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Applied Surface Science 253 (2007) 7755–7760
Matrix assisted pulsed laser evaporation of cinnamate-pullulan and
tosylate-pullulan polysaccharide derivative thin films for
pharmaceutical applications
M. Jelinek a,b,*, R. Cristescu a,c, E. Axente c, T. Kocourek a, J. Dybal d, J. Remsa a,J. Plestil d, D. Mihaiescu e, M. Albulescu f, T. Buruiana g, I. Stamatin h,
I.N. Mihailescu c, D.B. Chrisey i
a Institute of Physics ASCR, Na Slovance 2, 18221 Prague 8, Czech Republicb Faculty of Biomedical Engineering CVUT, nam. Sitna 3105, 27201 Kladno, Czech Republic
c National Institute for Laser, Plasma and Radiation Physics, MG-36, RO-77125 Bucharest, Romaniad Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 16206 Prague 6, Czech Republic
e University of Agriculture Sciences and Veterinary Medicine, 59 Marasti, Bucharest, Romaniaf Institute for Chemical-Pharmaceutical R&D, 112 Vitan, 74373 Bucharest 3, Romania
g Petru Poni Institute of Macromolecular Chemistry, Iasi 6600, Romaniah 3Nano-SAE Research Center, University of Bucharest, P.O. Box MG-38, Bucharest-Magurele, Romania
i Rensselaer Polytechnic Institute, Department of Material Science, 110 8th Street, Troy, NY 12180-3590, USA
Available online 25 February 2007
Abstract
We have demonstrated the successful thin film growth of two pullulan derivatives (cinnamate-pullulan and tosylate-pullulan) using matrix
assisted pulsed laser evaporation (MAPLE). Our MAPLE system consisted of a KrF* laser, a vacuum chamber, and a rotating target holder cooled
with liquid nitrogen. Fused silica and silicon (1 1 1) wafers were used as substrates. The MAPLE-deposited thin films were characterized by
transmission spectrometry, profilometry, atomic force microscopy (AFM), Fourier transform infrared (FTIR) spectroscopy and Raman spectro-
scopy. The deposited layers ranged from 250 nm to 16.5 mm in thickness, depending on the laser fluence (0.065–0.5 J cm�2) and number of pulses
applied for the deposition of one structure (1500–13,300). Our results confirmed that MAPLE was well-suited for the transfer of cinnamate-
pullulan and tosylate-pullulan.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Drug delivery; Polysaccharide; Pullulan derivatives; Thin films; Matrix assisted pulsed laser evaporation
1. Introduction
There is a growing interest in biomaterials in most branches
of science. One of the prospecting bio-polymers that have
proved to be of interest for pharmaceutical applications is
pullulan [1–3]. This is mostly because pullulan is an edible,
biodegradable, biocompatible, water soluble extracellular poly-
saccharide produced by strains of Aureobasidium pullulans.
* Corresponding author at: Institute of Physics ASCR, Na Slovance 2, 18221
Prague 8, Czech Republic. Tel.: +420 2 6605 2733; fax: +420 2 28689 0527.
E-mail address: jelinek@fzu.cz (M. Jelinek).
0169-4332/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsusc.2007.02.085
Due to its non-toxic, non-irritating properties and oxygen
impermeability, pullulan is used for producing film binders,
adhesives, viscosity improvers, and coating agents. Various
protocols have been applied in order to perform the desired
experiments, since pullulan, like most polysaccharides, has
poor solubility in common organic solvents. By introducing
functional groups into the pullulan macromolecule, it is
possible to improve its performance and extend the range of its
potential applications.
Cinnamate-pullulan (CP) and tosylate-pullulan (TP) are new
pullulan tailor-made biomaterials with desirable functional
groups, aimed not only to enhance their role in innovative drug
delivery systems, but also to make them usable as linings for
Table 1
Summary of deposition conditions
Sample symbol Fluence
(J cm�2)
Thickness
(nm)
Growth rate
(nm/pulse)
Cinnamate-pullulan
Si-30; N30 0.5 16,440 4.11
Si-31; N31 0.3 7,790 1.29
Si-32; N32 0.2 4,850 0.60
Si-33; N33 0.1 630 0.07
Si-34; N34 0.075 525 0.04
Tosylate-pullulan
Si-35; N35 0.5 3,870 2.58
Si-36; N36 0.3 2,150 0.72
Si-37; N37 0.2 830 0.40
Si-38; N38 0.1 96 0.19
Si-39; N39 0.09 224 0.16
M. Jelinek et al. / Applied Surface Science 253 (2007) 7755–77607756
artificial organs, substrates for cell growth, and immunology
testing agents [4].
Pullulan derivatives may be processed into thin films for
drug delivery systems. These polysaccharide thin films should
be rigorously similar to the starting material, with minimal
fragmentation. The pulsed laser deposition of biomaterials was
especially applied as it mostly relies upon the high power or
short penetration depth character of the laser-material inter-
action [5]. A drawback of using standard pulsed laser
deposition to produce film coatings is that direct ablation of
the target can be stressful to fragile materials that may break in
the process. To decrease the photochemical damage caused by
the direct interaction of the UV laser light with the organic or
biomaterial target, a novel deposition technique, known as
matrix assisted pulsed laser evaporation (MAPLE), was
demonstrated [6]. MAPLE was developed to overcome the
difficulties in solvent-based coating technologies such as
inhomogeneous films, inaccurate placement of material, and
difficult or erroneous thickness control. The process uses a low
fluence pulsed UV laser and a frozen composite target
consisting of a dilute mixture of the material to be deposited
and a high vapor-pressure solvent. The low-fluence laser pulse
interacts mainly with the volatile solvent, causing its
evaporation. During the process, the solute desorbs intact,
i.e., without any significant decomposition, and is then
uniformly deposited on the substrate. Various materials
dissolved in different solutions were tested till now [7–10].
By MAPLE processing it is possible to modulate the release of
drug particles by varying the thickness of biodegradable
polymer in a multilayer implementation of pullulan derivatives.
We tried to demonstrate in this work that MAPLE was able
to provide an improved approach to growing high quality thin
films of cinnamate-pullulan (CP) and tosylate-pullulan (TP),
including an accurate thickness control highly required in
targeted drug delivery.
2. Materials and methods
2.1. Materials
Our option for this work went to cinnamate-pullulan (CP)
and tosylate-pullulan (TP). CP can form highly stabilized
nanostructures because like other monocinnamate-block
copolymers it contains a photoreactive functionality [11]. TP
can be considered a precursor for obtaining methacrylated
pullulan. It is capable to copolymerize with other vinyl
monomers to form a great structural variety of hydrophilic
polymer networks with applications in controlled drug delivery
and immobilization of enzymes and cells [12–16].
2.2. MAPLE set-up
Our system basically consisted of a KrF* excimer laser
(248 nm and t = 20 ns), a vacuum chamber, and a rotating
target holder cooled to liquid nitrogen (LN) temperature. The
laser beam was focused on a frozen target of investigated
biomaterials. The target was prepared from a 2% solution of
either CP (or TP) in chloroform. We have chosen chloroform
for experiments in this work because it represents the best
compromise as solvent for both CP and TP. This is different to
the case of simple pullulan for which the best solvent was
proved to be dimethyl sulfoxide [3]. The colloidal solution was
poured into a pre-formed cast and frozen by embedding in LN.
The frozen disc was placed on a rotating holder (0.25 Hz, Kurt
J. Lesker Co.) to prevent excessive damage of the target. The
laser beam was incident at 458 on target surface, while the
target-substrate distance was 3 cm. The substrate was main-
tained at the room temperature. The incident fluence was set
within the range (0.065–0.5) J cm�2 inside a spot of 20 mm2.
The number of pulses applied to deposit one structure varied
from 1500 to 13,300. Before deposition the chamber was
evacuated down to a residual pressure of 5 � 10�4 Pa. Pressure
increased up to 10�1 Pa (for 0.5 J cm�2) or 3 � 10�3 Pa (for
0.07 J cm�2) during deposition, due to gas leakage from the
composite target. Two sets of substrates were used: fused silica
(FS) or Si (1 1 1), respectively. The deposition details are
summarized in Table 1.
2.3. Post-deposition characterization
Performances of MAPLE-deposited layers were investi-
gated by transmission spectrometry, profilometry, atomic force
microscopy (AFM), Fourier transform infrared (FTIR) spectro-
scopy and Raman spectroscopy. Transmission measurements
were performed with a UV 1601 Shimadzu spectrometer. The
thickness and growth rate of the deposited films were monitored
using a mechanical profilometer Talystep Tencor 500. The
morphology of the deposited films was imaged by AFM
(Quesant system, resolution of 500 cm�1, 1 Hz scan fre-
quency). FTIR spectra of CP and TP pullulan thin films and
nanostructures were recorded with a Thermo Nicolet Nexus
apparatus with 8 cm�1 resolution. A Renishaw in Via
spectrometer (Renishaw, U.K.) was used to collect Raman
spectra. The samples were excited with a HeNe laser beam
(632.8 nm) that was focused with a 50� microscope objective.
A 10 s integration time was used, and the signal was summed
over 20 scans in the extended scan mode. The reference
samples were prepared by dropcast method.
Fig. 1. Transmittance spectra for: (a) FS substrate and solutions of CP and TP in
chloroform, (b) TP starting material (dropcast) and thin films obtained by
MAPLE at fluences of 0.5 J cm�2 (symbol Si-35), 0.3 J cm�2 (symbol Si-36),
0.2 J cm�2 (symbol Si-37) and 0.07 J cm�2 (symbol Si-39), and (c) CP starting
material (dropcast) and thin films obtained by MAPLE at fluences of 0.5 J cm�2
(symbol Si-30), 0.3 J cm�2 (symbol Si-31), 0.2 J cm�2 (symbol Si-32),
0.1 J cm�2 (symbol Si-33) and 0.08 J cm�2 (symbol Si-34).
M. Jelinek et al. / Applied Surface Science 253 (2007) 7755–7760 7757
3. Results and discussion
3.1. Spectrometry studies
Transmissions of chloroform and CP and TP liquid solutions
are presented in Fig. 1a. We notice a high absorption of KrF*
radiation (248 nm) by chloroform. It is sharply increasing when
pullulan derivatives were added in solution. The dependencies
of the transmission curves for CP and TP mixtures mostly
differed in the region from 350 to 600 nm. Transmissions of TP
grown layers were close to dropcast for the lowest value of
fluence used in our experiments, 0.09 J cm�2—see Fig. 1b.
From Fig. 1c we observe that the transmission of CP MAPLE-
deposited structures is rather different. Thus, the transmission
spectra of films obtained at larger fluencies (0.3–0.5) J cm�2
are closer to that of the dropcast. This behavior could be a
combined effect of different absorption mechanisms of the two
derivatives at this wavelength and/or a consequence of a
different thickness of the structures obtained under experi-
mental conditions.
3.2. Profilometric measurements
We determined the ablation thresholds of CP to be
�0.075 J cm�2 and TP � 0.09 J cm�2. The growth rates of
the films varied from 0.04 to 4.11 nm/pulse for CP, and from
0.16 to 2.58 nm/pulse for TP (see Table 1). We note that these
rates were relatively higher as compared to the average growth
rate reported in MAPLE [7].
3.3. AFM observations
In Fig. 2 we present AFM micrographs of CP and TP thin
films obtained by MAPLE with different incident fluencies. The
general features fully support the previous observations
reported in [17,18] when evidenced surface morphology
strongly depends on the laser fluence. When increasing the
fluence the surface morphology changes from small globular
structures uniformly distributed to quite big clusters (in case of
MAPLE-deposited CP thin films) or large porous compact
regions (in case of MAPLE-deposited TP thin films). The
observed small globular structures uniformly distributed having
an average diameter of 300 nm (symbol N34) or 100 nm
(symbol N39) indicate an intact structure in case of the lowest
fluences (�0.075 J cm�2 for CP and �0.09 J cm�2 for TP,
respectively. When further increasing the fluence up to
0.5 J cm�2 in case of MAPLE-deposition of CP quite big
clusters are formed. This is typical for a global melting/
crystallization process [19,20] followed by solidification over
large domains with an average dimension of about 6 mm (for
symbol N30). We mention that it is indicative for an advanced
degradation process becaused by the impact of the CP clusters
with the substrate at higher kinetic energies. In case of MAPLE-
deposition of TP at the same fluence (0.5 J cm�2) the
aforementioned agglomerated structures get compact exhibit-
ing, nevertheless, a high porosity (with an approximate
dimension of about 100 nm (symbol N35) pointing to an
advanced degradation tendency. We notice that this morphol-
ogy modification could be the effect of thickness increase with
the incident laser fluence. Indeed, as known, thicker films are
usually rougher.
Fig. 2. AFM micrographs of (a) CP thin films MAPLE-deposited on Si at fluences of 0.5 J cm�2 (symbol N30), 0.3 J cm�2 (symbol N31), and 0.075 J cm�2 (symbol
N34), and (b) TP thin films MAPLE-deposited on Si at fluences of 0.5 J cm�2 (symbol N35), 0.3 J cm�2 (symbol N36), 0.2 J cm�2 (symbol N37), and 0.09 J cm�2
(symbol N39).
M. Jelinek et al. / Applied Surface Science 253 (2007) 7755–77607758
3.4. FTIR studies
As previously shown [3], pullulan consisting of maltotriose
units linked through alpha 1,6-glucosidic bonds has a
characteristic FTIR spectrum. On the other hand, we show
in Fig. 3 characteristic FTIR spectra recorded in case of (a) CP
starting material (dropcast) and thin films obtained by MAPLE
at fluences of 0.2 J cm�2 (symbol Si-32) and 0.3 J cm�2
(symbol Si-31), or (b) TP starting material (dropcast) and thin
films obtained by MAPLE at fluences of 0.2 J cm�2 (symbol Si-
37), 0.3 J cm�2 (symbol Si-36), and 0.5 J cm�2 (symbol Si-35).
We observe that in the (1175–975) cm�1 region the spectra of
pullulan comprised a number of highly fused bands. The main
bands found in the deconvoluted spectra of pullulan 1204,
1169, 1069, 1026 and 976 cm�1 were assigned to stretching
vibrations of the C–O and C–C bonds and bending vibrations of
CCH, COH and HCO, respectively [21]. CP groups had
distinctive lines at 976 and 1577 cm�1, while TP groups
showed distinctive asymmetric and symmetric bands at 1452
and 1177 cm�1, respectively. Fig. 3a and b proves a good
preservation of all structural characteristics from dropcast to
deposited films with minor changes reflecting conformations,
rearrangements, but no significant structural modifications. The
abovementioned bands are specific to polysaccharide chains (in
Fig. 3. Characteristic FTIR spectra recorded for (a) CP starting material
(dropcast) and thin films obtained by MAPLE at fluences of 0.3 J cm�2 (symbol
Si-31) and 0.2 J cm�2 (symbol Si-32), and (b) TP starting material (dropcast)
and thin films obtained by MAPLE at fluences of 0.5 J cm�2 (symbol Si-35),
0.3 J cm�2 (symbol Si-36), and 0.2 J cm�2 (symbol Si-37). Fig. 4. Raman spectra of (a) CP dropcast and CP thin films MAPLE-deposited
on Si at a fluence of 0.075 J cm�2 (symbol 34), and (b) TP dropcast and TP thin
films MAPLE-deposited on Si at fluences of 0.2 J cm�2 (symbol 37) and
0.09 J cm�2 (symbol 39).
M. Jelinek et al. / Applied Surface Science 253 (2007) 7755–7760 7759
our case, CP and TP). In samples Si-30 and Si-35 (Fig. 3a and
b), respectively, the major changes were in chain conformation
and the formation of glycosidic bond and glucose residues.
3.5. Raman investigations
Raman spectra of the CP dropcast and CP MAPLE-
deposited thin films on Si at a fluence of 0.075 J cm�2 (symbol
N34) are shown in Fig. 4a. The results of Raman investigations
of CP MAPLE-deposited thin films are consistent with AFM
images and entirely agree with the previous observations
reported in Ref. [17] according to which the surface
morphology evolves from globular material to large clusters.
The Raman studies revealed that spectra of samples deposited
at fluence of 0.075 J cm�2 (symbol N34) were the closest to CP
dropcast spectra. Similar features were noticed by examination
of Raman spectra recorded in case of samples N30–N33. The
Raman spectra of the TP dropcast and TP thin films deposited
by MAPLE at 0.09 J cm�2 (symbol N39), and 0.2 J cm�2
(symbol N37) are shown in Fig. 4b. We notice that the surface
morphology changes in this case as well from globular material
to quite large compact regions revealing a high porosity [18].
On the other hand, Raman studies of samples deposited at
0.09 J cm�2 (symbol N39) and 0.2 J cm�2 (symbol N37), have
shown identical features to TP dropcast spectra.
4. Conclusions
Cinnamate-pullulan (CP) and tosylate-pullulan (TP) dis-
solved in chloroform were successfully deposited by MAPLE in
vacuum at room temperature. We demonstrated that MAPLE is
suitable for producing CP and TP thin films with close
resemblance to the starting structures. AFM investigations
revealed in case of CP that the surface morphology strongly
depends on the laser fluence. It evolves from small globular
structures uniformly distributed to rather big clusters typical for a
global melting/crystallization process followed by solidification
over large domains. AFM investigations showed in case of TP
M. Jelinek et al. / Applied Surface Science 253 (2007) 7755–77607760
that the surface morphology is strongly determined by the laser
fluence. It evolves from small globular structures uniformly
distributed to quite large compact regions revealing a high
porosity. This is indicative for an advanced degradation tendency.
FTIR investigations prove a good preservation of all structural
characteristics from dropcast to MAPLE-deposited films with
minor changes reflecting conformations, rearrangements, but no
significant structural modifications. A good agreement was
found between the Raman and FTIR spectra recorded in the case
of dropcasts, and, respectively, CP and TP structures at lowest
fluences (�0.075 and�0.09 J cm�2, respectively). We conclude
that MAPLE can provide an appropriate approach to grow high
quality thin films of pullulan derivatives, allowing for an accurate
thickness control, highly required in targeted drug delivery.
These new pullulan-tailor-made derivatives can be used not only
for innovative drug delivery systems but also as potential linings
for artificial organs, substrates for cell growth, and agents in
immunology testing.
Acknowledgments
Experiments were carried out in the frame of the exchanges
Program between Romanian and Czech Republic Academies.
This research was supported by the Grant Agency of the Czech
Republic under grant No. 202/06/0216-1 and by the Institu-
tional Research Plan AS CR No. AVOZ 10100522. RC, EA,
DM, MA, TB, IS and INM acknowledge with thanks financial
support under Contracts CERES 4-178/15.11.2004.
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