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1-Dimensional nanoparticles – A brief critical review on biological, medical, and toxicological aspects
Applied Surface Science 275 (2013) 2–6
Contents lists available at SciVerse ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
1-Dimensional nanoparticles – A brief critical review on biological, medical, and toxicological aspects B.M. Popescu a , N. Ali b , G. Basturea c , G.I. Comsa a , L.A. Materon d , M. Chipara d,∗ a
Ovidius University, Faculty of Medicine, Constanta, Romania CNC Coatings, Rochdale, United Kingdom University of Miami, Biochemistry and Molecular Biology Department, FL, USA d University of Texas Pan American, Edinburg, TX, USA b c
a r t i c l e
i n f o
Article history: Received 1 October 2012 Received in revised form 15 January 2013 Accepted 16 January 2013 Available online 30 January 2013 Keywords: One dimensional materials Nanomaterials Carbon nanotubes Toxicology Biological and medical aspects Asbestos Amyloids Partial phagocytosis
a b s t r a c t A critical review of the biological and medical effects of one dimensional nanoparticles is presented. The review focuses on the toxicity of carbon nanotubes – a subject under strong debate and discusses briefly the most important concepts. It is tentatively suggested that the huge aspect ratio and the high Young modulus of carbon nanotubes are playing an important role in the biological, medical, and toxicological aspects of carbon nanotubes despite their low intrinsic biochemical activity. The most suitable biomedical model for the biological and medical effects of carbon nanotubes is described by the so-called impaling mode which appears to be qualitatively consistent with partial phagocytosis. Published by Elsevier B.V.
1. Introduction Nanomaterials are invading our current life. There are still questions raised about their biological and medical effects on humans and on the environment, with conflicting reports and points of view. The goal of this brief review is to summarize and crystallize recent research reports into a general picture focused on the biological and medical effects of one-dimensional nanomaterials, with a special focus on carbon nanotubes. Within this paper, carbon based materials (CBM) will include fullerenes, carbon nanotubes (single walled carbon nanotubes: SWCNTs, double walled carbon nanotubes: DWCNTs, multiwalled carbon nanotubes: MWCNTs, helical multiwalled carbon nanotubes: HMWCNTs), carbon fibers (CFs), carbon nanofibers (CNFs), graphene, nanographite, and nanodiamonds. The one dimensional carbon materials (1DCMs) will include SWCNTs, DWCNTs, MWCNTs, HMWCNTs, and CNF. For comparison, the analysis will include halloysite, which is a naturally
∗ Corresponding author at: The University of Texas Pan American, Department of Physics and Geology, 1201W, University Drive, Edinburg, TX 78541, USA. Tel.: +1 9566055123. E-mail addresses: [email protected], [email protected] (M. Chipara). 0169-4332/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.apsusc.2013.01.122
occurring tubular alumino-silicate (clay), and crysolite, which is also a nanotube (actually the most dangerous type of asbestos). In addition, the study will contain also references to the amyloid fiber protein (with an average diameter of 5–10 nm [1,2]) and an average length of about 100 nm. In the one dimensional form, this protein is responsible for a lethal malady: amyloidosis. All these materials have some common features; they are onedimensional, their diameter is confined at the nanometer scale, they have a rather large aspect ratio (the ratio between their length and their diameter is usually greater than 10). The amyloid fiber is an insoluble stiff lethal (nano)protein. Asbestos are crossing into the inorganic world as one-dimensional materials with recognized toxicity. Isolated asbestos fibrils are in the nanometer range but they are frequently occurring in bundles with diameters over 100 nm. Consequently, some simple obvious questions are emerging: Are carbon nanotubes and all one-dimensional materials toxic? Is there a correlation between shape (or aspect ratio) and toxicity? 1. What is the role of the size? 2. What are the consequences of the mechanical properties of one dimensional materials on their toxicity? 3. What is the effect of the surface on the toxicity of one dimensional nanomaterials? Within the category of one dimensional nanomaterials, carbon nanotubes play an important role because they are the most
B.M. Popescu et al. / Applied Surface Science 275 (2013) 2–6
frequently investigated one dimensional materials and because carbon is perceived as an element with no intrinsic toxicity due to its reduced chemical (and in particular biochemical) reactivity. The addition of carbon nanotubes can initiate or accelerate several activities within biological cells and organisms such as inflammatory, genotoxicity, and/or carcinosis activities [3]. Most studies are showing a certain degree of inflammatory activities (resulting eventually in the formation of granulomas [4]) due to the presence of carbon nanotubes. The induced genotoxicity of carbon nanotubes is still under debate, and there is a strong controversy regarding the carcinogen features of carbon nanotubes. Typically, 1DCMs are either tubular or cylindrical structures with the ends opened or closed. Their cross section can be assimilated with ellipses but in most cases it is assumed – for simplicity – that their cross sections are circular. In all cases, such nanostructures are confined at the nanometer scale in two dimensions, which define their (cross) section. The length of 1DCMs extends frequently beyond the nanometer scale and for most 1DCMs is within the micron scale (typically up to 102 m). The nanometer scale confinement is described by a single parameter (the radius in the case of nanocylinders) or by two parameters (internal and external radii (in the case of nanotubes). The theoretical situation of carbon fibers characterized by an external radius at the micron scale will not be analyzed in detail. While the discussion is focused on carbon nanotubes (i.e. on 1DCMs with an internal diameter that defines a lumen, which eventually it is open) the analysis will assume that actually all these 1DCMs are cylinders-like nanomaterials (i.e. the effects of the inner surface will be neglected). Formally, this is identical with the situation in which all nanotubes are completely closed (sealed). Scale is a very important parameter in science and in particular in nanoscience but traditionally is disregarded in toxicity related studies. There are several definitions for the nanometer scale. Here, an extended nanometer scale ranging from 0.1 nm to 1000 nm will be considered. Most known viruses will be confined within this extended nanometer range (which will contain typically DNA, RNA, and proteins). Some bacteria will also fall within this extended nanometer range but most will populate the micron scale that ranges from 1.000 nm to 1,000,000 nm. The millimeter-meter scale (or briefly the meter scale) will range from 106 nm to 1011 nm and will include complex organisms, plants, animals, and humans. Scales in excess of 100 m are of limited interest from biological, toxicological, and medical perspectives but may play an important role in environmental studies. The interaction between an 1DCM and a biologic target (or prey) presents specific features such as: 1.1. Intrinsic (chemical and biochemical) toxicity of CBM Ideal CBMs contain only carbon atoms and consequently are supposed to be intrinsically non-toxic, due to their reduced biochemical (and chemical) reactivity at usual temperatures and pressures. Carbon materials are typically stable in air up to temperatures higher than 300 ◦ C and in nitrogen or vacuum above 500 ◦ C. While carbon is not expected to initiate or participate in biochemical reactions, carbon materials can be functionalized by acidic or basic treatments. Functionalization of carbon nanotubes has been reported elsewhere in both basic and acid (HCl) media [5–7]. For example, ingested not functionalized 1DCM can be functionalized by the acidic pH within stomach, opening new paths for biochemical reactions [6]. Real CBMs, and in particular real 1DCMs, present chemical impurities (typically represented by catalyst residues) and structural defects [7]. Both of them can contribute to a certain chemical or biochemical activity. Typically, catalysts residues and structural defects are located at the extremities of 1DCMs. The mechanism of the intrinsic toxicity is represented by chemical and biochemical reactions, which are occurring irrespective of the nanometer-sized confinement. It is expected that this
3
mechanisms are not very efficient, at least for short retention times of carbonaceous nanomaterials within the biological target. Experimental in vivo studies [8] on the toxicity of fullerenes nanoparticles (diameter 55 nm and 930 nm) to rats exposed to aerosols containing fullerenes (3 h per day during 10 days) did not reveal significant toxicological issues. This supports the low biochemical reactivity of carbon based materials and nanomaterials. The table shows various parameters of different one dimensional materials and carbon based nanomaterials. It is noticed that fullerenes are characterized by a rather large surface to area value (comparable to SWCNTs) but a very small aspect ratio. This suggests that the biological and medical effects of 1DCMs are not surface effects but merely aspect ratio controlled effects. 1.2. Biodegradability and biopersistence Organisms and cells are using a variety of techniques to remove foreign (nano)materials. As a consequence, the nanomaterials that invaded a cell or an organism will be removed after a certain time. The average time during which the foreign nanomaterial is excreted is defining the biopersistence. Due to their reduced chemical and biochemical reactivity, 1DCMs are expected to have a low biodegradability. In the case of fullerenes, which are confined at nanometer scale along all three directions, the cells can excrete relatively easy such impurities. The huge aspect ratio of 1DCMs makes difficult their transport and finally their excretion. Hence, in the case of 1DCMs the excretion is particularly difficult, resulting in a long biopersistence. This raises a very particular issue: the biotoxicity of a nanomaterial should be estimated over at time that is comparable to the average time required to excrete or destroy 66% of the initial nanoparticles, which will be named biotoxicity lifetime. In the case of fullerene this time can be easily estimated and should not be very large. In the case of 1DCMs and in particular of CNTs with a high aspect ratio, the geometry of nanotubes, the reduced dimensions of the cell, and geometrical restrictions make difficult the excretion of 1DCMs and hence at least theoretically, the biotoxicity lifetime is limited by the lifetime of the target cell or organism. Projected onto medical studies this imposes to extend the investigation of the toxicity of 1 DCMs to the whole lifetime of the organism or of the patient. However, it is important to notice that carbon nanotubes have certain elasticity and a marked tendency to agglomerate, which may help isolating nanotube aggregates within various organisms, reducing thus their toxicity. Due to the huge Young modulus of 1DCMs [9], the destruction, breaking, or fragmentation of such materials within cells or organisms is highly improbable. The very long biotoxicity lifetime (theoretically infinite) and consequently the large residence time of 1DCMs compensate the rather low chemical and biochemical reactivity. Hence, during the biotoxicity lifetime of 1DCMs their (biochemical) functionalization is possible and consequently local chemical and biochemical modifications may occur and play an important role. Such functionalization can occur in certain environments such as the acidic environment of the stomach. 1.3. Morphological changes including conformational transitions The contact between the cellular membrane and 1DCMs can induce structural reorganization within the membrane of biological cells. Typically, such changes are not expected from a fresh pure 1DCM. However, due to the long lifetime of 1DCMs within biological targets, the functionalization of 1DCMs may result further in morphological phase transitions (such as protein collapse, protein folding, or protein misfolding) occurring on the surface of the cells and eventually later to chemical or biochemical reactions. Carbon nanotubes have the tendency to agglomerate and bundle.
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Fig. 1. Graphical representation of the three modes that describe the interaction between nanotubes and biological cells (in this case blood cells). The top panel describes the safe mode SM), the middle panel is representing the fakir mode (FM), and the bottom mode the impale mode (IM). To the left of each mode is shown qualitatively the stress ()–strain (ε) dependence for the cellular membrane with a localization of the mechanical domain associated to each mode.
This physical tendency may contribute also to the toxicity of some one-dimensional materials. 1.4. Size effects Typically the virus has a characteristic size of about 100 nm. Assuming that the virus and 1DCMs have the same density, the mass of such a virus is up to 100 times smaller than the mass of a SWNT. Even in an empty space, at thermal equilibrium, the virus will be subjected to more ample thermal equilibrium motions. Hence, the 1DCM will eventually operate in the impaling mode, penetrating the membrane of the virus (see the last panel of Fig. 1). In the absence of the thermal motion, local healing with a partial loss of membrane permeability is expected. In the presence of the thermal motion the hole produced within the viral membrane cell will increase up to the point at which the content is completely lost. This mechanisms was already documented and named partial phagocytosis [10,11]. The biological and medical effects of 1DCM can be described within few simple models: I The Scale-Less (SL) model, according to which the size does not affect the biological and medical activity. The model is oversimplified and denies the three important features that distinguish between bulk materials and nanomaterials: The quantum effects (due to the confinement of the nanomaterials), the surface effect (due to the huge surface to volume ratios of nanomaterials compared to bulk materials), and the role of geometry and spatial restrictions. According to this model, 1DCM are not toxic and have no biological or medical effects. The model exploits the reduced chemical and biochemical activity of carbon. II The Continuous Historical Approach (CHA) is technically reducible to the SL model. Several authors argue that atoms and molecules were interacting with biological system since life appeared on Earth, and hence nanomaterials are not a new threat to the life on Earth. While this is true, we need to face the reality that in the size range 100 nm down to about 1 nm there is a drastic change in many if not all physical properties of the matter and that this “nanomatter” has neither the physical and chemical properties of the bulk matter nor the features of the corresponding
isolated atoms or molecules. Few authors speculated about the fact that some nanomaterials such as carbon nanotubes were reported in antique artifacts such as the Toledo saber or that halloysite is a naturally occurring nanotube (aluminosilicate). The presence of fullerene in volcanic ashes or in about 10,000-year-old ice cores was reported elsewhere [1]. While these are scientifically proven, we can argue that the expected concentration of these nanomaterials was significantly low, and that naturally occurring nanomaterials (such as halloysite, carbon nanotubes, etc) were sporadically used. The model suggests that no (new) biological or medical effects are expected from nanomaterials. III The Impale Mode (via the partial phagocytosis mechanism). The static partial phagocytosis mechanism for biological and medical effects of 1DCMs can be noticed even for larger cells including bacteria. As long as the biological target is not able to engulf completely the 1DCM, the partial phagocytosis has been recognized as a potential explanation for the toxicity of 1DCMs. Due to their huge aspect ratios (see Table 1), carbon nanotubes are not completely engulfed by viruses, bacteria, and most biological cells. Consequently, a complete phagocytosis as a healing mechanism is almost impossible. Within the static (frozen) partial phagocytosis model, the 1DCM impales the biological target. Neither the 1DCMs nor the biological targets are moving one relative to other. This eventually allows for the biological target to heal and makes the impaling effect eventually improbable but not impossible. While it is expected that the tissue that will surround the impaling 1DCM will present slightly different physical, chemical, or morphological features such as a reduced elasticity and permeability, the biological target will eventually survive to the “attack” of 1DCM. The dynamic partial phagocytosis model describes more realistic situations. As in the previous model, the biological target did not engulf completely the 1DCM. At the extreme of very small biological targets such as viruses, due to the thermal equilibrium, the biological target will be subjected to more ample motions than the 1DCMs, which will result in the tearing of the viral cellular membrane. As the size of the biological target increases, the thermal motion of the 1DCM becomes more ample than the thermal motion of the biological target, and hence the control of the destruction process
B.M. Popescu et al. / Applied Surface Science 275 (2013) 2–6
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Table 1 Physical properties of some 1 dimensional nanomaterials.
SWCNT DWCNT MWCNT HMWCNT CNF CF Fullerene C60 Halloysite Asbetos (chrysotile)
Average external diameter [nm]
Average internal diameter [nm]
1.2 3.0 40 150 150 5000 1.0 50 25 500
1.0 1.5 10
Average length [nm] 16,000 50,000 15,000 5000 100,000 107 1.0 50,000 107 107
Average external side area [nm2 ]
Average external volume [nm3 ]
Surface/ volume [nm−1 ]
Young modulus [GPa]
5 × 104 5 × 105 106 106 5 × 107 5 × 107 5 107 104 5 × 105
104 5 × 105 107 108 109 109 1 108 5 × 104 107
5 1 0.1 0.05 0.05 0.01 5 0.1 0.1 0.05
1000 1000 1200 900
is transferred to the 1DCMs. The confinement and interlocking of 1DCM can contribute significantly to the amplification of biological and medical effects, via additional or accelerated ripping. For the one dimensional nanomaterials lung is a preferred organ. Consequently an important effort was directed towards the understanding of the pulmonary toxicity of such materials. However, the in vivo studies of the pulmonary toxicity of 1DCMs are facing complex issues. The use of aerosols does not allow an accurate estimation of nanoparticles actually reaching the lung. Thus, instillations appear more accurate. First in vivo studies on the toxicity of 1DCMs suggested huge effects. Later, it was understood that actually the nanomaterial aggregated and blocked the airways. In vivo studies of the effects of DWCNTs on rats [12] confirmed their toxicity and revealed a decrease of the local oxidative stress assigned to scavenger capabilities of 1DCMs. In an excellent review on the toxicity of carbon nanotubes [13], it was concluded that carbon nanotubes have an enhanced toxicity compared to the toxicity of carbon or even quartz particles, suggesting the importance of the aspect ratio. The authors noticed that 1DCM stimulate the growth of mesenchymal stem cells growth resulting in inflammation, fibrosis, and finally in cancer. Some authors [14] were trying to make a distinction between not functionalized 1DCM and functionalized 1DCM, aiming towards the insolubility of pristine SWCNTs or MWCNTs. Why the experimental data on the toxicity of 1DCM are so scattered? We speculate that the main reasons for the discrepancies noticed in the reported toxicity of 1DCM derive from the experimental design. A bed of 1DCM is not expected to show any significant biological effect on cells. A very dense forest of 1DCM will fall in the same category (see the top panel of Fig. 1). By decreasing the density of the forest, the weight of the cell and the elasticity of the cell, combined with the mechanical strength of carbon nanotubes can result in local modification, while the cell is not yet perforated. This corresponds to the fakir mode (FM) shown in Fig. 1. Only if the pressure exerted on the cell exceeds a certain critical value p*, the cellular membrane is penetrated, the cell is impaled and the healing mechanism (partial phagocytosis) is ignited, with all its subsequent secondary effects. Typically this is not a robust healing but rather a temporary patch. The toxicity of 1DCM on organism can be amplified (compared to the cellular toxicity) by geometrical restrictions and hindrances, which make difficult the motion and finally the excretion of the 1DCM. Aspect ratio now becomes a very important parameter. This description explains the reason for which important differences are expected between the results of the in vitro and in vivo studies on the toxicity of 1DCMs. The in vitro studies have the tendency to distort the results towards the SM or FM mode show weak or no toxic effects. The time scale of the experiment is extremely important and should scale with the biotoxicity lifetime of the 1DCM, which is expected to be very long. Consequently, such
300 300
Aspect ratio
104 –105 104 102 –103 102 103 103 –104 1.0 103 105 –106 104 –105
OBS
Individual fibrils Bundle
toxicity studies on animals should be extended over the whole lifetime of the animal. 2. Conclusions The general question is about the existence of an 1 dimensional toxicity. The case of asbestos has been confirmed since decades. The fibrous structure of amyloids proteins suggests the possibility of an one dimensional toxicity. Experimental data on carbon nanotubes appears to converge towards an enhanced toxicity compared to C60 . The impale model describes better the biological and medical effects of 1DCM. The main biological process is the partial phagocytosis, and the dominant mechanism is the impaling of the biological target by the 1DCM. Hence, while carbon-based nanomaterials are expected to have a reduced toxicity (such as C60 ) the large aspect ratio and the huge Young modulus of the 1DCMs ignite a specific mechanism for cell deterioration that activates a strong toxic response. Shape-induced or enhanced toxicity can explain the adverse effects of asbestos [15] and of amyloid proteins. It is the authors’ point of view that the actual debate regarding the effect of 1DCMs derives from sensible differences in the experimental details. As long as 1DCMs are allowed to sediment during the experiment, the impaling mechanism is deactivated (or frozen) and the adverse effect of 1DCM is masked by their reduced chemical and biological activity (at least at short term). Such a situation occurs dominantly during in vitro experiments. During experiments that allows the motion of 1DCM and in vivo studies, the motions, the confinement restrictions, and the (inter)locking of biological target-1DCM activates or amplifies the impale mechanism and eventually turn on the destruction of the biological target. Of a particular importance is the biotoxicity lifetime of the nanomaterials. In the case of 1DCM the theoretical value is suggested to be infinite, and hence it is considered the biotoxicity lifetime of carbon nanotubes should be set equal to the natural average lifetime of the target. There is a converging agreement according to which the toxicity of 1DCM depends on their length [4]. This brief review went beyond by indicating that the toxicity of 1DCM is controlled by the aspect ratio, diameter, and mechanical properties (with Young modulus as one of the most important parameters). In the last years, important research efforts have been done to use 1DCM as vectors for drug release, antigens, and genes delivery [16]. The final demonstration of the adverse effects of the impale mechanism would affect dramatically these projects. An asbestoslike behavior of 1DCM has been speculated by many authors [11,17] as a potential scenario for the toxicity of 1DCM. A final step in this analysis is to demonstrate if the toxicity is due to the pair aspect ratio-mechanical properties (large Young modulus) or if it is triggered just by the aspect ratio. If mechanical properties play an important role, then it is possible to find some soft nanotubes,
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which can replace 1DCM in medical applications. However, the story of amyloids proteins leaves too little hope. References [1] M.F. Perutz, J.T. Finch, J. Berriman, A. Lesk, Amyloid fibers are water-filled nanotubes, Proceedings of the National Academy of Sciences of the United States of America 99 (2002) 5591–5595. [2] J.D. Sipe, M.D. Benson, J.N. Buxbaum, S.-I. Ikeda, G. Merlini, M.J.M. Saraiva, et al., Amyloid fibril protein nomenclature: 2010 recommendations from the nomenclature committee of the International Society of Amyloidosis, Amyloid: The International Journal of Experimental and Clinical Investigation: the Official Journal of the International Society of Amyloidosis 17 (2010) 101–104. [3] J. Muller, M. Delos, N. Panin, V. Rabolli, F. Huaux, D. Lison, Absence of carcinogenic response to multiwall carbon nanotubes in a 2-year bioassay in the peritoneal cavity of the rat, Toxicological Sciences: An Official Journal of the Society of Toxicology 110 (2009) 442–448. [4] C.A. Poland, R. Duffin, I. Kinloc, A. Maynard, W.A.H. Wallace, A. Seaton, et al., Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study, Nature Nanotechnology 3 (2008) 423–428. [5] D. Tasis, N. Tagmatarchis, V. Georgakilas, M. Prato, Soluble carbon nanotubes, Chemistry (Weinheim an Der Bergstrasse, Germany) 9 (2003) 4000–4008. [6] H. Kuzmany, a. Kukovecz, F. Simon, M. Holzweber, C. Kramberger, T. Pichler, Functionalization of carbon nanotubes, Synthetic Metals 141 (2004) 113–122. [7] N. Ali, M. Chipara, J.B. Bai, Spectroscopic investigations on epoxy–multiwall carbon nanotubes composites, Journal of Nanoscience and Nanotechnology 11 (2011) 9050–9056.
[8] G.L. Baker, A. Gupta, M.L. Clark, B.R. Valenzuela, L.M. Staska, S.J. Harbo, et al., Inhalation toxicity and lung toxicokinetics of C60 fullerene nanoparticles and microparticles, Toxicological Sciences: An Official Journal of the Society of Toxicology 101 (2008) 122–131. [9] M. Terrones, Science And Technology of the twenty-first century: synthesis, properties, and applications of carbon nanotubes, Annual Review of Materials Research 33 (2003) 419–501. [10] J.C. Bonner, Lung fibrotic responses to particle exposure, Toxicologic Pathology 35 (2007) 148–153. [11] J.C. Bonner, J.W. Card, D.C. Zeldin, Nanoparticle-mediated drug delivery and pulmonary hypertension, Hypertension 53 (2009) 751–753. [12] D. Crouzier, S. Follot, E. Gentilhomme, E. Flahaut, R. Arnaud, V. Dabouis, et al., Carbon nanotubes induce inflammation but decrease the production of reactive oxygen species in lung, Toxicology 272 (2010) 39–45. [13] K. Donaldson, R. Aitken, L. Tran, V. Stone, R. Duffin, G. Forrest, et al., Carbon nanotubes: a review of their properties in relation to pulmonary toxicology and workplace safety, Toxicological Sciences: An Official Journal of the Society of Toxicology 92 (2006) 5–22. [14] H. Dumortier, S. Lacotte, G. Pastorin, R. Marega, W. Wu, D. Bonifazi, et al., Functionalized carbon nanotubes are non-cytotoxic and preserve the functionality of primary immune cells, Nano Letters 6 (2006) 1522–1528. [15] M.-C.F. Jaurand, A. Renier, J. Daubriac, Mesothelioma, Do asbestos and carbon nanotubes pose the same health risk? Particle and Fibre Toxicology 6 (2009) 16. [16] A. Bianco, K. Kostarelos, C.D. Partidos, M. Prato, Biomedical applications of functionalised carbon nanotubes, Chemical Communications (Cambridge, England) 41 (2005) 571–577. [17] C.P. Firme, P.R. Bandaru, Toxicity issues in the application of carbon nanotubes to biological systems, Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 245–256.
Contents lists available at SciVerse ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
1-Dimensional nanoparticles – A brief critical review on biological, medical, and toxicological aspects B.M. Popescu a , N. Ali b , G. Basturea c , G.I. Comsa a , L.A. Materon d , M. Chipara d,∗ a
Ovidius University, Faculty of Medicine, Constanta, Romania CNC Coatings, Rochdale, United Kingdom University of Miami, Biochemistry and Molecular Biology Department, FL, USA d University of Texas Pan American, Edinburg, TX, USA b c
a r t i c l e
i n f o
Article history: Received 1 October 2012 Received in revised form 15 January 2013 Accepted 16 January 2013 Available online 30 January 2013 Keywords: One dimensional materials Nanomaterials Carbon nanotubes Toxicology Biological and medical aspects Asbestos Amyloids Partial phagocytosis
a b s t r a c t A critical review of the biological and medical effects of one dimensional nanoparticles is presented. The review focuses on the toxicity of carbon nanotubes – a subject under strong debate and discusses briefly the most important concepts. It is tentatively suggested that the huge aspect ratio and the high Young modulus of carbon nanotubes are playing an important role in the biological, medical, and toxicological aspects of carbon nanotubes despite their low intrinsic biochemical activity. The most suitable biomedical model for the biological and medical effects of carbon nanotubes is described by the so-called impaling mode which appears to be qualitatively consistent with partial phagocytosis. Published by Elsevier B.V.
1. Introduction Nanomaterials are invading our current life. There are still questions raised about their biological and medical effects on humans and on the environment, with conflicting reports and points of view. The goal of this brief review is to summarize and crystallize recent research reports into a general picture focused on the biological and medical effects of one-dimensional nanomaterials, with a special focus on carbon nanotubes. Within this paper, carbon based materials (CBM) will include fullerenes, carbon nanotubes (single walled carbon nanotubes: SWCNTs, double walled carbon nanotubes: DWCNTs, multiwalled carbon nanotubes: MWCNTs, helical multiwalled carbon nanotubes: HMWCNTs), carbon fibers (CFs), carbon nanofibers (CNFs), graphene, nanographite, and nanodiamonds. The one dimensional carbon materials (1DCMs) will include SWCNTs, DWCNTs, MWCNTs, HMWCNTs, and CNF. For comparison, the analysis will include halloysite, which is a naturally
∗ Corresponding author at: The University of Texas Pan American, Department of Physics and Geology, 1201W, University Drive, Edinburg, TX 78541, USA. Tel.: +1 9566055123. E-mail addresses: [email protected], [email protected] (M. Chipara). 0169-4332/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.apsusc.2013.01.122
occurring tubular alumino-silicate (clay), and crysolite, which is also a nanotube (actually the most dangerous type of asbestos). In addition, the study will contain also references to the amyloid fiber protein (with an average diameter of 5–10 nm [1,2]) and an average length of about 100 nm. In the one dimensional form, this protein is responsible for a lethal malady: amyloidosis. All these materials have some common features; they are onedimensional, their diameter is confined at the nanometer scale, they have a rather large aspect ratio (the ratio between their length and their diameter is usually greater than 10). The amyloid fiber is an insoluble stiff lethal (nano)protein. Asbestos are crossing into the inorganic world as one-dimensional materials with recognized toxicity. Isolated asbestos fibrils are in the nanometer range but they are frequently occurring in bundles with diameters over 100 nm. Consequently, some simple obvious questions are emerging: Are carbon nanotubes and all one-dimensional materials toxic? Is there a correlation between shape (or aspect ratio) and toxicity? 1. What is the role of the size? 2. What are the consequences of the mechanical properties of one dimensional materials on their toxicity? 3. What is the effect of the surface on the toxicity of one dimensional nanomaterials? Within the category of one dimensional nanomaterials, carbon nanotubes play an important role because they are the most
B.M. Popescu et al. / Applied Surface Science 275 (2013) 2–6
frequently investigated one dimensional materials and because carbon is perceived as an element with no intrinsic toxicity due to its reduced chemical (and in particular biochemical) reactivity. The addition of carbon nanotubes can initiate or accelerate several activities within biological cells and organisms such as inflammatory, genotoxicity, and/or carcinosis activities [3]. Most studies are showing a certain degree of inflammatory activities (resulting eventually in the formation of granulomas [4]) due to the presence of carbon nanotubes. The induced genotoxicity of carbon nanotubes is still under debate, and there is a strong controversy regarding the carcinogen features of carbon nanotubes. Typically, 1DCMs are either tubular or cylindrical structures with the ends opened or closed. Their cross section can be assimilated with ellipses but in most cases it is assumed – for simplicity – that their cross sections are circular. In all cases, such nanostructures are confined at the nanometer scale in two dimensions, which define their (cross) section. The length of 1DCMs extends frequently beyond the nanometer scale and for most 1DCMs is within the micron scale (typically up to 102 m). The nanometer scale confinement is described by a single parameter (the radius in the case of nanocylinders) or by two parameters (internal and external radii (in the case of nanotubes). The theoretical situation of carbon fibers characterized by an external radius at the micron scale will not be analyzed in detail. While the discussion is focused on carbon nanotubes (i.e. on 1DCMs with an internal diameter that defines a lumen, which eventually it is open) the analysis will assume that actually all these 1DCMs are cylinders-like nanomaterials (i.e. the effects of the inner surface will be neglected). Formally, this is identical with the situation in which all nanotubes are completely closed (sealed). Scale is a very important parameter in science and in particular in nanoscience but traditionally is disregarded in toxicity related studies. There are several definitions for the nanometer scale. Here, an extended nanometer scale ranging from 0.1 nm to 1000 nm will be considered. Most known viruses will be confined within this extended nanometer range (which will contain typically DNA, RNA, and proteins). Some bacteria will also fall within this extended nanometer range but most will populate the micron scale that ranges from 1.000 nm to 1,000,000 nm. The millimeter-meter scale (or briefly the meter scale) will range from 106 nm to 1011 nm and will include complex organisms, plants, animals, and humans. Scales in excess of 100 m are of limited interest from biological, toxicological, and medical perspectives but may play an important role in environmental studies. The interaction between an 1DCM and a biologic target (or prey) presents specific features such as: 1.1. Intrinsic (chemical and biochemical) toxicity of CBM Ideal CBMs contain only carbon atoms and consequently are supposed to be intrinsically non-toxic, due to their reduced biochemical (and chemical) reactivity at usual temperatures and pressures. Carbon materials are typically stable in air up to temperatures higher than 300 ◦ C and in nitrogen or vacuum above 500 ◦ C. While carbon is not expected to initiate or participate in biochemical reactions, carbon materials can be functionalized by acidic or basic treatments. Functionalization of carbon nanotubes has been reported elsewhere in both basic and acid (HCl) media [5–7]. For example, ingested not functionalized 1DCM can be functionalized by the acidic pH within stomach, opening new paths for biochemical reactions [6]. Real CBMs, and in particular real 1DCMs, present chemical impurities (typically represented by catalyst residues) and structural defects [7]. Both of them can contribute to a certain chemical or biochemical activity. Typically, catalysts residues and structural defects are located at the extremities of 1DCMs. The mechanism of the intrinsic toxicity is represented by chemical and biochemical reactions, which are occurring irrespective of the nanometer-sized confinement. It is expected that this
3
mechanisms are not very efficient, at least for short retention times of carbonaceous nanomaterials within the biological target. Experimental in vivo studies [8] on the toxicity of fullerenes nanoparticles (diameter 55 nm and 930 nm) to rats exposed to aerosols containing fullerenes (3 h per day during 10 days) did not reveal significant toxicological issues. This supports the low biochemical reactivity of carbon based materials and nanomaterials. The table shows various parameters of different one dimensional materials and carbon based nanomaterials. It is noticed that fullerenes are characterized by a rather large surface to area value (comparable to SWCNTs) but a very small aspect ratio. This suggests that the biological and medical effects of 1DCMs are not surface effects but merely aspect ratio controlled effects. 1.2. Biodegradability and biopersistence Organisms and cells are using a variety of techniques to remove foreign (nano)materials. As a consequence, the nanomaterials that invaded a cell or an organism will be removed after a certain time. The average time during which the foreign nanomaterial is excreted is defining the biopersistence. Due to their reduced chemical and biochemical reactivity, 1DCMs are expected to have a low biodegradability. In the case of fullerenes, which are confined at nanometer scale along all three directions, the cells can excrete relatively easy such impurities. The huge aspect ratio of 1DCMs makes difficult their transport and finally their excretion. Hence, in the case of 1DCMs the excretion is particularly difficult, resulting in a long biopersistence. This raises a very particular issue: the biotoxicity of a nanomaterial should be estimated over at time that is comparable to the average time required to excrete or destroy 66% of the initial nanoparticles, which will be named biotoxicity lifetime. In the case of fullerene this time can be easily estimated and should not be very large. In the case of 1DCMs and in particular of CNTs with a high aspect ratio, the geometry of nanotubes, the reduced dimensions of the cell, and geometrical restrictions make difficult the excretion of 1DCMs and hence at least theoretically, the biotoxicity lifetime is limited by the lifetime of the target cell or organism. Projected onto medical studies this imposes to extend the investigation of the toxicity of 1 DCMs to the whole lifetime of the organism or of the patient. However, it is important to notice that carbon nanotubes have certain elasticity and a marked tendency to agglomerate, which may help isolating nanotube aggregates within various organisms, reducing thus their toxicity. Due to the huge Young modulus of 1DCMs [9], the destruction, breaking, or fragmentation of such materials within cells or organisms is highly improbable. The very long biotoxicity lifetime (theoretically infinite) and consequently the large residence time of 1DCMs compensate the rather low chemical and biochemical reactivity. Hence, during the biotoxicity lifetime of 1DCMs their (biochemical) functionalization is possible and consequently local chemical and biochemical modifications may occur and play an important role. Such functionalization can occur in certain environments such as the acidic environment of the stomach. 1.3. Morphological changes including conformational transitions The contact between the cellular membrane and 1DCMs can induce structural reorganization within the membrane of biological cells. Typically, such changes are not expected from a fresh pure 1DCM. However, due to the long lifetime of 1DCMs within biological targets, the functionalization of 1DCMs may result further in morphological phase transitions (such as protein collapse, protein folding, or protein misfolding) occurring on the surface of the cells and eventually later to chemical or biochemical reactions. Carbon nanotubes have the tendency to agglomerate and bundle.
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Fig. 1. Graphical representation of the three modes that describe the interaction between nanotubes and biological cells (in this case blood cells). The top panel describes the safe mode SM), the middle panel is representing the fakir mode (FM), and the bottom mode the impale mode (IM). To the left of each mode is shown qualitatively the stress ()–strain (ε) dependence for the cellular membrane with a localization of the mechanical domain associated to each mode.
This physical tendency may contribute also to the toxicity of some one-dimensional materials. 1.4. Size effects Typically the virus has a characteristic size of about 100 nm. Assuming that the virus and 1DCMs have the same density, the mass of such a virus is up to 100 times smaller than the mass of a SWNT. Even in an empty space, at thermal equilibrium, the virus will be subjected to more ample thermal equilibrium motions. Hence, the 1DCM will eventually operate in the impaling mode, penetrating the membrane of the virus (see the last panel of Fig. 1). In the absence of the thermal motion, local healing with a partial loss of membrane permeability is expected. In the presence of the thermal motion the hole produced within the viral membrane cell will increase up to the point at which the content is completely lost. This mechanisms was already documented and named partial phagocytosis [10,11]. The biological and medical effects of 1DCM can be described within few simple models: I The Scale-Less (SL) model, according to which the size does not affect the biological and medical activity. The model is oversimplified and denies the three important features that distinguish between bulk materials and nanomaterials: The quantum effects (due to the confinement of the nanomaterials), the surface effect (due to the huge surface to volume ratios of nanomaterials compared to bulk materials), and the role of geometry and spatial restrictions. According to this model, 1DCM are not toxic and have no biological or medical effects. The model exploits the reduced chemical and biochemical activity of carbon. II The Continuous Historical Approach (CHA) is technically reducible to the SL model. Several authors argue that atoms and molecules were interacting with biological system since life appeared on Earth, and hence nanomaterials are not a new threat to the life on Earth. While this is true, we need to face the reality that in the size range 100 nm down to about 1 nm there is a drastic change in many if not all physical properties of the matter and that this “nanomatter” has neither the physical and chemical properties of the bulk matter nor the features of the corresponding
isolated atoms or molecules. Few authors speculated about the fact that some nanomaterials such as carbon nanotubes were reported in antique artifacts such as the Toledo saber or that halloysite is a naturally occurring nanotube (aluminosilicate). The presence of fullerene in volcanic ashes or in about 10,000-year-old ice cores was reported elsewhere [1]. While these are scientifically proven, we can argue that the expected concentration of these nanomaterials was significantly low, and that naturally occurring nanomaterials (such as halloysite, carbon nanotubes, etc) were sporadically used. The model suggests that no (new) biological or medical effects are expected from nanomaterials. III The Impale Mode (via the partial phagocytosis mechanism). The static partial phagocytosis mechanism for biological and medical effects of 1DCMs can be noticed even for larger cells including bacteria. As long as the biological target is not able to engulf completely the 1DCM, the partial phagocytosis has been recognized as a potential explanation for the toxicity of 1DCMs. Due to their huge aspect ratios (see Table 1), carbon nanotubes are not completely engulfed by viruses, bacteria, and most biological cells. Consequently, a complete phagocytosis as a healing mechanism is almost impossible. Within the static (frozen) partial phagocytosis model, the 1DCM impales the biological target. Neither the 1DCMs nor the biological targets are moving one relative to other. This eventually allows for the biological target to heal and makes the impaling effect eventually improbable but not impossible. While it is expected that the tissue that will surround the impaling 1DCM will present slightly different physical, chemical, or morphological features such as a reduced elasticity and permeability, the biological target will eventually survive to the “attack” of 1DCM. The dynamic partial phagocytosis model describes more realistic situations. As in the previous model, the biological target did not engulf completely the 1DCM. At the extreme of very small biological targets such as viruses, due to the thermal equilibrium, the biological target will be subjected to more ample motions than the 1DCMs, which will result in the tearing of the viral cellular membrane. As the size of the biological target increases, the thermal motion of the 1DCM becomes more ample than the thermal motion of the biological target, and hence the control of the destruction process
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Table 1 Physical properties of some 1 dimensional nanomaterials.
SWCNT DWCNT MWCNT HMWCNT CNF CF Fullerene C60 Halloysite Asbetos (chrysotile)
Average external diameter [nm]
Average internal diameter [nm]
1.2 3.0 40 150 150 5000 1.0 50 25 500
1.0 1.5 10
Average length [nm] 16,000 50,000 15,000 5000 100,000 107 1.0 50,000 107 107
Average external side area [nm2 ]
Average external volume [nm3 ]
Surface/ volume [nm−1 ]
Young modulus [GPa]
5 × 104 5 × 105 106 106 5 × 107 5 × 107 5 107 104 5 × 105
104 5 × 105 107 108 109 109 1 108 5 × 104 107
5 1 0.1 0.05 0.05 0.01 5 0.1 0.1 0.05
1000 1000 1200 900
is transferred to the 1DCMs. The confinement and interlocking of 1DCM can contribute significantly to the amplification of biological and medical effects, via additional or accelerated ripping. For the one dimensional nanomaterials lung is a preferred organ. Consequently an important effort was directed towards the understanding of the pulmonary toxicity of such materials. However, the in vivo studies of the pulmonary toxicity of 1DCMs are facing complex issues. The use of aerosols does not allow an accurate estimation of nanoparticles actually reaching the lung. Thus, instillations appear more accurate. First in vivo studies on the toxicity of 1DCMs suggested huge effects. Later, it was understood that actually the nanomaterial aggregated and blocked the airways. In vivo studies of the effects of DWCNTs on rats [12] confirmed their toxicity and revealed a decrease of the local oxidative stress assigned to scavenger capabilities of 1DCMs. In an excellent review on the toxicity of carbon nanotubes [13], it was concluded that carbon nanotubes have an enhanced toxicity compared to the toxicity of carbon or even quartz particles, suggesting the importance of the aspect ratio. The authors noticed that 1DCM stimulate the growth of mesenchymal stem cells growth resulting in inflammation, fibrosis, and finally in cancer. Some authors [14] were trying to make a distinction between not functionalized 1DCM and functionalized 1DCM, aiming towards the insolubility of pristine SWCNTs or MWCNTs. Why the experimental data on the toxicity of 1DCM are so scattered? We speculate that the main reasons for the discrepancies noticed in the reported toxicity of 1DCM derive from the experimental design. A bed of 1DCM is not expected to show any significant biological effect on cells. A very dense forest of 1DCM will fall in the same category (see the top panel of Fig. 1). By decreasing the density of the forest, the weight of the cell and the elasticity of the cell, combined with the mechanical strength of carbon nanotubes can result in local modification, while the cell is not yet perforated. This corresponds to the fakir mode (FM) shown in Fig. 1. Only if the pressure exerted on the cell exceeds a certain critical value p*, the cellular membrane is penetrated, the cell is impaled and the healing mechanism (partial phagocytosis) is ignited, with all its subsequent secondary effects. Typically this is not a robust healing but rather a temporary patch. The toxicity of 1DCM on organism can be amplified (compared to the cellular toxicity) by geometrical restrictions and hindrances, which make difficult the motion and finally the excretion of the 1DCM. Aspect ratio now becomes a very important parameter. This description explains the reason for which important differences are expected between the results of the in vitro and in vivo studies on the toxicity of 1DCMs. The in vitro studies have the tendency to distort the results towards the SM or FM mode show weak or no toxic effects. The time scale of the experiment is extremely important and should scale with the biotoxicity lifetime of the 1DCM, which is expected to be very long. Consequently, such
300 300
Aspect ratio
104 –105 104 102 –103 102 103 103 –104 1.0 103 105 –106 104 –105
OBS
Individual fibrils Bundle
toxicity studies on animals should be extended over the whole lifetime of the animal. 2. Conclusions The general question is about the existence of an 1 dimensional toxicity. The case of asbestos has been confirmed since decades. The fibrous structure of amyloids proteins suggests the possibility of an one dimensional toxicity. Experimental data on carbon nanotubes appears to converge towards an enhanced toxicity compared to C60 . The impale model describes better the biological and medical effects of 1DCM. The main biological process is the partial phagocytosis, and the dominant mechanism is the impaling of the biological target by the 1DCM. Hence, while carbon-based nanomaterials are expected to have a reduced toxicity (such as C60 ) the large aspect ratio and the huge Young modulus of the 1DCMs ignite a specific mechanism for cell deterioration that activates a strong toxic response. Shape-induced or enhanced toxicity can explain the adverse effects of asbestos [15] and of amyloid proteins. It is the authors’ point of view that the actual debate regarding the effect of 1DCMs derives from sensible differences in the experimental details. As long as 1DCMs are allowed to sediment during the experiment, the impaling mechanism is deactivated (or frozen) and the adverse effect of 1DCM is masked by their reduced chemical and biological activity (at least at short term). Such a situation occurs dominantly during in vitro experiments. During experiments that allows the motion of 1DCM and in vivo studies, the motions, the confinement restrictions, and the (inter)locking of biological target-1DCM activates or amplifies the impale mechanism and eventually turn on the destruction of the biological target. Of a particular importance is the biotoxicity lifetime of the nanomaterials. In the case of 1DCM the theoretical value is suggested to be infinite, and hence it is considered the biotoxicity lifetime of carbon nanotubes should be set equal to the natural average lifetime of the target. There is a converging agreement according to which the toxicity of 1DCM depends on their length [4]. This brief review went beyond by indicating that the toxicity of 1DCM is controlled by the aspect ratio, diameter, and mechanical properties (with Young modulus as one of the most important parameters). In the last years, important research efforts have been done to use 1DCM as vectors for drug release, antigens, and genes delivery [16]. The final demonstration of the adverse effects of the impale mechanism would affect dramatically these projects. An asbestoslike behavior of 1DCM has been speculated by many authors [11,17] as a potential scenario for the toxicity of 1DCM. A final step in this analysis is to demonstrate if the toxicity is due to the pair aspect ratio-mechanical properties (large Young modulus) or if it is triggered just by the aspect ratio. If mechanical properties play an important role, then it is possible to find some soft nanotubes,
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