Applied Surface Science 255 (2009) 5491–5495

Multifunctional thin films of lactoferrin for biochemical use depositedby MAPLE technique

Catalin Constantinescu a,*, Alexandra Palla-Papavlu a, Andrei Rotaru a, Paula Florian b, Florica Chelu b,Madalina Icriverzi b, Anca Nedelcea a, Valentina Dinca a, Anca Roseanu b, Maria Dinescu a

a INFLPR, National Institute for Laser, Plasma and Radiation Physics, 409 Atomistilor Boulevard, PO Box MG-16, RO-077125 Magurele, Bucharest, Romaniab IB, Institute of Biochemistry of the Romanian Academy, 296 Splaiul Independentei, RO-060031 Bucharest, Romania

A R T I C L E I N F O

Article history:

Available online 3 August 2008

Keywords:

MAPLE

Lactoferrin

Thin films

Proteins

A B S T R A C T

Lactoferrin (Lf) is an iron-binding glycoprotein present in almost all mammalian secretions which plays

an important role in host defense against microbial and viral infections. The protein has been reported to

also have anti-inflammatory activity and antitumoral effects in vitro and in vivo.

Thin films of Lf were deposited on silicon, quartz and Thermanox plastic coverslip substrates by Matrix

Assisted Pulsed Laser Evaporation (MAPLE) technique, using a Nd:YAG laser working at 266 nm, at

different laser fluences (0.1–0.8 J cm�2). The deposited layers have been characterized by Fourier

Transformed Infra-Red spectroscopy (FTIR), and the morphology of the various substrates was

investigated by Atomic Force Microscopy (AFM). The biocompatibility of lactoferrin thin films was

evaluated for each substrate, by in vitro biochemical tests.

� 2008 Elsevier B.V. All rights reserved.

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1. Introduction

Conventional pulsed laser deposition (PLD) using ultraviolet(UV) laser sources has become a successful, widespread techniquefor fabricating inorganic thin films of well-controlled thickness andcomposition [1]. Unfortunately, this technique cannot be appliedto most organic materials, since irradiation by UV light induces asubstantial decomposition of the target molecules [2]. Softinorganic, organic, polymer and biomaterial films can be producedby an alternative technique, known as matrix assisted pulsed laserevaporation (MAPLE) [3,4], which has the potential to create thinfilms of controlled thickness at nanometer scale (10–500 nm) onsurfaces of various substrates [5–10]. Therefore, this technique isof large interest in the area of protein thin film processing forvarious applications, such as: slow drug release, implants or thepreparation of sensitive layers on biosensors or biocompatiblecoating of medical materials [10–19]. For most of the applicationsit is essential to preserve the chemical composition and naturalconformation of deposited protein molecules, so that biologicalactivity is not altered. Protein structure can be impaired by stronglaser irradiation during MAPLE, either due to an increase in localtemperature of the aqueous solution in the vicinity of the molecule

* Corresponding author. Tel.: +40 722 120991; fax: +40 318 115383.

E-mail address: catalin.constantinescu@inflpr.ro (C. Constantinescu).

0169-4332/$ – see front matter � 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.apsusc.2008.07.163

or due to direct absorption of light by protein chromophores, if thelaser wavelength is within protein absorption bands (i.e. shorterthan 300 nm) [3,12,16–18].

Lactoferrin (Lf) is an iron-binding glycoprotein that is closelyrelated to the plasma iron-transport protein transferrin [20]. Sinceits discovery and characterization, a large amount of research hasbeen carried out concerning its structure and function. Lf has beenreported to play an important role in host defense againstmicrobial and viral infections, exhibiting also anti-inflammatoryand antitumoral activities [21–28]. Lf offers the advantage of beinga natural compound, non-toxic and thus more biocompatible thansynthetic products. Coated implants or different biomedicaldevices proved to improve the release and delivery of biologicaland pharmaceutical agents to the sites of interest. These studiesaim to provide films of active Lf covering specific materials (silicon,quartz and Thermanox), in order to evaluate their biocompatibilityby in vitro tests.

2. Experimental

MAPLE is a physical vapor deposition technique, that involveshomogenizing a material in a solid matrix or solvent (0.1–2 wt %),compacting or freezing the mixture to create a solid target. Whenthe system is irradiated by laser beam, the solvent evaporateswhereas the guest molecules are collected on a substrate [3,4,29].A successful deposition by MAPLE requires a highly absorbing

C. Constantinescu et al. / Applied Surface Science 255 (2009) 5491–54955492

matrix and a relatively low absorption by the guest material. It isalso important that possible reactions between the matrix andguest materials should be avoided or considerably reduced[9,12,30,31]. However, all these requirements are sometimesdifficult to be completely fulfilled.

2.1. Target and sample preparation

The targets were prepared by freezing a solution of 1.5% oflactoferrin (bovine or human recombinant) dissolved in doubledistilled water. First, 500 ml of the solution were poured onto thecopper target holder, using a micropipette. The target holder wascooled by gently adding liquid nitrogen, until reaching 0 8C or less.The substrates previously mentioned were firstly cleaned in anultrasonic bath for 15 minutes, using acetone and isopropanol ascleaning mediums, and then dried under nitrogen pressured gas.Ten series of depositions (30 samples) were prepared by MAPLE atdifferent laser fluences (0.1–0.8 J cm�2), with three differentsubstrates (silicon, quartz and Thermanox) on each round.

2.2. Method

A laser beam from ‘‘Surelite II’’ pulsed Nd:YAG laser system(Continuum Company, 266 nm wavelength, 5 ns duration of thepulse, 10 Hz) was focused on the lactoferrin frozen target. Bovinelactoferrin (BLf) with very low iron content was kindly provided byProf. R. Evans, Univ. Brunel, UK. Recombinant human lactoferrin(rHLf) was provided by Ventria Bioscience, USA. As substrates, weused silicon and quartz (fused silica) square slides of about 1 cm2

and Thermanox plastic coverslips round shaped of 1.3 cmdiameter, placed at a distance of 4 cm from the target (Fig. 1).The substrates were kept at room temperature during thedeposition. The number of pulses was 20,000. In order to haveuniform evaporation, the target was rotated with a motionfeedthrough driven by a motor, the laser beam describing a ringonto the sample and in order to control the temperature, twothermocouples were placed in two different positions of the targetholder. The background pressure (air) ranging from 7�10–5 to2�10–4 mbar, were obtained with a Pfeiffer-Balzers TPU 170turbomolecular pump (170 L s�1).

2.3. Thin film analysis and testing

The films’ surface aspect and roughness were analysed usingatomic force microscopy (AFM), on several different areas anddimensions, with a ‘‘Nomad’’ AFM setup produced by ‘‘QuesantInstrument Corporation’’. The chemical composition was investi-gated by Fourier transformed infrared (FTIR) spectroscopy with aJasco FT/IR-6300 type A spectrometer in the range 400–7800 cm�1.All spectra were obtained by transmission measurements, 16 scansand with CO2/H2O correction. Only the 500–5500 cm�1 interval of

Fig. 1. (a) A frozen target of lactoferrin, immediately after being produced, and (b) image

and the substrate holder.

the spectra was chosen for comparison; the signal intensity is notrelevant because the thicknesses of the measured samples weredifferent.

Biochemical tests were performed on B16-F10 murine mela-noma cells (ECACC, Porton Down, U.K), that were cultured in RPMI-1640 medium (EuroClone) containing 10% (v/v) fetal bovine serumFBS (Biochrom), L-glutamine 20 mM and penicillin/streptomycin1%, at 37 8C in a 95% humidified air/5% CO2 atmosphere. MTSCellTiter 961 Aqueous non-radioactive cell proliferation assay(MTS kit, Promega) was performed to determine cell viability.Briefly, the cells were seeded in 96-well microtiter plates (NUNC)and incubated for 72 h at 37 8C; then, the medium was removedand the adherent cells were further incubated for 24 h in theculture medium resulted from 72 h incubation with thin films ofBLf/rHLf deposited on different substrates. 20 ml of MTS was addedto each well and all samples incubated for 1.5 h at 37 8C. Cellsgrown in standard conditions for 72 h represented the control. Theabsorbance values read at 450 nm (Anthos II/III Lab TechInstruments) are directly proportional to the number of livingcells. Cell viability was expressed as percent of control (untreatedcells). In order to determine cell morphology, B16-F10 cells wereincubated in 24-well plates with thin films of BLf/rHLf deposited ondifferent substrates for 72 h at 37 8C and then stained with ERTracker Blue-White DPX (Molecular Probes). This lipid is aphotostable probe that is selective for the endoplasmic reticulum(ER) in live cells and allows the visualisation of the cells’morphology on the biomaterial surface. Live cells were visualizedeither with a fluorescence Nikon Eclipse E600 microscope or aninverted phase-contrast optical Nikon Eclipse TS100 microscope.Images were captured with a Lucia Net software controlled camera(all photos at 10�).

3. Results and discussion

Most of the samples that were deposited resulted in uniformand complete thin films of lactoferrin, the best depositions being atfluences of 0.4–0.7 J cm�2. Below this range there is no visualdetection of a deposited thin film, and above this range the thinfilms present a burned aspect (that can also be associated with atypical burn odour!). The protein is adherent to all three differentsubstrates we used, but a hard tip (such as tweezers’) can leavescratches on the thin films. Protein interaction with the substrate isa phenomenon that has been studied in the literature underdifferent conditions and means of experimental setups; thenucleation, orientation and structure of the protein crystals arepresented in detail, but mostly on organic substrates. In our case,the possible chemical interactions are due to the hydrogen bondingand polarization at the substrate’s surface [32–34]. During thedeposition, the frozen target presents a blue glow in the area whereits irradiated, and because of the target’s rotation this area has acircular (ring) shape, as can be seen in Fig. 1b.

during the thin film deposition (with the target exhibiting blue light fluorescence),

C. Constantinescu et al. / Applied Surface Science 255 (2009) 5491–5495 5493

The AFM images (Fig. 2) reveal low roughness on all scannedareas, typically of nanometer size. Droplets are present, but rareand of low dimensions (the differences between the highest andthe lowest points being smaller than 200 nm. A comparisonbetween a sample deposited at 0.1 J cm�2 and one at 0.7 J cm�2

(Fig. 2a and b, respectively) emphasizes that there is no depositionat very low fluence. The roughness, or RMS deviation on a20 mm � 20 mm area is 7.8 nm approximately (mean deviation is3.7 nm), which is extremely low for such type of materials.

FTIR analysis was used to investigate the chemical structure oflactoferrin [35]; this procedure is used to identify the bestexperimental conditions for obtaining thin films and in order toavoid the degradation of lactoferrin’s molecular structure duringits transfer by MAPLE. Fig. 3 presents two FTIR spectra: in the firstone, the carbohydrate’s C–O molecular stretching vibrationabsorption bands appear from 1000 cm�1 up to 1160 cm�1 inthe FTIR spectra, and all samples appear to preserve it well, at allapplied fluences. The band from 1440 cm�1 up to 1465 cm�1 isrelated to the C–H deformation (asymmetrical) vibration in R–CH3,best preserved in the sample deposited at 0.5 J cm�2 (Fig. 3a.). Theband of 1490 cm�1 up to 1580 cm�1 is related to the N–Hdeformation vibrations, again best preserved in the sampledeposited at 0.5 J cm�2. Another similar absorption band ispresented in Fig. 3b, from 3200 cm�1 up to 3400 cm�1 and it isrelated to the N–H molecular stretching vibration; the samplesthat exhibit this were the ones obtained under fluences of 0.4 –0.7 J cm�2 (Fig. 3b.). From 2500 cm�1 and up to 3000 cm�1 theaminoacids C–H molecular stretching vibration appears. O–H

Fig. 2. AFM images of the lactoferrin thin films: (a) deposited at 0.7 J/cm2; area of

the sample is 20 mm � 20 mm, RMS = 5.6 A; (b) deposited at 0.1 J/cm2, area of the

sample is 20 mm � 20 mm.

stretching vibration, and intermolecular hydrogen bonds alsoappear under this band, so this can be considered as a typicalsignature of lactoferrin.

The development of new biomaterials implies the testing oftheir biocompatibility and activity in order to avoid cellularadverse reactions. Since biocompatibility can be linked to thebehaviour of cells when they are cultured on the samples, the effectof the thin films of Lf on murine melanoma B16-F10 cell viabilityand morphology was evaluated. First, we investigated whethersoluble compounds released by the thin films following incubationwith culture medium were toxic for the B16-F10 cells. This is auseful test to discriminate between the effect on cell viability ofeither the components released into the culture medium (i.e. Lf) orthe substrate properties. After 72 h incubation of the films withculture medium, the supernatant was collected and furtherincubated with B16-F10 cells (previously plated) for another24 h. The cytotoxicity was evaluated by MTS assay. Changes in themorphology of the cells incubated for 72 h with thin films of BLf/rHLf were visualised by either fluorescence microscopy, using anER marker, or by inverted optical microscopy. ER is an intracelullarorganelle spread all over the cytoplasm and its labeling allows theevaluation of the morphology of live cells. The viability of cellsgrown on thin films of BLf deposited on silicon substrate was 85%,whereas in samples coated with BLf on Thermanox and on quartzsubstrate it was 65% and 60% respectively compared to the control

Fig. 3. FTIR spectra of the deposited samples compared to the drop cast film.

Fig. 4. Effects of thin films of Lf on cell viability and morphology. B16-F10 cells grown in medium resulted from 72 h incubation with different films of Lf (a) were assessed for

viability by MTS test. Results are mean of three independent experiments. Fluorescence and/or optical microscopy images (10�) of B16-F10 cells grown on silicon (b), BLf–

silicon (c), quartz (d), BLf –quartz (e), Thermanox (f), BLf –Thermanox (g).

C. Constantinescu et al. / Applied Surface Science 255 (2009) 5491–54955494

(untreated cell, 100% viability) (Fig. 4a). The morphology of B16-F10 cells (Fig. 4b and c) incubated with thin films of BLf depositedon silicon substrate revealed a decrease in the number of livingcells, round-shaped cells clustered in small groups, compared tosilicon substrate alone where the morphology of living cells wasnot altered. Similar effects were observed for Thermanox samples(Fig. 4f and g). In the case of quartz substrate films (Fig. 4d and e), aless uniform distribution of living cells was observed. Cellspresented either a round shape or dendrite-like projections with

membrane-bound vesicles in their cytoplasm in both control andBLf samples. Same results were obtained for rHLf in the case ofquartz substrate (data not shown).

4. Conclusion

We have demonstrated that thin films of lactoferrin can bedeveloped by the MAPLE technique. Very smooth and uniform thinfilms can be obtained from the same targets and for specific sets of

C. Constantinescu et al. / Applied Surface Science 255 (2009) 5491–5495 5495

experimental conditions. It was demonstrated that for laserfluences in the range of 0.4–0.7 J/cm2 smooth and uniformlactoferrin layers result, with similar chemical structure as inbulk, and with optimum activity in cell viability. Such depositionsare suitable for being used in further applications includingscaffolding, tissue engineering, implants, and possibly biosensors.In vitro biochemical test revealed a good correlation between laserfluence, thin film quality and cell viability and morphology of cellsgrown on substrates covered with Lf. Albeit silicon and Thermanoxseem to be promising biomaterials, further studies are needed tovalidate their practical use as vehicle for lactoferrin delivery at thesite of interest.

Acknowledgments

The authors would like to acknowledge the support of Dr. NicuScarisoreanu and Ion ‘‘Felix’’ Nistorescu for the help they providedduring the vacuum chamber’s development and depositionexperiments. We also want to thank to Antoniu Moldovan andMihaela Filipescu for providing the AFM images. We thank VentriaBioscience for donating rHLf for this study.

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