| Nano silver usually refers to the silver particles which size is less than 100 nm. Since the surface effect of nanosilver,it’s antibacterial ability was much better than larger-size silver particle.In addition, nano scale silver also has better antibacterial effect than silver ion.In the field of medical treatment,antibiotics might lose it’s antibacterial activity because of the resistance of bacteria,but nano silver will not.For these reason,in recent years,nano silver became a focus in the field of health care.At present, nano-silver antibacterial fiber, nano-silver antibacterial gel,nano silver antimicrobial dressings and other medical products have come out.Their applications included the treatment of acne, prostatitis, psoriasis, gynecological problems and burns, trauma and so on.However,some studies evaluated the in vitro cytotoxicity of nano-silver medical products in present Chinese market,and found that the vast majority of medical products containing nano silver had poor biocompatibility,most of the products’in vitro cytotoxicity were more than level Ⅲ.In this regard, the purpose of this study was to prepare nano-silver medical products with lower cytotoxicity and certain antibacterial activity.The main research direction was for nano silver gel, secondary research direction was for nano silver dressing.Gel and dressing can be used tor the treatment oft skin burn(degrade Ⅱ) m exudation period.This is mainly due to the following six characteristics of the gel and dressing:① good biocompatibility; ② well ability to absorb wound exudate; ③ ability to keep the wound moist, thus contributing to the growth of granulation tissue and wound healing; ④ ability to anti-bacteria and prevent external bacteria which might arising infections; ⑤ ability to adhere well to the wound; ⑥ has a certain mechanical strength. In humoral exudate period of grade II skin burning,the wound will secrete large amounts of body fluids, not only to transfusion to replace lost fluids in clinical, but also need to clean up the secreted fluids, secretions containing rich nutrients will breed a lot of bacteria, leading to infection of the wound, thereby affecting the recovery of wound and even the lives of patients. In addition,because the wound without the protection of skin,the active ingredient of the drug and other low molecular weight components will be with the damaged skin into the body’s blood circulation and wound cells. If the ingredients of drugs have a higher cytotoxic components at the time of treatment, the patient will be caused secondary damage. Therefore,it will be a great benefit for patients’prognosis if the gel or dressing which had well performance in anti-infection and biocompatibility had been prepared and used.Usually,there are mainly two cytotoxic sources in gels and dressings containing nanosilver,one is nanosilver particles,and the other is the matrix of gels and dressings.For the same concentration of nano silver,the smaller the size of nano silver, the stronger the ability of antibacterial,but at the same time, the in vitro cytotoxicity was also greater.Similar to the previous, the higher the concentration of silver nanoparticles, the stronger its antibacterial activity, while its in vitro cytotoxicity is greater.Therefore,in this study,poloxamer 407 had been used as a kind of dispersant to dispersed nano-silver,and medium-sized nano-silver particles had been prepared by using photo induced method to balanced anti-bacterial effects and cytotoxicity in vitro.The introduction of cross-linking agents (such as glutaraldehyde) is often the main source of cytotoxicity for the matrix of gels and dressings.So this study used a physical cross-linking method to form gel and dressing with three-dimensional network structure through the electrostatic interaction, hydrogen bonding interaction, mutual entanglement of the molecular chain and other physical effects.Thus,the problem of increasing cytotoxicity of gels and dressings which due to the using of cytotoxic cross-linking agent had been resolved. In this study,chitosan had been chose to be the main matrix component in gel because of several characteristics: ① It could form a gel by adjusting the pH value; ② Chitosan could be formed by the deacetylation of chitin,which was a kind of wide-range-sources natural polymer, and it was degradable under natural conditions; ③ Chitosan itself had a certain antibacterial activity. Furthermore, poloxamer 407 had been added into matrix of gels and dressings to enhance its capability of absorbing moisture.This study had prepared nanosilver by using photo-induced method, and characterized by UV-visible absorption spectrum (UV-vis) test, X-ray powder diffraction (XRD) and scanning electron microscopy test (SEM) tests to analyzed the absorption spectral properties of nano silver solution, physical properties of nano-silver particles and microstructure of nano silver particles separately.Gels and dressing containing nano-silver had been prepared by using physical cross-linking method.Prepared gels had been characterized by pH test, swelling ratio test, scanning electron microscopy test, viscosity test, inhibition zone test and in vitro cytotoxicity test to analyzed the acid-base characteristics, water absorption capacity, micro morphology, viscosity, antibacterial ability and biocompatibility separately.Prepared dressings had been characterized by scanning electron microscope test, tensile test, swelling ratio test and inhibition zone test to analyzed the microstructure and mechanical properties, water absorption capacity and antibacterial activity.In the preparation of nano-silver,two dispersants--- polyvinylpyrrolidone (PVP) and poloxamer 407 had been added to dispersed nano-silver respectively in distilled water or ultra pure water.This means that four-group nano-silver samples---distilled water-PVP group, distilled water-ploloxamer group, ultra pure water-PVP group and ultra pure water-poloxamer group had been prepared.The above four samples were tested by UV-vis absorption spectroscopy. The results showed that the maximum absorption wavelength was 442 nm,431 nm,431 nm and 414 nm respectively.Since the maximum absorption wavelength and the average diameter of the nano silver was proportional, the above results showed that the ultra-pure water-poloxamer group had the smallest average particle diameter.This result also suggested that poloxamer compared to PVP had better effect to reduce the average particle size of nano silver in the same solvent,and the nano-silver which prepared in ultra pure water had smaller average diameter than prepared in distilled water in the same dispersion agent. According to relevant literature reports,the average diameter of nano-silver was about 60nm when the maximum absorption wavelength was about 416 nm.The average size above was medium,so the ultra pure water group had been chose and SEM test was carried out in this study.By SEM test,we confirmed that the nano-silver average diameter of ultra pure water-poloxamer group (particle size distribution of 45-85 nm) was smaller than ultra pure water-PVP group(particle size distribution of 50-120 nm).It also showed that ultra pure water- poloxamer group compared to the group of ultra pure water-PVP had a narrower particle size distribution.Finally,in order to confirm the particles which had been prepared was nano-silver,XRD experiment was carried out to test the sample of ultra pure water group.According to the X-ray powder diffraction pattern, the software of jade 5.0 was used to match the map in the map database, and the results confirmed that the prepared samples were nano silver.In the preparation of nano silver gel, six groups of nano silver gel samples were prepared according to the nano silver concentration gradient of 0,3,6,12,18,24 μg/ml, and the inhibition zone test and in vitro cytotoxicity test were carried out.The result of inhibition zone test showed that three groups samples(0,3,6 μg/ml) had almost same ability to inhibit Escherichia coli and Staphylococcus aureus and their inhibitory capacity had no significant difference, in addition, with the increase of the concentration of nanosilver,the antibacterial activity also significantly increased.When the concentration reached to 18,24μg/ml,the size of inhibition zone had no significant difference.Furthermore, every gel sample’s inhibitory effect on Staphylococcus aureus was better than Escherichia coli.The results of in vitro cytotoxicity test showed that three groups samples (0,3,6 μg/ml) had level 1 cytotoxicity rating (corresponding relative growth rate was between 80% and 90%; 88.1%,82.5% and 81.4% respectively),and another three groups samples (12,18,24 μg/ml) had level 2 cytotoxicity rating(corresponding relative growth rate was between 50% and 79%; 72.9%、 61.5% and 52.2% respectively).In this study,the nano-silver concentration of 12 μg/ml had been chose as the optimal formula by balancing the antibacterial ability and in vitro cytotoxicity.In order to test the influence on gel properties when adding poloxamer into chitosan matrix,dry chitosan gel and dry chitosan-poloxamer gel had been prepared respectively in the next,and SEM test and swelling ratio test were carried out.The result of SEM test suggested that,the cross-section of dry chitosan gel was relatively smooth, and the cross-section of dry chitosan-poloxamer gel had more pore structure and pore size was almost less than 20 μm.It could be known that the adding of poloxamer might change the interior microstructure of the gel in chitosan gel system,poloxamer could make gel became more fluffy.Therefore, we hypothesized that the interaction between molecules of poloxamer and chitosan molecules will lead to the formation of pores.In addition, according to the SEM images of chitosan and chitosan-poloxamer gel, we could roughly judged that the swelling property of chitosan-poloxamer gel had better than that of chitosan gel.The results of swelling ratio test confirm that the water absorbing capacity of chitosan-poloxamer gel was better than the chitosan gel whether in PBS solution (pH=7.3) or physiological saline solution.Finally,pH test and viscosity test had been carried out to test the sample of chitosan-poloxamer gel.pH-test results showed that,the pH of chitosan-poloxamer gel was between 5.8 and 6.1,weakly acidic.It’s suitable for weakly acidic environment of wound.Viscosity test results showed that the viscosity was 21.115 Pa.s when the rotor speed was 1 r/min.The viscosity was moderate, and with the increase of the rotor speed, the apparent viscosity of the gel was getting smaller and smaller. And the greater the rotor speed, the more obvious the effect of pole-climbing, so the prepared gel belonged to non Newtonian fluid.In the preparation of nano silver dressing, in order to test the influence on dressing properties(water absorption capacity and mechanical properties) when adding different concentration of poloxamer or glycerol into chitosan-poloxamer dressing,five groups with different poloxamer-concentration of 0‰、2‰、4‰、6‰ and 8%o had been prepared, and another five groups with different glycerol-concentration of 2%、4%、6%、8% and 10% had been prepared too.And swelling ratio test and tensile test (including tensile strength and elongation at break) had been carried out.The results of swelling ratio test showed that 4%o and 6%o poloxamer groups and 2% glycerol group had better water absorption capacity after 24 hours immersed in PBS solution,and maximum swelling ratio could exceed 100%,100% and 200% respectively;8‰ poloxamer group and 2% glycerol group had better water absorption capacity after 24 hours soaked in physiological saline solution, and the maximum swelling ratio could exceed 100% and 130% separately.In the tensile test,the tensile strength of the prepared dressing was proportional to the poloxamer concentration in the range of 2%o to 8%o,and the tensile strength of the prepared dressing was inversely proportional to the glycerol concentration in the range of 2% to 10%.Unlike the tensile strength,the elongation at break of the dressing reached to maximum value at 4‰ poloxamer concentration,and the elongation at break of the dressing was proportional to the glycerol concentration in the range of 2% to 10%.Furthermore,in order to test the influence on microstructure of dressing when adding different content of poloxamer,5 groups of samples with different concentration of poloxamer had been selected to test.According to the cross-sectional SEM test of dressing,we found that added poloxamer would promote the emergence of pores.The largest diameter of the pores was less than lOujm.By observing the microstructure of dressing,the phenomenon had been visually explained---the water absorption capacity of added poloxamer group was better than the group without poloxamer.Finally,in order to test the influence on its antimicrobial ability when adding different concentration of poloxamer,5 groups of samples with different content of poloxamer had been chose to test.It was found that the prepared samples had obvious inhibition zone in the different strains substantially.The difference of inhibition-zone size of samples of 0%o and 2%o poloxamer were all 2.5mm in different strains,which was larger than other three groups.Other three groups’difference of inhibition-zone size were all less than or equal to lmm in different strains.By balancing the water absorption capacity,mechanical properties and antimicrobial activity,this study selected poloxamer concentration of 6% and glycerol concentration of 2% as the optimal concentration of chitosan-poloxamer dressing. |
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