Assessing Methods for Blood Cell Cytotoxic Responses to Inorganic Nanoparticles and Nanoparticle Aggregates

Assessing Methods for Blood Cell Cytotoxic Responses to Inorganic Nanoparticles

DOI: 10.1002/smll.200800199

Assessing Methods for Blood Cell Cytotoxic Responses toInorganic Nanoparticles and Nanoparticle AggregatesBelen Dıaz, Christian Sanchez-Espinel, Manuel Arruebo,* Jose Faro, Encarnacion deMiguel, Susana Magadan, Clara Yague, Rodrigo Fernandez-Pacheco,M. Ricardo Ibarra, Jesus Santamarıa, and Africa Gonzalez-Fernandez

Keywords:� cytotoxicity

� magnetic nanoparticles

� reactive oxygen species

� silica

Inorganic nanoparticles (NPs) show great potential for medicinal therapy.

However, biocompatibility studies are essential to determine if they are safe.

Here, five different NPs are compared for their cytotoxicity, internalization,

aggregation in medium, and reactive oxygen species (ROS) production,

using tumoral and normal human blood cells. Differences depending on the

cell type are analyzed, and no direct correlation between ROS production

and cell toxicity is found. Results are discussed with the aim of standardizing

the procedures for the evaluation of the toxicity.

1. Introduction

The great potential of nanoparticles (NPs) in medical

applications justifies the tremendous growth in the number of

scientific publications in this area. NPs find application in areas

as diverse as diagnosis (e.g., contrast agents for magnetic

resonance imaging and biosensing),[1] prevention of infectious

diseases (e.g., vaccines),[2] and therapy (e.g., targeted drug

delivery, gene therapy, hyperthermia).[3]

[�] Dr. M. Arruebo, C. Yague, R. Fernandez-Pacheco, Prof. M. R. Ibarra,

Prof. J. Santamarıa

Aragon Nanoscience Institute (INA)

University of Zaragoza

Pedro Cerbuna 12, 50009 Zaragoza (Spain)

E-mail: [email protected]

B. Dıaz, C. Sanchez-Espinel, Dr. J. Faro, Prof. A. Gonzalez-Fernandez

Immunology Area, Faculty of Biology

University of Vigo

Campus As Lagoas–Marcosende

36310 Vigo, Pontevedra (Spain)

Dr. J. Faro

Estudos Avancados de Oeiras

Instituto Gulbenkian de Ciencia

Oeiras (Portugal)

Dr. E. de Miguel

Cellular Biology Area, Faculty of Biology

University of Vigo, Campus As Lagoas–Marcosende

36310 Vigo, Pontevedra (Spain)

Dr. S. Magadan

Instituto Superior de Saude do Alto Ave (ISAVE)

Quinta de Matos, Geraz do Minho, 4830-31 PVL (Portugal)

small 2008, 4, No. 11, 2025–2034 � 2008 Wiley-VCH Verlag Gmb

In this last area, NPs can offer clear advantages compared

to traditional therapies in at least three aspects: i) fewer side

effects of delivered drugs; ii) increased versatility in formula-

tion[4] (e.g., conjugation to drugs, antibodies, peptides, DNA,

carbohydrates, etc); and iii) display of optical, electrical, or

magnetic properties that can be tuned to cause damage to

inflammatory or tumoral cells (for instance, by means of

magnetic or optical hyperthermia in the near infrared),[5,6] or

used to activate a beneficial response.

There are, however, at least two important flaws in the

current analyses of potential health risks fromNPs. First, while

many NPs are intended to be used in humans intravenously,

where they will interact with peripheral blood cells before

reaching other cells in different tissues, only limited informa-

tion can be found in the literature regarding the effects of NPs

on normal human cells. Second, there are no standardized

procedures for toxicological studies of NPs. In this latter

aspect it is important to realize that the three key factors that

will determine the toxicological evaluation of the NPs are: i)

their physical/chemical characteristics; ii) the cell type(s)

tested; and iii) the method used to evaluate cytoxicity. Thus,

when some of those parameters differ between research

groups, it becomes difficult, or even impossible, to compare

the data from these groups.

The biological activity of NPs depends on chemical and

physical factors such as size and shape, surface area,

agglomeration state, chemical composition, surface chemistry

(charge and hydrophilicity), surface activity, solubility, dose,

and so on.[7] The most extensively analyzed biological effects

of NPs are those resulting from their interaction with the

innate immune system. Among them, the stimulation of

H & Co. KGaA, Weinheim 2025

full papers M. Arruebo et al.

Table 1. Scanning electron microscopy (SEM) and Transmission elec-tron microscopy (TEM) images of: iron/graphite magnetic particles(Type 1); superparamagnetic magnetite/silica nanoparticles (Type 2);bare (Type 3a) and PEG-ylated (Type 3b) silica particles; magneticcomposites magnetite/FAU zeolite (Type 4).

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phagocytosis and the potential induction of cell stress are

considered highly relevant. A particularly useful method to

assess potential cell stress is to determine changes in the levels

of reactive oxygen species (ROS) in the cell.[8] Although these

are essential intermediates in oxidative metabolism,[9] when

generated in excess ROS can become toxic and damage cells

by peroxidizing lipids and disrupting structural proteins,

enzymes, and nucleic acids.[10] ROS in excess are generated

during a variety of cell stresses, including ischemia/reperfu-

sion, exposure to ionizing and ultraviolet radiation, and

inflammation.[11]

Inorganic NPs, such as silica, gold, carbon-based materials,

layered double hydroxides, and so on, are a large group among

those designed to transport drugs and bioactive molecules

(peptides, proteins, enzymes, DNA, etc.) into cells.[12] Our

present work describes the interaction in vitro of five of those

NPs (magnetic iron/graphite, magnetite/silica, bare and

poly(ethylene glycol)(PEG)-ylated silica, and magnetite/

FAU zeolite) with different human peripheral blood cells

(lymphocytes, monocytes, granulocytes and erythrocytes), as

well as mouse macrophages and several human tumor cell

lines. We compare here the kinetics of several functional

activities such as phagocytosis, cell growth, viability, and ROS

production, at varying doses of NPs, for each of the different

combinations of cell types and NPs used.

Our results reveal different effects depending on the cell

analyzed for any given NP. For instance, while all NPs are

phagocytosed and able to induce ROS bymousemacrophages,

they behave differently on distinct human cell lines and in

human peripheral blood cells, both in respect to internaliza-

tion and ROS induction. Moreover, contrary to previous

reports, we have not found always a positive correlation

between toxicity and ROS production induced by NPs.

2. Results

2.1. Aggregation of NPs in Culture Medium

The five kinds of inorganic particle used in this work are

shown in Table 1.

Once the NPs were added to the culture medium in the

presence of 10 wt% of fetal calf serum, they aggregated in all

cases, probably due to protein adsorption, except for PEG-

ylated silica NPs (Table 2); however, the size and morphology

of those aggregates were different for each particle type tested.

Thus, there was no uniformity in the size of the nanoparticle-

based agglomerates formed. The aggregation is also caused

by magnetic attraction forces (for types 1, 2, and 4) and

hydrophobic–hydrophobic interactions (for type 1); hydrogen

bonding between the silica surface hydroxyl groups and

nearby silica particles (for types 2, 3a, and 4) also contributes

to agglomeration.[13] Agglomeration may also occur through

the condensation of silanol groups on the surface of individual

particles.[14]

The availability of the particles to be internalized by the

cells seems to be strongly dependent on the size and

morphology of the aggregates. Brown et al.[15] showed that

agglomeration leads to reduced cytotoxicity, due to a decrease

www.small-journal.com � 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim small 2008, 4, No. 11, 2025–2034


Table 2. General description of the nanoparticles used in this work. Theimages show the agglomeration caused after adding them to the culturemedium in the presence of 10 wt % of fetal calf serum.

Type of NP Composition

Size

(nm) Aggregation[a] Image

1 Iron/graphite 200 þþþ

2 Iron oxide/

silica shell

80 þþþ

3a Silica 371 þþþ

3b PEG-ylated silica 151 �

4 Magnetite/

aluminosilicate

600 þþþ

[a](�) no aggregation, (þþþ) high aggregation.

in the availability of the inorganic nanoparticles in contact

with the cells and that, when the size of the agglomerate is

similar or larger than that of the cell, there is a physical

restriction on the internalization of the particles.

2.2. Uptake of NPs

We studied the uptake of the five types of NPs by normal

human monocytes and mouse macrophages. All NPs were

rapidly taken in by human monocytes, resulting in the

appearance of groups of NPs inside the cells that could be

easily visualized using a conventional optical microscopy

(Figure 1). Due to the Rayleigh criterion it is impossible to see

particles smaller than 200 nm using optical microscopy; hence,

all the observed agglomerates must be micrometric or larger.

Figure 1. Nanoparticle uptake into human monocytes, mouse perito-

neal macrophages and human cell lines U937 and PC3. Cells were

incubated in medium (control) or with Iron/graphite magnetic particles

(NP1); superparamagnetic magnetite/silica nanoparticles (NP2); bare

silica particles (NP3a); PEGylated/silica particles (NP3b) or magnetic

composites magnetite/FAU zeolite (NP4).


As expected, mouse peritoneal macrophages seem to be the

more active cells in the process of NP uptake (Figure 1).

We also tested the NP uptake by the human U937

(myeloid-monocytic) and PC3 (prostatic) cell lines. While

U937 did not show internalization of NPs, PC3 showed very

similar behavior to that of mouse macrophages (Figure 1). It

has been reported that the uptake of mesoporous silica NPs

(�100 nm) by cells can be regulated by a threshold of positive

surface charge, and also that the modulation of surface charge

on those NPs is specific to the cell type.[16]

On the other hand, human lymphocytes, erythrocytes

and other human cell lines such as Jurkat (T cell line) or HMY

(B cell line) are not phagocytic cells, although some NPs could

be seen on the surface (especially in the case of HMY) or even

in the cytoplasm (data not shown). It is important to point

out that the loading of tumor-specific T and B cells with

nanoparticles has been postulated as a strategy for targeted

drug delivery against tumors, but the number of internalized

NPs in the tested cell lines is very low.

Further verification of uptake of all NPs by macrophages

was performed by SEM. This is shown in Figure 2 for NPs 3a,

3b, and 4, where several NPs appear attached to the surface of

the macrophage. In the case of NP type 3a (silica), several

beads, showing their round shape, appear inside the macro-

phage (Figure 2a), and the silicon-based composition of these

structures was confirmed by back-scattered-electron imaging

(SEM-BEI) (Figure 2a). In some cases, dead cells can be

visualized with many NPs inside, showing a honeycomb

pattern (Figure 2b). Similar energy-dispersive X-ray (EDX)

spectroscopy analysis was performed for the rest of NPs

focusing the electron beam on the areas of the cells showing

nanoparticles, confirming the presence of iron in the NPs (data

not shown).

2.3. Cytotoxicity Analysis

For toxicological analysis induced by NPs in human cells, it

is crucial to calculate the correct concentration of cells to be

added per well, because a combination of factors such as

different rate of proliferation, cellular concentration, propor-

tion of death cells, and limited nutrients, affects cell viability

and interferes with the viability assays (e.g., MTT or Trypan

Blue). For this reason, for each cell line to be tested, different

concentrations of cells in the absence of NPs were plated in

media supplemented with FCS and analyzed at 24, 48, and 72 h

for cell viability. Figure 3A shows the large differences found

between the U937 and Jurkat cell lines. In the case of U937, a

maximum of 25 000 cells per well should be used, because

above this number cell viability decreases dramatically after

two days, mainly due to the consumption of nutrients.

However, Jurkat cells grow much more slowly and the

number of cells to be analyzed could go up to 75 000–100 000

cells per well.

Another important issue is that NPs can interfere with the

viability assay. Some NPs in culture medium (in the absence of

cells) showed absorbance at the same wavelength (525 nm)

used in the MTT assay, and this absorbance increases with the

NP concentration, depending on the type of NP. Figure 3B

shows the absorbance of the NPs alone after 72 h in culture.

H & Co. KGaA, Weinheim www.small-journal.com 2027


Figure 2. A) Morphology of peritoneal mouse macrophages incubated with different

nanoparticles. Samples were examined by SEM or by SEM-BEI. B) Dead peritoneal mouse

macrophage showing silica particles (Type 3 a) within the cell.

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The experiment was performed at 24, 48 and 72 h, but nomajor

differences were found at any of these times (data not shown).

The NP that showed the highest absorbance was type 4,

composed of magnetite/aluminosilicate, and the PEG-ylated

silica NP showed the lowest level of interference. Thus, in

order to standardize cytotoxic techniques it is crucial to

consider parameters such as the contribution of the NPs to the

total absorbance, the appropriate number of cells to be used

per well, the type of cell to be analyzed, and, finally, the time

schedule.

The toxicity of NPs for the different cell lines at different

NP concentrations is shown in Figures 4 and 5, according to the

MTT assay (which measures levels of metabolically active

mitochondrial dehydrogenase enzymes) and the Trypan Blue

exclusion assay (which detects loss of cell-membrane integ-

www.small-journal.com � 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinh

rity), respectively. In the case of the Trypan

Blue method the NPs’ absorbance does not

interfere with the assay.

In the case of MTT assay, PEG-ylated

silica NPs (type 3b) were not cytotoxic in any

of the cell lines tested at concentrations

ranging from 0.5 to 32mgmL�1. Indeed some

of them even proliferated (Figure 4).

The results indicate that the type 4 NPs

were the most toxic, but did not affect all cell

lines in the same way; HMY and U937 were

the most sensitive, especially at shorter times

(24 h), and even at low NP concentration.

In some cases, after this initial decrease

in the metabolic rate, there is a cell recovery

at 48–72 h, suggesting that those NPs

affect some cells negatively, but only at

early times.

The results obtained with the MTT assay

were further verified by cell counting using

the Trypan Blue exclusion assay (Figure 5).

Although this method is not very accurate,

especially when cells forming a monolayer

are tested,[17] it was only used with non-

adherent cells (HMY and Jurkat cells).

The results indicate again that the PEG-

ylated NPs show the lowest level of toxicity

in both cell lines. However, important

differences are found regarding the cell type

analyzed. While HMY (B cell line) cells

die mainly with NPs 1, 2, and 3a in a dose-

dependent manner, Jurkat cells seem to

be more sensitive to NP 4 (Figure 5).

These differences between cell lines could

be attributed to several factors, such as

surface charge. In fact, B cells (HMY-2)

show higher levels of syalic acid on their

membranes than T cells (Jurkat).

Crystalline silica particles have been

widely studied to understand the mechanism

that causes silicosis, an occupational lung

disease. It was reported that aluminium free

zeolites in microcrystalline form exhibit no

significant cytotoxicity, while FAU zeolites

exhibit high toxicity to macrophages at high concentration.[18]

Although the mechanism by which aluminium is toxic has not

been fully established, it is no longer in question that

aluminium is a cytotoxic element,[19] which interrupts peptide

synthesis. Therefore, the high toxicity of type 4NPs can be

attributed to the possible leaching of aluminium to the culture.

2.4. ROS Induction

It has been described that NPs can be toxic, not only

affecting cells in a direct way, but also indirectly by the

induction of excess ROS.[22,23] The production of ROS

induced by NPs can be due to either external (membrane)

or internal (after NP uptake) interactions with the cells.

eim small 2008, 4, No. 11, 2025–2034


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Figure 3. A) Calculation of number of cells to be used in cytotoxicity

tests. U937 and Jurkat cells were incubated for 24, 48, 72, and 96 hours

and cell viability was measured by MTT assay. One example of

three different experiments performed is shown, with each well

measured in triplicate. All cell lines were analyzed in the same way.

B) Nanoparticles can affect cytotoxicity tests. Nanoparticles in the

absence of cells showed light absorbance at the same wavelength

than the MTT assay.

Interestingly, it has been demonstrated that the toxicity in

animal models of instilled crystalline silica NPs is more

dependent on particle surface activity effects (i.e., formation

of H2O2 at the particle surface) than on particle size, or

particle surface area.[20] When generated in excess, ROS can

damage membrane cells and can also contribute to inflamma-

tion. The analysis of ROS by flow cytometry using the 20-

70dichlorfluorescein-diacetate (DCHF-DA) in human periph-

eral blood cells was performed at 5, 15, 30, and 60 minutes.

Mouse macrophages, used as a positive control, and four

different human cell lines were also tested. The results, with

only the two most representative times (5 and 30 minutes),

are shown in Figure 6. Mouse peritoneal macrophages

produced ROS at high levels after 5minutes in the presence

of all NPs, including PEG-ylated silica NPs (NP type 3b).

However, at 30min, the level of ROS decreased, especially

with type 1 NPs, followed next by type 2 NPs, and then type 3b

(Figure 6A).

A very different result was obtained with human

peripheral blood cells. Erythrocytes did not produce ROS

at any time (Figure 6B), granulocytes produced low levels but

only with type 4 NPs, and lymphocytes and monocytes

produced very low levels of ROS with NP types 3a, 3b, and 4.

Incubation periods of 1 h with NPs and the DCFH-DA


solution showed a pattern of ROS production similar to

those at 30 minutes, which is in agreement with the data

of some authors that showed a response plateau at 30–

60 minutes.[21]

In the case of cell lines, a large variation was found

depending on the cell tested. U937 and Jurkat cells produced

low levels of ROS (Figure 6C), but HMY and PC3 cells

expressed ROS with all NPs except with NP type 1. In the case

of HMY the production of ROS decreased significantly at

30 minutes with NP types 3b and 2, but NP types 3a, 3b and

4 maintained the production of ROS in the PC3 cell line even

after 30 minutes, showing a similar pattern to peritoneal

macrophages. Longer periods of incubation (24, 48, or 72 h)

using Jurkat cells with NPs did not show any production of

ROS (data not shown). It is important to indicate that NP type

3b was able to induce ROS in many of the cells tested,

especially in the tumoral cell lines HMY and PC3, but also

induced the lowest level of cytoxicity measured either byMTT

or Trypan Blue assays, indicating that production of ROS is

not related a priori to cell toxicity, or to cell death, as other

authors have indicated.[22,23]

3. Discussion

Our present results indicate that different techniques are

required for a sensible evaluation of the biocompatibility of

NPs, and that using a single cell line, and only one time point

and technique, could result in misleading results and incorrect

interpretations. For instance, according to our results NP type

3b can induce high levels of ROS in some cell lines, but not in

others, and was not toxic for any of the cells tested, contrary to

what has been stated by some authors, who have suggested

that induction of ROS leads to cell death.[22,23] Moreover,

some NPs affected cell metabolism at 24 h but not in longer

time periods. Several factors such as the target cell analyzed

(directly extracted from blood, type of cellular line), the assay

performed (MTT, Trypan Blue, ROS production), time

schedule, and moreover the nanoparticle’s characteristics

(hydrophobicity, charge, size, etc.) are elements that must be

taken into account when biocompatibility is to be analyzed.

Thus, comparison of the behavior of several NPs analyzed at

the same time, using different procedures and type of cells,

seems to be the best way to standardize biocompatibility

studies.

The necessity of the development of standardized proto-

cols that test NP cytotoxicity has also been proposed by other

authors. Sayes et al.[24] conclude that, given the enormous

range of NP types, morphologies, and surface chemistries,

toxicological testing that gives only a measure of hazard is

not useful. Instead, it is necessary to consider how a NP’s

biological behavior relates to its structure, composition, and

morphology. On the other hand, Hurt et al.,[25] in dealing with

carbon-based nanomaterials, point out several recommenda-

tions pertaining to the evaluation of NP toxicity and health

risks. They mention that prior to the toxicological study it is

necessary to completely characterize the particles, something

that is frequently not carried out. Also, they stress the

necessity for realistic exposure scenarios, and the develop-



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Figure 4. Cellular metabolism measured by MTT assay in different cell lines (HMY, U937, PC3 and Jurkat) after incubation 24, 48, or 72 hours (dot,

line and black columns, respectively) in the presence of different concentration of nanoparticles (Types 1, 2, 3a, 3b, and 4, see text). The mean of

three independent experiments are shown; each data point was assayed in triplicate.

2030

ment of methods to track the nanomaterials in biological

experiments. Finally, they point out that the toxicological

studies need to be evaluated considering not only the mass of

the nanoparticles but also their number and their surface area.

The necessity of standardized sample-preparation protocols,

reference materials, and characterization methodologies, as

well as standardized methods for assessing whether the

particles are taken up by or adsorbed into cells, has also

been stated by several other authors.[26–28]

In addition to what has been indicated by those authors,

and in view of our results, it is important to note that due to

differences in cell physiology depending on their origin

(epithelial, lymphoid, etc.), proliferation state (tumoral or

resting cells), membrane characteristics, phagocyte character-

istics, and so on, different type of cells should be analyzed for

each NP, in order to standardize protocols. Thus, in this study,

we have compared the behavior of lymphoid, myeloid, and

epithelia cell lines, but also normal cells from peripheral

blood. Special care should also be taken with themethods used

for cell viability studies, such as MTT, because some NPs show

absorbance at the same wavelength as the analysis; further-

more, the number of cells to be analyzed is crucial in this

technique (Figure 3).

www.small-journal.com � 2008 Wiley-VCH Verlag Gm

Not only the concentration but also the number of NPs

may be an important parameter to take into account in toxicity

studies. In fact, we realized that both the mass of particles (as

an indication of the dose delivered) and the number of

particles are important. For the same mass, an increase in the

number of nanoparticles leads to a larger interface area, and

therefore it is reasonable to speculate that the results could be

different. In this work, even though the number of particles per

gram is different in each case studied, a separate cytotox-

icological study was carried out, evaluating the number of

particles per cell in each culture (manuscript under prepara-

tion). The same trends in cytotoxicity for each cell type and the

same lack of direct correlation between ROS production and

cell toxicity were found.

The first step towards normalization and standardization of

toxicological testing for NPs has been taken by the National

Characterization Laboratory (US National Cancer Institute),

who are developing and performing a standardized analytical

cascade that tests the pre-clinical toxicology, pharmacology,

and efficacy of NPs. The characterization involves evaluations

of the physico-chemical nature of nanoparticles, their in vitro

biological properties, and their in vivo compatibility through

the use of animal models. The assay cascade is designed to

bH & Co. KGaA, Weinheim small 2008, 4, No. 11, 2025–2034


JurkatHMY

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Figure 5. Cell viability measured by Trypan blue exclusion of HMY and Jurkat cell lines

after incubation 24, 48, or 72 hours in the presence of different concentration of

nanoparticles (Type 1, 2, 3a, 3b, and 4). A representative example (mean of four

measurements) is shown.

provide data and information related to the NPs’ interaction

and compatibility with biological systems.[29]

4. Conclusions

For a given NP its cytotoxicity depends on the cell type

tested, and it is not possible to find a direct correlation between

ROS production and cell toxicity. PEG-ylation avoids protein

adsorption on the external surface of the NPs and conse-

quently avoids agglomeration in culture medium. The

availability of the particles to be internalized by the cells

seems to be strongly dependent on the size and morphology of

the aggregates. For cytotoxicological analysis it is necessary to

optimize the number of test cells to be used, because a

combination of factors, such as different rate of proliferation,

cellular concentration, proportion of death cells, and limited

nutrients, affects cell viability and interferes with the viability

small 2008, 4, No. 11, 2025–2034 � 2008 Wiley-VCH Verlag GmbH & Co. KGaA, We

assays. In summary, it is necessary to develop

standardized protocols that test the cytotoxicity

of nanoparticles.

5. Experimental

Nanoparticles: The five different inorganic NPs

used in this work have been described in detail

elsewhere.[15,19,20,30] A brief description of their

preparation is as follows:

Type 1: Iron/graphite core/shell NPs (hydro-

dynamic size 300 nm) were synthesized by using

the arc-discharge method.[30] This procedure uses

a cylindrical chamber with two graphite electrodes;

a stationary anode containing iron powder with an

initial size of 10mm, and a moveable graphite

cathode.[31] An electric arc was produced between

the graphite electrodes in a helium atmosphere.

The graphite electrode sublimated under the

influence of the arc and built up a deposit on the

inner surface of the chamber. The material

collected from this deposit consisted of carbon

nanostructures, amorphous carbon, iron, and iron

oxide NPs encapsulated within graphitic layers.

Noncoated or partially coated magnetic particles

were eliminated by chemical etching. The particles

agglomerated; the size of each agglomerate-form-

ing subunit was �10 nm (measured by TEM).[30]

The sizes of the agglomerates ranged above

300 nm.

Type 2: Superparamagnetic magnetite/silica nano-

particles (�80 nm) were prepared as described in

our previous work.[32] They consist of an inner iron

oxide coated by a silica shell. The mean nanopar-

ticle size was approximately 80 nm for the compo-

site material, while the average size for the

magnetic cores varied between 6 and 20 nm (both

measured by TEM). The particle-size distribution for the silica-

coated nanoparticles at pH 7.3 measured by dynamic light

scattering yielded an average value of 98W15 nm.

Types 3a and 3b: Bare and PEG-ylated silica particles (Types 3a

and 3b, respectively). Mesoporous silica particles were prepared

following the synthesis procedure described by Zeng et al.[33]

using a precursor gel with molar composition 1TEOS:0.035C-

TABr:0.0175NaOH:692.5H2O and by stirring at 353 K for 3 h. The

synthesis of PEG-ylated dense silica particles was carried out

following the synthesis procedure described by Xu et al.[34] The

complete characterization and standard protein adsorption

experiments with those NPs were described in our previous

work.35] The bare silica particles show an average particle size of

130W 20 nm (measured by SEM) and a hydrodynamic particle size

of 371W 65 nm (in phosphate buffered saline (PBS) at pH 7.4).

The PEG-ylated silica nanoparticles show an average particle size

of 155W 20 nm (measured by SEM) and a hydrodynamic particle

size of 151W23 nm (in PBS at pH 7.4).

inheim www.small-journal.com 2031


Figure 6. ROS production by mouse macrophages (A), different human

blood cell types (B), and human cell lines (C), after incubation with

different NPs during 5 or 30 minutes. In the case of human blood cells,

a representative analysis of experiments performed from blood

obtained from three different donors is shown. In the case of human cell

lines, they were analyzed twice.

2032 www.small-journal.com � 2008 Wiley-VCH Verlag Gm

Type 4: Magnetite/FAU Zeolite: Magnetic composites of magnetite

embedded in an aluminosilicate-based matrix were prepared

using high-energy milling at room temperature according to our

previous experiment.[36] The range of hydrodynamic particle sizes

(volume average) extends from 20 to 600 nm although micro-

metric particles are also obtained.

Cells: Due to large differences in the physiology of cells

depending on their tissue origin (lymphoid, epithelial, etc.),

proliferation state (tumoral or normal cells), phagocyte character-

istics (macrophages versus non-phagocytic cells), we decided to

include several cells in this study to cover all those aspects. Thus,

for toxicology analysis, the following human tumor cell lines were

used: Hmy2,[37] Jurkat,[38] U937,[39] and PC3,[40] which correspond

to lymphoblastoid B and acute T lymphoblastic leukemia

(lymphoid origin), myeloid-monocytic lymphoma (myeloid origin)

and human prostate adenocarcinoma (eptithelial origin) cell lines,

respectively. All cell lines were maintained in RPMI (Gibco, Life

Technologies, Grand Island, NY) supplemented with 10 wt% heat

inactivated fetal calf serum (FCS) (PAA, Linz, Austria), penicillin

(100 U mLS1), streptomycin (100mg mLS1) and glutamine (2 mM)

(Gibco) at 37 -C in a humidified atmosphere containing 5 vol%

CO2.

Peripheral blood cells were obtained from heparinized whole

blood from healthy donors and several populations (erythrocytes,

lymphocytes, granulocytes and monocytes) were studied. Human

monocytes and lymphocytes were obtained by density-gradient

centrifugation over Ficoll-Paque solution (Amersham Pharmacia

Biotech AB, Uppsala, Sweden). The interphase was washed

three times with PBS. The purity of those cell preparations

was analyzed independently by size/complexity in the flow

cytometer. Human monocytes were isolated by plastic adhesion

to Petri dishes. Briefly, the plates were covered with medium

supplemented with 10% FCS for 1 h and then the mixture of

lymphocytes and monocytes was added and incubated for 2 h.

After several washes, human monocytes were recovered as

adherent cells.

Mouse peritoneal macrophages were collected by flushing the

peritoneal cavity of normal C57Bl/6 and Fox nudeR/S mice with

PBS. Recovered cells were centrifuged for 5 min at 231g and

resuspended in RPMI containing 10% heat-inactivated FCS. A

number of cells were plated in 96- or 24-well plates (Becton

Dickinson, Franklin Lakes, USA) or in chamber slides (Nalge Nunc

International, Rochester). After either 1 h or overnight incubation

at 37 -C in a humidified atmosphere containing 5 vol% CO2, NPs

were added to the cells at different concentrations and incubated

overnight at 37 -C with 5 vol% CO2. Sampling homogeneity was

achieved by sonicating the samples for 15 min and mixing

repeatedly, by loading and unloading the pipette at least three

times, before adding the dispersion to the cultured cells. In any

case, the suspensions were stable during the time required for the

supply of suspension to the cells, and no sedimentation or

preferential enrichment of particles was observed. Cells were

visualized with inverted and direct microscopes (Olympus IX50,

and BX51, respectively, Olympus Optical. Tokyo, Japan) and

photos were taken with an Olympus DP71 camera.

Microscopy: The morphology of the incubated cells with or

without nanoparticles was investigated by using scanning electron

microscopy (SEM). Cells were attached to sterile coverlids on 24-

bH & Co. KGaA, Weinheim small 2008, 4, No. 11, 2025–2034


well plates, and 2 h later the NPs were added. After overnight

incubations, cells were washed with PBS and fixed with 2.5 wt%

glutaraldehyde in 0.05 M cacodylate buffer (plus KCl 0.7 M, CaCl21.24 mM, and MgCl2 1.24 mM) for 2 h at 4 -C. Cells were washed

three times (ten minutes each) with 0.05 M cacodylate buffer and

incubated with 2 wt% OsO4 in ultrapure water during 1 h at 4 -C.

Then the cells were washed three times again with ultrapure

water, followed by dehydration in a graded series of ethanol

solutions (30, 50, 70, 90, and 100 vol%) and then incubated in

amilacetate/ethanol (25%/75%, 50%/50%, 75%/25%) and twice

in 100 vol% amilacetate for 15 min. The dehydration was finished

with the critical point dryer (Polaron E300, Polaron Equiment, UK).

The samples were mounted on microscope slides and sputtered

with gold, forming a conductive layer of approximately 10 nm

(Sputter Polar SC500, England). The cellular uptake of NPs was

evaluated by scanning electron microscopy (Philips XL30 and Jeol

JSM 6700F, The Netherlands) operating at 10–15 kV.

Viability Testing: Cell viability and proliferation were analyzed

by the MTT (3-[4,5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium

bromide) colorimetric assay[41]. Different human cell lines were

incubated in medium supplemented with 10 wt% FCS in the

absence or presence of various concentrations of NPs (from 0.5

to 32 mg mLS1) and cells were incubated for 24, 48, and 72 h at

37 -C, with 5 vol% CO2 in 96-well plates. The reaction product was

spectrophotometrically measured at 525 nm with a microplate

reader (Multiskan EX, BioAnalysis Labsytems. Barcelona, Spain).

All experiments were repeated at least three times and in

triplicates. Results are shown as percent of viability (%V)

according to the following formula (blank discounted):

%V ¼ absorbanceðcellsþmediumþNPsÞ�absorbanceðmediumþNPsÞabsorbanceðmedium þ NPsÞ

� 100

ð1Þ

Cell viability was also confirmed by Trypan Blue exclusion.

Aliquots of cells incubated with different concentrations of NPs

were counted at least four times in a Neubauer Chamber in the

presence of 1 wt% Trypan Blue in PBS to identify dead (stained

blue) and living (non-stained) cells under an optical microscope

(Nikon SE Type 102. Tokyo, Japan). The intracellular ROS

generation was investigated using 2(,7(-dichlorofluorescein-di-

acetate (DCFH-DA) to detect and quantify intracellular production

of H2O2. Briefly, a stock solution of DCFH-DA was prepared in

ethanol and stored at –20-C in the dark. Different cells were

exposed to NPs for 5, 15, 30 and 60 minutes, and even longer

periods of time (24, 48, and 72 h). The green fluorescence of DCF

was recorded by exciting the solution with a 488 nm argon laser

(FL1) using a FACS Coulter (FC500 MPL).

Acknowledgements

The authors would like to thank the Spanish Nanoscience

Action NAN200409270-C3-1/2, Consolider Ingenio 2010 pro-

gram (CSD2006-00012), and Xunta de Galicia, Spain (PGI-


DIT06TMT31402PR) for the support of this study. MA and JF

acknowledge support from the 2006 Ramon y Cajal program

(order ECI/158/2005) and from the 2004 Isidro Parga Pondal

program (Xunta de Galicia), respectively. We thank Jesus

Mendez for his valuable help in the SEM analysis.

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Received: February 4, 2008Revised: May 20, 2008Published online: October 15, 2008

small 2008, 4, No. 11, 2025–2034

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Assessing Methods for Blood Cell Cytotoxic Responses to Inorganic Nanoparticles and Nanoparticle Aggregates