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

# Research

Humans have always been exposed to air

borne particles through natural sources such as volcanoes, forest fires , or desert dust. After the Industrial Revolution in the 19 th cen tury, 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 UFP s on human health (Kreyling et al. 2004; Oberdörster and Utell 2002). When inhaled, UFPs are depos

ited in the respiratory tract and may provoke inflammation or granuloma formation in the lung and induce systemic effects such as pro thrombotic 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 treatment s (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 expo

sure to nanoparticles during pregnancy or early childhood (Lacasana et al. 2005). A recent ly performed cohort study suggests that prena

tal exposure to air pollution might be associ

ated 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 pla

centa or by mediators induced in the maternal circulation such as pro inflammatory 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 extrap

olated 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 syncy

tium with no lateral cell membranes, whereas in rats or mice three trophoblast layers are pres

ent between maternal blood and fetal blood capillaries ( for comprehensive review s, see Enders and Blankenship 1999 ; Takata et al. 1997). Thus, the transport efficiency for xeno

biotics 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). Th is 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 capillar

ies 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 dif

fusion, active transport, biotransformation through metabolic enzymes, and phagocytosis or pinocytosis (Syme et al. 2004). The plasma membrane of the syncytiotrophoblast is polar

ized, consisting of the brush border mem

brane 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 bor

der 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 manu

script, 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 Mandach 2

1 Empa, Swiss Federal Laboratories for Material Testing and Research, Laboratory for Materials–Biology Interactions, St. Gallen, Switzerland; 2 University Hospital Zurich, Department of Obstetrics, Zurich, Switzerland; 3 Institute of Pathology, Cantonal Hospital, St. Gallen, Switzerland

B ackground : Humans have been exposed to fine and ultrafine particles throughout their history. Since the Industrial Revolution, sources, doses , and types of nanoparticles have changed dramati

cally. 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 engi

neered 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. O b jective : In this study we investigated whether particles can cross the placental barrier and affect the fetus. M ethods : We used the ex vivo human placental perfusion model to investigate whether nanopar ticles can cross this barrier and whether this process is size dependent. Fluorescently labeled polysty

rene beads with diameter s of 50, 80, 240 , and 500 nm were chosen as model particles. R

esults : 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. C onclusions : The findings suggest that nanomaterials have the potential for transplacental transfer and underscore the need for further nanotoxicologic studies on this important organ system. K

ey 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 transporter s 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 re circulation 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 pharma

cokinetic 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 fluo

rescently 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 avail

able 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 ana

lyze 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 con

centration of about 10 6 to 10 9 particle s /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 com pared 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% etha

nol , and perfusion medium to a concentra

tion of 10 μg/mL and incubated for 24 h r at 37°C. The fluorescence of 100 μL suspen

sion was measured before and after filtra

tion 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 zetame

ter (Malvern Instruments Ltd, Malvern, Worcestershire, UK). First, PS beads were dis

persed 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 per

fusion medium for 10 min. Ex vivo dual re circulating human pla

cental perfusion model. The placental perfu

sion technique was originally developed by Panigel et al. ( 1967) and was further improved by Schneider et al. ( 1972). The dual re cir

culating perfusion was performed according to Malek et al. ( 2009). Intact placentas were obtained from uncomplicated term pregnan

cies 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 placen

tas in this study. To minimize damage, the placentas were placed in 200 mL of a 0.9% sodium chloride solution pre heated to 37° C, and then transported in a Styrofoam box to the research laboratory within 20 min of deliv

ery. 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 decid

ual plate. Furthermore, a venous drain, con

nected 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% N 2 and 5% CO 2 for the fetal circuit , and one with 95% air and 5% CO 2 for the mater

nal 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 ( pre phase) 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 pre phase, the maternal perfusate was exchanged with a perfusate containing PS beads of 50, 80, 240 , or 500 nm , respectively , at a final concen

tration of 25 μg/mL and 14 C-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 ) 14 C-antipyrine values: equilibrium between maternal and fetal circuit after 4–6 h r

(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 placen

tal tissue. We evaluated the permeability of the placenta by monitoring the transplacen

tal transfer of 14 C- antipyrine as described pre

viously (Challier 1985). Briefly, 50 nCi/mL 14 C-antipyrine were added to the maternal circuit, and 300 μL of the perfusion medium from each reservoir was transferred into sepa

rate scintillation tubes and mixed with 3 mL scintillation cocktail (Irgasafe Plus Scintillation Cocktail, Zinsser Analytic, Frankfurt, Germany). 14 C 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 cho

rionic gonadotropin using an enzyme-linked immunosorbent assay as described previously (Malek et al. 2000, 2001). Briefly, the net pro

duction (NP) of human choriongonadotropin or leptin was estimated as NP = ( end tissue content minus initial tissue content ) plus accu mulation. To determine the metabolic activity of the placenta, we used the sum of changesWick 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 cal

culate glucose consumption or lactate produc

tion, 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 cir

cuit after perfusion. A 1. 5- mL sample of the fetal perfusion medium was taken after 6 h r 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 fluores

cent 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 2 00 40 0 6 00 **Size (nm)** 80 0 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 14 C-antipyrine. After the pre phase , PS beads of different sizes were added to the maternal circuit to a final concen

tration of 25 μg/mL and the amount of 14 C-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 14 C-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** **( 14** **C counts/ 0.3 mL)**

0 6 0 120 180 240 300 360 **T ime (min)**  0 6 0 120 180 240 300 360 **T ime (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 (nm 2 ) c 1.88E+03 5.41E+03 4.52E+04 2.00E+05 Internal volume (nm 3 ) c 6.16E+04 2.99E+05 7.24E+06 6.70E+07 Density of PS (mg/nm 3 ) 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 PM c 3.87E+11 7.95E+10 3.29E+09 3.55E+08 PS particle surface/mL PM (nm 2 ) 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.
a According to manufacturer’s information

. b Experimentally determined (mean ± SE). c Calculated values

. d From 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 trans mission electron microscopy ( TEM ) analysis

. Statistical analysis. Data are shown as mean ± S E 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 con

sidered to be statistically relevant. 

### Results PS particle characterization. Table 1 sum

marizes detail s 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 perfu

sion medium. Size distribution analysis con

firmed that suspensions of 50 -nm , 80 -nm, and 240 -nm beads were relatively monodisperse (Figure 1A–C , E). However, we observed par ticles 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, espe

cially of PS beads, has been shown to cause experimental problems because of a possible transfer of the lipophilic dye to the surround

ing tissue and/or cells (Kanno et al. 2007; Pietzonka et al. 2002). To exclude false- positive results , we measured the stability of the fluores

cent 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 condition s, includ

ing our perfusion medium (data not shown). We experimentally determined the detec

tion 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 fluo

rescent 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 14 Cantipyrine , which is known to rapidly cross the placenta and reach equal concentration in the maternal and fetal circuit after 4

– 6 h r (Challier 1985) ( Figure 2 B). If we started the perfusion with 25 μg/mL 50 -nm PS beads in the mater

nal 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 adminis

tration of the same amount of beads to test whether the number of beads in the fetal cir

cuit could be further increased. Similar to the first injection of PS beads, we observed a gradual decrease of the particles in the mater

nal perfusion medium but detected no sig

nificant 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 circula

tion 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 par

ticles < 500 nm in diameter (Figure 1D). We used the ratio of fetal-to-maternal concentra

tion of PS beads detected after 180 min to esti

mate 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 14 C-anti

pyrine (Figure s 2 B, 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 flu

orescence 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 tis

sue did not affect the viability of the placental explant, because the consumption of glucose and the production of lactate, human chori

onic gonadotropin , and leptin were not altered ( Figure 4 ). In addition, structural analysis of the explant after perfusion showed no altera

tions of the tissue, cells , or subcellular struc

tures (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 fl uores cently 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 treat

ment 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 ) concentration s of 14 C-antipyrine (open bars) and PS beads ( blue bars) were calculated after 180 min of perfusion. The 14 C-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 choriongonado

tropin and leptin hormone net production during perfusion [NP divided by initial tissue content (T0)]

. Data represent mean ± SE of at least four indepen

dent 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 LeptinWick 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 limi

tation 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 concentra

tion of nanoparticles is often criticized, and instead surface concentration or particle num

ber 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 par

ticle characteristics are summarized in Table 1 for later comparison and interpretation. Possible transport routes for nanoparticles across the placenta are diffusion, vesicular trans

port, transmembranal transporter proteins , and the transtrophoblastic channel system. The per

fusion profiles obtained for the 50

- and 80 -nm beads could be interpreted to suggest that these particles cross the placenta very quickly by sim

ple diffusion. If so, the second application of PS beads should lead to a continuous translo

cation of the beads, which is not the case. The high doses applied could have induced dra

matic clogging of the intervillous space on the maternal side, such that transfer routes were nearly blocked. Vesicle transport such as clath

rin- 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 review s, see Conner and Schmid 2003; Hillaireau and Couvreur 2009). The extent to which placenta transporter pro

teins 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 H 2 O (cor

responding to around 60 mmHg), this chan

nel 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 consis

tent with observations made at the air–blood barrier , where fluorescent PS beads of around 200 nm diameter penetrate d 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 transplacen

tal transfer mechanism(s) has to be assessed separately for each type of nanoparticle. 

### Conclusion Our study showed a clear size-dependent bar

rier 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 under

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