Comparative assessment of the stability of nonfouling poly(2-methyl-2-oxazoline) andpoly(ethylene glycol) surface films: An in vitro cell culture studyYin Chen, Bidhari Pidhatika, Thomas von Erlach, Rupert Konradi, Marcus Textor, Heike Hall, and Tessa

Lühmann

Citation: Biointerphases 9, 031003 (2014); doi: 10.1116/1.4878461 View online: http://dx.doi.org/10.1116/1.4878461 View Table of Contents: http://scitation.aip.org/content/avs/journal/bip/9/3?ver=pdfcov Published by the AVS: Science & Technology of Materials, Interfaces, and Processing Articles you may be interested in Hydrogel/poly-dimethylsiloxane hybrid bioreactor facilitating 3D cell culturing J. Vac. Sci. Technol. B 31, 06F903 (2013); 10.1116/1.4831762 Fabrication of three-dimensional structures for the assessment of cell mechanical interactions within cellmonolayers J. Vac. Sci. Technol. B 28, C6K1 (2010); 10.1116/1.3511435 Self-assembled monolayers of poly(ethylene glycol) siloxane as a resist for ultrahigh-resolution electron beamlithography on silicon oxide J. Vac. Sci. Technol. B 27, 2292 (2009); 10.1116/1.3212899 Enhancement of hemocompatibility on titanium implant with titanium-doped diamond-like carbon film evaluatedby cellular reactions using bone marrow cell cultures in vitro J. Vac. Sci. Technol. B 27, 1559 (2009); 10.1116/1.3077271 Biofunctionalized magnetic nanoparticles for in vitro labeling and in vivo locating specific biomolecules Appl. Phys. Lett. 92, 142504 (2008); 10.1063/1.2907486

Comparative assessment of the stability of nonfouling poly(2-methyl-2-oxazoline) and poly(ethylene glycol) surface films: An in vitro cellculture study

Yin Chena) and Bidhari Pidhatikab)

BioInterface Group, Laboratory for Surface Science and Technology, Department of Materials, ETH Zurich,8093 Zurich, Switzerland

Thomas von Erlachc)

Cells and Biomaterials, Department of Materials, ETH Zurich, 8093 Zurich, Switzerland

Rupert Konradid) and Marcus TextorBioInterface Group, Laboratory for Surface Science and Technology, Department of Materials, ETH Zurich,8093 Zurich, Switzerland

Heike Halle) and Tessa L€uhmannf)

Cells and Biomaterials, Department of Materials, ETH Zurich, 8093 Zurich, Switzerland

(Received 26 March 2014; accepted 7 May 2014; published 23 May 2014)

Poly(ethylene glycol) (PEG) has been the most frequently reported and commercially used polymer

for surface coatings to convey nonfouling properties. PEGylated surfaces are known to exhibit

limited chemical stability, particularly due to oxidative degradation, which limits long-term

applications. In view of excellent anti-adhesive properties in the brush conformation and resistance

to oxidative degradation, poly(2-methyl-2-oxazoline) (PMOXA) has been proposed recently as an

alternative to PEG. In this study, the authors systematically compare the (bio)chemical stability of

PEG- and PMOXA-based polymer brush monolayer thin films when exposed to cultures of human

umbilical vein endothelial cells (HUVECs) and human foreskin fibroblasts (HFFs). To this end, the

authors used cell-adhesive protein micropatterns in a background of the nonfouling PEG and

PMOXA brushes, respectively, and monitored the outgrowth of HUVECs and HFFs for up to 21

days and 1.5 months. Our results demonstrate that cellular micropatterns spaced by PMOXA

brushes are significantly more stable under serum containing cell culture conditions in terms of

confinement of cells to the adhesive patterns, when compared to corresponding micropatterns

generated by PEG brushes. Moreover, homogeneous PEG and PMOXA-based brush monolayers

on Nb2O5 surfaces were investigated after immersion in endothelial cell medium using

ellipsometry and x-ray photoelectron spectroscopy. VC 2014 American Vacuum Society.

[http://dx.doi.org/10.1116/1.4878461]

I. INTRODUCTION

In the past few decades, due to the fast growth in the

demand for biomedical products such as medical devices,

drug-delivery systems, imaging contrast agents, and biosen-

sors, the need for nonadhesive polymer coatings has been

increasing.1,2 Hydrophilic, highly hydrated, uncharged,

brushlike polymeric surfaces are the most frequently used

approach to prevent spontaneous protein adsorption with

polymer grafting density and molecular weight being impor-

tant design criteria.3,4

It is widely known that the adsorption of proteins onto the

surface of artificial implants plays a crucial role in biological

processes such as blood clot formation, foreign body reac-

tions, and bacterial infections in vivo.5–16 In the fields of

drug delivery systems and imaging contrast, nanoparticles or

nanocapsules being shielded by nonfouling polymers were

shown to have prolonged blood circulation and remain in the

human circulatory system for a comparatively long period

before being removed by the body’s reticuloendothelial sys-

tem (RES) or by renal clearance.1,17

Among those polymers, poly(ethylene glycol) (PEG) is

most frequently used.1,11,12,14,17–23 The success of PEG is

based on its hydrophilicity, decreasing interaction with blood

components combined with high biocompatibility.1 Although

it shows excellent protein resistance properties, scientific

results obtained in recent years suggest that it may also have

possible drawbacks. For example, complement activation by

PEG attached to liposomes was observed though the mecha-

nism has not yet been elucidated. Moreover, hypersensitivity

a)Present address: Bioengineering Program, Division of Biomedical

Engineering, The Hong Kong University of Science and Technology,

Clear Water Bay, Kowloon, Hong Kong, China.b)Present address: Department of Industrial Chemical Engineering, College

of Industrial Management, Ministry of Industry, Jl. Letjen Suprapto No. 26

Cempaka Putih Jakarta 10510, Indonesia.c)Present address: Department of Materials, Department of Bioengineering

and Institute for Biomedical Engineering, Imperial College London,

Exhibition Road, London SW7 2AZ, United Kingdom.d)Present address: BASF SE, Advanced Materials and Systems Research,

D-67056 Ludwigshafen, Germany.e)Deceased.f)Present address: Institute for Pharmacy and Food Chemistry, Chair of

Pharmaceutics and Biopharmacy, University of W€urzburg, Am Hubland,

W€urzburg D-97074, Germany. Author to whom correspondence should be

addressed; electronic mail: t.luehmann@pharmazie.uni-wuerzburg.de

031003-1 Biointerphases 9(3), September 2014 1934-8630/2014/9(3)/031003/10/$30.00 VC 2014 American Vacuum Society 031003-1

reactions were observed, which could provoke an anaphylac-

tic shock in vivo.24 Besides, accelerated blood clearance after

the second injection of PEG-attached liposomes was found,

which might be triggered by the immune reaction to the pres-

ence of PEG.25–28 PEG coatings have also been reported to

lose their function in vivo during long-time application since

they can undergo oxidative degradation.29–31 The degradation

mechanism of PEG in vivo is complex. Some cells, such as

macrophages and polymorphonuclear leukocytes, can secret

reactive oxygen species such as hydroxyl radicals that can

degrade PEG through oxidation when they contact the poly-

mer.29,32 During the fabrication of PEG or PEGylated prod-

ucts, PEG might undergo oxidative thermodecomposition in

air at high temperature.33 In addition, it has been reported that

PEG can undergo nonoxidative thermodecomposition.34

Therefore, efforts have been taken to seek for nonfouling

polymers to substitute PEG. As alternatives polysaccharides,

peptidomimetic, and zwitterionic polymers have been

developed.16,35–37

Recently, we have become interested in poly(2-methyl-2-

oxazoline) (PMOXA), which has been shown to have prom-

ising nonfouling properties in previous studies.3,38,39

Structurally, PMOXA could be described as a poly(ethylene

imine) backbone with an acetyl group bound to the nitrogen

atom in each repeating unit3 (Fig. 1). Our previous study has

demonstrated much better chemical stability of PMOXA in

comparison to PEG brush surfaces in a range of different

(oxidative) aqueous solutions,40 likely related to the C-H

being less polarized in the backbone of PMOXA and hence

less prone to oxidation in comparison to the C-H bond in

PEG. In view of its favorable protein resistance property and

stability, PMOXA has promise as an alternative polymer to

PEG, but the biological stability of PMOXA versus PEG

in vitro requires more systematic studies including testing

under cell culture conditions.

In the last decades, numerous micropatterning techniques

have been deployed in order to generate cell-adhesive and

cell-repellent regions in various geometries.41,42 The pattern

stability was demonstrated to be dependent on the cell-type

used, on the material background and on the passivation

chemistry used under cell culture conditions.42–44 Thus,

micropatterning is an elegant approach to analyze and com-

pare pattern (bio)-stability by analyzing the time-dependent

outgrowth of cells from the adhesive patterns into the nonad-

hesive background.

In this study, micropatterned substrates of adhesive

squares surrounded by non-adhesive regions were employed

as a platform to compare the long-term stability of PMOXA

versus PEG under cell culture conditions. Copolymers

poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-

PMOXA) and poly(L-lysine)-graft-poly(ethylene glycol)

(PLL-g-PEG) were synthesized and immobilized onto

Nb2O5 surfaces, which were subsequently micropatterned by

direct lithography. In order to elucidate the stability of the

copolymers under cell culture conditions, human umbilical

vein endothelial cells (HUVECs) and human foreskin fibro-

blasts (HFF) were cultured on cell adhesive micropatterns

for various time points at 37 �C. Cellular outgrowth into non-

adhesive areas of the patterns, cell number, and cell viability

were determined.

II. MATERIALS AND METHODS

A. PLL-g-PMOXA and PLL-g-PEG copolymers

PLL-g-PMOXA copolymers were synthesized as previ-

ously published.39 PLL-g-PEG was purchased from Surface

Solution AG (Zurich, Switzerland). Table I summarizes the

polymers used in the present study.

B. Preparation of graft-copolymer films on Nb2O5

surfaces

Silicon wafers (1� 1 cm2) used as the substrates were

purchased from WaferNet GmbH (M€unchen, Germany). The

wafers were coated with a 15-nm-thick Nb2O5 layer using

reactive magnetron sputtering at Paul Scherrer Institute

(Villigen, Switzerland). Graft-copolymer films were coated

onto the Nb2O5 surfaces by dip and rinse procedures as previ-

ously described.45 Nb2O5 has a high negative surface density

resulting in strong electrostatic interaction and immobilization

of the polycationic copolymers.

FIG. 1. Chemical structures of PLL-g-PMOXA and PLL-g-PEG.

TABLE I. List of polymers. The grafting density (a) of copolymer is defined

as the ratio of the number of side chains to the number of L-lysine residues;

Mn, number-average molecular weight; DP, degree of polymerization.

Polymers

Mn of PLL

backbone

(kDa)

Grafting

density

Mn of

side chains

(kDa)

DP of side

chains

PLL(20)-a(0.19)-PMOXA(4) 20 0.19 4 47

PLL(20)-a(0.29)-PMOXA(8) 20 0.29 8 94

PLL(20)-a(0.28)-PEG(5) 20 0.28 5 113

031003-2 Chen et al.: Comparative assessment of the stability of nonfouling PMOXA and PEG surface films 031003-2

Biointerphases, Vol. 9, No. 3, September 2014

C. Preparation of micropatterns using directphotolithography

Micropatterns created by direct photolithography were

used to investigate the biological stability of the copolymer

thin films under cell culture conditions. Copper grids (Agar

Scientific, Germany) for transmission electron spectroscopy

(TEM) were used as masks for micropatterning, the geome-

try of which is displayed in Fig. S1(A). The polymer coated

substrates were placed as a straight line on a sample holder

made of stainless steel (14� 14 cm2) and each substrate

was covered with two masks prior to be sealed with an opti-

cal glass plate (borosilicate). The whole device was fixed by

pressing the sample holder and a stainless steel frame on top

of the glass plate with screws. Subsequently, the sample

holder was placed onto a lab jack in a dark box, in which a

UV lamp with a cooling system was installed. The distance

between the UV lamp and the samples was adjusted to

around 15 cm. To degrade the polymers not protected by the

TEM grids, the samples were exposed to UV light for 1 h.

After that, the wafers were removed from the sample holder,

followed by rinsing with absolute ethanol (Scharlau, Spain)

and ultrapure water before being blow-dried with a stream of

nitrogen gas. 50 lg/ml of Oregon green-labeled fibrinogen

(Invitrogen, F7496) or Alexa 488-labeled fibronectin (label-

ing method described in Ref. 46) solution was dripped onto

each wafer for 1 h to backfill the degraded area of the poly-

mer film and then these wafers were rinsed with ultrapure

water and blow-dried with nitrogen gas again prior to use.

The process of micropatterning using direct photolithogra-

phy is illustrated in Fig. S1(B), and further information are

available in the supplementary material.47

D. Cell culture

HUVECs and normal HFFs were harvested from expo-

nentially growing subconfluent monolayers and were pur-

chased from PromoCell, Germany. HUVECs were cultured

in 25 cm2 culture flasks in the endothelial cell (EC) medium

(PromoCell GmbH, Cat. No.: C-22010) with Supplement

Mix (PromoCell GmbH, Cat. No.: C-39215) and 1% ampho-

tericin B (Cat. No.: 15240, Gibco) at 37 �C and 5% CO2.

Passages 3–8 were used.

HFFs were maintained in 75 cm2 culture flasks in the

Dulbecco’s modified Eagle medium (DMEM, Cat. No.:

21885, low glucose, glutamax I, GibcoBRL) supplemented

with 10% heat inactivated fetal bovine serum (FBS, Cat. No.:

F7524, Sigma) and 1% ABAM (Cat. No. 15340, GibcoBRL)

at 37 �C and 5% CO2. Cells were used until passage 20.

E. Cell culture and actin staining on micropatternedsurfaces

Micropatterned silicon wafers were placed into 24 well

plates (TPP, Switzerland) and were washed carefully with

phosphate buffered saline (PBS) prior to use. 30 000

HUVECs were added on micropatterned surfaces and cultured

for various time points (3 h to 21 days). In a similar way,

40 000 HFF cells were seeded onto micropatterned substrates.

For further analysis, the cells were fixed for 30 min at room

temperature with 2% formaldehyde solution prepared by dilut-

ing formalin solution (4% w/v formaldehyde, Cat. No.:

HT501128, Sigma-Aldrich) in the PBS buffer. After washing

three times with PBS buffer, the fixed samples were permeab-

ilized with 0.1% Triton X-100 (Cat. No.: T8787, Sigma-

Aldrich) in PBS buffer for 10 min at room temperature fol-

lowed by blocking with 5% bovine serum albumin (Cat. No.:

A3311, Sigma-Aldrich) and 5% donkey serum (DS, Cat. No.:

D9663, Sigma-Aldrich) in PBS buffer for 1 h at room tempera-

ture. The actin cytoskeleton was stained by Alexa 546-labelled

phalloidin (1 mg/ml, dilution 1:200, Cat. No.: A2283,

Invitrogen) dissolved in PBS for 30 min, and then, the cell

nuclei were stained by 40,6-diamidino-2-phenylindole (DAPI,

1 mg/ml, dilution 1:1000, Cat. No.: D3571, Invitrogen) dis-

solved in PBS for 20 min. All the samples were rinsed with

PBS buffer three times and transferred into Lab-TekVR

two-well

chamber slides (Nunc, Thermo Fischer Scientific, USA) with

PBS buffer. The chamber slides were placed into Petri dishes

and images of the samples were taken with an inverted epi-

fluorescent microscope (Axiovert 200M, Carl Zeiss, Germany)

or with a high-resolution TCS-SP5 laser scanning confocal

microscope (Leica Microsystems, Mannheim, Germany).

F. Quantification of cellular outgrowth onmicropatterns

In order to compare the stabilities of the different poly-

mers, cellular outgrowth from the adhesive fibrinogen or

fibronectin-coated areas onto the non-adhesive polymer-

coated region of the micropatterns were analyzed. Images

with at least ten cells were analyzed with the software

Image J (NIH, USA). The outgrowth ratio of cells onto the

non-adhesive region was defined as

Outgrowth ratio

¼ Area of actin on the nonadhesive region

Area of the nonadhesive region� 100%:

(1)

The definitions of the parameters are displayed in Fig. 2. The

proliferation of cells cultured on the micropatterns was also

determined by counting the number of cell nuclei within the

FIG. 2. Schematic illustration of cells cultured on micropatterns. Adhesive

patterns are 90� 90 lm2 with a spacing region of 30 lm in width. Cellular

outgrowth into the nonadhesive area (gray area) was quantified.

031003-3 Chen et al.: Comparative assessment of the stability of nonfouling PMOXA and PEG surface films 031003-3

Biointerphases, Vol. 9, No. 3, September 2014

adhesive squares. For each polymer at each time point, more

than 200 squares from eight patterns of duplicate experi-

ments were analyzed.

G. Viability of HUVECs cultured on micropatterns

In order to evaluate the biocompatibilities of the copoly-

mers, the cell viability of HUVECs cultured on micropat-

terned surfaces was analyzed using the fluorescent stains

fluorescein diacetate (FDA, Sigma F7378) and Hoechst

33342 (Invitrogen). Living cells metabolite FDA to fluores-

cein, whereas Hoechst 33342 intercalates within the DNA of

cell nuclei. HUVECs were incubated with the mixture solu-

tion of fluorescein diacetate (1:1000 dilution, 1 mg/ml stock

solution in acetone) and Hoechst 33342 (1:1000 dilution,

10 mg/ml stock solution in water) in the incubator containing

5% CO2 at 37 �C for 5 min after the cell medium had been

carefully removed. The wafers were transferred into

two-well chamber slides (Lab-Tek Chambered #1.0

Borosilicate Cover glass System, Nunc) and imaged with an

inverted epifluorescent microscope (Axiovert 200M, Carl

Zeiss, Germany). Cell viability of cells cultured on the

micropatterns was determined by counting the number of

green fluorescent cells versus the total cell number.

H. Stability study of the homogeneous copolymerfilms in EC medium

In order to investigate if there were losses of polymers

during cell culture, homogeneous PLL-g-PMOXA and PLL-

g-PEG films on the Nb2O5-coated substrates were prepared

as above and immersed in EC medium at 37 �C for 10 days.

Subsequently, the wafers were rinsed with ultrapure water

and then blow-dried with a stream of nitrogen gas before fur-

ther analysis.

The thickness of dry copolymer film was determined

under ambient conditions by variable-angle spectroscopic

ellipsometry (VASE) using the M-2000F variable-angle

spectroscopic ellipsometer (J. A. Woollam Co., Inc., USA).

The measurements were performed at an angle of 65�, 70�,and 75� relative to the surface using light in the spectral

range of 370–1000 nm. The data obtained by the ellipsome-

ter were fitted with a Cauchy multilayer model (An¼ 1.45,

Bn¼ 0.01, and Cn¼ 0) to obtain the dry thickness of the co-

polymer film using the analysis software WVASE 32 devel-

oped for the ellipsometer by the manufacturer.

X-ray photoelectron spectroscopy (XPS) was used to study

the elemental information of dry copolymer films on

Nb2O5-coated wafers freshly prepared and after immersion in

EC. The measurements were carried out using a Sigma2

instrument (Thermo Fisher Scientific, Loughborough, Great

Britain), equipped with a UHV chamber (pressure<10�6 Pa

during measurements) and an Al-Ka nonmonochromatic

x-ray source (300 W, h�¼ 1486.6 eV) illuminating the sam-

ple at 54� to the surface normal. A hemispherical analyzer is

mounted at 0� with respect to the surface normal, thus operat-

ing at the magic source-analyzer angle, which eliminates the

need for angular-distribution correction.48 The spot size of the

analyzed region was about 400 lm in diameter (large-area

mode), so that each measurement represents laterally aver-

aged chemical composition of the surface measured. Standard

measurements comprise averaged data over nine scans for C

and N, and three scans for Nb and O with pass energy of

25 eV, as well as survey scans with pass energy of 50 eV. The

interval of the binding energies measured was 0.05 eV, and

the dwell time was 100 ms at each binding energy. The data

obtained were analyzed using the software CasaXPS (Casa

Software Ltd., UK). The spectra were calibrated by setting

the binding energy of aliphatic carbon (C-C-C) to 285.0 eV

and the assignments of C1s, N1s, and Nb3d were determined

by referring to previously published data.40

I. Statistical analysis

Statistical significance was calculated by Student t-test

for pairwise comparison. P values <0.01 were considered

statistically significant and were assigned by an asterisk “*.”

III. RESULTS

PMOXA has recently been proposed as an alternative to

PEG due to its excellent antifouling properties in combina-

tion with more advantageous chemical stability.40 However,

it is not known whether PMOXA is also superior in terms of

antifouling properties than the gold standard PEG in more

complex in vitro cell culture.

To contribute to this question, we investigated and com-

pared the stability of coatings prepared by self-assembly from

aqueous solution of two comb copolymers, namely, poly(L-

lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA)

and poly(L-lysine)-graft-poly-(ethylene glycol) (PLL-g-PEG).

These two copolymers have an analogous architecture and

essentially differ only in the side-chain polymer chemistry,

which, after surface self-assembly, leads to PEG- or

PMOXA-polymer brush monolayers.

A. Polymer properties

Table I displays the polymer composition of the

copolymers PLL(20)-a(0.19)-PMOXA(4), PLL(20)-a(0.29)-

PMOXA(8), and PLL(20)-a(0.28)-PEG(5) used in this study.

Synthesis and characterization of PLL(20)-a(0.19)-

PMOXA(4) and PLL(20)-a(0.29)-PMOXA(8) have been

given in detail elsewhere.39,40,49 The architecture of the poly-

mers was designed for comparing PMOXA and PEG moieties

as follows: PMOXA(4) is considered to be comparable to

PEG(5) in terms of the side chain molecular weight (4 kDa

and 5 kDa for PMOXA and PEG, respectively), whereas,

PMOXA(8) is considered comparable to PEG(5) in terms of

monomer repeating units (approximately 90 and 113 units for

PMOXA and PEG, respectively). All copolymers tested in

this study have identical PLL backbones of 20 kDa, with

approximately 96 lysine-repeating units. The grafting density

(a) defines the number of PEG or PMOXA side chains per

number of lysine units. All three copolymers have been found

to be protein resistant against full human serum (data not

shown) and resemble a brush architecture on the surface, with

031003-4 Chen et al.: Comparative assessment of the stability of nonfouling PMOXA and PEG surface films 031003-4

Biointerphases, Vol. 9, No. 3, September 2014

controlled thicknesses and surface densities.40 Table II sum-

marizes the properties of the copolymer monolayer films.

B. Pattern stability with endothelial cells

In order to compare the stability of non-adhesive

PMOXA-based films with the gold standard PEG-based

films with respect to cellular adhesion, micropatterned ad-

hesive substrates were produced by direct photolithogra-

phy. The micropattern consisted of adhesive squares (90

lm� 90 lm) spaced out by non-adhesive areas of �30 lm

width. The adhesive squares were backfilled with Oregon-

green labeled fibrinogen on the Nb2O5 surface while the

non-adhesive areas comprised PLL(20)-a(0.19)-PMOXA(4),

PLL(20)-a(0.29)-PMOXA(8) or PLL(20)-a(0.28)-PEG(5)

copolymers immobilized onto the Nb2O5 surface (Fig. S1).

The cell-adhesive molecule fibrinogen, containing the integrin

binding RGD site, has been previously described as an

excellent cell adhesive substrate for endothelial cells.50 All

experiments were performed with EC medium supplemented

with 2% fetal calf serum. The biological stability of the

copolymers was characterized by the outgrowth of HUVECs

into the non-adhesive regions since the anti-adhesive proper-

ties of the latter would be compromised by their degradation

and/or desorption. Figure 3 shows the fluorescent images

of HUVECs cultured on patterns of the Oregon-green

labeled fibrinogen with the protein repellent background of

PLL(20)-a(0.19)-PMOXA(4), PLL(20)-a(0.29)-PMOXA(8)

or PLL(20)-a(0.28)-PEG(5) for 16 h, 5 days, and 21 days,

respectively. The resulting cellular patterns were in line with

the designed geometries with respect to size and spacing of

the masks applied. After 16 h of incubation, HUVECs were

exclusively found to adhere onto adhesive micropatterns

generated by all tested conditions [Figs. 3(A)–3(C), (a)–(d)],

indicating that both types of copolymers inhibit cell adhesion

within non-adhesive regions for early time points after cell

seeding. HUVECs were observed to spread to some extent

into the non-adhesive regions of the micropattern generated

by all tested polymers after 5 days in culture [Figs.

3(A)–3(C), (e)–(h)]. However, after a culture period of 21

days HUVECs adhered regionally onto the adhesive

squares generated by PLL(20)-a(0.19)-PMOXA(4) and

PLL(20)-a(0.29)- PMOXA(8), whereas HUVECs cultured

onto micropatterns formed by PLL(20)-a(0.28)-PEG(5) were

found to grow and proliferate without any more restriction

into the non-adhesive areas as displayed in Fig. 3(C) [(i)–(l)].

In order to quantify the outgrowth of cells into the non-

adhesive region of the micropatterns, an outgrowth ratio was

defined and determined. In Fig. 4, the outgrowth ratio versus

the incubation time is presented for HUVECs cultured on

micropatterned substrates of PLL(20)-a(0.19)-PMOXA(4),

PLL(20)-a(0.29)-PMOXA(8), and PLL(20)-a(0.28)-PEG(5),

respectively.

No significant difference regarding the outgrowth ratio

among the different copolymers at early time points of incu-

bation was found. All the polymers showed good antifouling

properties, i.e., absence of cell adhesion, during the initial

seeding of HUVECs onto the adhesive areas of Oregon-

green labeled fibrinogen. Sixteen hours after seeding, the

outgrowth ratio of HUVECs cultured on micropatterns gen-

erated by PLL(20)-a(0.28)-PEG(5) increased to 11%

whereas for patterns with both PLL-g-PMOXA copolymers

outgrowth ratios were found to be below 5%. In line with the

fluorescent images shown in Fig. 3, the outgrowth ratio of

HUVECs cultured on PLL-g-PEG generated surfaces, signif-

icantly increased to 74% after 21 days incubation. In contrast

the outgrowth ratio of PLL-g-PMOXA-micropatterned surfa-

ces raised to only 11% for PLL(20)-a (0.19)-PMOXA(4) and

17% for PLL(20)-a(0.29)-PMOXA(8), respectively.

In order to quantify cell proliferation on the adhesive

micropatterns, the numbers of HUVECs on single squares

were counted over a total period of 21 days and the mean val-

ues 6 SD calculated and depicted in Fig. 5. After seeding

from 3 to 16 h, the cell number per single pattern (square)

remained constant with approximately two cells/square on av-

erage. This finding indicates that within early time points of

incubation, the process of cell adhesion onto the cell adhesive

pattern is predominant. In contrast, the cell numbers on all

tested substrates increased in a uniform manner from 16 h to 5

days on all surfaces analyzed as expected for cellular prolifera-

tion. After 10 days in culture, cell division of HUVECs was

observed to be arrested with a maximum of three to four

cells/square in average. The number of cells/square after 21

days of culture showed an average decrease of one to two cells

for all tested conditions, which might point to cellular degen-

erative processes such as apoptosis after long incubation peri-

ods. Cell detachment after 21 days might be one of the main

reasons why the average cell number/square diminished. In

order to elucidate if HUVECs cultured onto the adhesive

squares remain alive over the analyzed culture period, we per-

formed a live and dead assay, in which living cells were

stained with FDA and Hoechst 33342 was used for the deter-

mination of the total cell number. In Fig. 6, the viability of

HUVECs is presented with respect to the incubation time. Cell

viability of adherent HUVEC cultured on adhesive squares of

fibrinogen was found to remain constant with more than 90%

of living cells over a period of 21 days, indicating that all

tested surface films are biocompatible with endothelial cells.

C. Pattern stability with human fibroblasts

In order to investigate whether the observed stabilities

of the copolymers PLL-g-PEG and PLL-g-PMOXA are

TABLE II. Properties of the copolymer monolayer films used in this study.

Values of PLL(20)-a(0.28)-PEG(5) have been kindly taken from Ref. 40.

Polymers

Surface

chain

density (nm�2)

Monomer

surface

density (nm�2)

Film

thickness

(nm)

PLL(20)-a(0.19)-PMOXA(4) 0.22 6 0.01 11.43 6 0.57 1.31 6 0.03

PLL(20)-a(0.29)-PMOXA(8) 0.15 6 0.04 13.68 6 3.34 1.43 6 0.03

PLL(20)-a(0.28)-PEG(5) 0.27 6 0.09 30.85 6 9.66 1.71 6 0.03

031003-5 Chen et al.: Comparative assessment of the stability of nonfouling PMOXA and PEG surface films 031003-5

Biointerphases, Vol. 9, No. 3, September 2014

cell-type specific, analogous micropatterns generated with

either PLL(20)-a(0.28)-PEG(5) or PLL(20)-a(0.29)-

PMOXA(8) were incubated with HFFs. All experiments

were performed in 10% heat inactivated calf serum and the

cellular adhesion molecule fibronectin was used as cell ad-

hesive substrate for micropatterning. Figure 7(A) displays

the quantification of cellular outgrowth of HFFs into the

non-adhesive regions surrounding the fibronectin squares.

FIG. 3. Representative images of HUVECs cultured on micropatterned surfaces with adhesive squares of Oregon-green labeled fibrinogen and a passivated

background of (A) PLL(20)-a(0.19)-PMOXA(4), (B) PLL(20)-a(0.29)-PMOXA(8), (C) or PLL(20)-a(0.28)-PEG(5) for various time points as indicated (16 h,

5 days, and 21 days). (a), (e) and (i) corresponds to cell nuclei, (b), (f) and (j) to fibrinogen, and (d), (h) and (l) to the actin cytoskeleton. Scale bar is 100 lm.

031003-6 Chen et al.: Comparative assessment of the stability of nonfouling PMOXA and PEG surface films 031003-6

Biointerphases, Vol. 9, No. 3, September 2014

Two hours after seeding, 28.0% 6 9.8% of all HFFs were

observed to grow out of the micropatterns into the non-

adhesive regions generated by PLL(20)-a(0.28)-PEG(5)

but not into the non-adhesive regions generated by

PLL(20)-a(0.29)-PMOXA(8). Moreover, HFFs cultured on

fibronectin squares surrounded by PLL(20)-a(0.29)-

PMOXA(8) showed no significant increase in outgrowth

ratio (<10%) after 16 h in culture in comparison to micro-

patterns generated with PLL(20)-a(0.28)-PEG(5), where

HFF growth was not any longer restricted to cell adhesive

squares (100% outgrowth rate) as displayed in Fig. 7(C)

[(e)–(h)]. Remarkably, HFF cell growth was restricted to

fibronectin pattern spaced by PLL(20)-a(0.29)-PMOXA(8)

for the total analyzed time period of 1.5 months [Fig. 7(C),

(i)–(l)].

The number of cells seeded per single fibronectin square

was determined with respect to culture time in order to

investigate cellular distribution and proliferation on the pat-

terns [Fig. 7(B)]. Proliferation of HFFs was observed to be

similar relative to total incubation time on fibronectin micro-

patterns of PLL(20)-a(0.28)-PEG(5) and PLL(20)-a(0.29)-

PMOXA(8), respectively. On both substrates, HFFs were

found to proliferate constantly from approximately four

cells/square in average 2 h after seeding to approximately

nine cells/square in average after 40 h of incubation time, as

evidence for cell viability.

D. Stabilities of the homogeneous copolymer films inEC medium in the absence of cells

In order to elucidate if the observed loss of pattern stabil-

ity of PLL-g-PEG is a consequence of cellular metabolism or

is independent from cellular processes, homogeneous PLL-g-

PEG and PLL-g-PMOXA thin films on Nb2O5 surfaces after

immersion in EC medium were investigated using VASE and

XPS, respectively. Samples were freshly prepared and were

directly compared to similar films, which were immersed in

EC medium for 10 days at 37 �C. VASE was used to study

the stabilities of the copolymer films by observing the

changes of the dry thicknesses before and after immersion in

EC medium for 10 days, which are depicted in Table III.

PLL(20)-a(0.19)-PMOXA(4) monolayers showed the best

stability with respect to the relative remaining thickness among

all three copolymers tested with 86% 6 9% of their initial

thickness, whereas in comparison the remaining thickness of

PLL(20)-a(0.29)-PMOXA(8) was found to be 76% 6 9%. In

contrast to the two analyzed PLL-g-PMOXA based copoly-

mers, thin films generated by PLL(20)-a(0.28)-PEG(5)

degraded significantly faster to about half of the initial thick-

ness after storage in EC medium for 10 days at 37 �C.

In order to confirm the results obtained by VASE, similar

samples, which were either freshly prepared or stored in EC

FIG. 4. Quantification of cellular outgrowth of HUVECs into the non-

adhesive regions of the micropatterns generated by PLL(20)-a(0.19)-

PMOXA(4), PLL(20)-a(0.29)-PMOXA(8), and PLL(20)-a(0.28)-PEG(5).

The values shown are mean values 6 SD, and they were obtained by analyz-

ing 20 squares, where eight patterns from duplicate experiments were

included. Statistical significance was accepted for P< 0.01 by Student’s ttest and assigned by an asterisk “*” for differences between different

copolymers.

FIG. 5. Cell numbers of HUVECs cultured onto adhesive squares after dif-

ferent time points. The values shown are mean values 6 SD, and each value

was obtained by analyzing more than 200 squares, where eight patterns

from duplicate experiments were included.

FIG. 6. Cell viability of HUVECs cultured onto adhesive micropatterns. The

values shown are mean values 6 SD and each value was obtained by ana-

lyzing over 100 cells from eight samples from duplicate experiments.

031003-7 Chen et al.: Comparative assessment of the stability of nonfouling PMOXA and PEG surface films 031003-7

Biointerphases, Vol. 9, No. 3, September 2014

medium for 10 days at 37 �C, were examined with XPS as

previously described.40 CasaXPS was used for peak assign-

ment C1s, N1s, and Nb3d. Synthetic curves (GL(30)) were

used to fit the peaks based on nonlinear peak fitting. The

C/Nb and N/Nb ratios were calculated according to the

atomic concentrations and the results are summarized in

Table IV. Full spectra are displayed in the supplementary

material Fig. S2 and in Tables S1–S4.47

The C/Nb and N/Nb intensities after immersion in EC

medium for 10 days at 37 �C declined by 20%–28% for

PLL(20)-a(0.19)-PMOXA(4)-coated and PLL(20)-a(0.29)-

PMOXA(8)-coated Nb2O5 surfaces. This result might be

explained by the fact that a change of ionic strength from the

polymer assembly solution to serum containing EC-medium

results in less polymer coverage as previously described.40

Using the same experimental conditions PLL(20)-a(0.28)-

PEG(5) resulted in a decrease of C/Nb intensities nearly twice

as those observed for PLL-g-PMOXAs. The N/Nb ratio of

PLL(20)-a(0.28)-PEG(5) increased by 125%, indicating pro-

ceeding degradation of the PEG chains of the copolymer with

remaining PLL absorbed onto the surface. These results imply

the loss of antifouling property of PLL(20)-a(0.28)-PEG(5) af-

ter long-time immersion in EC medium.

IV. DISCUSSION

Micropatterning techniques provide a simple and versatile

platform for the precise patterning of molecules on surfa-

ces.51 To study cellular behavior on preformed patterns,

long-term stability of the background passivation is critical.

FIG. 7. (A) Quantification of cellular outgrowth of HFFs into the nonadhesive regions of the micropatterns generated with PLL(20)-a(0.29)-PMOXA(8) and

PLL(20)-a(0.28)-PEG(5) and (B) the number of cells per fibronectin square after different time points. The values correspond to mean values 6 SD. (C)

Fluorescent images of HFF on PLL(20)-a(0.28)-PEG(5) and PLL(20)-a(0.29)-PMOXA(8) patterned surfaces after 16 h incubation and after 1–5 months on

PLL(20)-a(0.29)-PMOXA(8) patterned surfaces in culture. (a), (e), and (i) Alexa-488 labeled fibronectin square structure, (b), (f), and (j) nuclei, (c), (g), and

(k) actin cytoskeleton, and (d), (h), and (l) overlay of images (a)–(c), (e)–(g), and (i)–(k), respectively.

TABLE III. Thickness change of the copolymer films in endothelial cell medium at 37 �C after 10 days in culture. Each value was calculated with four measure-

ments on two individual samples.

Film PLL(20)-a(0.19)-PMOXA(4) PLL(20)-a(0.29)-PMOXA(8) PLL(20)-a(0.28)-PEG(5))

Thickness of the fresh film (nm) 1.31 6 0.03 1.43 6 0.03 1.71 6 0.03

Thickness of the film after immersion (nm) 1.13 6 0.12 1.09 6 0.12 0.91 6 0.06

Relative remaining thickness (%) 86 6 9 76 6 9 53 6 4

031003-8 Chen et al.: Comparative assessment of the stability of nonfouling PMOXA and PEG surface films 031003-8

Biointerphases, Vol. 9, No. 3, September 2014

To this end, various parameters such as geometry, substrate,

cell-type, serum content of the culture medium, and polymer

types have been shown to contribute to long-term pattern sta-

bility in prolonged adherent cell pattern experiments.41–43

In this study, we systematically compared the influence of

the polymer architecture on pattern stability with two differ-

ent human cell types, namely, HUVEC and HFF seeded on

adhesive pattern with 90 � 90 lm2 dimensions. HUVECs

were chosen in order to work with an environmental sensi-

tive model cell type and were cultivated on adhesive patterns

of Oregon-green labeled fibrinogen. All polymers were capa-

ble to passivate the non-adhesive background regions against

HUVEC adhesion and outgrowth for 5 days in culture.

Interestingly, both types of PLL-g-PMOXA exhibited good

anti-adhesive properties for a total period of 21 days of incu-

bation in direct comparison to PLL-g-PEG. The observed

results for HUVECs are in line with the pattern stability

found for HFFs, seeded on similar fibronectin squares in

10% serum containing medium. Pattern stability was signifi-

cantly reduced after 16 h when HFFs were seeded on

PLL-g-PEG spaced pattern, whereas patterns produced by

PLL-g-PMOXA were shown to prevent cell growth for 1.5

month in culture. The differences in pattern stability

observed between HUVECs and HFFs clearly indicate that

pattern stability strongly depends on the used cell-type.

Other studies in this field observed that patterns with 200 �200 lm2 dimensions produced by MAPL and lcP remained

stable for 13 days when NIH-3T3 fibroblasts were seeded.43

Huang and colleagues used three different cell types

[NIH-3T3 fibroblasts, PC12, bone marrow derived mesen-

chymal stem cells (MCS)] for the analysis of pattern stability

generated with UV-irradiation using a photomask with spac-

ing distances of either 200 lm or 250 lm, respectively.52

The authors observed no loss in pattern stability generated

by oligo(ethylene glycol) methyl ether metacrylate

(OEGEMA) over different times points of incubation (19

days for MCS; 21 days for NIH-3T3 fibroblasts).

Degradation of micropatterns has been previously

described as interplay between cell dependent and cell-

independent mechanisms.53 Beside chemical alterations of

the polymer, cellular outgrowth on patterns spaced by non-

adhesive polymers could occur due to matrix deposition of

cell secreted extracellular matrix (ECM) proteins or by

cellular bridging between two adjoining adhesive pattern.

We studied the elongation of HFFs cultured on fibrin hydro-

gels recently.54 Cell length of individual fibroblasts was

160 lm in average, indicating that single cells are physically

able to bridge between adjacent adhesive patterns spaced by

a distance of 30 lm. Fibroblasts are known to be highly

active metabolic and mobile cells, which are responsible for

structural protein turnover within the ECM. In our study,

cell numbers of HFFs cultured on Alexa-488 labeled fibro-

nectin adhesive squares in medium supplemented with 10%

serum were found to double after 40 h in culture whereas en-

dothelial cells were displayed to reduplicate after 10 days in

culture in medium, containing 2% serum. This finding

clearly indicates that endothelial cells are less active in terms

of cell division on similar patterns compared to fibroblasts.

Moreover, this result might explain why endothelial cells

were five times longer restricted to pattern spaced with

PLL-g-PEG than similar surfaces cultured with HFFs. In

addition to cellular ECM secretion, breakdown of surfaces

can be caused by the activity of secreted proteases, which

actively modulate the cellular environment.53 It might be of

interest to compare the pattern stability of PMOXA and PEG

based thin films with cells treated with specific protease

inhibitors in order to investigate how polymer chemistry pre-

vents pattern failure in respect to enzymatic activity.

The type of the ECM adhesive proteins chosen has been

reported to have no significant influence on the stability of

the generated pattern.43 However, we observed that Alexa-

488 labeled fibronectin was partially endocytosed by HFFs

after long time of incubation of 1.5 months as pointed out

previously.42,43 This observation indicates that the initial ad-

hesive fibronectin layers have been replaced by cellular

derived proteins and/or have been rearranged due to cellular

forces.

The fact that pattern stability of both types of PLL-g-

PMOXAs was significant enhanced implies that the stability

variations are due to chemical degradation mechanisms of

the respective polymer side chains as indicated by the loss of

monolayer film thickness of PLL-g-PEG after exposure to

cell free EC medium. This hypothesis is supported by the ob-

servation that PEG degradation has been previously deter-

mined in aqueous solution and within surface bound

monolayers.2 A recent study indicates that PEG degradation

involved primarily the degradation of the polyether side-

chain and not the desorption of the entire molecule from the

Nb2O5-surface as determined by XPS measurements.40 Such

altered PLL-g-PEG monolayers showed strong protein

absorption after exposure to full human serum while similar

thin films of PLL-g-PMOXA remained largely protein resist-

ant for two weeks.40 Complementary to this finding, our

XPS measurements indicate that PMOXA side-chains are

more chemically resistant to exposure to EC-medium in con-

trast to PEG-moieties. We therefore speculate that patterns

formed by PLL-g-PEG polymers are more prone to chemical

degradation under cell culture conditions than similar pat-

terns generated by PLL-g-PMOXA. Serum proteins

adsorbed from the medium onto degraded PLL-g-PEG

TABLE IV. Characteristics of the copolymer films on the Nb2O5 surface

freshly prepared and after incubation in EC medium for 10 days: C/Nb and

N/Nb ratios.

Substrates

Analyzed

parameter

Freshly

prepared

In EC

medium

for 10 days

Relative

change

(%)

PLL(20)-a(0.19)-PMOXA(4) C/Nb 1.42 1.09 �23

N/Nb 0.36 0.26 �28

PLL(20)-a(0.29)-PMOXA(8) C/Nb 1.52 1.20 �21

N/Nb 0.37 0.29 �22

PLL(20)-a(0.28)-PEG(5) C/Nb 1.72 1.02 �41

N/Nb 0.04 0.09 125

031003-9 Chen et al.: Comparative assessment of the stability of nonfouling PMOXA and PEG surface films 031003-9

Biointerphases, Vol. 9, No. 3, September 2014

polymer layers might thus be responsible for raised cellular

outgrowth from adhesive patterns and therefore contribute to

pattern failure.

V. CONCLUSION

This study clearly confirms that micropatterns generated

by PLL-g-PMOXA provide better long-term stability than

similar patterns generated by PLL-g-PEG under cell culture

conditions. Moreover, PLL-g-PMOXA monolayers showed

superior chemical stabilities than PLL-g-PEG monolayers,

when homogeneous copolymer films in cell free EC me-

dium using XPS and VASE were analyzed. We therefore

conclude that micropatterns generated by PLL-g-PEG are

more prone to chemical degradation and subsequent serum

adsorption under cell culture conditions than similar pat-

terns generated by PLL-g-PMOXA copolymers. We antici-

pate that biomedical applications, in which long-term

stability in terms of antifouling properties are crucial, might

highly benefit from PMOXA based surface modifications in

the future.

ACKNOWLEDGMENTS

The authors gratefully acknowledge financial support

from the Competence Center for Materials Science and

Technology CCMX MatLife (H.H., Grant No. 0-21154-07).

The authors thank Mathias Rodenstein for excellent help

with XPS analysis. This work is dedicated to Heike Hall.

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