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Applied Surface Science

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Functionalized antibiolm thin coatings based on PLAPVA microspheres loaded with usnic acid natural compounds fabricated by MAPLE
Valentina Grumezescu a,b , Gabriel Socol a , Alexandru Mihai Grumezescu b, , Alina Maria Holban c , Anton Ficai b , Roxana Trus c a d , Coralia Bleotu e , Paul C at alin Balaure f , Rodica Cristescu a , Mariana Carmen Chiriuc c
National Institute for Lasers, Plasma & Radiation Physics, Lasers Department, PO Box MG-36, Bucharest-Magurele, Bucharest, Romania University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Department of Science and Engineering of Oxidic Materials and Nanomaterials, Polizu Street no 1-7, 011061 Bucharest, Romania c Faculty of Biology, University of Bucharest, Microbiology Immunology Department, Aleea Portocalelor 1-3, Sector 5, 77206 Bucharest, Romania d S.C. Metav-CD S.A., 31Rosetti Str., 020015 Bucharest, Romania e Stefan S Nicolau Institute of Virology, Bucharest, Romania f Department of Organic Chemistry, Faculty of Applied Chemistry and Materials Science, Politehnica Universitiy of Bucharest, Polizu Street no 1-7, 011061 Bucharest, Romania
b a

a r t i c l e

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a b s t r a c t
We report the fabrication of thin coatings of PLAPVA microspheres loaded with usnic acid by matrix assisted pulsed laser evaporation (MAPLE) onto Ti substrate. The obtained coatings have been physicochemically characterized by scanning electron microscopy (SEM) and infrared microscopy (IRM). In vitro biological assays have been performed in order to evaluate the inuence of fabricated microsphere thin coatings on the Staphylococcus aureus biolm development as well as their biocompatibility. SEM micrographs have revealed a uniform morphology of thin coatings, while IRM investigations have proved both the homogeneity and functional groups integrity of prepared thin coatings. The obtained microspherebased thin coatings have proved to be efcient vehicles for usnic acid natural compound with antibiolm activity, as demonstrated by the inhibitory activity on S. aureus mature biolm development, opening new perspectives for the prevention and therapy associated to biolm related infections. 2013 Elsevier B.V. All rights reserved.

Article history: Received 29 June 2013 Received in revised form 13 September 2013 Accepted 15 September 2013 Available online xxx Keywords: MAPLE Microspheres thin coatings PLAPVA Usnic acid Anti-biolm Staphylococcus aureus

1. Introduction Biodegradable polymers have attracted increased attention in the last decade due to the great potential for biomedical applications [15]. Current surface engineering approaches have moved towards the design of micro or nano-platforms with multifunctional therapeutic properties [6]. These platforms are intended to concentrate a synthetic or natural drug, but also to exhibit additional activities as complementary therapeutic effects [610]. Due to its good mechanical properties and enhanced biocompatibility, polylactic acid (PLA) has been extensively studied for biomedical applications including sutures, bone screws, drug delivery systems or tissue engineering scaffolds [1120].

Corresponding author. Tel.: +40765349326. E-mail address: grumezescu@yahoo.com (A.M. Grumezescu). 0169-4332/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsusc.2013.09.081

Polyvinyl alcohol (PVA) has been used in biomedical applications due to a combination of properties such as biocompatibility, availability, relative low price and low toxicity [21,22]. PVA has been frequently combined with natural molecules that can be recognized by cells favoring the preliminary adhesion and therefore the subsequent colonization of the scaffold [23,24]. It is known that lichens synthesize organic natural compounds by several biologic pathways [25]. The most extensively studied product is the dibenzofuran derivative usnic acid (UA) [26]. Due to its reported biological characteristics, UA has been used worldwide as an anti-microbial, anti-inammatory, anti-viral, antifungal and anti-mitotic agent [27]. The antimicrobial activity of UA against highly versatile human pathogen Staphylococcus aureus was already demonstrated [28]. Because of high incidence, associated morbidity, and antimicrobial resistance, S. aureus infections are a growing concern for clinicians. S. aureus is a common pathogen in skin, soft-tissue, catheter-related, bone, joint, pulmonary, and central nervous

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system infections. S. aureus bacteremias are particularly challenging because of the high incidence of associated complicated infections, including infective endocarditis [29,30]. Approximately 20% of healthy persons are persistent carriers of S. aureus, while 60% are intermittent carriers. Colonization rates are high in infants, hemodialysis patients, illicit injection drug users, surgical patients, and patients with insulin-dependent or poorly controlled diabetes [31,32]. Persistent microbial infections, including S. aureus infections are associated with either biolms or the occurrence of metabolically modied persister cells, named small colony variants (SCVs). SCVs are naturally occurring subpopulations of S. aureus that differ from the normal-phenotype by their small colony sizes (because of their slow rate of growth) and also by decreased pigmentation and hemolysis [33]. SCVs are frequently associated with biolm infections especially in prosthesized patients, where these infections are difcult to treat because of their increased resistance [34]. Titanium implants are routinely used for bone fractures as well as dental work. Recent studies have demonstrated that bacteria attach and develop biolms on titanium implants, degrading the prosthetic device faster and determining an enhanced response in the host [35]. Actual concern in biomedical and implanted device eld supports the development of novel antimicrobial strategies in order to reduce biolm formation on prosthetic devices [36]. We have explored deposition of thin coatings containing biopolymers that serve as carriers for antimicrobial natural compounds by means of matrix assisted pulsed laser evaporation (MAPLE). MAPLE is a laser technique that is based on a cryogenic approach for transferring organic and polymeric materials onto a substrate in a preserved manner [37,38]. This paper reports the MAPLE-deposition of PLAPVAUA microsphere-based thin coatings with enhanced biocompatibility and great anti-microbial adherence. The chemical structure was investigated by IRM, while the surface morphology was tested by SEM. Their inhibitory activity on S. aureus mature biolm development was studied. The coatings cytotoxicity was assessed on mesenchymal stem cells cultures.

2.3. MAPLE target preparation and deposition of microsphere-based thin coatings Hexane solutions of 1% (w/v) PLAPVAUA have been prepared. All MAPLE solutions have been poured into a pre-cooled target holder at 173 K and subsequently immersed in liquid nitrogen for 30 min. MAPLE depositions have been performed using a KrF* ( = 248 nm and FWHM = 25 ns) laser source COMPexPro 205 model, Lambda Physics-Coherent, that was operated at the repetition rate of 10 Hz. The laser uence was within the range of 100400 mJ/cm2 whereas the laser spot area was set at 38 mm2 . A laser beam homogenizer was used to improve the energy distribution of the laser spot. In order to avoid the target heating and subsequent drilling, the frozen target was rotated at a rate of 0.4 Hz during coating deposition. All depositions were conducted at room temperature into a background pressure of 1 Pa. All coatings were grown at a target-substrate separation distance of 4 cm by applying (10,00020,000) subsequent laser pulses. During deposition, the target was kept at a temperature of 173 K by active liquid nitrogen cooling. Thin coatings were deposited onto CP grade 4 Titanium with 8 mm diameter, both side polished (1 0 0) silicon and glass substrates for IRM, SEM and biological assays. Prior to introduction inside the deposition chamber, the substrates were successively cleaned into an ultrasonic bath with acetone, ethanol and deionized water for 15 min, dried in a jet of high purity nitrogen and then plasma-cleaned into oxygen atmosphere for 15 min with a plasma system type FEMTO from Diener electronic GmbH. For comparison data, a control set of lms were prepared by drop casting on the (1 0 0) silicon. 2.4. Characterization methods IR mapping were recorded on a Nicolet iN10 MX FT-IR Microscope with MCT liquid nitrogen cooled detector in the measurement range 4000600 cm1 . Spectral collection was made in reection mode at 4 cm1 resolution. For each spectra, 32 scans were co-added and converted to absorbance using Ominc Picta software (Thermo Scientic). SEM analysis was performed on a FEI electron microscope, using secondary electron beams with energies of 30 keV, on samples covered with a thin gold layer. Biolm formation was analyzed in 6 multi-well plates (Nunc), using a static model for monospecic biolms development. Coated and uncoated sterile titanium discs were distributed in 6 well plates (one per well). Two milliliter S. aureus inoculum with standard density (0.5 McFarland) were added in each well, to completely cover the discs. Samples were incubated for 24 h at 37 C. Biolms formation was assessed after 24 h, 48 h and 72 h by viable cell count assay. Viable cell counts (VCCs) analysis of microorganisms grown in biolms was assessed following an adapted, previously described protocol [41]. Briey, after 24 h incubation the culture medium was removed and the titanium discs were washed with sterile PBS, in order to remove the unattached bacteria. Titanium discs were placed in fresh medium and incubated for other additional 24 h, 48 h and 72 h. After the incubation, the discs were gently washed with sterile PBS to remove the non-adherent cells and placed in 1.5 mL microcentrifuge tubes containing 750 L PBS. Samples were vigorously mixed by vortexing for 30 s and sonicated for 10 s in order to disperse biolm cells into the suspension. Serial 10-fold dilutions were achieved and plated on LB Agar for viable cell counts assay. Experiments were performed in triplicate and repeated on three separate occasions. The biocompatibility was assessed by cytotoxicity assay using mesenchymal stem cells. 2 105 mesenchymal cells were seeded

2. Materials and methods 2.1. Materials The polylactic acid (PLA), polyvinyl alcohol (PVA), usnic acid (UA), chloroform and n-hexane were purchased from SigmaAldrich. Commercial pure (CP) grade 4 Titanium with 8 mm diameter and both side polished (1 0 0) silicon were used as substrates for the MAPLE coatings.

2.2. Chemical synthesis of PLAPVAUA microspheres PLAPVAUA microspheres were prepared using a solvent evaporation method [39,40]. Thus, 400 mg PLA and 50 mg UA were solubilized in 3 mL CHCl3 by sonication. The organic phase was emulsied with a sonicator model SONIC-1200WT from MRC for 5 min, in ON/OFF steps of 5 s and 3 s with a limitation temperature of max 40 C in 5 mL aqueous phase containing 2% (w/v) PVA. After sonication, the emulsion was added in 200 mL deionized water and stirred for 4 h until the CHCl3 was completely evaporated. The microsphere emulsion was centrifuged at 6000 rpm for 10 min. The obtained microspheres were washed four times with ultrapure water, collected by ltration, and then subjected to freeze drying. PLAPVAAU microspheres were used to prepare thin lms by MAPLE technique.

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Fig. 1. Second derivate IR mappings and IR spectra of dropcast surface: intensity distribution of (a) 3500 cm1 (OH stretch, end group), (b) 2924 cm1 (CH3 stretch), (c) 1750 cm1 (C=O carbonyl group), (d) 1090 (CO stretch contributing to alcohols).

Fig. 2. Second derivate IR mappings and IR spectra of thin coating (F = 200 mJ/cm2 ) surface: intensity distribution of (a) 3500 cm1 (OH stretch, end group), (b) 2924 cm1 (CH3 stretch), (c) 1750 cm1 (C=O carbonyl group), (d) 1090 (CO stretch contributing to alcohols).

Fig. 3. Second derivate IR mappings and IR spectra of thin coating (F = 300 mJ/cm2 ) surface: intensity distribution of (a) 3500 cm1 (OH stretch, end group), (b) 2924 cm1 (CH3 stretch), (c) 1750 cm1 (C=O carbonyl group), (d) 1090 (CO stretch contributing to alcohols).

Fig. 4. Second derivate IR mappings and IR spectra of thin coating (F = 500 mJ/cm2 ) surface: intensity distribution of (a) 3500 cm1 (OH stretch, end group), (b) 2924 cm1 (CH3 stretch), (c) 1750 cm1 (C=O carbonyl group), (d) 1090 (CO stretch contributing to alcohols).

Please cite this article in press as: V. Grumezescu, et al., Functionalized antibiolm thin coatings based on PLAPVA microspheres loaded with usnic acid natural compounds fabricated by MAPLE, Appl. Surf. Sci. (2013), http://dx.doi.org/10.1016/j.apsusc.2013.09.081

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Fig. 5. SEM micrographs of dropcasts ((a)(c)) and fabricated thin coatings ((d)(f)).

in each well of a 6 well plate (Nunc), containing a 0.5 cm diameter (coated/uncoated) titanium disc sterile slide, in triplicate, using minimal essential medium (GIBCO, USA) supplemented with 10% foetal bovine serum (GIBCO, USA) and 10 ng/mL basic broblast growth factor (bFGF) (Sigma, USA). Plates were cultured at 37 C in a humidied atmosphere containing 5% CO2 for 24 h. After incubation, titanium discs containing adherent cells were removed, xed in 70% ethanol and stained with 50 g/mL FDA (uorescein diacetate). The morphology of the stained cells was observed using an Observer.D1 Carl Zeiss microscope in order to evaluate cytotoxic potential of tested materials. 2.5. Statistical analysis The statistical signicance of the obtained results was analyzed using GraphPad Prism version 5.04 for Windows, GraphPad Software, San Diego, CA, USA, www.graphpad.com. We used for comparison the number of CFU mL1 as revealed by the readings of three values/experimental variant. Two-way ANOVA and Tukeys multiple comparison tests were used for revealing signicant differences among the analyzed groups. 3. Results and discussion The optimum deposition laser uence was identied using comparative IRM analysis (Figs. 14) between PLAPVAUA microsphere dropcast sample and PLAPVAUA microspheres thin coatings deposited by MAPLE at 200 mJ/cm2 , 300 mJ/cm2 and 400 mJ/cm2 . Figs. 14 show the second derivative infrared maps of all PLAPVAUA surfaces involved in this study. Second derivative infrared mapping can be used as a quick, easy, reproducible, inexpensive, and non-destructive tool to evaluate the purity and structural integrity of samples [42]. Absorbance intensities of IR spectra maps are proportional to color changes starting with blue (the lowest intensity) and gradually increasing through green, yellow to nally red (the highest

intensity) [43]. Approximately 600 spectra were analyzed for each sample. Analyzing the integrity of IR spectra for each sample deposited at different laser uences and the corresponding dropcast coating we selected 300 mJ/cm2 laser uence value as the best compromise between the deposition rate and the stoichiometric transfer to deposit the microsphere lms for biological assays. In Fig. 1 (dropcast) and Fig. 3 (microspheres thin lms prepared at F = 300 mJ/cm2 ), IR spectra are given at different points in plotted maps. The characteristic peaks of the prepared PLAPVAUA microspheres drop-cast (reference material) are assigned to: (a) 3500 cm1 (OH stretch, end group), (b) 2924 cm1 (CH3 stretch), (c) 1750 cm1 (C=O carbonyl group), (d) 1090 (CO stretch contributing to alcohols). As a general remark, typical IR spectra of MAPLE-deposited thin coatings at F = 300 mJ/cm2 show a very close similarity to the corresponding drop-cast IR spectra. Microsphere dimensions, as well as the integrity of prepared microspheres thin lms, were given by SEM analysis. PLAPVAUA microspheres and PLAPVAUA microspheres thin coatings have possessed a homogeneous shape, showing no concavities or distortions on their surface (Fig. 5). Fabricated microspheres and microsphere-based thin coatings have show a 5 m average diameter. Although a tendency to form aggregates cannot be observed, the microspheres have maintained their small dimensions. Biocompatibility results revealed that microsphere coatings are adequate supports for human eukaryotic cells growth, allowing for the normal development of mesenchymal stem cells (MSC). MSC have exhibited a normal morphology and growth on both uncoated and thin lm microsphere-based thin coated slides (Fig. 6a and c), demonstrating that the additional microspheres coatings have neither cytotoxic nor inhibitory effects. Micrographs have revealed that MSC cells adhere normally to plain and micro-coated titanium slides and are stained by FDA, demonstrating their viability (Fig. 6b and d).

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Fig. 6. Mesenchymal stem cells grown on coated and uncoated titanium slides. Typical inverted microscopy images in visible ((a) and (c)) and UV light ((b) and (d)) (100) for dropcasts (controls) ((a) and (b)) and thin coatings ((c) and (d)) proved the usefulness of fabricated thin lm for the fabrication of biocompatible surfaces for implantable prostheses.

Biolm assays have demonstrated that the newly fabricated micro-biocoatings have revealed a great antibiolm effect against S. aureus biolm formation. Staphylococcal biolms are impaired starting with their initialization, differences observed by viable analysis after 24 h of biolm growth being signicant (Fig. 7). These results demonstrate that the micro-biocoated titanium samples provide inappropriate surfaces for adhesion and biolm formation. Furthermore, the mature biolm formation is also altered when they are growing on thin-coated titanium slides, S. aureus biolms

proving a much weaker development after 4872 h. The biolm inhibition can be evaluated by the controlled release of active UA contained into the prepared thin coatings, natural compound that has been previously proved to act as an antimicrobial agent against Gram-positive species [44,45]. 4. Conclusions This paper reports on a newly fabricated micro-biocoating used to improve the surface of titanium prosthesis. Thin coatings of PLAPVA microspheres loaded with UA, deposited by MAPLE on inert substrates have been successfully fabricated and characterized by SEM and IRM. These bio-active thin coatings containing UA exhibited a great antimicrobial effect, inhibiting the adherence and biolm formation by S. aureus. Furthermore, the fabricated microcoating does not alter titanium slides biocompatibility, human mesenchymal cells exhibiting a normal growth and viability pattern on the thin coated titanium slides. Thus, among the advantages offered by PLAPVA microspheres containing UA, we could include improved antibiolm efciency, high biocompatibility, maintenance of UA concentrations within acceptable therapeutic limits, replacement of synthetic antibiolm compounds by natural compounds, lower therapeutic doses that also reduce dose-related side effects and provide a prolonged biological activity. This strategy using bio-active micro-coatings may be developed for clinical studies, improving the surface and lifetime of titanium prosthetic implantable devices by increasing their antimicrobial properties and biocompatibility.

Fig. 7. Graphic representation of viable cell counts analysis after removing S. aureus biolm embedded cells at 24 h, 48 h and 72 h post infection; * p < 0.05, ** p < 0.01, *** p < 0.001 (thin coating vs. uncoated control).

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V. Grumezescu et al. / Applied Surface Science xxx (2013) xxxxxx [21] A. Idris, E. Misran, N. Hassan, A.A. Jalil, C.E. Seng, Modied PVA-alginate encapsulated photocatalyst ferro photo gels for Cr(VI) reduction, J. Hazard. Mat. 227228 (2012) 309316. [22] J. Shen, D.J. Burgess, Accelerated in vitro release testing of implantable PLGA microsphere/PVA hydrogel composite coatings, Int. J. Pharm. 422 (2012) 341348. [23] S.H. Oh, J.H. Lee, Hydrophilization of synthetic biodegradable polymer scaffolds for improved cell/tissue compatibility, Biomed. Mater. 8 (2013) 014101. [24] Y. Wang, K. Kho, W.S. Cheow, K. Hadinoto, A comparison between spray drying and spray freeze drying for dry powder inhaler formulation of drug-loaded lipidpolymer hybrid nanoparticles, Int. J. Pharm. 424 (2012) 98106. [25] K. Molnr, E. Farkas, Current results on biological activities of lichen secondary metabolites: a review, Z. Naturforsch., C: Biosci. 65 (2010) 157173. [26] J.W. Bjerke, A. Elvebakk, E. Domnguez, A. Dahlback, Seasonal trends in usnic acid concentrations of Arctic, alpine and Patagonian populations of the lichen Flavocetraria nivalis, Phytochemistry 66 (2005) 337344. [27] A. Joseph, T. Lee, C.L. Moland, W.S. Branhamc, J.C. Fuscoe, J.E.A. Leakey, W.T. Allaben, S.M. Lewis, A.A. Ali, V.G. Desai, Effect of (+)-usnic acid on mitochondrial functions as measured by mitochondria-specic oligonucleotide microarray in liver of B6C3F1 mice, Mitochondrion 9 (2009) 149158. [28] B. Segatore, P. Bellio, D. Setacci, F. Brisdelli, M. Piovano, J.A. Garbarino, M. Nicoletti, G. Amicosante, M. Perilli, G. Celenza, In vitro interaction of usnic acid in combination with antimicrobial agents against methicillin-resistant Staphylococcus aureus clinical isolates determined by FICI and E model methods, Phytomedicine 19 (2012) 341347. [29] D.M. Bamberger, S.E. Boyd, Management of Staphylococcus aureus infections, Am. Fam. Physician 72 (2005) 24742481. [30] S.P. Chakraborty, S.K. Sahu, P. Pramanik, S. Roy, In vitro antimicrobial activity of nanoconjugated vancomycin against drug resistant Staphylococcus aureus, Int. J. Pharm. 436 (2012) 659676. [31] P. Moreillon, Y.A. Que, M.P. Glauser, Staphylococcus aureus (including staphylococcal toxic shock), in: G.L. Man-dell, J.E. Bennett, R. Dolin (Eds.), Mandell, Douglas, and Bennetts Principles and Practice of Infectious Diseases, sixth ed., Elsevier Churchill Livingstone, Philadelphia, PA, 2005, pp. 23212351. [32] A. Wattanasatcha, S. Rengpipat, S. Wanichwecharungruang, Thymol nanospheres as an effective anti-bacterial agent, Int. J. Pharm. 434 (2012) 360365. [33] P. Sendi, M. Rohrbach, P. Graber, R. Frei, P.E. Ochsner, W. Zimmerli, Staphylococcus aureus small colony variants in prosthetic joint infection, Clin. Infect. Dis. 43 (2006) 961967. [34] C. von Eiff, K. Becker, D. Metze, Intracellular persistence of Staphylococcus aureus small-colony variants within keratinocytes: a cause for antibiotic treatment failure in a patient with Dariers disease, Clin. Infect. Dis. 32 (2001) 16431647. [35] R. Brgers, T. Gerlach, S. Hahnel, F. Schwarz, G. Handel, M. Gosau, In vivo and in vitro biolm formation on two different titanium implant surfaces, Clin. Oral Implants Res. 21 (2010) 156164. [36] A.M. Grumezescu, M.C. Chiriuc, C. Saviuc, V. Grumezescu, R. Hristu, D.E. Mihaiescu, G.A. Stanciu, E. Andronescu, Hybrid nanomaterial for stabilizing the antibiolm activity of Eugenia carryophyllata essential oil, IEEE Trans. Nanobiosci. 11 (2012) 360. [37] R.A. McGill, D.B. Chrisey, Method of producing a lm coating by matrix assisted pulsed laser deposition, in: Patent number 6,025,036, 2000. [38] D.B. Chrisey, R.A. McGill, J.S. Horwitz, A. Pique, B.R. Ringeisen, D.M. Bubb, P.K. Wu, Laser Deposition of Polymer and Biomaterial Films, Chem. Rev. 103 (2003) 553576. [39] T.V.P. Doan, W. Couet, J.C. Olivier, Formulation and in vitro characterization of inhalable rifampicin-loaded PLGA microspheres for sustained lung delivery, Int. J. Pharm. 414 (2011) 112117. [40] C. Hu, H. Feng, C. Zhu, Preparation and characterization of rifampicin-PLGA microspheres/sodium alginate in situ gel combination delivery system, Colloids Surf., B 95 (2012) 162169. ar, E. Andronescu, [41] C. Chiriuc, V. Grumezescu, A.M. Grumezescu, C. Saviuc, V. Laz Hybrid magnetite nanoparticles/Rosmarinus ofcinalis essential oil nanobiosystem with antibiolm activity, Nanoscale Res. Lett. 7 (2012) 209. [42] D.M. Byler, R.M. Wilson, C.S. Randall, T.D. Sokoloski, Second derivative infrared spectroscopy as a nondestructive tool to assess the purity and structural integrity of proteins, Pharm. Res. 12 (1995) 446450. [43] A.M. Grumezescu, E. Andronescu, A.M. Holban, A. Ficai, D. Ficai, G. Voicu, V. Grumezescu, P.C. Balaure, M.C. Chiriuc, Water dispersible cross-linked magnetic chitosan beads for increasing the antimicrobial efciency of different therapeutic agents, Int. J. Pharm. (2013), in press. [44] A.M. Grumezescu, A.I. Cotar, E. Andronescu, A. Ficai, C.D. Ghitulica, V. Grumezescu, B.S. Vasile, M.C. Chiriuc, In vitro activity of the new waterdispersible Fe3 O4 @usnic acid nanostructure against planktonic and sessile bacterial cells, J. Nano. Res. 15 (2013) 1766. [45] A.M. Grumezescu, C. Saviuc, M.C. Chiriuc, R. Hristu, D.E. Mihaiescu, P. Balaure, G. Stanciu, V. Lazar, Inhibitory activity of Fe3 O4 /oleic acid/usnic acidcore/shell/extra-shell nanouid on S. aureus biolm development, IEEE Trans. Nanobiosci. 10 (2011) 269.

Acknowledgments This work was supported by the Romanian National Authority for Scientic Research, CNCS-UEFISCDI, project number PCCA 153/02.07.2012. References
[1] A.M. Grumezescu, E. Andronescu, A. Ficai, D. Ficai, K.S. Huang, I. Gheorghe, M.C. Chiriuc, Water soluble magnetic biocomposite with potential applications for the antimicrobial therapy, Biointerface Res. Appl. Chem. 2 (2012) 469475. [2] A.M. Grumezescu, E. Andronescu, A. Ficai, C. Bleotu, M.C. Chiriuc, Chitin based biomaterial for antimicrobial therapy: fabrication, characterization and in vitro prole based interaction with eukaryotic and prokaryotic cells, Biointerface Res. Appl. Chem. 2 (2012) 438446. [3] E. Andronescu, A.M. Grumezescu, A. Ficai, I. Gheorghe, M. Chiriuc, D.E. Mihaiescu, V. Lazar, In vitro efcacy of antibiotic magnetic dextran microspheres complexes against Staphylococcus aureus and Pseudomonas aeruginosa strains, Biointerface Res. Appl. Chem. 2 (2012) 332338. [4] A.M. Grumezescu, A.M. Holban, E. Andronescu, A. Ficai, C. Bleotu, M.C. Chiriuc, Water dispersible metal oxide nanobiocomposite as a potentiator of the antimicrobial activity of kanamycin, Lett. Appl. NanoBioSci. 1 (2012) 7782. [5] A.M. Grumezescu, A. Holban, E. Andronescu, A. Ficai, C. Bleotu, M.C. Chiriuc, Microbiological applications of a new water dispersible magnetic nanobiocomposite, Lett. Appl. NanoBioSci. 1 (2012) 8390. [6] B. Prez-Artacho, V. Gallardo, M.A. Ruiz, J.L. Arias, Maghemite/poly(d,llactide-co-glycolyde) composite nanoplatform for therapeutic applications, J. Nanopart. Res. (2012), 10.1007/s11051-012-0768-x. [7] P. Pouponneau, V. Segura, O. Savadogo, J.C. Leroux, S. Marte, Annealing of magnetic nanoparticles for their encapsulation into microcarriers guided by vascular magnetic resonance navigation, J. Nanopart. Res. (2012), 10.1007/s11051-012-1307-5. [8] R. Jeetah, A. Bhaw-Luximon, D. Jhurry, New amphiphilic PEG-b-P(esterether) micelles as potential drug nanocarriers, J. Nanopart. Res. (2012), 10.1007/s11051-012-1168-y. [9] Z. Zhao, A. Chen, Y. Li, J. Hu, X. Liu, J. Li, Y. Zhang, G. Li, Z. Zheng, Fabrication of silk broin nanoparticles for controlled drug delivery, J. Nanopart. Res. (2012), http://dx.doi.org/10.1007/s11051-012-0736-5. [10] Q. Miao, S. Li, S. Han, Z. Wang, Y. Wu, G. Nie, Construction of hydroxypropyl-cyclodextrin copolymer nanoparticles and targeting delivery of paclitaxel, J. Nanopart. Res. (2012), http://dx.doi.org/10.1007/s11051-012-1043-x. [11] H. Zhao, Z. Cui, X. Wang, L.S. Turng, X.F. Peng, Processing and characterization of solid and microcellular poly(lactic acid)/polyhydroxybutyrate-valerate (PLA/PHBV) blends and PLA/PHBV/Clay nanocomposites, Composites Part B 51 (2013) 7991. [12] Y.M. Corre, A. Maazouz, J. Duchet, J. Reignier, Batch foaming of chain extended PLA with supercritical CO(2): inuence of the rheological properties and the process parameters on the cellular structure, J. Supercrit. Fluids 58 (2011) 177188. [13] C. Thammawong, P. Sreearunothai, A. Petchsuk, P. Tangboriboonrat, N. Pimpha, P. Opaprakasit, Preparation and characterizations of naproxen-loaded magnetic nanoparticles coated with PLA-g-chitosan copolymer, J. Nano. Res. (2012), http://dx.doi.org/10.1007/s11051-012-1046-7. [14] E.K. Efthimiadou, L.A. Tziveleka, P. Bilalis, G. Kordas, Novel PLA modication of organic microcontainers based on ring opening polymerization: synthesis, characterization, biocompatibility and drug loading/release properties, Int. J. Pharm. 428 (2012) 134142. [15] L.M. Matuana, Solid state microcellular foamed poly(lactic acid): morphology and property characterization, Bioresour. Technol. 99 (2008) 36433650. [16] D. Jeevitha, K. Amarnath, Chitosan/PLA nanoparticles as a novel carrier for the delivery of anthraquinone: synthesis, characterization and in vitro cytotoxicity evaluation, Colloids Surf., B 101 (2013) 126134. [17] C. Giovino, I. Ayensu, J. Tetteh, J.S. Boateng, Development and characterisation of chitosan lms impregnated with insulin loaded PEG-b-PLA nanoparticles (NPs): a potential approach for buccal delivery of macromolecules, Int. J. Pharm. 428 (2012) 143151. [18] H. Xia, X. Gao, G. Gu, Z. Liu, Q. Hu, Y. Tu, Q. Song, L. Yao, Z. Pang, X. Jiang, J. Chen, H. Chen, Penetratin-functionalized PEGPLA nanoparticles for brain drug delivery, Int. J. Pharm. 436 (2012) 840850. [19] Q. Miao, S. Li, S. Han, Z. Wang, Y. Wu, G. Nie, Construction of hydroxypropyl-cyclodextrin copolymer nanoparticles and targeting delivery of paclitaxel, J. Nano. Res. (2012), http://dx.doi.org/10.1007/s11051-012-1043-x. [20] A. El Fagui, C. Amiel, PLA nanoparticles coated with a -cyclodextrin polymer shell: preparation, characterization and release kinetics of a hydrophobic compound, Int. J. Pharm. 436 (2012) 644651.

Please cite this article in press as: V. Grumezescu, et al., Functionalized antibiolm thin coatings based on PLAPVA microspheres loaded with usnic acid natural compounds fabricated by MAPLE, Appl. Surf. Sci. (2013), http://dx.doi.org/10.1016/j.apsusc.2013.09.081