The preparation of PE-g-HEMA (polyethylene-grafted-hydroxyethyl methacrylate) films by 60Co gamma irradiation has been optimized to produce a new material suitable for bioapplications.http://www.sciencedirect.com/science/article/B6TJN-4PKFGWY-1/1/ce67dece44bee190c7fdab19460ed6d

Ferreira, L. M.

Falcão, A. N.

Gil, M. H.

Estudo Geral

Available online at www.sciencedirect.comwww.elsevier.com/locate/nimb
Nuclear Instruments and Methods in Physics Research B 265 (2007) 193–197
NIMB
Beam Interactions
withMaterials &AtomsElemental and topographic characterization of LDPE
based copolymeric ﬁlms obtained by gamma irradiation
L.M. Ferreira a,*, A.N. Falca˜o a, M.H. Gil b
a Department of Physics, Nuclear and Technological Institute, E.N 10, 2686-953 Sacave´m, Portugal
b Department of Chemical Engineering, Faculty of Science and Technology, University of Coimbra, Po´lo II – Pinhal deMarrocos,
3030-290 Coimbra, Portugal
Available online 5 September 2007Abstract
The preparation of PE-g-HEMA (polyethylene-grafted-hydroxyethyl methacrylate) ﬁlms by 60Co gamma irradiation has been opti-
mized to produce a new material suitable for bioapplications.
This paper reports the elemental and topographic analysis of PE-g-HEMA ﬁlms with diﬀerent grafting degrees, obtained under opti-
mized experimental conditions, with the purpose to appraise their possible toxicological risk and their surface roughness properties.
In this way a Nuclear Microprobe using PIXE (Proton Induced X-ray Emission) technique was used to assess the elemental analysis
of the ﬁlm samples prepared. Trace contaminations of elements heavier than Si (Ca, Cl, Fe, K, P, S, Si and Zn) were found, but their
concentrations do not pose toxicological risk.
Atomic Force Microscopy (AFM) and Scanning Electronic Microscopy (SEM) techniques were used to assess the morphological
characterization of the ﬁlms, revealing the roughness and 3D porous structure of their surface, which are ones of essential characteristics
for bio-interaction or cell adhesion.
 2007 Elsevier B.V. All rights reserved.
PACS: 28.52.Fa; 82.35.Jk; 82.50.Kx; 82.80.Ej; 68.37.Hk; 68.37.Ps
Keywords: Graft copolymers; Gamma radiation; Polymers for bioapplications; Polymer elemental and topographic characterization1. Introduction
The selection of materials for bioapplications requires
proper considerations of its mechanical, physical and bio-
logical properties, and very rarely one ﬁnds a single natural
or synthetic material with all the adequate properties. The
development of new materials by manipulating their molec-
ular structure by means of gamma or high-energy electrons
irradiation has been proved to be successful in producing
materials with adequate surface and bulk properties, which
make their use promising notably for medical implant0168-583X/$ - see front matter  2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.nimb.2007.08.047
* Corresponding author. Tel.: +351 21 994 6066; fax: +351 21 994 1525.
E-mail address: ferreira@itn.pt (L.M. Ferreira).devices. These techniques are being used with increasing
success with polymeric based systems [1].
To adequate the material for bioapplications (health and
environment), the molecular structures of the precursors are
generally modiﬁed to improve mechanical resistance,
hemocompatibility, hydrophylicity and bio-interaction
ability (cell adhesion and/or proliferation) [2]. Important
questions to address are: (i) once implanted or when in con-
tact with body ﬂuids the new polymeric material can be
toxic? How clean and safe is it? (ii) Is the material surface
adequate for the required application? To answer these
questions a careful characterization of the new material is
necessary.
In our studies, using 2-hydroxyethyl methacrylate
(HEMA) as grafting monomer, we have optimized the
preparation of new grafted copolymeric Low Density
194 L.M. Ferreira et al. / Nucl. Instr. and Meth. in Phys. Res. B 265 (2007) 193–197Polyethylene (LDPE) based ﬁlms by means of 60Co gamma
irradiation. These copolymeric ﬁlms developed show, at a
macroscopic scale, a homogeneous graft distribution
(independent of the grafting degree) reaching hydration
levels up to 95% [3,4].
In this work stage the characterization of these ﬁlms was
done using Nuclear Microprobe (NM), Atomic Force
Microscopy (AFM) and Scanning Electron Microscopy
(SEM). These techniques reveal to be very important
for their elemental and topographic characterization. Ele-
mental analysis performed by Nuclear Microprobe using
PIXE (Proton Induced X-ray Emission), allowed evalua-
tion of the toxicological risk in the possible uses of the
new ﬁlms, while AFM and SEM techniques revealed the
topography and 3D porous structure of the polyHEMA
grafted surface that grows over the LDPE matrix ﬁlm.
Morphological analysis was important to inform about
polyHEMA grafted chain scission processes, which may
occur with increasing radiation dose above a certain
threshold [3].
2. Experimental
2.1. Materials
PE-g-HEMA copolymeric ﬁlms grafted at 237%
(d = 46 lm), 403% (d = 73 lm), 166% (d = 40 lm) and
163% (d = 38 lm), were obtained by mutual gamma irradi-
ation method (in the absence of air) at a dose rate (DR) of
0.3 kGy h1, during 25 h (D = 7.5 kGy), 30 h (D =
9.0 kGy), 35 h (D = 10.5 kGy) and 40 h (D = 12.0 kGy),
respectively, using a [HEMA]i = 15% (V/V in methanol).
Details of the experimental preparation conditions can be
found elsewhere [3,4].
The matrix used for the grafting process was bioriented
ﬁlm of LDPE (q = 0.920 g cm3) with 15 lm of thickness
commonly used for food packaging applications.
Fig. 1 shows a piece of LDPE matrix and the four
grafted ﬁlms analyzed.
The irradiation experiments were performed at the Por-
tuguese 60Co Facility (UTR) installed at the Nuclear and
Technological Institute (ITN), in Lisbon.Fig. 1. Analyzed ﬁlms. copolymeric ﬁlms are presented2.2. Samples characterization
2.2.1. Nuclear microprobe techniques
Analyses were performed with a Nuclear Microprobe
using simultaneously the techniques of PIXE (Proton
Induced X-ray Emission), RBS (Rutherford Backscattering
Spectrometry) and STIM (Scanning Transmission Micros-
copy). A 2 MeV energy proton beam focused to 3 · 4 lm2,
with beam currents of about 200 pA was used. Scanning
proton beam amplitudes of 530 · 530 lm2 was used to
obtain elemental distributions tables. Details of the instru-
mental settings can be found elsewhere [5].
2.2.2. Atomic force microscopy
Tapping mode atomic force microscopy (TMAFM)
ﬁlms images were acquired with a Veeco di CP-II System
and standard etched silicon probes (nominal spring con-
stant 50 N/m, resonance frequency 300 kHz).
2.2.3. Scanning electronic microscopy
LDPE and PE-g-HEMA ﬁlms were coated with a thin
layer of gold under reduced pressure and their scanning
electron micrographs were obtained using a JEOL (JSM
5310) scanning electron microscope.
Porous dimension and distribution analysis were made
using IMAGE J (V1.36b) software from WCIF (NIH-
USA).
3. Results and discussion
3.1. Nuclear microprobe techniques
In spite of diﬀerent nuclear microprobe techniques have
been used, in this section only data from PIXE will be dis-
cussed since analysis of the RBS and STIM data is still
ongoing.
Proton irradiation of samples, performed at the nuclear
microbe, result in the emission of X-rays characteristics
of the chemical elements present in the materials.
Under the experimental conditions used for the PIXE
experiments [5], it is only possible to detect elements
with atomic number equal or higher than Si. This meansin their ﬁnal standard analysis format (8 · 2 cm2).
L.M. Ferreira et al. / Nucl. Instr. and Meth. in Phys. Res. B 265 (2007) 193–197 195that, even though being unable to give information about
the thirteen chemical elements lighter than Si, the technique
is sensitive to the elements that can be the most eﬀectively
toxic contaminants of a biomaterial (metals and transition
metals).
Quantitative analysis performed on the PIXE data is
shown in Table 1. The results refer to a 530 · 530 lm2 scan-
ning area for each sample.
The results show traces of impurity contaminations (Si,
P, S, Cl, K, Ca, Ti, Fe and Zn) in the LDPE ﬁlm matrix
and in the copolymeric ﬁlms prepared, although their con-
centrations do not pose human toxicological risk [6–10].
The concentrations found are of ppm (lg g1) order, and
it was observed a reduction in Cl, K and Ca concentration,
a part from a ﬁlm (25 h, 237%), relatively to those found in
the LDPE ﬁlm matrix.
P, Cl, K and Ca are elements which are needed in the
human diet in amounts of 100 mg or more each day [7].
Phosphorus was only detected in the ﬁlm matrix
(72 ± 27 lg g1), and the highest concentration of Cl, K
and Ca found in the copolymeric ﬁlms were 171 ± 3,
31 ± 2 and 159 ± 2 lg g1 respectively.
Si, Fe and Zn are elements which the human body
requires in very small quantities, generally less than
100 mg/day [7]. Silicon is non-toxic as element and in all
its natural forms, namely silica and silicates, which are
the most abundant. The only way, medically reported, that
silicon may cause diseases is by inhalation in great quanti-
ties of crystalline silica (silicon dioxide) dust (e.g. miners
and stonecutters). In that case it is a potent respiratory haz-
ard (LD50(oral) = 3160 mg/kg bw) resulting in a serious
lung disease known as silicosis [8]. In our case the most sil-
icon contaminated copolymeric ﬁlm shows a concentration
of 752 ± 30 lg g1.
Humans might experience iron toxicity above 20 mg/kg
bw, and 60 mg/kg bw is a lethal dose [9], though our most
iron contaminated ﬁlm shows a value of 18 ± 1 lg g1. For
zinc the higher concentration found in the new ﬁlms is
51 ± 4 lg g1, and it is reported that evidences of adverse
eﬀects in health appears with intakes of 100–300 mg/day
[10].
Elemental sulphur is not toxic and is an essential
component of all living cells. It is especially important
for humans because of its role in protein metabolisms [8].
In our samples only one copolymeric ﬁlm shows a contam-
ination of 16 ± 5 lg g1 of S, value that can be
disregarded.Table 1
Average impurities concentration values found in the analyzed samples (lg g
Material Impurities
Si P S Cl
LDPE matrix 324 ± 84 72 ± 27 – 236 ± 1
Film (25 h, 237%) 323 ± 32 – – 22 ± 3
Film (30 h, 403%) 394 ± 39 – 16 ± 5 171 ± 3
Film (35 h, 166%) 752 ± 30 – – 71 ± 3
Film (40 h, 163%) 528 ± 37 – – 25 ± 3Titanium was detected only in one the new ﬁlms with a
concentration of 2 ± 1 lg g1. Ti is not a poison metal and
the human body can tolerate it in large doses being one of
the elected materials for hard body implants [6].
The contaminants present in the PE-g-HEMA ﬁlms that
are not present in the LDPE matrix (S, Ti and Zn), were
identiﬁed to come from the reagents used during the prep-
aration of the copolymeric ﬁlms. However it can not be
directly related with the grafting degree since there are
some oscillations in the concentration of the elements iden-
tiﬁed in the copolymeric ﬁlms. This fact suggests that, dur-
ing the grafting process, some impurities may become
trapped. The eﬀectiveness of the puriﬁcation stage to
remove them will depend on the speciﬁc position where
trapping occurs and of the speciﬁc polymeric network envi-
ronment surrounding it.
Data obtained from the RBS and STIM techniques are
expected to help to clarifying this issue. Thickness/density
maps as well as impurity distribution maps will be the
object of a forthcoming paper.3.2. Topographic analysis
3.2.1. Atomic force microscopy
AFM micrographs (Fig. 2) show that, while the LDPE
matrix presents a quite ﬂat and smooth surface, the pre-
pared ﬁlms show a rough surface. The estimated roughness
(peak-valley) for LDPE matrix is about 5 nm (evidence of
nanoscale structure at surface), and vary from 7.56 lm to
6.03 lm in the copolymeric ﬁlms.
The ﬁlms grafted at 243% (tirrad. = 25 h) and 403%
(tirrad. = 30 h) show a surface with broad peaks and valleys,
suggesting the presence of a dense grafted layer. In opposi-
tion, the ﬁlms grafted at 166% (tirrad. = 35 h) and 163%
(tirrad. = 40 h) show a sharp peak-valley surface.
In earlier studies, it was observed that the grafting yield
goes through a clear maximum at a certain irradiation
dose, after which begins to decrease with the increase of
total absorbed dose, even with a bulk monomer concentra-
tion enough to carry-over the grafting reaction. It was sug-
gested that this happens as result of the destructive eﬀect of
gamma radiation, for doses above the maximum grafting
yield over polyHEMA branches grafted onto LDPE sur-
face [3]. Judging from the morphology revealed by AFM
images (Fig. 2(d) and (e)), this partial destructive eﬀect,
that dominates over the chain linking process, works like1)
K Ca Ti Fe Zn
2 77 ± 9 148 ± 7 – 7 ± 4 –
7 ± 2 159 ± 2 – – 17 ± 2
18 ± 2 20 ± 21 – 2 ± 1 51 ± 4
31 ± 2 56 ± 1 – 18 ± 1 21 ± 2
5 ± 21 56 ± 2 2 ± 1 – 19 ± 2
Fig. 2. Topographic 3D projection of ﬁlms surface. (a) LDPE ﬁlm (scanned area: 20 · 20 lm2); (b) PE-g-HEMA ﬁlm – 25 h, 237% (scanned area:
35 · 35 lm2); (c) PE-g-HEMA ﬁlm – 30 h, 403% (scanned area: 33 · 33 lm2); (d) PE-g-HEMA ﬁlm – 35 h, 166% (40 · 40 lm2); (e) PE-g-HEMA ﬁlm –
40 h, 163% (scanned area: 50 · 50 lm2).
196 L.M. Ferreira et al. / Nucl. Instr. and Meth. in Phys. Res. B 265 (2007) 193–197an abrasive tool, leading to a thinner ﬁlm with a very
homogeneous rough surface.
Roughness observed in the copolymeric ﬁlms make
them very interesting and promising for bioapplications.
Peaks and valleys deﬁne a surface adequate for bio-interac-
tion for cell adhesion and/or proliferation, and physical or
chemical immobilization of active biomolecules (enzymes,
drugs, thromboresistant agents, etc.) [2].
3.2.2. Scanning electronic microscopy
SEM micrographs showing the surface morphology of
the ﬁlms are presented in Fig. 3. LDPE ﬁlm micrograph
show that it has a quite ﬂat surface, conﬁrming AFM
results. The images obtained from the new material reveal
the porous structure resulting from the graft process. It
may be observed the existence of rounded blocks, at diﬀer-
ent levels of profundity, linked by ﬁlaments of diﬀerentFig. 3. SEM micrographs of LDPE ﬁlm (500·) athicknesses that depend on radiation dose (or grafting
yield), forming a 3D porous network.
Whereas the lower grafted ﬁlms shows large porous
paths at the upper surface level, the highest grafted ﬁlm
show an obstructed porous surface structure with dense
rounded polymeric aggregates.
The high grafted ﬁlm (403%) seems to suﬀer a porous
agglutination process as a result of the high yield of graft.
This process leads to a partial blockage of the porous sur-
face structure with the consequent reduction of its hydra-
tion capacity. In fact, in previous hydration studies [3,4],
it was observed that the maximum hydration capacity
(up to 95%) does not correspond to the highest grafted ﬁlm
but to ﬁlms grafted at 230–250% (20–25 h of irradia-
tion). The ﬁlm grafted at 403% (30 h of irradiation) only
reaches a hydration level of 45%. The ﬁlms irradiated dur-
ing 35 h (166%) and 40 h (163%), present a hydrationnd PE-g-HEMA copolymeric ﬁlms (2000·).
0 1 2 3 4 5 6
25h; 237%
30h; 403%
35h; 166%
40h; 163%
PE
-g
-H
EM
A
 fi
lm
Average porous dimensions (µm)
Fig. 4. Average porous dimensions found in LDPE copolymeric ﬁlms;
(bars represent the dimensions range observed in each sample).
L.M. Ferreira et al. / Nucl. Instr. and Meth. in Phys. Res. B 265 (2007) 193–197 197capacity of 25% and 20%, respectively. The observed aver-
age porous dimensions and length spread is in agreement
with this behavior (Fig. 4). It can be seen that the high
grafted ﬁlm presents the lower values. The ﬁlms grafted
at 166% and 163%, although presenting good porosity
are thinner in consequence of the destructive eﬀect of
gamma irradiation, and cannot reach the hydration levels
obtained with 20–25 h of irradiation.
This behavior can now be better understood based on
morphological analysis obtained by SEM.
The inﬂuence of the porous structure on hydration/
dehydration kinetics of the grafted ﬁlms will be addressed
in a forthcoming work.
4. Conclusions
Obtained results evidence that the experimental protocol
developed and optimized to prepare these new copolymeric
ﬁlms is eﬀective. Data collected also shows that the external
fonts of ﬁlm contamination are controlled and that the
ﬁnal puriﬁcation stage leads to a general contamination
reduction relatively to that found in the initial LDPE ﬁlm
used as backbone. The result is a ﬁnal material with non-
toxicological risk.
The ﬁlms surface roughness revealed by AFM technique
meets one of the essential conditions required for bio-inter-action and the 3D porous structure of the copolymeric
ﬁlms observed by SEM allowed a better understanding of
the hydrophilic behavior already observed in previous
work.
To sum up it can be said that the disorder promoted in
the molecular structure of the LDPE ﬁlm by the gamma
irradiation grafting process tailored its surface, improving
the diﬀusion coeﬃcients of other species deep inside the
polymeric matrix and increasing the hydrophilic properties
of the material. In other words, the surface morphological
features of the grafted polyHEMA layer onto LDPE ﬁlm
leads to a new LDPE copolymeric based material very
promising for multiple bioapplications.
Acknowledgements
The authors wish to acknowledge L.C. Alves and C.P.
Marques from ITN Physics Department, for NM (PIXE)
and AFM measurements, and to P. Alves from University
of Coimbra, for SEM images.
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Elemental and topographic characterization of LDPE based copolymeric films obtained by gamma irradiation

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Elemental and topographic characterization of LDPE based copolymeric films obtained by gamma irradiation

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