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Carbon Nanotubes for Enhanced Biopharmaceutical Delivery
Abstract
Carbon nanotubes (CNTs) can be applied as versatile biopharmaceutical delivery systems due to their high drug-loading capacity, excellent cell-penetrating ability, and customizable surface chemistry. Biopharmaceuticals are potent therapeutics when applied correctly, but are challenged by significant drawbacks. The coupling of biopharmaceuticals to CNTs can mitigate these issues, thus additively or synergistically improving their performance. Improved therapeutic effects, enhanced (targeted) delivery to specific tissue types, highly controlled release profiles, and even the visualization of in-vivo biodistribution comprise just a few examples of the benefits of such complexes. Furthermore, novel complexes have been tailored to combat cancer and other difficult-to-treat diseases, with promising success. In this chapter, the application of CNTs for the delivery of biopharmaceuticals to disease sites in vitro and in vivo will be discussed.
... The pre-clinical studies for biomedical applications began at the beginning of the 2000's. CNT can be used as a reservoir hosting a molecule, within the tube or in between the walls in the case of Multi-Walled Carbon Nanotubes (MWCNT) [62], but it is mostly functionalized by covalent or non-covalent addition of functional groups [63]. It can also be used in scaffolds for stem cells growth [64] or tissue engineering [65]. ...
Ewing Sarcoma is a rare pediatric cancer, caused in the majority of the cases by the expression of the fusion oncogene EWS-Fli1. Current treatments have not much evolved over the past decades. We are investigating a new therapy based on siRNA specifically targeting the oncogene and inhibiting the tumor growth. During my PhD thesis, I have tested different types of synthetic nanodiamonds (ND) used to vectorize siRNA electrostatically bound at their surface: ND produced by detonation (DND) or by High Pressure-High Temperature synthesis (NDHPTH). Their surfaces have been cationized by various processes: (i) plasma or (ii) thermal hydrogenation, (ii) chemical treatment, or (iv) covalent grafting of a copolymer (COP-NDHPHT).My PhD work included two main axis: (i) in vitro study of ND:siRNA complexes (NDs physico-chemical characterization and oncogene inhibition efficacy by the complexes); (ii) tissue distribution of COP-NDHPHT, injected into mice, using fluorescent NDHPHT containing nitrogen-vacancy defects. To detect them individually in sections of mouse organs carrying a subcutaneous xenograft tumor, we developed an epifluorescence imaging system with large numerical aperture and resolved in time to reject tissue autofluorescence (of a shorter lifetime than NDs). We quantified the number, the aggregation state and the cell localization (thanks to simultaneous histopathological imaging) of these vectors 24 hours after injection. NDs have been clearly detected in different organs, including the tumor, paving the way for tumor progression control with siRNA.
... Graphene-based nanosheets, including graphenes and graphene oxides, have properties suitable for delivery of various molecules (Shim et al., 2016). CNTs are utilized as versatile biopharmaceutical delivery systems, due to the excellent cellpenetrating ability and high drug-loading capacity (Rallapalli and Smith, 2016). ...
... Graphene-based nanosheets, including graphenes and graphene oxides, have properties suitable for delivery of various molecules (Shim et al., 2016). CNTs are utilized as versatile biopharmaceutical delivery systems, due to the excellent cellpenetrating ability and high drug-loading capacity (Rallapalli and Smith, 2016). ...
Most bacterial pathogens have evolved as extensively drug resistant against wide classes of antimicrobials and cause a high threat to pharmaceutical sectors. As a result, there is a high necessity to address the issue and screen novel antimicrobials. This chapter focuses on the therapeutic potential of nanoparticles, especially carbon nanotubes and fullerenes, towards the probable drug targets of bacterial pathogens through computational drug discovery approaches. The chapter emphases the gene network analysis to identify probable drug targets, a need for three-dimensional (3D) structures, major 3D structure depositories, and approaches for modeling and validation. It further emphasizes various approaches for screening carbon fullerenes and nanotubes, and the need for structure-based virtual screening and molecular docking studies. Furthermore, prediction of the binding potential of carbon fullerene and nanotubes against potential drug targets of multidrug resistant pathogens. This chapter integrates the use of computational biology, proteomics and pharmaceutical biotechnology for the screening of novel carbon nanoleads, especially fullerenes and nanotubes, as ideal lead molecules against extreme drug resistant bacterial pathogens.
Novel nanomaterials for bioassay applications represent a rapidly progressing field of nanotechnology and nanobiotechnology. Here, we present an exploration of single-walled carbon nanotubes as a platform for investigating surface-protein and protein-protein binding and developing highly specific electronic biomolecule detectors. Nonspecific binding on nanotubes, a phenomenon found with a wide range of proteins, is overcome by immobilization of polyethylene oxide chains. A general approach is then advanced to enable the selective recognition and binding of target proteins by conjugation of their specific receptors to polyethylene oxide-functionalized nanotubes. This scheme, combined with the sensitivity of nanotube electronic devices, enables highly specific electronic sensors for detecting clinically important biomolecules such as antibodies associated with human autoimmune diseases.
Current interest in the pulmonary toxicity of carbon nanotubes (CNTs) has resulted in a need for an aerosol generation system that is capable of consistently producing a CNT aerosol at a desired concentration level. This two-part study was designed to: (1) assess the properties of a commercially-available aerosol generator when producing an aerosol from a purchased powder supply of double-walled carbon nanotubes (DWCNTs); and (2) assess the pulmonary sub-acute toxicity of DWCNTs in a murine model during a 5-day (4 h/day) whole-body exposure. The aerosol generator, consisting of a novel dustfeed mechanism and venturi ejector was determined to be capable of producing a DWCNT consistently over a 4 h exposure period at an average level of 10.8 mg/m 3 . The count median diameter was 121 nm with a geometric standard deviation of 2.04. The estimated deposited dose was 32 µg/mouse. The total number of cells in bronchoalveolar lavage (BAL) fluid was significantly (p < 0.01) increased in exposed mice compared to controls. Similarly, macrophages in BAL fluid were significantly elevated in exposed mice, but not neutrophils. All animals exposed to CNT and euthanized immediately after exposure had changes in the lung tissues showing acute inflammation and injury; however these pathological changes resolved two weeks after the exposure.
Carbon nanotubes (CNTs) possess unique physical and chemical properties and can serve as a platform for transporting a variety of bioactive molecules, such as drugs, proteins, and genes, given appropriate surface modifications. Here, we present an overview of the progress in applying CNTs as therapeutic agent carriers. Drugs can be attached to CNTs either through supramolecular chemistry to form noncovalent assembly or via covalent linkage to the functional groups preinstalled on CNTs. In addition to surface loading, packing of molecules inside the internal cavity of CNTs to protect less stable entities has also been achieved. Besides drugs, the high specific surface area of CNTs can also allow the installation of multiple molecules with different functions, e.g. target recognition and optical imaging, simultaneously to achieve synergistic effects. The drug release process tends to be gradual and sustained after being attached to CNTs, and could be tuned by various factors, such as pH, diameter of CNTs, and target recognition. The content throughout this review is mainly focused on the different protocols of loading drugs onto or into CNTs as well as how to control the drug release.
Multiwalled carbon nanotubes (MWCNTs) are cut short and grafted with polyethylenimine (PEI) for further covalent conjugation to fluorescein isothiocyanate (FITC) and prostate stem cell antigen (PSCA) monoclonal antibody (mAb). The in vitro and in vivo toxicity data reveal that the as-prepared CNT-PEI(FITC)-mAb has good biocompatibility. Combined flow cytometry and confocal luminescence imaging experiments confirm that the CNT-PEI(FITC)-mAb can specifically target the cancer cells which overexpress PSCA. The results of in vitro and in vivo ultrasound (US) imaging indicate that CNT-PEI(FITC)-mAb has great potential to be used as a targeted US contrast agent. The in vivo anti-cancer efficacy testing using PC-3 tumor-bearing mice as animal models demonstrates that CNT-PEI(FITC)-mAb can targetedly deliver drug to the tumors and suppress tumor growth. Findings from this study suggest that the CNT-PEI(FITC)-mAb could be used as a multifunctional platform for simultaneous US imaging and drug delivery applications.
In cancer imaging, nanoparticle biodistribution is typically visualized in living subjects using 'bulk' imaging modalities such as magnetic resonance imaging, computerized tomography and whole-body fluorescence. Accordingly, nanoparticle influx is observed only macroscopically, and the mechanisms by which they target cancer remain elusive. Nanoparticles are assumed to accumulate via several targeting mechanisms, particularly extravasation (leakage into tumour). Here, we show that, in addition to conventional nanoparticle-uptake mechanisms, single-walled carbon nanotubes are almost exclusively taken up by a single immune cell subset, Ly-6C(hi) monocytes (almost 100% uptake in Ly-6C(hi) monocytes, below 3% in all other circulating cells), and delivered to the tumour in mice. We also demonstrate that a targeting ligand (RGD) conjugated to nanotubes significantly enhances the number of single-walled carbon nanotube-loaded monocytes reaching the tumour (P < 0.001, day 7 post-injection). The remarkable selectivity of this tumour-targeting mechanism demonstrates an advanced immune-based delivery strategy for enhancing specific tumour delivery with substantial penetration.
Carbon nanotubes can be either single-walled or multi-walled, each of which is known to have a different electron arrangement and as a result have different properties. However, the shared unique properties of both types of carbon nanotubes (CNT) allow for their potential use in various biomedical devices and therapies. Some of the most common properties of these materials include the ability to absorb near-infra-red light and generate heat, the ability to deliver drugs in a cellular environment, their light weight, and chemical stability. These properties have encouraged scientists to further investigate CNTs as a tool for thermal treatment of cancer and drug delivery agents. Various promising data have so far been obtained about the usage of CNTs for cancer treatment; however, toxicity of pure CNTs represents a major challenge for clinical application. Various techniques both in vivo and in in vitro have been conducted by a number of different research groups to establish the factors which have a direct effect on CNT-mediated cytotoxicity. The main analysis techniques include using Alamar blue, MTT, and Trypan blue assays. Successful interpretation of these results is difficult because the CNTs can significantly disrupt the emission of the certain particles, which these assays detect. In contrast, in vivo studies allow for the measurement of toxicity and pathology caused by CNTs on an organismal level. Despite the drawbacks of in vitro studies, they have been invaluable in identifying important toxicity factors, such as size, shape, purity, and functionalisation, the latter of which can attenuate CNT toxicity.
A new design of fully automatic system was built up to produce multiwalled carbon nanotube (MWCNT) using arc discharge technique in deionized water and extra pure graphite multiple electrodes (99.9% pure). The goal of the experimental research is to determine the yield of CNT in two different cases: (a) single plasma electrodes and (b) multiplasma electrodes, particularly 10 electrodes. The experiments were performed at constant parameters (75 A, 238 V). The obtained CNT was examined by scanning electron microscope (SEM), transmission electron microscope (HRTEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). The results showed that the produced CNT is of type MWCNT, with a diameter of 5 nm, when using multiplasma electrodes and 13 nm when using single plasma electrodes. The yield of MWCNT was found to be 320% higher in case of comparing multielectrodes to that of single plasma electrodes. Under the experimental test conditions, a yield of 0.6 g/hr soot containing 40% by mass nanotube was obtained in case of single plasma electrodes and above 60% in case of multiplasma electrodes.
Methods which solubilize single-walled carbon nanotubes (SWNTs) in water as individuals, not bundles, while retaining their unique electronic, photonic and mechanical properties are highly desirable. Furthermore, functionalization with a diverse array of selectable chemical moieties would allow the range of useful applications to be significantly extended and may permit the designed assembly of SWNT networks. This paper presents a series of peptides that non-covalently solubilize carbon nanotubes in water using a design motif that combines a combinatorial library sequence to bind to nanotubes with a rationally designed section to create environmentally tuned solubility characteristics. The ability of the peptides to individually disperse carbon nanotubes without altering their electronic structure is shown by vis-NIR absorbance, fluorescence, and regular and vitreous ice cryo-TEM. Identification of the species composition of each sample by NIR fluorescence reveals that the peptides exhibit some diameter selectivity. Additionally, one of the rationally designed modifications addresses the poor stability of non-covalently solubilized SWNT suspensions by including cysteine residues for covalent crosslinking between adjacent peptides.
Carbon nanotubes (CNTs) have attracted great interdisciplinary interest due to their peculiar structural, mechanical and electronic properties. Applications of CNTs in biomedical research are being actively explored by many scientists worldwide. However, manipulation of CNTs is impeded by several problems, such as 1) formation of complex and entangled bundles; 2) very low solubility of CNTs in organic solvents and water; 3) inert properties of pristine CNTs under many chemical reaction conditions, etc. Chemical modification of CNTs has partly solved the above issues and is still one of the most effective means of manipulating and processing CNTs. Many bioapplications of CNTs rely on successful outer/inner surface functionalizations. This Feature Article is comprised of two main parts. In the first part, we briefly review the covalent surface chemistry for the CNT functionalization; in the second part, we focus on the biomedical applications of surface chemistry for CNTs, in particular, the chemistry for controlling biomedical functions and meanwhile lowering nanotoxicity of CNTs. We also analyze the underlying factors that led to the controversy in the previous experimental data of safety studies of CNTs.
A series of multi-walled carbon nanotube (MWCNT) conjugates is described, functionalized with different dendrons bearing positive charges at their termini (i.e. ammonium or guanidinium groups). The dendrimeric units are anchored to the nanotube scaffolds using two orthogonal synthetic approaches, amidation and click reactions. The final nanohybrids are characterized by complementary analytical techniques, while their ability to interact with siRNA is investigated by means of agarose gel electrophoresis. The demonstration of the cell uptake capacity, the low cytotoxicity, and the ability of these cationic conjugates to silence cytotoxic genes suggests them to be promising carriers for genetic material.
In spite of the extreme rise to the knowledge of nanotechnology in pharmaceutical sciences, there are currently limited experimental works studying the interactions between nanoparticles (NPs) and the biological system. Adjustment of size and surface area plays the main role in the reaction between NPs and cells leading to their increased entrance into cells through skin, gastrointestinal and respiratory system. Moreover, change in physicochemical reactivity of NPs causes them to interact with circulatory and cellular proteins differentially leading to the altered parameters of their biokinetics, including adsorption, distribution, translocation, transformation, and elimination. A direct relationship between the surface area, reactive oxygen species generating capability, and proinflammatory effects of NPs have been found in respiratory tract toxicity. Additionally, complement-mediated hypersensitivity reactions to liposomes and other lipid-based nanodrugs have been well defined. Inhalation studies of some NPs have confirmed the translocation of inhaled materials to extra pulmonary organs such as central nervous system (CNS) via olfactory neurons and induction of inflammatory response. Injectable uncoated NPs have a tendency to remain on the injection site while the poly ethanol glycol (PEG)-coated NPs can be notably drained from the injection site to get as far as the lymph nodes where they accumulate. This confirms the existence of channels within the extracellular matrix for NPs to move along. Furthermore, induction of DNA strand breaks and formation of micronuclei have been recorded for exposure to some NPs such as single-walled carbon nanotubes.
In the recent years, most of the studies have simply outlined better efficacy of nanodrugs, but few discussed their possible toxic reactions specially if used chronically. Therefore, we emphasize that this part of the nanoscience must not be undermined and toxicologists must be sensitive to set up suitable in vivo or in vitro toxicity models. A system for collecting data about the relationships between NPs’ structure-size-efficacy-toxicity (SSET) should be specified with special regard to portal of entry and target organ.
Given the increasing use of carbon nanotubes (CNT) in composite materials and their possible expansion to new areas such as nanomedicine which will both lead to higher human exposure, a better understanding of their potential to cause adverse effects on human health is needed. Like other nanomaterials, the biological reactivity and toxicity of CNT were shown to depend on various physicochemical characteristics, and length has been suggested to play a critical role.We therefore designed a comprehensive study that aimed at comparing the effects on murine macrophages of two samples of multi-walled CNT (MWCNT) specifically synthesized following a similar production process (aerosol-assisted CVD), and used a soft ultrasonic treatment in water to modify the length of one of them.We showed that modification of the length of MWCNT leads, unavoidably, to accompanying structural (i.e. defects) and chemical (i.e. oxidation) modifications that affect both surface and residual catalyst iron nanoparticle content of CNT. The biological response of murine macrophages to the two different MWCNT samples was evaluated in terms of cell viability, pro-inflammatory cytokines secretion and oxidative stress. We showed that structural defects and oxidation both induced by the length reduction process are at least as responsible as the length reduction itself for the enhanced pro-inflammatory and pro-oxidative response observed with short (oxidized) compared to long (pristine) MWCNT.In conclusion, our results stress that surface properties should be considered, alongside the length, as essential parameters in CNT-induced inflammation, especially when dealing with a safe design of CNT, for application in nanomedicine for example.
To determine whether carbon nanotubes can induce any significant health hazards we applied methods routinely used in the pathophysiological testing of asbestos-induced disease to show that the soot with a high content of CNTs does not induce any abnormalities of pulmonary function or measurable inflammation in guinea pigs treated with carbon nanotubes.
Despite the utility and promise of carbon nanotubes (CNTs), their production is generally based on empirical principles. There are only a few CNT formation models that predict the dependence of their growth on various synthesis parameters. Typically, these do not incorporate a detailed mechanistic consideration of the various processes that are involved during CNT synthesis. We address this need and present a model for catalytic CNT growth that integrates various interdependent physical and chemical processes involved in CNT production. We validate the model by comparing its predictions with one set of experimental measurements from a previous study for cobalt (Co) catalyzed growth. A brief parametric study is presented subsequently. From an application perspective, the model is able to predict the growth rate of the CNT length and its dependence on the ambient temperature and gas-phase feedstock partial pressure.
Carbon nanotubes (CNTs) have attracted great interest of the nano community and beyond. However, the biomedical applications of CNTs arouse serious concerns for their unknown in vivo consequence, in which the information of pharmacokinetics, metabolism and toxicity of CNTs is essential. In this review, we summarize the updated data of CNTs from the biomedical view. The information shows that surface chemistry is crucial in regulating the in vivo behaviors of CNTs. Among the functionalization methods, PEGylation is the most efficient one to improve the pharmacokinetics and biocompatibility of CNTs. The guiding effects of the pharmacokinetics, metabolism and toxicity information on the biomedical applications of CNTs are discussed.
Cancer is a generic term that encompasses a group of diseases characterized by an uncontrolled proliferation of cells. There are over 200 different types of cancer, each of which gains its nomenclature according to the type of tissue the cell originates in. Many patients who succumb to cancer do not die as a result of the primary tumor, but because of the systemic effects of metastases on other regions away from the original site. One of the aims of cancer therapy is to prevent the metastatic process as early as possible. There are currently many therapies in clinical use, and recent advances in biotechnology lend credence to the potential of nanotechnology in the fight against cancer. Nanomaterials such as carbon nanotubes (CNTs), quantum dots, and dendrimers have unique properties that can be exploited for diagnostic purposes, thermal ablation, and drug delivery in cancer. CNTs are tubular materials with nanometer-sized diameters and axial symmetry, giving them unique properties that can be exploited in the diagnosis and treatment of cancer. In addition, CNTs have the potential to deliver drugs directly to targeted cells and tissues. Alongside the rapid advances in the development of nanotechnology-based materials, elucidating the toxicity of nanoparticles is also imperative. Hence, in this review, we seek to explore the biomedical applications of CNTs, with particular emphasis on their use as therapeutic platforms in oncology.
Carbon nanotubes are increasingly being tested for use in cellular applications. Determining the mode of entry is essential to control and regulate specific interactions with cells, to understand toxicological effects of nanotubes, and to develop nanotube-based cellular technologies. We investigated cellular uptake of Pluronic copolymer-stabilized, purified ~145 nm long single wall carbon nanotubes (SWCNTs) through a series of complementary cellular, cell-mimetic, and in vitro model membrane experiments.
SWCNTs localized within fluorescently labeled endosomes, and confocal Raman spectroscopy showed a dramatic reduction in SWCNT uptake into cells at 4°C compared with 37°C. These data suggest energy-dependent endocytosis, as shown previously. We also examined the possibility for non-specific physical penetration of SWCNTs through the plasma membrane. Electrochemical impedance spectroscopy and Langmuir monolayer film balance measurements showed that Pluronic-stabilized SWCNTs associated with membranes but did not possess sufficient insertion energy to penetrate through the membrane. SWCNTs associated with vesicles made from plasma membranes but did not rupture the vesicles.
These measurements, combined, demonstrate that Pluronic-stabilized SWCNTs only enter cells via energy-dependent endocytosis, and association of SWCNTs to membrane likely increases uptake.
Nanomaterials are frontier technological products used in different manufactured goods. Because of their unique physicochemical, electrical, mechanical, and thermal properties, single-walled carbon nanotubes (SWCNT) are finding numerous applications in electronics, aerospace devices, computers, and chemical, polymer, and pharmaceutical industries. SWCNT are relatively recently discovered members of the carbon allotropes that are similar in structure to fullerenes and graphite. Previously, we (47) have reported that pharyngeal aspiration of purified SWCNT by C57BL/6 mice caused dose-dependent granulomatous pneumonia, oxidative stress, acute inflammatory/cytokine responses, fibrosis, and decrease in pulmonary function. To avoid potential artifactual effects due to instillation/agglomeration associated with SWCNT, we conducted inhalation exposures using stable and uniform SWCNT dispersions obtained by a newly developed aerosolization technique (2). The inhalation of nonpurified SWCNT (iron content of 17.7% by weight) at 5 mg/m(3), 5 h/day for 4 days was compared with pharyngeal aspiration of varying doses (5-20 microg per mouse) of the same SWCNT. The chain of pathological events in both exposure routes was realized through synergized interactions of early inflammatory response and oxidative stress culminating in the development of multifocal granulomatous pneumonia and interstitial fibrosis. SWCNT inhalation was more effective than aspiration in causing inflammatory response, oxidative stress, collagen deposition, and fibrosis as well as mutations of K-ras gene locus in the lung of C57BL/6 mice.
Carbon nanotubes have distinctive characteristics, but their needle-like fibre shape has been compared to asbestos, raising concerns that widespread use of carbon nanotubes may lead to mesothelioma, cancer of the lining of the lungs caused by exposure to asbestos. Here we show that exposing the mesothelial lining of the body cavity of mice, as a surrogate for the mesothelial lining of the chest cavity, to long multiwalled carbon nanotubes results in asbestos-like, length-dependent, pathogenic behaviour. This includes inflammation and the formation of lesions known as granulomas. This is of considerable importance, because research and business communities continue to invest heavily in carbon nanotubes for a wide range of products under the assumption that they are no more hazardous than graphite. Our results suggest the need for further research and great caution before introducing such products into the market if long-term harm is to be avoided.
Carbon nanotubes have remarkably attractive mechanical, thermal, and electrical properties. The synergistic incorporation of the nanotubes in the polymer matrices can thus lead to tremendous enhancements in the composite properties even at very low filler fractions. The nanotubes are inert at surface; therefore, their dispersion in various polymer matrices at the nanometer level is not straightforward. Many methods for the organic modification of the nanotubes have been developed in the recent years to improve the compatibility between the polymer and nanotubes. These methods are aimed in two directions: noncovalent functionalization and covalent functionalization. Both the techniques lead to polymer chains either physically adsorbed or chemically grafted with the surface; however, both of the methods have their own advantages and limitations. Therefore, the choice of the functionalization is primarily based on the intended applications of the nanotube nanocomposite materials.
Abstract Cancer development due to fiber-like straight type of multi-walled carbon nanotubes (MWCNTs) has raised concerns for human safety because of its shape similar to asbestos. To set concentrations of MWCNT for a rat carcinogenicity study, we conducted a 13-week whole body inhalation study. F344 male and female rats, 6-week-old at the commencement of the study, were exposed by whole-body inhalation to MWCNT at concentrations of 0, 0.2, 1 and 5 mg/m(3) with a generation and exposure system utilizing the cyclone sieve method. Measured concentrations in the exposure chambers were 0.20 ± 0.02, 1.01 ± 0.11 and 5.02 ± 0.25 mg/m(3) for 13 weeks. The MMAD (GSD) of MWCNT were 1.4-1.6 μm (2.3-3.0), and mean width and length were 94.1-98.0 nm and 5.53-6.19 μm, respectively, for each target concentration. Lung weights were increased 1.2-fold with 1 mg/m(3) and 1.3-fold with 5 mg/m(3) in both sexes compared to the controls. In the bronchoalveolar lavage fluid (BALF) analyses, inflammatory parameters were increased concentration-dependently in both sexes from 0.2 mg/m(3). Granulomatous changes in the lung were induced at 1 and 5 mg/m(3) in females and even at 0.2 mg/m(3) in males. Focal fibrosis of the alveolar wall was observed in both sexes at 1 mg/m(3) or higher. Inflammatory infiltration in the visceral pleural and subpleural areas was induced only at 5 mg/m(3). In conclusion, we determined 0.2 mg/m(3) as the low-observed-adverse-effect level (LOAEL) for respiratory tract toxicity in the present inhalation exposure study of rats.
Because of their unique physical, chemical, electrical, and mechanical properties, carbon nanotubes (CNTs) have attracted a great deal of research interest and have many potential applications. As large-scale production and application of CNTs increases, the general population is more likely to be exposed to CNTs either directly or indirectly, which has prompted considerable attention about human health and safety issues related to CNTs. Although considerable experimental data related to CNT toxicity at the molecular, cellular, and whole animal levels have been published, the results are often conflicting. Therefore, a systematic understanding of CNT toxicity is needed but has not yet been developed.
Carbon nanotubes (CNT) are structurally described as sheets of six-membered carbon atom rings rolled up into cylinders. CNTs with only one layer are known as single-walled CNTs (SWCNT), and those with two or more layers are known as multi-walled CNTs (MWCNT). The reasons are that CNTs have useful electrical, thermal, and mechanical characteristics, and their base material performance can be improved by combination with other materials. A recent industrial application of CNTs as an electrode additive to lithium-ion batteries is based on their excellent electrical characteristics. One reason for the intense competition to find biomaterial applications of CNTs and for the great potential of CNTs to advance medical care is their small size, which makes them suitable to react with living organisms.
Carbon nanotubes (CNTs) have the potential to overcome significant challenges related to vaccine development and immunotherapy. Central to these applications is an improved understanding of CNT interactions with the immune system. Unique properties such as high aspect ratio, flexible surface chemistry, and control over structure and morphology may allow for enhanced target specificity and transport of antigens across cell membranes. Although recent work has demonstrated the potential of CNTs to amplify the immune response as adjuvants, other results have also linked their proinflammatory properties to harmful health effects. Here, we review the recent advances of CNT-based immunological research, focusing on current understandings of therapeutic efficacy and mechanisms of immunotoxicology.
Background: The production and use of carbon nanotubes (CNTs) is rapidly growing. With increased production, there is potential that the number of occupational exposed workers will rapidly increase. Toxicological studies on rats have shown effects in the lungs, e.g. inflammation, granuloma formation, and fibrosis after repeated inhalation exposure to some forms of multi-walled CNTs (MWCNTs). Still, when it comes to health effects, it is unknown which dose metric is most relevant. Limited exposure data for CNTs exist today and no legally enforced occupational exposure limits are yet established. The aim of this work was to quantify the occupational exposures and emissions during arc discharge production, purification, and functionalization of MWCNTs. The CNT material handled typically had a mean length <5 μm. Since most of the collected airborne CNTs did not fulfil the World Health Organization fibre dimensions (79% of the counted CNT-containing particles) and since no microscopy-based method for counting of CNTs exists, we decided to count all particle that contained CNTs. To investigate correlations between the used exposure metrics, Pearson correlation coefficient was used.
Exposure measurements were performed at a small-scale producer of MWCNTs and respirable fractions of dust concentrations, elemental carbon (EC) concentrations, and number concentrations of CNT-containing particles were measured in the workers' breathing zones with filter-based methods during work. Additionally, emission measurements near the source were carried out during different work tasks. Respirable dust was gravimetrically determined; EC was analysed with thermal-optical analysis and the number of CNT-containing particles was analysed with scanning electron microscopy.
For the personal exposure measurements, respirable dust ranged between <73 and 93 μg m(-3), EC ranged between <0.08 and 7.4 μg C m(-3), and number concentration of CNT-containing particles ranged between 0.04 and 2.0cm(-3). For the emission measurements, respirable dust ranged between <2800 and 6800 μg m(-3), EC ranged between 0.05 and 550 μg C m(-3), and number concentration of CNT-containing particles ranged between <0.20 and 11cm(-3).
The highest exposure to CNTs occurred during production of CNTs. The highest emitted number concentration of CNT-containing particles occurred in the sieving, mechanical work-up, pouring, weighing, and packaging of CNT powder during the production stage. To be able to quantify exposures and emissions of CNTs, a selective and sensitive method is needed. Limitations with measuring EC and respirable dust are that these exposure metrics do not measure CNTs specifically. Only filter-based methods with electron microscopy analysis are, to date, selective and sensitive enough. This study showed that counting of CNT-containing particles is the method that fulfils those criteria and is therefore the method recommended for future quantification of CNT exposures. However, CNTs could be highly toxic not only because of their length but also because they could contain, for example transition metals and polycyclic aromatic hydrocarbons, or have surface defects. Lack of standardized counting criteria for CNTs to be applied at the electron microscopy analysis is a limiting factor, which makes it difficult to compare exposure data from different studies.
With large current interest in nanomedicine, there has been rapid progress during the last couple of years in the understanding of opportunities offered by advanced materials in diagnostics, drug delivery, functional biomaterials, and biosensors, as well as combinations of these, e.g., theranostics. In the present overview, focus is placed on drug delivery aspects of inorganic nanomaterials, notably as carriers for proteins, peptides, DNA, and siRNA. Throughout, an attempt is made to illustrate how structure and interactions affect loading and release of such biomacromolecular drugs in various inorganic delivery systems, and how this translates into functional advantages.
Single-walled carbon nanotubes (SWCNTs) are known to have great potential for biomedical applications such as photothermal ablation of tumor cells in combination with near-infrared (NIR) irradiation. In this study, the photothermal activity of a novel SWCNTs composite with a designed peptide having a repeated structure of H-(-Lys-Phe-Lys-Ala-)7-OH [(KFKA)7] against tumor cells was evaluated in vitro and in vivo. The SWCNTs-(KFKA)7 composite demonstrated high aqueous dispersibility that enabled SWCNTs to be used in tumor ablation. The NIR irradiation of SWCNTs-(KFKA)7 solution resulted in a rapid temperature increase dependent on the SWCNTs concentration up to 50μg/ml. Three minutes of NIR irradiation of a colon26 or HepG2 cell culture incubated with SWCNTs-(KFKA)7 resulted in remarkable cell damage, while that by single treatment with SWCNTs-(KFKA)7 or NIR irradiation alone was moderate. Intratumoral injection of SWCNTs-(KFKA)7 solution followed by NIR irradiation resulted in a rapid increase of the temperature to 43°C in the subcutaneously inoculated colon26 tumor based on thermographic observation and remarkable suppression of tumor growth compared with treatment with only SWCNTs-(KFKA)7 injection alone or NIR irradiation alone. These results suggest the great potential of a SWCNTs-peptide composite for use in photothermal cancer therapy.
Promising therapeutic and prophylactic effects have been achieved following impressive advances in the gene therapy research arena, giving birth to the new generation of disease-modifying therapeutics. The greatest challenge that gene therapy vectors still face is the ability to deliver sufficient genetic payloads in order to enable efficient gene transfer into target cells. A whole host of viral and non-viral gene therapy vectors have been explored over the past 10years, including carbon nanotubes. In this review we will address the application of carbon nanotubes in gene therapy with the aim to give a perspective on the past achievements, present challenges and future goals. A series of important topics concerning carbon nanotubes as gene therapy vectors will be addressed, including the benefits that carbon nanotubes offer over other non-viral delivery systems, furthermore, a perspective is given on what the ideal genetic cargo to deliver with carbon nanotubes is and finally the geno-pharmacological impact of carbon nanotube-mediated gene therapy is discussed.
Single-walled carbon nanotubes (SWNTs) can deliver imaging agents or drugs to tumours and offer significant advantages over approaches based on antibodies or other nanomaterials. In particular, the nanotubes can carry a substantial amount of cargo (100 times more than a monoclonal antibody), but can still be rapidly eliminated from the circulation by renal filtration, like a small molecule, due to their high aspect ratio. Here we show that SWNTs can target tumours in a two-step approach in which nanotubes modified with morpholino oligonucleotide sequences bind to cancer cells that have been pretargeted with antibodies modified with oligonucleotide strands complementary to those on the nanotubes. The nanotubes can carry fluorophores or radioisotopes, and are shown to selectively bind to cancer cells in vitro and in tumour-bearing xenografted mice. The binding process is also found to lead to antigen capping and internalization of the antibody-nanotube complexes. The nanotube conjugates were labelled with both alpha-particle and gamma-ray emitting isotopes, at high specific activities. Conjugates labelled with alpha-particle-generating (225)Ac were found to clear rapidly, thus mitigating radioisotope toxicity, and were shown to be therapeutically effective in vivo.
Abstract An aspargine-glycine-arginine (NGR) peptide modified single-walled carbon nanotubes (SWCNTs) system, developed by a simple non-covalent approach, could be loaded with the anticancer drug tamoxifen (TAM). This TAM-loaded NGR modified SWCNTs (TAM/NGR-SWCNTs) not only retained both optical properties of SWCNTs and cytotoxicity of TAM, but also could accumulate in tumors and enter into 4T1 cells, which facilitated combination chemotherapy with photothermal therapy in one targeting system. Enhanced cellular uptake, antitumor effect and cell apoptosis of TAM/NGR-SWCNTs on 4T1 cells were observed in vitro, compared with the TAM solution, TAM/SWCNTs and photothermal therapy alone. In vivo investigation of TAM/NGR-SWCNTs in tumor-bearing mice further confirmed that this system possessed much higher tumor targeting capacity and antitumor efficacy than the control, especially with the near-infrared-laser irradiation treatment. Moreover, it demonstrated negligible systematic toxicity through the histopathological analysis. All these results suggest TAM/NGR-SWCNTs are promising for high targeted efficiency and treatment efficacy and low side effects of future cancer therapy by synergistic effect of chemo-photothermal combination.
In the present study, we report the design and synthesis of peptide-based-multi-walled carbon nanotubes (MWCNTs) to target mitochondria. Targeting these intracellular organelles might open the way to develop alternative systems to address diseases related to genetic mutations in mitochondrial (mt)-DNA, by delivering therapeutic oligonucleotides. The first step towards mitochondrial delivery of this type of nucleic acid was to target MWCNTs to mitochondria by covalent functionalization with a well-known endogenous mitochondrial targeting sequence (MTS). The subcellular localization of the conjugates, which were fluorescently labeled, in murine RAW 264.7 macrophages and human HeLa cells was then studied using different microscopy techniques, such as wide-field epifluorescence microscopy, confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). The localization of the MTS-MWCNT conjugates into mitochondria was further confirmed by analyzing the isolated organelles using TEM.
The enhancement of the conventional synthesis of carbon nanotubes by means of catalytic chemical vapor deposition (CCVD), was investigated. This synthesis route is more efficient than conventional and commercial ones employing metal oxide catalyst supports. Electron microscopy investigations were made using a Philips XL30 scanning electron microscope and a Philips transmission electron microscope. The results show that NaCl catalysts support has an inhibiting effect on the growth of carbon nanotubes.
Small interfering RNA (siRNA) has the potential to influence gene expression with a high degree of target gene specificity. However, the clinical application of siRNA therapeutics has proven to be less promising as evidenced by its poor intracellular uptake, instability in vivo, and nonspecific immune stimulations. Recently, we have demonstrated that single-walled carbon nanotube (SWNT)-mediated siRNA delivery can enhance the efficiency of siRNA-mediated gastrin-releasing peptide receptor (GRP-R) gene silencing by stabilizing siRNA while selectively targeting tumor tissues. Based on our recent findings, we introduce a novel technique to silence specific gene(s) in human neuroblastoma through SWNT-mediated siRNA delivery in vitro and in vivo.
Carbon nanotubes (CNTs) are nanomaterials with interesting emerging applications. Their properties makes CNTs excellent candidates for use as new nanovehicles in drug delivery, immunization and diagnostics. In the current study, we assessed the immune-response-amplifying properties of CNTs to haptens by using azoxystrobin, the first developed strobilurin fungicide, as a model analyte. An azoxystrobin derivative bearing a carboxylated spacer arm (hapten AZc6) was covalently coupled to bovine serum albumin (BSA), and the resulting BSA-AZc6 conjugate was covalently linked to four functionalized CNTs of different shapes and sizes, varying in diameter and length. These four types of CNT-based constructs were obtained using efficient, fast, and easy functionalization procedures based on microwave-assisted chemistry. New Zealand rabbits and BALB/c mice were immunized with BSA-AZc6 alone and with the four CNT-BSA-AZc6 constructs, both with and without Freund's adjuvant. The IgG-type antibody responses were assessed in terms of the titer and affinity, paying special attention to the relationship between the immune response and the size and shape of the employed CNTs. Immunization with CNT-BSA-AZc6 resulted in enhanced titers and excellent affinities for azoxystrobin. More important, remarkable IgG responses were obtained even in the absence of an adjuvant, thus proving the self-adjuvanting capability of CNTs. Immunogens were able to produce strong anti-azoxystrobin immune responses in rabbits even when administered at a BSA-AZc6 conjugate dose as low as 0.05 μg. The short and thick CNT-BSA-AZc6 construct produced the best antibody response under all tested conditions.
Sometimes shorter is better: The apparent similarity between multi-walled carbon nanotubes (MWNTs) and asbestos fibers has generated serious concerns about their safety profile. The asbestos-like pathogenicity observed for long, pristine nanotubes (NTlong, see scheme) can be completely alleviated if their effective length is decreased as a result of chemical functionalization, such as with tri(ethylene glycol) (TEG).
INTEREST in carbon fibres1,2 has been stimulated greatly by
the recent discovery of hollow graphitic tubules of nanometre dimensions3.
There has been much speculation about the properties and potential application of these
nanotubes4–8. Theoretical studies predict that their electronic
properties will depend on their diameter and degree of helicity4,5.
Experimental tests of these ideas has been hampered, however, by the lack of macroscopic
quantities of the material. Here we report the synthesis of graphitic nanotubes in gram
quantities. We use a variant of the standard arc-discharge technique for fullerene
synthesis under a helium atmosphere. Under certain conditions, a carbonaceous deposit
forms on one of the graphite rods, consisting of a macroscopic (diameter of about 5 mm)
cylinder in which the core comprises pure nanotubes and nanoscale particles in high
yield. The purity and yield depend sensitively on the gas pressure in the reaction
vessel. Preliminary measurements of the conductivity of the bulk nanotube material
indicate a conductivity of about 100 S cm–1
1.
Single-walled carbon nanotubes (SWNTs) are special nano-materials which exhibit interesting physical and chemical properties, presenting new opportunities for biomedical research and applications. In this study, we have successfully adopted a novel strategy to chemically functionalize SWNTs with polyethylenimine (PEI) through purification, oxidation, amination and polymerization, which were then bound by DSPE-PEG2000-Maleimide for further conjugation with the tumor targeting NGR (Cys-Asn-Gly-Arg-Cys-) peptide via the maleimide group and sulfhydryl group of cysteine, and finally hTERT siRNA was loaded to obtain a novel tumor targeting siRNA delivery system, designated as SWNT-PEI/siRNA/NGR. The results showed that SWNT-PEI/siRNA/NGR could efficiently cross cell membrane, induced more severe apoptosis and stronger suppression in proliferation of PC-3 cells in vitro. Furthermore, in tumor-bearing mice model the delivery system exhibited higher antitumor activity due to more accumulation in tumor without obvious toxicity in main organs. The combination of RNAi and near-infrared (NIR) photothermal therapy significantly enhanced the therapeutic efficacy. In conclusion, SWNT-PEI/siRNA/NGR is a novel and promising anticancer system by combining gene therapy and photothermal therapy.
Two distinct proteins, streptavidin and HupR, bind and form regular helical arrays on the surface of multiwalled carbon nanotubes under appropriate conditions (see picture). The decoration of the outer surface of these nanostructures with biological macromolecules was investigated by electron microscopy.
Multi-walled carbon nanotubes functionalized with diethylentriaminepentaacetic dianhydride (DTPA-MWNT) and radiolabeled with Indium-111, having therapeutic and diagnostic applications, were tracked in the systemic blood circulation and the excretory system using a microSingle Photon Emission Tomography (microSPECT) scanner. Quantitative Kaiser test was used to determine the number of free amino groups on the DTPA-MWNT. A suspension of PBS and DTPA-MWNT was intravenously injected by tail vein in the Wistar rats and the urine production, water consumption and body weight were observed for 24 hours. The rat was necropised and tissues of various organs were fixed and observed with a Nikon Microphot-FXA microscope coupled with digital camera. The Length of DTPA-MWNT used in the study is larger than the dimensions of the glomerular capillary wall, so it was excreted through urine after 24 hours post-administration.
Delivery is one of the most critical obstacles confronting nanoparticle use in cancer diagnosis and therapy. For most oncological applications, nanoparticles must extravasate in order to reach tumor cells and perform their designated task. However, little understanding exists regarding the effect of nanoparticle shape on extravasation. Herein we use real-time intravital microscopic imaging to meticulously examine how two different nanoparticles behave across three different murine tumor models. The study quantitatively demonstrates that high-aspect ratio single-walled carbon nanotubes (SWNTs) display extravasational behavior surprisingly different from, and counterintuitive to, spherical nanoparticles although the nanoparticles have similar surface coatings, area, and charge. This work quantitatively indicates that nanoscale extravasational competence is highly dependent on nanoparticle geometry and is heterogeneous.
Since their first observation nearly a decade ago by Iijima (Iijima S. Helical microtubules of graphitic carbon Nature. 1991; 354:56–8), carbon nanotubes have been the focus of considerable research. Numerous investigators have since reported remarkable physical and mechanical properties for this new form of carbon. From unique electronic properties and a thermal conductivity higher than diamond to mechanical properties where the stiffness, strength and resilience exceeds any current material, carbon nanotubes offer tremendous opportunities for the development of fundamentally new material systems. In particular, the exceptional mechanical properties of carbon nanotubes, combined with their low density, offer scope for the development of nanotube-reinforced composite materials. The potential for nanocomposites reinforced with carbon tubes having extraordinary specific stiffness and strength represent tremendous opportunity for application in the 21st century. This paper provides a concise review of recent advances in carbon nanotubes and their composites. We examine the research work reported in the literature on the structure and processing of carbon nanotubes, as well as characterization and property modeling of carbon nanotubes and their composites.
Direct laser vaporization of transition-metal/graphite composite rods produced single-walled carbon nanotubes (SWT) in the condensing vapor in a heated flow tube. A much higher yield of nanotubes was found, with little of the amorphous overcoating on those produced by the metal-catalyzed arc-discharge method. A mixture of Co with Ni catalyzed about 50% of all the carbon vaporized to SWT. A model for SWT growth is presented for both the present case and the arc in which the metal particle size is limited due to the concurrent carbon condensation.
Mammalian cell expression has become the dominant recombinant protein production system for clinical applications because of its capacity for post-translational modification and human protein-like molecular structure assembly. While expression and production have been fully developed and Chinese hamster ovary cells are used for the majority of products both on the market and in clinical development, significant progresses in developing and engineering new cell lines, introducing novel genetic mechanisms in expression, gene silencing, and gene targeting, have been reported in the last several years. With the latest analytical methods development, more attention is being devoted towards product quality including glycol profiling, which leads to better understanding the impact of culture condition during production. Additionally, transient gene expression technology platform plays more important role in biopharmaceutical early development stages. This review focused on the latest advancements in the field, especially in active areas such as expression systems, glycosylation impact factors, and transient gene expression.