432 volume 118 | number 3 | March 2010 • Environmental Health Perspectives

Research

Humans have always been exposed to airborne particles through natural sources such 

as volcanoes, forest fires, or desert dust. After 

the Industrial Revolution in the 19th century, emission of anthropogenic particles into 

the atmosphere significantly increased. This 

increased exposure to respirable fine particles

(< 2.5 μm in diameter) and ultrafine particles 

(UFPs; < 0.1 μm in diameter). Epidemiologic 

and in vivo studies have shown potential toxic 

effects of fine particles and UFPs on human 

health (Kreyling et al. 2004; Oberdörster and 

Utell 2002). When inhaled, UFPs are deposited in the respiratory tract and may provoke 

inflammation or granuloma formation in the 

lung and induce systemic effects such as prothrombotic responses (Nemmar et al. 2008) 

or cardiovascular changes (Alfaro-Moreno et 

al. 2007; Kreyling et al. 2004; Nemmar et al. 

2007, 2008). Despite the presence of several 

highly efficient lung clearance mechanisms, 

different studies have shown that the transfer 

of nanoparticles from the lung to the blood 

is possible (Geiser et al. 2005; Nemmar et al. 

2005; Rothen-Rutishauser et al. 2006).

With the broad use of nanotechnology, 

intentional production of nanoobjects in the 

size range of 1–100 nm has further increased the 

number and variety of nanoparticles to which 

humans can be exposed. New applications of 

engineered nanoparticles in nanomedicine such 

as nanoparticulate contrast agents (Rudin and 

Weissleder 2003), cancer treatments (Thiesen 

and Jordan 2008), and vaccines (Reddy et al. 

2007) are in development that will be directly 

injected in the bloodstream of patients.

Most of the recent research dealing with 

potential health hazards of nanoparticles has 

focused on cells and tissues that are likely to 

come into immediate contact with airborne 

particles. There is a growing concern about 

the possible influence on health of the exposure to nanoparticles during pregnancy or early 

childhood (Lacasana et al. 2005). A recently

performed cohort study suggests that prenatal exposure to air pollution might be associated with higher respiratory need and airway 

inflammation in newborns (Latzin et al. 2008). 

However, it remains unclear whether these 

effects were caused by particles crossing the placenta or by mediators induced in the maternal 

circulation such as proinflammatory factors.

First indications that nanosized materials 

may cross the placental tissue came from a 

recent study in rats, showing that nanosized 

gold was transferred to the embryos 24 hr after 

intravenous injection (Semmler-Behnke et al. 

2007). However, these data cannot be extrapolated to humans because the anatomy and 

physiology of the human placenta are unique. 

One main difference is that in humans the 

syncytiotrophoblasts arise from the fusion of 

cytotrophoblast cells and form a true syncytium with no lateral cell membranes, whereas 

in rats or mice three trophoblast layers are present between maternal blood and fetal blood 

capillaries (for comprehensive reviews, see 

Enders and Blankenship 1999; Takata et al. 

1997). Thus, the transport efficiency for xenobiotics or nanoparticles across the placenta has 

to be defined for humans explicitly.

In detail, the cellular barrier between the 

maternal and the fetal blood is formed by 

the syncytiotrophoblast layer, which faces the 

maternal environment, and the endothelial 

cell layer of the fetal microcapillaries. Between 

these two cell layers there are several stromal 

cells such as cytotrophoblasts, fibroblasts, and 

Hofbaur cells (placental macrophages). This 

layer is relatively thick in early pregnancy and 

becomes progressively thinner with gestational 

age. This reduction in thickness together with 

an increase in the number of fetal capillaries enhances the efficiency of maternal–fetal 

exchange during the development of the fetus 

(Enders and Blankenship 1999). The transfer 

of substances and the amount that enters the 

fetal circulation are tightly controlled by at 

least four different mechanisms: simple diffusion, active transport, biotransformation 

through metabolic enzymes, and phagocytosis 

or pinocytosis (Syme et al. 2004). The plasma 

membrane of the syncytiotrophoblast is polarized, consisting of the brush border membrane that is in direct contact with maternal 

blood and the basal membrane that faces the 

fetal circulation (Ganapathy et al. 2000). The 

two membranes are further distinguishable 

from each other by their protein, receptor,

and transporter composition. The brush border membrane contains specific transporters,

such as those for serotonin (SER transporter) 

Address correspondence to P. Wick, Laboratory for 

Materials-Biology Interactions, Lerchenfeldstr. 5, 9014 

St. Gallen, Switzerland. Telephone: 41-71-274-76-84. 

Fax: 41-71-274-76-94. E-mail: peter.wick@empa.ch

*These authors contributed equally to this work.

We dedicate this article to our colleague Andreas 

Zisch, who died during the preparation of the manuscript, after an exemplary fight against his disease. 

We thank T. Thurnherr for critical reading of the 

manuscript and R. Arrigo from the Fritz Haber Institute 

for technical support in particle characterization.

This work was financially supported by an Empa 

internal grant and by the Medical Faculty Research 

of the University of Zurich. 

The authors declare they have no competing 

financial interests.

Received 14 July 2009; accepted 12 November 2009.

Barrier Capacity of Human Placenta for Nanosized Materials

Peter Wick,1,* Antoine Malek,2,* Pius Manser,1 Danielle Meili,2 Xenia Maeder-Althaus,1 Liliane Diener,1

Pierre-Andre Diener,3 Andreas Zisch,2 Harald F. Krug,1 and Ursula von Mandach2

1Empa, Swiss Federal Laboratories for Material Testing and Research, Laboratory for Materials–Biology Interactions, St. Gallen, 

Switzerland; 2University Hospital Zurich, Department of Obstetrics, Zurich, Switzerland; 3Institute of Pathology, Cantonal Hospital, 

St. Gallen, Switzerland

Background: Humans have been exposed to fine and ultrafine particles throughout their history. 

Since the Industrial Revolution, sources, doses, and types of nanoparticles have changed dramatically. In the last decade, the rapidly developing field of nanotechnology has led to an increase of 

engineered nanoparticles with novel physical and chemical properties. Regardless of whether this 

exposure is unintended or not, a careful assessment of possible adverse effects is needed. A large 

number of projects have been carried out to assess the consequences of combustion-derived or engineered nanoparticle exposure on human health. In recent years there has been a growing concern 

about the possible health influence of exposure to air pollutants during pregnancy, hence an implicit 

concern about potential risk for nanoparticle exposure in utero. Previous work has not addressed the 

question of whether nanoparticles may cross the placenta.

Objective: In this study we investigated whether particles can cross the placental barrier and affect 

the fetus.

Methods: We used the ex vivo human placental perfusion model to investigate whether nanoparticles can cross this barrier and whether this process is size dependent. Fluorescently labeled polystyrene beads with diameters of 50, 80, 240, and 500 nm were chosen as model particles.

Results: We showed that fluorescent polystyrene particles with diameter up to 240 nm were taken 

up by the placenta and were able to cross the placental barrier without affecting the viability of the 

placental explant.

Conclusions: The findings suggest that nanomaterials have the potential for transplacental transfer 

and underscore the need for further nanotoxicologic studies on this important organ system.

Key words: barrier tissue, ex vivo perfusion, human placenta, nanoparticles, nanotoxicity. Environ 

Health Perspect 118:432–436 (2010). doi:10.1289/ehp.0901200 available via http://dx.doi.org/ 

[Online 12 November 2009]
Placental barrier capacity for nanoparticles

Environmental Health Perspectives • volume 118 | number 3 | March 2010 433

or carnitine (novel organic cation transporter 

OCTN2), or unspecific transporters such as 

P-glycoprotein, a member of the multidrugresistance gene family. The folate transporter 

FOLT1, together with the folate receptor 

of the brush boarder membrane, is typically 

expressed in basal membrane and mediates 

the transport of vitamins across the placenta 

(Ganapathy et al. 2000).

The dual recirculation human placental 

perfusion model (Panigel et al. 1967; Schneider 

et al. 1972) provides a controlled system for 

studying the transplacental transport of various 

substances and is commonly used for pharmacokinetic studies. In the present study we used 

this model to investigate whether nanoparticles 

can cross the placental barrier and whether 

this process is size dependent. We chose fluorescently labeled polystyrene (PS) beads with 

diameters of 50, 80, 240, or 500 nm as model 

particles. In addition, we assessed the viability 

and functionality of the placenta after particle 

perfusion.

Materials and Methods

Polystyrene beads. Fluorescent PS particles 

of 50 and 240 nm were commercially available from Kisker (PFP-00552 and PFP-0252; 

G. Kisker GbR, Steinfurt, Germany), and 

80- and 500-nm beads were purchased from 

Polyscience (catalog nos. 17150 and 17152; 

Polyscience Europe GmbH, Eppelheim, 

Germany). The particle stock solution was 

vortexed 15 min before each perfusion assay.

Characterization of PS bead suspension.

We used a scanning electron microscope 

(SEM; S-4800, Hitachi High-Technologies 

Europe GmbH, Krefeld, Germany) to analyze the visual appearance of the as-received 

PS beads. We measured the size distribution 

of the PS beads in perfusion medium with a 

NanoSight LM 20 System (NanoSight Ltd., 

Amesbury, Wiltshire, UK) at a particle concentration of about 106 to 109 particles/mL 

and are presented as size–density plots. The 

density plot of the perfusion medium without 

beads has been reduced by a factor of 5 compared with particle measurement in relation to 

the number of counting events measured.

We tested the stability of the fluorescent 

PS beads in different solutions, including the 

perfusion medium. Briefly, the beads were 

diluted with phosphate-buffered saline (pH 

4), 1% sodium dodecyl sulfate, 100% ethanol, and perfusion medium to a concentration of 10 μg/mL and incubated for 24 hr

at 37°C. The fluorescence of 100 μL suspension was measured before and after filtration with a 0.1-μm Pall Acrodisc hydrophilic 

polyethersulfone filter (Pall Corporation, Port 

Washington, NY, USA), using a microplate 

reader (BioTex FLx800; Witec AG, Littau 

LU, Switzerland) with excitation and emission 

wavelengths of 485 and 528 nm, respectively.

We determined the detection limit of PS 

beads for each type of particles separately by 

serial dilution in perfusion medium in the range 

of 0.02–10 μg/mL. The detection limit was 

defined as the lowest concentration that showed 

a significant increase in the fluorescent intensity 

compared with pure perfusion medium.

We measured zeta potentials of different 

PS bead suspensions using a Malvern zetameter (Malvern Instruments Ltd, Malvern, 

Worcestershire, UK). First, PS beads were dispersed in water to a concentration of 1 mg/mL 

by vortexing for 10 min. A second suspension 

was obtained by vortexing the PS beads in perfusion medium for 10 min.

Ex vivo dual recirculating human placental perfusion model. The placental perfusion technique was originally developed by 

Panigel et al. (1967) and was further improved 

by Schneider et al. (1972). The dual recirculating perfusion was performed according 

to Malek et al. (2009). Intact placentas were 

obtained from uncomplicated term pregnancies either after vaginal or cesarean delivery at 

the Department of Obstetrics of the University 

Hospital Zurich. All mothers gave written 

informed consent for the use of the placentas in this study. To minimize damage, the 

placentas were placed in 200 mL of a 0.9% 

sodium chloride solution preheated to 37°C,

and then transported in a Styrofoam box to 

the research laboratory within 20 min of delivery. To perfuse the fetal side, the artery and 

vein of an intact cotyledon were cannulated. 

Subsequently, the placental tissue was fixed in 

a tissue holder and then placed into a perfusion 

chamber. The maternal side was perfused by 

introducing three blunt metal cannulas into the 

intervillous space by penetration of the decidual plate. Furthermore, a venous drain, connected with a peristaltic pump (Reglo Digital; 

Ismatec, Glattbrug, Switzerland) removed the 

perfusate from the chamber and returned it 

to the maternal reservoir. Fetal and maternal 

cannulas were connected to separate perfusion 

circuits. Peristaltic pumps held the fetal circuit 

flow at a rate of 6.0 mL/min and the maternal 

circuit at 12.0 mL/min.

The perfusion medium is composed of 

NCTC-135 tissue culture medium (ICN 

Biomedicals, Inc., Irvine, CA, USA) diluted 

with Earl’s buffer (1:1) supplemented with 

glucose (G 7528, 2 g/L; Sigma Switzerland, 

Buchs SG, Switzerland), dextran 40 (10 g/L;

Sigma 31389), bovine serum albumin 

(8 g/L; Sigma), sodium heparin (2,500 IU/L;

GlaxoSmithKline AG, Münchenbuchsee, 

Switzerland), and amoxicillin (Clamoxyl, 500 

mg/L; GlaxoSmithKline). Two oxygenators 

(LSI-OX, Living Systems Instrumentation Inc., 

Burlington, NH, USA) were used, one with 

95% N2 and 5% CO2 for the fetal circuit, and 

one with 95% air and 5% CO2 for the maternal perfusate. Both perfusates were maintained 

at a temperature of 37°C by a water bath (Glas 

water bath; UNI-GLAS, Zürich, Switzerland). 

The experiments included a 20-min period of 

open perfusion of both compartments (prephase) to flush the blood out of the intervillous 

space and the villous vascular compartment and 

to allow recovery of the placental tissue from 

the ischemic period after delivery. After the 

prephase, the maternal perfusate was exchanged 

with a perfusate containing PS beads of 50, 80, 

240, or 500 nm, respectively, at a final concentration of 25 μg/mL and 14C-antipyrine (50 

nCi/mL; specific activity, 4.7 mCi/mmol). The 

experiment was continued in closed maternal 

and fetal circuits with equal starting volumes 

of 120 mL. After 180 min the same amount of 

beads was added to the maternal circuit. The 

integrity of the fetal circulation was monitored 

by assessing the leak in the fetal-to-maternal 

direction, including the measurement of the 

volume loss of the fetal perfusate.

The criteria for a successful perfusion were

a) visual control: intact membranes, no lesion, 

no disruption of the placenta; b) leak from 

fetal to maternal side < 4 mL/min (Malek et 

al. 1994); c) 14C-antipyrine values: equilibrium 

between maternal and fetal circuit after 4–6

hr (Challier 1985); d) fetal perfusion pressure 

< 70 mm Hg at all times; and e) fetal pH at 

physiologic range of 7.2–7.4. To determine 

the concentration of fluorescent PS particles 

in the maternal and fetal circuit, we removed 

100 μL perfusion medium from each circuit at 

the indicated time points. After centrifugation 

at 800 rpm to remove residual erythrocytes, 

fluorescence was read at 485-nm excitation 

and 528-nm emission in a microplate reader 

(BioTex FLx800, Witec AG).

Permeability and viability of the placental tissue. We evaluated the permeability of 

the placenta by monitoring the transplacental transfer of 14C-antipyrine as described previously (Challier 1985). Briefly, 50 nCi/mL 

14C-antipyrine were added to the maternal 

circuit, and 300 μL of the perfusion medium 

from each reservoir was transferred into separate scintillation tubes and mixed with 3 mL 

scintillation cocktail (Irgasafe Plus Scintillation 

Cocktail, Zinsser Analytic, Frankfurt, 

Germany). 14C radioactivity was immediately 

measured for 5 min in a beta counter (Packard 

Tri-Carb 2200 liquid scintillation analyzer;

GMI, Ramsey, MN, USA). Maintenance of 

placental function was assessed by measuring 

the production and accumulation of the two 

placental hormones leptin and human chorionic gonadotropin using an enzyme-linked

immunosorbent assay as described previously 

(Malek et al. 2000, 2001). Briefly, the net production (NP) of human choriongonadotropin 

or leptin was estimated as NP = (end tissue 

content minus initial tissue content) plus accumulation. To determine the metabolic activity 

of the placenta, we used the sum of changes 
Wick et al.

434 volume 118 | number 3 | March 2010 • Environmental Health Perspectives

in total (maternal and fetal) content over time 

divided by the perfused cotyledon weight to calculate glucose consumption or lactate production, which were measured with an automated 

blood gas system (ABL800 FLEX automated 

benchtop analyzer; Radiometer Medical ApS, 

Copenhagen, Denmark).

Detection of PS beads in the fetal circuit after perfusion. A 1.5-mL sample of the 

fetal perfusion medium was taken after 6 hr

of perfusion and was centrifuged at 25,000 

relative centrifugal force for 30 min at 4°C. 

This allowed the concentration of PS beads 

> 50 nm in the pellet, because no fluorescent signals were detected in the supernatant 

after centrifugation. The pellet was washed 

Figure 1. SEM and size distribution of PS beads. (A–D) SEM micrographs depict the beads with diameters of 50 nm (A), 80 nm (B), 240 nm (C), and 500 nm (D). 

(E) The size distributions of all applied particles were measured in the perfusion medium, presented as a size–density plot.

0.025

0.020

0.015

0.010

0.005

0.000

Density

0 200 400 600

Size (nm)

800 1,000

50 nm PS beads

80 nm PS beads

240 nm PS beads

500 nm PS beads

Perfusion medium

Figure 2. Perfusion profiles of PS beads and 14C-antipyrine. After the prephase, PS beads of different sizes were added to the maternal circuit to a final concentration of 25 μg/mL and the amount of 14C-antipyrine and PS beads in both maternal circuits (M, solid symbols) and fetal circuits (F, open symbols) were measured 

after the indicated time points. (A) In the fetal circuit, significantly increased levels of 50-, 80-, and 240-nm PS beads were measured after only a few minutes 

of perfusion, whereas the 500-nm beads were retained in the placental tissue and maternal circuit. A second addition of particles after 180 min (arrow) did not 

further increase the amount of beads in the fetal circuit. (B) The perfusion profiles of 14C-antipyrine were not affected by the presence of beads and maintained 

equilibrium until the end of the perfusion assay. Data represent mean ± SE of at least four independent experiments.

50

25

0

5,000

2,500

0

PS particle (μg/mL)

Antipyrine (14C counts/ 0.3 mL)

0 60 120 180 240 300 360

Time (min)

0 60 120 180 240 300 360

Time (min)

50 M

80 M

240 M

500 M

50 F

80 F

240 F

500 F

Table 1. Summary of PS bead characteristics.

PS beads

Characteristic 50 nm 80 nm 240 nm 500 nm

Diameter (nm)a 49 83 240 504

Diameter (nm)b 43 ± 35 81 ± 44 220 ± 60 370 ± 96

Surface area (nm2)

c 1.88E+03 5.41E+03 4.52E+04 2.00E+05

Internal volume (nm3)

c 6.16E+04 2.99E+05 7.24E+06 6.70E+07

Density of PS (mg/nm3)

d 1.05E–18 1.05E–18 1.05E–18 1.05E–18

Mass of one PS particle (mg)c 6.47E–14 3.14E–13 7.60E–12 7.04E–11

Initial PS particles/mL PMc 3.87E+11 7.95E+10 3.29E+09 3.55E+08

PS particle surface/mL PM (nm2)

c 7.28E+14 4.30E+14 1.49E+14 7.10E+13

Detection limit of PS (μg/mL)b < 1.25 < 1.64 < 0.63 < 0.21

Detection limit of PS (no./mL)c 1.94E+10 5.22E+09 8.22E+07 2.96E+06

Zeta potential in DD water (mV)b –58.7 ± 2.26 –56.4 ± 2.12 –32.7 ± 0.78 –42.3 ± 0.49

Zeta potential in PM (mV)b –6.23 ± 0.20 –7.40 ± 0.70 –8.91 ± 0.74 –12.8 ± 1.20

Abbreviations: DD, double distilled; PM, perfusion medium. 

aAccording to manufacturer’s information. bExperimentally determined (mean ± SE). cCalculated values. dFrom literature 

(Sharp and Beard 1950). 
Placental barrier capacity for nanoparticles

Environmental Health Perspectives • volume 118 | number 3 | March 2010 435

once with Millipore water, applied onto a 

carbon-coated copper grid (400 mesh; Pelco,

Redding, CA, USA) and processed for transmission electron microscopy (TEM) analysis.

Statistical analysis. Data are shown as 

mean ± SE from at least four independent 

experiments. Statistical analysis was made 

with the Mann–Whitney two-sample test and 

the Wilcoxon test. A p-value < 0.05 was considered to be statistically relevant.

Results

PS particle characterization. Table 1 summarizes details of particle characterization,

such as particle size, surface area, and total 

surface charge in pure water and in perfusion 

medium. The zeta potential measurement of 

the PS beads in perfusion medium resulted in 

slightly negative values ranging from –12.8 ± 

1.20 to –6.23 ± 0.20 mV, indicating that the 

albumin stabilizes the spheres in the perfusion medium. Size distribution analysis confirmed that suspensions of 50-nm, 80-nm, and 

240-nm beads were relatively monodisperse 

(Figure 1A–C, E). However, we observed particles of various sizes in SEM images as well as 

in size–density plots of 500-nm suspensions 

(Figure 1D, E).

The use of fluorescent nanoparticles, especially of PS beads, has been shown to cause 

experimental problems because of a possible 

transfer of the lipophilic dye to the surrounding tissue and/or cells (Kanno et al. 2007; 

Pietzonka et al. 2002). To exclude false-positive 

results, we measured the stability of the fluorescent dye in different solutions, including our 

perfusion medium. Except for pure ethanol,

where we detected a release of 33.7 ± 0.74% of 

the fluorescent dye for 240-nm beads and 35.9 

± 10.2% for 500-nm beads, the fluorescence 

remained stable in all tested conditions, including our perfusion medium (data not shown).

We experimentally determined the detection limit of the fluorescent PS particles in 

perfusion medium for each particle size. The 

minimum concentration of particles that still 

allowed the detection of a significant fluorescent signal was 1.25 μg/mL for 50-nm, 

1.64 μg/mL for 80-nm, 0.63 μg/mL for 

240-nm, and 0.21 μg/mL for 500-nm beads 

(Table 1).

Perfusion profiles of PS beads of different 

sizes. Another important control was to assess 

the quality of the placental explant, which we 

achieved by monitoring the diffusion of 14Cantipyrine, which is known to rapidly cross the 

placenta and reach equal concentration in the 

maternal and fetal circuit after 4–6 hr (Challier 

1985) (Figure 2B). If we started the perfusion 

with 25 μg/mL 50-nm PS beads in the maternal circuit, the level of particles measured after 

180 min was 8.90 ± 1.80 μg/mL in the fetal 

perfusion medium and 18.1 ± 5.64 μg/mL in 

the maternal perfusion medium (Figure 2A). 

Because these concentrations remained stable 

thereafter, we performed a second administration of the same amount of beads to test 

whether the number of beads in the fetal circuit could be further increased. Similar to 

the first injection of PS beads, we observed a 

gradual decrease of the particles in the maternal perfusion medium but detected no significant increase of beads in the fetal perfusion 

medium. Administration of 80-nm particles 

resulted in a perfusion profile similar to the 

one for 50-nm beads, and the concentration of 

80-nm particles measured in the fetal circulation after 180 min was 7.47 ± 1.77 μg/mL. 

After perfusion with 240-nm PS particles for 

180 min, the bead concentration on the fetal 

side had dropped to 2.03 ± 0.29 μg/mL. Using 

500-nm PS beads, nearly all particles were 

retained in the maternal circulation or placental 

tissue, and the concentration of beads detected 

in the fetal circulation was 0.31 ± 0.21 μg/mL 

(Figure 2A). The level of particles was low most 

likely because the 500-nm bead suspension was 

not monodisperse but rather contained particles < 500 nm in diameter (Figure 1D). We 

used the ratio of fetal-to-maternal concentration of PS beads detected after 180 min to estimate the amount of PS particles that crossed 

the placenta (Figure 3). These measurements 

showed that the translocation of PS beads 

across the placenta is clearly size dependent,

and particles larger than 240 nm hardly pass 

this tissue barrier. The presence of PS beads 

did not affect the diffusion kinetic of 14C-antipyrine (Figures 2B, and 3), indicating that PS 

particles did not alter the transfer properties of 

the placental tissue.

At the end of each perfusion experiment, 

we took samples from the fetal circuit to check 

for the presence of beads. TEM micrographs 

indeed showed beads with diameters of up to 

240 nm in the fetal perfusion medium (data 

not shown). This confirms the results of the fluorescence measurements that beads of around 

240 nm were able to cross the human placenta.

Viability of placental tissue after perfusion.

The presence of PS beads in the placental tissue did not affect the viability of the placental 

explant, because the consumption of glucose 

and the production of lactate, human chorionic gonadotropin, and leptin were not altered 

(Figure 4). In addition, structural analysis of 

the explant after perfusion showed no alterations of the tissue, cells, or subcellular structures (data not shown).

Discussion

The main findings of this study are that PS 

beads up to a diameter of 240 nm were taken 

up by the placenta and, further, were able to 

cross the placental barrier without affecting 

the viability of the explant. We used fluorescently labeled negatively charged PS beads of 

50, 80, 240, and 500 nm as model particles 

because of their known biocompatibility and 

easy detection. This study provides evidence

that the translocation of these beads across the 

placenta is size dependent.

We applied the particles at a single dose and 

at a high concentration, which can be assumed 

to be more representative of an intravenous 

injection, as reported for glioblastoma treatment with magnetic nanoparticles (Thiesen and 

Jordan 2008), than an environmental exposure 

scenario. With the ex vivo perfusion model, the 

mechanism of nanoparticle transport through 

the placenta very close to the in vivo situation

can be studied, but it is limited to a perfusion 

period of a few hours: Chronic treatments with 

low doses over a long period are not possible 

Figure 3. Size-dependent barrier capacity of the 

placental tissue. The ratios between fetal (F) and 

maternal (M) concentrations of 14C-antipyrine (open 

bars) and PS beads (blue bars) were calculated 

after 180 min of perfusion. The 14C-antipyrine values 

remain unchanged, whereas the perfusion rate of 

the beads showed size dependence. Data represent 

mean ± SE of at least four independent experiments. 

*p < 0.05 compared with 240-nm ratio value; **p < 0.05

compared with 80-nm ratio value; #p < 0.05 compared with 

50-nm ratio value.

0.75

0.50

0.25

0 Control

F/M ratio after 3 hr 

of perfusion

50 nm 80 nm 240 nm 500 nm

#

*

#

**

Figure 4. Viability and functionality of the placenta 

were not affected after perfusion with PS beads. 

(A) Glucose consumption and lactate production 

in the perfused tissue. (B) Human choriongonadotropin and leptin hormone net production during 

perfusion [NP divided by initial tissue content (T0)]. 

Data represent mean ± SE of at least four independent experiments.

0.20

0.15

0.10

0.05

0.00

400

200

0

Control

μmol/g/min NP/TO (%)

50 nm 80 nm 240 nm 500 nm

Control 50 nm 80 nm 240 nm 500 nm

Glucose consumption Lactate production

Human choriongonadotropin Leptin
Wick et al.

436 volume 118 | number 3 | March 2010 • Environmental Health Perspectives

because of tissue degradation (Panigel et al. 

1967; Schneider et al. 1972). A further limitation of the model is that the perfusion rate 

measured in the explant represents only the late 

phase of pregnancy, for which the thickness of 

the barrier layer between the maternal and the 

fetal circuit is reduced and the number of fetal 

capillaries is increased (Enders and Blankenship 

1999). Thus, the perfusion rates obtained with 

the ex vivo perfusion model may be higher than 

during the greater part of pregnancy in vivo.

The consideration of the mass concentration of nanoparticles is often criticized, and 

instead surface concentration or particle number concentration are suggested as a model 

(Stoeger et al. 2006). In the present study we 

intentionally selected a highly biocompatible 

nanomaterial to specifically examine the barrier 

capacity of the placenta without inducing toxic 

side effects. Thus, in this particular case, particle 

surface–dependent or mass-dependent toxicities 

play minor roles. Nevertheless, all relevant particle characteristics are summarized in Table 1 

for later comparison and interpretation.

Possible transport routes for nanoparticles 

across the placenta are diffusion, vesicular transport, transmembranal transporter proteins, and 

the transtrophoblastic channel system. The perfusion profiles obtained for the 50- and 80-nm

beads could be interpreted to suggest that these 

particles cross the placenta very quickly by simple diffusion. If so, the second application of 

PS beads should lead to a continuous translocation of the beads, which is not the case. The 

high doses applied could have induced dramatic clogging of the intervillous space on the 

maternal side, such that transfer routes were 

nearly blocked. Vesicle transport such as clathrin- or caveolin-mediated uptake is likely for the 

smaller PS particles because of their restricted 

size of 60 nm for clathrin-coated vesicles and 

120 nm for caveolin-coated vesicles but rather 

questionable for bigger spheres of 240 nm 

(for comprehensive reviews, see Conner and 

Schmid 2003; Hillaireau and Couvreur 2009). 

The extent to which placenta transporter proteins in the basal- or brush-border membrane 

(e.g., of the syncytiotrophoblast) are involved in 

the nanoparticle transport remains unclear. The 

transtrophoblastic channel system represents a 

continuous membrane-lined tubular structure 

of about 20 nm in diameter that connects the 

intervillous space with the stroma and may act 

as a pressure-dependent valve regulating fetal 

water balance (Kertschanska et al. 1997). If 

the arterial pressure exceeds 80 cm H2O (corresponding to around 60 mmHg), this channel system is dilated, allowing the transport of 

liquid and small molecules. Whether this is true 

for the PS beads in our perfusion experiments 

requires further detailed investigations.

The critical size for PS nanoparticles to 

cross a placental barrier seems to be consistent with observations made at the air–blood 

barrier, where fluorescent PS beads of around 

200 nm diameter penetrated cells in an energyindependent way (Geiser et al. 2005). But the 

capability of nanomaterials to cross the placenta 

appears to depend not only on particle size but 

also on the material composition or surface 

coating. It has been shown that polyethylene 

glycol–coated gold particles up to 30 nm in 

diameter were not able to cross the placenta 

with the same experimental setup as used in the 

present study (Myllynen et al. 2008). The fact 

that PS beads and gold nanoparticles interact 

differently with biological compounds suggests 

that, in general, the capability for transplacental transfer mechanism(s) has to be assessed 

separately for each type of nanoparticle.

Conclusion

Our study showed a clear size-dependent barrier capacity of a healthy human placenta for 

PS nanoparticles, including an analysis of the 

viability and functionality of the tissue after 

the perfusion. We suggest that nanoparticles, 

in general, therefore have the potential for 

transplacental transfer. This potential underscores the need for further nanotoxicologic 

studies on this important organ system.

References

Alfaro-Moreno E, Nawrot TS, Nemmar A, Nemery B. 2007. 

Particulate matter in the environment: pulmonary and cardiovascular effects. Curr Opin Pulm Med 13(2):98–106.

Challier JC. 1985. Criteria for evaluating perfusion experiments and presentation of results. Contrib Gynecol Obstet 

13:32–39.

Conner SD, Schmid SL. 2003. Regulated portals of entry into the 

cell. Nature 422(6927):37–44.

Enders AC, Blankenship TN. 1999. Comparative placental structure. Adv Drug Deliv Rev 38(1):3–15.

Ganapathy V, Prasad PD, Ganapathy ME, Leibach FH. 2000. 

Placental transporters relevant to drug distribution across 

the maternal-fetal interface. J Pharmacol Exp Ther 

294(2):413–420.

Geiser M, Rothen-Rutishauser B, Kapp N, Schurch S, Kreyling W, 

Schulz H, et al. 2005. Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells. Environ Health Perspect 113:1555–1560.

Hillaireau H, Couvreur P. 2009. Nanocarrier’s entry into the cell: 

relevance to drug delivery. Cell Mol Life Sci 66:2873–2896.

Kanno S, Furuyama A, Hirano S. 2007. A murine scavenger 

receptor MARCO recognizes polystyrene nanoparticles. 

Toxicol Sci 97(2):398–406.

Kertschanska S, Kosanke G, Kaufmann P. 1997. Pressure 

dependence of so-called transtrophoblastic channels during fetal perfusion of human placental villi. Microsc Res 

Tech 38(1–2):52–62.

Kreyling WG, Semmler M, Moller W. 2004. Dosimetry and toxicology of ultrafine particles. J Aerosol Med 17(2):140–152.

Lacasana M, Esplugues A, Ballester F. 2005. Exposure to ambient air pollution and prenatal and early childhood health 

effects. Eur J Epidemiol 20(2):183–199

Latzin P, Roosli M, Huss A, Kuehni CE, Frey U. 2008. Air pollution during pregnancy and lung function in newborns: a 

birth cohort study. Eur Respir J 33(3):594–603

Malek A, Obrist C, Wenzinger S, von Mandach U. 2009. The 

impact of cocaine and heroin on the placental transfer of 

methadone. Reprod Biol Endocrinol 7:61–70.

Malek A, Sager R, Schneider H. 1994. Maternal-fetal transport of 

immunoglobulin G and its subclasses during the third trimester of human pregnancy. Am J Reprod Immunol 32(1):8–14.

Malek A, Sager R, Willi A, Muller J, Hanggi W, Leiser R, et al. 

2000. Production of protein hormones by cultured trophoblast cells isolated from term and early placentae. Am J 

Reprod Immunol 43(5):278–284.

Malek A, Willi A, Muller J, Sager R, Hanggi W, Bersinger N. 

2001. Capacity for hormone production of cultured trophoblast cells obtained from placentae at term and in early 

pregnancy. J Assist Reprod Genet 18(5):299–304.

Myllynen PK, Loughran MJ, Howard CV, Sormunen R, Walsh AA, 

Vahakangas KH. 2008. Kinetics of gold nanoparticles in the 

human placenta. Reprod Toxicol 26(2):130–137.

Nemmar A, Al-Maskari S, Ali BH, Al-Amri IS. 2007. Cardiovascular and lung inflammatory effects induced by systemically administered diesel exhaust particles in rats. Am 

J Physiol Lung Cell Mol Physiol 292(3):L664–L670.

Nemmar A, Hamoir J, Nemery B, Gustin P. 2005. Evaluation of 

particle translocation across the alveolo-capillary barrier in isolated perfused rabbit lung model. Toxicology 

208(1):105–113.

Nemmar A, Melghit K, Ali BH. 2008. The acute proinflammatory and prothrombotic effects of pulmonary exposure 

to rutile TiO2 nanorods in rats. Exp Biol Med (Maywood) 

233(5):610–619.

Oberdörster G, Utell MJ. 2002. Ultrafine particles in the urban 

air: to the respiratory tract—and beyond? Environ Health 

Perspect 110:A440–A441.

Panigel M, Pascaud M, Brun JL. 1967. Radioangiographic study 

of circulation in the villi and intervillous space of isolated human placental cotyledon kept viable by perfusion

[Letter] [in French]. J Physiol (Paris) 59(1 suppl):277. 

Pietzonka P, Rothen-Rutishauser B, Langguth P, WunderliAllenspach H, Walter E, Merkle HP. 2002. Transfer of lipophilic markers from PLGA and polystyrene nanoparticles 

to caco-2 monolayers mimics particle uptake. Pharm Res 

19(5):595–601.

Reddy ST, van der Vlies AJ, Simeoni E, Angeli V, Randolph GJ, 

O’Neil CP, et al. 2007. Exploiting lymphatic transport and 

complement activation in nanoparticle vaccines. Nat 

Biotechnol 25(10):1159–1164.

Rothen-Rutishauser BM, Schurch S, Haenni B, Kapp N, Gehr P. 

2006. Interaction of fine particles and nanoparticles with 

red blood cells visualized with advanced microscopic 

techniques. Environ Sci Technol 40(14):4353–4359.

Rudin M, Weissleder R. 2003. Molecular imaging in drug discovery and development. Nat Rev Drug Discov 2(2):123–131.

Schneider H, Panigel M, Dancis J. 1972. Transfer across the 

perfused human placenta of antipyrine, sodium and leucine. Am J Obstet Gynecol 114(6):822–828.

Semmler-Behnke M, Fertsch S, Schmid G, Wenk A, 

Kreyling WG. 2007. Uptake of 1.4 nm versus 18 nm gold 

nanoparticles in secondary target organs is size dependent 

in control and pregnant rats after intratrecheal or intravenous application. In: EuroNanoForum 2007—Nanotechnology in Industrial Applications. Luxembourg:European 

Communities, 102–104.

Stoeger T, Reinhard C, Takenaka S, Schroeppel A, Karg E, 

Ritter B, et al. 2006. Instillation of six different ultrafine carbon particles indicates a surface area threshold dose for 

acute lung inflammation in mice. Environ Health Perspect 

114:328–333.

Syme MR, Paxton JW, Keelan JA. 2004. Drug transfer and 

metabolism by the human placenta. Clin Pharmacokinet 

43(8):487–514.

Takata K, Fujikkura K, Shin BC. 1997. Ultrastructure of the 

rodent placental labyrinth: a site of barrier and transport. 

J Reprod Dev 43(1):13–24.

Thiesen B, Jordan A. 2008. Clinical applications of magnetic nanoparticles for hyperthermia. Int J Hyperthermia 24(6):467–474.