Antimicrobial nanospheres thin coatings prepared by advanced pulsed laser technique

Summary We report on the fabrication of thin coatings based on polylactic acid-chitosan-magnetite-eugenol (PLA-CS-Fe3O4@EUG) nanospheres by matrix assisted pulsed laser evaporation (MAPLE). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) investigation proved that the homogenous Fe3O4@EUG nanoparticles have an average diameter of about 7 nm, while the PLA-CS-Fe3O4@EUG nanospheres diameter sizes range between 20 and 80 nm. These MAPLE-deposited coatings acted as bioactive nanosystems and exhibited a great antimicrobial effect by impairing the adherence and biofilm formation of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) bacteria strains. Moreover, the obtained nano-coatings showed a good biocompatibility and facilitated the normal development of human endothelial cells. These nanosystems may be used as efficient alternatives in treating and preventing bacterial infections.


Introduction
Driven by more and more microbial antibiotic resistance, alternative therapeutic approaches are emerging [1][2][3][4]. Polar and nonpolar, functionalized and non-functionalized magnetite nanostructures have proven successfully in combating microbial infections both in vitro and in vivo [5,6]. In the past years a series of papers have been published in prestigious journals highlighting the relevance of magnetite nanostructures in preventing the development of microbial biofilm and the opportunity of utilizing these nanosystems to obtain improved, antimicrobial coatings for biomedical applications [7,8]. Nonpolar functionalized magnetite nanostructures alone [9,10] or combined with different natural products, such as usnic acid (UA) [11] or essential oils (Mentha piperita [12], Anethum graveolens [13], Salvia officinalis [14], Eugenia carryophyllata [15]), showed improved antibiofilm effects on different types of microbial strains. Usually, these types of phyto-nano-coatings have been applied to a variety of medical surfaces in order to improve their resistance to microbial colonization [16].
Our recent reports have highlighted the capability of the laser processing technique to prepare thin coatings based on polymeric microspheres. Thus, Socol et al., [43], firstly reported the novel deposition of PLGA-PVA, PLGA-PVA-BSA (bovine serum albumin) and PLGA-PVA-CS microspheres by matrix assisted pulsed laser evaporation (MAPLE) technique. SEM images of thin coatings reveal homogeneous and sphericalshaped particles in the micrometric range. The average diameter of PLGA-PVA, PLGA-PVA-BSA (bovine serum albumin) and PLGA-PVA-CS particles ranged from 180 to 250 nm. Grumezescu et al., [34], reported the MAPLE fabrication of PLA-PVA-UA microsphere thin coatings. These thin coatings possessed a homogeneous shape and showed no concavities or distortions on their surface within an average diameter of 1 μm of the deposited spheres. It is noteworthy that the microspheres maintain their initial size and do not show an aggregative behavior [34]. All these type of microspheres have been prepared by an oil-in-water emulsion-diffusion-evaporation method.
Here, we report the fabrication of thin coatings based on magnetic PLA-CS-Fe 3 O 4 @EUG nanospheres with an average diameter of the deposed spheres between 20 and 80 nm. This is the first study that reports the MAPLE processing of thin coatings containing spheres with a diameter of less than 100 nm. The thin coating is composed of nanospheres based on magnetite nanostructures and biocompatible polymers. The thin coating also exhibited antibiofilm activity, thereby opening a new perspective for the prevention of medical surfaces infections.

Preparation of magnetite nanostructures
A well-known procedure described in our previous work was used to synthesize the magnetite nanostructures [44]. Briefly, EUG and NH 4 OH (25%) were added in deionized water under vigorous stirring. Then, FeCl 3 and FeSO 4 ·7H 2 O were dissolved in deionized water, and Fe 2+ /Fe 3+ solution was dropped into the basic solution of EUG. After precipitation, magnetite-eugenol nanopowder (Fe 3 O 4 @EUG) were repeatedly washed with methanol and separated with a strong NdFeB permanent magnet.

Preparation of nanospheres
PLA-CS-Fe 3 O 4 @EUG nanospheres were prepared by means of a solvent evaporation method [34,45]. Thus, 4 mL PLA/chloroform solution (10 wt %) and 5 mL aqueous solution of PVA (2 wt %), CS (10 wt %) and Fe 3 O 4 @EUG (1 wt %) were emulsified with a SONIC-1200WT sonicator model from MRC for 6 min, in ON/OFF steps of 5 s and 3 s with a limitation temperature of max 40 °C, followed by solvent evaporation in 100 mL deionized water with mechanical stirring at 1000 rpm. The prepared nanospheres were thoroughly washed with deionized water and then lyophilized. PLA-CS-Fe 3 O 4 @EUG nanopheres were further used to deposit thin films by using the MAPLE technique.

MAPLE thin coating deposition
MAPLE targets were prepared by freezing them for 30 min at the temperature of liquid nitrogen using a suspension of 1.5% (w/v) PLA-CS-Fe 3 O 4 @EUG microspheres in n-hexane.
The radiation of a KrF* (λ = 248 nm, τ FWHM = 25 ns) COMPexPro 205 Lambda Physics-Coherent excimer laser source model impinged the frozen targets at a laser fluence of 300-500 mJ/cm 2 and a repetition rate of 15 Hz. In order to assure the reproducibility of the nanosphere thin film deposition, the energy distribution of the laser spot was improved by using a laser beam homogenizer. During the deposition, the target was rotated with 0.4 Hz to avoid target heating and subsequent drilling. All depositions were conducted at room temperature under 0.1 Pa background pressure and a target-substrate separation distance of 4 cm by applying 45,000-160,000 subsequent laser pulses. During deposition, the MAPLE target was kept at low temperature by continuous liquid nitrogen cooling. The coatings were deposited onto glass, both sides polished (100) silicon for IRM, SEM, and biological assays. Prior to placing the substrates inside the deposition chamber, they were cleaned in an ultrasonic bath with acetone, ethanol and deionized water for 15 min, and then dried in a jet of high purity nitrogen.

Characterization Transmision electron microscopy
The transmission electron microscopy (TEM) images were obtained on finely powdered samples by using a Tecnai TM G2 F30 S-TWIN high resolution transmission electron microscope manufactured by FEI Company (OR, USA). The microscope operated in transmission mode at 300 kV with a TEM point resolution of 2 Å and a line resolution of 1 Å. The prepared powder was dispersed into pure ethanol and ultrasonicated for 15 min. After that, the diluted sample was poured onto a holey carbon-coated copper grid and left to dry before TEM analysis.

Infrared Microscopy
IR mappings were recorded on a Nicolet iN10 MX FT-IR Microscope with an MCT liquid nitrogen cooled detector in the measurement range 4000-600 cm −1 . Spectral collection was carried out in reflection mode at 4 cm −1 resolution. For each spectrum, 32 scans were co-added and converted to absorbance by means of the OmincPicta software (Thermo Scientific). Approximately 600 spectra were analyzed for each coating and drop cast. Four absorptions peaks known as being characteristics for the PLA-CS-Fe 3 O 4 @EUG were selected as spectral markers for the presence of nanospheres in the prepared coatings.
Scanning electron microscopy SEM analysis was performed on a FEI electron microscope by using secondary electron beams with energies of 30 keV on samples covered with a thin gold layer.

In vitro microbial biofilm development
Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 strains were purchased from American Type Cell Collection (ATCC, USA). For the biofilm assays, fresh bacteria cultures were obtained in Luria Broth. Bacteria cultures were subsequently diluted as mentioned below.
The biofilm formation was assessed by using 6 multi-well plates (Nunc) in a static model for monospecific biofilm development. Coated and uncoated glass substrates were distributed in the plates containing 2 mL of microbial inoculum diluted to 10 4 -10 5 colony forming units/mL (CFU/mL) in Luria Broth. Samples were incubated for 24 h at 37 °C. The biofilm formation was assessed after 24 h, 48 h and 72 h by a viable cell counts (VCC) assay [46]. After 24 h of incubation time, the culture medium was removed and the samples were washed with sterile PBS to remove the unattached bacteria. Coated and uncoated substrates were placed in fresh medium and incubated for an additional 24 h, 48 h and 72 h. After the incubation the samples were gently washed with sterile PBS to remove the non-adherent cells and placed in 1.5 mL micro-centrifuge tubes (Eppendorf) containing 750 μL PBS. In order to disperse biofilm cells into the suspension, the samples were vigorously mixed by vortexing for 30 s and sonicated for 10 s. Serial ten-fold dilutions were prepared and plated on Luria-Bertani (LB) agar for VCC. Experiments were performed in triplicate and repeated on three separate occasions [12,47].

Statistical analysis
The statistical significance of the obtained results was analyzed by using GraphPad Prism version 5.04 for Windows, GraphPad-Software, San Diego, CA, USA. For comparison, we used the number of CFU/mL as revealed by the readings of three values/ experimental variants. Two-way ANOVA and Tukey's multiple comparison tests were used for revealing significant differences among the analyzed groups.

Results and Discussion
The morphology and size of magnetite nanoparticles was analyzed by TEM. We confirmed the nanometric dimensions of used powder in order to prepare PLA-CS-Fe 3 O 4 @EUG nanospheres. TEM images of Fe 3 O 4 @EUG at different magnification ( Figure 1) show that the prepared powder has a spherical shape with a narrow size distribution of approximately 7 nm.
Infrared microscopy was used to demonstrate the integrity of functional groups after MAPLE processing. The visible spectrum images and infrared maps based on full spectral intensity of drop cast and MAPLE thin coatings overlain on the surface are plotted in Figure 2. The prepared polymeric spheres thin coatings are distributed on the entire surface of the substrate without any free spots as can be observed on the maps of drop cast (Figure 3 a 1 , b 1 , c 1 and d 1 ). Figure 3 shows the second derivative infrared maps of PLA-CS-Fe 3 O 4 @EUG surfaces involved in this study. Second derivative infrared mapping is used to evaluate the structural integrity of samples [42]. Absorbance intensities of IR spectra maps commensurate with the color changes starting with blue (lowest intensity) and gradually increasing through green and yellow to red (highest intensity) [43]. 600 IR spectra were analyzed for each thin coating [34].
According to Figure 3 areas with moderate (green) and high intensity (red) of selected absorption bands can be observed. The tendency of nanospheres to form aggregates gives rise to the red areas. In the case of the drop cast maps, it can be concluded that there is no uniformity in the sampleand little high intensity can be observed. According to Figure 4, the thin films deposited by MAPLE (F = 300 mJ/cm 2 ) revealed no degradation of functional groups during the laser processing.
The thin coatings deposited at 300 mJ/cm 2 laser fluence with an estimated average thickness of (≈2 μm) were analyzed by SEM    ( Figure 5). It can be seen that the thin coatings contain higher numbers of nanospheres on top of their surfaces with diameters between 20 and 80 nm. This is the first study that reports the MAPLE processing of thin coatings containing spheres with a diameter lower than 100 nm. Previous studies have reported the MAPLE processing of thin coatings containing spheres with diameters within the range of 180-1,000 nm [34,38].
Cytotoxicity assays revealed that the prepared nano-coatings have a great biocompatibility, and support the growth of endothelial cell cultures. The cell tracker RED CMTPX fluo-rophore showed that the endothelial cells are viable and exhibit a normal grow and proliferation capacity in the presence of modified nano-coated bioactive surfaces. Furthermore, the cell monolayers developed on the thin coating surfaces have a normal morphology and architecture after five days of incubation ( Figure 6).
Despite its good biocompatibility with human cells, the newly synthesized nano-active thin coating exhibited a great antimicrobial activity. The surface inhibited both S. aureus and P. aeruginosa attachment and also the formation of non-specific  Even though magnetite nanoparticles displayed a great antimicrobial effect, many studies reported that these nanostructures may be highly toxic for hosts in higher concentrations or even active doses [48][49][50]. Our results demonstrate that the novel synthesized PLA-CS-Fe 3 O 4 @EUG complex nanosystems combine the proven efficacy of Fe 3 O 4 and eugenol [40] with the biocompatibility and biodegradability of PLA and CS polymers resulting in a novel safe nanobiocomposite. Due to these characteristics PLA-CS-Fe 3 O 4 @EUG thin films represent a competitive candidate for the development of novel biomedical surfaces or devices with low costs and a high efficiency.

Conclusion
This paper reports the successful MAPLE deposition of bioactive thin films based on PLA-CS-Fe 3 O 4 @EUG magnetic nanospheres with diameters between 20 and 80 nm. These nanocoatings displayed great antimicrobial colonization and antibiofilm formation properties, inhibiting S. aureus and P. aeruginosa biofilms. Due to the biocompatibility of this material it as a suitable candidate in developing nanostructured bioactive materials for biomedical applications.