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Neutron reflectometry characterization of PEI–PSS polyelectrolytemultilayers for cell culture

Saurabh Singh,†*a Ann Junghans,†a Mary J. Waltman,b Amber Nagy,b Rashi Iyerb and Jaroslaw Majewski*a

Received 20th June 2012, Accepted 31st August 2012

DOI: 10.1039/c2sm26433a

UsingNeutron Reflectometry (NR), polyelectrolyte multilayer (PEM) films made by layer-by-layer (LbL)

deposition of a strong polycation (polyethylene imine [PEI]) and a polyanion (polystyrene sulfonate [PSS])

have been characterized. PEI terminated sampleswith a total of 5, 7, and 9 layersweredeposited on aquartz

substrate and studied under three different environmental conditions (i.e., dry air, 100% relative humidity,

and bulk water). We were able to model all the measurements at three different contrast conditions using

one simple, physically reasonable and consistent model, which led to a firm understanding of the structure

of the systems.ThePEMthicknesswas found to vary linearlywith thenumberof layersdeposited.Thinfilm

structures formedusing theLbLmethodwere constitutedof twodistinctive regions, i.e., the bottomand top

strata. When measured in dry air and D2O vapors, the �30 to 50 �A thick bottom stratum was found to

consist of loosely packed polymers (i.e. 30% polymer by volume). This region could have resulted from an

island type of deposition during the initial stages of LbL assembly. In contrast, the thickness of the top

strata, which consisted of densely packed polymers (i.e. 100% polymer by volume when measured in dry

air), was found to vary linearly with the number of layers deposited. Upon exposure to D2O saturated

vapors, it was observed that the top and bottom strata absorbed significant quantities of heavy water,

accompanied with PEM swelling. We estimated that in this case, the top strata comprise ca. 57% (v/v)

polymer and 43% (v/v) D2O for 7- and 9-layered samples. No further swelling of the PEM samples was

observed when they were exposed to bulk D2O. Nevertheless, the entire polymeric system underwent a

rearrangement leading towards the homogenization of the multilayered structure, suggested by the

decreased scattering contrast between the top and bottom strata. We also performed studies to assess the

cytocompatibility of 7-layered PEM structures. Two different cell types, fibroblasts (3T3) and human

embryo kidney cells (HEK-293), were seeded on the polyelectrolyte multilayer, and the cell coverage was

monitored by optical microscopy at varying times. Our observations confirmed that cells adhered and

spreadonPEMsubstrates, which showedno signof immediate toxicity. Therefore, suchmultilayers proved

to be a suitable support for 3T3 and HEK-293 cell growth.

1. Introduction

Since the pioneering work of Iler, Decher and Loesche et al.,1–3

thin polyelectrolyte multilayer (PEM) films deposited by the

layer-by-layer (LbL) self-assembly technique have been employed

for a wide variety of applications, such as targeted drug delivery,

water desalination, and in biosensors.4–6 The LbL self-assembly

technique enables the deposition of ultrathin films by a sequential

deposition driven by electrostatic interactions of charged poly-

mers, nanoparticles, biological templates, or biologically active

species.7An inherently charged substrate is consecutively exposed

to solutions of oppositely charged species (i.e., polycations and

aManual Lujan Jr Neutron Scattering Center, Los Alamos NationalLaboratory, NM 87545, USA. E-mail: jarek@lanl.gov; saurabh@lanl.govbBioscience Division, Los Alamos National Laboratory, NM 87545, USA

† These authors contributed equally to this work.

11484 | Soft Matter, 2012, 8, 11484–11491

polyanions), which adsorb to the developing film at rates that

enable nanometer-scale control of film thickness.2 The possibility

to combine different polycations and polyanions extends the

number of applications where such thin films can be used. LbL

films can be made biocompatible by terminating the outermost

layer, also termed as the capping layer, with a stealth polymer

such as polyethylene glycol (PEG).8 The facile deposition of

polyelectrolyte nanofilms, the ability to control the thickness with

nanometer precision, and the possibility of tailoring the surface

properties (e.g., wettability, surface charge, roughness, etc.) make

such films highly attractive for biomedical and other applications.

For instance, PEMs have been employed in the fabrication of

implantable glucose sensing beads that can be used to continu-

ously monitor blood glucose levels in diabetics.5,6,9 In another

case, for targeted drug delivery, the outer surface of a therapeutic

drug containing microsphere was coated with an arginine–

glycine–aspartic acid (RGD) grafted polyelectrolyte – the RGD

This journal is ª The Royal Society of Chemistry 2012

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sequence has a high affinity towards integrin which is overex-

pressed in tumor/cancer cells.10

While successful LbL deposition has been demonstrated using

a wide variety of materials (e.g., polymers, proteins, DNA,

nanoparticles, etc.), in this work, we focus on polymeric systems.

Depending on the strength of the ionization of the components

(i.e. polycations and polyanions), such films can be prepared by

employing any desired combination of strong–strong, weak–

strong, and weak–weak interactions.11 Note that ‘‘strong poly-

cations’’ refer to polyelectrolytes that exist in a ‘‘protonated’’

state, even at extremely high pH values. Likewise, ‘‘strong poly-

anions’’ refer to polymers that can exist in a ‘‘de-protonated’’

state, even at extremely low pH values.

Techniques such as Quartz Crystal Microbalance (QCM),

ellipsometry, and UV-vis spectroscopy have been extensively

used in the past to confirm the structure of deposited multilayers.

However, they are frequently unable to provide information

regarding the layer intermixing, swelling and change in the

roughness under varying environmental conditions, including

hydration. Such information will be very useful in the design of

bio-implantable devices whose surfaces are modified by the LbL

method. For example, it has been proven that protein adsorp-

tion, which is followed by cell adhesion, is significantly influenced

by surface roughness.12

Neutron Reflectometry (NR) is a very effective technique for

studying the structures and properties of thin layers on solid

support, especially polymeric systems.2,13,14 The method has been

used to characterize PEMs deposited using a weak polycation

(poly(allyl amine) hydrochloride [PAH]) and a strong polyanion

(PSS).13,14 Nevertheless, swelling and roughness characteristics of

the LbL deposited system resulting from PEI and PSS have

hitherto not been reported. In this work, using NR, we have

investigated LbL deposited structures formed by a strong poly-

cation (polyethylene imine [PEI]) and a strong polyanion (PSS).

In addition, we performed studies to assess the cytocompatibility

of PEI–PSS multilayers.

It is well established that cell lines and primary cultures benefit

from the use of positively charged extracellular matrix proteins

or polymers that enhance their ability to attach to culture

plates.15,16 In recent years, PEI has gained attention as a trans-

fection reagent and an attachment factor.17,18 For example, PEI

coating is used to passivate negatively charged plastic/glass

surfaces to enhance cell attachment, since the outermost cell

membrane is negatively charged. It has been demonstrated for

two commonly used cell lines, MSY and HEK-293, that both

adhere very strongly to PEI-coated surfaces. Although MSY

cells (primary fibroblasts) can also strongly adhere to unmodified

surfaces, the HEK-293 (human embryo kidney) cells require PEI

modified surfaces for healthy cell growth and differentiation.18

Multilayered films deposited using the LbL method exhibit very

good pH and thermal stability for up to 200 �C.14,19 Therefore,this technique can be employed to obtain robust coatings.

Fig. 1 Structural formulas of the polyions used in the studies.

2. Experimental

2.1. Chemicals

Branched PEI (50% w/v in H2O) with a molecular weight of

750 kDa and PSS (powder form) of molecular weight 70 kDa

This journal is ª The Royal Society of Chemistry 2012

were purchased from Sigma Aldrich. The molecular structures of

the polyions used are depicted in Fig. 1. NaCl (99.5%) was

obtained from Fischer Scientific. All pH adjustments were per-

formed using 1 M HCl and NaOH (Fischer Scientific). D2O

(99.8% D) used in the NR experiments was obtained from

Acros�. Ultrapure DI water with a resistivity of 18.2MU cmwas

used throughout our studies.

Single crystal 300 dia. quartz (Institute of Electronic Materials

Technology, Warsaw, Poland) with a thickness of 0.50 0 was usedas a substrate for LbL film deposition. Surface roughness of the

quartz substrate was determined to be less than 5 �A using NR.

The quartz substrates were rinsed with a CHCl3 and MeOH

solution, followed by cleaning with a piranha solution

(H2SO4 : H2O2, 3 : 1) for 6 hours, and subsequently rinsed with

copious amounts of DI water. Finally, the substrates were

exposed to UV radiation for 20 minutes.

2.2. Neutron reflectometery

NRmeasurements were performed at the Surface Profile Analysis

Reflectometer (SPEAR) beam line at the Los Alamos Lujan

Neutron Scattering Center.20 The neutron beam is produced from

a spallation source and, after moderation by liquid H2, is directed

onto the sample at a very low angle while the specular reflection is

recorded by a time-of-flight (ToF), position-sensitive detector.

Reflectivity is defined as the ratio of the intensity of the reflected

beam to the incident beam, and is a function of the neutron

momentum transfer vectorQz, whereQz¼ 4psin(q)/l. q is the angle

of incidence of the beam, and l is the wavelength of the neutron.

Analysis of reflectivity vs. Qz enables development of the scat-

tering length density (SLD) distribution along normal to the

sample surface. SLD is a function of chemical composition and

density of the material. The SLD profile obtained from NR

measurements provides the structural information at very high

spatial resolution (<1nm). Specifications ofNRmeasurements and

modeling of the data have been detailed elsewhere.20,21Modeling of

the SLDwas performed using an open-source reflectivity package,

MOTOFIT, which runs in the IGOR Pro environment.22 MOTO-

FIT approximates the continuous SLD function by the number of

layerswith constant SLDs.An error function centered between two

adjacent interfaces is used to address the interfacial roughness. A

theoretical reflectometry curve can be calculated using the Abeles

matrix formalism.23 Both genetic optimization and Levenberg–

Marquardt nonlinear least-square methods were employed to

obtain the best fits for the NR data.

2.3. PEI–PSS multilayered films

The polyelectrolyte films were deposited using the LbL tech-

nique, detailed elsewhere.2 Both polycation and polyanion

Soft Matter, 2012, 8, 11484–11491 | 11485

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solutions were prepared by dissolving 2 mg mL�1 of respective

polyelectrolytes in DI water with 0.5 M NaCl. Finally, the pH

of PEI and PSS solutions was adjusted to 6.0 and 7.0, respec-

tively. Each deposition step lasted for ca. 10 minutes followed

by copious rinsing with DI water and drying under a N2 stream

after each step. Samples with 3 different total layer thicknesses

were prepared: (1) 5 layers [PEI–PSS]2–PEI, (2) 7 layers [PEI–

PSS]3–PEI, and (3) 9 layers [PEI–PSS]4–PEI. NR data for the

samples were collected in dry air, followed by the measurements

in D2O saturated vapors. Finally, NR measurements were

performed for LbL films in contact with bulk D2O.24 All

measurements were executed at room temperature. Measure-

ments with deuterated PSS (dPSS) intercalated in the LbL

structure were also performed, showing similar layer thickness

as well as high intermixing between layers, especially

pronounced in bulk liquids.

2.4. Cell culture

3T3 fibroblasts were purchased from the American Type Culture

Collection (ATCC) and maintained at 37 �C under a 5% CO2

atmosphere. Fibroblasts were propagated in T150 cm2 flasks

(Corning) using high glucose Dulbecco’s Modified Eagle’s Media

(DMEM, Hyclone) supplemented with 10% fetal calf serum

(FCS/Hyclone) and 1% penicillin/streptomycin (Gibco), until

�70 to 80% confluent. Cells were passaged weekly by trypsini-

zation and maintained by replacing culture media with fresh

media every 3 to 4 days.

HEK-293, human embryonic kidney cells (ATCC, CRL-

1573), were maintained at 37 �C under a 5% CO2 atmosphere and

were cultured in T75 cm2 flasks (Corning) in MEM-GlutaMAX

(Gibco), supplemented with 10% fetal bovine serum (Hyclone)

and 1% penicillin/streptomycin solution (Gibco). Cells at 60–70%

confluence were harvested and maintained by changing culture

media every 3–4 days.

Both cell cultures were harvested by trypsinization using

Lonza cell culture reagent packs. Briefly, cells were rinsed with

HEPES buffer (Sigma-Aldrich), and 1 to 3 mL of trypsin was

added to the cultures. Flasks were incubated at 37 �C in an

atmosphere of 5%CO2. Upon cell detachment (�5 minutes), cells

were collected and an equal amount of a trypsin neutralization

buffer was added. Cells were collected and centrifuged at 200� g

for 5 minutes. The media were aspirated and cells were resus-

pended in fresh media prior to counting using a Z2 Coulter

Counter.

Approx. 0.4 � 106 cells suspended in cell culture media were

seeded on a 30 0 dia. quartz crystal covered with [PEI–PSS]3–PEI

(7 layers). Subsequently, the crystal was placed in a cell culture

dish. To enable maximal adherence of cells, seeded cells were

allowed to adhere for 60 minutes prior to transfer to the incu-

bator (5% CO2 in air at 37 �C). After each microscopy imaging,

which took less than 10 minutes, the quartz crystals were

returned to the incubator.

Images were acquired using a Zeiss Axiophot microscope

with 10� magnification after 5, 25, 49, and 73 hours. Cell

surface coverage was calculated in Image J using the Thresh-

old_Colour Plugin by Gabriel Landini and the in-built particle

analyzer. The values are represented as the mean � standard

deviation.25

11486 | Soft Matter, 2012, 8, 11484–11491

3. Results and discussion

3.1. NR measurement of polyelectrolyte layers in dry air

NR profiles for 5, 7, and 9-layered samples in dry air are shown

in Fig. 2(A). Loesche et al., using PAH–deuterated-PSS polyions,

reported that the roughness between the adjacent layers is

around half of the thickness of a layer pair,13 which implies high

degree of interdigitation. Because of intermixing, we tried

modeling our data using a single stratummodel, where stratum is

defined as a region of constant SLD. However, such models did

not result in good fits. The simplest and most physically

reasonable model that fit the data for 5, 7, and 9-layered samples

consisted of two strata and resulted in lowest c2 values. The

obtained SLD profiles from the two strata models are shown in

Fig. 2(B). Previous research on LbL structures has shown that

the initial 3–4 layers exhibit an island type of deposition.26,27

It is not clear why LbL deposition results in a two strata

architecture. It is possible that the ‘‘bottom stratum’’ is formed

due to the island type of deposition of polymers in the initial

stages of LbL assembly (1–4 layers). This could be due to the

defects present on the quartz surface that carry additional

charges, thus strongly interacting with the first deposited poly-

electrolyte. As a result, the surface morphology changes,

exposing more charged binding sites of the first polymer to the

polyelectrolyte in the next deposition. This process of charge

over-expression is saturated after 3–4 layers and, subsequently,

more contiguous depositions are obtained.

QCM-D and ellipsometry measurements of two different

types of PEMs, constructed from PAH–PSS and poly-

(diallyldimethylammonium chloride) [PDDA]–PSS,27 found the

existence of two distinct regions. Similar to our studies, it was

discovered that the first few deposited layers exhibited higher

hydration than the layers deposited at later stages of LbL

assembly. For example, for PAH–PSS PEMs, the hydration was

found to be maximum for the first layer and smoothly decreased

as the number of deposited layers increased, reaching a satura-

tion after �4 layers.

In our models, these 3–4 layers are termed as ‘‘bottom strata’’,

which are in contact with the quartz crystal. Typically, the layers

deposited after initial 3–4 layers are more contiguous and exhibit

uniform layer thickness. In our model, this has been referred to

as ‘‘top strata’’.

Due to the limited Qz range (�0.16 �A�1) of our NR experi-

ments, the change in the interfacial roughness did not have a

significant effect on our fits. Therefore, it was constrained to 5 �1�A, which is similar to the roughness of the quartz substrate. The

roughness between the top strata and the air interfaces was found

to have an effect on the fits, and therefore was left open for

optimization for 9-layered samples. Since we know that after 3–4

layers, each subsequent layer results in contiguous and uniform

deposition of the polyelectrolyte, we fixed the film–air roughness

for 5- and 7-layered samples to the value that was obtained for

the 9-layered sample.

Fit parameters for the two strata model for PEM samples

consisting of 5, 7, and 9 layers are shown in Table 1. For 3

different samples, the thickness of the bottom strata was found to

vary in a narrow range from 38 to 48�A. In contrast, the thickness

of the top strata varied linearly with the number of layers. The

This journal is ª The Royal Society of Chemistry 2012

Fig. 2 Reflectivity (R vs. Qz) profiles for LbL deposited thin films (A) and SLD profiles (B) for 5, 7, and 9-layered samples measured in dry air. The total

PEM thickness vs. number of layers (n > 4) is shown in the inset. The error bars denote the standard deviation for each measurement.

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total thickness of the PEM structure (top + bottom strata) was

linearly related to the number of deposited layers (inset in

Fig. 2(B)). The SLD of the bottom stratum was ca. 0.0 �A�2,

which indicated a substantial amount of H2O trapped within it.

On the other hand, the SLD of the top strata ranged between

1.10 � 10�6 and 1.25 � 10�6 �A�2. We note that LbL assembly

was performed in an aqueous medium, and post-film deposition,

the samples were dried under a mild stream of N2 for ca. 5

minutes.

Depending on the deposition conditions (i.e. electrolyte and

buffer concentrations), densities of PAH–PSS multilayers have

been reported to range from 0.72 to 1.14 g cm�3.28 Assuming that

the densities of PEI and PSS are 1 g cm�3, the calculated SLD

values are 0.37 � 10�6 (bPEI) and 1.55 � 10�6 �A�2 (bPSS),

respectively.29 If the top stratum does not contain any H2O when

measured in air and its density is 1 g cm�3, we can use the

following equation to estimate the volume fraction, a, of PEI:

btop,exp ¼ abPEI + (1 � a)bPSS (1)

where btop,exp is the experimentally determined SLD of the top

stratum in dry air. Substituting a value of 1.25 � 10�6 �A�2 for

btop,exp in eqn (1), we can calculate a to be �0.25. Therefore, the

top stratum in dry air comprises ca. 25% PEI and 75% PSS by

volume. For polyelectrolyte multilayers constructed from PAH

and PSS, the volume percents of each component were 33% and

67%, respectively.13

Table 1 Fit parameters for 5, 7, and 9-layered samples measured in dry air

Total numberof layers

Bottom stratumthickness [�A]

Bottom stratumSLD � 106 [�A�2]

Top stratumthickness [�A]

5 43.8 � 0.7 0.0 � 0.1 33.9 � 1.77 38.4 � 0.4 0.0 � 0.1 90.5 � 0.69 48.3 � 0.3 0.0 � 0.1 141.1 � 0.4

This journal is ª The Royal Society of Chemistry 2012

Based on our previous assumptions, the following equation

can be used to estimate the volume fraction of water in the

bottom strata:

bbottom,exp ¼ awbw + (1 � aw)b

top,exp (2)

where bbottom,exp is the experimentally determined SLD of the

bottom strata, and bw is the SLD of water. The volume fraction

of water in the lower stratum is represented by aw. By

substituting 0 and �0.5 � 10�6 �A�2 for bbottom,exp and bw in eqn

(2), it was estimated that the lower strata consist of ca. 70% (v/v)

water and 30% (v/v) polyions (PEI + PSS). Thus, for the samples

measured in dry air, the packing density of the polymers in the

bottom strata was found to be substantially lower than that in

the top strata.

3.2. NR measurement of polyelectrolyte layers in D2O

saturated vapors

NR data for 5, 7, and 9 layered samples measured in D2O

saturated vapors are shown in Fig. 3(A), with solid lines showing

the fits for the respective datasets. The corresponding SLD

profiles resulting from NR fits are shown in Fig. 3(B). Again, a

two strata model was employed to fit the data collected in D2O

saturated vapors. The top stratum results from the contiguous

deposition of films, and again we can observe a consistent

increase in the thickness of this stratum with the number of layers

Top stratumSLD � 106 [�A�2]

Roughness of the topstratum (film–air interface) [�A]

Totalthickness [�A]

1.1 � 0.1 10 � 1 78 � 21.25 � 0.1 10 � 1 129 � 11.25 � 0.1 10 � 1 189 � 1

Soft Matter, 2012, 8, 11484–11491 | 11487

Fig. 3 Reflectivity (R vs. Qz) profiles for LbL deposited thin films (A) and SLD profiles (B) for 5, 7, and 9 layered samples measured in D2O saturated

vapors. A plot of total PEM thickness vs. number of layers (n > 4) is shown in the inset. The error bars denote the standard deviation for each

measurement.

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deposited (i.e., 5, 7, and 9). Fit parameters for the two strata

model for 5-, 7-, and 9-layered samples measured in D2O satu-

rated vapors are shown in Table 2. The SLD for the bottom

stratum was found to increase significantly from 0 to 4.5 � 10�6

�A�2, which indicates the exchange of H2O with D2O upon

exposure to D2O vapors. A slight reduction in the thickness of

the bottom strata can be observed (cf. Tables 1 and 2). For 5, 7,

and 9-layered samples, the top strata swelled by ca. 135, 75, and

42%, respectively. The difference in the swelling behaviors

between the bottom and top strata is consistent with the island

type deposition of polyelectrolytes in the bottom layer, which

results in less dense polymer packing. Therefore, during the

exposure to D2O vapors, polymers in the bottom strata can swell

in the lateral directions without affecting their thickness. On the

other hand, the more dense and homogeneous top strata can

swell along the normal direction only. In D2O vapors, the SLD of

the top strata increases to 3.7 � 10�6, 3.4 � 10�6, and 3.4 � 10�6

for 5-, 7-, and 9-layered samples, respectively. The increase in the

thickness and SLD of the top strata is due to the uptake of D2O.

By performing calculations similar to eqn (2), we estimated that

in this case the top strata comprise ca. 57% (v/v) polymer and

43% (v/v) D2O for 7- and 9-layered samples, respectively. The top

stratum of 5-layered samples consists of 50% (v/v) of both

polymer and D2O. A consistent and almost linear growth of the

total thickness of the multilayered structure can be observed in

the inset shown in Fig. 3(B). In agreement with the data obtained

for the samples measured in dry air, the less dense top stratum of

Table 2 Fit parameters for 5, 7, and 9-layered samples measured in D2O sa

Total numberof layers

Bottom stratumthickness [�A]

Bottom stratumSLD � 106 [�A�2]

Top stratumthickness [�A]

5 40 � 1.5 4.5 � 0.1 79.8 � 0.57 30 � 2.1 4.5 � 0.1 158.6 � 0.69 30 � 1.7 4.5 � 0.1 199.8 � 0.5

11488 | Soft Matter, 2012, 8, 11484–11491

the 5-layered sample intakes more water than do the more

densely packed top strata of 7- and 9-layered samples.

3.3. NR measurement of polyelectrolyte layers in bulk D2O

Finally, NR measurements were performed for all the samples in

contact with bulk D2O. The reflectivity profiles are shown in

Fig. 4(A), and the corresponding SLD profiles obtained from

modeling are shown in Fig. 4(B). Similar to previous cases for dry

air and D2O saturated vapor measurements, the two strata

models were found to be the simplest and physically most rele-

vant for 7- and 9-layered samples. Nevertheless, the SLD

contrasts between the two strata in these cases were not very

substantial, and, therefore, they could also be modeled as single

stratum systems with slightly higher c2. A single stratum model

resulted in a good fit for the 5-layered sample. Fit parameters

obtained for all the samples measured in bulk D2O are shown in

Table 3. The thicknesses obtained for the bottom strata of 7- and

9-layered samples were ca. 101 and 105 �A, respectively. In

addition, the SLD of the bottom strata decreased from 4.5 �10�6 to �3.57 � 10�6 �A�2, correspondingly. This suggests that

the entire polymeric system underwent a rearrangement in

contact with the bulk D2O. For 7- and 9-layered samples, the top

region exhibited a slightly higher polymer density than the lower

region, which resulted from less D2O intake in the top strata. In

the case of the 5-layered sample, a single stratum model resulted

in a good fit that was also physically relevant. A slight reduction

turated vapors

Top stratumSLD � 106 [�A�2]

Roughness of the topstratum (film–vapor interface) [�A]

Totalthickness [�A]

3.7 � 0.1 10 � 1 120 � 23.4 � 0.1 10 � 1 189 � 33.4 � 0.1 10 � 1 230 � 2

This journal is ª The Royal Society of Chemistry 2012

Fig. 4 Reflectivity (R vs. Qz) profiles for LbL deposited thin films (A) and SLD profiles (B) for 5, 7, and 9 layered samples measured in liquid D2O. A

plot of the total PEM thickness vs. number of layers (n > 4) is shown in the inset. The error bars denote the standard deviation for each measurement.

Table 3 Fit parameters for 5, 7, and 9-layered samples measured in liquid D2O

Total numberof layers

Bottom stratumthickness [�A]

Bottom stratumSLD � 106 [�A�2]

Top stratumthickness [�A]

Top stratumSLD � 106 [�A�2]

Roughness of the topstratum (film–D2O interface) [�A]

Totalthickness [�A]

5 86.3 � 0.4 3.5 � 0.1 — — 5 � 1 86 � 17 101.6 � 3.5 3.6 � 0.1 62.3 � 3.6 3.4 � 0.1 5 � 1 164 � 59 105.0 � 2.0 3.6 � 0.1 110.0 � 2.1 3.4 � 0.1 5 � 1 215 � 3

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in thickness in comparison to the D2O vapor measurements and

a corresponding decrease in the SLD clearly indicated the rear-

rangement of the polymer between the upper and lower regions.

Therefore, in all cases, we recorded a tendency towards the

homogenization of the multilayered structure when exposed to

bulk D2O, resulting in decreased distinction between the two

regions. Similar to the cases of measurements in dry air and D2O

saturated vapors, a consistent and almost linear increase in the

thickness of the PEI–PSS multilayered structure (n > 4) was

observed (inset Fig. 4(B)).

Fig. 5 Optical images of attachment and differentiation of 3T3 fibro-

blast cells on [PEI–PSS]3–PEI films deposited on a 30 0 quartz substrate.

The scale bar in the photomicrograph corresponds to 100 mm. Dividing

cells (circled) can be seen at all time points.

3.4. Cell culture experiments

To investigate the ability of a [PEI–PSS]3–PEI (7 layers) coated

quartz substrate to support cell growth, two different cell types

were seeded on the polyelectrolyte multilayer and cell coverage

was monitored by optical microscopy over time. Fig. 5 and 6

show representative images of 3T3 fibroblasts (Fig. 5) and HEK-

293 (Fig. 6) cells after 5, 25, 49, and 73 hours of incubation.

Fibroblasts, which secrete cellular fibronectin to bind to the

underlying support, attach and spread efficiently, showing their

characteristic spindle shape (Fig. 5). HEK cells demonstrated

normal growth and colony formation (Fig. 6). Both cell types

displayed normal morphology with higher surface coverage by

the fibroblasts, which, in general, adhere more strongly to

various surfaces. Over time, fibroblasts as well as HEK cells

covered previously empty areas of the substrate, and dividing

cells could be observed in both cell types, indicative of healthy

This journal is ª The Royal Society of Chemistry 2012

proliferating cells. We measured the level of cell coverage to test

whether any toxic effect was caused by the [PEI–PSS]3–PEI

coating. For both cell types, coverage increased (Fig. 7) over 3

days and no significant cell death could be observed by acridine

orange/ethidium bromide (AO/EB) staining (data not shown).

All of the above results confirm that these cells can adhere and

spread on [PEI–PSS]3–PEI substrates with no immediate

Soft Matter, 2012, 8, 11484–11491 | 11489

Fig. 6 Optical images of attachment and differentiation of HEK-293

cells on [PEI–PSS]3–PEI films deposited on a 30 0 quartz substrate. The

scale bar in the photomicrograph corresponds to 100 mm. Dividing cells

(circled) can be seen at all time points.

Fig. 7 Time evolution of surface coverage of 3T3 fibroblasts (A) and

HEK-293 (B) cells over 73 hour observation periods. Coverage increased

over 3 days and no significant cell death was observed.Publ

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toxicity, and therefore the substrates are suitable for the growth

of various cell types.

4. Conclusions

We successfully characterized the structure of PEM films

comprising PEI and PSS. NR measurements were performed by

exposing the samples to three different environmental conditions

(i.e. dry air, saturated water vapors, and bulk water). By

modeling all the measurements at three different contrast

conditions using one simple, physically relevant and consistent

model, we determined a well-founded structural interpretation of

PEM films formed by PEI and PSS. Modeling of NR data sug-

gested that the structure of PEI–PSS PEMs comprises two

distinct regions (i.e., bottom and top strata). The bottom

stratum, which is closest to the quartz substrate, consists of 30%

(v/v) polyions. On the other hand, the top stratum consists of

densely packed polymers when measured in dry air. The thick-

ness of these strata varied linearly with the number of layers

(for n > 4). These results are in concurrence with the previous

11490 | Soft Matter, 2012, 8, 11484–11491

finding of PEMs formed from the alternate deposition of PAH

and PSS.26,30 Upon exposure to D2O saturated vapors, PEMs

comprising PEI and PSS were found to swell by absorbing

substantial quantities of D2O. In this case, the top stratum

comprised ca. 57% polyions and 43% water. Similar values for

water vapor uptake have been reported in saturated vapor

conditions for PEMs comprising PAH and PSS.13 In our case,

after dry air and D2O saturated vapor measurements, the PEI–

PSS PEMs were exposed to bulk D2O and measured using NR.

No further swelling was observed in comparison to the PEMs

measured in D2O saturated vapors. Nevertheless, the entire

polymeric system underwent a rearrangement accompanied by a

slight reduction in the total thickness. Consequently, the SLD

differences between the top and bottom strata dramatically

reduced, which indicated a tendency towards the homogeniza-

tion of PEM films.

Seven-layered PEM structures comprising PEI and PSS, with

PEI as the capping layer, were also assessed for their ability to

serve as an attachment support for cell growth.18 Two different

cell types, 3T3 and HEK-293, were seeded on the polyelectrolyte

multilayer and the cell coverage was monitored by optical

microscopy at varying times. Our observations confirm that cells

can adhere and proliferate on such substrates. In addition, no

immediate toxicity was observed, providing evidence that the

surface modification of culture dishes with such PEMs could be

employed as a suitable support for the growth and differentiation

of different cell types.

Acknowledgements

This work benefited from the use of the LujanNeutron Scattering

Center at Los Alamos Neutron Science Center funded by the

DOE Office of Basic Energy Sciences and Los Alamos National

Laboratory under DOE contract DE-AC52-06NA25396. We are

thankful to SofiyaMicheva-Viteva andElisabethHong-Geller for

providing the HEK-293 cells.

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