Flow cytometry evaluation of in vitro cellular necrosis and apoptosis induced by silver nanoparticles

Particles possess unique properties in the nanoscale, e.g., enhanced catalytic activity, high surface area, and light emission/absorption properties, that might result in interference with colorimetric in vitro cytotoxicity assays such as MTT, XTT or MTS. Alternatively, assays that do not use spectrophotometric detection, such as trypan blue exclusion or flow cytometry (FC) based assays, are less likely to be influenced by nanoparticle interference. The aim of this study was to evaluate FC assays to assess the cytotoxicity of three different sizes (10, 100, or 200 nm) of silver nanoparticles (AgNPs) at different mass concentrations (1, 25, or 50 ug/ml) in L-929 fibroblast cells. After 4 h and 24 h exposure, cell necrosis and apoptosis were assessed using 7-AAD and Annexin V dyes, respectively, with FC. The data indicate that cell necrosis and apoptosis in AgNP-exposed fibroblasts depends on dose, exposure time, and AgNP size. The data indicate that AgNPs produced a dose- and time-dependent decrease in cell viability; however, 10 nm AgNPs were significantly more toxic than larger-sized particles. Thus, standard FC assays can be utilized to assess apoptosis and necrosis in response to nanomaterial exposure.     

Potential of biofluid components to modify silver nanoparticle toxicity

Establishing realistic exposure scenarios is critical for cytotoxic investigation of silver nanoparticles (AgNP) in the gastrointestinal tract. This study investigated the potential interaction with and effect of biofluid components, namely cholic acid, deoxycholic acid and ursodeoxycholic acid, on AgNP toxicity. Two cell lines corresponding to organs related to the biofluid components were employed. These were HepG‐2 a hepatocellular carcinoma derived from liver tissue and Hep2 an epithelial cell line. Physiochemical and cytotoxic screening was performed and the ability of biofluid components to modify AgNP cytotoxicity was explored. No alteration to the physiochemical characteristics of AgNP by biofluid components was demonstrated. However, biofluid component addition resulted in alteration of AgNP toxicity. Greater reactive oxygen species induction was noted in the presence of cholic acid and deoxycholic acid. Ursodeoxycholic acid demonstrated no modification of toxicity in HepG‐2 cells; however, significant modification was noted in Hep2 cells. It is concluded that biofluid components can modify AgNP toxicity but this is dependent on the biofluid component itself and the location where it acts. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparative toxicity of silicon dioxide,silver and iron oxide nanoparticles after repeated oral administration to rats

Although silicon dioxide (SiO₂), silver (Ag) and iron oxide (Fe₂O₃) nanoparticles are widely used in diverse applications from food to biomedicine, in vivo toxicities of these nanoparticles exposed via the oral route remain highly controversial. To examine the systemic toxicity of these nanoparticles, well‐dispersed nanoparticles were orally administered to Sprague–Dawley rats daily over a 13‐week period. Based on the results of an acute toxicity and a 14‐day repeated toxicity study, 975.9, 1030.5 and 1000 mg kg^–1 were selected as the highest dose of the SiO₂, Ag and Fe₂O₃ nanoparticles, respectively, for the 13‐week repeated oral toxicity study. The SiO₂ and Fe₂O₃ nanoparticles did not induce dose‐related changes in a number of parameters associated with the systemic toxicity up to 975.9 and 1000 mg kg^–1, respectively, whereas the Ag nanoparticles resulted in increases in serum alkaline phosphatase and calcium as well as lymphocyte infiltration in liver and kidney, raising the possibility of liver and kidney toxicity induced by the Ag nanoparticles. Compared with the SiO₂ and Fe₂O₃ nanoparticles showing no systemic distribution in all tissues tested, the Ag concentration in sampled blood and organs in the Ag nanoparticle‐treated group significantly increased with a positive and/or dose‐related trend, meaning that the systemic toxicity of the Ag nanoparticles, including liver and kidney toxicity, might be explained by extensive systemic distribution of Ag originating from the Ag nanoparticles. Our current results suggest that further study is required to identify that Ag detected outside the gastrointestinal tract were indeed a nanoparticle form or ionized form. Copyright © 2015 John Wiley & Sons, Ltd.     

Analysis of cytotoxic effects of silver nanoclusters on human peripheral blood mononuclear cells ‘in vitro’

The antimicrobial properties of silver nanoparticles (AgNPs) have made these particles one of the most used nanomaterials in consumer products. Therefore, an understanding of the interactions (unwanted toxicity) between nanoparticles and human cells is of significant interest. The aim of this study was to assess the in vitro cytotoxicity effects of silver nanoclusters (AgNC, < 2 nm diameter) on peripheral blood mononuclear cells (PBMC). Using flow cytometry and comet assay methods, we demonstrate that exposure of PBMC to AgNC induced intracellular reactive oxygen species (ROS) generation, DNA damage and apoptosis at 3, 6 and 12 h, with a dose‐dependent response (0.1, 1, 3, 5 and 30 µg ml^–1). Advanced electron microscopy imaging of complete and ultrathin‐sections of PBMC confirmed the cytotoxic effects and cell damage caused by AgNC. The present study showed that AgNC produced without coating agents induced significant cytotoxic effects on PBMC owing to their high aspect ratio and active surface area, even at much lower concentrations (<1 µg ml^–1) than those applied in previous studies, resembling what would occur under real exposure conditions to nanosilver‐functionalized consumer products. Copyright © 2015 John Wiley & Sons, Ltd.     

用PCR诊断不同虫荷量的大鼠卡氏肺孢子虫肺炎的价值

目的　评价在不同虫荷量条件下用 PCR诊断大鼠卡氏肺孢子虫肺炎 (PCP)的价值。　方法　将 PCP大鼠随机分为药物治疗组及未治疗组 ,测定其肺虫荷量 ,收集其支气管灌洗液 (BAL F)和血清标本 ,用 PCR和半套式 (Sn) PCR检测标本中的卡氏肺孢子虫基因 ,比较 PCR和环六亚甲基四胺银 (GMS)染色结果 ,及与病鼠肺虫荷量的关系。　结果　虫荷量高的未治疗组病鼠的 BAL F标本 PCR、Sn PCR及 GMS染色阳性率分别为 10 0 %、10 0 %和 92 .6 % ,差异无显著性 (P>0 .0 5 ) ;而虫荷量低的治疗组的 PCR(88.1% )、Sn PCR(94 .9% )阳性率均显著高于 GMS染色镜检阳性率 (30 .5 % ) (P<0 .0 5 )。肺虫荷量 >5 0 0个 /mg肺的 PCP大鼠 ,血清 PCR阳性率显著高于≤ 5 0 0者 (分别为 4 2 .9%和 1.7% )。　结论　PCR可用于虫株负荷量较低时的 PCP诊断 ,对判断 PCP病情及疗效考核亦有一定的帮助。     

Compatibility of Different Histochemical Techniques for the Demonstration of ATPase for Muscle Fiber Typing: A Review

Abstract

Several recent studies suggest a diagnostic and a possible prognostic role for silver stained nucleolar organizer region (AgNOR) determination in the assessment of human neoplasia. Technical pitfalls, especially silver grain precipitate, may hamper accurate counting of AgNORs. We have identified pH as a main factor causing silver precipitation and overstaining. An improved silver staining method, which raises the pH to 3.0, optimizes visualization by eliminating silver precipitation and improving contrast between AgNORs and surrounding tissue components. (The J Histotechnol 14:187, 1991)     

Antibacterial activities of hexadecanoic acid methyl ester and green-synthesized silver nanoparticles against multidrug-resistant bacteria

Antibacterial drug resistance is considered one of the biggest threats to human health worldwide, and the overuse of antibiotics accelerates this problem. Multidrug-resistant (MDR) bacteria are becoming harder to treat as the antibiotics used to treat them become less effective. Therefore, it is necessary to evaluate novel methods to control MDR bacteria. In this study, 40 bacterial isolates were collected from diabetic patients. The sensitivity of 40 bacterial isolates to seven antibiotics was evaluated. Four bacterial isolates were resistant to all antibiotic groups. The MDR pathogenic bacteria were selected and identified morphologically and biochemically and confirmed by VITEK® 2 system as follows: Staphylococcus aureus W35, Pseudomonas aeruginosa D31, Klebsiella pneumoniae DF30, and K. pneumoniae B40. Identification of the most resistant P. aeruginosa D31 was confirmed by the sequencing of a 16S ribosomal RNA gene with an accession number (MW241596). The inhibitory activity of eight types of native grown plant extracts against MDR bacteria was studied. Clove alcoholic extract (CAE) showed the highest inhibitory activity against MDR bacteria. Gas chromatography–mass spectrometry analysis of partially purified CAE at 0.9 Rf detected by thin-layer chromatography showed an active compound named hexadecenoic acid methyl ester with the highest antimicrobial effect against clinical pathogenic bacteria. The formation of silver nanoparticles (AgNPs) by CAE was studied. Evaluation of AgNPs was investigated by X-ray diffraction, UV–Vis, and transmission electron microscopy. The antibacterial effect of AgNPs after 2, 4, and 6 days in light and dark conditions was evaluated. Finally, the AgNPs synthesized using CAE possess good inhibition activity against the tested pathogenic bacteria. As a result, the bactericidal components listed above were promising in reducing MDR bacteria and can be used for treatments of bacterial infection and in the development of safe products with a natural base.     

The Kv channel blocker 4-aminopyridine enhances Ag+ uptake: a scanning electrochemical microscopy study of single living cells

We report that silver ion (Ag⁺) uptake is enhanced by 4-aminopyridine (4-AP), a well known voltage-sensitive potassium ion channel (K_v) blocker. Both bacterial (Escherichia coli) and mammalian (3T3 fibroblast) cells were used as model systems. Ag⁺ uptake was monitored with a scanning electrochemical microscope with an amperometric Ag⁺ ion-selective electrode (Ag⁺-ISE) and the respiration rates of E. coli cells were measured by oxygen reduction at an ultramicroelectrode. The results showed that not only the amount but also the rate of silver uptake by the cells increased significantly when 4-AP was added to the solution. For fibroblasts, the Ag⁺ uptake rate was 4.8 × 10⁷ ions per cell per sec without 4-AP compared with 1.0 × 10⁸ ions per cell per sec with 0.2 mM 4-AP. For E. coli cells, the uptake rate was 1.5 × 10⁴ ions per cell per sec without 4-AP vs. 3.5 × 10⁴ ions per cell per sec with 0.5 mM 4-AP and 5.9 × 10⁴ ions per cell per sec with 1 mM 4-AP. Thus, 4-AP might be useful where silver is used as antimicrobial agent to speed its uptake.     

多中心完全随机、标准治疗平行对照评价京万红软膏治疗糖尿病足慢性创面的临床研究

目的：：通过多中心完全随机、标准治疗平行对照方法评价京万红软膏治疗糖尿病足慢性创面的疗效。方法：本研究共有11家医院参加,采用多中心完全随机、标准治疗平行对照、前瞻性临床研究设计。131例糖尿病足溃疡患者随机分为京万红软膏组67例和对照组64例,两组创面面积分别为(16.7±6.1)cm2和(15.9±8.3) cm2,创面形成时间(45.7±68.3)d和(52.5±79.6)d 。两组分别用京万红软膏或复方磺胺嘧啶锌凝胶涂于创面,观察疗程均为20周。结果：两组患者年龄、糖尿病病程、血常规、肝功能、肾功能等数据差异无统计学意义。京万红组于2、5、10、15周创面愈合速率明显优于复方磺胺嘧啶锌凝胶组,以第5周、10周最为明显(P<0.01)。京万红组创面达到完全上皮化平均时间为(46.5±15.6)d,复方磺胺嘧啶锌凝胶组为(67.9±17.9)d,差异显著(P<0.05)。结论：京万红软膏与复方磺胺嘧啶锌凝胶均有促进糖尿病足创面愈合的作用,京万红软膏作用更优。