Study On The Characterization And Release Of Silve-Contained Wound Dressing And Thetoxicology Of Silvernanoparticles

Due to its broad-spectrum antimicrobial properties, silver nanoparticles (AgNPs) are widely used in medical devices, textile, food storage materials, cosmetics, water purification and other fields. However, its safety has attracted attention with the rapid increase in its manufacture andapplication, which leads to exposure increasingly for human and environment. Particularly, AgNPs and silver ions (Ag+) can shed or release, which exert antimicrobial effectswhen silver-contained wound dressing contact with body of patients. Meanwhile, shed or released AgNPs and Ag+ caused a potential risk of toxicity on patients. And as a resource of Ag+ release, AgNPs can release Ag+ constantly, which will result in further potential toxicity risk. Toxicity risk of silver to human body is contributed to the amount of released AgNPs and AgT, as well as exposure time and the concentration of free Ag+ and AgNPs. Therefore, it is a basic and key step to study the characterization of silver and silver release in silver-contaied wound dressing. And the major mechanism of AgNPs-induced cytotoxicity on L929 cells is still unknown.In this study, surface morphology, particles size and distribution of silver were charactierazed in three wound dressings (Anson, YB, AT dressings) by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). X-ray energy dispersive spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) were employed to study the surface element, valence and crystal structure of silver particles, respectively. The characterization results demonstrated that Ag0 was present on three dressings, and Ag+ was also observed in Anson dressing. The average silver particles size was 5-25 nm and 5-10 nm in Anson and YB dressing, respectively, and some large particles were found about 30-40 nm in YB dressing. And silver particles were amorphous silver in both dressings. The cubic silver particles size was 30-45 nm in AT dressing.Release property of silver from dressings was evaluated by reciprocating holder method in H2O and simulated body fluid (SBF) from dressings. Since the rich content of Cl- in SBF, soluble Ag+ presents as insoluble AgCl salts. It is difficult to separate the AgCl and AgNPs due to the similar particle size. In general, at high Cl- concentration, the insoluble AgCl salts can re-dissolve due to the formation of soluble anionic chlorocomplexes. Therefore, the effect of Ag+ concentration on the AgCl solubility was studied to solve the interference problem of AgCl particles in the separation process of AgNPs. And the release amount of AgNPs and Ag+ was further separated by ultrafiltration effectively. MTT assay and LDH leakage assay were employed to evaluate cytotoxicity of extracted solution of silver-contained wound dressings to L929 cells. The AgCl solubility results showed that when the Ag+ concentration decreased to 0.5 p.g/mL, the solubility of AgCl salts increased to over 96.7%. And no significant increase in solubility of AgCl salts was observed when further reducing the Ag+ concentration. Therefore,0.5 μg/mL of Ag+ was set as a threshold value of AgCl solubility. Release experiments of silver in Anson wound dressings demonstrated that the total silver release amount in SBF was 2 times higher than that in H2O. After dilution by SBF and ultrafiltration, the release amount of total silver was 12.40,49.23 and 59.90 μg/cm2 in Anson, YB, AT dressings, and the released AgNPs amount was 2.54, 17.86 and 29.04 μg/cm2, respectively. Cytotoxicity results showed that three kinds of dressing extractions caused strong toxic effect on L929 cells in MTT assay as well as LDH leakage assay. The toxic effect of AT dressing was the strongest, and Anson dressing was the weakest, which consistent with the results of silver release experiments (both total Ag and AgNPs).In toxicology study of AgNPs on L929 cell, TEM, Dynamic Light Scattering (DLS), Zeta potential and UV-visible spectroscopy were employed to study the physical and chemical properties of AgNPs. MTT assay and LDH leakage assay were used to evaluated the cytotoxicity of AgNPs. The toxicity mechanisms of AgNPs were studied by detecting the change of ROS, SOD, GSH-px and MDA in cells without or with N-acetyl cysteine (NAC, a kind of ROS inhitor) treatment. The characterization results demonstrated that AgNPs were spherical and distributed uniformly. The size of AgNPs was 4-30 nm,72% of which was 5-10 nm, and the average size was 7.25 nm. The zeta potential of AgNPs (100μg/mL) was-37.83±3.20 mV and characteristic peak was around 415 nm. The decrease of cell viability and the increase of LDH leakage were caused after L929 cells exposed to AgNPs, with a dose-depend manner. At the concentration of 1.25 and 1.50 μg/mL, the cell viabilities were 60% and 36%, and LDH leakage were 28% and 39%, respectively. And the half-maximum inhibitory of cell viability concentration was 1.38μg/mL. The cell viability and LDH leakage recovered to normal levels, when treated with NAC, which had no significant difference with control groups. The ROS leveland MDA contentwere also increased, SOD and GSH-px activity decreased when the cells were exposed to AgNPs. At the concentration of 1.25 and 1.50 μg/mL, the intracellular ROS levels were 1.8 times and 2.4 times higher compared with control group, respectively. The SOD activity decreased by 13.1% and 24.3%, the GSH-px activity decreased by 21.5% and 35.0%,the MDA content increased by 74.0% and 106.6%, respectively. These results indicated that AgNPs induced intracellular oxidative stress and cellular damage, which had a dose-depended manner. When treated with NAC, the ROS levels decreased, the SOD and the GSH-px activity recovered, and the MDA content decreased compared to AgNPs exposure group which without NAC treatment, suggesting that ROS-induced oxidative stress was a major mechanism of AgNPs-induced cytotoxicity.In this study, the characterization of silver and silver release in silver-contaied wound dressing and cytotoxicity mechanisms of AgNPs were evaluated. And the results would provide a scientific basis for risk assessment and supervision of silver-containing wound dressings in clinical.