Mechanisms of Radical Formation on Chemically Modified Graphene Oxide under Near Infrared Irradiation.

In this work, the mechanisms of radical generation on different functionalized graphene oxide (GO) conjugates under near-infrared (NIR) light irradiation are investigated. The GO conjugates are designed to understand how chemical functionalization can influence the generation of radicals. Both pristine and functionalized GO are irradiated by a NIR laser, and the production of different reactive oxygen species (ROS) is investigated using fluorimetry and electron paramagnetic resonance to describe the type of radicals present on the surface of GO. The mechanism of ROS formation involves a charge transfer from the material to the oxygen present in the media, via the production of superoxide and singlet oxygen. Cytotoxicity and effects of ROS generation are then evaluated using breast cancer cells, evidencing a concentration dependent cell death associated to the heat and ROS. The study provides new hints to understand the photogeneration of radicals on the surface of GO upon near infrared irradiation, as well as, to assess the impact on these radicals in the context of a combined drug delivery system and phototherapeutic approach. These discoveries open the way for a better control of phototherapy-based treatments employing graphene-based materials.

Artificial antibody created by conformational reconstruction of the complementary-determining region on gold nanoparticles.

To impart biomedical functions to nanoparticles (NPs), the common approach is to conjugate functional groups onto NPs by dint of the functions of those groups per se. It is still beyond current reach to create protein-like specific interactions and functions on NPs by conformational engineering of nonfunctional groups on NPs. Here, we develop a conformational engineering method to create an NP-based artificial antibody, denoted "Goldbody," through conformational reconstruction of the complementary-determining regions (CDRs) of natural antibodies on gold NPs (AuNPs). The seemingly insurmountable task of controlling the conformation of the CDR loops, which are flexible and nonfunctional in the free form, was accomplished unexpectedly in a simple way. Upon anchoring both terminals of the free CDR loops on AuNPs, we managed to reconstruct the "active" conformation of the CDR loops by tuning the span between the two terminals and, as a result, the original specificity was successfully reconstructed on the AuNPs. Two Goldbodies have been created by this strategy to specifically bind with hen egg white lysozyme and epidermal growth factor receptor, with apparent affinities several orders of magnitude stronger than that of the original natural antibodies. Our work demonstrates that it is possible to create protein-like functions on NPs in a protein-like way, namely by tuning flexible surface groups to the correct conformation. Given the apparent merits, including good stability, of Goldbodies, we anticipate that a category of Goldbodies could be created to target different antigens and thus used as substitutes for natural antibodies in various applications.

How macrophages respond to two-dimensional materials: a critical overview focusing on toxicity.

With wider use of graphene-based materials and other two-dimensional (2 D) materials in various fields, including electronics, composites, biomedicine, etc., 2 D materials can trigger undesired effects at cellular, tissue and organ level. Macrophages can be found in many organs. They are one of the most important cells in the immune system and they are relevant in the study of nanomaterials as they phagocytose them. Nanomaterials have multi-faceted effects on phagocytic immune cells like macrophages, showing signs of inflammation in the form of pro-inflammatory cytokine or reactive oxidation species production, or upregulation of activation markers due to the presence of these foreign bodies. This review is catered to researchers interested in the potential impact and toxicity of 2 D materials, particularly in macrophages, focusing on few-layer graphene, graphene oxide, graphene quantum dots, as well as other promising 2 D materials containing molybdenum, manganese, boron, phosphorus and tungsten. We describe applications relevant to the growing area of 2 D materials research, and the possible risks of ions and molecules used in the production of these promising 2 D materials, or those produced by the degradation and dissolution of 2 D materials.

Toxicological Effects of Caco-2 Cells Following Short-Term and Long-Term Exposure to Ag Nanoparticles.

Extensive utilization increases the exposure of humans to Ag nanoparticles (NPs) via the oral pathway. To comprehensively address the action of Ag NPs to the gastrointestinal systems in real situations, i.e., the long-term low-dose exposure, we evaluated and compared the toxicity of three Ag NPs (20-30 nm with different surface coatings) to the human intestine cell Caco-2 after 1-day and 21-day exposures, using various biological assays. In both the short- and long-term exposures, the variety of surface coating predominated the toxicity of Ag NPs in a descending order of citrate-coated Ag NP (Ag-CIT), bare Ag NP (Ag-B), and poly (N-vinyl-2-pyrrolidone)-coated Ag NP (Ag-PVP). The short-term exposure induced cell growth inhibition and death. The cell viability loss appeared after cells were exposed to 0.7 µg/mL Ag-CIT, 0.9 µg/mL Ag-B or >1.0 µg/mL Ag-PVP for 24 h. The short-term and higher-dose exposure also induced reactive oxygen species (ROS) generation, mitochondrial damage, cell membrane leakage, apoptosis, and inflammation (IL-8 level). The long-term exposure only inhibited the cell proliferation. After 21-day exposure to 0.4 µg/mL Ag-CIT, the cell viability dropped to less than 50%, while cells exposed to 0.5 µg/mL Ag-PVP remained normal as the control. Generally, 0.3 µg/mL is the non-toxic dose for the long-term exposure of Caco-2 cells to Ag NPs in this study. However, cells presented inflammation after exposure to Ag NPs with the non-toxic dose in the long-term exposure.

Biological effect of food additive titanium dioxide nanoparticles on intestine: an in vitro study.

Titanium dioxide nanoparticles (TiO2 NPs) are widely found in food-related consumer products. Understanding the effect of TiO2 NPs on the intestinal barrier and absorption is essential and vital for the safety assessment of orally administrated TiO2 NPs. In this study, the cytotoxicity and translocation of two native TiO2 NPs, and these two TiO2 NPs pretreated with the digestion simulation fluid or bovine serum albumin were investigated in undifferentiated Caco-2 cells, differentiated Caco-2 cells and Caco-2 monolayer. TiO2 NPs with a concentration less than 200 µg ml(-1) did not induce any toxicity in differentiated cells and Caco-2 monolayer after 24 h exposure. However, TiO2 NPs pretreated with digestion simulation fluids at 200 µg ml(-1) inhibited the growth of undifferentiated Caco-2 cells. Undifferentiated Caco-2 cells swallowed native TiO2 NPs easily, but not pretreated NPs, implying the protein coating on NPs impeded the cellular uptake. Compared with undifferentiated cells, differentiated ones possessed much lower uptake ability of these TiO2 NPs. Similarly, the traverse of TiO2 NPs through the Caco-2 monolayer was also negligible. Therefore, we infer the possibility of TiO2 NPs traversing through the intestine of animal or human after oral intake is quite low. This study provides valuable information for the risk assessment of TiO2 NPs in food.

Evaluation of the toxicity of food additive silica nanoparticles on gastrointestinal cells.

Silica nanoparticles (NPs) have been widely used in food products as an additive; however, their toxicity and safety to the human body and the environment still remain unclear. As a food additive, silica NPs firstly enter the human gastrointestinal tract along with food, thus their gastrointestinal toxicity deserves thorough study. Herein, we evaluated the toxicity of food additive silica NPs to cells originating from the gastrointestinal tract. Four silica NP samples were introduced to human gastric epithelial cell GES-1 and colorectal adenocarcinoma cell Caco-2 to investigate the effect of silica sample, exposure dose and exposure period on the morphology, viability and membrane integrity of cells. The cell uptake, cellular reactive oxygen species (ROS) level, cell cycle and apoptosis were determined to reveal the toxicity mechanism. The results indicate that all four silica NPs are safe for both GES-1 and Caco-2 cells after 24-h exposure at a concentration lower than 100 µg ml(-1) . At a higher concentration and longer exposure period, silica NPs do not induce the apoptosis/necrosis of cells, but arrest cell cycle and inhibit the cell growth. Notably, silica NPs do not pass through the Caco-2 cell monolayer after 4-h contact, indicating the low potential of silica NPs to cross the gastrointestinal tract in vivo. Our findings indicate that silica NPs could be used as a safe food additive, but more investigations, such as long-term in vivo exposure, are necessary in future studies.

Hazard assessment of hexagonal boron nitride and hexagonal boron nitride reinforced thermoplastic polyurethane composites using human skin and lung cells.

Hexagonal boron nitride (hBN) is an emerging two-dimensional material attracting considerable attention in the industrial sector given its innovative physicochemical properties. Potential risks are associated mainly with occupational exposure where inhalation and skin contact are the most relevant exposure routes for workers. Here we aimed at characterizing the effects induced by composites of thermoplastic polyurethane (TPU) and hBN, using immortalized HaCaT skin keratinocytes and BEAS-2B bronchial epithelial cells. The composite was abraded using a Taber® rotary abraser and abraded TPU and TPU-hBN were also subjected to photo-Fenton-mediated degradation mimicking potential weathering across the product life cycle. Cells were exposed to the materials for 24 h (acute exposure) or twice per week for 4 weeks (chronic exposure) and evaluated with respect to material internalization, cytotoxicity, and proinflammatory cytokine secretion. Additionally, comprehensive mass spectrometry-based proteomics and metabolomics (secretomics) analyses were performed. Overall, despite evidence of cellular uptake of the material, no significant cellular and/or protein expression profiles alterations were observed after acute or chronic exposure of HaCaT or BEAS-2B cells, identifying only few pro-inflammatory proteins. Similar results were obtained for the degraded materials. These results support the determination of hazard profiles associated with cutaneous and pulmonary hBN-reinforced polymer composites exposure.

Combined Photothermal and Photodynamic Therapy for Cancer Treatment Using a Multifunctional Graphene Oxide.

Graphene oxide (GO) is one of the most studied nanomaterials in many fields, including the biomedical field. Most of the nanomaterials developed for drug delivery and phototherapies are based on noncovalent approaches that lead to an unspecific release of physisorbed molecules in complex biological environments. Therefore, preparing covalently functionalized GO using straightforward and versatile methods is highly valuable. Phototherapies, including photothermal therapy (PTT) and photodynamic therapy (PDT), have shown great potential as effective therapeutic approaches against cancer. To overcome the limits of a single method, the combination of PTT and PDT can lead to a combined effect with a higher therapeutic efficiency. In this work, we prepare a folic acid (FA) and chlorin e6 (Ce6) double-functionalized GO for combined targeted PTT/PDT. This conjugate can penetrate rapidly into cancer cells and macrophages. A combined effect of PTT and PDT is observed, leading to a higher killing efficiency toward different types of cells involved in cancer and other diseases. Our work provides a simple protocol to prepare multifunctional platforms for the treatment of various diseases.

Hazard assessment of abraded thermoplastic composites reinforced with reduced graphene oxide.

Graphene-related materials (GRMs) are subject to intensive investigations and considerable progress has been made in recent years in terms of safety assessment. However, limited information is available concerning the hazard potential of GRM-containing products such as graphene-reinforced composites. In the present study, we conducted a comprehensive investigation of the potential biological effects of particles released through an abrasion process from reduced graphene oxide (rGO)-reinforced composites of polyamide 6 (PA6), a widely used engineered thermoplastic polymer, in comparison to as-produced rGO. First, a panel of well-established in vitro models, representative of the immune system and possible target organs such as the lungs, the gut, and the skin, was applied. Limited responses to PA6-rGO exposure were found in the different in vitro models. Only as-produced rGO induced substantial adverse effects, in particular in macrophages. Since inhalation of airborne materials is a key occupational concern, we then sought to test whether the in vitro responses noted for these materials would translate into adverse effects in vivo. To this end, the response at 1, 7 and 28 days after a single pulmonary exposure was evaluated in mice. In agreement with the in vitro data, PA6-rGO induced a modest and transient pulmonary inflammation, resolved by day 28. In contrast, rGO induced a longer-lasting, albeit moderate inflammation that did not lead to tissue remodeling within 28 days. Taken together, the present study suggests a negligible impact on human health under acute exposure conditions of GRM fillers such as rGO when released from composites at doses expected at the workplace.

Effects of carbon dots surface functionalities on cellular behaviors - Mechanistic exploration for opportunities in manipulating uptake and translocation.

Carbon dots (CDots) for their excellent optical and other properties have been widely pursued for potential biomedical applications, in which a more comprehensive understanding on the cellular behaviors and mechanisms of CDots is required. For such a purpose, two kinds of CDots with surface passivation by 3-ethoxypropylamine (EPA-CDots) and oligomeric polyethylenimine (PEI-CDots) were selected for evaluations on their uptakes by human cervical carcinoma HeLa cells at three cell cycle phases (G0/G1, S and G2/M), and on their different internalization pathways and translocations in cells. The results show that HeLa cells could internalize both CDots by different pathways, with an overall slightly higher internalization efficiency for PEI-CDots. The presence of serum in culture media could have major effects, significantly enhancing the cellular uptake of EPA-CDots, yet markedly inhibiting that of PEI-CDots. The HeLa cells at different cell cycle phases have different behaviors in taking up the CDots, which are also affected by the different dot surface moieties and serum in culture media. Mechanistic implications of the results and the opportunities associated with an improved understanding on the cellular behaviors of CDots for potentially the manipulation and control of their cellular uptakes and translocations are discussed.

Intestinal injury alters tissue distribution and toxicity of ZnO nanoparticles in mice.

The fast growing applications of ZnO nanoparticles (NPs) in food sector and other fields enhance the exposure possibility of human beings to ZnO NPs including via oral administration route. Although the oral toxicity of ZnO NPs has been studied, most of the research was performed on the normal animal models. Therefore, the understanding of the biological consequence of ZnO NPs on the population with diseases, especially gastrointestinal disease, is extremely limited. In this study, a mice model of inflammatory bowel disease (IBD) induced by indomethacin has been developed to comprehensively investigated the bioeffects of ZnO NPs on the specific population. The effect of the intestinal inflammation/injury on the distribution and toxicity of orally administrated ZnO NPs (nZnO, 20 nm × 100 nm and mZnO, ∼200 nm) in mice were analyzed. The results showed that there was a difference in the distribution of Zn and the essential trace elements (Fe and Cu) between the IBD mice and the normal mice. We also observed an obvious size effect. Higher hepatic Zn was detected in the IBD mice post-exposure to ZnO NPs, especially bigger ZnO NPs. In addition, the histopathological examination of main organs and biological parameters analysis showed that ZnO NPs caused slight toxicity to the liver and kidneys in the IBD mice. Our findings highlight the importance of the health status of animals on the bioeffects of nanomaterials.

Biological effects of agglomerated multi-walled carbon nanotubes.

The physicochemical properties of nanomaterials play crucial roles in determining their biological effects. Agglomeration of nanomaterials in various systems is a common phenomenon, however, how agglomeration affects the biological consequence of nanomaterials has not been well investigated because of its complexity. Herein, we prepared variable sized agglomerates of oxidized multi-walled carbon nanotubes (O-MWCNTs) by using Ca(2+) and studied their cellular uptake and cytotoxicity in HeLa cells. We found the altered property of O-MWCNTs agglomerates could be controlled and adjusted by the amount of Ca(2+). Agglomeration remarkably facilitated the cellular uptake of O-MWCNTs at the initial contact stage, due to the easy contact of agglomerates with cells. But agglomeration did not induce evident cytotoxicity when the concentration of O-MWCNTs was less than 150µg/mL. That was assayed by cell proliferation, membrane integrity, apoptosis and ROS generation. This study suggests us that the biological behaviors of nanomaterials could be altered by their states of agglomeration.

Lanthanide (Gd(3+) and Yb(3+)) functionalized gold nanoparticles for in vivo imaging and therapy.

Nanoparticles are regularly used as contrast agents in bioimaging. Unlike other agents such as composite materials, nanoparticles can also be used for treating as well as imaging disease. Here we synthesized lanthanide functionalized gold nanoparticles that can be used for both imaging and therapy in vivo. That is a multifunctional nanoplatform was developed based on a simple and versatile method, by incorporating 10-nm gold nanoparticles and lanthanide ions (Gd(3+) and Yb(3+)), denoted as LnAu nanoparticles hereby. The LnAu nanoparticles were then surface-modified using a PEGylated amphiphilic polymer (C18MH-mPEG), and the resulting PEG modified LnAu nanoparticles (PEG-LnAu) display good monodispersion in water and good solubility in biological media. Due to the low toxicity in vitro and in vivo (as determined by a cell viability assay and histological and serum biochemistry analysis), the PEG-LnAu nanoparticles can be successfully applied to in vivo magnetic resonance imaging (MRI), in vivo computed tomography (CT) imaging and photothermal therapy (PTT) for tumor-bearing mice. Therefore, the present work developed an easy yet powerful strategy to combine lanthanide ions and gold nanoparticles to a unified nanoplatform for integrating bioimaging and therapy.

New nanoplatforms based on UCNPs linking with polyhedral oligomeric silsesquioxane (POSS) for multimodal bioimaging.

A new and facile method was used to transfer upconversion luminescent nanoparticles from hydrophobic to hydrophilic using polyhedral oligomeric silsesquioxane (POSS) linking on the surface of upconversion nanoparticles. In comparison with the unmodified upconversion nanoparticles, the POSS modified upconversion nanoplatforms [POSS-UCNPs(Er), POSS-UCNPs(Tm)] displayed good monodispersion in water and exhibited good water-solubility, while their particle size did not change substantially. Due to the low cytotoxicity and good biocompatibility as determined by methyl thiazolyl tetrazolium (MTT) assay and histology and hematology analysis, the POSS modified upconversion nanoplatforms were successfully applied to upconversion luminescence imaging of living cells in vitro and nude mouse in vivo (upon excitation at 980 nm). In addition, the doped Gd(3+) ion endows the POSS-UCNPs with effective T1 signal enhancement and the POSS-UCNPs were successfully applied to in vivo magnetic resonance imaging (MRI) for a Kunming mouse, which makes them potential MRI positive-contrast agents. More importantly, the corner organic groups of POSS can be easily modified, resulting in kinds of POSS-UCNPs with many potential applications. Therefore, the method and results may provide more exciting opportunities for multimodal bioimaging and multifunctional applications.

Chitosan-coated red fluorescent protein nanoparticle as a potential dual-functional siRNA carrier.

AIMS: Developing safe and efficient nano vectors is critical for the success of siRNA therapy. MATERIALS & METHODS: By encapsulating red fluorescent protein (RFP) with chitosan (CS), a dual-functional siRNA delivery nano vector, RFP@CS, has been synthesized. RESULTS: RFP@CS has an optimum size of 7-23 nm for siRNA delivery; and the fluorescence of RFP, protected by CS coating, provides an excellent probe to track the delivery of siRNA. RFP@CS delivers siRNA efficiently into cells and the targeted gene could be completely silenced even after 48 h. No cytotoxicity or acute toxicity in mice was observed. CONCLUSION: The high transfection efficacy and safety demonstrate RFP@CS is a promising nano vector for the gene therapy.

Progress in the characterization and safety evaluation of engineered inorganic nanomaterials in food.

Nanotechnology has stepped into the food industry, from the farm to the table at home, in order to improve the taste and nutritional value, extend the shelf-life and monitor the food quality. In fact, as consumers we have already been in contact, via oral exposure, with a number of food products containing engineered nanomaterials (ENMs) more often than most people think. However, the fate of ENMs after entering the GI tract of the human body is not yet clearly understood. Hence, the related safety issue is raised, and attracts much attention and wide debate from the public, even including protest demonstrations against nanotechnology in food. In this review, we summarize the up-to-date information about the characterization and safety evaluation of common inorganic ENMs (with a focus on silver, titanium dioxide, silica and zinc oxide nanoparticles) in food. Based on the literature, a whole scenario of the safety issue of these ENMs in food and an outlook on the future studies are given.