www.elsevier.com/locate/apsusc

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.

References

[1] M. Moscovici, C. Ionescu, C. Oniscu, O. Fotea, P. Protopopescu, L.D.

Hanganu, Biotechnol. Lett. 18 (7) (1996) 787.

[2] K.I. Shingel, Carbohydrate Res. 339 (2004) 447.

[3] R. Cristescu, I. Stamatin, D.E. Mihaiescu, C. Ghica, M. Albulescu, I.N.

Mihailescu, D.B. Chrisey, Thin Solid Films 453–454C (2004) 262.

[4] L.B. Peppas, Medical Plastics and Biomaterials, 1997, p. 34.

[5] D.B. Chrisey, A. Pique, R.A. McGill, J.S. Horwitz, B.R. Ringeisen, D.M.

Bubb, P.K. Wu, Chem. Rev. 103 (2) (2003) 553.

[6] R.A. McGill, D.B. Chrisey, Method of producing a film coating by matrix

assisted pulsed laser deposition, Patent No. 6,025,036 (2000).

[7] M. Jelinek, R. Cristescu, T. Kocourek, V. Vorlicek, J. Remsa, L. Stamatin,

D. Mihaiescu, I. Stamatin, I.N. Mihailescu, D.B. Chrisey, J. Phys.: D, in

press.

[8] K. Rodrigo, B. Toftmann, J. Schou, R. Pedris, Chem. Phys. Lett. 399

(2004) 368.

[9] A.L. Mercado, C.E. Allmond, J.G. Hoekstra, J.M. Fitz-Gerald, Appl.

Phys. A 81 (2005) 591.

[10] A. Gutierrez-Llorente, G. Horowitz, R. Perez-Casero, J. Perriere, J.L.

Fave, A. Yassar, C. Sant, Org. Electron. 5 (2004) 29.

[11] T. Fujiwara, T. Iwata, Y. Kimura, J. Polym. Sci.: Part A: Polym. Chem. 39

(2001) 4249.

[12] G. Masci, D. Bontempo, V. Crescenzi, Polymer 43 (2002) 5587.

[13] R. Lamanna, A.P. Sobolev, G. Masci, D. Bontempo, V. Crescenzi, A.L.

Segre, Polym. Prepr. 44 (2003) 406.

[14] M.H. Wu, B.R. Bao, J. Chen, Y.J. Xy, S.R. Zao, Z.J. Ma, Radiat. Phys.

Chem. 56 (1999) 341.

[15] L.C. Dong, A.S. Hoffman, J. Controlled Release 13 (1990) 21.

[16] T.G. Park, A.S. Hoffmann, J. Biomed. Res. 24 (1990) 21.

[17] R. Cristescu, M. Jelinek, T. Kocourek, E. Axente, S. Grigorescu, A.

Moldovan, D.E. Mihaiescu, M. Albulescu, T. Buruiana, J. Dybal, I.

Stamatin, I.N. Mihailescu, D.B. Chrisey, J. Phys.: D, in press.

[18] R. Cristescu, M. Jelinek, T. Kocourek, E. Axente, A. Moldovan, J. Dybal,

M. Albulescu, T. Buruiana, I. Stamatin, I.N. Mihailescu, D.B. Chrisey, in:

J. Hozman, P. Kneppo (Eds.), IFMBE Proceedings in 3rd European

Medical & Biological Engineering Conference, vol. 11, 2005, p. 3579.

[19] S.N. Magonov, M.-H. Whangbo, Surface Analysis with STM and AFM,

VCH, Weinheim, 1996, p. 323.

[20] M.-H. Whangbo, S.N. Magonov, H. Bengel, Probe Microscopy 1 (1997)

23.

[21] S.P. Firsov, R.G. Zhbankov, P.T. Petrov, K.I. Shingel, V.M. Tsarenkov, in:

J. Greve, G.J. Puppels, C. Otto (Eds.), Carbohydrates. Proceedings of the

8th European Conference on the Spectroscopy of Biological Molecules,

Kluwer, Dodrecht, 1999, p. 323.