MAPLE fabricated coatings based on magnetite

Applied Surface Science 448 (2018) 230–236

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MAPLE fabricated coatings based on magnetite nanoparticles embedded into biopolymeric spheres resistant to microbial colonization Valentina Grumezescu a,b, Irina Negut a,c, Alexandru Mihai Grumezescu b,g, Anton Ficai b, Gabriela Dorcioman a,⇑, Gabriel Socol a, Florin Iordache f, Roxana Trusßca˘ b, Bogdan Stefan Vasile b, Alina Maria Holban d,e a

Lasers Department, National Institute for Lasers, Plasma & Radiation Physics, Magurele, Bucharest, Romania Department of Science and Engineering of Oxide Materials and Nanomaterials, Faculty of Applied Chemistry and Materials Science, Politehnica University of Bucharest, Bucharest, Romania c Faculty of Physics, University of Bucharest, 077125 Magurele, Ilfov, Romania d Microbiology Immunology Department, Faculty of Biology, University of Bucharest, Bucharest, Romania e Research Institute of the University of Bucharest, Bucharest, Romania f Department of Fetal and Adult Stem Cell Therapy, Institute of Cellular Biology and Pathology of Romanian Academy ‘‘Nicolae Simionescu”, Bucharest, Romania g Academy of Romanian Scientists (AOSR), Bucharest, Romania b

a r t i c l e

i n f o

Article history: Received 13 November 2017 Revised 3 April 2018 Accepted 6 April 2018 Available online 10 April 2018 Keywords: Gentamicin sulfate Spheres Laser processing MAPLE thin coatings Antimicrobial surfaces

a b s t r a c t The aim of this study was to obtain improved coatings for advanced surfaces with increased biocompatibility and resistance to microbial colonization and biofilm formation. The prepared magnetite nanoparticles functionalized with gentamicin (Fe3O4@G) have been embedded into poly(lactic-co-glycolic acid) (PLGA) spheres by oil-in-water emulsion. The PLGA-Fe3O4@G spheres were deposited on glass and silicone surfaces by Matrix Assisted Pulsed Laser Evaporation (MAPLE) technique. The obtained thin coatings were analyzed by Scanning Electron Microscopy (SEM) and Infrared Microscopy (IRM). The antimicrobial and antibiofilm efficiency of coatings was tested with respect to Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) clinical strains by viable cells counts assay, performed at different time intervals. The obtained results proved that coatings based on PLGA-Fe3O4@G spheres exhibited an efficient antimicrobial activity against both adherent and sessile bacterial cells. Besides their excellent anti-adherence and antibiofilm effect, the obtained MAPLE-deposited coatings were highly biocompatible, allowing the normal development and growth of cultured human amniotic fluid stem cells. This approach could be successfully applied for the optimization of medical surfaces in order to control and prevent microbial colonization and further development of biofilm associated infections. Ó 2018 Elsevier B.V. All rights reserved.

1. Introduction Gentamicin sulfate is a broad-spectrum aminoglycoside used to treat various types of infections, caused by Gram-positive and Gram-negative bacteria [1]. The antimicrobial mechanism of action of gentamicin (G) relies on the ability of this antibiotic to bind the 30S subunit of the bacterial ribosome and to inhibit protein synthesis [2]. G can be administered intramuscularly [3], intravenously [4] or topically [5] into the human organism. G has been administered for the treatment of general sepsis [6], respiratory and urinary tract infections [7], as well as for skin and soft tissues [8], stomach, bone [9,10] and heart infections [11].

⇑ Corresponding author. E-mail address: [email protected] (G. Dorcioman). https://doi.org/10.1016/j.apsusc.2018.04.053 0169-4332/Ó 2018 Elsevier B.V. All rights reserved.

In spite of unique pharmacokinetic effects of this drug, its medical applications are limited, due to notable deficiencies such as short half-life, low intestinal absorption and adverse side-effects (e.g. ototoxicity and nephrotoxicity) when utilized in high amounts of for prolonged time periods [12–14]. In order to minimalize complications associated with G administration concomitant with the improvement of its therapeutic efficiency, the use of adequate delivery systems to the infection site, have been suggested [15–17]. Infections are common complications when biomaterials are implanted into the human organism and are typically triggered by the adherence and colonization of implanted material with microorganisms. The adhesion of microorganisms on the surfaces of implants is a critical issue; causes failure and destabilizes their clinical applicability. Once attached to surfaces, bacteria forms

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biofilms, complex biological populations characterized by cells with an altered phenotype [18], highly tolerant to all known antimicrobial substances and host defense mechanisms. Numerous microorganisms can contaminate an implant, but most common pathogens involved in biofilm associated infections are Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa [19,20]. One of the most effective tactics for reducing the risk of biomaterial-associated infections is to avoid the adhesion and/or to delay the development of already adhered and/or inhabiting pathogens by coating the implants’ surface with bioactive films with adequate antibacterial properties [21,22]. The delivery of antibiotics at the implantation site represents a favorable route for repelling bacterial adhesion and the establishment of microbial biofilms [23,24]. Antibiotics contained in medical coatings can be tuned to offer a rapid release at early stages adequate for eliminating early infection, followed by a sustained release for several days/ weeks [25]. In recent decades, nanoparticles (NPs) have attracted immense attention to develop nanosized carriers capable of adjusting the release of various drugs [26]. Pharmaceutical NPs have been created as unique platforms capable of controlling drugs release into the body, shielding active compounds from enzymatic and/or chemical decomposition together with target site directed drug delivery [27,28]. Size of nanoparticles is very important for their pharmacology and biological impact. While small nanoparticles (usually under 30 nm) are able to penetrate the cells (both eukaryotic and prokaryotic) and have more severe biological impact, larger nanoparticles (more than 50 nm) have different biological impact and usually do not penetrate cellular membranes. Therefore, altering the size, shape and/or surface chemistry of nanoparticles allows their functionalities to be tailored to meet different requirements. Nanoparticle size, shape and core composition are strong determinants of the possible cellular uptake [29,30]. Nanoparticles ranging from 30 to 60 nm in size are able to produce endocytosis of mammalian cells, by determining the cell-wrapping process, while smaller nanoparticles could produce passive dislocation of membrane constituents and some nanostructures may even induce irreversible cell piercing and content leakage (leading to cell death) [31]. Antimicrobial effect of numerous nanoparticles was also proved to be size-dependent [32,33]. Numerous nanoparticles have been obtained to offer intrinsic antimicrobial effect and also to deliver various antimicrobial agents and to increase their effect [34]. Following the paths of eradicating biofilm associated infections and significantly diminishing bacterial attachment, many nanoparticle-based antibiotic delivery systems have been obtained [35–37]. Synthetic polymers are widely employed for medical coatings due to their outstanding features: biocompatibility, biodegradability, absence of immunogenicity, and flexibility to modify their physical-chemical chemical properties [38]. In order to strengthen the biocompatibility of inorganic magnetic iron oxide NPs, biodegradable polymers can be used [39,40]. Polylactide-coglycolide (PLGA) represents a Food and Drug Administration and European Medicine Agency approved biodegradable and biocompatible polymer frequently used in drug delivery systems [41–43]. One of the approaches employed for the engineering of nanostructured antimicrobial coatings is matrix-assisted pulsed laser evaporation (MAPLE) [44,45]. The elusive substances are suspended or diluted in a volatile solvent, which is then frozen in liquid nitrogen. The laser beam impinge the frozen target, which is evaporated, permitting the transfer of unharmed molecules to the collector substrate in the form of a thin film [46–48]. It has been reported that magnetite NPs functionalized with amoxicillin, benzyl-penicillin, cefotaxime and norfloxacin in form of coatings obtained by MAPLE exhibited an inhibitory activity against biofilm embedded S. aureus and P. aeruginosa cells [49].

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Considering the advantages of NPs, G and MAPLE fabricated coatings, in this study we aimed to prepare antimicrobial and biocompatible coatings consisting of magnetite NPs functionalized with Gentamicin sulfate (Fe3O4@G) embedded into the matrix of the polymeric spheres, and to further study in vitro antibacterial activity. 2. Materials Gentamicin sulfate was purchased from a commercial source (MP Biomedicals LLC, Germany). The poly(lactic-co-glycolic acid), FeCl3, FeSO4, NH3 and n-hexane were purchased from SigmaAldrich. 3. Methods 3.1. Synthesis of magnetic NPs functionalized with Gentamicin sulfate Magnetic nanoparticles were prepared by co-precipitation method from Fe2+ and Fe3+ (1:2 molar ration) according to our previously published papers [50,51]. The concentration of Gentamicin sulfate in aqueous NH4OH solution was 0.25 %. 3.2. Preparation of spheres PLGA/Fe3O4@G spheres were prepared using a solvent evaporation method [52,53]. Briefly, a compound consisits of PLGA and Fe3O4@G in a 4:1 mass ratio was dispersed in 2 mL chloroform by sonication. The PLGA/Fe3O4@G phase was emulsified with a sonicator model SONIC-1200WT from MRC for 5 min, in ON/OFF steps of 5 and 3 s with a limitation temperature of maximum 37 °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 chloroform was evaporated. The emulsion was centrifuged at 6000 rpm for 20 min. The obtained spheres were washed three times in deionized water, collected by filtration and then subjected to freeze-drying. PLGA/Fe3O4@G microspheres were used to prepare coatings by MAPLE technique. 3.3. MAPLE experimental conditions and deposition of Fe3O4@G thin coatings MAPLE targets were prepared by freezing at liquid nitrogen temperature a solution of 1% (w/v) PLGA/Fe3O4@G spheres in nhexane. A KrF⁄ (k = 248 nm, sFWHM = 25 ns) (COMPexPro 205 Lambda Physics-Coherent) excimer laser beam impinged the target at a laser fluence of 200–400 mJ/cm2, repetition rate of 20 Hz and for 20,000–40,000 pulses. During the laser irradiation, the cryogenic target was maintained at a temperature of 173 K by constant cooling with liquid nitrogen, rotated at a rate of 0.4 Hz and upheld at a 4 cm distance from substrates. All depositions were conducted at room temperature and at a background pressure of 10 2 mbar. The coatings were deposited onto double-side polished Si (1 0 0) substrates for FT-IR and SEM analyses and glass plates for antibacterial assays. Before the mounting into the stainless steel deposition chamber, substrates were successively cleaned for 15 min into an ultrasonic bath using acetone, ethanol and deionized water. Then, they were dried in a jet of high purity nitrogen. For comparison study a batch of drop-cast samples were prepared. 3.4. Characterization methods 3.4.1. TEM The transmission electron microscopy (TEM) images were performed on Fe3O4@G powdered samples using a TecnaiTM G2 F30 S-TWIN high resolution transmission electron microscope from

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FEI Company (OR, USA) equipped with SAED. The microscope operated in transmission mode at 300 kV with TEM point resolution of 2 Å and line resolution of 1 Å. The prepared powder was dispersed into pure ethanol and ultrasonicated for 15 min. Diluted sample was poured onto a holey carbon-coated copper grid and left to dry before TEM analysis. 3.4.2. IRM IR mapping was recorded on a Nicolet iN10 MX FT-IR Microscope with MCT liquid nitrogen cooled detector in the measurement range 4000–700 cm 1. Spectral collection was made in reflection mode at 4 cm 1 resolution. For each spectrum, 32 scans were co-added and converted to absorbance using Ominc Picta software (Thermo Scientific). The IR maps were built using the 1745 cm 1 absorption peak characteristic to the PLGA. 3.4.3. SEM The surface morphology of samples was investigated by scanning electron microscopy (SEM). The images were acquired on a FEI electron microscope, using secondary electron beams with energies of 30 keV, previously the samples were capped with a thin gold layer. 3.5. MTT assay The human amniotic fluid stem cells (AFSC) were used to evaluate the biocompatibility of prepared coatings. The cells were

cultured in DMEM medium (Sigma-Aldrich, Missouri, USA) supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin antibiotics (Sigma-Aldrich, Missouri, USA). To maintain optimal culture conditions, medium was changed twice a week. The biocompatibility was assessed using MTT assay (CellTiter 96Ò Non-Radioactive Cell Proliferation Assay, Promega, Wisconsin, USA). This assay is a colorimetric method that allows quantitative assessment of proliferation, cell viability and cytotoxicity, and is relies on reduction of yellow tetrazolium salt MTT (3-(4,5dimetiltia zoliu)-2,5-diphenyltetrazolium bromide) to a dark blue formazan by the mitochondrial enzymes. Briefly, the human AFSC were grown in 96-well plates, with a seeding density of 3000 cells/well in the presence of thin coatings for 72 h. Then 15 ml Solution I was added and incubated at 37 °C for 4 h. After that the Solution II was added and pipette vigorously to solubilise formazan crystals. After 1 h the absorbance was read using spectrophotometer at 570 nm (TECAN Infinite M200, Männedorf, Switzerland). 3.6. Biofilm development Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 (American Type Cell Collection, US) were utilized to assess biofilm formation. Monospecific biofilm development was analyzed at different times of exposure, using sterile 6 well plates (Nunc). UV sterilized MAPLE coated samples were added in 6 well plates containing 2 mL of nutritive broth inoculated with

Fig. 1. TEM images (a, b); corresponding histogram (c) and SAED pattern (d) of Fe3O4@G.


106 CFU/mL of microbial suspensions prepared in sterile saline solution. The samples were allowed to grow at 37 °C for three time points (24 h, 48 h and 72 h) to assess the time dynamic of biofilms developed on the obtained nanocoatings. After incubation, the samples were carefully washed with sterile saline buffer to remove any unattached microbial cells and then immersed in 1 mL sterile saline buffer. Biofilm cells were detached by vigorous vortexing and pipetting. The resulting biofilm – detached cell suspensions were further diluted and 10 mL of each serial dilution were plated in triplicate on nutritive agar. After 24 h of incubation at 37 °C, viable count was performed and the CFU/mL values for each sample were obtained.

4. Results and discussion Fe3O4@G nano-powders were characterized by HR-TEM and SAED in order to emphasize both the presence of magnetite as the only crystalline phase and the size of nanoparticles. The average size of the NPs evaluated by TEM examination (Fig. 1) was estimated at around 6.9 ± 0.23 nm. SAED pattern (Fig. 1d) certifies the crystalline structure of the Fe3O4@G powder. Fig. 2 shows the second derivative infrared micrographs of microsphere-based coatings deposited by MAPLE at laser fluencies

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of 200, 300 and 400 mJ/cm2, respectively. Based on IRM analysis, we established that the best uniformity is found for MAPLE samples synthesized at a laser fluence of 400 mJ/cm2. For the coatings deposited at 200 and 300 mJ/cm2 laser fluence values, the amount of the material transferred was rather low. In Fig. 3 are shown dropcast results of the spheres-containing thin coatings prepared at a laser fluence (F = 400 mJ/cm2), IR spectra are given at different points in plotted maps. The characteristic peaks of the prepared PLGA/Fe3O4@G spheres drop-cast (reference material) are assigned to: 3500 cm 1 (OH stretch, end group), 2996 cm 1 (CH3 stretch), 1778 cm 1 (C = O carbonyl group – stretching). As a general remark, typical IR spectra of MAPLEdeposited thin coatings at F = 400 mJ/cm2 show a very close similarity to the corresponding drop-cast IR spectra. The shape and size of the PLGA/Fe3O4@G as well as their integrity after the laser transfer was investigated by SEM analysis. SEM micrographs at different magnifications given in Fig. 4(a–d) show PLGA/Fe3O4@G structures with almost compact spherical shape. The size distribution of the pristine PLGA-Fe3O4@G particles varies from submicron up to several microns with an average of around 3 lm. Moreover, the morphological features of the microspheres were conserved after the laser transfer in form of coatings. Cytotoxicity was assessed with respect to human amniotic fluid stem cells grown at 24 h, 48 and 72 h, respectively, in the presence

Fig. 2. Second derivate IR mappings of drop-cast (a) and the thin coatings (b, c, d), F = 200 mJ/cm2 (b), F = 300 mJ/cm2 (c), F = 400 mJ/cm2 (d).

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of the obtained materials. Viability assay results (Fig. 5) showed that the morphology of human amniotic fluid stem cells was not altered in the presence of PLGA-Fe3O4@G. Our results show that after 3 day of human amniotic fluid stem cells culturing with PLGA-Fe3O4@G, cultured cells remained viable. The cells shape, adherence properties and cells distribution on the surface of the PLGA-Fe3O4@G coatings are comparable with control samples revealing an appropriate viability of human amniotic fluid stem cells, good attachment on the substrate and normal cell morphology.

Antimicrobial results were performed using a Gram positive (S. aureus) and a Gram negative (P. aeruginosa) microbial model, with proved clinical impact [54]. S. aureus is one of the most frequent etiology of skin and wound related infections. Since 1 of 3 people are estimated to bear S. aureus in their nose and pharynx, community and hospital acquired S. aureus infections with resistant strains (especially MRSA = methicillin resistant S. aureus) are very frequent, this opportunistic species also representing a leading cause of device associated infections [55]. On the other

Fig. 3. IR spectra of PLGA/Fe3O4@G spheres drop-cast and thin coatings prepared at F = 400 mJ/cm2.

Fig. 4. Typical SEM images at different magnifications of PLGA-Fe3O4@G spheres after laser transfer at 400 mJ/cm2 fluence.


Fig. 5. MTT results after 24, 48 and 72 h of incubation in presence of human amniotic fluid stem cells.

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both in the case of S. aureus and P. aeruginosa. The obtained coatings seem to limit the formation and maturation of biofilms, altering various steps of biofilm development, starting with the attachment of cells and initiation of biofilms (seen at 24 h of incubation) and continuing with the maturation stages (Figs. 6 and 7) at 48 and 72 h. For both S. aureus and P. aeruginosa tested strains it can be observed a significant biofilm development inhibition on the MAPLE nano-modified, as compared with uncoated control samples. The most significant biofilm inhibition was observed for both tested strains at 72 h of contact, suggesting that late biofilm formation and maturation is highly impaired. The antimicrobial efficiency of the tested surfaces is maintained for at least 3 days, with significant impact in biofilm development limitation (Figs. 6 and 7). Antimicrobial results suggest that the PLGA-Fe3O4@G microsphere based coatings may be efficiently used for developing bioactive surfaces for various biomedical devices, tailored to limit microbial colonization and subsequent biofilm formation. 5. Conclusions

Fig. 6. Viability of the S. aureus strains on the modified surface and control samples for 24, 48 and 72 h.

This paper reports on the successful development of PLGAFe3O4@G coatings with excellent anti-adherence and antibiofilm effect against opportunistic bacteria model species, currently involved in device-associated infections. The prepared coatings showed a uniform distribution and a fine structure, being highly biocompatible, allowing for the normal development and growth of cultured human amniotic fluid stem cells. This nanomodified coating could be successfully applied for the optimization of medical devices surfaces to control and prevent microbial colonization and further development of biofilm associated infections. The approach has the potential to be extended and applied for a variety of medical and industrial interest surfaces, to limit biofilm formation. Acknowledgments The work has been funded by the Romanian National Authority for Scientific Research, CNCS-UEFISCDI, under project no. TE 188/2014, PN-II-RU-TE-2014-4-1590 and Nucleu Programme 3N/2018. The SEM analyses on samples were possible due to EUfunding grant POSCCE-A2-O2.2.1-2013-1/ Priority direction 2, Project No.638/12.03.2014, cod SMIS-CSNR 48652. References

Fig. 7. Viability of the P. aeruginosa strains on the modified surface and control samples for 24, 48 and 72 h.

hand, P. aeruginosa is the most versatile opportunistic pathogen, causing a wide range of infections in hospitalized patients, representing a leading cause of mortality in critical care units. Because of its natural resistance, ability to acquire resistance gene with an increased rate and also to form specialized multicellular communities, called biofilms, infections involving this species being very difficult to treat [56]. Our results demonstrated that the obtained coatings containing PLGA-Fe3O4@G microspheres are able to limit biofilm formation

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