Accumulation and cytotoxicity assessment of TAT-IONPs on cancerous mammalian cells.

Nanotechnology is a fast-growing research technology. Nanoparticles have intensive scientific applications in many fields. Depending on the physical and chemical characteristics of a nanoparticle, it can be used either as a treatment agent to fight disease or as a delivery vehicle to transport the therapeutic drug to a specified biological organ, tissue, and cell. Cytotoxicity evaluation of nanoparticles is one of the primary concerns in clinical practices to avoid unpredicted or undesirable interactions that could worsen the case. Iron oxide nanoparticle (IONP) is the most utilized nanoparticle in medical fields for treatment, diagnostic, and imaging. This paper is designated to investigate the cytotoxicity of IONPs that decorated with Trans-Activator of Transcription (TAT) protein. WST-1 assay and flow cytometry were used to assess the cytotoxicity of TAT-IONPs, which showed no significant cytotoxic effect on mammalian breast cancer cells (MCF-7). Nanoparticles accumulation in the cell's cytoplasm was evaluated from TEM images by measuring the size of the endosome. The results indicate that TAT-IONPs can be used as a safe and non-toxic nanoplatform for targeted delivery at 50 µg/ml or less. Also, they present an approach by which the area of intracellular endosome can be assessed from the TEM images of fixed cells. In this study, the endosome size increased in a time-dependent manner.

p53-competent cells and p53-deficient cells display different susceptibility to oxygen functionalized graphene cytotoxicity and genotoxicity.

Due to the distinctive physical, electrical, and chemical properties of graphene nanomaterials, numerous efforts pursuing graphene-based biomedical and industrial applications are underway. Oxidation of pristine graphene surfaces mitigates its otherwise hydrophobic characteristic thereby improving its biocompatibility and functionality. Yet, the potential widespread use of oxidized graphene derivatives raises concern about adverse impacts on human health. The p53 tumor suppressor protein maintains cellular and genetic stability after toxic exposures. Here, we show that p53 functional status correlates with oxygen functionalized graphene (f-G) cytotoxicity and genotoxicity in vitro. The f-G exposed p53-competent cells, but not p53-deficient cells, initiated G0 /G1 phase cell cycle arrest, suppressed reactive oxygen species, and entered apoptosis. There was p53-dependent f-G genotoxicity evident as increased structural chromosome damage, but not increased gene mutation or chromatin loss. In conclusion, the cytotoxic and genotoxic potential for f-G in exposed cells was dependent on the p53 functional status. These findings have broad implications for the safe and effective implementation of oxidized graphene derivatives into biomedical and industrial applications. Published 2017. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Titanium Dioxide Nanoparticles Evoke Proinflammatory Response during Murine Norovirus Infection Despite Having Minimal Effects on Virus Replication.

Noroviruses (NoV) have enhanced tropism for the gastrointestinal (GI) tract and are the major cause of nonbacterial gastroenteritis in humans. Titanium dioxide (TiO2) nanoparticles (NPs) used as food additives, dietary supplements, and cosmetics accumulate in the GI tract. We investigated the effect anatase TiO2 NPs on NoV replication and host response during virus infection, using murine norovirus (MNV-1) infection of RAW 264.7 macrophages. Pretreatment with 20 µg/ml anatase NPs significantly reduced the viability of macrophages alone or during virus infection, but did not alter virus replication. In contrast, pre-incubation with 2 µg/ml anatase NPs reduced virus replication fivefold at 48 h. The presence of anatase NPs during MNV-1 infection evoked a pro-inflammatory response, as measured by a significant increase in expression of cytokines, including IL-6, IFN-γ, TNFα and the TGFß1. No genotoxic insults due to anatase TiO2 NPs alone or to their presence during MNV-1 infection were detected. This study highlights important safety considerations related to NP exposure of the GI tract in individuals infected with noroviruses or other foodborne viruses.

Valved Polytetrafluoroethylene Conduits for Right Ventricular Outflow Tract Reconstruction.

BACKGROUND: The purposes of this study were to review our early outcomes using valved expanded polytetrafluoroethylene (ePTFE) conduits, with or without bulging sinus structure, for right ventricular outflow tract reconstruction and to examine the mechanical properties of the ePTFE material after bulging sinuses were created. METHODS: A retrospective review was performed of all patients who received the valved ePTFE conduit between 2008 and 2014 at a single institution. The surface morphologies and the mechanical strengths of the ePTFE conduit with bulging sinuses examined by scanning electron microscopy and unidirectional pull test were compared with those of the original ePTFE material. RESULTS: There were 120 operations with the valved ePTFE conduit (60 with bulging sinuses). The patients median age and weight were 6.9 years and 23.7 kg. The conduits were a median size of 22 mm. At 5 years, freedom from conduit reoperation was 92.7% (95% confidence interval, 82.7% to 97.0%), and freedom from severe conduit insufficiency or more than a 50 mm Hg gradient was 74.8% (95% confidence interval, 60.8% to 84.4%). No significant differences in the surface morphologies were observed by the scanning electron microscopy or in the maximum tolerated loads obtained by the pull test between the original ePTFE material and the ePTFE with bulging sinuses (121 and 122 N in longitudinal direction and 115 and 121 N in circumferential direction; p = 0.88 and p = 0.68). CONCLUSIONS: The valved ePTFE conduits demonstrated excellent early clinical outcomes. The mechanical property examinations showed no obvious difference after bulging sinuses were created on the ePTFE material.

Calcium-channel blocking and nanoparticles-based drug delivery for treatment of drug-resistant human cancers.

BACKGROUND: Cancer cell chemoresistance is one of the major limitations to successful cancer treatment and one of the factors that is responsible for the possible recurrence of the disease. Here, we aimed to combine a calcium-channel blocker, verapamil, with an alternative delivery of the anti-cancer drug, doxorubicin, using nanostructural materials. This approach could reduce the cellular resistance to chemotherapeutics agents. RESULTS: The outcome of this complex approach on cellular viability was investigated by using various assays in both a time- and concentration-dependent manner: WST-1, flow cytometry cell viability assay, fluorescence microscopy, DNA fragmentation, and TUNEL labeling of apoptotic cells. CONCLUSION: All of these analytical assays confirmed the ability to reduce the chemoresistance of the cancer cells based on the proposed procedure.

Single-walled carbon nanotube and graphene nanodelivery of gambogic acid increases its cytotoxicity in breast and pancreatic cancer cells.

Graphene and single-walled carbon nanotubes were used to deliver the natural low-toxicity drug gambogic acid (GA) to breast and pancreatic cancer cells in vitro, and the effectiveness of this complex in suppressing cellular integrity was assessed. Cytotoxicity was assessed by measuring lactate dehydrogenase release, mitochondria dehydrogenase activity, mitochondrial membrane depolarization, DNA fragmentation, intracellular lipid content, and membrane permeability/caspase activity. The nanomaterials showed no toxicity at the concentrations used, and the antiproliferative effects of GA were significantly enhanced by nanodelivery. The results suggest that these complexes inhibit human breast and pancreatic cancer cells grown in vitro. This analysis represents a first step toward assessing their effectiveness in more complex, targeted, nanodelivery systems.

Circulating tumor cell identification by functionalized silver-gold nanorods with multicolor, super-enhanced SERS and photothermal resonances.

Nanotechnology has been extensively explored for cancer diagnostics. However, the specificity of current methods to identify simultaneously several cancer biomarkers is limited due to color overlapping of bio-conjugated nanoparticles. Here, we present a technique to increase both the molecular and spectral specificity of cancer diagnosis by using tunable silver-gold nanorods with narrow surface-enhanced Raman scattering (SERS) and high photothermal contrast. The silver-gold nanorods were functionalized with four Raman-active molecules and four antibodies specific to breast cancer markers and with leukocyte-specific CD45 marker. More than two orders of magnitude of SERS signal enhancement was observed from these hybrid nanosystems compared to conventional gold nanorods. Using an antibody rainbow cocktail, we demonstrated highly specific detection of single breast cancer cells in unprocessed human blood. By integrating multiplex targeting, multicolor coding, and multimodal detection, our approach has the potential to improve multispectral imaging of individual tumor cells in complex biological environments.

Single-walled carbon nanotubes as specific targeting and Raman spectroscopic agents for detection and discrimination of single human breast cancer cells.

Raman active nano-complex agents based on single-walled carbon nanotubes (SWCNTs) are prepared and used for the swift and specific detection of breast cancer cells. SWCNTs are functionalized to bond covalently with the anti-epithelial cell adhesion molecule (anti-EpCAM) antibody, which is specific to the highly expressed EpCAM antigen on the surface of breast cancer cells (MCF-7), but not on normal cells. The Raman nano-complexes demonstrate excellent ability to detect in vitro single breast cancer cells (MCF-7) and discriminate between them and normal fibroblast cells during the first 30 min of the targeting process. Raman linearity scanning is collected from a monolayer cell mixture, including both cancer cells and normal cells incubated with anti-EpCAM-SWCNTs, using a 633-nm laser excitation. The results shows that the Raman signal collected from targeted MCF-7 cells is extremely high, whereas there is little signal from the normal cells.

Photothermal nanodrugs: potential of TNF-gold nanospheres for cancer theranostics.

Nanotechnology has been extensively explored for drug delivery. Here, we introduce the concept of a nanodrug based on synergy of photothermally-activated physical and biological effects in nanoparticle-drug conjugates. To prove this concept, we utilized tumor necrosis factor-alpha coated gold nanospheres (Au-TNF) heated by laser pulses. To enhance photothermal efficiency in near-infrared window of tissue transparency we explored slightly ellipsoidal nanoparticles, its clustering, and laser-induced nonlinear dynamic phenomena leading to amplification and spectral sharpening of photothermal and photoacoustic resonances red-shifted relatively to linear plasmonic resonances. Using a murine carcinoma model, we demonstrated higher therapy efficacy of Au-TNF conjugates compared to laser and Au-TNF alone or laser with TNF-free gold nanospheres. The photothermal activation of low toxicity Au-TNF conjugates, which are in phase II trials in humans, with a laser approved for medical applications opens new avenues in the development of clinically relevant nanodrugs with synergistic antitumor theranostic action.

Raman spectroscopy analysis and mapping the biodistribution of inhaled carbon nanotubes in the lungs and blood of mice.

Because of their small size, robust structure and unique characteristics, carbon nanotubes (CNTs) are increasingly being used in a variety of biomedical applications, materials and products. As their use increases, so does the probability of their unintended release and human exposure. Therefore, it is important to establish their potential biodistribution and biopersistence to better understand the potential effects of their exposure to humans. This study examines the distribution of CNTs in CD-1 mice after exposure by inhalation of single-walled carbon nanotubes (SWCNTs) and investigates the possibility that inhaled nanoparticles could enter the circulatory system via the lungs. Raman spectroscopy was employed for the detection of CNTs in lung tissue and blood based on their unique spectroscopic signatures. These studies have important implications concerning the potential effects of exposure to SWCNTs and their use as potential transport vehicles in nanomedicine.

Carbon nanotubes as plant growth regulators: effects on tomato growth, reproductive system, and soil microbial community.

Multi-walled carbon nanotubes (CNTs) can affect plant phenotype and the composition of soil microbiota. Tomato plants grown in soil supplemented with CNTs produce two times more flowers and fruit compared to plants grown in control soil. The effect of carbon nanotubes on microbial community of CNT-treated soil is determined by denaturing gradient gel electrophoresis and pyrosequencing analysis. Phylogenetic analysis indicates that Proteobacteria and Bacteroidetes are the most dominant groups in the microbial community of soil. The relative abundances of Bacteroidetes and Firmicutes are found to increase, whereas Proteobacteria and Verrucomicorbia decrease with increasing concentration of CNTs. The results of comparing diversity indices and species level phylotypes (OTUs) between samples showed that there is not a significant affect on bacterial diversity.

Iron oxide nanoparticle-based radio-frequency thermotherapy for human breast adenocarcinoma cancer cells.

Iron oxide nanoparticles (IONPs) with diameters of 15, 25, and 41 nm were evaluated as mediators of thermal cytotoxicity under radio-frequency (RF) exposure. The 25 nm IONPs were found to be the most efficient of the three in killing cancer cells at 350 kHz low-frequency RF irradiation. However, at a higher frequency of 13.56 MHz, 15 nm IONPs produced the highest percentage of cell death. Moreover, the killing effect was concentration-dependent in that a higher concentration of IONPs resulted in increased cellular death. Size-dependent internalization of IONPs in MCF-7 cells was quantified by using inductively coupled-plasma mass spectrometry (ICP-MS). Dark-field microscopy and transmission electron microscopy (TEM) revealed that MCF-7 cells internalize IONPs through endocytosis after 24 hours of incubation. In addition, after RF treatment, the cancer cells underwent the apoptosis process, and the level of reactive oxygen species (ROS) increased significantly after hyperthermia. Scanning electron microscopy (SEM) and TEM further established that the ultrastructure morphological changes in the cancer cells originated from the apoptosis process.

Role of carbonaceous nanomaterials in stimulating osteogenesis in mammalian bone cells.

In this work, we demonstrate that graphitic nanomaterials-carboxylated multi-walled carbon nanotubes (MWCNTs) and carboxylated graphenes (Gn)-have the ability to stimulate the process of osteogenesis in mammalian bone cells and significantly increase the level of bone mineralization. Exposure of MC3T3-E1 bone cells to carboxylated MWCNTs-nano-sized (nano-Gn) and micro-sized (micro-Gn) in concentrations of 1-10 µg ml-1-resulted in the enhancement of mineralization in a time-dependent manner for the cells exposed to the nanomaterials, as compared to unexposed cells. However, the graphitic nanomaterials did not show significant toxicity in the concentration levels that were studied. Gene expression analysis revealed that the MWCNTs activated expression of the mid-stage osteogenic marker, Col I, on the 12th day of cell incubation. The gene expression of the earliest osteogenic marker, Cbfa-1, and the downstream effector of BMP signaling, SMAD1, were significantly increased in bone cells exposed to both materials (MWCNTs and nano-Gn) as compared to unexposed control cells. Our data clearly demonstrate the ability of graphitic nano-materials to penetrate bone cells and regulate deposition of minerals in an in vitro model system. Our findings highlight the potential use of such materials in regenerative nanomedicine.

Multifunctional magnetic nanoparticles for synergistic enhancement of cancer treatment by combinatorial radio frequency thermolysis and drug delivery.

Few-layer, carbon-coated, iron (C/Fe) magnetic nanoparticles (MNPs) were synthesized with controlled sizes ranging from 7 to 9 nm. The additional loading of two anti-cancer drugs, doxorubicin and erlotinib, was achieved through - stacking onto the carbon shells. Controlled release of the drugs was successfully triggered by radio frequency (RF) heating or pH variation. Based on the experimental results, C/Fe MNPs act as heat-inducing agents and are able to thermally destroy cancer cells when RF is applied. It was found that the combination of anti-cancer drugs (in particular a low dose of doxorubicin) and RF treatment demonstrates a synergistic effect in inducing cell death in pancreatic cancer cells. Our findings demonstrate that MNPs can be used as highly efficient multimodal nanocarrier agents for an integrated approach to cancer treatment involving triggered delivery of antineoplastic drugs and RF-induced thermal therapy.

Genotoxicity of TiO(2) anatase nanoparticles in B6C3F1 male mice evaluated using Pig-a and flow cytometric micronucleus assays.

In vivo micronucleus and Pig-a (phosphatidylinositol glycan, class A gene) mutation assays were conducted to evaluate the genotoxicity of 10 nm titanium dioxide anatase nanoparticles (TiO(2)-NPs) in mice. Groups of five 6-7-week-old male B6C3F1 mice were treated intravenously for three consecutive days with 0.5, 5.0, and 50 mg/kg TiO(2)-NPs for the two assays; mouse blood was sampled one day before the treatment and on Day 4, and Weeks 1, 2, 4, and 6 after the beginning of the treatment; Pig-a mutant frequencies were determined at Day -1 and Weeks 1, 2, 4 and 6, while percent micronucleated-reticulocyte (%MN-RET) frequencies were measured on Day 4 only. Additional animals were treated intravenously with three daily doses of 50 mg.kg TiO(2)-NPs for the measurement of titanium levels in bone marrow after 4, 24, and 48 h of the last treatment. The measurement indicated that the accumulation of the nanoparticles reached the peak in the tissue 4 h after the administration and the levels were maintained for a few days. No increase in either Pig-a mutant frequency of the frequency of %MN-RETs was detected, although the %RETs was reduced in the treated animals on Day 4 in a dose-dependent manner indicating cytotoxicity of TiO(2)-NPs in the bone marrow. These results suggest that although TiO(2)-NPs can reach the mouse bone marrow and are capable of inducing cytotoxicity, the nanoparticles are not genotoxic when assessed with in vivo micronucleus and Pig-a gene mutation tests.

Raman spectroscopy as a detection and analysis tool for in vitro specific targeting of pancreatic cancer cells by EGF-conjugated, single-walled carbon nanotubes.

Single-walled carbon nanotubes (SWCNTs) were covalently linked to epidermal growth factor (EGF) proteins through an esterification process that was found to be responsible for the docking of SWCNTs on the human pancreatic cancer cells (PANC-1) surface, thus providing a mechanism for the enhanced delivery and internalization of the nanotubes. Micro Raman spectroscopy and enzyme-linked immunosorbent assay were used to evaluate the delivery process and kinetics of the SWCNTs. In vitro studies indicated that the delivery kinetics of SWCNT-EGF conjugates, at a concentration of 85 µg ml(-1), to the PANC-1 cell surfaces was significant in the first 30 min of incubation, but reached a plateau with time in accordance with the establishment of equilibrium between the association and the dissociation of EGF with the cell receptors. SWCNT-EGF conjugates could act as strong thermal ablation agents and could induce higher percentages of cellular death compared with the nontargeted SWCNTs alone.

Mechanistic toxicity evaluation of uncoated and PEGylated single-walled carbon nanotubes in neuronal PC12 cells.

We investigated and compared the concentration-dependent cytotoxicity of single-walled carbon nanotubes (SWCNTs) and SWCNTs functionalized with polyethylene glycol (SWCNT-PEGs) in neuronal PC12 cells at the biochemical, cellular, and gene expressional levels. SWCNTs elicited cytotoxicity in a concentration-dependent manner, and SWCNT-PEGs exhibited less cytotoxic potency than uncoated SWCNTs. Reactive oxygen species (ROS) were generated in both a concentration- and surface coating-dependent manner after exposure to these nanomaterials, indicating different oxidative stress mechanisms. More specifically, gene expression analysis showed that the genes involved in oxidoreductases and antioxidant activity, nucleic acid or lipid metabolism, and mitochondria dysfunction were highly represented. Interestingly, alteration of the genes is also surface coating-dependent with a good correlation with the biochemical data. These findings suggest that surface functionalization of SWCNTs decreases ROS-mediated toxicological response in vitro.

Ethylenediamine functionalized-single-walled nanotube (f-SWNT)-assisted in vitro delivery of the oncogene suppressor p53 gene to breast cancer MCF-7 cells.

A gene delivery concept based on ethylenediamine-functionalized single-walled carbon nanotubes (f-SWCNTs) using the oncogene suppressor p53 gene as a model gene was successfully tested in vitro in MCF-7 breast cancer cells. The f-SWCNTs-p53 complexes were introduced into the cell medium at a concentration of 20 µg mL(-1) and cells were exposed for 24, 48, and 72 hours. Standard ethidium bromide and acridine orange assays were used to detect apoptotic cells and indicated that a significantly larger percentage of the cells (approx 40%) were dead after 72 hours of exposure to f-SWCNTs-p53 as compared to the control cells, which were exposed to only p53 or f-SWCNTs, respectively. To further support the uptake and expression of the genes within the cells, green fluorescent protein-tagged p53, attached to the f-SWCNTs was added to the medium and the complex was observed to be strongly expressed in the cells. Moreover, caspase 3 activity was found to be highly enhanced in cells incubated with the f-SWCNTs-p53 complex, indicating strongly induced apoptosis. This system could be the foundation for novel gene delivery platforms based on the unique structural and morphological properties of multi-functional nanomaterials.

Nanostructural materials increase mineralization in bone cells and affect gene expression through miRNA regulation.

We report that several nanomaterials induced enhanced mineralization (increased numbers and larger areas of mineral nests) in MC3T3-E1 bone cells, with the highest response being induced by silver nanoparticles (AgNPs). We demonstrate that AgNPs altered microRNA expression resulting in specific gene expression associated with bone formation. We suggest that the identified essential transcriptional factors and bone morphogenetic proteins play an important role in activation of the process of mineralization in bone cells exposed to AgNPs.