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shell nanoparticle thin films deposited by MAPLE: Investigation by chemical, morphological and in vitro biological assays
Applied Surface Science 258 (2012) 9250–9255
Contents lists available at SciVerse ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Magnetic core/shell nanoparticle thin films deposited by MAPLE: Investigation by chemical, morphological and in vitro biological assays R. Cristescu a,∗ , C. Popescu a , G. Socol a , I. Iordache a , I.N. Mihailescu a , D.E. Mihaiescu b , A.M. Grumezescu b , A. Balan c , I. Stamatin c , C. Chifiriuc d , C. Bleotu e , C. Saviuc d , M. Popa d , D.B. Chrisey f a
National Institute for Lasers, Plasma & Radiation Physics, Lasers Department, P.O. Box MG-36, Bucharest-Magurele, Romania Faculty of Applied Chemistry and Materials Science, “Politehnica” University of Bucharest, 1–7 Polizu Street, 011061 Bucharest, Romania c University of Bucharest, 3Nano-SAE Research Center, PO Box MG-38, Bucharest-Magurele, Romania d Faculty of Biology, University of Bucharest, Microbiology Immunology Department, Aleea Portocalilor 1-3, Sector 5, 77206 Bucharest, Romania e Stefan S. Nicolau Institute of Virology, 285 Mihai Bravu, 030304 Bucharest, Romania f Rensselaer Polytechnic Institute, School of Engineering, Departments of Materials Science & Biomedical Engineering, Troy, 12180-3590, NY, USA b
a r t i c l e
i n f o
Article history: Available online 18 February 2012 Keywords: Fe3 O4 /oleic acid/cephalosporin nanoparticles Core/shell Magnetic nanoparticle Matrix assisted pulsed laser evaporation Antimicrobial activity
a b s t r a c t We report on thin film deposition of nanostructured Fe3 O4 /oleic acid/ceftriaxone and Fe3 O4 /oleic acid/cefepime nanoparticles (core/shell/adsorption-shell) were fabricated by matrix assisted pulsed laser evaporation (MAPLE) onto inert substrates. The thin films were characterized by profilometry, Fourier transform infrared spectroscopy, atomic force microscopy, and investigated by in vitro biological assays. The biological properties tested included the investigation of the microbial viability and the microbial adherence to the glass coverslip nanoparticle film, using Gram-negative and Gram-positive bacterial strains with known antibiotic susceptibility behavior, the microbial adherence to the HeLa cells monolayer grown on the nanoparticle pellicle, and the cytotoxicity on eukaryotic cells. The proposed system, based on MAPLE, could be used for the development of novel anti-microbial materials or strategies for fighting pathogenic biofilms frequently implicated in the etiology of biofilm associated chronic infections. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The technological importance of nanoparticles has driven efforts to fabricate novel core/shell nanomaterials, which are of fundamental interest to modern materials science due to their vast applications in controlled drug release [1], magnetic drug targeting [2], inhibition of microbial biofilm growth [3], biosensors [4], antimicrobial therapy [5] or medical diagnostics [6–12]. Several synthesis methods have been used in the past to obtain nanoparticles with core/shell morphology [1–5]. To examine the efficacy of magnetic core/shell nanoparticles for antimicrobial activity, our research focused on the transfer of these magnetic nanoparticles, functionalized with antibiotics, onto assays
∗ Corresponding author at: National Institute for Lasers, Plasma & Radiation Physics, Lasers Department, P.O. Box MG-36, Bucharest, Magurele, Romania. Tel.: +40 21 4574491; fax: +40 21 4574243. E-mail addresses: rodica.cristescu@inflpr.ro, [email protected] (R. Cristescu). 0169-4332/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2012.02.055
for further structural, morphological and antibacterial investigations. The envisaged antibiotics for this study are ceftriaxone [13] and cefepime [14]; two cephalosporins (beta-lactam derivatives) used for the treatment of a number of common bacterial infections, exhibiting a bactericidal effect by preventing bacteria from building and maintaining their cell walls [15]. Laser processing offers an attractive alternative because of several advantages including compatibility with non-contact masking techniques and minimal degradation. Matrix assisted pulsed laser evaporation (MAPLE) [16] was developed to overcome the difficulties in solventbased coating technologies and has been successfully applied to many different systems [17–22]. Herein, thin films of Fe3 O4 /oleic acid/ceftriaxone and Fe3 O4 /oleic acid/cefepime nanoparticles have been produced by MAPLE. The chemical structure and surface morphology of MAPLE-obtained thin films and structures were investigated by profilometry, Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM). The antibacterial activity of Fe3 O4 /oleic acid/ceftriaxone and Fe3 O4 /oleic acid/cefepime nanoparticle thin films was tested against Grampositive and Gram-negative bacterial strains.
R. Cristescu et al. / Applied Surface Science 258 (2012) 9250–9255
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Fig. 1. Typical FTIR spectra (from down to top) of (a) Fe3 O4 /oleic drop-cast and (b) Fe3 O4 /oleic acid thin films; (c) Fe3 O4 /oleic acid/ceftriaxone drop-cast and (d) Fe3 O4 /oleic acid/ceftriaxone thin films; (e) Fe3 O4 /oleic acid/cefepime drop-cast and (f) Fe3 O4 /oleic acid/cefepime thin films. All MAPLE films were deposited at an incident laser fluence of 200 mJ/cm2 .
2. Experimental 2.1. Materials The preparation protocols of Fe3 O4 /oleic acid core/shell and Fe3 O4 /oleic acid/cephalosporins core/shell/adsorption-shell are described in detail in Ref. [23]. It was shown by HRTEM investigations that the dimensions of Fe3 O4 nanoparticles were within the 5–20 nm range. In this work, we used two cephalosporins as adsorption-shells, ceftriaxone [Hoffman-La Roche] and cefepime [Bristol-Myers Squibb], respectively. Solutions consisting of 1% (wt./vol.) of Fe3 O4 /oleic acid/cephalosporin core-shell/adsorptionshell in chloroform were prepared and tested. All solutions were poured into a pre-cooled target holder at 173 K and subsequently immersed in liquid nitrogen for 30 min. 2.2. MAPLE experimental conditions Thin films were fabricated by MAPLE and drop-casting. All MAPLE depositions were conducted using a KrF* 248 nm laser which was operated at a fluence of 65–300 mJ/cm2 , a repetition rate of 10 Hz, and for 7200–20,000 pulses. The target was rotated at a rate of 0.4 Hz during deposition and the laser beam scanned the entire target surface at an angle of 45◦ . All depositions were conducted with a background pressure of 30–100 Pa and a substrate-to-target distance of 4 cm. The target was maintained at a temperature of ∼173 K by active liquid nitrogen cooling. Thin films were deposited onto one-side polished Si 1 0 0 substrates for FTIR, AFM, and optic glass for antibacterial assays/strains. All of the substrates were ultrasonically cleaned prior to deposition by immersion in ethanol, and then dried in air under UV exposure from a VL-115UV lamp.
deflection height-measurement sensor and stylus with 2.5 m radius and 0.1 mg force. Thin film composition and morphology was investigated by FTIR and AFM, respectively. FTIR spectra were recorded by a Thermo 6700 FTIR/Raman system with a resolution 4 cm−1 that operated at 4000–650 cm−1 in transmission and/or micro-ATR/ZnSe modes. AFM micrographs were obtained with an Integrated Platform SPM-NTegra model Prima in phase contrast mode. All AFM images were recorded on 5 × 5 m2 areas. 2.4. Microbial strains used for antimicrobial activity assay The antimicrobial activity of the obtained systems (drop-casts and MAPLE-deposited thin films) was tested against Gram-positive (Staphylococcus aureus (S. aureus), and Bacillus subtilis (B. subtilis)) and Gram-negative (Pseudomonas aeruginosa (Ps. aeruginosa), Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli)) reference strains. The Enterobacteriaceae strains were identified by API 20E biochemical tests, while Ps. aeruginosa, B. subtilis and S. aureus were identified by VITEK I automatic system. VITEK cards for identification and susceptibility testing (GNS-522) were inoculated and then incubated according to the manufacturer’s recommendations [24,25]. These cards contain different lyophilized biochemical substrates with various antibiotic concentrations. In the presence of certain substrate and antibiotic concentration, the microbial growth is measured with a spectrophotometer that provides the biochemical identification of strain and its susceptibility to different antibiotics. Bacterial suspensions of 0.5 McFarland IU density obtained from 18 h bacterial cultures developed on solid media were used in the experiments [26]. 2.5. Microbiological assay investigation procedure
2.3. Profilometry FTIR, and AFM investigations Thin film thickness was recorded using a Stylus Profiler XP2 system (Ambios) with 0.1 nm vertical resolution, an optical
The pelliculised samples were placed in 2 ml nutrient broth and incubated for 24 h at 37 ◦ C. After 24 h of incubation, the samples were washed in sterile saline solution in order to remove
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Fig. 2. Atomic force micrographs of (a) Fe3 O4 /oleic drop-cast and (b) Fe3 O4 /oleic acid thin films; (c) Fe3 O4 /oleic acid/ceftriaxone drop-cast and (d) Fe3 O4 /oleic acid/ceftriaxone thin films; (e) Fe3 O4 /oleic acid/cefepime drop-cast and (f) Fe3 O4 /oleic acid/cefepime thin films. All thin films have been deposited by MAPLE at 20 mJ laser energy.
the non-adherent bacteria, while the adherent bacteria remained attached to the respective samples. After washing, the samples were placed in fresh medium and incubated for another 24 h. After incubation the turbidity of the culture medium was determined by measuring the absorbance at 600 nm, the obtained values being proportional to the percentage of viable bacterial cells adhered on the pelliculised slide [27]. In order to measure the influence of the tested hybrid system on the cell wall properties, we have also characterized the ability of viable bacterial cells to adhere to glass substrate. The bacterial cells secrete an exopolysaccharide substance implicated in the bacterial adherence to inert substrates known as slime. When bacterial cultures are obtained in liquid media, the slime production is followed by the adherence of bacterial cells to the glass walls and formation of a ring at the interface liquid–air. The intensity of the ring occurred on the culture tubes walls was appreciated by 0, 1 or 2 “++”.
2.6. Microbial adhesion to eukariotic cells grown on the pelliculised coverslips The Human epithelial carcinoma (HeLa) cell line was cultivated on the pelliculised samples and incubated for 24 h at 37 ◦ C in 5% CO2 atmosphere. Thereafter, the microbial suspensions were added and incubated for 2 h at 37 ◦ C. After incubation, the samples were fixed by ethanol, Giemsa stained and examined using an Olympus inverted microscope in order to appreciate the degree of cell confluency and indirectly, the cytotoxic effect of nanoparticles, as well as the number of adherent bacteria and the adherence pattern. Three distinct patterns of adherence have been investigated during this study: localized adherence (LA), in which bacteria attach to and form microcolonies in distinct regions of the surface; diffuse adherence (DA), in which bacteria adhere evenly to the whole cell surface, and aggregative adherence (AggA), in which aggregated bacteria attach to the cell in a stacked-brick arrangement.
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Fig. 3. Influence of the tested drop-cast/MAPLE-deposited thin films on the intensity of microbial growth quantified by measuring the absorbance at 600 nm of the liquid cultures.
Fig. 4. Influence of the drop-cast/MAPLE-deposited thin films on the ability of the tested bacterial strains to adhere to the inert substrate represented by the glass wall, quantified by the slime production by measuring the absorbance at 490 nm.
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The adherence index was expressed as the ratio between the numbers of the eukaryotic cells and adhered bacteria: 100 eukaryotic cells counted within the microscopic field (examined with the 100× immersion objective) [28]. 3. Results and discussion
roughness values for films versus drop-casts. It is generally known that a larger roughness means an extended active surface which is considered beneficial for any biological applications including the antimicrobial activity. A larger active surface acts strongly and more efficiently against microbes of any sort, i.e., the ones studied in this manuscript.
3.1. Profilometry and FTIR investigations
3.3. Biological activities
Optimal deposition laser fluence was identified using comparative FTIR analysis between spectra of the reference core/shell material and thin films deposited by MAPLE at 65, 100, 200 and 300 mJ/cm2 . Moreover, the typical thickness values for films deposited at 65 and 100 mJ/cm2 are about 250 and 300 nm, respectively. At 200 mJ/cm2 fluence, the film thickness is about 400 nm, while the films deposited at 300 mJ/cm2 have 1.6 m thickness. Corresponding to the best compromise between chemical structure and growth rate, we decided to continue our further investigations with a 200 mJ/cm2 fluence. In Fig. 1, FTIR spectra are given of Fe3 O4 /oleic drop-cast (a), Fe3 O4 /oleic acid thin films (b), Fe3 O4 /oleic acid/ceftriaxone drop-cast (c), Fe3 O4 /oleic acid/ceftriaxone thin films (d), Fe3 O4 /oleic acid/cefepime drop-cast (e), and Fe3 O4 /oleic acid/cefepime thin films (f). The characteristic peaks of the Fe3 O4 /oleic drop-cast (reference material) are assigned to: ferrite characteristic vibrations (∼610 cm−1 ), CH2 and CH3 stretching (∼2950, 2923 and 2853 cm−1 ) correlated to deformation modes (1300 and 1400 cm−1 ), oleic acid OH stretching (3424 cm−1 ), and silicon substrate peak (1108 cm−1 ). The specific peaks of ceftriaxone are centered at about 1038, 1781, and 3197 cm−1 (Fig. 1c and d), while the specific ones of cefepime are 729, 1364, 1742 and 3251 cm−1 (Fig. 1e–f). The chloroform dissociation products are volatile and expected to be pumped away by the vacuum system. As a general remark, all MAPLE-deposited thin film spectra show a very close similarity to the corresponding drop-cast FTIR spectra.
Our results showed that the nanoparticles coated with antibiotics inhibited the microbial growth and the adherence of the tested bacterial strains to the inert substrates. However, the intensity of the inhibitory effect varied with the tested strain. Concerning the interference with the microbial growth, the drop-cast procedure proved to be much more efficient than MAPLE in what concerns the preservation of the antibiotic activity. Concerning the microbicidal effect, the most evident results were obtained in case of Gram-positive microorganisms (i.e., B. subtilis and S. aureus) as well as for E. coli for both antibiotics and, in case of Ps. aeruginosa and K. pneumoniae for ceftriaxone only (Fig. 3). Thus, with few exceptions, the inclusion of beta-lactam antibiotics in the nanofilm lead to an increased antimicrobial activity as compared to that exhibited by the antibiotic alone. These results could be due either to an intrinsic antimicrobial effect of the nanoparticles or to an improved diffusion rate of the antibiotic in the liquid medium. Concerning the influence of the tested nanosystems on the ability of microbial strains to colonize the pelliculised glass coverslips our results showed an improved anti-biofilm activity for cefepime against the inert substrate colonization by the Gram-negative strains, probably due to the interference of the tested nanosystems with the production of the bacterial extracellular polysaccharide called slime known to be implicated in the microbial adherence to different substrates (Fig. 4). In this experiment, the MAPLE deposition technique proved to be more efficient than drop-cast concerning the intensity of the anti-biofilm effect. The hybrid nanosystems did not inhibit, but stimulated the colonization of the inert substrate by the Gram-positive strains, demonstrating the specific interaction of the nanosystems components with the bacterial function. Our results have shown that the nanoparticles did not significantly influence the adherence of HeLa cells to the pelliculised slides that developed a confluent monolayer after 24 h of incubation. The examination of the cell monolayer by inverted microscopy evidenced no morphological changes in HeLa cell morphology as compared to the control, demonstrating that the nanofilms have no cytotoxic effect on the eukaryotic cells. Neither the adherence pattern, nor the adherence index was affected by the pelliculised nanoparticles.
3.2. AFM studies AFM images of both drop-cast and MAPLE-deposited thin film at 200 mJ/cm2 in case of Fe3 O4 /oleic acid, Fe3 O4 /oleic acid/ceftriaxone, and Fe3 O4 /oleic acid/cefepime are given in Fig. 2. One first observation is that the density and size of the grains are larger on the films than the case of corresponding drop-casts. This slight change of morphology by MAPLE transfer is well known in literature and is demonstrated to depend on solvent and solute concentration and the laser deposition parameters [18]. In Fig. 2a, the surface morphology of Fe3 O4 /oleic acid drop-cast (13 nm RMS) shows a random distribution of coarse grains with diameters within the 1–300 nm range. In Fig. 2c, the surface morphology of Fe3 O4 /oleic acid/ceftriaxone drop-cast displays a network of small grains (5.6 nm RMS) while the surface of Fe3 O4 /oleic acid/cefepime drop-cast (7.1 nm RMS) exhibits small separated grains (Fig. 2e). The thin films of Fe3 O4 /oleic acid show a uniform morphology with big grains with diameters within the range of 0.5–1.5 m (61 nm RMS, Fig. 2b). Thin films of Fe3 O4 /oleic acid/ceftriaxone exhibit a morphology with smaller rounded grains with diameters within the range of 0.2–1 m not uniformly distributed on the surface (46 nm RMS, Fig. 2d). Although Fe3 O4 /oleic acid/cefepime thin film morphology puts in evidence much smaller elongated grains with 0.1–0.5 m diameters uniformly distributed (31 nm RMS, Fig. 2f), the cephalosporin drug has an aditive effect on the grain size and even a modification in grain shape in the specific case of cefepime drug. Grains of a larger density and size were observed on MAPLE films by comparison with the corresponding drop-casts (particularly visible in Fig. 2). This reflects in the higher
4. Conclusions We have demonstrated in the case of magnetic Fe3 O4 /oleic acid/ceftriaxone and Fe3 O4 /oleic acid/cefepime core/shell/adsorption-shell nanoparticles that MAPLE is a very efficient technique for obtaining thin films having a chemical structure similar to the starting material. The surface morphology of Fe3 O4 /oleic acid/cephalosporin thin films evidenced by AFM studies revealed that the cephalosporin drug has an aditive effect on the grain size and even a modification in grain shape in the specific case of cefepime drug. We demonstrated that the antibiotic charged nanoparticles exhibited a strong inhibitory effect on the viability as well as on the adherence to the cellular monolayer and to the inert substrate of the tested strains. This improvement in the anti-biofilm activity of the cephalosporin antibiotics correlated, with equal or superior rates to the minimal inhibitory concentration of the antibiotic suspensions allow us to conclude that
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MAPLE-deposited magnetic nanoparticle thin films could be used for the development of novel anti-microbial materials or strategies for fighting medical biofilms pathogens frequently implicated in the etiology of biofilm associated chronic infections, having the advantage of low cytotoxicity on the eukaryotic cells. Acknowledgments This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS-UEFISCDI, project no. PN-II-ID-PCE-2011-3-0888 (209/5.10.2011). This work was also supported by the CNCSIS Human Resources, project no. 135/2010 (contract no. 76/2010). One of the authors, Gabriel Socol, gratefully acknowledges the European Social Fund POSDRU 2007-2013 through the contract POSDRU/89/1.5/S/60746. References [1] D.E. Mihaiescu, A.M. Grumezescu, D.E. Mogosanu, V. Traistaru, P.C. Balaure, A. Buteica, Biointerface Res. Appl. Chem. 1 (2011) 41–47. [2] A.M. Grumezescu, C. Saviuc, A. Holban, R. Hristu, C. Croitoru, G. Stanciu, C. Chifiriuc, D. Mihaiescu, P. Balaure, V. Lazar, Biointerface Res. Appl. Chem. 1 (2011) 160–165. [3] C. Saviuc, A.M. Grumezescu, M.C. Chifiriuc, C. Bleotu, G. Stanciu, R. Hristu, D. Mihaiescu, V. Laz˘ar, Biointerface Res. Appl. Chem. 1 (2011) 31–40. [4] M. Chirea, E.M. Pereira, C.M. Pereira, F. Silva, Biointerface Res. Appl. Chem. 1 (2011) 119–126. [5] A.S. Buteic˘a, D.E. Mihaiescu, A.M. Grumezescu, B.S¸. Vasile, A. Popescu, D. C˘alina, O.M. Mihaiescu, Digest J. Nanomater. Biostruct. 5 (2010) 651–655. [6] S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L.V. Elst, R.N. Muller, Chem. Rev. 108 (2008) 2064–2110. [7] A.K. Gupta, M. Gupta, Biomaterials 26 (2005) 3995–4021. [8] T.-J. Yoon, K. Yu, E. Kim, J. Kim, B. Kim, S.-H. Yun, B.-H. Sohn, M.-H. Cho, J.-K. Lee, S. Park, Small 2 (2006) 209–215. [9] J. Zhang, R.D.K. Misra, Acta Biomater. 3 (2007) 838–850.
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Contents lists available at SciVerse ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Magnetic core/shell nanoparticle thin films deposited by MAPLE: Investigation by chemical, morphological and in vitro biological assays R. Cristescu a,∗ , C. Popescu a , G. Socol a , I. Iordache a , I.N. Mihailescu a , D.E. Mihaiescu b , A.M. Grumezescu b , A. Balan c , I. Stamatin c , C. Chifiriuc d , C. Bleotu e , C. Saviuc d , M. Popa d , D.B. Chrisey f a
National Institute for Lasers, Plasma & Radiation Physics, Lasers Department, P.O. Box MG-36, Bucharest-Magurele, Romania Faculty of Applied Chemistry and Materials Science, “Politehnica” University of Bucharest, 1–7 Polizu Street, 011061 Bucharest, Romania c University of Bucharest, 3Nano-SAE Research Center, PO Box MG-38, Bucharest-Magurele, Romania d Faculty of Biology, University of Bucharest, Microbiology Immunology Department, Aleea Portocalilor 1-3, Sector 5, 77206 Bucharest, Romania e Stefan S. Nicolau Institute of Virology, 285 Mihai Bravu, 030304 Bucharest, Romania f Rensselaer Polytechnic Institute, School of Engineering, Departments of Materials Science & Biomedical Engineering, Troy, 12180-3590, NY, USA b
a r t i c l e
i n f o
Article history: Available online 18 February 2012 Keywords: Fe3 O4 /oleic acid/cephalosporin nanoparticles Core/shell Magnetic nanoparticle Matrix assisted pulsed laser evaporation Antimicrobial activity
a b s t r a c t We report on thin film deposition of nanostructured Fe3 O4 /oleic acid/ceftriaxone and Fe3 O4 /oleic acid/cefepime nanoparticles (core/shell/adsorption-shell) were fabricated by matrix assisted pulsed laser evaporation (MAPLE) onto inert substrates. The thin films were characterized by profilometry, Fourier transform infrared spectroscopy, atomic force microscopy, and investigated by in vitro biological assays. The biological properties tested included the investigation of the microbial viability and the microbial adherence to the glass coverslip nanoparticle film, using Gram-negative and Gram-positive bacterial strains with known antibiotic susceptibility behavior, the microbial adherence to the HeLa cells monolayer grown on the nanoparticle pellicle, and the cytotoxicity on eukaryotic cells. The proposed system, based on MAPLE, could be used for the development of novel anti-microbial materials or strategies for fighting pathogenic biofilms frequently implicated in the etiology of biofilm associated chronic infections. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The technological importance of nanoparticles has driven efforts to fabricate novel core/shell nanomaterials, which are of fundamental interest to modern materials science due to their vast applications in controlled drug release [1], magnetic drug targeting [2], inhibition of microbial biofilm growth [3], biosensors [4], antimicrobial therapy [5] or medical diagnostics [6–12]. Several synthesis methods have been used in the past to obtain nanoparticles with core/shell morphology [1–5]. To examine the efficacy of magnetic core/shell nanoparticles for antimicrobial activity, our research focused on the transfer of these magnetic nanoparticles, functionalized with antibiotics, onto assays
∗ Corresponding author at: National Institute for Lasers, Plasma & Radiation Physics, Lasers Department, P.O. Box MG-36, Bucharest, Magurele, Romania. Tel.: +40 21 4574491; fax: +40 21 4574243. E-mail addresses: rodica.cristescu@inflpr.ro, [email protected] (R. Cristescu). 0169-4332/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2012.02.055
for further structural, morphological and antibacterial investigations. The envisaged antibiotics for this study are ceftriaxone [13] and cefepime [14]; two cephalosporins (beta-lactam derivatives) used for the treatment of a number of common bacterial infections, exhibiting a bactericidal effect by preventing bacteria from building and maintaining their cell walls [15]. Laser processing offers an attractive alternative because of several advantages including compatibility with non-contact masking techniques and minimal degradation. Matrix assisted pulsed laser evaporation (MAPLE) [16] was developed to overcome the difficulties in solventbased coating technologies and has been successfully applied to many different systems [17–22]. Herein, thin films of Fe3 O4 /oleic acid/ceftriaxone and Fe3 O4 /oleic acid/cefepime nanoparticles have been produced by MAPLE. The chemical structure and surface morphology of MAPLE-obtained thin films and structures were investigated by profilometry, Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM). The antibacterial activity of Fe3 O4 /oleic acid/ceftriaxone and Fe3 O4 /oleic acid/cefepime nanoparticle thin films was tested against Grampositive and Gram-negative bacterial strains.
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Fig. 1. Typical FTIR spectra (from down to top) of (a) Fe3 O4 /oleic drop-cast and (b) Fe3 O4 /oleic acid thin films; (c) Fe3 O4 /oleic acid/ceftriaxone drop-cast and (d) Fe3 O4 /oleic acid/ceftriaxone thin films; (e) Fe3 O4 /oleic acid/cefepime drop-cast and (f) Fe3 O4 /oleic acid/cefepime thin films. All MAPLE films were deposited at an incident laser fluence of 200 mJ/cm2 .
2. Experimental 2.1. Materials The preparation protocols of Fe3 O4 /oleic acid core/shell and Fe3 O4 /oleic acid/cephalosporins core/shell/adsorption-shell are described in detail in Ref. [23]. It was shown by HRTEM investigations that the dimensions of Fe3 O4 nanoparticles were within the 5–20 nm range. In this work, we used two cephalosporins as adsorption-shells, ceftriaxone [Hoffman-La Roche] and cefepime [Bristol-Myers Squibb], respectively. Solutions consisting of 1% (wt./vol.) of Fe3 O4 /oleic acid/cephalosporin core-shell/adsorptionshell in chloroform were prepared and tested. All solutions were poured into a pre-cooled target holder at 173 K and subsequently immersed in liquid nitrogen for 30 min. 2.2. MAPLE experimental conditions Thin films were fabricated by MAPLE and drop-casting. All MAPLE depositions were conducted using a KrF* 248 nm laser which was operated at a fluence of 65–300 mJ/cm2 , a repetition rate of 10 Hz, and for 7200–20,000 pulses. The target was rotated at a rate of 0.4 Hz during deposition and the laser beam scanned the entire target surface at an angle of 45◦ . All depositions were conducted with a background pressure of 30–100 Pa and a substrate-to-target distance of 4 cm. The target was maintained at a temperature of ∼173 K by active liquid nitrogen cooling. Thin films were deposited onto one-side polished Si 1 0 0 substrates for FTIR, AFM, and optic glass for antibacterial assays/strains. All of the substrates were ultrasonically cleaned prior to deposition by immersion in ethanol, and then dried in air under UV exposure from a VL-115UV lamp.
deflection height-measurement sensor and stylus with 2.5 m radius and 0.1 mg force. Thin film composition and morphology was investigated by FTIR and AFM, respectively. FTIR spectra were recorded by a Thermo 6700 FTIR/Raman system with a resolution 4 cm−1 that operated at 4000–650 cm−1 in transmission and/or micro-ATR/ZnSe modes. AFM micrographs were obtained with an Integrated Platform SPM-NTegra model Prima in phase contrast mode. All AFM images were recorded on 5 × 5 m2 areas. 2.4. Microbial strains used for antimicrobial activity assay The antimicrobial activity of the obtained systems (drop-casts and MAPLE-deposited thin films) was tested against Gram-positive (Staphylococcus aureus (S. aureus), and Bacillus subtilis (B. subtilis)) and Gram-negative (Pseudomonas aeruginosa (Ps. aeruginosa), Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli)) reference strains. The Enterobacteriaceae strains were identified by API 20E biochemical tests, while Ps. aeruginosa, B. subtilis and S. aureus were identified by VITEK I automatic system. VITEK cards for identification and susceptibility testing (GNS-522) were inoculated and then incubated according to the manufacturer’s recommendations [24,25]. These cards contain different lyophilized biochemical substrates with various antibiotic concentrations. In the presence of certain substrate and antibiotic concentration, the microbial growth is measured with a spectrophotometer that provides the biochemical identification of strain and its susceptibility to different antibiotics. Bacterial suspensions of 0.5 McFarland IU density obtained from 18 h bacterial cultures developed on solid media were used in the experiments [26]. 2.5. Microbiological assay investigation procedure
2.3. Profilometry FTIR, and AFM investigations Thin film thickness was recorded using a Stylus Profiler XP2 system (Ambios) with 0.1 nm vertical resolution, an optical
The pelliculised samples were placed in 2 ml nutrient broth and incubated for 24 h at 37 ◦ C. After 24 h of incubation, the samples were washed in sterile saline solution in order to remove
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Fig. 2. Atomic force micrographs of (a) Fe3 O4 /oleic drop-cast and (b) Fe3 O4 /oleic acid thin films; (c) Fe3 O4 /oleic acid/ceftriaxone drop-cast and (d) Fe3 O4 /oleic acid/ceftriaxone thin films; (e) Fe3 O4 /oleic acid/cefepime drop-cast and (f) Fe3 O4 /oleic acid/cefepime thin films. All thin films have been deposited by MAPLE at 20 mJ laser energy.
the non-adherent bacteria, while the adherent bacteria remained attached to the respective samples. After washing, the samples were placed in fresh medium and incubated for another 24 h. After incubation the turbidity of the culture medium was determined by measuring the absorbance at 600 nm, the obtained values being proportional to the percentage of viable bacterial cells adhered on the pelliculised slide [27]. In order to measure the influence of the tested hybrid system on the cell wall properties, we have also characterized the ability of viable bacterial cells to adhere to glass substrate. The bacterial cells secrete an exopolysaccharide substance implicated in the bacterial adherence to inert substrates known as slime. When bacterial cultures are obtained in liquid media, the slime production is followed by the adherence of bacterial cells to the glass walls and formation of a ring at the interface liquid–air. The intensity of the ring occurred on the culture tubes walls was appreciated by 0, 1 or 2 “++”.
2.6. Microbial adhesion to eukariotic cells grown on the pelliculised coverslips The Human epithelial carcinoma (HeLa) cell line was cultivated on the pelliculised samples and incubated for 24 h at 37 ◦ C in 5% CO2 atmosphere. Thereafter, the microbial suspensions were added and incubated for 2 h at 37 ◦ C. After incubation, the samples were fixed by ethanol, Giemsa stained and examined using an Olympus inverted microscope in order to appreciate the degree of cell confluency and indirectly, the cytotoxic effect of nanoparticles, as well as the number of adherent bacteria and the adherence pattern. Three distinct patterns of adherence have been investigated during this study: localized adherence (LA), in which bacteria attach to and form microcolonies in distinct regions of the surface; diffuse adherence (DA), in which bacteria adhere evenly to the whole cell surface, and aggregative adherence (AggA), in which aggregated bacteria attach to the cell in a stacked-brick arrangement.
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Fig. 3. Influence of the tested drop-cast/MAPLE-deposited thin films on the intensity of microbial growth quantified by measuring the absorbance at 600 nm of the liquid cultures.
Fig. 4. Influence of the drop-cast/MAPLE-deposited thin films on the ability of the tested bacterial strains to adhere to the inert substrate represented by the glass wall, quantified by the slime production by measuring the absorbance at 490 nm.
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The adherence index was expressed as the ratio between the numbers of the eukaryotic cells and adhered bacteria: 100 eukaryotic cells counted within the microscopic field (examined with the 100× immersion objective) [28]. 3. Results and discussion
roughness values for films versus drop-casts. It is generally known that a larger roughness means an extended active surface which is considered beneficial for any biological applications including the antimicrobial activity. A larger active surface acts strongly and more efficiently against microbes of any sort, i.e., the ones studied in this manuscript.
3.1. Profilometry and FTIR investigations
3.3. Biological activities
Optimal deposition laser fluence was identified using comparative FTIR analysis between spectra of the reference core/shell material and thin films deposited by MAPLE at 65, 100, 200 and 300 mJ/cm2 . Moreover, the typical thickness values for films deposited at 65 and 100 mJ/cm2 are about 250 and 300 nm, respectively. At 200 mJ/cm2 fluence, the film thickness is about 400 nm, while the films deposited at 300 mJ/cm2 have 1.6 m thickness. Corresponding to the best compromise between chemical structure and growth rate, we decided to continue our further investigations with a 200 mJ/cm2 fluence. In Fig. 1, FTIR spectra are given of Fe3 O4 /oleic drop-cast (a), Fe3 O4 /oleic acid thin films (b), Fe3 O4 /oleic acid/ceftriaxone drop-cast (c), Fe3 O4 /oleic acid/ceftriaxone thin films (d), Fe3 O4 /oleic acid/cefepime drop-cast (e), and Fe3 O4 /oleic acid/cefepime thin films (f). The characteristic peaks of the Fe3 O4 /oleic drop-cast (reference material) are assigned to: ferrite characteristic vibrations (∼610 cm−1 ), CH2 and CH3 stretching (∼2950, 2923 and 2853 cm−1 ) correlated to deformation modes (1300 and 1400 cm−1 ), oleic acid OH stretching (3424 cm−1 ), and silicon substrate peak (1108 cm−1 ). The specific peaks of ceftriaxone are centered at about 1038, 1781, and 3197 cm−1 (Fig. 1c and d), while the specific ones of cefepime are 729, 1364, 1742 and 3251 cm−1 (Fig. 1e–f). The chloroform dissociation products are volatile and expected to be pumped away by the vacuum system. As a general remark, all MAPLE-deposited thin film spectra show a very close similarity to the corresponding drop-cast FTIR spectra.
Our results showed that the nanoparticles coated with antibiotics inhibited the microbial growth and the adherence of the tested bacterial strains to the inert substrates. However, the intensity of the inhibitory effect varied with the tested strain. Concerning the interference with the microbial growth, the drop-cast procedure proved to be much more efficient than MAPLE in what concerns the preservation of the antibiotic activity. Concerning the microbicidal effect, the most evident results were obtained in case of Gram-positive microorganisms (i.e., B. subtilis and S. aureus) as well as for E. coli for both antibiotics and, in case of Ps. aeruginosa and K. pneumoniae for ceftriaxone only (Fig. 3). Thus, with few exceptions, the inclusion of beta-lactam antibiotics in the nanofilm lead to an increased antimicrobial activity as compared to that exhibited by the antibiotic alone. These results could be due either to an intrinsic antimicrobial effect of the nanoparticles or to an improved diffusion rate of the antibiotic in the liquid medium. Concerning the influence of the tested nanosystems on the ability of microbial strains to colonize the pelliculised glass coverslips our results showed an improved anti-biofilm activity for cefepime against the inert substrate colonization by the Gram-negative strains, probably due to the interference of the tested nanosystems with the production of the bacterial extracellular polysaccharide called slime known to be implicated in the microbial adherence to different substrates (Fig. 4). In this experiment, the MAPLE deposition technique proved to be more efficient than drop-cast concerning the intensity of the anti-biofilm effect. The hybrid nanosystems did not inhibit, but stimulated the colonization of the inert substrate by the Gram-positive strains, demonstrating the specific interaction of the nanosystems components with the bacterial function. Our results have shown that the nanoparticles did not significantly influence the adherence of HeLa cells to the pelliculised slides that developed a confluent monolayer after 24 h of incubation. The examination of the cell monolayer by inverted microscopy evidenced no morphological changes in HeLa cell morphology as compared to the control, demonstrating that the nanofilms have no cytotoxic effect on the eukaryotic cells. Neither the adherence pattern, nor the adherence index was affected by the pelliculised nanoparticles.
3.2. AFM studies AFM images of both drop-cast and MAPLE-deposited thin film at 200 mJ/cm2 in case of Fe3 O4 /oleic acid, Fe3 O4 /oleic acid/ceftriaxone, and Fe3 O4 /oleic acid/cefepime are given in Fig. 2. One first observation is that the density and size of the grains are larger on the films than the case of corresponding drop-casts. This slight change of morphology by MAPLE transfer is well known in literature and is demonstrated to depend on solvent and solute concentration and the laser deposition parameters [18]. In Fig. 2a, the surface morphology of Fe3 O4 /oleic acid drop-cast (13 nm RMS) shows a random distribution of coarse grains with diameters within the 1–300 nm range. In Fig. 2c, the surface morphology of Fe3 O4 /oleic acid/ceftriaxone drop-cast displays a network of small grains (5.6 nm RMS) while the surface of Fe3 O4 /oleic acid/cefepime drop-cast (7.1 nm RMS) exhibits small separated grains (Fig. 2e). The thin films of Fe3 O4 /oleic acid show a uniform morphology with big grains with diameters within the range of 0.5–1.5 m (61 nm RMS, Fig. 2b). Thin films of Fe3 O4 /oleic acid/ceftriaxone exhibit a morphology with smaller rounded grains with diameters within the range of 0.2–1 m not uniformly distributed on the surface (46 nm RMS, Fig. 2d). Although Fe3 O4 /oleic acid/cefepime thin film morphology puts in evidence much smaller elongated grains with 0.1–0.5 m diameters uniformly distributed (31 nm RMS, Fig. 2f), the cephalosporin drug has an aditive effect on the grain size and even a modification in grain shape in the specific case of cefepime drug. Grains of a larger density and size were observed on MAPLE films by comparison with the corresponding drop-casts (particularly visible in Fig. 2). This reflects in the higher
4. Conclusions We have demonstrated in the case of magnetic Fe3 O4 /oleic acid/ceftriaxone and Fe3 O4 /oleic acid/cefepime core/shell/adsorption-shell nanoparticles that MAPLE is a very efficient technique for obtaining thin films having a chemical structure similar to the starting material. The surface morphology of Fe3 O4 /oleic acid/cephalosporin thin films evidenced by AFM studies revealed that the cephalosporin drug has an aditive effect on the grain size and even a modification in grain shape in the specific case of cefepime drug. We demonstrated that the antibiotic charged nanoparticles exhibited a strong inhibitory effect on the viability as well as on the adherence to the cellular monolayer and to the inert substrate of the tested strains. This improvement in the anti-biofilm activity of the cephalosporin antibiotics correlated, with equal or superior rates to the minimal inhibitory concentration of the antibiotic suspensions allow us to conclude that
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