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Nanotoxicology, December 2009; 3(4): 276–283
The relationship between pH and zeta potential of ~30 nm metal oxide
nanoparticle suspensions relevant to in vitro toxicological evaluations
J. MICHAEL BERG, AMELIA ROMOSER, NIVEDITA BANERJEE, REMA ZEBDA &
CHRISTIE M. SAYES
Department of Veterinary Physiology & Pharmacology, Texas A&M University, College Station, Texas, USA
(Received 30 April 2009; accepted 19 August 2009)
Abstract
Zeta potential measurements are common in nanotoxicology. This research probes the effects of pH and time on nanoparticle
zeta potential, agglomerate size, and cellular viability. The nanoparticles TiO
2
,Fe
2
O
3
,Al
2
O
3
, ZnO, and CeO
2
, were titrated
from pH 12.0–2.0. The isoelectric points (IEP) of the nanoparticles were near neutral with the exception of TiO
2
(IEP =5.19)
and Fe
2
O
3
(IEP =4.24). Nanoparticle agglomerates were largest at the IEP. TiO
2
and Fe
2
O
3
increased in zeta potential and
agglomerate size over time; while Al
2
O
3
and ZnO displayed little change. CeO
2
increased in zeta potential; however, the net
charge remained negative. Cytotoxicity studies revealed that TiO
2
and Fe
2
O
3
caused decreasing cellular viability over 48 h.
Al
2
O
3
, ZnO, and CeO
2
cellular viability remained similar to control. Results indicate that alterations in the pH have a large
effect on zeta potential and agglomerate size which may be used as a predictive measure of nanotoxicity.
Keywords: Metal oxide nanoparticles,zeta potential,agglomeration,pH,cellular viability
Introduction
Within the nanotoxicology field, increased findings
demonstrate the ability of nanoparticle charge (zeta
potential) to influence corresponding cellular
responses ranging from particulate endocytosis to
cytotoxicity (Xia et al. 2006; Nan et al. 2008; Lundq-
vist et al. 2009). Reviews of current literature suggest
a need for careful characterization of all classes of
nanomaterials including metal colloids, metal oxides,
and carbonaceous particles, prior to their use in
toxicity testing (Karakoti et al. 2006; Handy
et al. 2008). The relationship between zeta potential
and ambient conditions surrounding the nanoparti-
cles remains a largely unexplored area which will
require researchers from many scientific disciplines
to answer. This research probes the effects of pH on
nanoparticle zeta potential and size using ~30 nm
particles in aqueous suspension. The nanoparticles
include titanium dioxide (TiO
2
), iron oxide (Fe
2
O
3
),
aluminum oxide (Al
2
O
3
), zinc oxide (ZnO), and
cerium dioxide (CeO
2
). In these nanoparticle
systems, which include both the nanoparticle and
the suspension medium, features such agglomeration,
dispersion, and suspension stability may be
influenced by external factors such as the flux of
hydrogen ions (H+) in solution.
The zeta potential represents the charge of a nano-
particle in relation to the surrounding conditions.
Nevertheless, the zeta potential is not an actual mea-
surement of the individual molecular surface charge;
rather, it is a measurement of the electric double layer
produced by the surrounding ions in solution (i.e.,
counter ions) (Malvern Instruments 2008). These
counter ions play a role in the calculation of zeta
potential measurement (Figure 1). All particle systems
in an aqueous media carry an electric charge which
may be positive, negative, or neutral. For surface-
derived nanoparticles, dissociation of an acidic group,
such as a carboxylic acid moiety on a nanoparticle
surface will yield a negatively charged surface; while
dissociation of a basic group on a nanoparticle surface
will yield a positively charged surface. For unmodified
nanoparticles, the individual atoms that comprise the
surface of the particle dictate its charge.
Relevance to toxicology
Agglomeration of nanoparticles occurs when individ-
ual particles are held together by weak inter-particle
Correspondence: Christie M. Sayes, Department of Veterinary Physiology & Pharmacology, TAMU 4466, College Station, Texas 77843, USA. Tel: +1 979 862
2682. Fax: +1 979 862 4929. E-mail: csayes@cvm.tamu.edu
ISSN 1743-5390 print/ISSN 1743-5404 online 2009 Informa UK Ltd.
DOI: 10.3109/17435390903276941
interactions including solvation forces, van der Waals
forces, electrostatic attractions, and hydrophobic
interactions (Hakim et al. 2007; Fichthorn and
Qin 2008; Min et al. 2008). In most instances,
agglomeration state is reversible, but only if additional
entropy (e.g., sonication or homogenization) or ions
(changing H+) are added to the system. It is suspected
that this methodology could be used to size select for
certain nano-populations within a particle suspension.
Limitations of utilizing changes in pH for biological
applications are that the resultant suspension could
have a pH that is outside of the physiological range
(7.35–7.45), may alter chemistry of drug delivery
vehicles, or change conditions in cell culture media
for in vitro use (Klaassen and Watkins 2003). Further,
this methodology could yield insights into the fate and
transport of nanoparticle aggregates after an exposure
has occurred.
A likely exposure to nanoparticles may occur orally
(Gatti et al. 2009). This route of exposure would
ensure that nanoparticles enter an environment yield-
ing a wide range of pH values. Initially, the nanopar-
ticle would be exposed to a low pH in the stomach
(pH =~2) (Rodriguez et al. 1999; Beak et al. 2006).
Following acidic conditions in the stomach, the envi-
ronment is once again altered in the intestinal tract,
yielding a more basic pH combined with peristaltic
movement. Under these physiological situations,
physical properties of the agglomerates such as size,
surface area, and zeta potential will change. This
alteration in physical properties will yield nanoagglo-
merates of differing size and may increase likelihood
that these nanoparticles could translocate through the
endothelial lining and enter the circulatory system.
This in turn may induce a systemic and/or chronic
effect (Lockman et al. 2004; Oberdörster 2007;
Semmler-Behnke et al. 2007; Panessa-Warren
et al. 2008; Santhanam et al. 2008).
Altering the pH of the medium in which the nano-
particle is suspended will also yield differences in
nanoparticle dissolution into ions or alter the surface
chemistry of a nanoparticle (Guo et al. 2009). Altering
the surface chemistry of nanoparticles may in turn
effect how a nanoparticle binds cellular substituents
such as proteins, and may affect the mechanisms by
which these nanoparticles enter cells (Al-Jamal
et al. 2008; Lundqvist et al. 2009).
Discovering the fundamental relationships between
the properties of nanomaterials and certain toxicolog-
ical responses will require the separation of the com-
plex kinetics of nanoparticle delivery in vitro from the
dynamics of response. This could be made possible by
integrated computational and experimental dose-
response analyses.
Experimental procedures
Particle preparation
Titanium dioxide (TiO
2
,~27 nm, 99.8%) particles
were acquired from Evonik, Hanau-Wolfgang, Ger-
many. Aluminum oxide, zinc oxide, and cerium diox-
ide (Al
2
O
3
,<50 nm, 99.5%; ZnO, <50 nm, 99.5%;
CeO
2
,<25 nm, 99.5%) nanoparticles were purchased
from Sigma Aldrich, St Louis, Missouri, USA. Iron
oxide (Fe
2
O
3
,23–35 nm, 98%) nanoparticles were
synthesized using a flame synthesis method that has
been described in detail elsewhere (Yang et al. 2001;
Guo and Kennedy 2007). The stock solutions of each
of the five ~30 nm particles were prepared in Milli-Q
ultrapure water (18.2 mW). The resultant suspensions
Figure 1. Schematic diagram of the effects of pH on a metal oxide nanoparticle. The zeta potential plane is measured as the primary indicator
of surface charge. Surface charge is altered when the pH is increased or decreased. The downstream effect of altered zeta potential is a change in
agglomeration state, which influences the cytotoxicity.
Nanoparticle pH, charge, toxicity 277
were bath sonicated for 30 min prior to material
characterization and in vitro exposures. Nanoparticle
size was visualized using dynamic light scattering
(DLS). Zeta potential was measured via electropho-
retic light scattering (ELS) combined with phase
analysis light scattering (PALS) (Malvern Zetasizer
Nano ZS). Size and zeta potential parameters were
measured over various time points ranging from
t=0 h to t =1 wk.
Titration
Nanoparticle titration was performed using the Mal-
vern MPT-2 Autotitrator in parallel with the Malvern
Zetasizer Nano-ZS. This combination allowed auto-
mated titration over a wide pH range and thus made it
possible to determine the isoelectric point (IEP).
Nanoparticles were titrated from basic pH
(pH =12) to an acidic pH (pH =2) utilizing HCl
and NaOH. At every pH unit (±0.2 U) the zeta
potential and size were determined. The IEP was
determined using the Malvern Software Version 5.03.
The concentration of the nanoparticles used was
dependent upon the turbidity of the sample. The zeta
potential and agglomerate size of ZnO, Al
2
O
3
and
CeO
2
were measured at a concentration of 200 ppm
(mg/l); TiO
2
was measured at a concentration of 50
ppm (mg/l); and Fe
2
O
3
was measured at a concentra-
tion of 25 ppm (mg/l). The differences in nanoparticle
concentration for IEP measurements were necessary
due to the refractive indices of the nanoparticles
probed; however, as a note, a higher concentration
of nanoparticles would facilitate increased incidence of
collision, thus leading to agglomeration when com-
pared to a lower concentration of nanoparticles.
Cytotoxicity
Cytotoxicity studies were performed by dosing cul-
tured mouse hepatocytes (AML12, American Type
Tissue Collection, Manassas, VA, USA) with 10 mg/l
nanoparticles in 24-well plates. Dosed wells differed
only by the type of nanoparticles used and not the
concentration of nanoparticles. A control was added
for comparison. DMEM/F-12 media was used for all
cells. The dosed-cell plates were incubated at 37C.
Cell viability was determined using a Countess Auto-
mated Cell Counter (Invitrogen Corp., Carlsbad, CA,
USA) in combination with trypan blue dye at 1 h, 24
h, and 48-h post-exposure time points. Dead cells
exhibit a compromised membrane, which allows the
dye to penetrate, providing for differentiation from
live cells.
Results
Results indicate that the pH has a pronounced effect on
the zeta potential of each nanoparticle tested in this
study. The change in zeta potential was found to alter
the stability of the nanoparticle suspension. Figure 2B–
E illustrates the titration curves of TiO
2
,ZnO,Al
2
O
3
,
CeO
2
,andFe
2
O
3
. A hypothetical model nanoparticle
exhibits the largest agglomerate size at the point where
its zeta potential is 0 mV (Figure 2A), as was deter-
mined empirically for the remainder of the tested nano-
particles (Figure 2B–E).Thepointatwhichthe
nanoparticle exhibits no net charge is termed the iso-
electric point (IEP). ZnO, Al
2
O
3
,andCeO
2
each
display an IEP (IEP =7.13, 7.06, and 6.71, respectively)
at a pH relevant to interstitial fluid, broncheoalveloar
lavage fluid, lymph, and blood (Klaassen and Wat-
kins 2003). However, this trend does not continue
for TiO
2
and Fe
2
O
3
nanoparticles. The TiO
2
and
Fe
2
O
3
nanoparticles exhibited an IEP at pH 5.19
and 4.24, respectively. The largest nanoparticle agglom-
erate size was dependent upon chemical composition
and ranged from 1,772 ±47.56 nm in the Fe
2
O
3
sample
to 3,185 ±541.0 nm in the Al
2
O
3
suspension. Further-
more, the smallest agglomerate size throughout each
nanoparticle suspension existed at the pH where the
nanoparticle displayed a strongly charged surface. The
intensechargeonthesurfaceincreasesthecharge-
charge repulsions between the particles, thus maintain-
ing a more stable and monodisperse suspension. The
smallest nanoparticle size was found in the ZnO sample
(203.8 nm). This size is indicative of the hydrodynamic
radius of the nanoparticle.
In addition to changes in pH, the zeta potential of a
nanoparticle changes over time when held in aqueous
suspension. Figure 3 demonstrates that the nanopar-
ticles tested demonstrate a wide range of agglomera-
tion states and zeta potentials over a time period t =1h
to t =1 wk. Over the course of this experiment, TiO
2
and Fe
2
O
3
nanoparticles displayed a large zeta poten-
tial increase, which consequently affected their agglom-
eration state. TiO
2
and Fe
2
O
3
nanoparticles originally
exhibited zeta potentials of -29 mV and -32 mV
respectively; however, after the one-week time point,
the zeta potential rose to -15 mV. As witnessed in both
of these nanoparticles, a more neutral surface charge
led to larger agglomeration state. On the contrary, ZnO
and Al
2
O
3
nanoparticles retained their negative surface
charge over time. In these samples, stability was due to
the strongly negative-charged zeta potential and the
lack of agglomeration. CeO
2
nanoparticles demon-
strated a slight increase in zeta potential over the
one-week time course. Interestingly, this slight increase
still led to a very negative zeta potential of -27.8 mV.
Because the zeta potential remained close to -30 mV, a
278 J.M. Berg et al.
number commonly used to indicate solution stability,
the agglomeration state of the nanoparticles decreased
slightly.
Cellular viability in the AML12 mouse liver hepa-
tocyte was examined at 24 and 48-h time points. It was
noted that while the TiO
2
- and Fe
2
O
3
-dosed samples
exhibited heightened cellular viability at the 1 h time
point, they showed a decrease in viability each time
thereafter. The ZnO and Al
2
O
3
samples displayed
increasing cell viability at 48 h, with viability of 91.5%
and 91.75%, respectively. This percentage is com-
pared to the control cell viability of 96.0% after 48 h.
Figure 2. Titration of nanoparticles in ultrapure water (18.2 mW). (A) In a model nanoparticle system, the largest aggregate size would be
observed at its isoelectric point (zeta potential =0 mV). The farther the zeta potential deviates from 0 mV, the smaller the particle agglomerate
due to increasing repulsive forces. Titrations of (B) TiO
2
, (C) ZnO, (D) Al
2
O
3
, (E) CeO
2
, and (F) Fe
2
O
3
from basic (pH >10) to acidic
(pH <3) conditions. All nanoparticles exhibit an isoelectric point (IEP). Results indiciate that zeta potential and size are dependent upon pH.
Dashed vertical line represents isoelectric point.
Nanoparticle pH, charge, toxicity 279
CeO
2
nanoparticles, which exhibited only a slight
change in cell viability, initially yielded 85.25% with a
final 48 h post-exposure time point viability of 90%.
Discussion
Results indicate that alterations in the pH have a large
effect on zeta potential and agglomerate size which
may be used a predictive measure of nanoparticle
toxicity. The suspension stability is dependent upon
physical characteristics of both the suspended nano-
particles and their suspension medium. One of the
major factors involved in the agglomeration process is
electrostatic stabilization. Altering the zeta potential
to the point at which it exhibits zero net charge, or the
IEP of the nanoparticle, decreases stabilization forces
and promotes agglomeration.
Figure 3. Alterations in zeta potential change over time. The zeta potential of various metal oxide nanoparticles were measured over a time
period of t =0tot=1 week. Here, the TiO
2
nanoparticles (A) and the Fe
2
O
3
nanoparticles (B) had an increasing zeta potential value (smaller
absolute value). This increasing zeta potential corresponds with an increase in aggregate size. Both the Al
2
O
3
nanoparticles (C) and the ZnO
nanoparticles (D) remained highly negative, thus their aggregate size remained the same or decreased. CeO
2
nanoparticles (E) tend to decrease
in size slowly over time despite the apparent small increase in zeta potential (smaller absolute value).
280 J.M. Berg et al.
In accordance with Figure 2, all of the nanoparticles
tested displayed unique isoelectric points. While the
ZnO, CeO
2
,andAl
2
O
3
nanoparticles possessed IEP at
a physiologically-relevant pH, such as blood or inter-
stitial fluid (pH =7.4), Fe
2
O
3
and TiO
2
nanoparticles
possessed IEP at acidic conditions. Conversely, the
Fe
2
O
3
and TiO
2
nanoparticles exhibited a charged
surface at pH =7.4. This data, when compared with
the difference in cellular viability seen in Figure 4,
suggests that the presence of a charged surface on the
nanoparticle agglomerate at physiological pH of 7.4
may correlate with a decrease in cellular viability in
hepatocytes.
The study is pertinent due the possibility that many
environments present in the body such as gastric
secretions, urine, and lysosomal fluid are known to
have a varying degree of pH which correlate with our
findings regarding nanoparticle surface charge. Table I
shows the representative pH of various bodily com-
partments which the particle would potentially be
exposed to over the course of the pharmacodynamic
process. Specifically, a pH of 2 present in the acidic
Figure 4. Cellular viability after nanoparticle exposure (10 mg/l). Cellular viability correlates with the nanoparticle aggregation trend. AML12
cells were analyzed for cellular viabiliy at times t =0tot=48 hours post exposure. Both the TiO
2
nanoparticles (A) and the Fe
2
O
3
nanoparticles
(B) displayed a decrease in cell viability until the 48 hr timepoint. This contrasts the Al
2
O
3
(C) and the ZnO (D) nanoparticle data, which
implies that cell viability increases over time. CsO nanoparticles (E) displayed no significant change in viability. Control cells increased in
cellular viability. *p<0.05 vs. 1 h post-exposure time point within the same graph.
Nanoparticle pH, charge, toxicity 281
gastric secretions from parietal cells, would likely alter
the agglomeration state of the nanoparticles which have
been examined in this study. Physiological conditions
such as these would yield the smallest nanoparticle
agglomerates seen by the body. In addition, for many
of the nanoparticles tested, a strongly positive zeta
potential may further yield electrostatic or covalent
interactions with cellular components such as DNA
or proteins as well as determine the specificrouteof
uptake into the cell (Dausend et al. 2008).
Table I indicates that agglomerate size varies sig-
nificantly, depending upon a variety of factors, includ-
ing both pH and nanoparticle chemical composition.
One possible condition not explored in this paper is
the influence of ionic strength due to the addition
of Na
+
and Cl
-
ions introduced during the titration.
Increasing the ionic strength of the suspension
medium may lead to an altered agglomeration state
through possible charge shielding and condensation of
the charge at the electric double layer (Tiyaboonchai
and Limpeanchob 2007; Handy et al. 2008). This
often poses a challenge when working with a biocom-
patible suspension medium. In addition, a buffered
suspension medium (such as phosphate buffered
saline) influences the particle agglomeration state
(Sager et al. 2007). While these agglomerate sizes
seen in the nanoparticle titration are representative
of what would be seen in an aqueous suspension, it is
important to note that both in vitro and in vivo con-
ditions contain biomaterial, such as proteins and/or
lipids that may coat the nanoparticle surface, altering
both the chemistry and agglomeration state. This
emerging hypothesis has proven valuable as shifts in
the zeta potential and the isoelectric point have been
reported after proteins adsorb onto the particle sur-
face (Cael et al. 2003; Xia et al. 2006). It has been
suggested that this protein adsorption is just as sig-
nificant to toxicology as the inherit physico-chemical
characteristics of the nanoparticles themselves
(Lundqvist et al. 2009). Even in an in vivo model,
specific nanoparticle-protein interactions will not only
influence the agglomeration state of nanoparticles,
butalsoprovideapossiblerouteofentryinto
cells. Although many reports have cited interactions
at the nanomaterial-biological interface, these dyn-
amic properties are influenced by the zeta potential
and agglomeration state. This research strives to
make a connection between these physico-chemical
characteristics as a predictive measure of nanoma-
terial toxicity.
Cellular viability was influenced by a variety of
factors including zeta potential and agglomeration
state. Results indicate that, of the nanoparticles
tested, cellular exposure to Fe
2
O
3
and TiO
2
yielded
a decrease in viability over time. This alteration in
cellular viability correlates with the charged surface
and altered agglomeration state demonstrated by only
these nanoparticles. It is hypothesized that the poten-
tial toxicity of nanoparticles is due to not just one, but
many characteristics of the nanoparticle system. Our
data suggests that both pH and agglomeration state
show an association with cytotoxicity. Figure 3 shows
that these nanoparticles increase in agglomeration size
over time, coupled with a zeta potential trending
towards neutral. These experiments were carried
out in ultrapure water (pH =5.9). When suspended
in a solution where pH =7.4, as that which is seen in
cell culture media, Fe
2
O
3
and TiO
2
were the only
materials which were found not to be at or near their
IEP. These results indicate that surface charge has the
potential to influence cell viability.
Overall, it is important that nanoparticles be clas-
sified as an entire system that encompasses the nano-
particle, its suspension medium, and the ions in
solution. This careful characterization is necessary
to interpret which components of a nanoparticle
may contribute to alterations in surface charge and
toxicological effects. We have shown that factors such
as pH can influence the zeta potential of different
nanoparticles. Additionally, it was observed that
changes in zeta potential lead to a change in cellular
viability. In the future it will be important for research-
ers to carefully observe the conditions under which the
zeta potential is measured when reporting results.
Table I. Prediction of metal oxide nanomaterial properties in various pharmacodynamic compartments. The nanomaterials exhibit different zeta
potentials and agglomerate sizes at various physiological pH. TiO
2
and Fe
2
O
3
nanoparticles demonstrate strongly charged agglomerates at
pH =7.4. The pH values of 2.00, 4.50, 5.00, and 7.40 were chosen to represent various physiological matrices,such as gastric acid, lysosomal fluid,
intestinal fluid or urine, and blood or interstitial fluid, respectively. Values are reported as zeta potential (mV) / average agglomerate size (nm).
pH
Metal oxide nanomaterials <2.00 4.50 5.00 7.40
ZnO +50.0/360 +44.0/945 +16.0/1200 -3.00/1170
Al
2
O
3
+45.0/561 +38.0/1750 +27.0/2400 -4.00/3050
CeO
2
+32.6/1440 +26.0/2340 +20.0/2590 -6.00/2850
Fe
2
O
3
+25.4/1800 -9.00/1740 -15.0/1700 -47.0/830
282 J.M. Berg et al.
Acknowledgments
The authors thank Dr Bing Guo of the Department of
Mechanical Engineering at Texas A&M University
for supplying the Fe
2
O
3
nanoparticle samples. We
also thank the Department of Veterinary Physiology
and Pharmacology for supporting this work.
Declaration of interest:The authors report no
conflicts of interest. The authors alone are responsible
for the content and writing of the paper.
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