Antonio Nunes

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.

Functional motor recovery from brain ischemic insult by carbon nanotube-mediated siRNA silencing

Stroke is the second cause of death worldwide with ischemic stroke accounting for 80% of all stroke insults. Caspase-3 activation contributes to brain tissue loss and downstream biochemical events that lead to programmed cell death after traumatic brain injury. Alleviation of symptoms following ischemic neuronal injury can be potentially achieved by either genetic disruption or pharmacological inhibition of caspases. Here, we studied whether silencing of Caspase-3 using carbon nanotube-mediated in vivo RNA interference (RNAi) could offer a therapeutic opportunity against stroke. Effective delivery of siRNA directly to the CNS has been shown to normalize phenotypes in animal models of several neurological diseases. It is shown here that peri-lesional stereotactic administration of a Caspase-3 siRNA (siCas 3) delivered by functionalized carbon nanotubes (f-CNT) reduced neurodegeneration and promoted functional preservation before and after focal ischemic damage of the rodent motor cortex using an endothelin-1 induced stroke model. These observations illustrate the opportunity offered by carbon nanotube-mediated siRNA delivery and gene silencing of neuronal tissue applicable to a variety of different neuropathological conditions where intervention at well localized brain foci may offer therapeutic and functional benefits.

Asbestos-like Pathogenicity of Long Carbon Nanotubes Alleviated by Chemical Functionalization

Carbon nanotubes (CNTs) are considered one of the most popular types of nanomaterials and in the last few years have gained tremendous interest in a wide range of applications due to their unique physical, chemical, and electronic properties. Multi-walled carbon nanotubes (MWNTs) consist of sheets of carbon atoms rolled up into multiple concentric hollow tubular structures. The lack of dispersibility of pristineMWNTs in most solvents is owing to strong inter-tube van der Waals forces and this has been an obstacle for their effective use in biological applications and material sciences (i.e. composites). This may be largely overcome by surface modification of the nanotube backbone, allowing application of CNTs in biomedical applications. Some types of chemically functionalized CNTs have shown great advantages for use as delivery systems because of their capacity to pierce cellular membranes and translocate directly into the cytoplasm, providing a method for effective drug and macromolecule intracellular transport. Moreover, chemical surface-functionalization strategies can improve the colloidal properties of the CNT dispersions and result in populations of individualized MWNTs in physiological environments that have the capacity for glomerular translocation, leading to rapid urinary excretion. Such biokinetic processes are also extremely important to determine the biopersistence and ultimately the potential risk from medical use of carbon nanotubes. The use of CNTs—particularly in mass-scale, industrial applications—is currently considered with apprehension owing to their yet undefined safety profile and their potential environmental and health risks, especially given their structural resemblance to asbestos fibers. Several research groups have attempted to determine the carcinogenic risks that may be associated with intended or unintentional exposure to CNTs using various in vivo models. The first study that highlighted the importance of carbon nanotube length characteristics was carried out by Poland et al. using pristine (non-functionalized), long CNTs in a structure– toxicity study, which was originally validated with asbestos fibers. According to this method, which relates length and biopersistence of asbestos fibers to the development of mesothelioma (cancer of the pleural membrane), non-functionalized MWNTs longer than 20 mm were found to trigger an inflammatory response and result in granuloma formation seven days after intra-peritoneal exposure, similar to long asbestos fibers (LFA, long fiber amosite). This was thought to be due to induction of a process termed “frustrated phagocytosis” as resident and recruited macrophages attempt unsuccessfully to remove the long fibers from the mesothelium. Similar conclusions regarding the risk of unwanted

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.

Hybrid Polymer-Grafted Multiwalled Carbon Nanotubes for In vitro Gene Delivery

Carbon nanotubes (CNTs) consist of carbon atoms arranged in sheets of graphene rolled up into cylindrical shapes. This class of nanomaterials has attracted attention because of their extraordinary properties, such as high electrical and thermal conductivity. In addition, development in CNT functionalization chemistry has led to an enhanced dispersibility in aqueous physiological media which indeed broadens the spectrum for their potential biological applications including gene delivery. The aim of this study is to determine the capability of different cationic polymer-grafted multiwalled carbon nanotubes (MWNTs) (polymer-g-MWNTs) to efficiently complex and transfer plasmid DNA (pCMV-βGal) in vitro without promoting cytotoxicity. Carboxylated MWNT is chemically conjugated to the cationic polymers polyethylenimine (PEI), polyallylamine (PAA), or a mixture of the two polymers. In order to explore the potential of these polymer-g-MWNTs as gene delivery systems, we first study their capacity to complex plasmid DNA (pDNA) using agarose gel electrophoresis. Gel migration studies confirm pDNA binding to polymer-g-MWNT with different affinities, highest for PEI-g-MWNT and PEI/PAA-g-CNT constructs. β-galactosidase expression is assessed in human lung epithelial (A549) cells, and the cytotoxicity is determined by modified LDH assay after 24 h incubation period. Additionally, PEI-g-MWNT and/or PEI/PAA-g-MWNT reveal an improvement in gene expression when compared to the naked pDNA or to the equivalent amounts of PEI polymer alone. Mechanistically, pDNA was delivered by the polymer-g-MWNT constructs via a different pathway compared to those used by polyplexes. In conclusion, polymer-g-MWNTs may be considered in the future as a versatile tool for efficient gene transfer in cancer cells in vitro, provided their toxicological profile is established.

In vivo degradation of functionalized carbon nanotubes after stereotactic administration in the brain cortex

AIM Carbon nanotubes (CNTs) are increasingly being utilized in neurological applications as components of implants, electrodes or as delivery vehicles. Any application that involves implantation or injection of CNTs into the CNS needs to address the distribution and fate of the material following interaction and residence within the neuronal tissue. Here we report a preliminary study investigating the fate and structural integrity of amino-functionalized CNTs following stereotactic administration in the brain cortex. MATERIALS & METHODS The CNTs investigated had previously shown the capacity to internalize in various cell types of the CNS. An aqueous suspension of multiwalled CNT-NH(3) (+) was stereotactically injected into the mouse brain cortex. Their interaction with neural cells and consequent effects on the CNT structural integrity was investigated by optical, transmission electron microscopy and Raman spectroscopy of brain tissue sections for a period between 2 and 14 days post cortical administration. RESULTS & DISCUSSION The occurrence of severe nanotube structure deformation leading to partial degradation of the chemically functionalized-multiwalled CNT-NH(3) (+) in vivo following internalization within microglia was revealed even at early time points. Such initial observations of CNT degradation within the brain tissue render further systematic investigations using high-resolution tools imperative.

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 nano‐tubes 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.—Al‐Jamal, K. T., Toma, F. M., Yilmazer, A., Ali‐Boucetta, H., Nunes, A., Herrero, M.‐A., Tian, B., Eddaoudi, A., Al‐Jamal, W. T., Bianco, A., Prato, M., Kostarelos, K. Enhanced cellular internalization and gene silencing with a series of cationic dendron‐multiwalled carbon nanotube:siRNA complexes. FASEB J. 24, 4354–4365 (2010). www.fasebj.org

Therapeutics, imaging and toxicity of nanomaterials in the central nervous system

Treatment and diagnosis of neurodegenerative diseases and other CNS disorders are nowadays considered some of the most challenging tasks in modern medicine. The development of effective strategies for the prevention, diagnosis and treatment of CNS pathologies require better understanding of neurological disorders that is still lacking. The use of nanomaterials is thought to contribute to our further understanding of the CNS and the development of novel therapeutic and diagnostic modalities for neurological interventions. Even though the application of nanoparticles in neuroscience is still embryonic, this article attempts to illustrate the use of different types of nanomaterials and the way in which they have been used in various CNS applications in an attempt to limit or reverse neuropathological processes.

Functionalized Carbon Nanotubes in the Brain: Cellular Internalization and Neuroinflammatory Responses

The potential use of functionalized carbon nanotubes (f-CNTs) for drug and gene delivery to the central nervous system (CNS) and as neural substrates makes the understanding of their in vivo interactions with the neural tissue essential. The aim of this study was to investigate the interactions between chemically functionalized multi-walled carbon nanotubes (f-MWNTs) and the neural tissue following cortical stereotactic administration. Two different f-MWNT constructs were used in these studies: shortened (by oxidation) amino-functionalized MWNT (oxMWNT-NH3 +) and amino-functionalized MWNT (MWNT-NH3 +). Parenchymal distribution of the stereotactically injected f-MWNTs was assessed by histological examination. Both f-MWNT were uptaken by different types of neural tissue cells (microglia, astrocytes and neurons), however different patterns of cellular internalization were observed between the nanotubes. Furthermore, immunohistochemical staining for specific markers of glial cell activation (GFAP and CD11b) was performed and secretion of inflammatory cytokines was investigated using real-time PCR (qRT-PCR). Injections of both f-MWNT constructs led to a local and transient induction of inflammatory cytokines at early time points. Oxidation of nanotubes seemed to induce significant levels of GFAP and CD11b over-expression in areas peripheral to the f-MWNT injection site. These results highlight the importance of nanotube functionalization on their interaction with brain tissue that is deemed critical for the development nanotube-based vector systems for CNS applications.

Eigenspectra optoacoustic tomography achieves quantitative blood oxygenation imaging deep in tissues

Light propagating in tissue attains a spectrum that varies with location due to wavelength-dependent fluence attenuation, an effect that causes spectral corruption. Spectral corruption has limited the quantification accuracy of optical and optoacoustic spectroscopic methods, and impeded the goal of imaging blood oxygen saturation (sO2) deep in tissues; a critical goal for the assessment of oxygenation in physiological processes and disease. Here we describe light fluence in the spectral domain and introduce eigenspectra multispectral optoacoustic tomography (eMSOT) to account for wavelength-dependent light attenuation, and estimate blood sO2 within deep tissue. We validate eMSOT in simulations, phantoms and animal measurements and spatially resolve sO2 in muscle and tumours, validating our measurements with histology data. eMSOT shows substantial sO2 accuracy enhancement over previous optoacoustic methods, potentially serving as a valuable tool for imaging tissue pathophysiology.