Nanoparticles and the Brain: Cause for Concern?

Günter Oberdörster*, Alison Elder, and Amber Rinderknecht

University of Rochester Department of Environmental Medicine 575 Elmwood Avenue Rochester,

NY 14642 USA

Abstract

Engineered nanoparticles (NPs) are in the same size category as atmospheric ultrafine particles,

<100 nm. Per given volume, both have high numbers and surface areas compared to larger

particles. The high proportion of surface atoms/molecules can give rise to a greater chemical as

well as biological activity, for example the induction of reactive oxygen species in cell-free

medium as well as in cells. When inhaled as singlet particles, NPs of different sizes deposit

efficiently in all regions of the respiratory tract by diffusion. A major difference to larger size

particles is the propensity of NPs to translocate across cell barriers from the portal of entry (e.g.,

the respiratory tract) to secondary organs and to enter cells by various mechanisms and associate

with subcellular structures. This makes NPs uniquely suitable for therapeutic and diagnostic uses,

but it also leaves target organs such as the central nervous system (CNS) vulnerable to potential

adverse effects (e.g., oxidative stress). Neuronal transport of NPs has been described, involving

retrograde and anterograde movement in axons and dendrites as well as perineural translocation.

This is of importance for access of inhaled NPs to the central nervous system (CNS) via sensory

nerves existing in the nasopharyngeal and tracheobronchial regions of the respiratory tract. The

neuronal pathway circumvents the very tight blood brain barrier. In general, translocation rates of

NP from the portal of entry into the blood compartment or the CNS are very low. Important

modifiers of translocation are the physicochemical characteristics of NPs, most notably their size

and surface properties, particularly surface chemistry. Primary surface coating (when NPs are

manufactured) and secondary surface coating (adsorption of lipids/proteins occurring at the portal

of entry and during subsequent translocation) can significantly alter NP biokinetics and their

effects. Implications of species differences in respiratory tract anatomy, breathing pattern and

brain anatomy for extrapolation to humans of NP effects observed in rodents need to be

considered. Although there are anecdotal data indicating a causal relationship between long-term

ultrafine particle exposures in ambient air (e.g., traffic related) or at the workplace (e.g., metal

fumes) and resultant neurotoxic effects in humans, more studies are needed to test the hypothesis

that inhaled nanoparticles cause neurodegenerative effects. Some, but probably not the majority of

NPs, will have a significant toxicity (hazard) potential, and this will pose a significant risk if there

is a sufficient exposure. The challenge is to identify such hazardous NPs and take appropriate

measures to prevent exposure.

Keywords

Nanoparticle; inhalation; translocation; brain; neurodegeneration

(tele: 585-275-3804; fax: 585-256-2631) (gunter_oberdorster@urmc.rochester.edu).

*This review article was in part presented at the Satellite Symposium: Neurobiology of the 10th International Congress on Amino

Acids and Proteins (ICAAP), August, 2007, Kallithea, Greece.

NIH Public Access

Author Manuscript

J Nanosci Nanotechnol. Author manuscript; available in PMC 2013 October 21.

Published in final edited form as:

J Nanosci Nanotechnol. 2009 August ; 9(8): 4996–5007.

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1. Introduction

Engineered nanoparticles (NPs, <100 nm in size in any dimension) are manufactured at an

increasing rate, characterized by diverse chemistries, different crystalline or amorphous

states, and different shapes, and feature desirable superior mechanical, chemical, electrical

or optical properties for use in multiple applications, including diverse consumer and

industrial products, and for medical purposes. However, in the wake of these exciting

developments and discoveries are mounting concerns that inadvertent or unavoidable

exposures of humans and the environment to NPs will give rise to undesirable effects for

human and environmental health. That is because the same properties that make NPs so

desirable for numerous applications may also enhance their potential to induce toxicity in

living systems. For example, the exponentially increasing surface area per unit volume with

decreasing nanoparticle size is equivalent to an increasing ratio of the number of particle’s

surface atoms (or molecules) to the number of total atoms (or molecules) of the particle.

This greater surface area per volume has the potential to make the smaller NPs chemically

more reactive and to render them biologically more active per given mass. Obviously, this

general surface area concept is modified by other particle properties, e.g., chemistry or

shape, so that even small chemical alterations or defects of the NP surface can change its

activity (Jiang et al., 2008). A specific concern is that any contact with NPs – whether by

inhalation, ingestion or dermal – is thought to result in significant uptake and internal

exposure of sensitive organs, such as the central nervous system (CNS), and cause

permanent damage (Minkel, 2007). These concerns are based on studies showing high

toxicity of some NPs when administered at very high doses; however, a high hazard

potential is not equivalent to a high risk; thus an understanding of NP biokinetics relevant to

exposure levels is essential. Some of the underlying concepts are discussed in this paper.

2. Concepts of inhalation nanotoxicology

Among the different routes of exposure to NPs, the respiratory tract is considered to be a

major portal-of-entry because of the likelihood that NPs will become airborne during

handling. Results from numerous in vitro and in vivo toxicological studies as well as

epidemiological studies of susceptible populations revealed adverse effects of ambient

atmospheric ultrafine particles (<100 nm) in toxicological studies and in susceptible parts of

the population (EPA, 2004). Knowledge acquired from these studies as well as from in vitro

and in vivo studies with NPs clearly shows that the interactions of NPs with the organism,

cells and tissues can be very different from those of larger particles. Table 1 contrasts some

of these differences, but also shows similarities with respect to physicochemical properties

and biological/toxicological effects, assuming for the latter the respiratory tract as portal-ofentry.

Although NPs are considered to be particles <100 nm in diameter by toxicologists and

material scientists, this definition should not imply a rigid division between nano-sized

particles and larger ones, but more so a vague transitional phase in physicochemical terms

that is material-specific. For example, 240 nm polystyrene particles deposited in the alveolar

region were not found to translocate across the alveolo-capillary barrier into the blood

circulation; however, when coated with lecithin these particles did translocate and appeared

in blood monocytes (Kato et al., 2003). Thus, even particles larger than 100 nm can

translocate across the alveolo-capillary barrier depending on surface modifications.

Therefore this transitional phase between NPs <100 nm and particles >500 nm is left open in

Table 1.

Some fundamental differences between NPs and larger particles include deposition behavior

in the respiratory tract and mechanisms related to clearance, cell entry and translocation to

secondary organs. With respect to the type of effects that have been observed in studies with

nano-sized particles and larger particles, there does not appear to be a major difference.

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However, one key difference is related to the uniqueness of NPs to translocate from the

respiratory tract to secondary target organs. Thus, effects such as inflammation, oxidative

stress and molecular cell activation are likely to occur not only in the primary organ of entry,

but also in secondary target organs. Such effects are unlikely to occur with larger particles,

except under lung particle overload conditions (silicosis of the liver, spleen, bone marrow

[Eide et al., 1984; Slavin et al., 1985]) and in the case of asbestos-induced mesothelioma and

associated lympho-hematogenic spread of asbestos fibers (Brown, 1974). An important

caveat to keep in mind is that Table 1 refers to the particles themselves and not to any

soluble fractions, either of the particle or of adsorbed materials.

2.1 Dose and dose rate as key concepts of nanotoxicology

The aforementioned studies on particle lung overload in rats (Morrow, 1992) demonstrate

that overwhelming the capacity of alveolar macrophages to phagocytize and clear retained

particles from the alveolar region of the lung results in severe lung injury by mechanisms

that are not operational at lower doses. Dose, dose rate and dose metrics are critical

determinants of effects which need to be considered when designing toxicological studies.

This is indicated in Table 1 with an important cautionary note regarding the issue of dose.

High doses administered as a bolus in an animal study (e.g., via intratracheal instillation) or

to cell cultures can readily identify a NP as hazardous on the basis of observed significant

inflammatory/oxidative stress responses. Although such studies are valuable and may be

used for ranking the toxicity of newly developed NPs against a reference or benchmark

particle, observed effects may not be directly extrapolated as occurring under in vivo

exposure conditions (Driscoll et al., 2000). A key difference is the dose rate (dose per unit

time) in addition to the amount of the delivered dose (dose per unit surface area of the

respiratory tract). The mechanisms underlying effects induced by a high dose rate (bolus

delivery) are likely very different from those induced when the same dose is delivered by

inhalation over days, weeks or months. Results from bolus type dose delivery should not be

used for purposes of risk assessment; however, when designed as dose-response studies

including reasonably low doses, they can be very valuable as hypothesis forming or proof of

principle studies, to be validated in vivo.

As an example, the oxidative stress-inducing capacity of nano-TiO2 (~25 nm primary

particle size with ~150 nm aggregates [Jiang et al., 2008]) was described in a well-designed

in vitro dose-response study using mouse brain microglia cells, showing that concentrations

of 10 ppm of nano-TiO2 induced reactive oxygen species (Long et al., 2006). This is an

intriguing finding; however, the result was unfortunately misinterpreted by the popular

press, despite the cautionary tone of the study’s authors, with the eye-catching headline

Nanoparticles in sun creams can stress brain cells” (Ball, 2006). Instead, the results need to

be considered in the context of the biokinetics of NPs from the portal of entry to the brain.

Even if nano-TiO2 in skin care products could enter the blood circulation by crossing the

skin barrier – which has not yet been demonstrated despite concerted efforts (Lademann et

al., 2007) – it would be only miniscule amounts. In order to get to the brain, the very tight

blood brain barrier (BBB) must be overcome next, which is another event of low

probability. Any amount of nano-TiO2 that may travel from skin to brain will result in

orders of magnitude lower concentrations than used in in vitro studies. Thus, confirmation of

in vitro results through realistic in vivo studies is mandatory to test hypotheses generated

from in vitro studies.

The generally held view that a non-cytotoxic in vitro dose is realistic and expected to occur

under relevant in vivo exposure conditions needs to be critically assessed. The relevancy of

doses and the impact of dose rate should carefully be considered and discussed each time.

The usually very low dose rates experienced in vivo are likely to induce adaptive

physiologic (often protective) responses which can render the organism unresponsive to

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even high doses. For example, a 15-minute inhalation exposure of rats to highly toxic PTFE

(polytetrafluoroethylene) fumes (ultrafine particles, ~20 nm) caused severe lung damage and

mortality; however, exposures for 5 minutes on 3 consecutive days resulted in adaptive

responses (e.g., up-regulation of antioxidants) that completely protected the rats from the

toxicity of a subsequent 15-minute exposure, whereas all simultaneously exposed nonadapted

rats died (Figure 1; Johnston et al., 2000).

With respect to the BBB, on-going research is aimed at developing strategies to overcome

this hurdle so NPs administered into the blood circulation can be used for efficient drug

delivery to the brain (Kreuter, 2007). One strategy involves adsorbing specific proteins,

peptides or surfactants onto the surface of biodegradable NPs in order to prolong particle

circulation in blood, as well as augment specific interactions with endothelial transport

receptors, thereby optimizing NP crossing potential without altering normal BBB function.

For example, a recent study showed that only cholesterol-terminated polyethylene glycol

nanoparticles (150-200 nm) functionalized with a tyrosine aminotransferase peptide were

shown to localize after tail-vein injection in the neural stroma of the rat hippocampus (Liu et

al., 2008). In addition, the upper respiratory tract can also serve as a portal-of-entry for NPs

to the blood and to the CNS, which will be discussed in the following section.

3. NP Translocation to the Brain and Effects

3.1 Deposition of inhaled NPs and translocation pathways

The deposition of inhaled NPs in the respiratory tract is governed by random motion due to

bombardment by gas molecules, known as Brownian motion or diffusion. Deposition by this

mechanism is greatest for the smallest NPs, as shown in Figure 2 for the adult human

respiratory tract, assuming nose breathing at 10 L/min (ICRP, 1994). Thus, about 85% of

airborne NPs of about 1 nm in size will be deposited by this diffusional deposition in the

upper respiratory tract, whereas in the tracheobronchial and alveolar regions of the lower

respiratory tract, peaks of deposition are for NPs of around 5 nm (~35%) and of 20 nm

(~50%), respectively. Of course, if the primary particles of NPs are agglomerated, the

deposition efficiency is a function of the larger agglomerate size. Gravitational

(sedimentation) and inertial (impaction) forces increasingly become determinants of

deposition for particle sizes above 200 nm.

The high diffusional deposition of the smallest NPs in the upper respiratory tract has

significant biological/toxicological implications because NPs of this size range behave

similarly to smell molecules in the inspired air that are directed at the olfactory mucosa

(Figure 3). For efficient smell recognition, numerous neurons embedded in the olfactory

mucosa are connected to the nasal lumen through their dendrites. The significance of

sensory nerve structures lies in the potential that neuronal axons and dendrites can transport

nanoparticles in retrograde and anterograde directions (Adams and Bray, 1983). Indeed, this

was demonstrated for the olfactory nerve by the recent confirmation of a little noticed earlier

discovery that nanoparticles deposited in the nasal cavity can translocate with amazing

velocity along this sensory neuronal pathway to the olfactory bulb (DeLorenzo, 1970;

Oberdörster et al., 2004). The nasopharyngeal airways and the tracheobronchial airways are

supplied with sensory nerves as well, whereas the presence of sensory nerves in the alveolar

region of the lung is less defined (Figure 4). These sensory nerves have either direct

(olfactory and trigeminus nerve) or indirect (tracheobronchial, via vagus) connections to

specific areas of the CNS.

Thus, translocation of NPs depositing in the respiratory tract can occur along different

routes, as indicated in the previous paragraphs and illustrated in Fig. 5. NPs depositing in the

upper and lower respiratory tract may translocate directly to the blood compartment or via

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the nasal and lung lymphatic circulation. Based on studies published thus far, translocation

rates for NPs into the blood circulation appear to be very low (Kreyling et al., 2002).

Subsequent translocation of blood-borne NPs across the tight junctions of the BBB in vivo

has not been conclusively demonstrated; although experimental drug delivery via

intravenously administered NPs to the brain has been reported (Kreuter, 2007; Liu et al.,

2008), it is still an open question as to whether the NPs in those studies did localize in brain

tissue or had just entered the endothelial cells of the CNS vasculature and the drug was

released to the brain from there. NP translocation across the BBB is more likely to occur at

sites where this barrier is less well developed or injured, and thus leaky, i.e, at the

circumventricular areas. For example, destroying the integrity of the BBB by osmotic

pressure or by extremely high doses of metal NPs (30 mg/kg, i.v. or 50 mg/kg i.p. of ~50 nm

Cu or Ag or 60 nm Al particles) can deliver NPs to the brain (Sharma, 2007). The doses

used in these studies cannot be achieved under realistic in vivo conditions, and results need

to be interpreted with caution. The mechanisms of NP transfer across the intact BBB remain

elusive and there are still open questions as to the long-term consequences of NP

accumulation in the CNS and their fate within CNS structures.

Translocation to CNS structures from the upper respiratory tract via neuronal pathways is

now well established but seems to be of low efficiency. Table 2 summarizes studies that

demonstrated olfactory nerve translocation of different nano-sized particles following dosing

of several animal species by intranasal instillation or whole body inhalation. Estimates from

instillation and inhalation studies of the amount translocated from deposits on the nasal

olfactory mucosa range from <1% to more than 10%. This is apparently dependent on NP

surface chemistry, size (primary particle vs. agglomerate), dose and exposure method as

discussed below. It is interesting to note that a loss in olfactory function is a common feature

of neurodegenerative diseases such as Parkinson’s, Alzheimer’s and Huntington’s Disease

(Barrios et al., 2007; Doty, 2008; Kovács, 2004; Moberg and Doty, 1997), raising the

possibility that olfactory translocation of inhaled NPs and olfactory neuropathology might

be etiologically linked.

An interesting translocation pathway depicted in Figure 5 is suggested by the study of

Czerniawska (1970), which describes the appearance of radioactive nanogold (198Au)

particles in the cerebrospinal fluid (CSF) following submucosal injection into the nasal

olfactory area of rabbits. This author observed highest radioactivity in the CSF adjacent to

the cribriform plate of the skull, the area where olfactory nerve axons emerge and connect to

the olfactory bulb (Figure 6). High 198Au activity was also measured in CSF surrounding the

olfactory bulb and the corpus callosum cistern. Czerniawska (1970) confirmed from these

results earlier findings that there is a direct connection between the olfactory mucosa and the

CSF (Orosz et al., 1957). 198Au activity in CSF can be interpreted as input from perineural

transport of nanogold particles which has been described as a very rapid translocation route

from nose to brain (Illum, 2000). In order to access the brain from the CSF compartment, the

cerebrospinal fluid brain barrier has to be overcome (Figure 5). An interesting alternative to

this interpretation is that nanogold particles after axonal transport to the olfactory bulb cross

into the CSF space and are distributed to different brain areas (Segal, 2000). Further

research is needed to identify the biokinetics of NPs within the CNS and associated

structures.

The importance of the CSF compartment for delivery of nanoparticles and nanomedicines

from the nose to the brain was recently demonstrated when researchers showed that a

neuroprotective drug encapsulated within 80 nm MPEG-PLA NPs was significantly and

directly transported from the olfactory mucosa to the olfactory bulb, CSF, and other brain

regions, i.e., bypassing systemic transport from the blood (Zhang et al., 2006). CSF fluid had

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the highest direct transport percentage of the drug after nasal NP delivery, it was even higher

than in the olfactory bulb; all CNS compartments received 1.6 – 3.3-fold more of the drug

when bound to the NP than when the drug was delivered in solution (Figure 7).

3.2 Neuronal NP translocation to olfactory bulb and CNS effects

Elder et al. (2006) carried out several inhalation studies in rats with Mn-oxide nanoparticles

(33 nm count median diameter in the airborne state) to test olfactory nerve translocation and

the potential of such exposure to induce effects. After occlusion of the right nostrils of the

rats, they were exposed to Mn-oxide NPs by inhalation on 2 consecutive days for 6 hours

each day. Only the left olfactory bulb showed a large increase in Mn, with no increase of Mn

in the right olfactory bulb (Figure 8). This result confirmed the olfactory neuronal pathway

of NP translocation from nose to brain and excluded the possibility that a significant

contribution may have come from blood-borne Mn. An estimation of the amount depositing

on the olfactory mucosa using a computerized Multiple Path Particle Deposition Model

(Asgharian et al., 1999) showed that the amount of Mn translocated to the olfactory bulb

was 11% of the deposit on the olfactory mucosa. In a subsequent 12-day (6 hrs/day)

inhalation exposure with both nostrils open, Mn concentration in the olfactory bulbs

increased almost 4-fold, with slight increases also in other brain regions, whereas lung Mn

concentrations only increased a little more than 2-fold (Figure 9). Inflammatory responses

were also observed in those brain regions that had increased Mn levels, with the olfactory

bulb showing an almost 30-fold increase in TNFa (Figure 10). This may not be surprising,

given that manganese is well recognized as a neurotoxicant and that dissolution of Mn-oxide

NPs in brain tissue may reach very high local intracellular levels. It should be mentioned hat

these inhalation exposures were performed at airborne concentrations of Mn that are similar

to what is generated by arc welding (0.01-5 mg/m3). Indeed, altered locomotion has been

described in a cohort of welders (Finkelstein et al., 2007).

Obviously, the inflammatory response induced in rats by Elder et al. (2006) through realistic

and relevant inhalation exposures to Mn-oxide NPs should be regarded as a serious health

concern. However, it would be premature to suggest that all NPs have the same neuroinflammatory

potential. There are many significant differences among NPs in terms of

physicochemical characteristics that influence both NP biokinetics as well as responses, and

this has to be evaluated on a case-by-case basis. However, particular caution has to be

exercised to avoid situations where exposures to high concentrations of airborne NPs occur.

Prudence calls for personal protection equipment and engineering controls to guard against

such exposures.

There is an urgent need for identifying hazardous NPs and characterizing exposures and the

biokinetics of NPs. For example, highly reactive inhaled materials such as the

aforementioned PTFE fumes comprising nano-sized particles (Figure 1) which induce

severe acute lung injury, are likely to also damage CNS structures. Indeed, gene array

analysis of olfactory bulb tissue of PTFE-exposed rats revealed increased levels of

inflammatory and oxidative stress-related genes (Figure 11, unpublished data). A large

decrease in glutamate transporter stands out, possibly indicating an adverse effect on

excitatory neurotransmitter removal which may result in toxic glutamate buildup and

dysregulation of glutathione levels in the extracellular space of CNS tissue. The specificity

of this response and its implications need to be examined in further studies. Of course,

because of the high acute pulmonary and associated systemic toxicity the observed gene

expression changes in the olfactory bulb may just be a manifestation of the general high

systemic toxicity induced in the PTFE-exposed rats. Exposures of humans to metal and

polymer fumes (all consisting of nano-sized particles) are well known to induce CNS

symptoms such as headaches; however, there are no data in humans demonstrating

translocation of fume particles to the brain.

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As pointed out above, significant differences among NPs with regard to their translocation

kinetics to and effects in the CNS should be expected because of the vast differences in

physico-chemical properties of different NPs. For example, in contrast to the result with

inhaled Mn-oxide NP (Figures 8 and 9) only a very small fraction of gold NPs deposited by

intranasal instillation onto the olfactory mucosa was translocated to the olfactory bulb in rats

(Figure 12). This was found with different sizes of nanogold particles that were coated with

rat serum albumin to avoid agglomeration when these particles were suspended in

physiological saline (Rinderknecht et al., 2007). There appears to be a size dependency,

though, with the larger 20 nm gold particles translocating less than the 2 and 5 nm particles.

Surface coating also appears to affect translocation rate since more of the uncoated 35 nm

Mn-oxide particles translocated to the olfactory bulb compared to the 35 nm PEG-coated Qdots

and similar-sized albumin-coated Au particles. Overall, the translocation rates at 24-

hours post exposure were less than 0.01% of the instilled dose. This low translocation rate

may be due to several factors: (i) intranasal instillation may not dose all areas of the

olfactory mucosa; (ii) in contrast, inhalation of singlet NPs optimizes deposition on the

olfactory mucosa in terms of total amount and evenness of distribution; and (iii) perhaps

more importantly, nasal instillation is equivalent to a bolus administration to the nasal

mucosa, where efficient ciliary clearance mechanisms exist. The high dose rate of

instillation is very different from the low dose rate of inhalation which represents continuous

dosing over many hours.

The importance of dose rate for affecting not only the response but also NP biokinetics is

supported by results from studies in which the same NP was administered by both methods.

As described above, Elder et al., (2006) estimated that 11% of Mn-oxide NPs deposited on

the olfactory mucosa by inhalation had translocated to the olfactory bulb in rats. In a

separate study, the same Mn oxide particles were administered by intranasal instillation and

only 0.01% of the intranasally administered amount accumulated in the olfactory bulb

(Figure 12). Moreover, intranasal instillation of differently coated CdSe-ZnS polymercapped

quantum dots (Elder et al., 2007) in rats revealed even lower translocation rates

which were similar to the nanogold particles (Figure 12). In contrast, estimates of nose-toolfactory

bulb translocation from another short-term inhalation study in rats with inhaled

carbon NPs (count median diameter 36 nm) were ~20%, although this value is probably

associated with considerable uncertainty(Oberdörster et al., 2004).

A reasonable conclusion from these diverse results of nose-to-brain translocation seems to

be that the physiological route of inhalation (assuming nasal breathing) is a more effective

way of dosing the olfactory mucosa with NPs for subsequent translocation to the brain than

intranasal instillation. Of course, this conclusion has to be considered in the context of the

deposition efficiency of inhaled NPs in the nose, which is very high for 1 nm particles but an

order of magnitude lower for 70 nm particles (Figure 2). As discussed before, NP

translocation to the CSF seems to be more efficient than directly to the olfactory bulb.

Regardless, highest NP concentrations in the CNS – which could be either toxic or

therapeutic – are expected to be achieved with the smallest inhaled NPs, whereas larger

inhaled NPs predictably result in higher concentrations in other parts of the body.

3.3 NP surface properties affecting translocation

Particle size is only one of several NP properties that determine their kinetics and effects in

the organism. Other physicochemical characteristics, in particular those related to NP

surface, are decisive as is illustrated by the concept of differential adsorption. This concept

states that physicochemical properties of NPs and conditions at the portal-of-entry determine

adsorption of proteins/lipids on the NP surface, which in turn determines their biokinetics

and effects (Figure 13). For example, NPs depositing in the respiratory tract will come into

contact with the epithelial lining fluid containing proteins and lipids that can adsorb onto the

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NPs. This alters NP properties and, as a result, affects their biokinetic behavior and cell

entry. Subsequent translocation to the interstitium, lymph or blood circulation may result in

successive protein coatings. This secondary coating process is dynamic and depends on

adsorption and desorption constants of the different proteins and their relative concentrations

in the surrounding milieu and, therefore, changes with time. Upon cell entry of NPs, coating

with the cytoskeletal proteins, actin and vimentin has been observed (Ehrenberg and

McGrath, 2005), raising the question that this may affect cytoskeletal networks.

Cedervall et al. (2007) have identified a number of human plasma proteins that bind to copolymer

NPs. They found different classes of proteins, most prominently apolipoproteins,

albumin, fibrinogen and others, which formed a protein corona on NPs during incubation

with plasma. It is of interest to note that in other studies coating of polymer NPs with

apolipoprotein E was found to enhance delivery across the BBB of drug-loaded NPs,

probably via LDL receptors on endothelial cells of brain capillaries (Kreuter, 2007). An

interesting finding with respect to NP-protein association and corona formation was reported

by Linse et al. (2007) who found that under specific in vitro conditions different types of

NPs accelerated fibrillation of an amyloid protein (Figure 14). A general hypothesis derived

from these proof-of-principle studies is that NPs may be an etiological factor for amyloid

diseases, such as Parkinson’s, Alzheimer’s, and Creutzfeld-Jakob diseases and dialysisrelated

amyloidosis. Proving this hypothesis that NPs may cause protein fibrillation under

realistic in vivo conditions would have far-reaching consequences, in particular with respect

to underlying mechanisms of neurodegenerative diseases (Colvin and Kulinowski, 2007).

The necessity for more research in this area is obvious.

3.4 NP biokinetics based on low translocation rates

From studies like these, researchers become increasingly aware of the concept that coating

of NPs with specific proteins, lipids or polymers can significantly alter their kinetics in the

organism and their interactions with cells. Studies evaluating this concept of differential

adsorption revealed significant differences between gold particles coated with either PEG or

albumin, administered either to the lower respiratory tract or directly intravenously into rats

(Rinderknecht et al., 2007). These studies confirmed that NP biokinetics can be modified by

their coating as well as the portal-of-entry, as is schematically illustrated in Figures 15 for

two target organs: brain and liver. Of course, major differences between the two routes of

exposure exist with respect to the input into the blood compartment. While both represent

bolus-type dosing, input of NPs into the blood compartment from deposits in the respiratory

tract occurs (i) at a much lower overall dose; (ii) at a very low dose rate; and (iii) into the

pulmonary venous circulation (oxygenated blood, entering systemic arterial circulation)

which is in contrast to systemic venous circulation of i.v. injection. The secondary coating of

NPs along this translocation pathway will very likely affect their fate once they enter the

blood circulation, as discussed above. In addition, NP size also modifies NP kinetics, as will

be described in a detailed forthcoming paper.

Protein coating of NPs during incubation in biological media depends on particle surface

properties and on the components in the biological medium. For example, it is conceivable

that incubation of NPs in blood plasma from humans, rats, or mice leads to different NPprotein

complexes. Whether this causes differences with respect to NP biodistribution and

effects between these species is unknown. However, the possibility of such differences

might be an important factor to be considered for extrapolation of results from rodent studies

to humans.

Although translocation rates of NP from the portal-of-entry to secondary organs may be very

low, a continuous exposure may result in significant accumulation of NPs in a secondary

target organ. Thus, it is important to obtain data on the retention characteristics of NPs in

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