AgNP Treatment Research Articles

Toxicity of internalized laser generated pure silver nanoparticles to the isolated rat hippocampus cells.

Silver nanoparticles (AgNPs) are the most commonly used nanoparticles (NPs) in medicine, industry and cosmetics. They are generally considered as biocompatible. However, contradictory reports on their biosafety render them difficult to accept as 'safe'. In this study, we evaluated the neurotoxicity of direct AgNP treatment in rat hippocampal slices. We produced pure uncoated AgNPs by a pulsed laser ablation method. NP characterization was performed by Ultraviolet (UV) visible spectrophotometer, scanning electron microscope, transmission electron microscope (TEM) and energy-dispersive X-ray spectroscopy. Rat hippocampal slices were treated with AgNPs for an hour. AgNP exposure of hippocampal tissue resulted in a significant decrease in cell survival in a dose-dependent manner. Our TEM results showed that AgNPs were distributed in the extracellular matrix and were taken into the cytoplasm of the neurons. Moreover, we found that only larger AgNPs were taken into the neurons via phagocytosis. This study showed that the pure AgNPs produced by laser ablation are toxic to the neural tissue. We also found that neurons internalized only the large NPs by phagocytosis which seems to be the major mechanism in AgNP neurotoxicity.

Effects of silver nanoparticles on the interactions of neuron- and glia-like cells: Toxicity, uptake mechanisms, and lysosomal tracking.

Silver nanoparticles (AgNPs) are commonly used nanomaterials in consumer products. Previous studies focused on its effects on neurons; however, little is known about their effects and uptake mechanisms on glial cells under normal or activated states. Here, ALT astrocyte-like, BV-2 microglia and differentiated N2a neuroblastoma cells were directly or indirectly exposed to 10 nm AgNPs using mono- and co-culture system. A lipopolysaccharide (LPS) was pretreated to activate glial cells before AgNP treatment for mimicking NP exposure under brain inflammation. From mono-culture, ALT took up the most AgNPs and had the lowest cell viability within three cells. Moreover, AgNPs induced H2 O2 and NO from ALT/activated ALT and BV-2, respectively. However, AgNPs did not induce cytokines release (IL-6, TNF-α, MCP-1). LPS-activated BV-2 took up more AgNPs than normal BV-2, while the induction of ROS and cytokines from activated cells were diminished. Ca2+ -regulated clathrin- and caveolae-independent endocytosis and phagocytosis were involved in the AgNP uptake in ALT, which caused more rapid NP translocation to lysosome than in macropinocytosis and clathrin-dependent endocytosis-involved BV-2. AgNPs directly caused apoptosis and necrosis in N2a cells, while by indirect NP exposure to bottom chamber ALT or BV-2 in Transwell, more apoptotic upper chamber N2a cells were observed. Cell viability of BV-2 also decreased in an ALT-BV-2 co-culturing study. The damaged cells correlated to NP-mediated H2 O2 release from ALT or NO from BV-2, which indicates that toxic response of AgNPs to neurons is not direct, but indirectly arises from AgNP-induced soluble factors from other glial cells.

Abstract B47: Silver nanoparticles exhibit subtype specific cytotoxic and therapeutic effects in claudin low breast cancer in vitro and in vivo

Abstract Triple negative breast cancers (TNBC) are characterized by loss of expression of hormone receptors and decreased expression of the human epidermal growth factor receptor 2 (HER2). TNBC patients do not benefit from current targeted breast cancer (BC) treatments. Molecular profiling of breast cancer has found that TNBC is largely comprised of basal-like and claudin-low intrinsic molecular subtypes. Claudin-low breast cancer (CLBC) accounts for approximately one third of TNBCs, and early evidence suggests CLBC tumors may be more resistant to neoadjuvant anthracycline/taxane-based chemotherapy compared to basal-like tumors. For the development of novel breast cancer therapeutics, attention must be paid to therapeutic efficacy in specific sub-types of the disease. We discovered a type of silver nanoparticle (AgNP) that is selectively cytotoxic for treatment of CLBC. We find that the increased sensitivity of CLBC cells as compared to non-cancerous cells was independent of nanoparticle size, and CLBC cell lines (MDA-MB-231, BT-549, SUM-159) are more sensitive to AgNP exposure than luminal A BC (MCF-7), HER2 positive BC (SKBR3), and basal-like BC (MDA-MB-468, BT-20), or non-cancerous breast cells (MCF-10A, 184B5, HMT-3522 S1) via MTT assay. By treating CLBC cells and non-cancer breast cells with AgNPs or silver ions, in the form of silver nitrate, we demonstrated that intact AgNPs are necessary for selective cytotoxicity in CLBC. To determine the mechanism of cell death caused by AgNPs, annexin V (AnnV) and propidium iodide (PI) co-staining was performed on non-cancerous MCF-10A breast cells and MDA-MB-231 CLBC cells treated with AgNPs for 48 hours. AgNPs induced a dose-dependent increase in late-stage apoptosis and an increase in necrosis in MDA-MB-231 compared to vehicle control. Conversely, AgNPs had a minimal effect on late-stage apoptosis and necrosis in non-cancerous MCF-10A. Utilizing western blots, we showed that AgNPs induce oxidative damage to protein thiols and activate the unfolded protein response (UPR) in CLBC, but not in non-cancerous breast cells. To better recapitulate the tumor volume in an in vitro setting, multicellular tumor nodules from MDA-MB-231 TNBC cells or HMT-3522 S1 non-transformed mammary epithelial cells were formed by culturing the cells on basement membrane. When grown using 3D culture techniques, HMT-3522 S1 cells can develop growth arrested, polarized spherical structures containing lumens resembling a normal breast acinar structure characterized by a central lumen, cell-cell junctional complexes (including apical tight junctions, TJ), and basal expression of hemidesmosomal α6/β4 integrins ligating an endogenous basement membrane. 48h treatment with AgNPs did not prevent apical localization of the TJ marker ZO-1, alter the basal deposition of collagen-IV, induce proliferation of the growth arrested acini, or induce DNA damage. Conversely, significant amounts of DNA damage were observed in the MDA-MB-231 tumor nodules following 48h AgNP treatment. We conducted a 3 month in vivo safety/efficacy study in CLBC tumor-bearing mice where we showed that AgNPs can be delivered repeatedly at substantial doses intravenously and are effective for treatment of tumors without systemic toxicity. This is the first time any nanomaterial has been demonstrated subtype-specific therapeutic efficacy. This work demonstrates that AgNPs may provide a significant benefit for the CLBC patient population. Citation Format: Jessica L. Swanner, Iliana Tenvooren, Brian W. Bernish, Cale D. Fahrenholtz, Pierre A. Vidi, Katherine L. Cook, Ravi N. Singh. Silver nanoparticles exhibit subtype specific cytotoxic and therapeutic effects in claudin low breast cancer in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B47.

Alteration of Cytokine Profiles Inhibits Efficacy of Silver Nanoparticle-based Neutralization of arenaviruses

Background: Arenaviruses are important pathogens that can cause hemorrhagic fever or meningoencephalitis. There is not an effective treatment targeting arenaviruses, which necessitates a search for therapies against this virus family, as well as others. Silver nanoparticles (Ag-NPs) have been shown to have effective antiviral activity in cell culture models against arenaviruses, and therefore it was hypothesized that they may make an effective therapeutic against viral meningitis. However, the silver nanoparticles interfered with normal cytokine profiles produced in response to the virus infection, which led to exacerbated pathology. Methods: Mice were infected with Tacaribe virus, a mouse-adapted arenavirus that causes lethal encephalitis. The virus was left untreated or treated with 10 nm Ag-NPs, which were demonstrated previously to inhibit virus replication in cell culture. Virus replication, brain pathology, and cytokine profiles (pro-inflammatory interleukins and type I interferons), as well as over morbidity and mortality were assessed in the mice with and without treatment. Results: Although the viral loads appeared to be reduced in the Ag-NP treated mice, mortality was observed in all virus-infected animals, regardless of whether or not they received nanoparticle therapy. The Ag-NP treatments also the innate immune response against the virus infection and unregulated the production of interleukin-1 beta, which likely contributed to the observed mortality in the mice. Conclusions: This study confirms why it is important to assess promising in vitro results using in vivo models. Biological barriers can impact the efficacy of therapeutics in mammals. Although Ag-NPs may still have success as an antiviral therapeutic in other areas of the body that are less constricted, in sensitive areas such as the brain, the nanoparticles can cause very adverse reactions.

Two-Phase Bactericidal Mechanism of Silver Nanoparticles against Burkholderia pseudomallei.

Silver nanoparticles (AgNPs) have a strong antimicrobial activity against a variety of pathogenic bacteria. The killing mechanism of AgNPs involves direct physical membrane destruction and subsequent molecular damage from both AgNPs and released Ag+. Burkholderia pseudomallei is the causative agent of melioidosis, an endemic infectious disease primarily found in northern Australia and Southeast Asia. B. pseudomallei is intrinsically resistant to most common antibiotics. In this study, the antimicrobial activity and mechanism of AgNPs (10–20 nm) against B. pseudomallei were investigated. The MIC and MBC for nine B. pseudomallei strains ranged from 32–48 μg/mL and 96–128 μg/mL, respectively. Concentrations of AgNPs less than 256 μg/mL were not toxic to human red blood cells. AgNPs exhibited a two-phase mechanism: cell death induction and ROS induction. The first phase was a rapid killing step within 5 min, causing the direct damage of the cytoplasmic membrane of the bacterial cells, as observed by a time-kill assay and fluorescence microscopy. During the period of 5–30 min, the cell surface charge was rapidly neutralized from -8.73 and -7.74 to 2.85 and 2.94 mV in two isolates of B. pseudomallei, as revealed by zeta potential measurement. Energy-dispersive X-ray (EDX) spectroscopy showed the silver element deposited on the bacterial membrane, and TEM micrographs of the AgNP-treated B. pseudomallei cells showed severe membrane damage and cytosolic leakage at 1/5 MIC and cell bursting at MBC. During the killing effect the released Ag+ from AgNPs was only 3.9% from the starting AgNPs concentration as observed with ICP-OES experiment. In the second phase, the ROS induction occurred 1–4 hr after the AgNP treatment. Altogether, we provide direct kinetic evidence of the AgNPs killing mechanism, by which cell death is separable from the ROS induction and AgNPs mainly contributes in the killing action. AgNPs may be considered a potential candidate to develop a novel alternative agent for melioidosis treatment with fast action.

Impact assessment of silver nanoparticles on plant growth and soil bacterial diversity.

The present study was carried out to investigate the impact of silver nanoparticles (AgNPs) on the growth of three different crop species, wheat (Triticum aestivum, var. UP2338), cowpea (Vigna sinensis, var. Pusa Komal), and Brassica (Brassica juncea, var. Pusa Jai Kisan), along with their impact on the rhizospheric bacterial diversity. Three different concentrations (0, 50 and 75 ppm) of AgNPs were applied through foliar spray. After harvesting, shoot and root parameters were compared, and it was observed that wheat was relatively unaffected by all AgNP treatments. The optimum growth promotion and increased root nodulation were observed at 50 ppm treatment in cowpea, while improved shoot parameters were recorded at 75 ppm in Brassica. To observe the impact of AgNPs on soil bacterial community, sampling was carried out from the rhizosphere of these crops at 20 and 40 days after the spraying of AgNPS. The bacterial diversity of these samples was analyzed by both cultural and molecular techniques (denaturing gradient gel electrophoresis). It is clearly evident from the results that application of AgNPs changes the soil bacterial diversity and this is further influenced by the plant species grown in that soil. Also, the functional bacterial diversity differed with different concentrations of AgNPs.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0567-7) contains supplementary material, which is available to authorized users.

Silver nanoparticle treatment ameliorates biliary atresia syndrome in rhesus rotavirus inoculated mice

Biliary atresia (BA) is a neonatal biliary system disease closely associated with viral infection and bile duct inflammation. Silver nanoparticles (AgNps) have previously revealed antiviral and anti-inflammatory properties. In this study, we have investigated the effects of AgNps in the treatment of the Rhesus rotavirus inoculation induced BA in mice. The morphology, liver histopathology, clinical biochemistry examination, and inflammatory cells were analyzed in BA mice. Results indicated that AgNps could significantly increase the survival rate of BA mice, and reduce jaundice and weight lost and the liver enzymes and bilirubin metabolism clinical parameters were close to the normal levels. Diminished numbers of NK cells were observed by flow cytometry analysis and immunohistochemical staining. Furthermore, the viral load was reduced and transcripts for TGF-β mRNA were augmented after AgNps treatment. Collectively, our results suggest that AgNps treatment has beneficial effects on the BA mouse model partially through upregulation of TGF-β.

Anticancer activity of silver nanoparticles from Panax ginseng fresh leaves in human cancer cells.

The pharmaceutical role of silver nanoparticles has been increased over the last decades, especially those synthesized through herbal medicinal plants, due to their variety of pharmacological importance. Panax ginseng Meyer (P. ginseng) has been widely used as a therapeutic herbal medicine for a long time in cancer treatment. In this study, the cytotoxic and oxidative effect of a novel silver nanoparticles synthesized from P. ginseng fresh leaves (P.g AgNPs) were evaluated in different human cancer cell lines. In addition, the effect of P.g AgNPs on cell migration, apoptosis and the determination of the mechanism involve was determinate by the use of A549 lung cancer cell line. It was found that P.g AgNPs treatment inhibited cell viability and induced oxidative stress in A549, MCF7 and HepG2 cancer cell lines. Likewise, P.g AgNPs treatment inhibited the epidermal growth factor (EGF)-enhanced migration, as well as decreased the mRNA levels and phosphorylation of EGF receptors in A549 cells. Moreover, P.g AgNPs modified the morphology of the cell nucleus and increase apoptosis percentage; this effect was linked to the stimulation of p38 MAPK/p53 pathway. Taken together, our results showed that P.g AgNPs exhibited anti-cancer activity in A549 and the regulation of EGFR/p38 MAPK/p53 pathway might be the possible mechanism of its anti-activity. Further experiments are suggested to determinate the mechanism by which P.g AgNPs induce cytotoxicity and ROS generation in MCF-7 and HepG2 cells.

Eco-Friendly Synthesis of Silver Nanoparticles Through Economical Methods and Assessment of Toxicity Through Oxidative Stress Analysis in the Labeo Rohita.

The physicochemical and biological properties of metals change as the particles are reduced to nanoscale. This ability increases the application of nanoparticles in commercial and medical industry. Keeping in view this importance, Silver nanoparticles (Ag-NPs) were synthesized by reduction methods using formaldehyde as reducing agent in the chemical route and lemon extracts in the biological route. The scanning electron microscope (SEM) images of nanoparticles suggested that the particles were either agglomerated or spherical in shape with mean diameter of 16.59nm in the chemical route and 42.93nm in the biological route. The particles were between 5 and 80nm with maximum frequency between 5 and 20nm in the chemical route and between 5 and 100nm with maximum frequency between 15 and 50nm in the biological method. In the second phase of the study, the effect of Ag-NPs on the oxidative stress was studied. For this purpose, Labeo rohita (20 ± 2.5g in weight and 12 ± 1.4cm in length) were involved. Six treatments were applied in three replicates having five fishes in each replicate. The first treatment was used as control group, and the other five treatments were exposed to either 10 or 20 or 30 or 45 or 55mgL-1 of Ag-NPs for 28days. The treatment of Ag-NPs caused oxidative stress in the liver and gill tissues, which induced alterations in the activities of antioxidant enzymes. The level of catalase (CAT) was decreased in response to Ag-NPs concentration in dose-dependent manner. Ag-NPs treatment stimulated the liver and gill tissues to significantly increase the level of superoxide dismutase (SOD), which might be due to synthesis of SOD and addition in the pre-existing SOD level. The level decreases again due to depletion of SOD level. There was a sharp decline in the activities of glutathione S-transferase (GST) in both gills and liver tissues even at lower concentration, and this decrease in the GST activity was significantly different at each treatment after 28days of treatment except 20mgL-1. The malondialdehyde (MDA) levels of gills and liver tissues were increased with the increase in the concentration. The elevated levels of glutathione (GSH) showed that the liver started defensive mechanism against the oxyradicals. This study finds out the cheap eco-friendly and economical method of Ag-NP synthesis. It is further revealed that Ag-NPs caused oxidative stress in the aquatic animals if exposure occurs at high concentrations.

Silver nanoparticles synthesized using aqueous leaf extract of Ziziphus oenoplia (L.) Mill: Characterization and assessment of antibacterial activity

Biological approach to synthesis of metal nanoparticles using aqueous leaf extract is a highly relevant and recent theme in nanotechnological research. Phytosynthesized AgNPs have better inhibitory and antimicrobial effects compared to aqueous leaf extract and silver nitrate. In the present investigation crystalline silver nanoparticles (AgNPs) with size of 10nm have been successfully synthesized using aqueous leaf extract (AQLE) of Ziziphus oenoplia (L.) Mill., which act as both reducing as well as capping agent. The particles were characterized using UV Visible spectroscopy, HRTEM-EDAX, XRD, FT-IR and DLS. An evaluation of the anti bacterial activity was carried out using Agar well diffusion method and MIC determination against four bacterial strains, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli and Salmonella typhi; the AgNPs exhibited quite high antibacterial activity. Furthermore, bactericidal studies with TEM at different time intervals after AgNPs treatment showed the presence of AgNPs near cell membrane of bacteria at about 30min exposure and the bacterial-lysis was found completed at 24h. This gave an insight on the mechanism of bacterial-lysis by direct damage to the cell membrane.

The Molecular Mechanisms of the Antibacterial Effect of Picosecond Laser Generated Silver Nanoparticles and Their Toxicity to Human Cells.

Silver nanoparticles (Ag NPs) are known to have antibacterial properties. They are commonly produced by chemical synthesis which involves the use of harmful reducing agents. Contras, the laser technique is able to generate high-purity Ag NPs in water with specified surface charge characteristics. In the past, the molecular mechanisms contributing to the bactericidal effects of Ag NPs have been investigated extensively, but little is known of the antibacterial and toxic effects and mechanisms involved in laser-generated Ag NPs. In the current study Ag NPs were generated by picosecond laser ablation. Their antibacterial activity was determined on the gram-negative bacteria E. coli and Pseudomonas aeruginosa, and the gram positive bacteria Staphylococcus aureus including the methicillin resistant strain MRSA. Results showed that the laser generated Ag NPs exhibited strong dose-dependent antibacterial activity against all the three bacterial strains tested. Using E.coli as a model system, the laser Ag NPs treatment induced significantly high levels of reactive oxygen species (ROS). These ROS did not include detectable hydroxyl radicals, suggesting for the first time the selective ROS induction in bacterial cells by laser generated Ag NPs. The increased ROS was accompanied by significantly reduced cellular glutathione, and increased lipid peroxidation and permeability, suggesting ROS related bacterial cell damage. The laser generated Ag NPs exhibited low toxicity (within 72 hours) to five types of human cells although a weak significant decrease in cell survival was observed for endothelial cells and the lung cells. We conclude that picosecond laser generated Ag NPs have a broad spectrum of antibacterial effects against microbes including MRSA with minimal human cell toxicity. The oxidative stress is likely the key mechanism underlying the bactericidal effect, which leads to lipid peroxidation, depletion of glutathione, DNA damages and eventual disintegration of the cell membrane.

Effects of silver nanoparticles on adult neural stem cell differentiation and beta-catenin signaling.

Silver nanoparticles (AgNP) are distinguished by their unique antimicrobial actions. Since AgNPs inhibit the growth of bacteria, they are incorporated into common consumer products such as clothing, electronics and medicine. In concentrations as high as 50-100μg/mL, AgNP cause cytotoxicity in neural cells; yet in low concentrations of 30μg/mL and below, cytotoxicity decreased and cell viability remained. Most people will not be exposed to toxic doses of AgNP, but rather a sub-lethal exposure which may cause subtle effects. AgNP can disrupt the blood brain barrier, cross cell membranes, and accumulate in the brain, resulting in chronic low-level exposure. My project used cultured adult neural stem cells (NCS) to test the effects of AgNP exposure at 1μg/mL, well below cytotoxic levels. NCS play a key role in learning, memory and repair and provide a cell culture model for brain cells. We previously found that low level AgNP exposure disrupted the cytoskeleton structure in cultured NSC, resulting in inhibition of neurite dynamics and the formation of f-actin puncta. This research aims to investigate β-catenin signaling pathways as a potential target of AgNP effects on NSC cytoskeleton function. We found that β-catenin and f-actin puncta co-localize after AgNP treatment. Also, the subcellular localization of β-catenin in differentiating NCS is altered after exposure to AgNP. This suggests an interaction between AgNP and β-catenin, resulting in the formation of cytoskeletal disruptions during NCS differentiation. Consequently, as β-catenin signaling contributes to healthy neural physiology and development, disruption by low-level AgNP could affect proper growth and function.

Abstract 5238: The chemopreventive potential of silver nanoparticles against UVB-induced skin carcinogenesis in mouse model

Abstract Solar ultraviolet (UV) radiation, particularly its UVB component, is an established cause of human skin carcinogenesis due to its ability to cause DNA damage in skin cells. Although several sunscreen formulations have been developed and commercialized to limit or minimize UVB exposure to skin cells, incidence of skin cancer has continued to increase suggesting their limited or no efficacy in preventing UV-induced skin cancer occurrence. Recently, we revealed the potential of silver nanoparticles (AgNPs; ≤ 50 nm) against UVB radiation-induced DNA damage in human keratinocytes (HaCaT). Here we performed preclinical evaluation of chemopreventive efficacy of AgNPs against UVB-induced skin tumorigenesis in SKH-1 hairless mouse model. The different concentrations (20 and 40 mg/kg) of AgNPs mixed with hydrophilic cream base were uniformly applied topically onto the dorsal area of mouse skin prior to UVB-irradiation to assess chemopreventive efficacy for several weeks. The UVB treatment group showed tumor formation within 13 weeks, and had high incidence (93.3%) rate by the end of the study (29 weeks). Interestingly, the pretreatment of mice with AgNPs significantly increased the latency period (6-9 weeks) of UVB-induced tumor formation. Moreover, overall tumor incidence was found to be significantly decreased (50 and 78%; in 20 and 40 mg/kg respectively) as compared to AgNPs-untreated mice. Tumor multiplicity and average tumor volume/tumor-bearing mouse were also observed to be significantly reduced in AgNPs-treated mice. Additionally, AgNPs treatment alone for 29 weeks did not induce any apparent signs of toxicity and changes in the body weight suggesting the safety of AgNPs. Altogether, these findings suggest that AgNPs are potential chemopreventive agents against UVB radiation-induced skin carcinogenesis and thus opening the ways for human applications. Citation Format: Nikhil Tyagi, Sumit Arora, Sanjeev K. Srivastava, James E. Carter, Ajay P. Singh, Seema Singh. The chemopreventive potential of silver nanoparticles against UVB-induced skin carcinogenesis in mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5238.

Phytogenic synthesis of silver nanoparticles, optimization and evaluation of in vitro antifungal activity against human and plant pathogens

An attempt was made to synthesis of biocompatible silver nanoparticles from ten different Cassia spp. Among them, Cassia roxburghii aqueous leaf extract supported the synthesis of highly efficient and stable AgNPs. The synthesis of AgNPs was optimized at different physico-chemical condition and highly stable AgNPs were synthesized with 1.0mL of C. roxburghii leaf extract, pH 7.0, 1.0mM AgNO3 and at 37°C. The synthesized AgNPs were characterized by XPS, DLS and ZETA potential. DLS and ZETA potential analysis, the average AgNPs size was 35nm and the zeta potential was −18.3mV. The AgNPs exhibit higher antifungal activity when compared with the conventional antifungal drug amphotericin B against all the tested human fungal pathogens such as Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Penicillium sp., Candida albicans and the plant pathogens such as Rhizoctonia solani, Fusarium oxysporum and Curvularia sp. Scanning electron microscope (SEM) analysis showed distinct structural changes in the cell membranes of C. albicans upon AgNPs treatment. These results suggest that phytosynthesized AgNPs could be used as effective growth inhibitors in controlling various human and plant diseases caused by fungi.

Prolonged Effects of Silver Nanoparticles on p53/p21 Pathway-Mediated Proliferation, DNA Damage Response, and Methylation Parameters in HT22 Hippocampal Neuronal Cells

It is widely accepted that silver nanoparticles (AgNPs) are toxic to biological systems. However, little is known about their actions at molecular level and the cytophysiological effects after AgNP removal. As nanoparticles are suggested a promising tool to transport drugs to the brain for use in neurological conditions, we used HT22 mouse hippocampal neuronal cells as a model to study AgNP-mediated effects after their removal from the cell culture medium. We selected a relatively low concentration of AgNPs, 5 μg/ml, treated the cells for 48 h, and evaluated AgNP-induced cytophysiological effects after 96 h of AgNP removal. AgNP removal did not result in cytotoxicity. In contrast, AgNPs modulated HT22 cell cycle and proliferation and induced oxidative stress and 53BP1 recruitment, which were accompanied by elevated levels of p53 and p21. AgNP-associated diminution in lamin B1 pools did not significantly affect the structure of the nucleus. No disruption in F-actin dynamics was observed upon AgNP treatment. Moreover, we showed for the first time that AgNPs stimulated changes in DNA methylation: the augmentation in 5-methylcytosine (5-mC) and DNMT1, DNMT2, DNMT3a, and DNMT3b levels were observed. The upregulation of DNMT2 may be a part of cellular stress response to AgNP treatment. Taken together, AgNP removal resulted in p53/p21-mediated inhibition of cell proliferation, oxidant-based DNA damage response, and changes in DNA methylation patterns, which suggests that more attention should be paid to the possible outcomes in individuals exposed to nano-sized biomaterials.

Hypoxia-mediated autophagic flux inhibits silver nanoparticle-triggered apoptosis in human lung cancer cells.

Solid tumors are frequently associated with resistance to chemotherapy because the fraction of hypoxic tumor cells is substantial. To understand the underlying mechanism of hypoxia on silver nanoparticle (AgNPs)-induced apoptosis, the expression of hypoxia-inducible factor (HIF)-1α, a hallmark of hypoxia, was measured in the presence and absence of AgNPs. The results showed that HIF-1α expression was upregulated after AgNPs treatment under both hypoxic and normoxic conditions. Cell viability assays showed that AgNPs promoted cell death in cancer cells but not in non-cancer cells, as cancer cells are slightly more acidic than normal cells. However, reactive oxygen species generation induced by AgNPs in lung cancer cells caused high susceptibility to oxidative stress, whereas pre-exposure to hypoxia blocked AgNPs-induced oxidative stress. Notably, HIF-1α inhibited AgNPs-induced mitochondria-mediated apoptosis by regulating autophagic flux through the regulation of ATG5, LC3-II, and p62. Further, cell viability after treatment of cancer cells with AgNPs under hypoxic conditions was lower in HIF-1α siRNA-transfected cells than in control siRNA-transfected cells, indicating that HIF-1α knockdown enhances hypoxia induced decrease in cell viability. Our results suggest that hypoxia-mediated autophagy may be a mechanism for the resistance of AgNPs-induced apoptosis and that strategies targeting HIF-1α may be used for cancer therapy.

Abstract B04: Systemic delivery of silver nanoparticles and targeting of the folate receptor alpha for the treatment of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) lacks expression of the estrogen and progesterone receptors and does not overexpress the HER-2 receptor. Thus, current targeted therapies are rendered ineffective against this subtype of breast cancer, leaving a gap in treatment options for these patients. The use of nanotechnology has the potential to dramatically enhance the way cancer is diagnosed and treated. However, concerns persist about the toxicity of nanomaterials, which may not degrade, or are only slowly degraded and excreted after long body residence time. For clinical translation to become a reality, the potential risk–benefit balance for these materials must be resolved. We recently identified that silver nanoparticles (AgNPs) have the capacity to act as a single, self-therapeutic agent with a desirable combination of selective cytotoxicity and radiation dose enhancement effects in TNBC cells in vitro and in vivo at doses that that are non-toxic to non-cancerous breast and other cells. This is the first time any nanomaterial has been demonstrated to possess a TNBC specific toxicity profile and provides evidence that a therapeutic window exists for the safe use of AgNPs. Our current studies focus on determining the properties of the nanomaterial that are important to retain or enhance this TNBC selective response. Nanoparticle size and aggregation can have a dramatic effect on the cytotoxic and tumor targeting properties of nanomaterials. We used AgNP preparations with varying diameters (5 nm, 25 nm, 50 nm, 75 nm) to determine the influence of AgNP size on the increased sensitivity of TNBC cell lines to AgNPs. Nanoparticles were characterized by transmission electron microscopy, dynamic light scattering, and nanoparticle tracking analysis. Using this combinatorial analysis, we identify spherical AgNPs preparations that resisted aggregation or dissolution in physiologic solutions for up to one month. We find that the increased sensitivity of TNBC cells as compared to non-cancerous cells was independent of nanoparticle size, and TNBC cell lines (MDA-MB-231, BT-549, SUM-159) are more sensitive to AgNP exposure than luminal A (MCF-7) or non-cancerous breast (MCF-10A, 184B5). We then performed tumor treatment studies in nude mice bearing MDA-MB-231 TNBC tumors in the 4th mammary fat pad. Mice were intravenously injected three times per week with AgNPs (6 mg/kg) or phosphate buffered saline (PBS). Remarkably, AgNP treatment significantly slows tumor TNBC tumor growth in vivo with no apparent systemic toxicity. Targeting has the potential to increase selectivity and uptake of AgNPs leading to increased therapeutic efficacy. Therefore, we functionalized our AgNPs with folic acid (FA) to exploit the folate receptor α (FRA), which is overexpressed in approximately 80% of TNBC. AgNPs were functionalized with a SH-PEG-FA targeting moiety to form FRA targeted AgNPs (FA-AgNPs). Using a modified dot blot probed with an anti-FA antibody, we demonstrate that FA-AgNPs were free of unbound FA and that AgNP bound- FA was available for binding with the FRA. In comparison to non-targeted AgNPs, FA-AgNPs significantly increase the cytotoxic effects of our AgNPs against TNBC in vitro. Furthermore, excess FA decreased the cytotoxicity of FA-AgNPs but had no effect on the cytotoxicity of the non-targeted AgNPs, indicating that the increased cytotoxicity of FA-AgNPs was FRA mediated. In summary, we identified that AgNPs exert significant anti-cancer activity toward TNBC cells in vitro and in vivo, and point to potential vulnerabilities in TNBC cells that could be exploited for the development of new therapeutic agents. Additionally, these AgNPs are suitable for systemic delivery and can be modified with FA to increase the specificity which further decreases the potential for off-target toxicity. Citation Format: Jessica L. Swanner, Cale D. Fahrenholtz, Ravi N. Singh. Systemic delivery of silver nanoparticles and targeting of the folate receptor alpha for the treatment of triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B04.

Silver nanoparticles composition for treatment of distemper in dogs

Canine distemper is caused by the canine distemper virus (CDV), a paramyxovirus of the genus Morbillivirus, with a worldwide distribution. Canine distemper is a multisystem disease, potentially fatal. Affected organisms may develop clinical or subclinical infections, among them respiratory, gastrointestinal and of the nervous system. In the present work, we use a veterinary pharmaceutical composition of silver nanoparticles (AgNPs) for the treatment of distemper in dogs with neurological and non-neurological symptoms. AgNPs treatment of animals with non-neurological distemper provides a very high rate of recovery without sequelae. However, in dogs with neurological symptoms, AgNPs treatment was not able to reverse the disease.

Silver nanoparticles: a mechanism of action on moulds.

Silver nanoparticles (AgNPs) are widely used in all branches of industry. However, their mechanisms of action towards moulds have not been studied yet. Thus we conducted this study in which we have used laser desorption/ionization time-of-flight mass spectrometry (LDI-ToF-MS) analysis to determine metabolomic changes, and microscopic analysis (transmission electron microscopy, fluorescent microscopy) to observe changes in mould cells. The AgNP treatment caused the downregulation of 162 (15 ppm) and 284 (62 ppm), and 19 (15 ppm) and 29 (62 ppm) metabolites of Aspergillus niger and Penicillium chrysogenum, respectively. All influenced features were below m/z 600 (mass-to-charge ratio). We have observed silver ions and their clusters (Ag, Ag2, and Ag3) accumulated in the mould mycelium. As well as, mono-silver ion adducts with nucleotide derivatives (Coenzyme A), amino acids (phenylglycine), peptides (LeuSerAlaLeuGlu) and lipids (fatty acids, diacylglycerophosphoglycerols, monoglicerides and glycerophospholipids). The ultrastructure analysis revealed many sever alterations due to the action of AgNPs, such us shortening and condensation of hyphae, ultrastructural reorganisation, cell plasmolysis, increased vacuolisation, numerous membranous structures, collapsed cytoplasm, accumulation of lipid material, condensed mitochondria, disintegration of organelles, nuclear deformation, condensation and fragmentation of chromatin, creation of apoptotic bodies, as well as a new inside cell wall in P. chrysogenum.

Distribution and accumulation of 10 nm silver nanoparticles in maternal tissues and visceral yolk sac of pregnant mice, and a potential effect on embryo growth

We examined the distribution of silver in pregnant mice and embryos/fetuses following intravenous injections of 10 nm silver nanoparticles (AgNPs) or soluble silver nitrate (AgNO3) at dose levels of 0 (citrate buffer control) or 66 µg Ag/mouse to pregnant mice on gestation days (GDs) 7, 8 and 9. Selected maternal tissues and all embryos/fetuses from control, AgNP- and AgNO3-treated groups on GD10 and control and AgNP-treated groups on GD16 were processed for the measurement of silver concentrations, intracellular AgNP localization, histopathology and gross examination of tissue morphology. Inductively-coupled plasma mass spectrometry revealed silver in all examined tissues following either AgNP or AgNO3 treatment, with highest concentrations of silver in maternal liver, spleen and visceral yolk sac (VYS), and lowest concentrations in embryos/fetuses. For VYS, mean silver concentration following AgNO3 treatment (4.87 ng Ag/mg tissue) was approximately two-fold that following AgNP treatment (2.31 ng Ag/mg tissue); for all other tissues examined, mean silver concentrations following either AgNP or AgNO3 treatment were not significantly different from each other (e.g. 2.57 or 2.84 ng Ag/mg tissue in maternal liver and 1.61 or 2.50 ng Ag/mg tissue in maternal spleen following AgNP or AgNO3 treatment, respectively). Hyperspectral imaging revealed AgNP aggregates in maternal liver, kidney, spleen and VYS from AgNP-treated mice, but not AgNO3-treated mice. Additionally, one or more embryos collected on GD10 from eight of ten AgNP-treated mice appeared small for their age (i.e. Theiler stage 13 [GD8.5] or younger). In the control group (N = 11), this effect was seen in embryos from only one mouse. In conclusion, intravenous injection of 10 nm AgNPs to pregnant mice resulted in notable silver accumulation in maternal liver, spleen and VYS, and may have affected embryonic growth. Silver accumulation in embryos/fetuses was negligible.