio is 1-3%, and carry out below precooling 8-16h at-18 ℃, then carry out lyophilization processing, subsequently products therefrom is immersed in and in alkali liquor, removes residual acetic acid, with deionized water, repeatedly rinse to neutrality again, obtain spongy chitosan.
10. the preparation method of chitosan-nanometer silver wound dressing according to claim 6, is characterized in that: described deacetylating degree of chitosan is 70-100%, and viscosity is 100-200cPas.
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