A Comparative Study of Cancer Cells Susceptibility to Silver Nanoparticles Produced by Electron Beam.

INTRODUCTION: Silver nanoparticles (AgNPs) have a wide range of bioactivity, which is highly dependent on particle size, shape, stabilizer, and production method. Here, we present the results of studies of AgNPs cytotoxic properties obtained by irradiation treatment of silver nitrate solution and various stabilizers by accelerating electron beam in a liquid medium. METHODS: The results of studies of morphological characteristics of silver nanoparticles were obtained by transmission electron microscopy, UV-vis spectroscopy, and dynamic light scattering measurements. MTT test, alamar blue test, flow cytometry, and fluorescence microscopy were used to study the anti-cancer properties. As biological objects for standard tests, adhesive and suspension cell cultures of normal and tumor origin, including prostate cancer, ovarian cancer, breast cancer, colon cancer, neuroblastoma, and leukemia, were studied. RESULTS: The results showed that the silver nanoparticles obtained by irradiation with polyvinylpyrrolidone and collagen hydrolysate are stable in solutions. Samples with different stabilizers were characterized by a wide average size distribution from 2 to 50 nm and low zeta potential from -7.3 to +12.4 mV. All AgNPs formulations showed a dose-dependent cytotoxic effect on tumor cells. It has been established that the particles obtained with the combination of polyvinylpyrrolidone/collagen hydrolysate have a relatively more pronounced cytotoxic effect in comparison to samples stabilized with only collagen or only polyvinylpyrrolidone. The minimum inhibitory concentrations for nanoparticles were less than 1 µg/mL for various types of tumor cells. It was found that neuroblastoma (SH-SY5Y) is the most susceptible, and ovarian cancer (SKOV-3) is the most resistant to the action of silver nanoparticles. The activity of the AgNPs formulation prepared with a mixture of PVP and PH studied in this work was higher that activity of other AgNPs formulations reported in the literature by about 50 times. CONCLUSIONS: The results indicate that the AgNPs formulations synthesized with an electron beam and stabilized with polyvinylpyrrolidone and protein hydrolysate deserve deep study for their further use in selective cancer treatment without harming healthy cells in the patient organism.

Silver nanoparticles induce a non-immunogenic tumor cell death.

Immunogenic cell death (ICD) is a form of cell death characterized by the release of danger signals required to trigger an adaptive immune response against tumor-associated antigens. Silver nanoparticles (AgNP) display anti-proliferative and cytotoxic effects in tumor cells, but it has not been previously studied whether AgNP act as an ICD inductor. The present study evaluated the in vitro release of calreticulin as a damage-associated molecular pattern (DAMP) associated with the cytotoxicity of AgNP and their in vivo anti-cancer effects. In vitro, mouse CT26 colon carcinoma and MCA205 fibrosarcoma cells were exposed to AgNP and then cell proliferation, adhesion, and release of calreticulin were determined. The results indicated there were time- and concentration-related anti-proliferative effects of AgNP in both the CT26 and MCA205 lines. Concurrently, changes in cell adhesion were detected mainly in the CT26 cells. Regarding DAMP detection, a significant increase in calreticulin was observed only in CT26 cells treated with doxorubicin and AgNP; however, no differences were found in the MCA205 cells. In vivo, the survival and growth of subcutaneous tumors were monitored after vaccination of mice with cell debris from tumor cells treated with AgNP or after intra-tumoral administration of AgNP to established tumors. Consequently, anti-tumoral prophylactic immunization with AgNP-dead cells failed to protect mice from tumor re-challenge; intra-tumor injection of AgNP did not induce a significant effect. In conclusion, there was a noticeable anti-tumoral effect of AgNP in vitro in both CT26 and MCA205 cell lines, accompanied by the release of calreticulin in CT26 cells. In vivo, immunization with cell debris derived from AgNP-treated tumor cells failed to induce a protective immune response in the cancer model mice. Clearly, further research is needed to determine if one could combine AgNP with other ICD inducers to improve the anti-tumor effect of these nanoparticles in vivo.

Chronic toxicity of shrimp feed added with silver nanoparticles (Argovit-4®) in Litopenaeus vannamei and immune response to white spot syndrome virus infection.

In recent years, the application of silver nanoparticles (AgNPs) as antibacterial compounds has been widely used in human and veterinary medicine. In this work, we investigated the effects of AgNPs (Argovit-4®) as feed additives (feed-AgNPs) on shrimp (Litopenaeus vannamei) using three different methods: 1) chronic toxicity after 28 days of feeding, 2) Effects against white spot syndrome virus (WSSV) challenged by oral route, and 3) transcriptional responses of immune-related genes (PAP, ProPO, CTL-3, Crustin, PEN3, and PEN4) following WSSV infection. The results showed that the feed-AgNPs did not interfere with the growth and survival of shrimp. Also, mild lesions in the hepatopancreas were recorded, proportional to the frequency of the feed-AgNP supply. Challenge test versus WSSV showed that feeding every 7 days with feed-AgNPs reduced mortality, reaching a survival rate of 53%, compared to the survival rates observed in groups fed every 4 days, daily and control groups of feed-AgNPs for the 30%, 10%, and 7% groups, respectively. Feed-AgNPs negatively regulated the expression of PAP, ProPO, and Crustin genes after 28 days of treatment and altered the transcriptional responses of PAP, ProPO, CTL-3, and Crustin after WSSV exposure. The results showed that weekly feeding-AgNPs could partially prevent WSSV infection in shrimp culture. However, whether or not transcriptional responses against pathogens are advantageous remains to be elucidated.

AgNPs Argovit™ Modulates Cyclophosphamide-Induced Genotoxicity on Peripheral Blood Erythrocytes In Vivo.

Silver nanoparticles (AgNPs) have been studied worldwide for their potential biomedical applications. Specifically, they are proposed as a novel alternative for cancer treatment. However, the determination of their cytotoxic and genotoxic effects continues to limit their application. The commercially available silver nanoparticle Argovit™ has shown antineoplastic, antiviral, antibacterial, and tissue regenerative properties, activities triggered by its capacity to promote the overproduction of reactive oxygen species (ROS). Therefore, in this work, we evaluated the genotoxic and cytotoxic potential of the Argovit™ formulation (average size: 35 nm) on BALB/c mice using the micronucleus in a peripheral blood erythrocytes model. Besides, we evaluated the capability of AgNPs to modulate the genotoxic effect induced by cyclophosphamide (CP) after the administration of the oncologic agent. To achieve this, 5-6-week-old male mice with a mean weight of 20.11 ± 2.38 g were treated with water as negative control (Group 1), an single intraperitoneal dose of CP (50 mg/kg of body weight, Group 2), a daily oral dose of AgNPs (6 mg/kg of weight, Group 3) for three consecutive days, or a combination of these treatment schemes: one day of CP doses (50 mg/kg of body weight) followed by three doses of AgNPs (one dose per day, Group 4) and three alternate doses of CP and AgNPs (six days of exposure, Group 5). Blood samples were taken just before the first administration (0 h) and every 24 h for seven days. Our results show that Argovit™ AgNPs induced no significant cytotoxic or acute genotoxic damage. The observed cumulative genotoxic damage in this model could be caused by the accumulation of AgNPs due to administered consecutive doses. Furthermore, the administration of AgNPs after 24 h of CP seems to have a protective effect on bone marrow and reduces by up to 50% the acute genotoxic damage induced by CP. However, this protection is not enough to counteract several doses of CP. To our knowledge, this is the first time that the exceptional chemoprotective capacity produced by a non-cytotoxic silver nanoparticle formulation against CP genotoxic damage has been reported. These findings raise the possibility of using AgNPs as an adjuvant agent with current treatments, reducing adverse effects.

Antitumor Activity against Human Colorectal Adenocarcinoma of Silver Nanoparticles: Influence of [Ag]/[PVP] Ratio.

Silver nanoparticles (AgNPs) not only have shown remarkable results as antimicrobial and antiviral agents but also as antitumor agents. This work reports the complete characterization of five polyvinylpyrrolidone-coated AgNP (PVP-AgNP) formulations, their cytotoxic activity against human colon tumor cells (HCT-15), their cytotoxic effect on primary mouse cultures, and their lethal dose on BALB/c mice. The evaluated AgNP formulations have a composition within the ranges Ag: 1.14-1.32% w/w, PVP: 19.6-24.5% and H2O: 74.2-79.2% with predominant spherical shape within an average size range of 16-30 nm according to transmission electron microscopy (TEM). All formulations assessed increase mitochondrial ROS concentration and induce apoptosis as the leading death pathway on HCT-15 cells. Except for AgNP1, the growth inhibition potency of AgNP formulations of human colon tumor cancer cells (HCT-15) is 34.5 times higher than carboplatin, one of the first-line chemotherapy agents. Nevertheless, 5-10% of necrotic events, even at the lower concentration evaluated, were observed. The cytotoxic selectivity was confirmed by evaluating the cytotoxic effect on aorta, spleen, heart, liver, and kidney primary cultures from BALB/c mice. Despite the cytotoxic effects observed in vitro, the lethal dose and histopathological analysis showed the low toxicity of these formulations (all of them on Category 4 of the Globally Harmonized System of Classification and Labelling of Chemicals) and minor damage observed on analyzed organs. The results provide an additional example of the rational design of safety nanomaterials with antitumor potency and urge further experiments to complete the preclinical studies for these AgNP formulations.

Hemolysis of Human Erythrocytes by Argovit™ AgNPs from Healthy and Diabetic Donors: An In Vitro Study.

The use of nanomaterials is becoming increasingly widespread, leading to substantial research focused on nanomedicine. Nevertheless, the lack of complete toxicity profiles limits nanomaterials' uses, despite their remarkable diagnostic and therapeutic results on in vitro and in vivo models. Silver nanoparticles (AgNPs), particularly Argovit™, have shown microbicidal, virucidal, and antitumoral effects. Among the first-line toxicity tests is the hemolysis assay. Here, the hemolytic effect of Argovit™ AgNPs on erythrocytes from one healthy donor (HDE) and one diabetic donor (DDE) is evaluated by the hemolysis assay against AgNO3. The results showed that Argovit™, in concentrations ≤24 µg/mL of metallic silver, did not show a hemolytic effect on the HDE or DDE. On the contrary, AgNO3 at the same concentration of silver ions produces more than 10% hemolysis in both the erythrocyte types. In all the experimental conditions assessed, the DDE was shown to be more prone to hemolysis than the HDE elicited by Ag+ ions or AgNPs, but much more evident with Ag+ ions. The results show that Argovit™ is the least hemolytic compared with the other twenty-two AgNP formulations previously reported, probably due to the polymer mass used to stabilize the Argovit™ formulation. The results obtained provide relevant information that contributes to obtaining a comprehensive toxicological profile to design safe and effective AgNP formulations.

New Protein-Coated Silver Nanoparticles: Characterization, Antitumor and Amoebicidal Activity, Antiproliferative Selectivity, Genotoxicity, and Biocompatibility Evaluation.

Nanomaterials quickly evolve to produce safe and effective biomedical alternatives, mainly silver nanoparticles (AgNPs). The AgNPs' antibacterial, antiviral, and antitumor properties convert them into a recurrent scaffold to produce new treatment options. This work reported the full characterization of a highly biocompatible protein-coated AgNPs formulation and their selective antitumor and amoebicidal activity. The protein-coated AgNPs formulation exhibits a half-inhibitory concentration (IC50) = 19.7 µM (2.3 µg/mL) that is almost 10 times more potent than carboplatin (first-line chemotherapeutic agent) to inhibit the proliferation of the highly aggressive human adenocarcinoma HCT-15. The main death pathway elicited by AgNPs on HCT-15 is apoptosis, which is probably stimulated by reactive oxygen species (ROS) overproduction on mitochondria. A concentration of 111 µM (600 µg/mL) of metallic silver contained in AgNPs produces neither cytotoxic nor genotoxic damage on human peripheral blood lymphocytes. Thus, the AgNPs formulation evaluated in this work improves both the antiproliferative potency on HCT-15 cultures and cytotoxic selectivity ten times more than carboplatin. A similar mechanism is suggested for the antiproliferative activity observed on HM1-IMSS trophozoites (IC50 = 69.2 µM; 7.4 µg/mL). There is no change in cell viability on mice primary cultures of brain, liver, spleen, and kidney exposed to an AgNPs concentration range from 5.5 µM to 5.5 mM (0.6 to 600 µg/mL). The lethal dose was determined following the OECD guideline 420 for Acute Oral Toxicity Assay, obtaining an LD50 = 2618 mg of Ag/Kg body weight. All mice survived the observational period; the histopathology and biochemical analysis show no differences compared with the negative control group. In summary, all results from toxicological evaluation suggest a Category 5 (practically nontoxic) of the Globally Harmonized System of Classification and Labelling of Chemicals for that protein-coated AgNPs after oral administration for a short period and urge the completion of its preclinical toxicological profile. These findings open new opportunities in the development of selective, safe, and effective AgNPs formulations for the treatment of cancer and parasitic diseases with a significant reduction of side effects.

Cytokinesis-Block Micronucleus Assay Using Human Lymphocytes as a Sensitive Tool for Cytotoxicity/Genotoxicity Evaluation of AgNPs.

Silver nanoparticles (AgNPs) are the most used nanomaterials worldwide due to their excellent antibacterial, antiviral, and antitumor activities, among others. However, there is scarce information regarding their genotoxic potential measured using human peripheral blood lymphocytes. In this work, we present the cytotoxic and genotoxic behavior of two commercially available poly(vinylpyrrolidone)-coated silver nanoparticle (PVP-AgNPs) formulations that can be identified as noncytotoxic and nongenotoxic by just evaluating micronuclei (MNi) induction and the mitotic index, but present enormous differences when other parameters such as cytostasis, apoptosis, necrosis, and nuclear damage (nuclear buds (NBUDs) and nucleoplasmic bridges (NPBs)) are analyzed. The results show that Argovit (35 nm PVP-AgNPs) and nanoComposix (50 nm PVP-AgNPs), at concentrations from 0.012 to 12 µg/mL, produce no changes in the nuclear division index (NDI) or micronuclei (MNi) frequency compared with the values found on control cultures of human blood peripheral lymphocytes from a healthy donor. Still, 50 nm PVP-AgNPs significantly decrease the replication index and significantly increase cytostasis, apoptosis, necrosis, and the frequencies of nuclear buds (NBUDs) and nucleoplasmic bridges (NPBs). These results provide evidence that the cytokinesis-block micronucleus (CBMN) assay using human lymphocytes and evaluating the eight parameters provided by the technique is a sensitive, fast, accurate, and inexpensive detection tool to support or discard AgNPs or other nanomaterials, which is worthwhile for continued testing of their effectiveness and toxicity for biomedical applications. In addition, it provides very important information about the role played by the [coating agent]/[metal] ratio in the design of nanomaterials that could reduce adverse effects as much as possible while retaining their therapeutic capabilities.

Antiproliferative and Antitumour Effect of Nongenotoxic Silver Nanoparticles on Melanoma Models.

During the last 3 decades, there has been a slow advance to obtain new treatments for malignant melanoma that improve patient survival. In this work, we present a systematic study focused on the antiproliferative and antitumour effect of AgNPs. These nanoparticles are fully characterized, are coated with polyvinylpyrrolidone (PVP), and have an average size of 35 ± 15 nm and a metallic silver content of 1.2% wt. Main changes on cell viability, induction of apoptosis and necrosis, and ROS generation were found on B16-F10 cells after six hours of exposure to AgNPs (IC50 = 4.2 µg/mL) or Cisplatin (IC50 = 2.0 µg/mL). Despite the similar response for both AgNPs and Cisplatin on antiproliferative potency (cellular viability of 53.95 ± 1.88 and 53.62 ± 1.04) and ROS production (20.27 ± 1.09% and 19.50 ± 0.35%), significantly different cell death pathways were triggered. While AgNPs induce only apoptosis (45.98 ± 1.88%), Cisplatin induces apoptosis and necrosis at the same rate (22.31 ± 1.72% and 24.07 ± 1.10%, respectively). In addition to their antiproliferative activity, in vivo experiments showed that treatments of 3, 6, and 12 mg/kg of AgNPs elicit a survival rate almost 4 times higher (P < 0.05) compared with the survival rate obtained with Cisplatin (2 mg/kg). Furthermore, the survivor mice treated with AgNPs do not show genotoxic damage determined by micronuclei frequency quantification on peripheral blood cells. These results exhibit the remarkable antitumour activity of a nongenotoxic AgNP formulation and constitute the first advance toward the application of these AgNPs for melanoma treatment, which could considerably reduce adverse effects provoked by currently applied chemotherapeutics.

Toxicity of silver nanoparticles in mouse bone marrow-derived dendritic cells: Implications for phenotype.

Silver nanoparticles (AgNP) are one of the most studied nanoparticles due to their anti-bacterial, -fungal, -viral, -parasitic, and -inflammatory properties. This raises the need to evaluate the toxicity and biological effects of AgNP in the immune system in order to develop new safer biomedical products. In this study, an AgNP formulation currently approved for veterinary applications was applied to mouse bone marrow-derived dendritic cells (BMDC), considered important antigen-presenting cells of the immune system, to evaluate cytotoxicity, genotoxicity, and any significant influence on expression of cellular markers associated with BMDC phenotype and maturation status. The results showed that after 12 h of AgNP exposure, a significant decrease in BMDC viability occurred at the highest concentration tested (1.0 µg AgNP/ml) and at lower doses, the cells maintained membrane integrity and metabolic activity. DNA damage was not significant with any AgNP level aside from the 1.0 µg AgNP/ml level. Regarding phenotype, no differences in expression of CD40 (co-stimulatory molecule highly present in mature BMDC) or in CD273 (a marker for inhibitory T-cell response) were observed. The current results showed that the toxicity of this AgNP formulation was dose-related. The findings also suggest BMDC could maintain structural conservation of co-stimulatory/co-inhibitory surface molecules after 12 h of exposure to this AgNP. This work represents the first step in identifying the toxic effects of this AgNP formulation on dendritic cells.

Toxicity of silver nanoparticles in biological systems: Does the complexity of biological systems matter?

Currently, nanomaterials are more frequently in our daily life, specifically in biomedicine, electronics, food, textiles and catalysis just to name a few. Although nanomaterials provide many benefits, recently their toxicity profiles have begun to be explored. In this work, the toxic effects of silver nanoparticles (35nm-average diameter and Polyvinyl-Pyrrolidone-coated) on biological systems of different levels of complexity was assessed in a comprehensive and comparatively way, through a variety of viability and toxicological assays. The studied organisms included viruses, bacteria, microalgae, fungi, animal and human cells (including cancer cell lines). It was found that biological systems of different taxonomical groups are inhibited at concentrations of silver nanoparticles within the same order of magnitude. Thus, the toxicity of nanomaterials on biological/living systems, constrained by their complexity, e.g. taxonomic groups, resulted contrary to the expected. The fact that cells and virus are inhibited with a concentration of silver nanoparticles within the same order of magnitude could be explained considering that silver nanoparticles affects very primitive cellular mechanisms by interacting with fundamental structures for cells and virus alike.