TOXICOLOGICAL SCIENCES 90(1), 23–32 (2006)
doi:10.1093/toxsci/kfj084
Advance Access publication January 4, 2006
FORUM SERIES
Research Strategies for Safety Evaluation of Nanomaterials,
Part V: Role of Dissolution in Biological Fate and
Effects of Nanoscale Particles
Paul Borm,* Frederick C. Klaessig,† Timothy D. Landry,‡,1 Brij Moudgil,§ Jürgen Pauluhn,¶
Karluss Thomas,k Remi Trottier,jjj and Stewart Woodkk
*Centre of Expertise in Life Sciences, Zuyd University, Heerlen, the Netherlands; †Aerosil & Silanes, Degussa Corporation, Piscataway, New Jersey
08855, USA; ‡Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674, USA;
§Particle Engineering Research Center, University of Florida, Gainesville, Florida 32611, USA; ¶Institut für Toxikologie, Bayer HealthCare, Wuppertal,
Germany; kILSI Health and Environmental Sciences Institute, Washington, DC 20005, USA; jjjAnalytical Sciences, The Dow Chemical Company,
Freeport, Texas 77541, USA; and kkAnalytical Sciences, The Dow Chemical Company, Midland, Michigan 48674, USA
Received September 7, 2005; accepted December 20, 2005
Dissolution, translocation, and disposition have been shown to
play a key role in the fate and effects of inhaled particles and
fibers. Concepts that have been applied in the micron size range
may be usefully applied to the nanoscale range, but new
challenges are presented based on the small size and possible
change in the dissolution:translocation relationship. The size of
the component molecule itself may be on the nanoscale. Solute
concentration, surface area, surface morphology, surface energy,
dissolution layer properties, adsorbing species, and aggregation
are relevant parameters in considering dissolution at the nanoscale. With regard to the etiopathology caused by these types of
particulates, the metrics of dose (particle number, surface area,
mass or shape) is not yet well defined. Analytical procedures for
assessing dissolution and translocation include chemical assay and
particle characterization. Leaching of substituents from particle
surfaces may also be important. Compartmentalization within the
respiratory tract may add another dimension of complexity.
Dissolution may be a critical step for some nanoscale materials
in determining fate in the environment and within the body. This
review, combining aspects of particle toxicology, material science,
and analytical chemistry, is intended to provide a useful basis for
developing relevant dissolution assay(s) for nanoscale particles.
Key Words: dissolution; nanotechnology; nanoscale particles;
ultrafine.
a dimension in the range of 1–100 nm in nanoscience and
nanotechnologies (Borm, 2002; Borm and Kreyling, 2004).
Nanoscale particles (NSP) have three or perhaps two dimensions in the range of 1–100 nm, and may be considered to be
a toxicological entity in their interactions with the environment, cells, and molecules within the cell. At these dimensions,
there is increased concern that nanoscale particles may translocate from the respiratory tract or other portal of exposure,
with potential for effects in other organ systems. In addition,
little is known about their environmental distribution, accumulation, and degradation (Colvin, 2003). Dissolution, translocation, and deposition have been shown to play a key role in
the fate and effects of conventional fibers and particles. The
concepts that have been applied to particles in the micron size
range may be usefully applied to the nanoscale range, but with
new challenges based on the very small size and possible
changes in the dissolution:translocation relationship.
Determining the colloidal and surface properties of particles
in this size range can be complex, but this state is likely to play
a key role in biological fate and effects. This review seeks to
combine materials science principles and analytical approaches
to nanoscale particle dissolution, with an objective that these
could be applied to assessment of dissolution as part of a screen
for environmental and or toxicological evaluation of nanoscale
materials.
INTRODUCTION
PARTICLE TOXICOLOGY: BASIC CONCEPTS
During the past decade, a challenge for modern particle
research has emerged by the increasing use of materials with
1
To whom correspondence should be addressed at Toxicology and
Environmental Research and Consulting, The Dow Chemical Company,
Midland, Michigan 48674. E-mail: [email protected].
Apart from the specific chemical surface reactivity of
nanoparticles, a particle load or burden in the lung can induce
toxicological response(s) that differ principally from soluble or
non-particulate toxicants. For the interpretation of inhaled
particle effects, dose, deposition, dimension, durability and
Ó The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For Permissions, please email: [email protected]
24
BORM ET AL.
defense are important determinants of response. First of all, the
dose at a specific site (in the lungs) determines the potential
toxicity of particles. Obviously, this deposited dose is dependent on the inhaled concentration as well as the dimensions
of the particle. As a result of diffusion processes, the deposition
probability of nanoparticles in the respiratory tract markedly
increases for ultrafine particles above that for larger particles
where convective flow dominates deposition. The dose metric
for nanoscale particles (NSP) is an additional complexity, as
particle number, surface area, and shape, among other factors,
may play a role in addition to the traditional mass-based metric
(Donaldson et al., 2002; Oberdorster et al., 2005).
The lung has extensive, location-specific defense systems
such as mucociliary clearance in the upper airways and
macrophage clearance in the lower, unciliated part of the
respiratory tract. Particle transport by macrophages from the
alveolar region toward the larynx is rather slow in humans, even
under normal conditions, and it eliminates only a fraction of the
deposited particles in the peripheral lung. The remainder may
accumulate unless the particles are biodegradable or cleared by
simple chemical dissolution in lung surfactant or interstitial
fluid. Therefore, the same deposition of NSP with a different
durability can lead to a different cumulative dose. The kinetics
of dissolution of inhaled particulates determine whether a lowtoxicity particle, such as amorphous silica, will dissolve in the
epithelial lining fluid or whether such particles as carbon blacks
or iron oxides are engulfed by alveolar macrophages. Thus,
solubility is a key driver for the clearance mechanisms involved
in their removal. The prediction of the in vivo dissolution
kinetics of particles is potentially affected by subcellular compartments with different pHs than the lining fluids. Moreover,
agglomerated airborne particles in the 1 lm range may be
broken down to the size of the primary nanoparticle within these
structures of the lung. Ultrafine particles may have constituents,
either as chemical components or as elements adsorbed to the
surface, and dissolution could release these components into the
lung tissues and circulation (Bagate et al., 2004). If a toxic
particle is neither soluble nor degradable in the lung, it is likely
to have high durability (biopersistence) with accumulation upon
sustained exposure. In addition to the known biological effects
of low-soluble particulate materials, dispositional factors of
NSP may also influence the effects.
Toxicology of Ultrafine Particles
Nanoscale particles, whether termed ultrafine, nano-, engineered, intentional, or incidental, pose challenges for physical,
chemical, and biological characterization. A variety of biological effects have been reported, although in some cases
potential hazards have been identified, generally without an
assessment of a relevant or likely exposure (Donaldson et al.,
2002; Borm and Kreyling, 2004; Oberdorster et al., 2005). A
variety of physico-chemical effects have also been reported,
such as strong interactions with other particles, either of the
same size or aggregated (cohesion), and with surrounding
media (adhesion). Toxic effects have been reported to include
an inflammatory response with exacerbation of airways
disease, cardiovascular events by hypercoagulabiliy or plaque
destabilization (Pope et al., 2004), or chronic effects in animal
models (Driscoll et al., 1996; Borm et al., 2004). Much of this
database is from research on combustion-derived nanoscale
particles and may not be generally characteristic of NSPs or
may be less relevant for engineered NSPs.
Realistic exposure assessments and test procedures will be
important for risk assessment of NSPs. Migration of NSPs to
a distance from their application or deposition sites is possible,
although the characterization of nanoscale particle pharmacokinetics is very limited, except for pharmaceutical-related
products. Discrete primary NSP may have relatively low
recognition and uptake by lung macrophages, and this could
facilitate movement from the lungs. Exposure to aggregates
(also termed ‘‘secondary particles’’), which may be the more
likely species of inhalation exposure, may have quite different
clearance kinetics. In addition to phagocytosis (cellular ingestion of relatively large objects), endocytotic processes of
pinocytosis and receptor-mediated endocytosis are likely to
play a role in translocation of NSP (Gumbleton, 2001;
Arredouani et al., 2004). Several authors have reported on
the movement of nanoscale particles from the lung to the blood
(Berry et al., 1977; Nemmar et al., 2002), and they hypothesized the potential for microthrombus formation, which has
become a focus of research attention in relation to fine
particulate air pollution (Nemmar et al., 2004). Oberdorster
et al. (2005) reviewed studies that indicated that various NSP,
including gold particles (50 nm), poliovirus (30 nm), MnO2,
and carbon-spark generated NSP may be directly transported
toward the brain via axonal transport, including trans-synaptic
transport. Nanoscale particles may affect movement of endogenous substances (proteins, enzymes) or exhibit increased
reactions with biological molecules.
The cumulative dose upon chronic exposure is dependent on
exposure level and clearance pathways. Apart from macrophage clearance, dissolution and biodegradation are important
components of (lung) clearance that may prevent accumulation
of NSP. This is reflected in the outcomes of a chronic rat
experiment, to which the tumor incidence of several durable
ultrafine particles (carbon black, TiO2, diesel) and soluble
(amorphous silica) ones, as well as some fine particles of the
same chemical composition, were compared (Borm et al.,
2004). Particles were instilled intratracheally in doses up to
60 mg. Lung tumor incidence induced by three different insoluble NSP was proportional to their surface area. The highsurface amorphous silica induced few lung neoplasms, possibly
because of its high solubility, i.e., low durability in vivo. These
data suggest that particle dissolution may be a useful in vitro
screening method for assessment of particulate nanomaterials.
In Figure 1, a schematic of the role of dissolution is illustrated
by contrasting the fate of soluble versus poorly soluble
ROLE OF DISSOLUTION IN BIOLOGICAL FATE: NANOTECHNOLOGY
FIG. 1. A graphic simplified relationship between exposure to nanoscale
particles, dissolution, effects, and clearance. This utilizes the hypothesis that
surface dose is critical to effects of a particle. ‘‘Large’’ dose implies
biologically excessive, with the potential for injury. Dissolved constituents of
the NSP may be present in excess (potential for injury), or dissolution may
provide an additional clearance route (biotransformation and/or excretion).
particles. Dissolution may yield constituents with biologic
activity, producing injury if present or accumulated in excessive amounts. For the poorly soluble nanoscale particles,
a ‘‘large’’ surface dose (‘‘large’’ equals potential to cause
injury) could cause injury, presumably on the basis of surface
effects. In the case of inhaled nanoscale particles, the effects
may be reflected in pulmonary inflammation. The diagram also
depicts potential accumulation, translocation, and distribution
of nanoscale particles.
Assessing durability and dissolution have been important for
the development and regulation of man-made vitreous fibers
(MMVF), with markedly reduced potential for pulmonary
toxicity. However, procedures for definition and measurement
of nanoscale particle dissolution are needed to allow evaluation
and implementation of such a method. Theoretical and
practical aspects of NSP dissolution and measurement are
discussed in the following sections.
MATERIALS SCIENCE ASPECTS
Before describing the dissolution process in more detail, it is
important to differentiate nanosized particulate materials from
nanosized molecules. As already mentioned, nanoparticles are
generally defined as particles that have at least one dimension in
the 1–100 nm size range. Though this definition is accurate by
contemporary standards, questions arise as to what differentiates a molecule in solution from a particle—many NSPs
such as fullerenes, dendrimers, and protein molecules are
composed of a single molecule but embody particle-like
characteristics; whereas others, such as polyethylene glycol
(PEG) molecules, arguably may not. One reasonable assessment is that if a molecule contains segments, or an internal core
that is insoluble (has an energetically unfavorable interaction
25
with the solvent promoting phase separation), then that
molecule as a whole should be classified as a particle. Because
insolubility restricts a molecule’s conformational and thermal
freedom, it can be argued that it contributes to a more particlelike behavior. In many cases, NSPs such as metal oxide and
polymeric nanoparticles, are composed of multiple molecular
constituents that exist as relatively rigid structures independent
of the solvent present. In contrast to solvated molecular species,
these molecules closely interact with each other and are tightly
packed together via intermolecular binding forces (e.g., metal
binding, van der Waal’s forces, ionic binding) that are often
heterodesmic. During the dissolution process, these intermolecular bonds are broken via a process that results in a lower free
energy of the system. It should be noted that particles and
aggregates of particles can also be broken down to smaller
particles via milling and/or dispersion—these phenomena are
distinct from dissolution in that they predominantly result in the
generation of smaller particles, rather than solvated molecules,
and typically require relatively large amount of mechanical
work to reduce the particle size. The change in free energy
involved in breaking apart particulate constituents or increasing
surface area may be an important consideration for assessing
dissolution of a species. However, issues surrounding differentiating spontaneous dispersion of single molecule nanoparticles
versus classical dissolution phenomena are yet to be resolved.
More definition is needed in this area. For the purpose of this
review, dissolution is defined as described in the next section.
Dissolution
Dissolution is the dynamic process by which a particle goes
into the solution phase to form a homogeneous mixture. For
a material to dissolve, its constituent molecules must have
some solubility in the local environment. For certain classes of
NSPs, as discussed above, the size of the component molecule
may be on the nanoscale. For example, a single C-60 is
approximately 1 nm and a hemoglobin molecule is approximately 6 nm. (It should be noted that further breakdown of
these NSPs results in degradation rather than dissolution.)
Material solubility is highly dependent on solvent properties
(e.g., pH, ionic strength, constituent solvated molecules, and
concentration), and therefore may vary with the region of
exposure in vivo. Both particle dissolution kinetics and solubility are size dependent, and nanoparticulate materials are
often expected to dissolve more quickly and to a greater extent
(i.e., higher equilibrium solubility) than macroscopic particles
of the same material. Because of this, the dissolution of nanoparticulate materials can govern their persistence in vivo and
should be considered an important factor when interpreting
biological responses to NSP exposures. Considering the difficulty in predicting dissolution behavior in complex systems
(e.g., in vivo), experimental toxicologists need to be aware of
fundamental aspects of the dissolution process as well as other
variables that can affect the phenomenon. Such knowledge will
26
BORM ET AL.
where Ct is the concentration of solute molecule in bulk
solution, Cs is the solubility or saturation concentration of
solute molecule in bulk solution, t is the time, and k is the rate
constant of dissolution. The dissolution rate constant depends
on the surface area of solute (A) as shown:
k¼
A3D
;
V 3h
ð2Þ
where D is the diffusion coefficient of solute molecule, V is
the volume of solution, and h is the thickness of diffusion layer.
Because of the lack of reliable theoretical approaches for their
prediction, diffusion coefficients, and layer thicknesses are
often experimentally determined (Macinnis and Brantley,
1992; Wang and Flanagan, 2002).
Role of Surface Morphology and Curvature
FIG. 2. Top. Simplified pictorial representation of the dissolution phenomenon denoting the concentration profile of solute molecules and ions.
Bottom. Relative concentration profile of solute molecules as a function of
distance from bulk particle to bulk solution.
be useful in designing more meaningful experimental protocols, characterizing systems, and interpreting data from particle exposure studies.
During particle dissolution, constituent molecules of the
dissolving solid migrate from the surface to the bulk solution
through a diffusion layer (Fig. 2). The diffusion layer
represents a region between the particle surface and bulk
solution environment that is densely populated by solvated
molecules (e.g., ions, solute molecules, biomolecules, etc.) and
is demarcated by the shear plane that separates the stagnant
fluid layer that travels with the particle, from the bulk solution
phase. The driving force for dissolution depends on the
materials solubility within a given environment as well as
the concentration gradient between the particle surface and
the bulk solution phase. Hence, the kinetics of dissolution of
soluble materials is surface area dependent. Nanoparticulate
materials are anticipated to dissolve faster than larger sized
materials of the same mass by surface area considerations
alone—although other factors such as surface curvature/
roughness also play a role. The dissolution process slows as
the concentration in the bulk solution phase reaches equilibrium solubility levels.
The kinetics of dissolution for particulate materials are often
described using the Noyes-Whitney equations, as shown below:
kt
Ct ¼ Cs 3 ð1 e Þ;
ð1Þ
Different sized and shaped particles of the same material at
equivalent surface area dosages and starting free (dissolved) solute concentrations undergo dissolution at different rates (Tang
et al., 2004). This phenomenon has been explained through
alterations in diffusion layer properties (Tinke et al., 2005), as
well as variations in equilibrium solubility (Iler, 1979).
Diffusion layer thickness has been experimentally found to
decrease with particle size leading to faster transport of
solvated molecules to bulk solution and hence more rapid
dissolution (Tinke et al., 2005). This consideration can also be
extended to surface morphological features, and may provide
explanations for the preferential dissolution of convex versus
concave surface structures. The overall hydrodynamic shape of
a particle defines its surrounding shear plane boundary;
therefore smaller particle surface features can have varying
local diffusion layer thicknesses. Intuitively, we can conclude
that convex features will have thinner diffusion layers because
they extend from the surface toward the shear plane, whereas
concave features will have thicker diffusion layers because
they are recessed on the surface—leading to relatively faster
and slower dissolution rates, respectively.
In addition to the kinetic assessment given above, thermodynamically, particles and surface features of different shape
and dimension can have different equilibrium solubilities,
which further modify their dissolution behavior. The GibbsThompson effect, which describes the relationship between
curvature, interfacial free energy, and solubility (Adamson
et al., 1997; Iler, 1979), predicts that particles and surface
features with a smaller radius of positive curvature (convex) are
energetically unfavorable. Therefore these particles and features are subject to preferential dissolution and have higher
equilibrium solubility—in some cases their equilibrium solubility can be above saturation concentrations, leading to the
precipitation and growth of larger particles (i.e., Ostwald
ripening). Following the same principles, it can be concluded
that surface features with a smaller radius of negative curvature (concave) are more favorable and have lower equilibrium
ROLE OF DISSOLUTION IN BIOLOGICAL FATE: NANOTECHNOLOGY
27
gists should be aware that particles of different size and/or
shape are often manufactured/generated through different
routes. The route of manufacture/generation often leaves
fingerprints of imperfections (e.g., localized stress, structural
defects, and dislocations), contaminants (e.g., dispersant
molecules and residual molecules from the synthesis process),
and surface features (e.g., dominant crystal orientation, roughness, passivation layers, and morphology) that can further
modify dissolution behavior (Adamson et al., 1997). Actual
particle surfaces tend to be complex and imperfect in many
ways and are often poorly characterized. To further complicate
the matter, for small NSPs (1–10 nm) quantum mechanical
effects and cooperative events from a limited number of atoms
may alter various physical properties (Adamson et al., 1997).
Our current understanding of nanoscale systems is inadequate
to predict the complex behavior of these materials. However, in
the literature there are reports of phenomena unique to small
NSPs. The size-dependant optical properties have been well
established for a variety of semiconductor materials, e.g.,
quantum dots (Alivisatos, 1996a, b). More recently, small NSP
crystallites have been shown to change crystalline structure in
different solvent environments, which may have a impact on
dissolution phenomena (Zhang et al., 2003).
Impact of Agglomeration
FIG. 3. Top. Pictorial representation of positive and negative curvature.
Bottom. Illustrative example of the solubility of amorphous silica, which has
saturation concentration of 77 ppm. Adapted from Iler (1979).
solubility (Fig. 3). The Gibbs-Thompson equation for the relative solubility of spherical particles of identical materials of
varying size is known as the Ostwald-Freundlich equation and
is given below:
RTq S1
1 1
ln ¼ 2r
;
ð3Þ
M S2
r1 r2
where R is the gas constant, T is the absolute temperature, M
the molecular weight of the solid in solution, r is the interfacial
tension between the solid and liquid, q is the density of the
solid, and S1 and S2 are the solubilities of particle of radius r1
and r2, respectively. The Ostwald-Freundlich equation predicts
that the equilibrium solubility of particles increases with
decreasing particle size. Theoretically, according to this
equation, NSP will have very high propensity for dissolution
relative to larger particles and therefore will not be stable in
solution. Experimentally this is often not the case, which has
been ascribed to kinetics surrounding the critical nucleus size
and energy barrier, non-ideal solution behavior, particle shape,
supersaturation, and the Ostwald Ripening phenomenon. For
certain NSP the measured dissolution is reported to be 25% of
Ostwald-Freundlich predictions (Tang et al., 2004).
Though there is some basic understanding of trends in
particle dissolution with size and surface curvature, toxicolo-
In addition to the complications described above, nanoparticles have a tendency to agglomerate, or form multiple
single particle clusters, in biological fluids. The formation of
agglomerates can hinder dissolution (increasing persistence)
by reducing the average equilibrium solubility of the particle
system and by introducing kinetic hindrance to the diffusion
process. Experimental toxicologists should be aware of the
potential role of agglomerates in dissolution, because the state
of agglomeration is affected in large part by the mode of
administration (i.e., internasal/intertracheal inhalation, insufflation, or instillation). The probability for agglomeration
increases with increased localized dose and for doses given
over shorter time frames.
When agglomerates form a neck between two or more
particles, this creates an area of negative surface curvature
(Fig. 3) (Israelachvili, 1992). Areas of negative curvature have
lower theoretical equilibrium solubility, as previously discussed. Under equilibrium conditions nucleation is predicted
to occur at this interface, which can result in fusion of the agglomerate and a reduction in the total particle surface area. This
process is most likely to occur toward the center of rather large
aggregates in vivo because saturated and quasi-equilibrium
solution conditions are more likely to occur in the agglomerate
interior due to restricted diffusion to the bulk solution phase.
Because the hydrodynamic shear plane is defined around the
agglomerate, the formation of a clump or cluster of particles
greatly increases the overall diffusion layer thickness. This significantly hinders the diffusion efficiency of solute molecules
28
BORM ET AL.
from interior particles to the bulk solution. The extent of this
hindrance process will ultimately be related to the aggregate
volume and packing factor, with larger, more densely packed
aggregates exhibiting slower dissolution.
Impact of Adsorbed Species on Dissolution
The adsorption of molecules and ions from solution can also
have a profound effect on dissolution probability and kinetics.
They can serve to catalyze or passivate dissolution in addition
to modifying diffusion layer characteristics (Adamson et al.,
1997; Fukushi and Sato, 2005). The adsorption of molecules at
the particle solid–liquid interface generally results in a lower
interfacial tension and adds an additional molecular diffusion
barrier, resulting in lower and slower solubility. However,
certain classes of molecules may specifically promote or
demote the dissolution process; e.g., chelating agents are
known to modify the solubility of various materials (Fredd
and Fogler, 1998; Gao et al., 2004; Perry et al., 2005).
Solubility and dissolution are enhanced when organic or ionic
molecules are able to form complexes with surface molecules
that collectively are soluble. In contrast, complexes that result
in a less soluble, ‘‘stickier’’ construct will tend to hinder
dissolution. The adsorption of molecules from solution is
a dynamic process, even at ‘‘equilibrium’’ conditions. Adsorbed ions and molecules are constantly exchanged at the
interface driven by thermal motion and their affinity to the
interface. More solvated molecules undergo faster and more
energetically favorable exchange processes and therefore are
able to enhance dissolution by essentially sequestering and
directly removing surface ions (Iler, 1979).
In biological systems, a vast variety of proteins, enzymes,
polysaccarides, and other molecules are present that are known
to adsorb to interfaces (e.g., opsonization) in a matter of seconds
(Ratner, 1996). Difficulties, however, are encountered when one
tries to rationalize what particular molecules are interacting
with the surface at any given time. In addition, components
from the biological system may react with surface components
on the particle, such as described for ascorbate and iron on the
silica surface (Fenoglio et al., 2000). Exposures to particles by
inhalation typically result in the triggering of a number biochemical pathways that ultimately result in a series of modified
solution conditions. For instance, particle exposures have been
shown to lead to the upregulation of genes that promote
fibronectin and collagen (potential ‘‘sticky’’ particle-coating
molecules) expression in the lung epithelium (Kuwahara et al.,
1994). At the same time the infiltration of neutrophils and
macrophages through chemotaxis may result in exposure to
various enzymes and acidic molecules or, perhaps, particle
uptake, but overall an interaction with a completely different
environment (Renwick et al., 2004). In addition to these considerations, the adsorption and denaturing of biomolecules to
interfaces is apparently also a size-dependent phenomenon—
adding further complexity to the nanoparticle dissolution pro-
cess (Borm and Kreyling, 2004; Vertegel et al., 2004). Tighter,
more packed biomolecule layers are typically found on larger
particles. Moreover, the formation of passivation layers (e.g.,
oxide films), and the presence of impurities, stress loci, and
dislocations can also modify the adsorption process, as well as
the dissolution phenomena in vivo.
Analytical Aspects
Particle dissolution studies can be performed through the
evaluation of particulate properties, such as particle size or
number, concentration, or weight, by assay of the concentration
of dissolved particulate (solvated molecular species) generated,
or through a combination of both. For toxicological interpretation, it may be relevant to try to simulate more than one
compartment within the body or in the environment. In some
cases, sinks and issues of non-steady-state thermodynamics
may also influence the fate of the particle. Monitoring of both
particulate and molecular parameters ultimately enables the
completion of a mass balance. Choice of technique to monitor
particle dissolution will be dependent on factors such as the
desired precision and/or accuracy, the particle properties
(composition, morphology, phase, size, and shape), the analytes, the chemical make-up of the dissolution fluid when
attempting to simulate biological systems, and the desired
cycle time of an analysis. As a result, each measurement
technique will have a discrete range of applicability. The
discussion of dissolution technologies will be limited to the
study of particles that have been dispersed in a fluid that may or
may not contain additives intended to simulate a biological
fluid, thus enabling measurements under well-controlled or
ideal conditions. When analyzing particle size distribution of
liquid-borne particulates or the concentration of dissolved
species, the response of the analysis system to either form or
to separate solid-phase or solubilized molecular species, must
be clearly understood. For instance, ultraviolet-visible (UVVIS) spectroscopy has been used extensively to determine the
concentration of molecular species in solution during drug
dissolution studies. In the UV-VIS spectrophotometric analysis
of submicron particle dispersions, modeling has been developed and implemented as a software package that enables
the output of a spectrophotometer to be converted into particle
size distribution information (Alba, 2004). The results of such
an analysis are dependent on the scattering from the particulate
present and will be greatly influenced by the presence of
absorbing molecular species as well. Whether determining
solubilized molecular species concentration or dispersed particle size with UV-VIS spectrophotometry and possibly other
chemical assay techniques, one must fully understand how the
simultaneous presence of undissolved particles and dissolved
molecular species affect the measurement. Sampling the
particle or solvent may affect dissolution kinetics. If sampling
volumes are not relatively small, and if dissolution kinetics is
not relatively slow, effects of sampling should be considered.
ROLE OF DISSOLUTION IN BIOLOGICAL FATE: NANOTECHNOLOGY
Under well-controlled conditions, filters may be used to
remove particulates that have been found to interfere with the
measurement of solubilized molecular species during particle
dissolution. Filters with high efficiency ratings at nanoparticle
sizes are available from several filter manufacturers.
Monitoring Dissolution via Chemical Assay
Chemical assays of molecular species in solution have been
performed using a wide variety of analytical techniques. The
applicability of any given technique will be dependent on the
particle’s chemical composition and/or the concentration of
soluble species that one is trying to detect. The concentration of
molecular species from organic particle dissolution can be
measured using instrumental techniques (Settle, 1997) such as
fluorescence spectroscopy, UV-VIS spectroscopy, liquid chromatography, and electrochemical techniques, to name a few.
The limit of detection and quantitation for each technique will
be dependent on the detection scheme used in the chromatographic techniques and on the chromophore or electroactive
species being monitored.
Techniques for monitoring inorganic molecular species
include x-ray fluorescence spectroscopy, inductively coupled
plasma spectroscopy, neutron activation analysis, and ion
chromatography, among others to name a few. Once again,
the technique to employ and the ultimate detection limit will be
dependent on the analyte of interest.
A sample preparation besides filtration, i.e., extraction, concentration, dilution, may be required in order to complete the assay.
Monitoring Dissolution via Particle Characterization
When it comes to the particle size analysis of nanoparticles
suspended in an aqueous fluid, there are numerous techniques
available. The goal of performing a particle size analysis would
be to monitor the particle size distribution as a function of time
to obtain a particle dissolution profile. Chromatographic
techniques such as field flow fractionation (Giddings, 1993;
Chen et al., 2005) and hydrodynamic chromatography (Small,
1974; DosRamos, 1991; Tribe et al., 2003) enable the
measurement of complex size distributions with relatively high
resolution in the <10 to >100 nanometer size range. Moreover,
during a field flow fractionation and hydrodynamic chromatography analysis, particulate and solubilized molecular species are separated, enabling one to determine the concentration
of both. Another particle size technique that is fractionation
based is photosedimentometry (Weiner, 1991, Fitzpatrick,
1998). The lower size limit of the photosedimentometer can
be limited by the density difference between the particulate of
interest and the analysis fluid. At low density differences, the
sedimentation of <100-nm-sized particles requires long analysis times, and when this occurs Brownian motion can
detrimentally affect the fractionation and results.
29
Light-scattering techniques have also been used to monitor
size and number concentration of particles present in a fluid.
Static scattering techniques have been applied to particle
characterization in a wide variety of forms including UV-VIS
spectroscopy (Alba, 2004), turbidimetric (Tucker, 2004), and
photon migration (Dali et al., 2005). The lower size and
concentration limits of detection for each of these techniques
should be evaluated for each particle system of interest. The
ability to monitor changes in size and concentration at 10 nm
may be limited for most of these techniques. Dynamic light
scattering (Allen, 1992) has been applied extensively in the
nanometer size range. However, its ability to accurately
characterize complex size distributions is somewhat limited.
Finally, microscopy techniques can be used to monitor
changes in particle size and particle concentration. Recently,
advances in visible-light-optical microscopy have enabled
visualization of particles and/or the light scattered by particles
with diameters less than 100 nm using a visible light microscope. Darkfield illumination techniques now commercially
available may be capable of enabling visualization of the
particle dissolution process using a visible light microscope
(Aetos Technologies, Inc., Auburn, AL, USA; Nanosight Ltd.,
Wiltshire, UK). Plasmon resonant particles (Schultz, 2003)
also have the unique characteristic of being ‘‘ultra-bright’’
when evaluated in light-scattering experiments thus enabling
viewing with a light microscope.
State of Aggregation and Agglomeration
Aggregates are defined as a secondary particle composed of
primary particles that are strongly bonded together (fused,
sintered) and acting as a unit. An agglomerate is defined as
a secondary particle which, upon handling, breaks into
aggregates. The degree of aggregation and agglomeration
may affect movement of the particle in external media (for
example, aggregation would influence aerodynamic diameter)
as well as internally. Agglomerates and aggregates act as large
particles with internal porosity in their aerodynamic properties.
It is difficult to determine the degree to which an agglomerate
will break apart and to calculate an aggregate size, as this
depends on the mechanical stresses to which the agglomerate is
subjected. It is unlikely that an aggregated secondary particle
will itself de-aggregate to become a collection of separate
primary particles (Rödelsperger et al., 2003), but there are no
recognized test standards to follow on this matter. A recent
study by Cherukuri et al. (2004) combined the use of fluorescence microscopy and near-infrared excitation to show that
morphological aggregates of carbon nanotubes in macrophages
were behaving chemically as simple primary carbon nanotubes. This suggests that physical properties of NSP may help
to characterize the fate of NSP in the body.
The parameters that change as primary particles aggregate
and agglomerate are the hydrodynamic size (becomes larger),
the number concentration (becomes smaller), and possibly the
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BORM ET AL.
specific surface area. The agglomerated particles will behave
as a population of larger particles, and the size distribution,
specific surface area, dissolution rate, or any other parameters
that can be used in toxicological assessment can be measured
on the agglomerated system, but it must be understood that the
results cannot be fully predictive of mobile, discrete NSP.
It may be possible to assess the state of agglomeration by
analyzing electron micrographs of the particles; however,
sample preparation often involves a dispersion step followed
by evaporation, and the mechanical stresses may break up
agglomerates. Any non-imaging techniques will provide a size
distribution but no direct measurement of the state of agglomeration. The state of agglomeration can be assessed with prior
knowledge of the size distribution of the primary particles.
All of the size characterization techniques outlined earlier
can be used to measure the size distribution of agglomerated
and non-agglomerated particles. Because the assumption of
sphericity is used in all particle-sizing technologies, with the
exception of imaging techniques, it is expected that the data
generated for non-spherical particles may be somewhat dependent on the particular technique used. In addition, when
sedimentation techniques are used, the density of the particles
needs to be known for accurate measurement, as Brownian
motion can affect hydrodynamic flow at the smallest measurable size. Furthermore, if the particles under measurement
consist of distinct populations having different densities, the
size distributions generated may lack accuracy.
DISCUSSION AND FUTURE DIRECTIONS
Dissolution assessment has been successfully applied to
macro-sized materials such as man-made mineral fibers that are
both slightly soluble and insoluble (Fubini et al., 1998). In a
very strict sense, the final form of any soluble solid material is
a nanoscale particle that becomes smaller than the classical
critical nucleus size. It is also a formalism that all materials that
precipitate from a fluid pass from the original soluble species
through the critical nucleus size up and through the nanosize,
until they attain the macro-sizes that allow sedimentation,
filtration, or centrifugation. However, the current interest in
nanoscale materials is for what are traditionally considered
insoluble materials. Furthermore, the use of dissolution data
when applied to toxicological investigations will focus on
degrees of insolubility. The difference between a persistent,
durable, solid nanoscale substance and a less durable, ‘‘biosoluble’’ nanoscale substance can be quite small and will
require discerning analysis.
Amorphous silica was used earlier in this article as an
example of a soluble nanoscale material that caused few
neoplasms following intratracheal instillation, relative to more
durable NSP (Borm et al., 2004). Amorphous silica has a solubility ranging from 2.00 mmol l1 at around pH 7 and 25°C to
4.5 mmol ll at the around pH 7 and 45°C (Vogelsberger et al.,
1995) which are discretely different than cristobalite at 0.45
mmol ll and quartz at 0.09 mmol ll (Iler, 1979). With silicas, in
addition to dissolution, surface reactivity has also been important in determining biological reactivity (Donaldson and Borm,
1998; Fubini et al., 2004; Schins et al., 2002). In Warheit’s
review of pulmonary responses to different forms of silica
(2001), cristobalite was indicated to produce the greatest
measure of lung injury; quartz produced intermediate effects,
and amorphous silica produced minimal pulmonary effects. In
terms of analytical technique, small differences in dissolution
exist among these polymorphs of silica, and dissolution, in turn,
influences pulmonary effects through the concept of persistence.
Synthetic amorphous silica has also been the subject of dissolution testing using a simulated biological medium (Roelofs
and Vogelsberger, 2004) at 37°C and pH near 7. The authors
report a maximum solubility of 2.3–2.7 mmol ll, depending on
the source of the commercial silica samples and on primary
particle size. These values are consistent with the same authors’
earlier study in water. Roelof and Vogelsberger also confirmed
the fact that silica dissolution rate is more rapid than the reverse
precipitation rate. In dissolution studies, this means that the solution becomes supersaturated, and the experimentalist would
need to wait a considerable time to reach equilibrium. Hence,
silica dissolves both in water and in simulated biological systems beyond the equilibrium concentration value.
Isolated NSP or agglomerates located in the alveoli will not
necessarily attain an equilibrium state in terms of dissolution.
Thermodynamically, the alveoli represent an open system
where pulmonary fluids circulate and can be replenished.
Some constituents of these fluids have ion-complexation/sequestration properties that enhance dissolution even further.
The subcompartmental kinetic rates of dissolution can therefore dominate over equilibrium values for solubility. This is
likely the situation observed with synthetic amorphous silica
particles, where the tendency to supersaturate aids in rapidly
dissolving and being eliminated from the lung. Other substances may have different properties. Their dissolution may be
inhibited by adsorbed biological and tramp species, or hydrous
surface layers may act to limit dissolution. All of these factors,
the equilibrium solubility value, the kinetic rates of dissolution
and precipitation, and the possibility of enhancement or
inhibition due to hydrated surface layers and adsorbed species
may be relevant when evaluating a particle’s dissolution in the
context of pulmonary persistence. Nevertheless, for man-made
vitreous fibers, mathematical modeling of dissolution and
subsequent biological responses in the rat lung has successfully
been done (Tran et al., 2003). Nanoscale particles may be
subject to endogenous breakdown mechanisms that are available for proteins, nucleic acids, or general xenobiotics.
However, few studies are available on the biotransformation
of NSP. Studies with different water-soluble fullerenes have
shown that minor alterations in the fullerene structure have
a large effect on the cytotoxicity in two different human cell
lines (Sayes et al., 2004). This suggests that biotransformation
ROLE OF DISSOLUTION IN BIOLOGICAL FATE: NANOTECHNOLOGY
of molecular components or NSP itself maybe a relevant
mechanism for the prevention of cytotoxicity.
In conclusion, dissolution may be among the critical steps
for some nanoscale materials in determining fate in the
environment and within the body. Translocation of insoluble
particles into cells is likely to be dependent on processes of
endocytosis (mainly pinocytosis and phagocytosis or receptormediated endocytosis). Similarly, movement of particles from
live cells may occur largely by exocytosis. Diffusional
movement of nanoparticles through cell membranes is likely
to be limited under normal conditions (Heckel et al., 2004;
Meiring et al., 2005), although more research in this area may
provide a better understanding of nanoscale particle translocation processes. There is potential for accumulation of
(insoluble) NSP in the body. Effects would be dependent on the
dose, the properties of the nanoscale material, and the location
of accumulation. Dissolution, even over the time frame of
weeks or months, may significantly enhance clearance of
nanoscale materials. Characterizing the state of the component
molecule or particle, and understanding the processes of
translocation, dissolution, and particle receptor interactions
are key challenges to defining the toxicology of NSPs.
Measuring fiber dissolution in vivo (Bellmann et al., 1986;
Warheit et al., 1994) and in vitro (Jaurand, 1994; Zoitos et al.,
1997) has been done to assess their biopersistence and predict
potential chronic effects. Both morphological biopersistence
(measuring fibers with microscopic techniques) and dissolution
rate (kdis, in units of mass per surface area of particle) have
been used. Biological fluid simulants at defined pH are used in
the in vitro assessment. These principles may be usefully
applied to nanoscale materials, although substantial technical
challenges exist. Following the morphological transformation
of a particle into single molecules will require resolution at the
nanometer scale. Measuring the appearance of the chemical in
the surrounding fluid will require analytical sensitivity and
resolution at extraordinarily low concentrations to evaluate
nanoparticles at low dissolution rates.
The description of material science concepts and analytical
approaches to dissolution at the nano scale is intended to
provide a useful basis for developing relevant dissolution
assay(s) for nanoscale particles and addressing the challenges
of how this may relate to the in vivo situation.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the ILSI-HESI organization, especially
M. P. Holsapple, for support of the ILSI-HESI Nanomaterials EHS Committee
and Dissolution group. We thank James E. Gibson for review of the manuscript
as part of the ILSI-HESI review process.
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