www.elsevier.com/locate/msec

Materials Science and Engineering C 23 (2003) 779–786

Ion beam induced nanometric structure and oligopeptide adsorption on

patterned polymer surfaces

C. Satrianoa,*, N. Spinellaa, M. Mansob, A. Licciardelloa, F. Rossib, G. Marlettaa

aLaboratory for Molecular Surfaces and Nanotechnologies (LAMSUN), Department of Chemical Sciences, University of Catania,

V. le Andrea Doria, 6, 95125 Catania, ItalybEuropean Commission, Joint Research Centre, 21020 Ispra VA, Italy

Abstract

The adsorption process of the H–Arg–Gly–Asp–OH oligopeptide (RGD sequence) onto surfaces of poly(ethyleneterephtalate) and

poly(caprolactone) patterned by 50 keVAr+ ion beams is discussed. The irradiation through masks patterned with micrometric features was

employed to create grooves and/or pits of nanometric depth. Both XPS and Imaging time-of-flight secondary-ion mass spectrometry (ToF-

SIMS) measurements showed that incubation of the polymer samples with RGD solutions produces a preferential adsorption on the ion-

irradiated areas of the PCL surfaces, while only a negligible adsorption is obtained onto PET. The adsorption behaviour of the RGD is found

to correlate with the surface nanostructuring induced by the ion irradiation.

D 2003 Elsevier B.V. All rights reserved.

PACS: 82.65.Dp; 85.40.Ux; 85.40.Hp; 82.80.Ms; 61.80.�x

Keywords: Surface nanostructuring; RGD adsorption; Ion beam modification; Imaging TOF-SIMS

1. Introduction

Polymers are most frequently used in biomaterial field

as substrates for cell adhesion and growing. In general,

many cellular properties, such as viability, shape, move-

ment (‘‘cell guidance’’), cell signaling and gene expression,

have been discussed in terms of chemistry-related proper-

ties and/or topography of the polymer substrate at the

micro- and nanoscale [1–6]. On the other hand, a very

promising approach to achieve the cytocompatibility of a

given material is to prompt the tissue formation by means

of the controlled immobilization of specific adhesion

sequences of the extracellular proteins [7]. Among these,

one of the most common adhesive sequences is formed by

Arg–Gly–Asp (RGD). This peptide is found in cell-

binding domains of many extracellular matrix proteins,

including fibronectin, vitronectin, and type I collagen [8]

and it has been shown to be the preferred site where

integrins on the surface of cells may bind through specific

0928-4931/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.msec.2003.09.095

* Corresponding author. Tel.: +39-095-7385075; fax: +39-095-336422.

E-mail address: csatriano@unict.it (C. Satriano).

ligand–receptor interactions, eliciting different cellular

responses, including adhesion, spreading and focal contact

adhesion [9].

In this paper, we report a bioengineering methodology

to promote selective cell response to patterned surfaces,

based on the preferential adsorption of the RGD tripeptide

on poly(ethyleneterephtalate) and poly(caprolactone) surfa-

ces irradiated by 50 keV Ar+ to obtain groove or pit arrays

of nanometer depth distributed in lateral micrometric

patterns.

2. Materials and methods

2.1. Preparation, surface modification and characterization

of the polymer films

Poly(q-caprolactone) (PCL) and poly(ethyleneterephta-

late) (PET) films (from Aldrich) were deposited on p-doped

silicon (100) wafers by spin coating (3000 rpm, 60 s, room

temperature) of a chloroform (PCL) or a 20/80 mixture of

chloroform/1,1,1,3,3,3-Hexafluoro-2-propanol (PET) solu-

tion of the dissolved powders. The thicknesses of the

deposited films were, respectively, 100(F 20) nm for PCL

C. Satriano et al. / Materials Science and Engineering C 23 (2003) 779–786780

and 600(F 50) nm for PET, as measured from an alpha-step

profilometer. The surface patterning was performed by

irradiation through nickel masks with 50 keV Ar+ at a

fluence of 1�1015 ions/cm2 (RT, chamber pressure better

than 10� 5 Pa, current density of 1.5 AA/cm2). Typical

examples of the standard patterns are reported in Fig. 1.

The diameters of the dots and the width of the stripes were

20, 60, 80, 160 and 360 Am, the pitch between the dots or

stripes ranged between 120 and 500 Am. Optical microsco-

py showed that a good control of the lateral dimension of

the features at the micrometer scale is obtained by Ar+

irradiation. After irradiation, the samples were aged in the

laboratory atmosphere for 1 week before both surface

physico-chemical characterization and peptide adsorption

experiments.

2.1.1. X-ray photoelectron spectroscopy (XPS)

XPS analysis was carried out with a PHI 5600 Multi

Technique Spectrometer equipped with a dual Al/Mg

anode, a hemispherical analyzer and an electrostatic lens

system (Omni Focus III). The electron take-off angle was

45j and the analyzer was operated in FAT mode by using

the Al Ka1,2 radiation with pass energies of 187.85 and

11.75 eV for survey and detailed scans, respectively.

Fig. 1. OM micrographs of PCL pattern

According to the attenuation length value of 2.8 nm for

N1s photoelectrons [10] in organic materials, the estimated

vertical sampling depth is about 6.0 nm. The spectra were

analyzed by using an iterative least squares fitting routine

based on Gaussian peaks and the Shirley background

subtraction. Binding energies (BEs) of all the spectra were

referenced to the intrinsic hydrocarbon-like C1s peak at

285 eV.

2.1.2. Optical microscopy (OM) and atomic force micro-

scopy (AFM)

Optical Microscopy was performed by means of a

Leika DM-RME microscope with 50 to 1000� magnifi-

cation equipped with a Polaroid digital camera. The

surface micro- and nano-topography as well as the rough-

ness were measured with a Multimode/Nanoscope IIIA

Atomic Force Microscope (Digital Instruments) in tapping

mode in air with a standard silicon tip. The relative room

humidity was 30% and the room temperature was 25 jC.Data were acquired on square frames having edges of 10

Am, 1 Am and 350 nm. Images were recorded using

height, phase-shift and amplitude channels with 512� 512

measurement points (pixels). Measurements were made

twice or three times on different zones of each sample.

ed surfaces with grooves and pits.

Table 1

XPS average atomic concentrations and characteristic atomic ratios for

untreated and 50 keV Ar+-irradiated PCL before and after incubation in

RGD solution at pH= 2, pH= 5 and pH= 10

C1s

(at.%)

O1s

(at.%)

N1s

(at.%)

C/O N/O

Untreated PCL 77.8 22.2 0.0 3.50

Untreated PCL+RGD pH= 2 77.6 22.4 0.0 3.46

Untreated PCL+RGD pH= 5 73.8 26.2 0.0 2.82

Untreated PCL+RGD pH= 10 77.3 22.1 0.3 3.50 0.01

50 keV Ar+ irr. PCL 85.3 14.5 0.2 5.88 0.01

50 keV Ar+ irr. PCL+RGD pH=2 81.5 17.4 1.1 4.68 0.06

50 keV Ar+ irr. PCL+RGD pH=5 81.8 16.3 2.7 5.02 0.17

50 keV Ar+ irr. PCL+RGD pH=10 82.5 16.4 1.1 5.03 0.07

Statistical error fF 1%.

C. Satriano et al. / Materials Science and Engineering C 23 (2003) 779–786 781

Surface roughness values were determined in five random

areas per sample, scanning across areas 10� 10 and 1�1

Am2. Roughness parameter (Ra and Rms) calculation and

image processing were performed using the Nanoscope III

software.

2.1.3. Time-of-flight secondary-ion mass spectrometry

(ToF-SIMS)

Time-of-Flight secondary-ion mass spectra were

recorded in a IONTOF TOF-SIMS IV instrument, using

25 keV Ga+ primary ions (PI). Secondary ion spectra and

mass resolved images were typically taken over areas in the

range 2� 10� 4–4� 10� 4 cm2. PI fluences were < 3� 1011

and < 1�1012 ions cm� 2 for spectra and image acquisition,

respectively, in both cases below the static SIMS limit.

Surface charge compensation was accomplished by pulsed

slow (20 eV) electron irradiation.

2.1.4. Zeta-potential measurements

Surface Z-potential (ZP) values were monitored along

a pH range of 4–10 by using a Bi-Eka electrokinetic

analyser (Anton Paar, Austria). A special cell for flat

surface analysis was used with half contribution form the

sample and half contribution from a capillary patterned

PMMA surface. Z-potential was calculated by the Fair-

brother–Mastin algorithm from the streaming curves

(max pressure 600 mbar, two measurements in two

flowing directions between the platinum electrodes). As

Zpot values were compared with those of untreated

polymer surfaces, the PMMA contribution was not sub-

tracted from the graphs.

2.1.5. Surface free energy (SFE) measurements

Measurements of Surface Free Energy were performed

by evaluating both static and dynamic contact angles of

three different liquids onto the untreated and treated

surfaces. Half-automatic video-based measurements of

contact angle were performed at 25 jC and 65% relative

humidity by using an OCA30 instrument (Dataphysics).

The advancing (hadv) and the receding (hrec) contact

angles were measured by the needle-syringe method

[11]. At least five measurements were made for each

sample and then averaged. The Surface Free Energies, in

terms of apolar Lifshitz–van der Waals (gLW) and polar

Lewis acid (g+) and basic (g�) components, were evalu-

ated by using the Good–van Oss model [12], with

ultrapure Millipore water, glycerol and tricresyl phosphate

(Aldrich).

2.2. Peptide adsorption experiments

Arg–Gly–Asp (RGD) sequence was purchased as pow-

der (Aldrich) and solubilized in ultrapure MilliQ water to a

final concentration of f 2 mM at the pH values, respec-

tively, of 5.35(F 0.02), 10.33(F 0.02) and 1.78(F 0.02).

According to the isoelectric pH values of arginine (Arg)

and asparagines (Asp) residuals, respectively, of 10.76 and

2.77, the RGD molecules in the used solutions are estimat-

ed to be prevalently: (a) for pHf 5, in the neutral

zwitterionic form, (b) for pHf 10, in the completely

deprotonated, negatively charged form and (c) for

pHf 2, in the protonated, positively charged form. The

RGD adsorption process was performed by soaking the

substrates for 1 h in the peptide solution, followed by

gentle rinsing with 1 ml of Millipore water, and drying step

in air.

3. Results and discussion

3.1. RGD adsorption on the ion-irradiated surfaces

The adsorption of RGD from aqueous solutions at

different pH on PCL and PET samples was investigated

by AFM and Imaging ToF-SIMS analysis for the surfaces

patterned with grooves and pits, and by means of XPS

analysis for laterally homogeneous surfaces.

Tables 1 and 2 report the average surface atomic compo-

sitions and the characteristic atomic ratios from XPS data,

before and after incubation with RGD, for both untreated

and 50 keV Ar+-irradiated surfaces.

It can be seen that for both polymers that the oxygen

content at surfaces decreases after irradiation. In fact, for PCL

the stoichiometry changes from C3.5O1 to C6O1, and for PET

it changes from C2.4O1 to C5.5O1. Furthermore, both polymer

surfaces exhibit very low traces ( < 1%) of nitrogen after

irradiation, probably due to the reaction of surfaces with

contaminants from atmosphere after irradiation. More in

details, the analysis of XPS high resolution spectra (not

shown) evidenced that for PET and PCL surfaces ion irradi-

ation induces a common carbonization pattern, basically

involving the marked decrease of COO� (at 289.7F 0.2

eV), and a persistence or slight increase of C–O–C or C–

OH groups (at 286.4F 0.2 eV) and C = O groups (at

288.3F 0.2 eV). In addition, for PET it is apparent the

disappearance of the spectral features characteristic of the

Table 2

XPS average atomic concentrations and characteristic atomic ratios for

untreated and 50 keV Ar+-irradiated PET before and after incubation in

RGD solution at pH= 2, pH= 5 and pH= 10

C1s

(at.%)

O1s

(at.%)

N1s

(at.%)

C/O N/O

Untreated PET 70.3 29.7 0.0 2.37 0.00

Untreated PET+RGD pH= 2 76.0 23.2 0.9 3.28 0.04

Untreated PET+RGD pH= 5 76.0 23.2 0.8 3.28 0.03

Untreated PET+RGD pH= 10 74.3 25.0 0.7 2.97 0.03

50 keV Ar+ irr. PET 85.6 14.0 0.4 6.11 0.03

50 keV Ar+ irr. PET+RGD pH= 2 82.4 16.1 1.5 5.12 0.09

50 keV Ar+ irr. PET+RGD pH= 5 84.6 14.4 1.0 5.88 0.07

50 keV Ar+ irr. PET+RGD pH= 10 84.0 15.6 0.4 5.38 0.03

Statistical error =F 1%.

C. Satriano et al. / Materials Science and Engineering C 23 (2003) 779–786782

aromatic structure (i.e., the shake-up bands at about 6–7 eV

from the main O1s and C1s peaks).

The analysis of nitrogen concentration in Tables 1 and 2

clearly shows that after incubation in RGD solution, only

irradiated PCL surfaces exhibit a preferential adsorption of

Fig. 2. Tapping mode AFM height images of: (a) a pit region on PCL; a region ou

before (d) and after (e) RGD incubation.

peptide onto irradiated surfaces, with a major effect at

pH = 5. For PET surfaces, the adsorption experiments show

no selectivity and very low adsorption (f 1% of N

content) going from the unirradiated samples to the irradi-

ated ones.

Fig. 2 shows the AFM height images of a pit patterned

by irradiation in a PCL surface (Fig. 2a), with the zoom

areas taken outside (Fig. 2b–c) and the ones taken inside

the pit (Fig. 2d–e), before and after RGD incubation. The

area outside the pit exhibits a very rough topography both

before and after incubation with RGD, the height of the

features being about 20 nm, with Rms values, respectively,

of f 4 nm before and f 3 nm after incubation. At

variance of this, the irradiated area inside the pit exhibits

much lower features, with a typical height of f 8 nm and

Rms f 1 nm. This surface becomes even smoother after

incubation, with features high f 5 nm and Rms values

of f 0.8 nm.

A confirmation of the occurrence of RGD preferential

adsorption in the irradiated grooves and pits was obtained

tside the pit before (b) and after (c) RGD incubation; a region inside the pit

C. Satriano et al. / Materials Science and Engineering C 23 (2003) 779–786 783

from Tof-SIMS imaging. Fig. 3 displays the ToF-SIMS

negative mass-resolved ion images of a pit-patterned PCL

and a groove-patterned PET sample. The images were

obtained by selecting characteristic fragments for RGD-

incubated samples, as well as those based on the lack of

the substrate-characteristic peaks. In particular, for both

PCL and PET irradiated surfaces the prevalence in the

irradiated areas of the peaks due to NH� (m/z= 15), CN�

(m/z = 26), C2H2� (m/z = 26) and CNO� (m/z = 42) was

clearly observed. On the other hand, in the maps of

fragments related to the polymer matrix, while a good

contrast was observed in the case of PET (images based

on C6H4� and C7H5O2

� at m/z 76 and 121, respectively), in

the case of PCL (images based on fragments at m/z 113

and 115, assigned to C6H9O2� and C6H11O2

�), contrast was

not very good. It is to note that, the ion images of the bare

samples before the incubation with RGD were qualitative-

Fig. 3. ToF-SIMS mass resolved images of RGD-incubated surfaces of PCL with

ion images (TIC) are shown as well.

ly similar to those of incubated samples, due to the

interference of nitrogen-containing species due to the

post-irradiation reaction in atmosphere of the surfaces

(see above).

In any case, significant differences between irradiated and

non-irradiated areas are evidenced from the high mass

resolution spectra. Fig. 4, for example, reports the regions

around nominal m/z 42 and 43, where CNO� and C2H3O�

fragments are observed. The CNO� to C2H3O� intensity

ratio is about 0.12 for both untreated PET (Fig. 4a–b) and

PCL (Fig. 4c–d) irrespectively of the fact that the sample

were or not incubated with RGD. The ratio increases to f 1

for both bare irradiated PET (Fig. 4e) and PCL (Fig. 4g), due

to the post-irradiation uptake of N-containing species at

surfaces. After the RGD incubation, a further increase of

CNO� intensity (relative to that of the C2H3O�) is observed,

leading to a ratio of f 4 for PCL (Fig. 4h) and f 2.5 for

a pit (upper panels) and PET with a groove (lower panels). Total secondary

Fig. 5. Zeta-potential curves for unirradiated and 50 keVAr+-irradiated PCL

(a) and PET (b) samples.

Fig. 4. Portion of high resolution spectra showing the intensity behaviour of

CNO� (m/z = 42) and C2H3O� (m/z 43) for: (a) PET untreated, (b) PET

untreated +RGD, (c) PCL untreated, (d) PCL untreated +RGD, (e) 50 keV

Ar+-irr. PET, (f) 50 keV Ar+-irr. PET+RGD, (g) 50 keV Ar+-irr. PCL, (h)

50 keV Ar+-irr. PCL+RGD.

C. Satriano et al. / Materials Science and Engineering C 23 (2003) 779–786784

PET (Fig. 4f). These findings indicate that actually, in the

limit of the ToF-SIMS sensitivity, ion irradiation prompts the

RGD adsorption on both kinds of polymers and that, in

agreement with the XPS data, the uptake of RGD is more

efficient for PCL than for PET.

3.2. Surface properties of the patterned areas

The very important issues concerning the properties of

the interface with an aqueous solution were studied by

means of zeta-potential measurements (ZP), providing the

surface charge in various pH conditions. Furthermore, also

the total surface free energy and the related polar and

dispersive components were investigated by using the

contact angle technique.

Fig. 5 reports the ZP curves for PCL and PET unirradi-

ated and irradiated surfaces, investigated both immediately

after the modification (fresh surfaces) and after 1 week of

aging in atmosphere (aged surfaces). One can see that the

behavior of the Ar+-irradiated PCL surfaces does not suffer

any drastic modifications of the surface charge with respect

to the untreated PCL film (Fig. 5a). At variance of this, PET

samples after Ar+ irradiation (Fig. 5b) showed clear eviden-

ces of a remarkable increase of the absolute surface charge

for pH higher than 6 with respect to the unirradiated one.

After the aging, the surface charge recovers the originary

value.

As to the surface free energy properties, the wettability

measurements did not show meaningful changes of both

static and dynamic water contact angle values. In fact both

polymers, having static water contact angles on the unirra-

diated surfaces, respectively, of 69.6F 0.7j for PCL and of

73.1F 0.1j for PET, still remain quite hydrophobic also

after Ar+ irradiation, exhibiting just a restricted change of

contact angle values in the range of 10j. However, the

thorough investigation of the surface free energies in terms

of the Lifshitz–van der Waals and Lewis acid–base com-

ponents indicated that for PCL the polar acid–base param-

eter increases from almost 0 for the untreated surface to f 3

mJ/m2 after irradiation, which is quite the same as the ones

measured for PET before and after irradiations. According-

ly, the preferential RGD adsorption onto PCL cannot be

justified in terms of the SFE effects.

The other important surface parameter possibly affect-

ing the adsorption behaviour is the nanometer-scale

morphology. This has been measured by AFM technique.

Fig. 6 reports the AFM height-images and the cor-

Fig. 8. AFM height images at 10 and 1 Am scale for PCL untreated (a) and

50 keV Ar+-irradiated (b).

Fig. 6. AFM height images and section analysis of a pit area for PCL (a)

and PET (b) surfaces.

C. Satriano et al. / Materials Science and Engineering C 23 (2003) 779–786 785

responding section analysis of the area around a pit

obtained by 50 keV Ar+-irradiation, respectively, for

PCL (a) and PET (b). It appears clearly that very

different depths are obtained for the two polymers, being

f 40 nm for PCL and f 70 nm for PET, respectively. It

is to recall that the huge decrease in thickness observed

for the irradiated areas is due to the densification pro-

cesses, related to the complex chemical reactions induced

by ion irradiation [13].

Fig. 6 shows also clearly that the surface roughness

changes drastically only for PCL surfaces, the PET samples

keeping a very similar smooth morphology before and after

irradiation. Accordingly, both untreated (Fig. 7a) and 50

keVAr+-irradiated PET (Fig. 7b) surfaces do not exhibit any

significant structuring on the nanometer scale. At variance

Fig. 7. AFM height images at 10 and 1 Am scale for PET untreated (a) and

50 keV Ar+-irradiated (b).

of this, for PCL (Fig. 8) characteristic features of nanometric

dimensions, of average height and diameter, respectively, of

3.8F 0.8 and 106F 18, are developed under irradiation

(compare Fig. 8a, for unirradiated surfaces, and Fig. 8b

for the irradiated ones). The measured values for Rms and

Ra are listed in Table 3. Accordingly, the RGD adsorption

behaviour could be phenomenologically related to the much

stronger morphology modification induced by ion irradia-

tion on PCL at the nanometer scale with respect to the

unchanging smoothness of PET surfaces.

Table 3

Roughness parameters Rms, Ra and Rmax values (F standard deviations)

for PCL and PET surfaces, untreated and 50 keV Ar+-irradiated

Rms (nm) Ra (nm) Rmax (nm)

Unirradiated PCL 4.43F 0.76 3.64F 0.71 24.68F 2.04

50 keV Ar+-irradiated PCL 0.94F 0.31 0.75F 0.26 6.05F 1.82

Unirradiated PET 0.27F 0.11 0.19F 0.06 1.35F 0.11

50 keV Ar+-irradiated PET 0.22F 0.05 0.16F 0.03 1.97F 0.85

C. Satriano et al. / Materials Science and Engineering C 23 (2003) 779–786786

4. Conclusions

In this paper, the RGD adsorption has been studied on

PCL and PET surfaces micropatterned with grooves or pits

by irradiation with 50 keV Ar+. The RGD is found to be

preferentially adsorbed only onto irradiated areas of PCL, as

evidenced by AFM, XPS and ToF-SIMS measurements.

According to the reported results, the observed preferen-

tial adsorption of RGD onto irradiated PCL surfaces seems to

be related to a kind of interlocking mechanism, depending on

the observed modification of the morphology at the nanome-

ter scale. At the present stage of the research, we cannot

exclude that a critical size of the surface features is needed to

observe the preferential RGD adsorption, as it is suggested

for instance by the fact that the PCL roughness is decreased

by irradiation.

In any case, further experiments need to be performed in

order to clarify the detailed ion-induced modification mech-

anism of PCL and PET, as well as the detailed nature and

stability of the species produced on the surfaces.

Acknowledgements

The technical assistance of H. Willers for ion implanta-

tion and M. Romor for Z-potential measurements are

gratefully acknowledged. The authors wish to acknowledge

the financial support of FIRB: RBAU01N79B (MIUR) and

PRIN 2001 (MIUR Rome).

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