Biotransformation modulates the penetration of metallic nanomaterials across an artificial blood-brain barrier model.

Understanding the potential of nanomaterials (NMs) to cross the blood-brain barrier (BBB), as a function of their physicochemical properties and subsequent behavior, fate, and adverse effect beyond that point, is vital for evaluating the neurological effects arising from their unintentional entry into the brain, which is yet to be fully explored. This is not only due to the complex nature of the brain but also the existing analytical limitations for characterization and quantification of NMs in the complex brain environment. By using a fit-for-purpose analytical workflow and an in vitro BBB model, we show that the physiochemical properties of metallic NMs influence their biotransformation in biological matrices, which in turn modulates the transport form, efficiency, amounts, and pathways of NMs through the BBB and, consequently, their neurotoxicity. The data presented here will support in silico modeling and prediction of the neurotoxicity of NMs and facilitate the tailored design of safe NMs.

Controlled Chemical Derivatisation of Carbon Nanotubes with Imaging, Targeting, and Therapeutic Capabilities.

In drug delivery, carbon nanotubes (CNTs) hold a great potential as carriers because of their ability to easily cross biological barriers and be internalised into cells. Their high aspect ratio allows multi-functionalisation and their development as a multimodal platform for targeted therapy. In this article, we report the controlled covalent derivatisation of triple-functionalised CNTs with the anticancer drug gemcitabine, folic acid as a targeting ligand and fluorescein as a probe. The anticancer activity of gemcitabine was maintained after covalent grafting onto the CNTs. The functionalised nanotubes were internalised into both folate-positive and negative cells, suggesting the passive diffusion of CNTs. Overall, our approach is versatile and offers a precise chemical control of the sidewall functionalisation of CNTs and the possibility to manoeuvre the types of functionalities required on the nanotubes for a multimodal therapeutic strategy.

Safety considerations for graphene: lessons learnt from carbon nanotubes.

Many consider carbon nanomaterials the poster children of nanotechnology, attracting immense scientific interest from many disciplines and offering tremendous potential in a diverse range of applications due to their extraordinary properties. Graphene is the youngest in the family of carbon nanomaterials. Its isolation, description, and mass fabrication has followed that of fullerenes and carbon nanotubes. Graphene's development and its adoption by many industries will increase unintended or intentional human exposure, creating the need to determine its safety profile. In this Account, we compare the lessons learned from the development of carbon nanotubes with what is known about graphene, based on our own investigations and those of others. Despite both being carbon-based, nanotubes and graphene are two very distinct nanomaterials. We consider the key physicochemical characteristics (structure, surface, colloidal properties) for graphene and carbon nanotubes at three different physiological levels: cellular, tissue, and whole body. We summarize the evidence for health effects of both materials at all three levels. Overall, graphene and its derivatives are characterized by a lower aspect ratio, larger surface area, and better dispersibility in most solvents compared to carbon nanotubes. Dimensions, surface chemistry, and impurities are equally important for graphene and carbon nanotubes in determining both mechanistic (aggregation, cellular processes, biodistribution, and degradation kinetics) and toxicological outcomes. Colloidal dispersions of individual graphene sheets (or graphene oxide and other derivatives) can easily be engineered without metallic impurities, with high stability and less aggregation. Very importantly, graphene nanostructures are not fiber-shaped. These features theoretically offer significant advantages in terms of safety over inhomogeneous dispersions of fiber-shaped carbon nanotubes. However, studies that directly compare graphene with carbon nanotubes are rare, making comparative considerations of their overall safety and risk assessment challenging. In this Account, we attempt to offer a set of rules for the development of graphene and its derivatives to enhance their overall safety and minimize the risks for adverse reactions in humans from exposure. These rules are: (1) to use small, individual graphene sheets that macrophages in the body can efficiently internalize and remove from the site of deposition; (2) to use hydrophilic, stable, colloidal dispersions of graphene sheets to minimize aggregation in vivo; and (3) to use excretable graphene material or chemically-modified graphene that can be degraded effectively. Such rules can only act as guidelines at this early stage in the development of graphene-based technologies, yet they offer a set of design principles for the fabrication and safe use of graphene material that will come in contact with the human body. In a broader context, the safety risks associated with graphene materials will be entirely dependent on the specific types of graphene materials and how they are investigated or applied. Therefore, generalizations about the toxicity of "graphene" as a whole will be inaccurate, possibly misleading, and should be avoided.

Modulation of Heat Shock Protein Expression in Alveolar Adenocarcinoma Cells through Gold Nanoparticles and Cisplatin Treatment.

Heat-shock proteins (HSPs) are stress-responsive molecules belonging to the family of evolutionary molecular chaperones known to be crucial in many cancer types, including human alveolar adenocarcinoma cells (A549). These proteins are highly overexpressed in cancers to support their ability to accommodate imbalances in cell signalling, DNA alterations, proteins, and energy metabolism associated with oncogenesis. The current study evaluated the effects of gold nanoparticles (AuNPs) combined with cisplatin (CDDP) on molecular chaperone HSPs in A549 cells. It was found that AuNPs:CDDP decreased the percentage of cell viability (38.5%) measured using the modified lactated dehydrogenase (mLDH) and 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. AuNPs:CDDP exposure caused a significant (p < 0.05) increase in reactive oxygen species (ROS) generation by 1.81-fold, apoptosis induction, and a decrease in the mitochondrial membrane potential (MMP) compared to AuNPs or CDDP alone. Similarly, exposure to the AuNPs:CDDP combination had pronounced cytotoxic effects on the expression of HSPs and PI3K/AKT/mTOR, as well as apoptosis-related proteins. The results demonstrate that the combination of AuNPs with CDDP might enhance the anticancer efficacy of CDDP.

Impact of Environmental Stresses on the Antibacterial Activity of Graphene Oxide (GO) Nanoparticles against P. putida Biofilms.

As the production of graphene-based nanomaterials such as GO is increasing, it is expected that a large amount of GO waste will be generated. The environment (i.e., soil and aquatic systems) will be amongst the final repositories of these wastes which means important natural microbial communities in such environments will be at risk of GO exposure. However, little is known about how these communities respond to environmental stresses in synergy with the presence of GO. In this study, the effect of three different stress conditions: temperature (5, 25 and 40 °C); pH (5 to 9) and osmotic stress (51, 219 and 320 mM NaCl) in addition to GO treatment was investigated on the viability and physiology of biofilms and planktonic cells of soil bacterium P. putida. It was found that planktonic cells were more resistant to GO alone compared to biofilms. However, the cells were sensitive to GO when exposed to pH or osmotic stresses. Temperature was not found to influence the survival of biofilm with or without exposure to GO. However, low pH caused a reduction in colony-forming units (CFU) at pHs 5 and 6 for the pre-treated samples, while biofilms at pH 7-9 did not show any decrease. Interestingly, the post-treatment of planktonic cells or biofilms with GO showed a significant reduction in CFU at all pH ranges. The effect of higher osmotic stress in combination with GO resulted in a significant reduction in biofilms. These results show that the effect of stresses naturally occurring in the environment can be affected and changed when in combination with GO and can potentially affect the balance of natural biofilms.

Gold Nanoparticles Induced Size Dependent Cytotoxicity on Human Alveolar Adenocarcinoma Cells by Inhibiting the Ubiquitin Proteasome System.

Gold nanoparticles (AuNPs) are widely used in biomedicine due to their remarkable therapeutic applications. However, little is known about their cytotoxic effects on the ubiquitin proteasome system (UPS). Herein, the cytotoxicity of different sizes of AuNPs (5, 10, and 80 nm) on the UPS was investigated with a particular focus on deubiquitinating enzymes (DUBs) such as ubiquitin-specific proteases (USP) and ubiquitin carboxyl-terminal hydrolases (UCHL-1) in human alveolar epithelial adenocarcinoma (A549). It was found that all sizes of AuNPs reduced the percentage of viable A549 cells and increased lactate dehydrogenase (LDH) release, measured using the MTT and LDH assays, respectively. Furthermore, the 5 nm AuNPs were found to exhibit greater cytotoxicity than the 10 and 80 nm AuNPs. In addition, apoptosis and necrosis were activated through reactive oxygen species (ROS) generation due to AuNPs exposure. The internalisation of AuNPs in A549 cells increased with increasing particle size (80 > 10 > 5 nm). Interestingly, the expression of USP7, USP8, USP10, and UCHL-1 was significantly (p < 0.001) downregulated upon treatment with 5-30 µg/mL of all the AuNPs sizes compared to control cells. Moreover, the inhibition of these proteins triggered mitochondrial-related apoptosis through the upregulation of poly (ADP-ribose) polymerase (PARP), caspase-3, and caspase-9. Collectively, these results indicate that AuNPs suppress the proliferation of A549 cells and can potentially be used as novel inhibitors of the proteasome.

Functionalized Gold Nanoparticles Suppress the Proliferation of Human Lung Alveolar Adenocarcinoma Cells by Deubiquitinating Enzymes Inhibition.

Functionalized gold nanoparticles (AuNPs) are widely used in therapeutic applications, but little is known regarding the impact of their surface functionalization in the process of toxicity against cancer cells. This study investigates the anticancer effects of 5 nm spherical AuNPs functionalized with tannate, citrate, and PVP on deubiquitinating enzymes (DUBs) in human lung alveolar adenocarcinoma (A549) cells. Our findings show that functionalized AuNPs reduce the cell viability in a concentration- and time-dependent manner as measured by modified lactate dehydrogenase (mLDH) and 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. An increased generation of intracellular reactive oxygen species (ROS) and depletion of glutathione (GSH/GSSG) ratio was observed with the highest AuNP concentration of 10 µg/mL. The expression of DUBs such as ubiquitin specific proteases (USP7, USP8, and USP10) was slightly inhibited when treated with concentrations above 2.5 µg/mL. Moreover, functionalized AuNPs showed an inhibitory effect on protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) and wingless-related integration site (Wnt) signaling proteins, and this could further trigger mitochondrial related-apoptosis by the upregulation of caspase-3, caspase-9, and PARP in A549 cells. Furthermore, our study shows a mechanistic understanding of how functionalized AuNPs inhibit the DUBs, consequently suppressing cell proliferation, and can be modulated as an approach toward anticancer therapy. The study also warrants the need for future work to investigate the effect of functionalized AuNPs on DUB on other cancer cell lines both in vitro and in vivo.

Nanotoxicity of Graphene Oxide - Gold Nanohybrid to Daphnia magna.

The expanding use of hybrid nanomaterials in many applications necessitates evaluation of their environmental risks. This study investigates the acute toxicity and bioaccumulation of graphene oxide - gold (GO-Au) nanohybrid in neonates (<24 hrs old) of Daphnia magna after exposure to a wide range of concentrations (1-100 mg/L). No significant mortality or immobilisation was observed after the exposure period. Microscopic observation showed an uptake of the nanohybrid and internal damage in the gut of the exposed organisms. Bioaccumulation of the GO-Au nanohybrid also occurred in a concentration-dependant manner. Continuous evaluation of the environmental risks from exposure to this nanohybrid and other advanced materials is imperative to avert disruption to the ecosystem.

Length-dependent retention of carbon nanotubes in the pleural space of mice initiates sustained inflammation and progressive fibrosis on the parietal pleura.

The fibrous shape of carbon nanotubes (CNTs) raises concern that they may pose an asbestos-like inhalation hazard, leading to the development of diseases, especially mesothelioma. Direct instillation of long and short CNTs into the pleural cavity, the site of mesothelioma development, produced asbestos-like length-dependent responses. The response to long CNTs and long asbestos was characterized by acute inflammation, leading to progressive fibrosis on the parietal pleura, where stomata of strictly defined size limit the egress of long, but not short, fibers. This was confirmed by demonstrating clearance of short, but not long, CNT and nickel nanowires and by visualizing the migration of short CNTs from the pleural space by single-photon emission computed tomographic imaging. Our data confirm the hypothesis that, although a proportion of all deposited particles passes through the pleura, the pathogenicity of long CNTs and other fibers arises as a result of length-dependent retention at the stomata on the parietal pleura.

Degree of chemical functionalization of carbon nanotubes determines tissue distribution and excretion profile.

Getting rid of the tubes: An assessment of the retention of functionalized multi-walled carbon nanotubes (MWNTs) in the organs of mice was carried out using single photon emission computed tomography and quantitative scintigraphy (see scheme). Increasing the degree of functionalization on MWNTs enhanced renal clearance, while lower functionalization promoted reticuloendethelial system accumulation.

Synthesis and Characterisation of a Graphene Oxide-Gold Nanohybrid for Use as Test Material.

This paper reports the synthesis and characterization of a graphene oxide-gold nanohybrid (GO-Au) and evaluates its suitability as a test material, e.g., in nano(eco)toxicological studies. In this study, we synthesised graphene oxide (GO) and used it as a substrate for the growth of nano-Au decorations, via the chemical reduction of gold (III) using sodium citrate. The GO-Au nanohybrid synthesis was successful, producing AuNPs (~17.09 ± 4.6 nm) that were homogenously distributed on the GO sheets. They exhibited reproducible characteristics when characterised using UV-Vis, TGA, TEM, FTIR, AFM, XPS and Raman spectroscopy. The nanohybrid also showed good stability in different environmental media and its physicochemical characteristics did not deteriorate over a period of months. The amount of Au in each of the GO-Au nanohybrid samples was highly comparable, suggesting a potential for use as chemical label. The outcome of this research represents a crucial step forward in the development of a standard protocol for the synthesis of GO-Au nanohybrids. It also paves the way towards a better understanding of the nanotoxicity of GO-Au nanohybrid in biological and environmental systems.

Cellular uptake and cytotoxic impact of chemically functionalized and polymer-coated carbon nanotubes.

The impact of nanomaterials such as carbon nanotubes on biological matter is a topic of increasing interest and concern and requires a multifaceted approach to be resolved. A modified cytotoxic (lactate dehydrogenase (LDH)) assay is developed in an attempt to offer a valid and reliable methodology for screening carbon nanotube toxicity in vitro. Two of the most widely used types of surface-modified multiwalled carbon nanotubes (MWNTs) are tested: ammonium-functionalized MWNTs (MWNT-NH3+ ) and Pluronic F127 coated MWNTs (MWNT:F127). Chemically functionalized MWNTs show significantly greater cellular uptake into lung epithelial A549 cells compared to the non-covalently Pluronic F127-coated MWNTs. In spite of this, MWNT:F127 exhibit enhanced cytotoxicity according to the modified LDH assay. The validity of the modified LDH assay is further validated by direct comparison with other less reliable or accurate cytotoxicity assays. These findings indicate the reliability of the modified LDH assay as a screening tool to assess carbon nanotube cytotoxicity and illustrate that high levels of carbon nanotube cellular internalization do not necessarily lead to adverse responses.

Filled and glycosylated carbon nanotubes for in vivo radioemitter localization and imaging.

Functionalization of nanomaterials for precise biomedical function is an emerging trend in nanotechnology. Carbon nanotubes are attractive as multifunctional carrier systems because payload can be encapsulated in internal space whilst outer surfaces can be chemically modified. Yet, despite potential as drug delivery systems and radiotracers, such filled-and-functionalized carbon nanotubes have not been previously investigated in vivo. Here we report covalent functionalization of radionuclide-filled single-walled carbon nanotubes and their use as radioprobes. Metal halides, including Na(125)I, were sealed inside single-walled carbon nanotubes to create high-density radioemitting crystals and then surfaces of these filled-sealed nanotubes were covalently modified with biantennary carbohydrates, improving dispersibility and biocompatibility. Intravenous administration of Na(125)I-filled glyco-single-walled carbon nanotubes in mice was tracked in vivo using single-photon emission computed tomography. Specific tissue accumulation (here lung) coupled with high in vivo stability prevented leakage of radionuclide to high-affinity organs (thyroid/stomach) or excretion, and resulted in ultrasensitive imaging and delivery of unprecedented radiodose density. Nanoencapsulation of iodide within single-walled carbon nanotubes enabled its biodistribution to be completely redirected from tissue with innate affinity (thyroid) to lung. Surface functionalization of (125)I-filled single-walled carbon nanotubes offers versatility towards modulation of biodistribution of these radioemitting crystals in a manner determined by the capsule that delivers them. We envisage that organ-specific therapeutics and diagnostics can be developed on the basis of the nanocapsule model described here.

Enhanced cellular internalization and gene silencing with a series of cationic dendron-multiwalled carbon nanotube:siRNA complexes.

One of the major obstacles to the clinical development of gene silencing by small interfering RNA (siRNA) is its effective cytoplasmic delivery. Carbon nanotubes have been proposed as novel nanomaterials that can offer significant advantages for the intracellular delivery of nucleic acids, such as siRNA. We recently demonstrated in a proof-of-principle study that amino-functionalized multiwalled carbon nanotubes (f-MWNT) can effectively deliver in vivo an siRNA sequence, triggering cell apoptosis that results in human lung xenograft eradication and prolonged survival. In the present study, we demonstrate how a newly synthesized series of polycationic dendron-MWNT constructs with a precisely tailored number of amino functions (dendron generations) can complex and effectively deliver double-stranded siRNA to achieve gene silencing in vitro. A systematic comparison between the f-MWNT series in terms of cellular uptake, cytotoxicity, and siRNA complexation is offered. Significant improvement in siRNA delivery with the dendron-MWNT conjugates is shown, and gene silencing was obtained in 2 human cell lines using 2 different siRNA sequences. The study reveals that through f-MWNT structure-biological function analysis novel nanotube-based siRNA transfer vectors can be designed with minimal cytotoxicity and effective delivery and gene-silencing capabilities.

Antitumor activity and prolonged survival by carbon-nanotube-mediated therapeutic siRNA silencing in a human lung xenograft model.

Carbon nanotubes are novel nanomaterials that are thought to offer potential benefits to a variety of biomedical and clinical applications. In this study, the treatment of a human lung carcinoma model in vivo using siRNA sequences leading to cytotoxicity and cell death is carried out using either cationic liposomes (DOTAP:cholesterol) or amino-functionalized multi-walled carbon nanotubes (MWNT - NH(+)(3)). Validation for the most cytotoxic siRNA sequence using a panel of human carcinoma and murine cells reveals that the proprietary siTOX sequence is human specific and can lead to significant cytotoxic activities delivered both by liposome or MWNT - NH(+)(3) in vitro. A comparative study using both types of vector indicates that only MWNT - NH(+)(3):siRNA complexes administered intratumorally can elicit delayed tumor growth and increased survival of xenograft-bearing animals. siTOX delivery via the cationic MWNT - NH(+)(3) is biologically active in vivo by triggering an apoptotic cascade, leading to extensive necrosis of the human tumor mass. This suggests that carbon-nanotube-mediated delivery of siRNA by intratumoral administration leads to successful and statistically significant suppression of tumor volume, followed by a concomitant prolongation of survival of human lung tumor-bearing animals. The direct comparison between carbon nanotubes and liposomes demonstrates the potential advantages offered by carbon nanotubes for the intracellular delivery of therapeutic agents in vivo. The present work may act as the impetus for further studies to explore the therapeutic capacity of chemically functionalized carbon nanotubes to deliver siRNA directly into the cytoplasm of target cells and achieve effective therapeutic silencing in various disease indications where local delivery is feasible or desirable.

Antibacterial effect of graphene oxide (GO) nano-particles against Pseudomonas putida biofilm of variable age.

Graphene oxide (GO) has been reported to possess antibacterial activity; therefore, its accumulation in the environment could affect microbial communities such as biofilms. The susceptibility of biofilms to antimicrobials is known to depend on the stage of biofilm maturity. The aim of this study was to investigate the effect of GO nano-particles on Pseudomonas putida KT2440 biofilm of variable age. FT-IR, UV-vis, and Raman spectroscopy confirmed the oxidation of graphene while XPS confirmed the high purity of the synthesised GO over 6 months. Biofilms varying in maturity (24, 48, and 72 h) were formed using a CDC reactor and were treated with GO (85 µg/mL or 8.5 µg/mL). The viability of P. putida was monitored by culture on media and the bacterial membrane integrity was assessed using flow cytometry. P. putida cells were observed using confocal microscopy and SEM. The results showed that GO significantly reduced the viability of 48-h biofilm and detached biofilm cells associated with membrane damage while the viability was not affected in 24- and 72-h biofilms and detached biofilm cells. The results showed that susceptibility of P. putida biofilm to GO varied according to age which may be due to changes in the physiological state of cells during maturation. Graphical abstract.

Multiwalled carbon nanotube-doxorubicin supramolecular complexes for cancer therapeutics.

Multiwalled carbon nanotube aqueous dispersions using block copolymers are able to form supramolecular complexes with the aromatic chromophore and anticancer agent doxorubicin via pi-pi stacking and enhance its cytotoxic activity.

Facile production of nanocomposites of carbon nanotubes and polycaprolactone with high aspect ratios with potential applications in drug delivery.

The geometries and surface properties of nanocarriers greatly influence the interaction between nanomaterials and living cells. In this work we combine multiwalled carbon nanotubes (CNTs) with poly-ε-caprolactone (PCL) to produce non-spherical nanocomposites with high aspect ratios by using a facile emulsion solvent evaporation method. Particles were characterised by dynamic light scattering (DLS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and asymmetric flow field flow fractionation (AF4). Different sizes and morphologies of nanoparticles were produced depending on the concentration of the sodium dodecyl sulphate (SDS), CNTs and PCL. Rod-like PCL-CNT nanostructures with low polydispersity were obtained with 1.5 mg mL-1 of SDS, 0.9 mg mL-1 of CNTs and 10 mg mL-1 PCL. AFM analysis revealed that the PCL and PCL-CNT nanocomposite had comparatively similar moduli of 770 and 560 MPa respectively, indicating that all the CNTs have been coated with at least 2 nm of PCL. Thermogravimetric analysis of the PCL-CNT nanocomposite indicated that they contained 9.6% CNTs by mass. The asymmetric flow field flow fractionation of the samples revealed that the PCL-CNT had larger hydrodynamic diameters than PCL alone. Finally, the drug loading properties of the nanocomposites were assessed using docetaxel as the active substance. The nanocomposites showed comparable entrapment efficiencies of docetaxel (89%) to the CNTs alone (95%) and the PCL nanoparticles alone (81%). This is a facile method for obtaining non-spherical nanocomposites that combines the properties of PCL and CNTs such as the high aspect ratio, modulus. The high drug entrapment efficiency of these nanocomposites may have promising applications in drug delivery.

Pharmacology of carbon nanotubes: toxicokinetics, excretion and tissue accumulation.

Carbon nanotubes (CNT) are increasingly being investigated for their use in biomedical applications and nanomedicine. An emergent need for the understanding of their in vivo biodistribution and pharmacokinetics is therefore needed to establish the essential properties and criteria for their further development as targeted CNT delivery systems to specific tissues for diagnostics and therapeutic purposes. Until their biodistribution and toxicoketic profiles are fully understood, their translation into the clinic will be hindered. This review will highlight the important factors affecting the biodistribution and pharmacokinetic profile of CNT and address their toxicokinetics following systemic, pulmonary and dermal exposure.