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Revista Portuguesa de Pneumología ISSN: 0873-2159 [email protected] Sociedade Portuguesa de Pneumologia Portugal

Ferreira, A.J.; Cemlyn-Jones, J.; Robalo Cordeiro, C. Nanoparticles, nanotechnology and pulmonary nanotoxicology Revista Portuguesa de Pneumología, vol. 19, núm. 1, enero-marzo, 2013, pp. 28-37 Sociedade Portuguesa de Pneumologia Lisboa, Portugal

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Rev Port Pneumol. 2013;19(1):28---37

www.revportpneumol.org

SPECIAL ARTICLE

Nanoparticles, nanotechnology and pulmonary nanotoxicology

∗

A.J. Ferreira , J. Cemlyn-Jones, C. Robalo Cordeiro

Centre of Pneumology, Coimbra University Medical School, Coimbra, Portugal

Received 9 April 2012; accepted 25 September 2012

KEYWORDS Abstract The recently emergent ﬁeld of Nanotechnology involves the production and use of

Nanoparticles; structures at the nanoscale. Research at atomic, molecular or macromolecular levels, has

Nanotoxicology; led to new materials, systems and structures on a scale consisting of particles less than

Nanotubes; 100 nm and showing unique and unusual physical, chemical and biological properties, which has

Fullerenes; enabled new applications in diverse ﬁelds, creating a multimillion-dollar high-tech industry.

Lung; Nanotechnologies have a wide variety of uses from nanomedicine, consumer goods, electron-

Toxicology ics, communications and computing to environmental applications, efﬁcient energy sources,

agriculture, water puriﬁcation, textiles, and aerospace industry, among many others.

The different characteristics of nanoparticles such as size, shape, surface charge, chemical

properties, solubility and degree of agglomeration will determine their effects on biological

systems and human health, and the likelihood of respiratory hazards. There are a number of

new studies about the potential occupational and environmental effects of nanoparticles and

general precautionary measures are now fully justiﬁed.

Adverse respiratory effects include multifocal granulomas, peribronchial inﬂammation, pro-

gressive interstitial ﬁbrosis, chronic inﬂammatory responses, collagen deposition and oxidative

stress.

The authors present an overview of the most important studies about respiratory nanotoxicol-

ogy and the effects of nanoparticles and engineered nanomaterials on the respiratory system.

PALAVRAS-CHAVE

Nanopartículas, nanotecnologia e nanotoxicologia pulmonar

Nanopartículas;

Nanotoxicologia; Resumo O campo recentemente emergente da nanotecnologia envolve a produc¸ão e o uso

Nanotubos; de estruturas em nanoescala. A pesquisa a níveis atómicos, moleculares e macro molecu-

Fulerenos; lares conduziu a novos materiais, sistemas e estruturas numa escala constituída por partículas

Pulmão; menores que 100 nm, apresentando propriedades físicas, químicas e biológicas únicas e inco-

Toxicologia muns, o que tem permitido novas aplicac¸ões em diversos campos, criando uma indústria de

alta tecnologia multimilionária. As nanotecnologias têm uma vasta variedade de usos, desde a

nano medicina, bens de consumo, eletrónica, comunicac¸ões e informática, até às aplicac¸ões

∗

Corresponding author.

E-mail address: [email protected] (A.J. Ferreira).

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Nanoparticles, nanotechnology and pulmonary nanotoxicology 29

ambientais, fontes de eficiência energética, agricultura, purificação de água, têxteis e indústria

aeroespacial, entre muitos outros.

As diferentes características das nanopartículas, tais como tamanho, forma, carga de superfí-

cie, propriedades químicas, solubilidade e grau de aglomerac¸ão, determinarão os efeitos sobre

os sistemas biológicos e na saúde humana e a probabilidade de riscos respiratórios. Existem

alguns novos estudos sobre os potenciais efeitos ocupacionais e ambientais das nanopartículas,

sendo totalmente justiﬁcadas as medidas gerais de precauc¸ão.

Os efeitos respiratórios adversos incluem granulomas multifocais, inﬂamac¸ão peribrônquica,

fibrose intersticial progressiva, respostas inflamatórias crónicas, deposição de colagénio e stress

oxidativo.

Os autores apresentam um resumo dos mais importantes estudos sobre nanotoxicologia

respiratória e dos efeitos das nanopartículas e dos nanomateriais artiﬁciais sobre o sistema

respiratório.

Introduction New applications are constantly emerging. The Nano-

technology Consumer Products Inventory, kept by the

The second half of the XX Century saw the emergence of a Woodrow Wilson Institute, listed over 1317 products or prod-

truly revolutionary era of nanotechnology. uct lines as of April 2012.

The development of new materials, systems and Nanomaterials vary widely in terms of their composition,

structures through research at atomic, molecular, or macro- properties, and uses. Common products include cosmetics

molecular levels has enabled new applications. and personal care products, wound dressing pads, pregnancy

The nanoscale is typically a scale of matter under 100 nm. tests, toothpaste, food supplements and food storage, appli-

According to several authors, unique and unusual physi- ances, clothing, coatings, electronics and computers and

cal, chemical, and biological properties can be seen at this sporting goods.

dimensional level. With the increasing technological complexity of nano-

Nanotechnology involves imaging, measuring, modeling, technology, several generations of NPs can be reported:

∼

and manipulating matter at this scale. Some of the most First generation 2001: Passive nanostructures (nano-

promising uses of these technologies have emerged in structured coatings, nanoparticles, nanostructured metals,

some fundamental sectors, such as energy, communications, polymers, ceramics, catalysts, composites, displays).

∼

water puriﬁcation, pollution reduction and environmental Second generation 2007: Active nanostructures (tran-

progress, improved materials and new products, medical sistors, ampliﬁers, targeted drugs and chemicals, actuators,

1,2

and biomedical applications. adaptive structures, sensors, diagnostic assays, fuel cells,

Besides industrial and household uses, nanoparticles solar cells, high performance nanocomposites, ceramics,

(NPs) can be used in Medicine (creating the new ﬁeld of metals).

∼

nanomedicine) for cancer treatment, infectious diseases, Third generation 2010: 3-D nanosystems and systems

immunization purposes and diagnostic procedures with new of nanosystems (various assembly techniques, networking at

3,4

imaging sensors and agents. the nanoscale and new architectures, biomimetic materials,

As nanotechnology penetrates the marketplace and novel therapeutics/targeted drug delivery).

∼

attracts attention, the public is starting to develop opinions Fourth generation 2015: Molecular nanosystems

about it. Those opinions will shape the market for consumer (molecular devices ‘‘by design’’, atomic design, emerging

5 9

goods using these new technologies. functions).

Nanotechnology is a sector of high-tech industry that has The expression ‘‘nanoparticle’’ is commonly used to

already created a multibillion $US market, and is widely describe engineered structures with diameters of <100 nm,

18 6

expected to grow to 1 trillion (10 ) US dollars by 2015. that are created by chemical and/or physical processes,

Nanotechnology research and development can integrate with very characteristic properties usually not present at

the nanoscale structures into larger material components, a macro-scale level.

systems, and architectures. Reducing the size of a particle increases the ratio of

The majority of the NPs currently in use today have been surface area to mass. Because the reactive portion of the

made from transition metals, silicon, different forms of car- particle is on the surface, increasing the relative surface

bon (carbon nanotubes; fullerenes), and metal oxides (such area will increase reactivity of a given amount of material.

as zinc dioxide and titanium dioxide). Also, at the nanoscale, both classical physics and quan-

In sensu lato, inorganic nanoparticles do not contain car- tum physics can direct the behaviour of a particle. The

bon molecules; carbon nanotubes and carbon fullerenes are inﬂuence of quantum effects can change important mate-

usually addressed as organic nanoparticles. But it must be rial properties, such as optical, magnetic, and electrical

stated that, strictly from a chemical point of view, these properties.

forms containing pure carbon molecules without bonds to NPs may be suspended in a gas (as a nanoaerosol),

hydrogen, constitute allotropes of carbon and are part of suspended in a liquid (as a colloid or nano-hydrosol), or

what is now known as the Inorganic Chemistry of Carbon. embedded in a matrix (as a nanocomposite).

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30 A.J. Ferreira et al.

Some authors state that the main difference between NPs low molecular weight drugs, nanoﬂuidics for targeted

and ultraﬁne particles (UFP) refers to the fact that the term synthesis, nanodetection for target identiﬁcation, the

UFP is commonly used to describe nanometer-size particles discovery of natural macromolecules, including anti-

that have not been intentionally produced, but are the inci- bodies, proteins and genes with biological activity and

dental products of industrial processes or different types of the creation of drug delivery systems (liposomes

combustion, and even volcanic activities. and nanoparticles---nanopharmaceuticals), promoting

UFP (or incidental nanoparticles) show more complex disease-speciﬁc targeting, in order to thoroughly control

chemical composition, irregular shapes, and polydispersed the release of the drugs over the desired period of time, or

size distribution as compared to engineered particles. even to supply suitable routes of administration that can

These particles could exert their effects per se or reach locations in the body that are traditionally difﬁcult

through a multitude of substances that could be adsorbed to access, such as the brain.

on their surfaces. For instance, some engineered NPs exist Nanotechnology has an enormous potential in the ﬁeld of

as nanocrystals composed of a number of compounds such human imaging and early recognition of disease, with the

as silicon and metals (quantum dots). tailoring of speciﬁc nanoagents for molecular imaging in

Carbon fullerenes represent NPs with identical dimen- the context of Magnetic Resonance Imaging, ultrasound,

18---23

sions in all directions (i.e., spherical). They are carbon optical imaging, and X-ray imaging.

allotropes rolled up to form closed-cage, hollow spheres. Other medical areas include gene and oncologic ther-

Some of their characteristics such as their small size, large apy using multicomponent, nanosized delivery vectors.

surface area and high reactivity make them interesting in Nanotube drug delivery is promising for cancer therapy

technological and medical ﬁelds. They were initially discov- with high treatment efﬁcacy and minimum side effects.

ered by Kroto, Smalley and Curl (who later won the 1996 There is an increasing use of NPs as carrier systems for

Nobel Prize in Chemistry) as a new form of carbon, buck- chemotherapeutic drugs because of the ability to speciﬁcally

5,14,15

minsterfullerene or C60. target cancer cells, improve efﬁcacy and reduce systemic

Single-walled carbon nanotubes (SWCNTs) typically form toxicity.

convoluted, ﬁbre-like NPs with a diameter below 100 nm. Noble metal NPs can efﬁciently target several kinds of

Many particle morphologies can be created at the nanoscale, tumours, as they present highly tunable optical properties

including ‘‘ﬂower’’ and ‘‘belt’’-like structures. that can be adjusted to desirable wavelengths according to

Multi-walled carbon nanotubes (MWCNT) are larger and their shape and composition with possible uses in tumour

consist of many single-walled tubes stacked one inside the targeting, gene silencing and drug delivery; they can also

other. efﬁciently convert light or radiofrequencies into heat, pro-

Carbon nanotubes (CNTs) are distinct from carbon ﬁbres, moting thermal ablation.

because the latter consist of strands of layered graphite CNTs can be effective in delivering drugs such as pacli-

sheets and are not single carbon molecules. CNTs are taxel to retard tumour growth in experimental models of

26,27

reported to be physically very strong and stiff. For exam- cancer.

ple a SWCNT can be up to 10 times as strong as steel and Solid lipid nanoparticles (SLN) are ideal carriers for

16,17

1.2 times as stiff as diamond. weakly soluble drugs, and are alternatives to current col-

Close-packed nanotube structures could have a yield loidal carriers. Inhaled SLN---paclitaxel could represent a

strength exceeding 45 7 GPa, which is over 20 times the potential system for regional delivery to the lungs, with spe-

yield strength of typical high-strength steels. cial efﬁcacy on the lymphatic system, fundamental in the

As would be expected from the above statements, it is progression of lung adenocarcinomas.

now understood that potential occupational and environ- Therefore, targeted nanoparticle delivery to the respi-

mental exposure to manufactured NPs is on the increase. ratory system is one of the future trends in nanomedicine,

as it can improve drug therapies systemically and locally

using advanced drug delivery systems based in more or less

Nanomedicine and the lung complex nanostructures. In the speciﬁc area of lung can-

cer treatment, NPs can revolutionize future chemotherapy

The ﬁeld of nanomedicine is the science and technology of options. Other areas of development are the improve-

diagnosing, treating and preventing disease and traumatic ment of monoclonal antibodies for lung targeting (attaching

injury, of relieving pain, and of preserving and improv- antibodies to drug molecules or drug delivery systems),

ing human health, using molecular tools and molecular lung imaging, gene delivery, cystic ﬁbrosis treatment (gene

knowledge of the human body. Its objectives encom- therapy and nano-selective and sustained delivery of pro-

pass monitoring, control, construction, repair, defense and teasome inhibitor drugs) and tuberculosis diagnosis and

improvement of human biological systems, using engineered treatment (as macrophage involvement make NPs a perfect

18 28---31

devices and nanostructures for medical beneﬁt. drug carrier).

Nanomedicine involves the best knowledge about the

science and technology of intricate systems of nanometre- Environmental challenges of exposure

scale size, with several components, one of which is an

to nanoparticles

active principle, with the whole system leading to a spe-

cial function connected to the diagnosis, treatment and/or Soon after the great development and commercial intro-

prevention of disease. duction of these materials, some authors raised signiﬁcant

Nanomedicine also includes the identiﬁcation of questions about the potential impact on human health and

new molecular targets, the creation of new synthetic the environment.

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Nanoparticles, nanotechnology and pulmonary nanotoxicology 31

In the 1990s, toxicology and epidemiology research on stress. The types of toxicological response will probably vary

ultraﬁne aerosols began developing in combination. How- between molecular and nano-sized forms.

ever, the emerging ﬁeld of nanotechnology stimulated the The size of the NPs suggests that the physiological

actions toward developing a more complete understanding responses could be of an immune or inﬂammatory nature.

of what unexpected and unanticipated impact nanoscale These adverse effects may not follow a classic dose-response

materials might have on health. curve, and can display high unpredictability within the pop-

In recent decades there has been human exposure to ulation, potentially dependent on individual sensitivity.

nanoscale particles in the form of diesel soot and bulk UFPs Nanoparticle toxicity is extremely complex and multi-

from diverse industrial procedures, essentially in the form factorial and depends on a multiplicity of physicochemical

of combustion-derived UFPs, but also from natural sources properties such as size and shape, as well as surface prop-

such as forest ﬁres and volcanoes. erties (charge, area, and reactivity). Ultraﬁne or nanosize

Exposure to these particles has been associated with range (<100 nm) particles seem to be more toxic on a

pulmonary inﬂammation, immune changes, and adverse mass-based exposure metric compared to larger particles

systemic effects including blood hypercoagulability, con- of identical chemical composition. Also, particle surface

32---35

tributing to undesirable cardiovascular effects. area dose is a better predictor of the toxic and pathological

9,40

Consistent with cross-sectional ﬁndings and animal stud- responses to inhaled particles than particle mass dose.

ies, there seems to be an association between exposure to Differences in physico-chemical properties between NPs

air pollution and the progression of atherosclerosis. Some and larger particles determine their behaviour as aerosol,

studies report the association of particulate matter <2.5 um their biodistribution following translocation from the portal

(PM2.5) and carotid intima-media thickness. of entry, their cellular interactions and effects. Secondary

Oxidative stress, inﬂammation, induction of a pro- organs are usually affected differently from the primary tar-

coagulatory state and dysfunction of the autonomic nervous get of these particles.

system can enhance respiratory and cardiovascular diseases. In 1990, the Journal of Aerosol Science published two

Changes in lung function, heart rate, blood pressure and of the ﬁrst cornerstone papers on a higher than expected

inﬂammatory state, and also respiratory symptoms, throm- effect on lung inﬂammation patterns in rats exposed to NPs,

bosis, myocardial infarction, arrhythmia, strokes, and death effects that could not be predicted by just taking the chem-

are more often seen in polluted environments, causing ical composition and the inhaled amounts of the particles

38 41---43

shorter life expectancy. into account.

A lot of the concern related to the exposure to nano- One of the ﬁrst important assumptions, that is still of

materials comes from our knowledge from the reports of great importance, is that NPs have the potential to show

inhalation of ultraﬁne particles found in occupational sett- previously unrecognized biological behaviour.

ings and also ultraﬁne aerosols resulting from combustion. A major report concerning the uncertainties of nanotech-

There seems to be an important link between several chronic nologies was published in 2004 by the Royal Society and

diseases and the inhalation of UFPs, such as Clara cell car- the Royal Academy of Engineering. This was one of the

cinomas (polycyclic aromatic hydrocarbons), mesothelioma ﬁrst reports to highlight the potential risks to health and

(asbestos), and berylliosis (beryllium). Some syndromes the environment that may arise from exposure to nanoma-

associated with exposure to aerosols include coal worker terials, especially NPs (which included nanomaterials such

pneumoconiosis, emphysema (combustion products), and as nanotubes). Since then, more than ﬁfty national and

metal fume fever (zinc, tin, and other transition metals). international reviews carried out by government depart-

However, the technical requirements for the detection ments, industry associations, insurance organizations and

and characterization of nanoparticles in the environ- researchers have considered nanoparticle risk issues.

ment push the limits of modern sampling techniques and A workshop co-sponsored by the National Science Foun-

instrumentation. dation and the US Environmental Protection Agency has

At present, there are no legal thresholds for nanoparticle identiﬁed a number of potential risks regarding manufac-

number concentrations in ambient air, so local observation tured NPs, such as exposure assessment of manufactured

networks do not generally monitor them. The development NPs; toxicology of manufactured NPs; the ability (or not)

of ambient particle regulations has been limited for tech- to extrapolate manufactured nanoparticle toxicity using

nical and practical reasons, such as the lack of standard existing particle and ﬁbre toxicological databases; envi-

methods and instrumentation, and the uncertainty about ronmental and biological fate, transport, persistence, and

repeatability and reproducibility of measurements. transformation of manufactured NPs, recyclability and over-

Assessment of the risk associated with nanoparticles all sustainability of manufactured nanomaterials.

requires knowledge about the ability of a material to reach The ability of absorbed particles to generate local toxic

a sensitive site of action, and the type and magnitude of the effects at the site of initial deposition as well as very

resultant response at the sensitive site. signiﬁcant systemic toxic responses shows how dangerous

they can be in real-life settings. The potential for adverse

health effects may arise from direct exposure to intention-

Nanotoxicology ally produced nanomaterials and/or byproducts associated

with their applications.

The lung is the primary and probably the most important tar- Toxicological assessment of manmade nanomateri-

get of nanomaterials, but NPs can enter the circulation and als requires information about the route (inhalation, oral,

migrate to various organs and tissues, where they can build dermal) of exposure, as well as their complete physicochem-

up and injure organ systems that are sensitive to oxidative ical characterization of them in order to provide thorough

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32 A.J. Ferreira et al.

information. The most common scenarios for human expo- composition, posing speciﬁcally a higher respiratory and sys-

sure to NPs are occupational, environmental and consumer temic health threat.

ones. At equivalent mass doses, insoluble ultraﬁne particles

cause more damage than larger particles of a similar compo-

sition in terms of pulmonary inﬂammation, tissue damage,

Inhalation hazards of nanoparticles 11

and lung tumours.

From the alveoli, NPs have the potential to enter the

One of the most widespread routes of human exposure to

bloodstream from the lungs and even translocate to other

airborne NPs is inhalation in the workplace and the environ- 11

organs. For instance, biokinetic studies show that inhaled

ment.

NPs can translocate via olfactory neurons from the nose to

As seen before, at present there are large numbers 40

the Central Nervous System.

of nanomaterials; as their technological and toxicological

The biokinetics of NPs in the body varies, depending

properties vary considerably, so will their risk proﬁles. We

on the portal of entry. The same NPs entering the lung

are now able to consider that the extensive and heteroge-

(via inhalation or intratracheal instillation) or intravenously,

neous group of NPs of diverse scale constitutes an inhalation

interact with different biological media and will receive

hazard of unknown potential.

different secondary coatings, affecting nanoparticle biodis-

The deposition of NPs in the respiratory tract is 27

tribution to target organs.

determined essentially by the particle aerodynamic or ther-

At present, carrying out risk assessment of NPs can only

modynamic diameter (depending on particle size). 6

be done sensibly on a case-by-case basis.

According to the classical model developed by the Inter-

Most of the studies about the effects of respiratory expo-

national Commission on Radiological Protection (ICRP), the

sure to NPs involve pulmonary models and are performed

probability of nanoparticles reaching the alveoli peaked at

through instillation, aspiration and inhalation of carbon

a size of approximately 20 nm, with lower probabilities of

nanotubes (the most studied NPs) in rodent species.

deposition in the alveoli for both smaller and larger nano-

Table 1 summarizes some of the main in vivo investiga-

particles. Nanoparticle deposition, in particular for smaller

tions regarding the respiratory effects of carbon nanotubes.

particles, is governed by Brownian movements.

Many of the most pressing health and safety concerns

There are still issues that have not yet been fully resolved

regarding NPs and nanotechnologies come from the lack of

about how exposure to NPs can be thoroughly measured and

knowledge about levels of occupational and other types of

quantiﬁed; they are usually described as mass concentration 61

exposure during their production and use.

−3 −3

(units mg m ); number concentration (units per m ) and

Among the particles with potential respiratory risks, CNTs

2 −3

surface area concentration units (m m ).

are one of the specialized structures of engineered NPs that

As stated before, the possible health effects arising from

are widespread. Discovered more than 20 years ago, they

exposure to NPs may be better correlated with surface area,

have increasing potential uses in biomedical, aeronautic,

6,16

rather than with mass concentration.

and industrial ﬁelds due to their unique conductive and elec-

NPs have some essential properties that could be directly 59

trochemical properties.

related to their pathogenicity:

As seen before, CNTs are classiﬁed according to their

structure: single-walled carbon nanotubes (SWCNT) and

- As particles less than 100 nm, they may have more toxicity

multi-walled carbon nanotubes (MWCNT).

than larger sized particles.

Nanotubes can have features of both NP and conventional

- Generally they are considered as ﬁbre shaped, and so

ﬁbres. The literature currently available suggests that CNT

might behave like some other pathogenic ﬁbres (asbestos,

may have toxic effects beyond those predictable for their

man-made ﬁbres).

mass exposure. For instance, they have more adverse effects

- As most of the NPs have an essentially graphitic constitu-

than the same mass of NP carbon and quartz, a commonly

tion, they are expected to be biopersistent in biological

used comparator amongst harmful inhalable particles.

settings, such as the respiratory system.

CNTs can induce oxidative stress and inﬂammation, and

several studies imply that they could cause granuloma for-

Commercial NPs could also contain some impurities, a mation and ﬁbrogenesis.

common result of the synthesis process, such as metals In occupational contexts, CNTs should be considered in

(Co, Fe, Ni, and Mo), organic compounds, and support the same way as other biopersistent ﬁbres in the workplace,

material. with implications for at least similar approaches to control

Smaller particles are likely to be more aggressive to the and assessment.

lung than larger particles; particles with more inert surfaces It would appear that the main mechanism of engineered

may be aggressive, exerting their effects on cells by reason nanomaterial toxicity is related to oxidative stress, caused

of having a large surface area. Particles with more reactive by the activation of responsive transcription factors. Chronic

surfaces can affect cells without necessarily having large inﬂammation and oxidative stress observed during and after

surface areas. exposure can induce adverse health effects such as ﬁbro-

Shape, biopersistence, presence of transition metals and sis, genotoxicity and cancer caused by ﬁbres or secondary

the power to generate reactive oxygen species also explain mutation.

the potential for lung damage. In fact, some of the main One of the ﬁrst studies on the subject of CNTs expo-

experimental studies in rodents and cell cultures have shown sure was performed by Lam et al. In this study mice

that the toxicity of UFPs or NPs is greater than that of were instilled intratracheally with 0, 0.1, or 0.5 mg of

the same mass of larger particles with a similar chemical carbon nanotubes, a carbon black negative control, or a

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Nanoparticles, nanotechnology and pulmonary nanotoxicology 33

Table 1 In vivo investigations of carbon nanotube toxicity.

Animal species, agent and Results Authors

method of administration

Intratracheal instillation of No induction of any abnormalities of pulmonary function or Huczko et al.

CNT containing soot on measurable inﬂammation. No lung pathology was performed (2001)

guinea pigs in this study.

Intratracheal instillation of All nanotube products induced dose-dependent persistent Lam e al. (2004)

different SWCNT samples epithelioid granulomas. The lungs of some animals also

(three SWCNT products revealed peribronchial inﬂammation and necrosis extending

made by different methods into the alveolar septa. The lungs of mice treated with carbon

and containing different black were normal, whereas those treated with high-dose

types and amounts of quartz revealed mild to moderate inﬂammation.

residual metals) on mice

Intratracheal instillation of Pulmonary exposures to SWCNT in rats produced a Warheit et al.

SWCNT on Sprague-Dawley non-dose-dependent series of multifocal granulomas, which (2004)

rats were evidence of a foreign tissue body reaction and were non

uniform in distribution and not progressive beyond 1 month

post exposure.

Intrapharyngeal aspiration Acute inﬂammation, early onset of formation of granulomas, Shvedova et al.

of puriﬁed SWCNT on mice and progressive ﬁbrosis. SWCNT-induced granulomas, mainly (2005)

(C57CL/6) associated with hypertrophied epithelial cells surrounding the

dust aggregates, and diffusive interstitial ﬁbrosis and alveolar

wall thickening. Lung lesions were dose-dependent and

progressive. Bronchial alveolar lavage ﬂuid (BALF) increases

in total protein concentration, cell counts, concentration

␤ of transforming growth factor beta (TGF- ), lactate

␥

dehydrogenase (LDH), and - glutamyltranspeptidase

activities, and glutathione depletion (biomarkers

of inﬂammation, oxidative stress, and cytotoxicity).

Intratracheal instillation of Inﬂammation, collagen rich granulomas, and ﬁbrosis. Muller et al.

MWCNT on Sprague-Dawley Hydroxyproline and soluble collagen, increased in the lung (2005)

Rats tissues in a dose-dependent fashion.

Intrapharyngeal instillation of Activation of heme-oxygenase-1 (HO-1), a marker of oxidative Li et al. (2007)

SWCNT on mice (C57CL/6) insult, in lung, aorta, and heart tissue. Aortic mtDNA damage

at 7, 28, and 60 days after exposure.

Intratracheal Difference in lung pathological lesions induced by instilled and Li et al. (2007)

Instillation + inhalation of inhaled MWCNTs probably due to different size and distribution

MWCNT on Kunming mice of aggregations of MWCNTs in lung.

Whole-body inhalation of The experiment did not result in signiﬁcant lung inﬂammation Mitchell et al.

MWCNT on mice (C57CL/6) or tissue damage, but caused systemic immune function (2007)

alterations. BALF demonstrated particle-laden macrophages.

Nonmonotonic systemic immunosuppression. Decreased NK cell

function. No changes in gene expression were observed in lung.

Intratracheal instillation Alveolar macrophage activation, several chronic inﬂammatory Chou et al.

of SWCNT on mice responses, and severe pulmonary granuloma formation. (2008)

Whole-body inhalation of Development of multifocal granulomatous pneumonia Shvedova et al.

SWCNT on mice (C57CL/6) and interstitial ﬁbrosis. SWCNT inhalation caused more (2008)

inﬂammatory response, oxidative stress, collagen deposition,

ﬁbrosis and mutations of K-ras gene when compared with

aspiration.

Wistar rats. MWCNT---Acute Pulmonary inﬂammogenicity following exposure to MWCNT was Ellinger-

inhalation study. Positive concentration-dependent with evidence of regression over Ziegelbauer et al.

control: alpha quartz. time. Alpha-quartz (positive controls) resulted in progressive (2009)

Negative control: air changes over time.

Wistar rats. 90-day inhalation Pronounced multifocal granulomatous inﬂammation, diffuse

toxicity study with MWCNT histiocytic and neutrophilic inﬂammation, and intra-alveolar

lipoproteinosis were observed in lung and lung-associated

lymph nodes, incidence and severity related.

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34 A.J. Ferreira et al.

Table 1 (cont.)

Animal species, agent and Results Authors

method of administration

Ma-Hock et al. (2009)

Wistar rats. MWCNT --- Acute Sustained pulmonary inﬂammation related to pulmonary Pauluhn (2010)

inhalation with 3 months overload of NPs resulting probably from stasis of clearance.

postexposure period; Macrophages with enlarged and/or foamy appearance;

Repeated inhalation increased inﬂux of inﬂammatory cells and septal thickening,

exposure with 6 months slight to moderate inﬂammation, focally with granulomatous

postexposure period appearance; interstitial ﬁbrosis. Thickening of the visceral

pleura. Inﬂammation did not decrease in severity with

increasing post-exposure duration.

C57BL/6 mice. Repeated The incidence and severity of inﬂammatory and ﬁbrotic Teeguarden et al.

pharyngeal aspiration responses were greatest in mice treated with SWCNTs. The (2010)

of SWCNTs, crocidolite, proteomic analysis suggested that lung tissue and lung

and ultraﬁne carbon black inﬁltrate responses to SWCNT and crocidolite asbestos were

similar, but as for several histopathological endpoints, the

response was generally greater on a mass dose basis for

SWCNT, when compared with asbestos or ultraﬁne carbon

black.

SWCNT-transformed cells One week post-injection, tumours were formed at the Wang et al.

injected in immunodeﬁcient injection site in mice receiving B-SWCNT cells, whereas mice (2011)

mice receiving control BEAS-2B cells did not develop tumours.

quartz positive control, and lung pathology studied 7 or C57BL/6 mice. This is one of the mutated genes that can

90 days after exposure. All nanotube products induced be implicated in pulmonary tumourigenesis.

dose-dependent epithelioid granulomas and, in some cases, Aerosolized inhalation in animal studies supports a com-

interstitial inﬂammation, which was more pronounced in the mon sequence of biological events following single CNT

90-day group. Some animals had peribronchial inﬂammation exposure: important acute phase inﬂammation similar to a

and necrosis, extending to the alveolar septa. On a same foreign body response, followed by formation of multifocal

weight basis, when CNTs reach the lungs, they are much granulomas, and early onset ﬁbrosis.

more toxic than carbon black and can be more toxic than MWCNTs and other carbon NPs in ﬁne (<2.5 ␮m) par-

quartz. ticulate matter (PM) aggregates have been related to the

Inhalation studies are able to provide more information combustion of methane, propane, and natural-gas ﬂames of

and reproducibility of occupational and environmental risks. some stoves; indeed, indoor and outdoor ﬁne PM samples

However, instillation studies are less complex and cheaper, were reported to contain signiﬁcant fractions of MWCNTs.

so they could represent a form of screening for the potential A very recent study from Teeguarden et al. used

harm and toxicity of NPs. high sensitivity based proteomics --- HPLC---FTICR---MS (high

In the experiments where intratracheal instillation of NPs performance liquid chromatography/Fourier transform ion

was performed, more pronounced and severe effects were cyclotron resonance mass spectrometry) to assess some

62,63

observed, when compared with inhalation studies. of the major differences in exposure to three materials

CNTs have toxicologically signiﬁcant structural and --- SWCNT, asbestos, and ultraﬁne carbon black (UFCB).

chemical similarities to asbestos, and experiments have Rodent exposure to SWCNT led to a signiﬁcantly greater

shown that they cause pulmonary inﬂammation, granuloma inﬂammatory response, when compared with crocidolite

formation and ﬁbrosis after entering the respiratory sys- asbestos and UFCB, with an increase in polymorphonuclear

tem of rodents, as observed after exposure to asbestos. neutrophils (PMNs) and total cell count of bronchoalveolar

Some studies confirm for MWCNTs a well established lavage fluid (BALF). Also, moderate multifocal inflamma-

asbestos-like pathogenicity that is associated with long tion was present in all lungs from SWCNT exposed mice,

59,64

ﬁbres. with granuloma formation near bronchioles and adjacent

In addition, the impact of some impurities, such as alveoli consisting of round clusters of large macrophages

metals, present in some MWCNTs cannot be underestimated and multinucleated giant cells. Statistically signiﬁcant dif-

in the forms in which the lung could be affected. ferences in some cytokines (TARC, IL-12 and MDC) were also

According to Pacurari et al., the ﬁbrous characteristics of seen, proving the high inﬂammogenic potential of SWCNT. In

MWCNT, their durability and their ability to generate reac- addition, the proteomic analysis conducted by the authors

tive oxygen species at low levels in cellular systems may supports the conclusion that lung tissue and lung inﬁltrate

contribute to the initiation and progression of asbestos-like responses were generally greater on a mass dose basis for 66

pathological responses. SWCNT.

Shvedova et al. demonstrated that SWCNT inhalation The potential for particles to cause ﬁbrotic reactions

resulted in mutations of K-ras gene locus in the lung of within the lung depends on the particle size, composition,

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Nanoparticles, nanotechnology and pulmonary nanotoxicology 35

surface activity, and retained dose. Particles containing A very wide range of endpoints have to be considered

certain transition metals have a greater capacity to gen- when testing potential risks derived from NPs. Hazards

erate reactive oxygen species, in addition to those already should be tested according to their potential routes within

generated by inﬂammatory neutrophils and activated alve- the human body.

olar macrophages. According to Oberdörster in a recent paper regarding

Recently, Wang at al. demonstrated that chronic expo- the safety assessment for nanotechnology, although many

sure to SWCNT can produce malignant transformation of of the current engineered nanomaterials with a potential for

human lung epithelial cells. Also, SWCNT-transformed cells human exposure are not likely to induce signiﬁcant adverse

injected in immunodeﬁcient mice led to tumour growth effects, some could cause an asbestos-type disaster if it is

at the injection site in mice receiving SWCNT-transformed not controlled.

BEAS-2B cells, whereas mice receiving control BEAS-2B cells There is, therefore, an urgent need for the development

did not develop tumours, showing also the potential role of of criteria for extrapolating toxicological data in biological

p53 in this process. systems in order to predict the risk of adverse outcomes in

Like other areas of knowledge regarding nanotech- humans.

nologies, studies of the carcinogenic potential of carbon Not only traditional tests, but also newer models reﬂect-

nanotubes are still in the early stages, but there are some ing the permanent evolution of nanotoxicology as an

data suggesting that these structures are not risk free, and emerging ﬁeld, should be used to shed light on the mech-

can be widely dependent on several other physico-chemical anisms of NP toxicity. The combination of in vitro and

and biological characteristics, and also speciﬁc composition in vivo tests should contribute to hazard assessment in the

and type and size of impurities. future. A multidisciplinary team approach is fundamental to

Also, fullerenes (NPs with identical dimensions in all addressing respiratory human risks related with Nanotech-

directions) following exposure via the pulmonary route are nology.

capable of eliciting localized responses that are pro- or anti- At present, there are several extremely relevant

inﬂammatory in nature, with the type of response initiated attempts currently under way in order to formulate

likely to be reliant on the fullerene in question, exposure a science-based risk management research framework.

method and the dose used. However, inhalation studies One of them is the US. National Nanotechnology Initia-

regarding the effects of fullerenes are still limited. tive/Environmental, Health, and Safety Research Strategy;

Fullerene toxicity probably involves an oxidant response, the main research needs to include, among other points, the

suggesting the potential of fullerenes to cause oxidative development of measurement tools for the determination

stress and related consequences (such as inﬂammation or of physico-chemical properties of engineered nanomate-

genotoxicity). Generally, the greater the water solubility rials (ENMs), their detection and monitoring in realistic

exhibited by a fullerene sample, the lower the toxicity asso- exposure media and conditions during the life cycle, the

ciated with exposure. evaluation of transformations of ENMs in relevant media,

In addition, recent studies in murine models prove that the further assessment of biological responses, the better

titanium dioxide (TiO2) or gold (Au) NPs can interfere with understanding of the processes and factors that determine

the modulation of the asthmatic response regarding diiso- exposures to nanomaterials, the identiﬁcation of popula-

cyanate induced asthma, as they can aggravate pulmonary tion groups exposed to ENMs and their health surveillance,

inﬂammation and airway hyperreactivity. and the development of appropriate, reliable, and repro-

There is still a shortage of publications addressing the ducible assays and models to predict human responses to

in vivo, real world effects of nanoparticle exposure. How- ENMs.

ever, some of these are very worrying; for instance, Chinese With appropriate strategies that integrate risk assess-

workers exposed to polyacrylate consisting of NPs in a print ment into decision-making frameworks for risk manage-

plant have shown shortness of breath, and the same clin- ment, and incorporate and standardize risk communication

ical ﬁndings of pleural effusion and pericardial effusion. within the risk management framework, better esults should

The study of these workers revealed nonspeciﬁc pulmonary be expected in the future.

inﬂammation, pulmonary ﬁbrosis and foreign-body granu- Now is the time to promote, on a global scale, an in-depth

lomas of the pleura; BALF elicited increased lymphocytes discussion and evaluation of the human and environmental

and neutrophil leukocytes. Tw o young patients subsequently risks of these new materials; as stated by Maynard et al.,

died from respiratory failure. these resources are ‘‘merely the vanguard of a new era of

complex materials, where novel and dynamic functional-

Conclusions and future trends ity is engineered into multifaceted substances. If we are to

meet the challenge of ensuring the safe use of this new gen-

Respiratory exposure to NPs can cause important adverse eration of substances, it is time to move beyond ‘‘nano’’

respiratory effects, such as multifocal granulomas, peri- toxicology and toward a new toxicology of sophisticated

bronchial inﬂammation, progressive interstitial ﬁbrosis, materials.’’

chronic inﬂammatory responses, collagen deposition, oxida-

tive stress, pleural lesions and gene mutations, at least in

experimental animal studies. Ethic disclosures

At this point in time, it is fundamental to know more

about the toxicological effects of NPs, to address the Protection of human and animal subjects. The authors

increased concern of potentially harmful public and occu- declare that no experiments were performed on humans or

pational exposures. animals for this investigation.

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36 A.J. Ferreira et al.

Conﬁdentiality of data. The authors declare that no patient 19. Kundu AK, Hazari S, Chinta DD, Pramar YV, Dash S, Mandal TK.

data appears in this article. Development of nanosomes using high-pressure homogenization

for gene therapy. J Pharm Pharmacol. 2010;62:1103---11.

20. Sandhiya S, Dkhar SA, Surendiran A. Emerging trends

Right to privacy and informed consent. The authors of nanomedicine --- an overview. Fundam Clin Pharmacol.

declare that no patient data appears in this article. 2009;23:263---9.

21. Jain KK. Advances in the ﬁeld of nanooncology. BMC Med.

2010;8:83.

22. Kojima C, Turkbey B, Ogawa M, Bernardo M, Regino CA,

Conﬂicts of interest

Bryant Jr LH, et al. Dendrimer-based MRI contrast agents:

the effects of PEGylation on relaxivity and pharmacokinetics.

The authors have no conﬂicts of interest to declare.

Nanomedicine. 2011;7:1001---8.

23. Frimpong RA, Hilt JZ. Magnetic nanoparticles in biomedicine:

synthesis, functionalization and applications. Nanomedicine

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