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Flexible camphor diamond-like carbon coating on polyurethane to prevent Candida albicans biofilm growth
Journal of the mechanical behavior of biomedical materials 68 (2017) 239–246
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Flexible camphor diamond-like carbon coating on polyurethane to prevent Candida albicans biofilm growth
MARK
Thaisa B. Santosa, Angela A. Vieiraa, Luciana O. Paulaa, Everton D. Santosa, Polyana A. Radia,b, ⁎ Sônia Khouria, Homero S. Maciela,b, Rodrigo S. Pessoaa,b, Lucia Vieiraa,b, a Universidade do Vale do Paraíba – UNIVAP, Grupo Nanotecplasma, Av. Shishima Hifumi, 2911 – Urbanova, CEP 12.244-000 São José dos Campos, SP, Brazil b Instituto Tecnológico de Aeronáutica – ITA, Laboratório de Processamento a Plasma, Praça Marechal Eduardo Gomes, 50 – Vila das Acácias, São José dos Campos, SP 12228-900, Brazil
A R T I C L E I N F O
A BS T RAC T
Keywords: Diamond-like carbon (DLC) Candida albicans Camphor Raman Biofilm growth inhibition
Camphor was incorporated in diamond-like carbon (DLC) films to prevent the Candida albicans yeasts fouling on polyurethane substrates, which is a material commonly used for catheter manufacturing. The camphor:DLC and DLC film for this investigation was produced by plasma enhanced chemical vapor deposition (PECVD), using an apparatus based on the flash evaporation of organic liquid (hexane) containing diluted camphor for camphor:DLC and hexane/methane, mixture for DLC films. The film was deposited at a low temperature of less than 25 °C. We obtained very adherent camphor:DLC and DLC films that accompanied the substrate flexibility without delamination. The adherence of camphor:DLC and DLC films on polyurethane segments were evaluated by scratching test and bending polyurethane segments at 180°. The polyurethane samples, with and without camphor:DLC and DLC films were characterized by Raman spectroscopy, scanning electron microscopy, atomic force microscopy, and optical profilometry. Candida albicans biofilm formation on polyurethane, with and without camphor:DLC and DLC, was assessed. The camphor:DLC and DLC films reduced the biofilm growth by 99.0% and 91.0% of Candida albicans, respectively, compared to bare polyurethane. These results open the doors to studies of functionalized DLC coatings with biofilm inhibition properties used in the production of catheters or other biomedical applications.
1. Introduction Diamond-Like Carbon (DLC) is a very attractive material for biological applications because it has physical and chemical properties similar to diamond such as hardness and relatively high modulus of elasticity. DLC is easily deposited on large and 3D areas (Hauert, 2003). Although, there are a significant number of manuscripts about DLC (Choudhury et al., 2016) and DLC with metallic nanoparticles (Widoniak et al., 2005; Marciano et al., 2009), little is known about the use of DLC in polymeric materials (Wang et al., 2004). Some studies mention silver used as a possible inhibitor of microbial growth and catheters coated with a mixture of silver and DLC (Dearnaley and Arps, 2005). On the other hand, only a few studies have used Camphor as a carbon precursor for DLC production, and these prioritized manufacturing process of only carbon bonds, and terpenes radicals were not preserved (Fadzilah et al., 2013; Liu and Kwek, 2008; Zani et al., 2013). No literature was found about camphor:DLC as a material to combat
candidiasis proliferation. Candidiasis contamination is related to parenteral nutrition, and it is transmitted through the hands of healthcare workers and especially the use of catheters [6]. Candidiasis contamination has been a big problem for hospitalized patients, especially those in critical condition, and is the fourth leading cause of bloodstream infection. Bloodstream infection increases hospital costs, patient's hospitalization time and mortality rate (Morrison et al., 2006; van Asbeck et al., 2007). Biofilm is responsible for conducting catheter infection, due to a set of micro-organisms that live in association with biofilm (Bazaka et al., 2012; Griffiths and Hall, 2010). Most microorganisms involved in colonization of catheters are not virulent in planktonic form but can cause persistent infection when they are in a group (Co-investigator, 2013). Camphor is cetonic terpenoid (C10H6O) obtained from the camphor tree that is native to China, and it is part of a class of bioactive compounds synthesized by plants (Frizzo et al., 2000; Gershenzon and Dudareva, 2007; Moreno et al., 2010). Terpenes are several open-
⁎ Corresponding author at: Universidade do Vale do Paraíba – UNIVAP, Grupo Nanotecplasma, Av. Shishima Hifumi, 2911 – Urbanova, CEP 12.244-000 São José dos Campos, SP, Brazil. E-mail address: [email protected] (L. Vieira).
http://dx.doi.org/10.1016/j.jmbbm.2017.02.013 Received 29 May 2016; Received in revised form 5 February 2017; Accepted 12 February 2017 Available online 16 February 2017 1751-6161/ © 2017 Elsevier Ltd. All rights reserved.
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chained or cyclic compounds, usually oxygenates such as aldehydes, alcohols, and ketones. Gershenzon and Dudareva (Gershenzon and Dudareva, 2007) reported previously that plants with terpenes are resistant to diseases, due to their action on fungi and bacteria. Terpenes have detergent properties that are toxic to fungi due to their ability to complex with sterols in fungi membranes, which leads to loss of membrane integrity (Gershenzon and Dudareva, 2007). The aim of this work was to develop a DLC film that comprises camphor with properties to prevent biofilm of Candida albicans for use on catheters and biomedical instruments. For this, we developed a simple apparatus that was coupled in a commercial plasma enhanced chemical vapor deposition (PECVD) reactor for camphor delivery during DLC production. This process allows the deposition of DLC films containing radicals for functionalized surfaces or nanoparticles, covering materials three-dimensionally at low temperature.
showed in Raman spectra by the terpene band centered on 1099 cm−1 (Fig. 1). Table 1 shows the parameters used for the camphor:DLC and DLC film deposition. The deposition plasma process started with argon plasma to remove oxides in (PU) surfaces. DLC films were produced using a vapor mixture of hexane and methane and for camphor:DLC was used camphor oil diluted in hexane (10% v.v.) with methane vapor. The glass tube apparatus containing the liquid precursor were sonicated using an ultrasound bath. The flow for each carbon precursor was controlled using flowmeters and pressure valves to maintain the work pressure inside the vacuum chamber at 7 Pa. The power supply was 200 W, and the temperature at substrate surface was kept at 25 °C by cathode refrigeration.
2. Material and methods
For microbiological evaluation, fungal suspensions were prepared of standard strain ATCC (American Type Culture Collection) of C. albicans (10231) in the concentration of 0.5 McFarland scale [106 colony forming units per milliliters (CFU/mL)] in Dextrose Sabouraud broth (Difco™). The samples (PU, camphor:DLC/PU and DLC/PU) were introduced into different Erlenmeyer flasks containing 15 mL of fungal suspension.They were incubated at 37 °C for 48 h under 110 rpm in constant stirring using an incubator shaker model MA 420 (Marconi). After incubation time, the fungal suspension was transferred to glass tubes with phosphate buffer (PBS pH 7.2 ± 0.1) (de Vasconcelos et al., 2014) and were homogenized in a vortex for 60 s. A 100 µL aliquot of this suspension were spread on Sabouraud agar solidified by “SpreadPlate” technique and incubated at 37 °C for 48 h. This incubation time was used to obtain a mature biofilm (Braga et al., 2008). After this time, the colony-forming units (CFU/mL) were counted and compared between PU, camphor:DLC/PU and DLC/PU samples.
2.2. Biofilm formation and microbiological evaluation
For this study, we used three samples: bare polyurethane (PU) and polyurethane covered with camphor:DLC (Camphor:DLC/PU) and DLC (DLC/PU) films. The PU sheets used in this work is smooth, flat, transparent, and has 2 mm thickness they are commercial and widely used to produce peripherally inserted central catheters (PICC) - Uni Lumen Biomedical®. Biological tests were performed in three triplicates for each sample. Each triplicate was carried out on different days. Mechanical adhesion tests were also done in triplicate. 2.1. DLC deposition process The PU substrates were cleaned using multi-enzymatic detergent and sterile distilled water in an ultrasound bath. After this, the samples were dried in an oven at 40°. The samples were placed in a reactor where they were cleaned by argon plasma prior to film deposition. The Fig. 1 shows a schematic drawing from PECVD reactor assisted by DC pulsed source, containing an apparatus for liquids deliver used to produce the films. The system is composed by an ultrasound bath, a container for liquid precursors, a needle valve and spherical valve to control liquid vapor flux, a vacuum chamber and a turbo pump were also used to control work pressure, as presented in Fig. 1 (1–6), respectively. Inside the vacuum chamber (Fig. 1 (5)), was used a metallic shield to confine the plasma ions. This ionization was controlled by a DC-pulsed power supply allowing the deposition of the Camphor:DLC films keeping some radicals from Camphor, as
2.3. Camphor:DLC and DLC films evaluation The modified PU substrates with and without camphor:DLC and DLC films were examined using Raman spectra to verify atomic arrangement. The films were analyzed by confocal Raman microscope Horiba-Model Lab Ram HR Evolution with laser wavelength (λ=514 nm) and calibrated about the diamond peak. The laser diameter spot has submicron lateral resolution and axial confocal performance better than 2 µm. The power on the sample was
Fig. 1. Schematic drawing of deposition reactor.
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Table 1 Parameters of camphor:DLC and DLC film deposition. Parameters Samples
Precursor
Flow (sccm)
Time (min)
Pressure (Pa)
Power Supply (W)
Temperature (°C)
Polyurethane Cleaning DLC deposition Camphor: DLC deposiion
Argon
30
10
7
200 W
25
Hexane and Methane Hexane, Camphor and Methane
5 5
180 180
7 7
200 W 200 W
25 25
polyuretane were record. A Bruker optical profile, model NT9100, with 20X objective magnitude was used to measure the roughness of the samples. The operation mode was vertical scanning interferometry (VSI) and this mode allows to measure rough surfaces with steps up to one millimeter high. Red interferometric light was used because it is indicated for poorly reflective samples and it is more sensitivity to lowcontrast fringes. AFM and optical profiler with VSI mode were used to compare Roughness Medium Square (RMS) results from the samples with and without films. 3. Results and discussion The PU, camphor:DLC/PU and DLC/PU samples were analyzed by Raman spectroscopy to verify their chemical composition. AFM and optical profilometry were used to evaluate roughness. SEM and AFM were used to analyze Candida albicans morphology, and counting of colony-forming units (CFU/mL) was used to analyze Candida albicans proliferation.
Fig. 2. Raman spectra plot with overlapping spectra of the DLC, Camphor:DLC, and camphor samples.
3.1. Raman spectroscopy
~0.6 mW, and the laser depth inserted into the film was approximately 1 µm. The film thickness was higher than 1.5 µm, which means that the laser did not reach the PU substrate. All measurements were carried out in air at room temperature. The morphological properties of camphor:DLC, DLC film, and biofilm were evaluated using electron microscopy technique (SEM) EVO MA 10 and Atomic Force Microscopy (AFM) Park NX10. In addition, AFM was used to analyze details from Candida albicans biofilm morphology on PU substrate, after 48 h immersed in fungal suspension. The scratching resistance of the films was evaluated by constant load from a Bruker micro-scratch test using a diamond stylus (Rockwell C 120°) with a 200 µm radius diamond tip. The test was performed in two steps: the first one the diamond tip touched the sample and the Z position was zeroed and then an increasing load was applied until reach the 5 N load (Fz); on the second step the sample was scratched with 5N as constant load for 10 mm with 0.1 mm/s. The tests were performed 5 times on different samples. Thus, the friction coefficient between the diamond tip and camphor:DLC films on
Fig. 2 shows a spectra plot containing the three Raman spectra overlapping from DLC/PU, camphor, and camphor:DLC/PU in blue, black, and green line spectra, respectively. The DLC films presented a band of disorder (D band) from sp, sp2, and sp3 bonding centered at 1350 cm−1 and a band from graphitic phase (G band) centered at 1580 cm−1 (Ferrari and Robertson, 2000). The presence of camphor in DLC films was evidenced by the presence of a terpene band centered at 1099 cm−1, which is characteristic of cyclic terpene bonds in Raman spectra, from theoretical vibrational studies in (Moreno et al., 2010). Camphor contribution also caused an enlargement of D and G bands, and a left shift was observed for both peaks. Fig. 3(a) shows an optical image from DLC film highlighting the area used to construct the Raman 2D map of 25 µm2 with 64×64 points; each point corresponds to one spectrum. Fig. 3(b) shows the optical image of the analyzed area overlapped with Raman spectra 2D map with the incidence regions of the terpene band centered at 1099 cm−1. In Fig. 3(c), the Raman spectra of camphor:DLC show
Fig. 3. Raman spectra images (a-b) constructed in 2D using an area of 25×25 µm2 (c) Raman shift plot with overlapping spectra of DLC/PU and camphor:DLC/PU.
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Fig. 4. Images obtained by atomic force microscopy (AFM) of (a) Bare polyurethane, (b) Polyurethane+camphor:DLC, and (c) DLC on silicon.
Fig. 5. Comparison between RMS from images obtained by optical profilometry of (a) PU, (b) DLC/PU, and (c) camphor:DLC/PU.
3.2. Roughness analysis
the area selected to construct the Raman map. The Raman map provides a way to visualize the contribution of terpene peaks in DLC films, and how camphor as a terpene bonds were distributed in the films. Previous studies about Raman spectra of camphor found peaks distribution between 1000 cm−1 and 1127 cm−1, the peaks were related with some cyclic terpene bonds (Moreno et al., 2010). Thus, the chemical contribution of camphor in DLC films indicated that some cyclic terpenes resisted becoming ions during camphor:DLC film growth, which was due to DC power supply of the system that allowed the transition between + and − pulses with a frequency of 4 µs. This pulse frequency was low enough for some molecules remained without total disintegration or ionization.
Fig. 4 contains three images of 50 µm2 obtained by AFM and shows roughness values from (a) bare PU, (b) camphor:DLC/PU, and (c) silicon covered with DLC. The bare PU sample presented Ra 50.0 nm and RMS 58.6 nm. The camphor:DLC film on PU presented Ra 650.0 nm and RMS 800.0 nm. Pure DLC on silicon substrate presented very low roughness with Ra 0.560 nm and RMS 1.251 nm. Comparing the roughness and morphology in Fig. 4 was possible to see that the camphor contributed to increase the roughness. In addition, optical profilometry was used to analyzed the PU, camphor:DLC/PU and DLC/ PU samples morphology and roughness in a larger area and to compare cracks in the film morphology. Fig. 5 shows three images obtained by using optical profilometry with an area 230.0×301.0 µm2 from samples: PU bare (Fig. 5(a)), DLC/ 242
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Fig. 6. Images from camphor:DLC/PU segment with film folded using a Mohr clamp and photomicrograph from the same surface morphology analyses during and after bending.
and 48 h is enough to develop a mature biofilm (Braga et al., 2008). From our results, we observed that the cracks and roughness increase did not increase delamination neither biofilm proliferation, as can be seen in Figs. 6–9.
PU (Fig. 5(b)), and camphor:DLC/PU (Fig. 5(c)), in addition it shows two profiles emphasizing cracks (Fig. 5(d-e)). The PU sample presented RMS 0.44 µm, DLC/PU presented RMS 0.87 µm, and camphor:DLC/ PU presented RMS 2.95 µm. The roughness from camphor:DLC/PU were 3.4 times higher than DLC/PU and 6.7 times higher than PU. From Fig. 5(b) and (c) is possible to see cracks in the film surfaces. Comparisons from these figures are shown in Fig. 5(d) and (e) using Z depth profile emphasized by red circle. The crack depth from camphor:DLC was 2 times higher than in DLC/PU. Others regions were measured and the depth presented same dimensions. The literature shows that shrinkage and cracks are common in the films deposited in polymeric substrates, caused by compression tension (Volynskii et al., 2000). The compression tension is due to the difference between the coefficients of linear thermal expansion, from PU is (~57.6×(10−6 m/(m K))) and from DLC is (~1×(10-6 m/(m K))). To minimize the difference and promoted a gradient of linear thermal expansion is a necessary interlayer with intermediary thermal expansion coefficient. Biofilms has been benefited by roughness surfaces, literature also brings examples from nature were surface roughness improves biofouling (Bazaka et al., 2012; Bixler and Bhushan, 2012). The surface roughness can influence the adhesion of proteins and microorganisms in many kinds of devices. The biofilm begins soon after surface contact,
3.3. Flexibility and adherence of camphor:DLC/PU Fig. 6 presents six images related to adherence and flexibility of camphor:DLC/PU segments. The film flexibility was evaluated by the persistence of the camphor:DLC/PU film after folding PU segments 180°. Fig. 6(a) is a picture from camphor:DLC/PU segment folded using a Mohr clamp to analyze camphor:DLC film morphology in SEM. Fig. 6(b) shows a photomicrograph of camphor:DLC/PU from folded region using a magnitude of 30×. From this image, the film morphology seems to be homogeneous; however, in Fig. 6(c) with a magnitude of 1.00 K ×, the same region shows fractures on the film after recovering its original position as shown in Fig. 6(d-e). Fig. 6(d) is a picture of the PU segments with camphor:DLC film after folding 180°; this image shows the results of the fold remaining in the resilient camphor:DLC film morphology. Fig. 6(e) presents a photomicrograph using a magnitude of 1.00 K × of the folded region of camphor:DLC/PU segment after the film recovered the original position. Fig. 6(f) shows a photomicrograph of camphor:DLC/PU sample also of the folded 243
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Fig. 7. (a) Track obtained from scratching tests performed with diamond tip on Camphor:DLC/PU, (b) Friction plot from Camphor:DLC/Pu (pink line) and constant normal force in (black line) and penetration depth in (gray line),(c) Friction coefficients curves recorded during tests.(d) Bar plot with the mean friction coefficient and the standard deviation for each and entire friction coefficient curves.
friction coefficient was recorded during all test and the five measurements can be observed on Fig. 7(c). The variation on the friction coefficient values were related with the resistance of the tip sliding on the film and the Z position variation occurred to keep the normal load constant. Fig. 7(d) shows the bar plot with the mean friction coefficient and the standard deviation for each and entire friction coefficient curve. The bars from Fig. 7(d) was related with mean friction coefficient curves by the color from Fig. 7(c). The overall mean friction coefficient was 0.2 ± 0.05 so these films can be considered as solid lubricant. Singer and co-author demonstrated that materials with friction coefficient lower than 0.3 would minimize the surface damage and they are considered solid lubricant (Singer and Pollock, 1992).
region using a magnitude of 1.5 K × to highlight the coating without delamination. From this image, it is possible to see that the camphor:DLC film recovered the original position in PU segment without any delamination just some cracks. The morphology of camphor:DLC/PU surface after recovering the initial position has similar cracks as surface micrographs of strained samples of Polyethylene (PE), Polyethylene Terephthalate (PET) (Tsubone et al., 2007). Fig. 7 shows a set of imagens obtained on constant load scratching test. Fig. 7(a) shows a SEM image from center of scratching track, the white lines mark the track edges and the track width was of 602.1 µm. This image shows a homogenous camphor:DLC surface without delamination with some cracks that are characteristics from the camphor:DLC films on PU. The diamond tip used on this test had 200 µm of diameter, so the tip passed over the cracks without change this form and size as can be seen in Fig. 7(a) comparing inside and outside the track. The image also shows that the film resisted to the scratch without delamination. Also was observed that the failures on camphor:DLC film did not affect the scratch and friction results in microscale, because the tip contact is 10 times higher than the camphor:DLC film crack enlargement. Fig. 7(b) shows the representative results obtained on second step of scratching test performed with constant load of 5 N (Fz - black line). The tip was pressed down; the depth during the test (Z - gray line) and the friction coefficient (olive green line) were shown in the same plot. Some variation on depth curve during the test was observed because the system makes this movement to keep the normal load constant as can be observed on gray line from Fig. 7(b). The maximum deformation depth of the diamond tip for all measurements was 0.73 ± 0.03 mm. This depth is about 5 times higher than the film thickness and occurred due to elastic deformation of the PU substrate. The camphor:DLC film accompanied this deformation and kept adhered on the surface. The friction coefficient on the Fig. 7(b) presents one of the friction curves (T5) from Fig. 7(c). The
3.4. Microbiology quantitative biofilm analysis Fig. 8(a) shows the results from microbiology data using PU surface and shows that almost 80% was covered with Candida albicans biofilm after immersion in fungal suspension for 48 h. Fig. 8(b) was obtained by AFM also from PU surface with Candida albicans and shows details of iiae (Calderone and Fonzi, 2001). The literature indicates that pseudohyphae are responsible for polarizing the cell division; in this process, the cell stretches from adjacent cells, and the cells remain attached to each other (Mayer et al., 2013). When Candida albicans finds favorable conditions, it changes from its unicellular yeast form to filamentous form because hyphal forms are required for tissue invasion and damage (Sajjad et al., 2010). In the same Fig. 8(b), it is possible to see chlamydospores with spherical structures, which are a type of refractory spore with a thick cell wall (Staib and Morschhäuser, 2007). Fig. 8(b) also shows Candida albicans blastospores, which act as a medium in their embryonic state and make contact with the external digestive cavity. The Candida albicans blastospores were formed in the embryonic period at the beginning of cell differentiation (“Fungi pathogenic to humans molecular bases of virulence of Candida.pdf”, 244
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Fig. 8. SEM and AFM images of PU with Candida albicans. (a) SEM image of PU with Candida albicans yeasts after immersed for 48 h in fungal suspension. (b) AFM image emphacizing pseudohypha, blastspore, and chlamydospore from mature Candida albicans yeasts. (c) SEM image of Candida albicans in DLC/PU, (d) SEM image of Candida albicans in camphor.:DLC/PU with some fungal outbreaks.
mature Candida albicans morphology with pseudohyphae, chlamydospores, and blastospores spread on the samples in areas higher than 20µm2 and from SEM images it was not observed. Fig. 9 shows a plot with the reduction of colony-forming units for each group studied. The PU group was used as control, because no additive was used to inhibit fungal growth in this sample. The camphor:DLC/PU and DLC/PU groups presented a reduction of 99% and 91% corresponding to 2 log and 1 log of CFUs, respectively. In previous literature, DLC was used as a bactericide surface, because its flat and smooth surface reduces the bacterial adherence (Terriza et al., 2010). Comparing surfaces roughness and CFUs results, were observed that camphor:DLC/PU roughness are higher than DLC/PU and in both surface the roughness did not increase CFU. 4. Conclusions
Fig. 9. Bar plot from number of colony-forming units as a function of the groups studied.
We produced DLC film and DLC containing terpenes from camphor deposited on polyurethane surface. Both films presented high adhesion even when the substrate was folded many times. The Raman spectra found the signature of DLC and showed that camphor peaks contributed to broadening DLC peaks on camphor:DLC film. The Raman map showed terpenoid bond from camphor distribution on DLC surface, which indicates that Raman in confocal images is a powerful tool to analyze mixed materials such as camphor:DLC. The Atomic Force Microscopy and optical profilometry technique elucidated the roughness of PU with and without DLC coating. The roughness increased for Camphor:DLC/PU and presented some cracks on the film morphology. The failures presented on Camphor:DLC films on PU
n.d.). Fig. 8(c-d) shows two images obtained by SEM from DLC/PU and camphor:DLC/PU both containing Candida albicans biofilm. Fig. 8(c) shows a small area with Candida albicans in DLC/PU sample that shows a low proliferation of the yeast on the DLC film when compared PU sample on Fig. 8(a). Fig. 8(d) shows camphor:DLC/PU from both figures and it is possible to see a slight region containing microorganisms. Only fungal outbreaks were observed on the entire camphor:DLC/PU surface. The results from Fig. 8 demonstrates that the presence of failures on the coatings did not provide support for cell adhesion because the set off 245
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and DLC films on PU were cracks with approximately 20 μm of enlargment and did not affect the scratching and friction results in microscale, because the tip contact was 10 times higher than the crack enlargement. In addition, the failures did not provide support for cell adhesion because the set off mature Candida albicans morphology with pseudohyphae, chlamydospores, and blastospores were found just on the bare PU samples in areas higher than 20µm2. Comparing surfaces roughness and CFUs results, was observed that camphor:DLC/PU roughness are higher than DLC/PU and in both surface the roughness did not increase CFU. camphor:DLC/PU presented 99% reduction of colonies using CFUs technique, and only fungal outbreaks of Candida albicans yeasts were found on the surface of the sample. These results indicate that camphor:DLC films avoided proliferation of Candida albicans biofilm and have great potential for depositing on polyurethane materials as medical instruments to prevent microorganism proliferation. In addition, scratching test under 5N constant normal load using a diamond tip was used to evaluate the film adherence, and it was kept in the track without delamination. The diamond tip pressed down the camphor:DLC/PU surface and the maximum deformation depth was 0.73 ± 0.03 mm. This deformation was about 5 times higher than the film thickness and occurred due to elastic deformation of the PU substrate. The camphor:DLC film accompanied this deformation and kept adhered on the surface as show in Fig. 7(a). Finally, from flexibility tests the camphor:DLC/PU film recovered the original position after folded in 180° on PU segment without any delamination and presented morphology just puzzle-like cracks.
coatings: a review. Surf. Coat. Technol. 200, 2518–2524. http://dx.doi.org/10.1016/ j.surfcoat.2005.07.077. de Vasconcelos, L.C., Sampaio, F.C., Albuquerque, A., de, J., dos, R., Vasconcelos, L.C., de, S., 2014. Cell viability of Candida albicans against the antifungal activity of thymol. Braz. Dent. J. 25, 277–281. http://dx.doi.org/10.1590/01036440201300052. Fadzilah, A.N., Dayana, K., Rusop, M., 2013. Fabrication and characterization of camphor-based amorphous carbon thin films. Procedia Eng. 56, 743–749. http:// dx.doi.org/10.1016/j.proeng.2013.03.188. Ferrari, A., Robertson, J., 2000. Interpretation of Raman spectra of disordered and amorphous carbon. Phys. Rev. B. http://dx.doi.org/10.1103/PhysRevB.61.14095. Frizzo, C.D., Santos, A.C., Paroul, N., Serafini, L. a, Dellacassa, E., Lorenzo, D., Moyna, P., 2000. Essential oils of camphor tree (cinnamomum camphora nees & eberm) cultivated in Southern Brazil. Braz. Arch. Biol. Technol. 43, 313–316. http:// dx.doi.org/10.1590/S1516-89132000000300011. Fungi pathogenic to humans molecular bases of virulence of Candida.pdf, n.d. Gershenzon, J., Dudareva, N., 2007. The function of terpene natural products in the natural world. Nat. Chem. Biol. 3, 408–414. http://dx.doi.org/10.1038/ nchembio.2007.5. Griffiths, R.D., Hall, J.B., 2010. Intensive care unit-acquired weakness. Crit. Care Med. 38, 779–787. http://dx.doi.org/10.1097/CCM.0b013e3181cc4b53. Hauert, R., 2003. A review of modified DLC coatings for biological applications. Diam. Relat. Mater. 12, 583–589. http://dx.doi.org/10.1016/S0925-9635(03)00081-5. Liu, E., Kwek, H.W., 2008. Electrochemical performance of diamond-like carbon thin films. Thin Solid Films 516, 5201–5205. http://dx.doi.org/10.1016/ j.tsf.2007.07.089. Marciano, F.R., Bonetti, L.F., Santos, L.V., Da-silva, N.S., Corat, E.J., Trava-airoldi, V.J., 2009. Diamond & Related Materials Antibacterial activity of DLC and Ag – DLC films produced by PECVD technique. Diam. Relat. Mater. 18, 1010–1014. http:// dx.doi.org/10.1016/j.diamond.2009.02.014. Mayer, F.L., Wilson, D., Hube, B., 2013. Candida albicans pathogenicity mechanisms. Virulence 4, 119–128. http://dx.doi.org/10.4161/viru.22913. Moreno, J.R.A., Ureña, F.P., González, J.J.L., 2010. Chiral terpenes in different matrices: r-(+)-camphor studied by IR-Raman-VCD spectroscopies and quantum chemical calculations. Asian J. Spectrosc. 14, 1–21. Morrison, M.L., Buchanan, R. a, Liaw, P.K., Berry, C.J., Brigmon, R.L., Riester, L., Abernathy, H., Jin, C., Narayan, R.J., 2006. Electrochemical and antimicrobial properties of diamondlike carbon-metal composite films. Diam. Relat. Mater. 15, 138–146. http://dx.doi.org/10.1016/j.diamond.2005.08.031. Sajjad, M., Khan, A., Ahmad, I., Aqil, F., Owais, M., 2010. Combating Fungal Infections. http://dx.doi.org/10.1007/978-3-642-12173-9. Singer, I.L., Pollock, H.M. (Eds.), 1992. Fundamentals of Friction: Macroscopic and Microscopic Processes. Springer, Netherlands, Dordrecht. http://dx.doi.org/ 10.1007/978-94-011-2811-7. Staib, P., Morschhäuser, J., 2007. Chlamydospore formation in Candida albicans and Candida dubliniensis - an enigmatic developmental programme. Mycoses 50, 1–12. http://dx.doi.org/10.1111/j.1439-0507.2006.01308.x. Terriza, A., Del Prado, G., Pérez, A.O., Martínez, M.J., Puértolas, J.A., Manso, D.M., González-Elipe, A.R., Yubero, F., Barrena, E.G., Esteban, J., 2010. Bacterial adherence on fluorinated carbon based coatings deposited on polyethylene surfaces. J. Phys. Conf. Ser.. http://dx.doi.org/10.1088/1742-6596/252/1/012013. Tsubone, D., Hasebe, T., Kamijo, A., Hotta, A., 2007. Fracture mechanics of diamond-like carbon (DLC) films coated on flexible polymer substrates. Surf. Coat. Technol. 201, 6423–6430. http://dx.doi.org/10.1016/j.surfcoat.2006.12.008. van Asbeck, E.C., Huang, Y.-C., Markham, A.N., Clemons, K.V., Stevens, D.A., 2007. Candida parapsilosis fungemia in neonates: genotyping results suggest healthcare workers hands as source, and review of published studies. Mycopathologia 164, 287–293. http://dx.doi.org/10.1007/s11046-007-9054-3. Volynskii, A.L., Bazhenov, S., Lebedeva, O.V., Bakeev, N.F., 2000. Mechanical buckling instability of thin coatings deposited on soft polymer substrates. J. Mater. Sci. 35, 547–554. http://dx.doi.org/10.1023/A:1004707906821. Wang, J., Huang, N., Yang, P., Leng, Y.X., Sun, H., Liu, Z.Y., Chu, P.K., 2004. The effects of amorphous carbon films deposited on polyethylene terephthalate on bacterial adhesion. Biomaterials 25, 3163–3170. http://dx.doi.org/10.1016/ j.biomaterials.2003.10.010. Widoniak, J., Eiden-Assmann, S., Maret, G., 2005. Silver particles tailoring of shapes and sizes. Colloids Surf. A Physicochem. Eng. Asp. 270–271, 340–344. http:// dx.doi.org/10.1016/j.colsurfa.2005.09.004. Zani, A., Dellasega, D., Russo, V., Passoni, M., 2013. Ultra-low density carbon foams produced by pulsed laser deposition. Carbon 56, 358–365. http://dx.doi.org/ 10.1016/j.carbon.2013.01.029.
Acknowledgements Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Project number 313280/2014-2, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Project number 05 Capes/ITA. The authors are also grateful to Evado José Corat from INPE for Raman support, Carlos Costa and Evandro Martin Lanzoni from the Brazilian Nanotechnology National Laboratory (LNNano) for AFM analysis support. References Bazaka, K., Jacob, M.V., Crawford, R.J., Ivanova, E.P., 2012. Efficient surface modification of biomaterial to prevent biofilm formation and the attachment of microorganisms. Appl. Microbiol. Biotechnol. 95, 299–311. http://dx.doi.org/ 10.1007/s00253-012-4144-7. Bixler, G.D., Bhushan, B., 2012. Biofouling: lessons from nature. Philos. Trans. R. Soc. A Math. Phys. Eng. Sci. 370, 2381–2417. http://dx.doi.org/10.1098/rsta.2011.0502. Braga, P.C., Culici, M., Alfieri, M., Dal Sasso, M., 2008. Thymol inhibits Candida albicans biofilm formation and mature biofilm. Int. J. Antimicrob. Agents 31, 472–477. http://dx.doi.org/10.1016/j.ijantimicag.2007.12.013. Calderone, R.A., Fonzi, W.A., 2001. Virulence factors of Candida albicans. Trends Microbiol. 9, 327–335. http://dx.doi.org/10.1016/S0966-842X(01)02094-7, [pii]. Choudhury, D., Lackner, J.M., Major, L., Morita, T., Sawae, Y., Bin Mamat, A., Stavness, I., Roy, C.K., Krupka, I., 2016. Improved wear resistance of functional diamond like carbon coated Ti–6Al–4V alloys in an edge loading conditions. J. Mech. Behav. Biomed. Mater. 59, 586–595. http://dx.doi.org/10.1016/j.jmbbm.2016.04.004. Co-investigator, N., 2013. Summary for policymakers. In: Intergovernmental Panel on Climate Change (Ed.), Climate Change 2013 – The Physical Science Basis. Cambridge University Press, Cambridge, 1–30. http://dx.doi.org/10.1017/ CBO9781107415324.004. Dearnaley, G., Arps, J.H., 2005. Biomedical applications of diamond-like carbon (DLC)
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Journal of the Mechanical Behavior of Biomedical Materials journal homepage: www.elsevier.com/locate/jmbbm
Flexible camphor diamond-like carbon coating on polyurethane to prevent Candida albicans biofilm growth
MARK
Thaisa B. Santosa, Angela A. Vieiraa, Luciana O. Paulaa, Everton D. Santosa, Polyana A. Radia,b, ⁎ Sônia Khouria, Homero S. Maciela,b, Rodrigo S. Pessoaa,b, Lucia Vieiraa,b, a Universidade do Vale do Paraíba – UNIVAP, Grupo Nanotecplasma, Av. Shishima Hifumi, 2911 – Urbanova, CEP 12.244-000 São José dos Campos, SP, Brazil b Instituto Tecnológico de Aeronáutica – ITA, Laboratório de Processamento a Plasma, Praça Marechal Eduardo Gomes, 50 – Vila das Acácias, São José dos Campos, SP 12228-900, Brazil
A R T I C L E I N F O
A BS T RAC T
Keywords: Diamond-like carbon (DLC) Candida albicans Camphor Raman Biofilm growth inhibition
Camphor was incorporated in diamond-like carbon (DLC) films to prevent the Candida albicans yeasts fouling on polyurethane substrates, which is a material commonly used for catheter manufacturing. The camphor:DLC and DLC film for this investigation was produced by plasma enhanced chemical vapor deposition (PECVD), using an apparatus based on the flash evaporation of organic liquid (hexane) containing diluted camphor for camphor:DLC and hexane/methane, mixture for DLC films. The film was deposited at a low temperature of less than 25 °C. We obtained very adherent camphor:DLC and DLC films that accompanied the substrate flexibility without delamination. The adherence of camphor:DLC and DLC films on polyurethane segments were evaluated by scratching test and bending polyurethane segments at 180°. The polyurethane samples, with and without camphor:DLC and DLC films were characterized by Raman spectroscopy, scanning electron microscopy, atomic force microscopy, and optical profilometry. Candida albicans biofilm formation on polyurethane, with and without camphor:DLC and DLC, was assessed. The camphor:DLC and DLC films reduced the biofilm growth by 99.0% and 91.0% of Candida albicans, respectively, compared to bare polyurethane. These results open the doors to studies of functionalized DLC coatings with biofilm inhibition properties used in the production of catheters or other biomedical applications.
1. Introduction Diamond-Like Carbon (DLC) is a very attractive material for biological applications because it has physical and chemical properties similar to diamond such as hardness and relatively high modulus of elasticity. DLC is easily deposited on large and 3D areas (Hauert, 2003). Although, there are a significant number of manuscripts about DLC (Choudhury et al., 2016) and DLC with metallic nanoparticles (Widoniak et al., 2005; Marciano et al., 2009), little is known about the use of DLC in polymeric materials (Wang et al., 2004). Some studies mention silver used as a possible inhibitor of microbial growth and catheters coated with a mixture of silver and DLC (Dearnaley and Arps, 2005). On the other hand, only a few studies have used Camphor as a carbon precursor for DLC production, and these prioritized manufacturing process of only carbon bonds, and terpenes radicals were not preserved (Fadzilah et al., 2013; Liu and Kwek, 2008; Zani et al., 2013). No literature was found about camphor:DLC as a material to combat
candidiasis proliferation. Candidiasis contamination is related to parenteral nutrition, and it is transmitted through the hands of healthcare workers and especially the use of catheters [6]. Candidiasis contamination has been a big problem for hospitalized patients, especially those in critical condition, and is the fourth leading cause of bloodstream infection. Bloodstream infection increases hospital costs, patient's hospitalization time and mortality rate (Morrison et al., 2006; van Asbeck et al., 2007). Biofilm is responsible for conducting catheter infection, due to a set of micro-organisms that live in association with biofilm (Bazaka et al., 2012; Griffiths and Hall, 2010). Most microorganisms involved in colonization of catheters are not virulent in planktonic form but can cause persistent infection when they are in a group (Co-investigator, 2013). Camphor is cetonic terpenoid (C10H6O) obtained from the camphor tree that is native to China, and it is part of a class of bioactive compounds synthesized by plants (Frizzo et al., 2000; Gershenzon and Dudareva, 2007; Moreno et al., 2010). Terpenes are several open-
⁎ Corresponding author at: Universidade do Vale do Paraíba – UNIVAP, Grupo Nanotecplasma, Av. Shishima Hifumi, 2911 – Urbanova, CEP 12.244-000 São José dos Campos, SP, Brazil. E-mail address: [email protected] (L. Vieira).
http://dx.doi.org/10.1016/j.jmbbm.2017.02.013 Received 29 May 2016; Received in revised form 5 February 2017; Accepted 12 February 2017 Available online 16 February 2017 1751-6161/ © 2017 Elsevier Ltd. All rights reserved.
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chained or cyclic compounds, usually oxygenates such as aldehydes, alcohols, and ketones. Gershenzon and Dudareva (Gershenzon and Dudareva, 2007) reported previously that plants with terpenes are resistant to diseases, due to their action on fungi and bacteria. Terpenes have detergent properties that are toxic to fungi due to their ability to complex with sterols in fungi membranes, which leads to loss of membrane integrity (Gershenzon and Dudareva, 2007). The aim of this work was to develop a DLC film that comprises camphor with properties to prevent biofilm of Candida albicans for use on catheters and biomedical instruments. For this, we developed a simple apparatus that was coupled in a commercial plasma enhanced chemical vapor deposition (PECVD) reactor for camphor delivery during DLC production. This process allows the deposition of DLC films containing radicals for functionalized surfaces or nanoparticles, covering materials three-dimensionally at low temperature.
showed in Raman spectra by the terpene band centered on 1099 cm−1 (Fig. 1). Table 1 shows the parameters used for the camphor:DLC and DLC film deposition. The deposition plasma process started with argon plasma to remove oxides in (PU) surfaces. DLC films were produced using a vapor mixture of hexane and methane and for camphor:DLC was used camphor oil diluted in hexane (10% v.v.) with methane vapor. The glass tube apparatus containing the liquid precursor were sonicated using an ultrasound bath. The flow for each carbon precursor was controlled using flowmeters and pressure valves to maintain the work pressure inside the vacuum chamber at 7 Pa. The power supply was 200 W, and the temperature at substrate surface was kept at 25 °C by cathode refrigeration.
2. Material and methods
For microbiological evaluation, fungal suspensions were prepared of standard strain ATCC (American Type Culture Collection) of C. albicans (10231) in the concentration of 0.5 McFarland scale [106 colony forming units per milliliters (CFU/mL)] in Dextrose Sabouraud broth (Difco™). The samples (PU, camphor:DLC/PU and DLC/PU) were introduced into different Erlenmeyer flasks containing 15 mL of fungal suspension.They were incubated at 37 °C for 48 h under 110 rpm in constant stirring using an incubator shaker model MA 420 (Marconi). After incubation time, the fungal suspension was transferred to glass tubes with phosphate buffer (PBS pH 7.2 ± 0.1) (de Vasconcelos et al., 2014) and were homogenized in a vortex for 60 s. A 100 µL aliquot of this suspension were spread on Sabouraud agar solidified by “SpreadPlate” technique and incubated at 37 °C for 48 h. This incubation time was used to obtain a mature biofilm (Braga et al., 2008). After this time, the colony-forming units (CFU/mL) were counted and compared between PU, camphor:DLC/PU and DLC/PU samples.
2.2. Biofilm formation and microbiological evaluation
For this study, we used three samples: bare polyurethane (PU) and polyurethane covered with camphor:DLC (Camphor:DLC/PU) and DLC (DLC/PU) films. The PU sheets used in this work is smooth, flat, transparent, and has 2 mm thickness they are commercial and widely used to produce peripherally inserted central catheters (PICC) - Uni Lumen Biomedical®. Biological tests were performed in three triplicates for each sample. Each triplicate was carried out on different days. Mechanical adhesion tests were also done in triplicate. 2.1. DLC deposition process The PU substrates were cleaned using multi-enzymatic detergent and sterile distilled water in an ultrasound bath. After this, the samples were dried in an oven at 40°. The samples were placed in a reactor where they were cleaned by argon plasma prior to film deposition. The Fig. 1 shows a schematic drawing from PECVD reactor assisted by DC pulsed source, containing an apparatus for liquids deliver used to produce the films. The system is composed by an ultrasound bath, a container for liquid precursors, a needle valve and spherical valve to control liquid vapor flux, a vacuum chamber and a turbo pump were also used to control work pressure, as presented in Fig. 1 (1–6), respectively. Inside the vacuum chamber (Fig. 1 (5)), was used a metallic shield to confine the plasma ions. This ionization was controlled by a DC-pulsed power supply allowing the deposition of the Camphor:DLC films keeping some radicals from Camphor, as
2.3. Camphor:DLC and DLC films evaluation The modified PU substrates with and without camphor:DLC and DLC films were examined using Raman spectra to verify atomic arrangement. The films were analyzed by confocal Raman microscope Horiba-Model Lab Ram HR Evolution with laser wavelength (λ=514 nm) and calibrated about the diamond peak. The laser diameter spot has submicron lateral resolution and axial confocal performance better than 2 µm. The power on the sample was
Fig. 1. Schematic drawing of deposition reactor.
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Table 1 Parameters of camphor:DLC and DLC film deposition. Parameters Samples
Precursor
Flow (sccm)
Time (min)
Pressure (Pa)
Power Supply (W)
Temperature (°C)
Polyurethane Cleaning DLC deposition Camphor: DLC deposiion
Argon
30
10
7
200 W
25
Hexane and Methane Hexane, Camphor and Methane
5 5
180 180
7 7
200 W 200 W
25 25
polyuretane were record. A Bruker optical profile, model NT9100, with 20X objective magnitude was used to measure the roughness of the samples. The operation mode was vertical scanning interferometry (VSI) and this mode allows to measure rough surfaces with steps up to one millimeter high. Red interferometric light was used because it is indicated for poorly reflective samples and it is more sensitivity to lowcontrast fringes. AFM and optical profiler with VSI mode were used to compare Roughness Medium Square (RMS) results from the samples with and without films. 3. Results and discussion The PU, camphor:DLC/PU and DLC/PU samples were analyzed by Raman spectroscopy to verify their chemical composition. AFM and optical profilometry were used to evaluate roughness. SEM and AFM were used to analyze Candida albicans morphology, and counting of colony-forming units (CFU/mL) was used to analyze Candida albicans proliferation.
Fig. 2. Raman spectra plot with overlapping spectra of the DLC, Camphor:DLC, and camphor samples.
3.1. Raman spectroscopy
~0.6 mW, and the laser depth inserted into the film was approximately 1 µm. The film thickness was higher than 1.5 µm, which means that the laser did not reach the PU substrate. All measurements were carried out in air at room temperature. The morphological properties of camphor:DLC, DLC film, and biofilm were evaluated using electron microscopy technique (SEM) EVO MA 10 and Atomic Force Microscopy (AFM) Park NX10. In addition, AFM was used to analyze details from Candida albicans biofilm morphology on PU substrate, after 48 h immersed in fungal suspension. The scratching resistance of the films was evaluated by constant load from a Bruker micro-scratch test using a diamond stylus (Rockwell C 120°) with a 200 µm radius diamond tip. The test was performed in two steps: the first one the diamond tip touched the sample and the Z position was zeroed and then an increasing load was applied until reach the 5 N load (Fz); on the second step the sample was scratched with 5N as constant load for 10 mm with 0.1 mm/s. The tests were performed 5 times on different samples. Thus, the friction coefficient between the diamond tip and camphor:DLC films on
Fig. 2 shows a spectra plot containing the three Raman spectra overlapping from DLC/PU, camphor, and camphor:DLC/PU in blue, black, and green line spectra, respectively. The DLC films presented a band of disorder (D band) from sp, sp2, and sp3 bonding centered at 1350 cm−1 and a band from graphitic phase (G band) centered at 1580 cm−1 (Ferrari and Robertson, 2000). The presence of camphor in DLC films was evidenced by the presence of a terpene band centered at 1099 cm−1, which is characteristic of cyclic terpene bonds in Raman spectra, from theoretical vibrational studies in (Moreno et al., 2010). Camphor contribution also caused an enlargement of D and G bands, and a left shift was observed for both peaks. Fig. 3(a) shows an optical image from DLC film highlighting the area used to construct the Raman 2D map of 25 µm2 with 64×64 points; each point corresponds to one spectrum. Fig. 3(b) shows the optical image of the analyzed area overlapped with Raman spectra 2D map with the incidence regions of the terpene band centered at 1099 cm−1. In Fig. 3(c), the Raman spectra of camphor:DLC show
Fig. 3. Raman spectra images (a-b) constructed in 2D using an area of 25×25 µm2 (c) Raman shift plot with overlapping spectra of DLC/PU and camphor:DLC/PU.
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Fig. 4. Images obtained by atomic force microscopy (AFM) of (a) Bare polyurethane, (b) Polyurethane+camphor:DLC, and (c) DLC on silicon.
Fig. 5. Comparison between RMS from images obtained by optical profilometry of (a) PU, (b) DLC/PU, and (c) camphor:DLC/PU.
3.2. Roughness analysis
the area selected to construct the Raman map. The Raman map provides a way to visualize the contribution of terpene peaks in DLC films, and how camphor as a terpene bonds were distributed in the films. Previous studies about Raman spectra of camphor found peaks distribution between 1000 cm−1 and 1127 cm−1, the peaks were related with some cyclic terpene bonds (Moreno et al., 2010). Thus, the chemical contribution of camphor in DLC films indicated that some cyclic terpenes resisted becoming ions during camphor:DLC film growth, which was due to DC power supply of the system that allowed the transition between + and − pulses with a frequency of 4 µs. This pulse frequency was low enough for some molecules remained without total disintegration or ionization.
Fig. 4 contains three images of 50 µm2 obtained by AFM and shows roughness values from (a) bare PU, (b) camphor:DLC/PU, and (c) silicon covered with DLC. The bare PU sample presented Ra 50.0 nm and RMS 58.6 nm. The camphor:DLC film on PU presented Ra 650.0 nm and RMS 800.0 nm. Pure DLC on silicon substrate presented very low roughness with Ra 0.560 nm and RMS 1.251 nm. Comparing the roughness and morphology in Fig. 4 was possible to see that the camphor contributed to increase the roughness. In addition, optical profilometry was used to analyzed the PU, camphor:DLC/PU and DLC/ PU samples morphology and roughness in a larger area and to compare cracks in the film morphology. Fig. 5 shows three images obtained by using optical profilometry with an area 230.0×301.0 µm2 from samples: PU bare (Fig. 5(a)), DLC/ 242
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Fig. 6. Images from camphor:DLC/PU segment with film folded using a Mohr clamp and photomicrograph from the same surface morphology analyses during and after bending.
and 48 h is enough to develop a mature biofilm (Braga et al., 2008). From our results, we observed that the cracks and roughness increase did not increase delamination neither biofilm proliferation, as can be seen in Figs. 6–9.
PU (Fig. 5(b)), and camphor:DLC/PU (Fig. 5(c)), in addition it shows two profiles emphasizing cracks (Fig. 5(d-e)). The PU sample presented RMS 0.44 µm, DLC/PU presented RMS 0.87 µm, and camphor:DLC/ PU presented RMS 2.95 µm. The roughness from camphor:DLC/PU were 3.4 times higher than DLC/PU and 6.7 times higher than PU. From Fig. 5(b) and (c) is possible to see cracks in the film surfaces. Comparisons from these figures are shown in Fig. 5(d) and (e) using Z depth profile emphasized by red circle. The crack depth from camphor:DLC was 2 times higher than in DLC/PU. Others regions were measured and the depth presented same dimensions. The literature shows that shrinkage and cracks are common in the films deposited in polymeric substrates, caused by compression tension (Volynskii et al., 2000). The compression tension is due to the difference between the coefficients of linear thermal expansion, from PU is (~57.6×(10−6 m/(m K))) and from DLC is (~1×(10-6 m/(m K))). To minimize the difference and promoted a gradient of linear thermal expansion is a necessary interlayer with intermediary thermal expansion coefficient. Biofilms has been benefited by roughness surfaces, literature also brings examples from nature were surface roughness improves biofouling (Bazaka et al., 2012; Bixler and Bhushan, 2012). The surface roughness can influence the adhesion of proteins and microorganisms in many kinds of devices. The biofilm begins soon after surface contact,
3.3. Flexibility and adherence of camphor:DLC/PU Fig. 6 presents six images related to adherence and flexibility of camphor:DLC/PU segments. The film flexibility was evaluated by the persistence of the camphor:DLC/PU film after folding PU segments 180°. Fig. 6(a) is a picture from camphor:DLC/PU segment folded using a Mohr clamp to analyze camphor:DLC film morphology in SEM. Fig. 6(b) shows a photomicrograph of camphor:DLC/PU from folded region using a magnitude of 30×. From this image, the film morphology seems to be homogeneous; however, in Fig. 6(c) with a magnitude of 1.00 K ×, the same region shows fractures on the film after recovering its original position as shown in Fig. 6(d-e). Fig. 6(d) is a picture of the PU segments with camphor:DLC film after folding 180°; this image shows the results of the fold remaining in the resilient camphor:DLC film morphology. Fig. 6(e) presents a photomicrograph using a magnitude of 1.00 K × of the folded region of camphor:DLC/PU segment after the film recovered the original position. Fig. 6(f) shows a photomicrograph of camphor:DLC/PU sample also of the folded 243
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Fig. 7. (a) Track obtained from scratching tests performed with diamond tip on Camphor:DLC/PU, (b) Friction plot from Camphor:DLC/Pu (pink line) and constant normal force in (black line) and penetration depth in (gray line),(c) Friction coefficients curves recorded during tests.(d) Bar plot with the mean friction coefficient and the standard deviation for each and entire friction coefficient curves.
friction coefficient was recorded during all test and the five measurements can be observed on Fig. 7(c). The variation on the friction coefficient values were related with the resistance of the tip sliding on the film and the Z position variation occurred to keep the normal load constant. Fig. 7(d) shows the bar plot with the mean friction coefficient and the standard deviation for each and entire friction coefficient curve. The bars from Fig. 7(d) was related with mean friction coefficient curves by the color from Fig. 7(c). The overall mean friction coefficient was 0.2 ± 0.05 so these films can be considered as solid lubricant. Singer and co-author demonstrated that materials with friction coefficient lower than 0.3 would minimize the surface damage and they are considered solid lubricant (Singer and Pollock, 1992).
region using a magnitude of 1.5 K × to highlight the coating without delamination. From this image, it is possible to see that the camphor:DLC film recovered the original position in PU segment without any delamination just some cracks. The morphology of camphor:DLC/PU surface after recovering the initial position has similar cracks as surface micrographs of strained samples of Polyethylene (PE), Polyethylene Terephthalate (PET) (Tsubone et al., 2007). Fig. 7 shows a set of imagens obtained on constant load scratching test. Fig. 7(a) shows a SEM image from center of scratching track, the white lines mark the track edges and the track width was of 602.1 µm. This image shows a homogenous camphor:DLC surface without delamination with some cracks that are characteristics from the camphor:DLC films on PU. The diamond tip used on this test had 200 µm of diameter, so the tip passed over the cracks without change this form and size as can be seen in Fig. 7(a) comparing inside and outside the track. The image also shows that the film resisted to the scratch without delamination. Also was observed that the failures on camphor:DLC film did not affect the scratch and friction results in microscale, because the tip contact is 10 times higher than the camphor:DLC film crack enlargement. Fig. 7(b) shows the representative results obtained on second step of scratching test performed with constant load of 5 N (Fz - black line). The tip was pressed down; the depth during the test (Z - gray line) and the friction coefficient (olive green line) were shown in the same plot. Some variation on depth curve during the test was observed because the system makes this movement to keep the normal load constant as can be observed on gray line from Fig. 7(b). The maximum deformation depth of the diamond tip for all measurements was 0.73 ± 0.03 mm. This depth is about 5 times higher than the film thickness and occurred due to elastic deformation of the PU substrate. The camphor:DLC film accompanied this deformation and kept adhered on the surface. The friction coefficient on the Fig. 7(b) presents one of the friction curves (T5) from Fig. 7(c). The
3.4. Microbiology quantitative biofilm analysis Fig. 8(a) shows the results from microbiology data using PU surface and shows that almost 80% was covered with Candida albicans biofilm after immersion in fungal suspension for 48 h. Fig. 8(b) was obtained by AFM also from PU surface with Candida albicans and shows details of iiae (Calderone and Fonzi, 2001). The literature indicates that pseudohyphae are responsible for polarizing the cell division; in this process, the cell stretches from adjacent cells, and the cells remain attached to each other (Mayer et al., 2013). When Candida albicans finds favorable conditions, it changes from its unicellular yeast form to filamentous form because hyphal forms are required for tissue invasion and damage (Sajjad et al., 2010). In the same Fig. 8(b), it is possible to see chlamydospores with spherical structures, which are a type of refractory spore with a thick cell wall (Staib and Morschhäuser, 2007). Fig. 8(b) also shows Candida albicans blastospores, which act as a medium in their embryonic state and make contact with the external digestive cavity. The Candida albicans blastospores were formed in the embryonic period at the beginning of cell differentiation (“Fungi pathogenic to humans molecular bases of virulence of Candida.pdf”, 244
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Fig. 8. SEM and AFM images of PU with Candida albicans. (a) SEM image of PU with Candida albicans yeasts after immersed for 48 h in fungal suspension. (b) AFM image emphacizing pseudohypha, blastspore, and chlamydospore from mature Candida albicans yeasts. (c) SEM image of Candida albicans in DLC/PU, (d) SEM image of Candida albicans in camphor.:DLC/PU with some fungal outbreaks.
mature Candida albicans morphology with pseudohyphae, chlamydospores, and blastospores spread on the samples in areas higher than 20µm2 and from SEM images it was not observed. Fig. 9 shows a plot with the reduction of colony-forming units for each group studied. The PU group was used as control, because no additive was used to inhibit fungal growth in this sample. The camphor:DLC/PU and DLC/PU groups presented a reduction of 99% and 91% corresponding to 2 log and 1 log of CFUs, respectively. In previous literature, DLC was used as a bactericide surface, because its flat and smooth surface reduces the bacterial adherence (Terriza et al., 2010). Comparing surfaces roughness and CFUs results, were observed that camphor:DLC/PU roughness are higher than DLC/PU and in both surface the roughness did not increase CFU. 4. Conclusions
Fig. 9. Bar plot from number of colony-forming units as a function of the groups studied.
We produced DLC film and DLC containing terpenes from camphor deposited on polyurethane surface. Both films presented high adhesion even when the substrate was folded many times. The Raman spectra found the signature of DLC and showed that camphor peaks contributed to broadening DLC peaks on camphor:DLC film. The Raman map showed terpenoid bond from camphor distribution on DLC surface, which indicates that Raman in confocal images is a powerful tool to analyze mixed materials such as camphor:DLC. The Atomic Force Microscopy and optical profilometry technique elucidated the roughness of PU with and without DLC coating. The roughness increased for Camphor:DLC/PU and presented some cracks on the film morphology. The failures presented on Camphor:DLC films on PU
n.d.). Fig. 8(c-d) shows two images obtained by SEM from DLC/PU and camphor:DLC/PU both containing Candida albicans biofilm. Fig. 8(c) shows a small area with Candida albicans in DLC/PU sample that shows a low proliferation of the yeast on the DLC film when compared PU sample on Fig. 8(a). Fig. 8(d) shows camphor:DLC/PU from both figures and it is possible to see a slight region containing microorganisms. Only fungal outbreaks were observed on the entire camphor:DLC/PU surface. The results from Fig. 8 demonstrates that the presence of failures on the coatings did not provide support for cell adhesion because the set off 245
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and DLC films on PU were cracks with approximately 20 μm of enlargment and did not affect the scratching and friction results in microscale, because the tip contact was 10 times higher than the crack enlargement. In addition, the failures did not provide support for cell adhesion because the set off mature Candida albicans morphology with pseudohyphae, chlamydospores, and blastospores were found just on the bare PU samples in areas higher than 20µm2. Comparing surfaces roughness and CFUs results, was observed that camphor:DLC/PU roughness are higher than DLC/PU and in both surface the roughness did not increase CFU. camphor:DLC/PU presented 99% reduction of colonies using CFUs technique, and only fungal outbreaks of Candida albicans yeasts were found on the surface of the sample. These results indicate that camphor:DLC films avoided proliferation of Candida albicans biofilm and have great potential for depositing on polyurethane materials as medical instruments to prevent microorganism proliferation. In addition, scratching test under 5N constant normal load using a diamond tip was used to evaluate the film adherence, and it was kept in the track without delamination. The diamond tip pressed down the camphor:DLC/PU surface and the maximum deformation depth was 0.73 ± 0.03 mm. This deformation was about 5 times higher than the film thickness and occurred due to elastic deformation of the PU substrate. The camphor:DLC film accompanied this deformation and kept adhered on the surface as show in Fig. 7(a). Finally, from flexibility tests the camphor:DLC/PU film recovered the original position after folded in 180° on PU segment without any delamination and presented morphology just puzzle-like cracks.
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