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Matrix assisted pulsed laser evaporation processing of triacetate-pullulan polysaccharide thin films for drug delivery systems
Applied Surface Science 252 (2006) 4647–4651 www.elsevier.com/locate/apsusc
Matrix assisted pulsed laser evaporation processing of triacetate-pullulan polysaccharide thin films for drug delivery systems R. Cristescu a,b,*, G. Dorcioman a, C. Ristoscu a, E. Axente a, S. Grigorescu a, A. Moldovan a, I.N. Mihailescu a, T. Kocourek b, M. Jelinek b, M. Albulescu c, T. Buruiana d, D. Mihaiescu e, I. Stamatin f, D.B. Chrisey g a
National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor, P.O. Box MG-36, RO-077125, Bucharest-Magurele, Romania b Institute of Physics, Academy of Sciences of Czech Republic, Na Slovance 2, 182 21 Prague 8, Czech Republic c National Institute for Chemical–Pharmaceutical R&D, 112 Vitan, 74373 Bucharest 3, Romania d Petru Poni Institute of Macromolecular Chemistry, Iasi 6600, Romania e University of Agriculture Sciences and Veterinary Medicine, 59 Marasti, Bucharest, Romania f University of Bucharest, Faculty of Physics, P.O. Box MG-38, 3 Nano-SAE Research Center, Bucharest-Magurele, Romania g US Naval Research Laboratory, Washington, DC 20375-5345, USA Received 3 May 2005; accepted 20 July 2005 Available online 8 November 2005
Abstract We report the first successful deposition of triacetate-pullulan polysaccharide thin films by matrix assisted pulsed laser evaporation. We used a KrF* excimer laser source (l = 248 nm, t 20 ns) operated at a repetition rate of 10 Hz. We demonstrated by FTIR that our thin films are composed of triacetate-pullulan maintaining its chemical structure and functionality. The dependence on incident laser fluence of the induced surface morphology is analysed. # 2005 Elsevier B.V. All rights reserved. Keywords: Polysaccharide; Pullulan; Thin films; Laser deposition; Matrix assisted pulsed laser evaporation; Drug delivery
1. Introduction Targeted drug delivery has gained recognition in modern therapeutics. Attempts are made to explore the possibility of cell biology related bioevents in the development of specific, programmed and target oriented systems. Oral solid dosage formulations are a convenient method of targeted drug delivery and account for a large proportion of pharmaceutical products. Drug substances are not routinely administered in the pure state and functional components are a heterogeneous mixture of many components, with individual particles of the order of micrometers in size. Additionally, solid dosage design can
* Corresponding author. Tel.: +40 21 4574491; fax: +40 21 4574243. E-mail addresses: [email protected], [email protected] (R. Cristescu). 0169-4332/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2005.07.152
include strategies to precisely control the rate of drug delivery (for example, sustained release), typically using one or more polymer coatings that must reach a specific body site and possess an appropriate release profile [1,2]. Strategies and protocols have been adopted in order to perform the desired experiments, since pullulan, like most polysaccharides, has poor solubility in common organic solvents. In this context, chemical modification of the polymer becomes a valuable method to improve the polymer properties including its solubility. Pullulan and its derivates may be processed into thin films for drug delivery systems. These polysaccharides thin films should closely resemble the starting material, with minimal fragmentation. Laser deposition of biomaterials presents an especially interesting case study because the pulsed laser deposition of materials usually uses the high power (e.g., pyrolytic chemical vapor deposition) or short penetration
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R. Cristescu et al. / Applied Surface Science 252 (2006) 4647–4651
depth character of the laser-material interaction, and these are usually incommensurate with their nondestructive processing [3]. A shortcoming to using conventional pulsed laser deposition in the creation of film coatings is that direct ablation of the target can be stressful and damaging to fragile materials. To diminish photochemical damage that results from 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), has been demonstrated at the Naval Research Laboratory [4]. MAPLE was developed to surmount the difficulties in solvent-based coating technologies such as inhomogeneous films, inaccurate placement of material, and intricate or erroneous thickness control. The process utilizes a low fluence pulsed UV laser and a frozen target consisting of a dilute mixture of the material to be deposited and a high vapor-pressure solvent. The lowfluence 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. In 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 and/or its derivates. In a previous work, we reported the succesful obtaining of pure pullulan thin films by MAPLE [5]. We demonstrate in this work that MAPLE can provide an improved approach to growing high quality thin films of triacetate-pullulan (a completely new derivate of pullulan), including an accurate thickness control required in targeted drug delivery. 2. Experimental 2.1. Materials We synthesized a new derivate of pullulan named triacetatepullulan. It is provided by the Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania, starting from pure pullulan made in this country (P-20 type) following a patented original method by the National Institute for ChemicalPharmaceutical R&D, Bucharest, Romania [6]. Triacetatepullulan with a substitution degree of about 2.9 was obtained by an esterification reaction of the hydroxyl groups with acetic anhydride in acetic acid medium and in the presence of sulphuric acid. This biopolymer is soluble in organic solvents as chloroform, methylene chloride, acetone, DMF and DMSO. In this study, we used the chloroform (melting point of 209 K) as a solvent for the MAPLE experiments because of good absorptivity at the 248 nm KrF* laser wavelength.
2.2. Experimental conditions Structures and thin films from triacetate-pullulan have been obtained by MAPLE technique [4]. The colloidal solutions containing less than 2% of triacetate-pullulan in chloroform were carefully mixed and then frozen to 77 K. After freezing, the target was rapidly mounted inside the deposition chamber and rotated at 0.25 Hz to avoid overheating and possible piercing by the laser beam. Before deposition, the chamber was evacuated down to a residual pressure of 3 104 Pa. Thin films of triacetate-pullulan were deposited using a pulsed KrF* laser with l = 248 nm, tFWHM 20 ns and operated at 10 Hz. The laser radiation was focused by a fused silica lens placed out of chamber. The incident angle of the laser beam was 458. During deposition the double face polished h1 0 0i single crystalline Si substrate was kept at room temperature. Prior to the introduction in the chamber, the substrates were cleaned with ethanol and acetone, respectively, for 10 min in an ultrasonic bath. The target–substrate distance was set at 3 cm. After preliminary tests, we used incident laser fluences set within the range 115– 500 mJ/cm2. The number of pulses applied for deposition of one film was within the range 500–30,000. The irradiation spot area was of 20 mm2. The MAPLE deposition conditions are summarized in Table 1. 2.3. Characterization methods The characterization of all MAPLE thin films was carried out by Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM) and optical microscopy (OM). FTIR spectra of triacetate-pullulan thin films and structures were recorded with a Thermo Nicolet Nexus apparatus with 8 cm1 resolution. The AFM micrographs of triacetate-pullulan thin films were performed with a Quesant Atomic Force Microscope with a resolution of 500 cm1 at a 1 Hz scan frequency. The OM investigations have been performed by a Neofot 32 instrument equipped with an Olympus E1 CCD camera. 3. Results and discussion 3.1. Fourier transform infrared spectroscopy FTIR is widely used to study the composition of complex carbohydrate systems, the molecular orientation, molecular interactions and conformational transitions of polysaccharides in solution or upon hydration. In Fig. 1, we give typical
Table 1 MAPLE experimental conditions of thin films of triacetate-pullulan Sample (FTIR, OM and AFM symbol Figs. 1–3)
Vacuum pressure [104 Pa]
Number of applied subsequent laser shots
Energy/pulse [mJ]
Incident fluence [mJ/cm2]
N22 N23 N21 N20
1.5 1.5 1.5 3
30000 10000 2000 500
23 44 64 100
115 220 320 500
R. Cristescu et al. / Applied Surface Science 252 (2006) 4647–4651
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(symbol N21), and (iii) for triacetate-pullulan thin films obtained by MAPLE at a fluence of 500 mJ/cm2 (symbol N20). Conformational and structural changes of MAPLE-deposited triacetate-pullulan thin films have been investigated pointing out the three main fingerprints: two bands assigned to C O (1747 cm1) and C–O (1220 cm1) from acetate goups, and another band centered at 1035 cm1 attributed to pollysacharide chains [7,8]. The aforementioned bands are specific to polysaccharide chains (in our case, pullulan) and any possible difference with increasing fluence should be present in recorded spectra either as steric and conformational changes or as structural modifications by new bonds or glucose residues. As we notice (Fig. 1) in case of samples N21 and N20, respectively, the major changes are in chain conformation and glycosidic bond and glucose residues manifest formation. That is clearly observed by enlarging the bands in FTIR spectra presented in Ref. [7]. In general, the structure of triacetat-pullulan deposited as thin films by MAPLE even for high fluences (i.e., 500 mJ/ cm2) is preserved even major changes in chain conformation and rearangemens are observed. Typically when all the important functional groups are maintained and there are no new degradation peaks, it can be assumed that the protein functionality is preserved in the film. These results confirm that MAPLE is suited for triacetate-pullulan transfer preserving its chemical structure and thus its functionality. Fig. 1. Typical transmission Fourier transform infrared spectra recorded for the triacetate-pullulan starting material (reference), for triacetate-pullulan thin films obtained by MAPLE at a fluence of 320 mJ/cm2 (symbol N21), and for triacetate-pullulan thin films obtained by MAPLE at a fluence of 500 mJ/ cm2 (symbol N20).
transmission FTIR spectra recorded for (i) the triacetatepullulan starting material (reference), (ii) for triacetate-pullulan thin films obtained by MAPLE at a fluence of 320 mJ/cm2
3.2. Atomic force microscopy Fig. 2 contains the AFM micrographs of triacetate-pullulan thin films deposited by MAPLE. The surface morphology strongly depends on the laser fluence and evolves from uniform small globular structures with an average diameter of 200 nm (at 115 mJ/cm2, symbol N22), to clusters with an average dimension of about 1 mm (for 220 mJ/cm2, symbol N23). We
Fig. 2. AFM micrographs of triacetate-pullulan thin films obtained by MAPLE at a fluence of 115 mJ/cm2 (symbol N22), at a fluence of 220 mJ/cm2 (symbol N23), at a fluence of 320 mJ/cm2 (symbol N21), and at a fluence of 500 mJ/cm2 (symbol N20).
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notice a fragmentation/dissociation of clusters (having an average size of about 500 nm) typical for a local melting/plastic deformation/crystallization process at 320 mJ/cm2 (symbol N21). We also observe a global melting/plastic deformation/ crystallization process followed by solidification over small domains at 500 mJ/cm2 (symbol N20). This means that deposited biopolymer is in fact found in so called ‘‘glassy polymer’’ state. FTIR investigations are consistent with AFM images where the transformation from triacetate-pullulan at fluence of 115 mJ/cm2 (N22) to the glycosidic bonds and glucose residues at fluence of 500 mJ/cm2 (N20) change the morphology from aggregates of globular material to platelets structures. AFM images clearly show that triacetate-pullulan has been changed presenting new bonds and fragmentated chains. This behaviour could be becaused by the impact of the triacetate-pullulan clusters with the substrate at higher kinetic energies. 3.3. Optical microscopy Fig. 3 contains the OM images of triacetate-pullulan thin films obtained by MAPLE. In all cases the magnification is 25. We remark a good correlation to the AFM results. The surface morphology evolves from uniform small globular structures at lower fluences (115 mJ/cm2, symbol N22) (see above) to clusters at higher fluences (220 mJ/cm2, symbol N23), and then to clusters which are slighthly underneath the percolation limit because of the small number of subsequent laser pulses—Table 1 (320 mJ/cm2, symbol N21). Finally, there is a global melting/plastic deformation/crystallization
process followed by solidification with increasing fluence (500 mJ/cm2, symbol N20). If we look at microscopic level the morphology surprised in Fig. 3 (phase contrast) we observe the presence of the aforementioned two phases (due to glycolise bonds and glucose residues—320 mJ/cm2, N21). The existence of glucose residues is more evident when fluence increases. 4. Conclusions We have demonstrated in the case of triacetate-pullulan polysaccharide that the structure of MAPLE-deposited thin films for high fluences as well (i.e., 500 mJ/cm2) is preserved even major changes in chain conformation and rearangemens are observed. Typically when all the important functional groups are maintained and there are no new degradation peaks, it can be assumed that the protein functionality is preserved in the film. These results show that MAPLE is well-suited for triacetatepullulan transfer preserving its chemical structure and thus its functionality. AFM and OM investigations revealed that the surface morphology strongly depends on the laser fluence and evolves from uniform small globular structures with an average diameter of 200 nm at a fluence of 115 mJ/cm2 to clusters with an average dimension of about 1 mm at a fluence of 220 mJ/cm2. Further on 320 mJ/cm2, we can notice a fragmentation/ dissociation of clusters (with an average dimension of about 500 nm) which are slighthly underneath the percolation limit because of the small number of subsequent laser pulses typical for a local melting/plastic deformation/crystallization process and even a global melting/plastic deformation/crystallization process followed by solidification on small domains at 500 mJ/ cm2. FTIR investigations are consistent with AFM images where the transformation from triacetate-pullulan at fluence of 115 mJ/ cm2 to the glycosidic bonds and glucose residues at fluence of 500 mJ/cm2 change the morphology from aggregates of globular material to platelets structures. AFM images clearly show that triacetate-pullulan has been changed presenting new bonds and fragmentated chains. This behaviour could be due to the impact of the triacetate-pullulan clusters with the substrate at higher kinetic energies. Acknowledgements Experiments were carried out in the frame of the scientific exchanges between Romanian and Czech Republic Academies. R.C., D.M, I.S, and I.N.M. acknowledge with thanks the financial support of Contracts CERES 4-178/15.11.2004 and LAPLAS PN 03/17.01.2003. One of us (DBC) gratefully acknowledges financial support from the Office of Naval Research. References
Fig. 3. Optical microscope images of triacetate-pullulan thin films obtained by MAPLE at a fluence of 115 mJ/cm2 (symbol N22), at a fluence of 220 mJ/cm2 (symbol N23), at a fluence of 320 mJ/cm2 (symbol N21), and at a fluence of 500 mJ/cm2 (symbol N20). In all cases the magnification is 25.
[1] R.K. Singh, W.S. Kim, M. Ollinger, V. Craciun, I. Coowantwong, G. Hochhaus, N. Koshizaki, Appl. Surf. Sci. 197 (2002) 610. [2] J.D. Talton, J.M. Fitz-Gerald, R.K. Singh, G. Hochhaus, Respir. Drug Deliv. VII (2000) 67. [3] 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.
R. Cristescu et al. / Applied Surface Science 252 (2006) 4647–4651 [4] R.A. McGill, D.B. Chrisey, Method of producing a film coating by matrix assisted pulsed laser deposition, Patent No. 6,025,036 (2000). [5] R. Cristescu, I. Stamatin, D.E. Mihaiescu, C. Ghica, M. Albulescu, I.N. Mihailescu, D.B. Chrisey, Thin Solid Films 453–454C (2004) 262.
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[6] M. Moscovici, C. Ionescu, C. Oniscu, O. Fotea, P. Protopopescu, L.D. Hanganu, Biotechnol. Lett. 18 (7) (1996) 787. [7] K.I. Shingel, Carbohydr. Res. 337 (2002) 1445. [8] KnowItAll Database by Bio-Rad Laboratories, Academic edition 4.1, http://www.bio-rad.com.
Matrix assisted pulsed laser evaporation processing of triacetate-pullulan polysaccharide thin films for drug delivery systems R. Cristescu a,b,*, G. Dorcioman a, C. Ristoscu a, E. Axente a, S. Grigorescu a, A. Moldovan a, I.N. Mihailescu a, T. Kocourek b, M. Jelinek b, M. Albulescu c, T. Buruiana d, D. Mihaiescu e, I. Stamatin f, D.B. Chrisey g a
National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor, P.O. Box MG-36, RO-077125, Bucharest-Magurele, Romania b Institute of Physics, Academy of Sciences of Czech Republic, Na Slovance 2, 182 21 Prague 8, Czech Republic c National Institute for Chemical–Pharmaceutical R&D, 112 Vitan, 74373 Bucharest 3, Romania d Petru Poni Institute of Macromolecular Chemistry, Iasi 6600, Romania e University of Agriculture Sciences and Veterinary Medicine, 59 Marasti, Bucharest, Romania f University of Bucharest, Faculty of Physics, P.O. Box MG-38, 3 Nano-SAE Research Center, Bucharest-Magurele, Romania g US Naval Research Laboratory, Washington, DC 20375-5345, USA Received 3 May 2005; accepted 20 July 2005 Available online 8 November 2005
Abstract We report the first successful deposition of triacetate-pullulan polysaccharide thin films by matrix assisted pulsed laser evaporation. We used a KrF* excimer laser source (l = 248 nm, t 20 ns) operated at a repetition rate of 10 Hz. We demonstrated by FTIR that our thin films are composed of triacetate-pullulan maintaining its chemical structure and functionality. The dependence on incident laser fluence of the induced surface morphology is analysed. # 2005 Elsevier B.V. All rights reserved. Keywords: Polysaccharide; Pullulan; Thin films; Laser deposition; Matrix assisted pulsed laser evaporation; Drug delivery
1. Introduction Targeted drug delivery has gained recognition in modern therapeutics. Attempts are made to explore the possibility of cell biology related bioevents in the development of specific, programmed and target oriented systems. Oral solid dosage formulations are a convenient method of targeted drug delivery and account for a large proportion of pharmaceutical products. Drug substances are not routinely administered in the pure state and functional components are a heterogeneous mixture of many components, with individual particles of the order of micrometers in size. Additionally, solid dosage design can
* Corresponding author. Tel.: +40 21 4574491; fax: +40 21 4574243. E-mail addresses: [email protected], [email protected] (R. Cristescu). 0169-4332/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2005.07.152
include strategies to precisely control the rate of drug delivery (for example, sustained release), typically using one or more polymer coatings that must reach a specific body site and possess an appropriate release profile [1,2]. Strategies and protocols have been adopted in order to perform the desired experiments, since pullulan, like most polysaccharides, has poor solubility in common organic solvents. In this context, chemical modification of the polymer becomes a valuable method to improve the polymer properties including its solubility. Pullulan and its derivates may be processed into thin films for drug delivery systems. These polysaccharides thin films should closely resemble the starting material, with minimal fragmentation. Laser deposition of biomaterials presents an especially interesting case study because the pulsed laser deposition of materials usually uses the high power (e.g., pyrolytic chemical vapor deposition) or short penetration
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R. Cristescu et al. / Applied Surface Science 252 (2006) 4647–4651
depth character of the laser-material interaction, and these are usually incommensurate with their nondestructive processing [3]. A shortcoming to using conventional pulsed laser deposition in the creation of film coatings is that direct ablation of the target can be stressful and damaging to fragile materials. To diminish photochemical damage that results from 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), has been demonstrated at the Naval Research Laboratory [4]. MAPLE was developed to surmount the difficulties in solvent-based coating technologies such as inhomogeneous films, inaccurate placement of material, and intricate or erroneous thickness control. The process utilizes a low fluence pulsed UV laser and a frozen target consisting of a dilute mixture of the material to be deposited and a high vapor-pressure solvent. The lowfluence 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. In 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 and/or its derivates. In a previous work, we reported the succesful obtaining of pure pullulan thin films by MAPLE [5]. We demonstrate in this work that MAPLE can provide an improved approach to growing high quality thin films of triacetate-pullulan (a completely new derivate of pullulan), including an accurate thickness control required in targeted drug delivery. 2. Experimental 2.1. Materials We synthesized a new derivate of pullulan named triacetatepullulan. It is provided by the Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania, starting from pure pullulan made in this country (P-20 type) following a patented original method by the National Institute for ChemicalPharmaceutical R&D, Bucharest, Romania [6]. Triacetatepullulan with a substitution degree of about 2.9 was obtained by an esterification reaction of the hydroxyl groups with acetic anhydride in acetic acid medium and in the presence of sulphuric acid. This biopolymer is soluble in organic solvents as chloroform, methylene chloride, acetone, DMF and DMSO. In this study, we used the chloroform (melting point of 209 K) as a solvent for the MAPLE experiments because of good absorptivity at the 248 nm KrF* laser wavelength.
2.2. Experimental conditions Structures and thin films from triacetate-pullulan have been obtained by MAPLE technique [4]. The colloidal solutions containing less than 2% of triacetate-pullulan in chloroform were carefully mixed and then frozen to 77 K. After freezing, the target was rapidly mounted inside the deposition chamber and rotated at 0.25 Hz to avoid overheating and possible piercing by the laser beam. Before deposition, the chamber was evacuated down to a residual pressure of 3 104 Pa. Thin films of triacetate-pullulan were deposited using a pulsed KrF* laser with l = 248 nm, tFWHM 20 ns and operated at 10 Hz. The laser radiation was focused by a fused silica lens placed out of chamber. The incident angle of the laser beam was 458. During deposition the double face polished h1 0 0i single crystalline Si substrate was kept at room temperature. Prior to the introduction in the chamber, the substrates were cleaned with ethanol and acetone, respectively, for 10 min in an ultrasonic bath. The target–substrate distance was set at 3 cm. After preliminary tests, we used incident laser fluences set within the range 115– 500 mJ/cm2. The number of pulses applied for deposition of one film was within the range 500–30,000. The irradiation spot area was of 20 mm2. The MAPLE deposition conditions are summarized in Table 1. 2.3. Characterization methods The characterization of all MAPLE thin films was carried out by Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM) and optical microscopy (OM). FTIR spectra of triacetate-pullulan thin films and structures were recorded with a Thermo Nicolet Nexus apparatus with 8 cm1 resolution. The AFM micrographs of triacetate-pullulan thin films were performed with a Quesant Atomic Force Microscope with a resolution of 500 cm1 at a 1 Hz scan frequency. The OM investigations have been performed by a Neofot 32 instrument equipped with an Olympus E1 CCD camera. 3. Results and discussion 3.1. Fourier transform infrared spectroscopy FTIR is widely used to study the composition of complex carbohydrate systems, the molecular orientation, molecular interactions and conformational transitions of polysaccharides in solution or upon hydration. In Fig. 1, we give typical
Table 1 MAPLE experimental conditions of thin films of triacetate-pullulan Sample (FTIR, OM and AFM symbol Figs. 1–3)
Vacuum pressure [104 Pa]
Number of applied subsequent laser shots
Energy/pulse [mJ]
Incident fluence [mJ/cm2]
N22 N23 N21 N20
1.5 1.5 1.5 3
30000 10000 2000 500
23 44 64 100
115 220 320 500
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(symbol N21), and (iii) for triacetate-pullulan thin films obtained by MAPLE at a fluence of 500 mJ/cm2 (symbol N20). Conformational and structural changes of MAPLE-deposited triacetate-pullulan thin films have been investigated pointing out the three main fingerprints: two bands assigned to C O (1747 cm1) and C–O (1220 cm1) from acetate goups, and another band centered at 1035 cm1 attributed to pollysacharide chains [7,8]. The aforementioned bands are specific to polysaccharide chains (in our case, pullulan) and any possible difference with increasing fluence should be present in recorded spectra either as steric and conformational changes or as structural modifications by new bonds or glucose residues. As we notice (Fig. 1) in case of samples N21 and N20, respectively, the major changes are in chain conformation and glycosidic bond and glucose residues manifest formation. That is clearly observed by enlarging the bands in FTIR spectra presented in Ref. [7]. In general, the structure of triacetat-pullulan deposited as thin films by MAPLE even for high fluences (i.e., 500 mJ/ cm2) is preserved even major changes in chain conformation and rearangemens are observed. Typically when all the important functional groups are maintained and there are no new degradation peaks, it can be assumed that the protein functionality is preserved in the film. These results confirm that MAPLE is suited for triacetate-pullulan transfer preserving its chemical structure and thus its functionality. Fig. 1. Typical transmission Fourier transform infrared spectra recorded for the triacetate-pullulan starting material (reference), for triacetate-pullulan thin films obtained by MAPLE at a fluence of 320 mJ/cm2 (symbol N21), and for triacetate-pullulan thin films obtained by MAPLE at a fluence of 500 mJ/ cm2 (symbol N20).
transmission FTIR spectra recorded for (i) the triacetatepullulan starting material (reference), (ii) for triacetate-pullulan thin films obtained by MAPLE at a fluence of 320 mJ/cm2
3.2. Atomic force microscopy Fig. 2 contains the AFM micrographs of triacetate-pullulan thin films deposited by MAPLE. The surface morphology strongly depends on the laser fluence and evolves from uniform small globular structures with an average diameter of 200 nm (at 115 mJ/cm2, symbol N22), to clusters with an average dimension of about 1 mm (for 220 mJ/cm2, symbol N23). We
Fig. 2. AFM micrographs of triacetate-pullulan thin films obtained by MAPLE at a fluence of 115 mJ/cm2 (symbol N22), at a fluence of 220 mJ/cm2 (symbol N23), at a fluence of 320 mJ/cm2 (symbol N21), and at a fluence of 500 mJ/cm2 (symbol N20).
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notice a fragmentation/dissociation of clusters (having an average size of about 500 nm) typical for a local melting/plastic deformation/crystallization process at 320 mJ/cm2 (symbol N21). We also observe a global melting/plastic deformation/ crystallization process followed by solidification over small domains at 500 mJ/cm2 (symbol N20). This means that deposited biopolymer is in fact found in so called ‘‘glassy polymer’’ state. FTIR investigations are consistent with AFM images where the transformation from triacetate-pullulan at fluence of 115 mJ/cm2 (N22) to the glycosidic bonds and glucose residues at fluence of 500 mJ/cm2 (N20) change the morphology from aggregates of globular material to platelets structures. AFM images clearly show that triacetate-pullulan has been changed presenting new bonds and fragmentated chains. This behaviour could be becaused by the impact of the triacetate-pullulan clusters with the substrate at higher kinetic energies. 3.3. Optical microscopy Fig. 3 contains the OM images of triacetate-pullulan thin films obtained by MAPLE. In all cases the magnification is 25. We remark a good correlation to the AFM results. The surface morphology evolves from uniform small globular structures at lower fluences (115 mJ/cm2, symbol N22) (see above) to clusters at higher fluences (220 mJ/cm2, symbol N23), and then to clusters which are slighthly underneath the percolation limit because of the small number of subsequent laser pulses—Table 1 (320 mJ/cm2, symbol N21). Finally, there is a global melting/plastic deformation/crystallization
process followed by solidification with increasing fluence (500 mJ/cm2, symbol N20). If we look at microscopic level the morphology surprised in Fig. 3 (phase contrast) we observe the presence of the aforementioned two phases (due to glycolise bonds and glucose residues—320 mJ/cm2, N21). The existence of glucose residues is more evident when fluence increases. 4. Conclusions We have demonstrated in the case of triacetate-pullulan polysaccharide that the structure of MAPLE-deposited thin films for high fluences as well (i.e., 500 mJ/cm2) is preserved even major changes in chain conformation and rearangemens are observed. Typically when all the important functional groups are maintained and there are no new degradation peaks, it can be assumed that the protein functionality is preserved in the film. These results show that MAPLE is well-suited for triacetatepullulan transfer preserving its chemical structure and thus its functionality. AFM and OM investigations revealed that the surface morphology strongly depends on the laser fluence and evolves from uniform small globular structures with an average diameter of 200 nm at a fluence of 115 mJ/cm2 to clusters with an average dimension of about 1 mm at a fluence of 220 mJ/cm2. Further on 320 mJ/cm2, we can notice a fragmentation/ dissociation of clusters (with an average dimension of about 500 nm) which are slighthly underneath the percolation limit because of the small number of subsequent laser pulses typical for a local melting/plastic deformation/crystallization process and even a global melting/plastic deformation/crystallization process followed by solidification on small domains at 500 mJ/ cm2. FTIR investigations are consistent with AFM images where the transformation from triacetate-pullulan at fluence of 115 mJ/ cm2 to the glycosidic bonds and glucose residues at fluence of 500 mJ/cm2 change the morphology from aggregates of globular material to platelets structures. AFM images clearly show that triacetate-pullulan has been changed presenting new bonds and fragmentated chains. This behaviour could be due to the impact of the triacetate-pullulan clusters with the substrate at higher kinetic energies. Acknowledgements Experiments were carried out in the frame of the scientific exchanges between Romanian and Czech Republic Academies. R.C., D.M, I.S, and I.N.M. acknowledge with thanks the financial support of Contracts CERES 4-178/15.11.2004 and LAPLAS PN 03/17.01.2003. One of us (DBC) gratefully acknowledges financial support from the Office of Naval Research. References
Fig. 3. Optical microscope images of triacetate-pullulan thin films obtained by MAPLE at a fluence of 115 mJ/cm2 (symbol N22), at a fluence of 220 mJ/cm2 (symbol N23), at a fluence of 320 mJ/cm2 (symbol N21), and at a fluence of 500 mJ/cm2 (symbol N20). In all cases the magnification is 25.
[1] R.K. Singh, W.S. Kim, M. Ollinger, V. Craciun, I. Coowantwong, G. Hochhaus, N. Koshizaki, Appl. Surf. Sci. 197 (2002) 610. [2] J.D. Talton, J.M. Fitz-Gerald, R.K. Singh, G. Hochhaus, Respir. Drug Deliv. VII (2000) 67. [3] 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.
R. Cristescu et al. / Applied Surface Science 252 (2006) 4647–4651 [4] R.A. McGill, D.B. Chrisey, Method of producing a film coating by matrix assisted pulsed laser deposition, Patent No. 6,025,036 (2000). [5] R. Cristescu, I. Stamatin, D.E. Mihaiescu, C. Ghica, M. Albulescu, I.N. Mihailescu, D.B. Chrisey, Thin Solid Films 453–454C (2004) 262.
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