Comment on âBactericidal Effects of Natural Nanotopography of

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Influence of superhydrophobicity on the bactericidal efficiency of black silicon surfaces Denver Paige Linklater, Saulius Juodkazis, Sergey Rubanov, and Elena P. Ivanova ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b05707 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

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ACS Applied Materials & Interfaces

Influence of Superhydrophobicity on the Bactericidal Efficiency of Black Silicon Surfaces Denver P. Linklater†, ‡, Saulius Juodkazis‡, Sergey Rubanov§ Elena P. Ivanova† †Faculty of Life and Social Sciences, Swinburne University of Technology, Hawthorn, VIC 3122, Australia, ‡Centre

for Micro-Photonics and Industrial Research Institute Swinburne, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC, 3122, Australia

§Advanced

Microscopy Facility, Bio21 Institute, University of Melbourne, 30 Flemington Rd, 3010, Victoria, Australia *correspondence should be addressed to Elena P. Ivanova: [email protected]

ABSTRACT The recent discovery of nanostructured surface induced rupturing of bacterial cells has seen a surge in the development of surfaces for the physical killing of bacteria, contributing to the fight against bacterial colonization of abiotic surfaces. It is established that the nanoarchitecture is directly responsible for the inactivation of bacterial cells; however, the mechanobactericidal action remains to be fully elucidated. Here we report the fabrication of superhydrophilic and superhydrophobic black silicon surfaces with well-defined surface geometries and wettability that are responsible for inactivating approximately 98% of P. aeruginosa cells and 97% of S. aureus cells. Increased adhesion of bacterial cells onto superhydrophobic surfaces was not accompanied by increased secretion of extracellular polymeric substances. Surface hydrophobicity has been demonstrated to be statistically unimportant in determining the bactericidal efficiency of nanostructured surfaces.

Investigation into biomimetic nano- and micro-structured surfaces for the control of adhesion and proliferation of bacteria has become a research priority for the development of advanced biomaterials, which will combat the prevalence of medical-device associated infections impacting upon our healthcare systems 1-4. Surface modifications for the minimization of 1 ACS Paragon Plus Environment


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bacterial colonization and biofilm formation has been largely pursued through antimicrobial drug-delivery carriers facilitated by polymers loaded with a variety of compounds such as antibiotics 5, heavy metals 6 and quaternary ammonium compounds 7 or direct, physical modification of surface structure 8-9. Recently, advancements in nanofabrication techniques has made it possible to design and fabricate surfaces, based upon nanotopologies found in nature, which are known to kill bacteria upon contact 10-12. These surfaces have, so far, been fabricated using materials such as silicon 13, titanium 11, 14, stainless steel 15, glass 16, polymers 10, 17

and more 18.

As it was first discovered that the cicada Psaltoda claripennis wing nanoarchitecture was responsible for the mechanical killing of Pseudomonas aeruginosa cells, synthetic surfaces bearing similar surface geometries have been developed. Black silicon (bSi), a self-organised nanotextured surface, has paved the way for an industrial upscaling of fabrication of such patterns and a significant number of investigations into the mechanobactericidal effect of nanotextured surfaces against bacterial cells 12-13, 19 although the killing mechanism remains to be clearly understood. It is currently established that as bacterial cells encounter a nanopillared surface, the membrane area that is suspended between the pillars is stretched. Once the stretching of the membrane is sufficient it will lead to rupturing and eventual cell death. This understanding is in line with typical mechanical behaviour of materials showing a much lower threshold for mechanical failure upon tensile stress as compared with a compressive stress exerted at the nano-needle’s tip (at the contact point of bSi and bacterial membrane). The involvement of surface chemistry in the bacterial killing efficiency of these surfaces has also been invalidated as gold-coating of cicada wing surfaces, which changed the surface chemistry but preserved the physical surface structure, resulted in no substantial differences in cell viability, demonstrating that the surface pattern is directly responsible for bacterial cell inactivation 12. 2 ACS Paragon Plus Environment

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Recently, a new mechanistic bactericidal model, which is in opposition to previous publications, has been proposed 20. The authors suggested that membrane damage of E. coli cells on nanostructured surfaces of dragonfly wings is achieved by means of a combination of strong adhesion between nanopillars and the bacterial extracellular polysaccharide substances (EPS) layer, as well as shearing of the membrane as the cell attempts to move across the nanotextured surface while it is immobilised. The strength of adhesion to nanotextured surfaces by staphylococci strains has also been investigated where some strains were observed to undertake ‘pressure-induced’ EPS secretion on nanopillared surfaces 21. These authors purported that cell death is a result of compromise of the bacterial membrane barrier function as the cell opens membrane efflux channels to allow excretion of EPS. This inconsistency in reports of the mechano-bactericidal action of nanotextured surfaces provides validity for further investigations and prompted this study. The aim of this work is to provide further insights towards understanding on whether adhesion affinity and production of EPS indeed influence the bactericidal efficiency of the nanostructured surfaces. It remains of significant interest to evaluate both attachment and killing patterns of nanotextured substrata for the design of bio-mimetic materials. Here, black silicon (bSi) surfaces, possessing three different nanotopologies, were fabricated using plasma etching of silicon wafers, using etch time to vary the resultant nanopattern and to generate differing heights, pillar-to-pillar spacing and pillar density. A detailed protocol is provided in the Supporting Information. The height of the nanopillars was controlled using etching time; an increase in etch time resulted in taller pillars as more material is removed from the substratum surface. SEM imaging was used to characterise the surface nanoarchitecture and to determine height, pillar-to-pillar spacing, pillar density, cap area and diameter. The samples were tilted at an angle of 45° in order to view the nanopillar profiles to measure heights for each sample (Figure 1). Pyramidal bSi nanopillars were reliably achieved via 3 ACS Paragon Plus Environment


a)

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180 160

Super-hydrophobic

140 120 100 80

Hydrophobic

bSi + PFTS bSi

60 40 20

Hydrophilic

0 Control 610 nm 475 nm 212 nm Figure 1. Representative superhydrophobic and superhydrophilic black silicon surfaces. Typical SEM images of the superhydrophobic (left column) and superhydrophilic (right column) surfaces with the nanopillars of (a) 610 nm (b) 475 nm (c) 212 nm. There is no observed change in nanopillar structure after the silane treatment, while the water contact angles changes dramatically from ~ 8 ° to ~ 160 ° for all bSi samples. (d) Graph showing the invariability between water drop contact angles for as-fabricated bSi of varying heights as well as the water contact angle achieved after adsorption of a monolayer of PFTS to each bSi surface. bSi is normally hydrophilic with a WCA of ~ 8 °. bSi + PFTS consistently achieved a WCA of ~160 ° despite variation in pillar height and surface geometry. All images are taken at ×50,000 magnification, scale bars are 100 nm. 4 ACS Paragon Plus Environment

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plasma etching with controlled heights between approximately 200 nm and 600 nm (results shown in Figure S1). Sample heights increased linearly from 213.3 ± 27.1 nm to 475.5 ± 52.1 nm and to 612.1 ±72.2 nm with etch times of 15, 30 and 45 minutes. The cap widths of each sample were 21.04 ± 7.5 nm, 93.4 ±44.2 nm and 113.8 ± 56.4 nm. Similarly, interpillar spacing also increased with time with pillar-pillar spacing of 49.9, 106.6 and 139.7 nm, respectively. Density of the pillars (per µm2) decreased with etch time due to an overall increase in the size of the nanofeatures: 212 nm – 29.6/µm2, 475 nm 9.84/µm2 and 610 nm – 7.07/µm2. All surfaces were a uniform black colour across the entirety of the sample due to

high anti-reflectiveness of the surface because of tapered Si pillars which render a gradual refractive index change, hence, reducing the reflectivity. The resultant nanotextured superhydrophilic bSi surfaces were further modified to convert them into superhydrophobic surfaces. A chemical vapour deposition method of placing a monolayer of trichloro(1H, 1H, 2H, 2H-perfluorooctyl) silane (PFTS) on bSi surfaces was followed according to the description outlined by Glass et al 22. Hydrophilic bSi surfaces were first exposed to 1 minute of O2 plasma to clean organic contaminants from the surface and to generate hydroxyl (Si-OH) surface groups. Then the substrata were placed in a glass Petri dish inside a desiccator at room temperature and 100 µL of PFTS was placed beside the bSi substrata. The desiccator was placed under vacuum and left for 1 h. After venting the desiccator, the samples were rinsed twice with chloroform, twice with ethanol and then dried with gentle nitrogen gas flow. XPS analysis of the surface showed the presence of C-F3 and C-F2 bonds typical of those found in fluorocarbon coatings such as PFTS (Figure S2). Wettability analysis of the two types of bSi surfaces revealed a significant difference between bSi and silane-coated bSi. Silanised bSi samples all achieved a water droplet contact angle of ~160 º or more, making them superhydrophobic 23. The bSi surfaces have often been reported to be highly hydrophobic 12, 24 due to both a low surface energy material and sufficient 5 ACS Paragon Plus Environment


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surface roughness. However, the bSi surfaces fabricated in this study achieved a superhydrophilic state due to the particular pyramidal shape of the nanostructures. For these surface structures, where the surface is randomly roughened resulting in non-vertical alignment of features to the substratum surface, the inclination of the side-walls plays an important part in determining the hydrophilicity/hydrophobicity of the surface. With significant side-wall inclination, there is a reduction in the Laplace pressure (the capillary difference pressure across the air/water interface and which confines the water droplet meniscus at this point due to surface tension) 25, sufficient enough that there is no air entrapment between features, resulting in complete wetting of the surface. Silanization of the surface using a vapour-phase deposition of PFTS introduces a selfassembled monolayer, which changes the surface chemistry by lowering the surface free energy. No visible layer was apparent under SEM of PFTS-coated bSi samples, causing no notable change in surface topography. Previous experimental and theoretical results have estimated the thickness of PFTS films deposited by a vapour phase method as approximately 1.6 nm 26 . Thus, hydrophilic and hydrophobic bSi samples, and an as-received silicon wafer and PFTS coated silicon wafer used as control surfaces, were employed in this study. All samples were rinsed once with ethanol and three times with deionised water and placed in a sterile 24-well plate (In Vitro Technologies). To assess the antibacterial effect of these surfaces, the samples were submerged in 1 mL of bacterial suspension. Both hydrophilic and hydrophobic bSi samples were incubated with bacteria (Staphylococcus aureus and Pseudomonas aeruginosa) for 24 hr at 25 °C under dark and static conditions. This eliminates any optical absorption effects which may impact on the killing efficiency of the surfaces. Confocal laser scanning microscopy (CLSM) imaging was performed using a Fluoview FV10i inverted microscope (Olympus, Tokyo, Japan) at ×100K magnification to visualise live and dead cells attached 6 ACS Paragon Plus Environment

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onto bSi and control surfaces. Prior to CLSM, samples were removed from bacterial suspensions and washed gently three times with Milli Q water. Surfaces were then stained for 25 min in the dark using LIVE/DEAD Baclight Bacterial Viability Kit, L7012 (Molecular Probes, Life Technologies). Staining of EPS was carried out using Alexa Fluor Concanavalin A 633 conjugate, followed by staining as above with LIVE/DEAD Baclight (Molecular Probes, Invitrogen). To assess cell morphology, the samples were viewed under SEM after sputter coating with gold for 2 min using a NeoCoater MP-19020NCTR. At least two technical replicates were performed for each experiment. Images were captured over a 76 µm2 field of view, assessing at least 10 different areas of the sample. MatLab image processing software, CellC, was used to count numbers of total and specifically stained cells. The observed attachment behaviour of two studied bacterial strains on superhydrophilic versus superhydrophobic bSi surfaces is in agreement with previously published reports 12

.The attachment of bacterial cells to substratum can be regulated by cell surface

hydrophobicity, as well as Brownian motion, and Van der Waals interactions 27. Most bacterial species have an enhanced ability to colonise abiotic and biotic surfaces through an extracellular capsule, which is composed of acidic polysaccharides that cause a significant increase in cell surface hydrophobicity 28-29. Cells with higher hydrophobicity of their outer layer will adhere in a greater degree to hydrophobic surfaces 16, 30-31. Cell surface characteristics for both P. aeruginosa and S. aureus used in this study can be found in Table S1. Here, silanization of superhydrophilic bSi to create superhydrophobic bSi resulted in an increase in bacterial cell attachment over an incubation period of 24 h. This increase in attachment to hydrophobic bSi surfaces was most obvious for P. aeruginosa as seen in both CLSM and SEM imaging (Figure 2 and 3). Here it was also found that an increase of the surface feature height resulted in a significant reduction in cell adhesion, compared to the non-modified substrata, for both Gram-positive and Gram-negative bacterial species.

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Superhydrophilic

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475 nm

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Figure 2. Attachment patterns of P. aeruginosa and S. aureus bacterial cells and bactericidal efficiency of superhydrophobic and superhydrophilic black silicon surfaces. Representative SEM and CLSM images of P. aeruginosa (left-hand side panels) and S. aureus (right-hand side panels) showing the loss of cellular morphology as a result of contacting with the nanostructured surfaces. Non-viable cells are fluoresced red. Hydrophilic bSi surfaces show low attachment propensity for both P. aeruginosa and S. aureus bacteria. Main image scale bar is 1 µm, inset scale bar is 100 nm.


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Attachment of P. aeruginosa cells to the nanostructured surfaces was dependent on height and as the height increases, so does the inability of the bacterial cell to attach onto the surface. There was a significant reduction in numbers of attached P. aeruginosa cells on the bSi surface with the tallest nanopillars (610 nm) compared to smaller structures with reduced feature size. The number of attached S. aureus cells increased slightly on the tallest nanopillar surface as seen in Figure 3. For the smallest pillar height (212 nm) there was a 34- fold increase in the number of total attached cells (3.8 × 103 – 1.03 × 105). For 475 nm, there was a 56-fold increase (2.79 × 103 – 1.58 × 105) and for 610 nm, a 3-fold increase in attached cell numbers (4.63 × 103 – 15.71 × 103 cells per mm2) was observed. S. aureus also possesses significant cell wall hydrophobicity (SI, Table S1) 32 and a small increase in attachment numbers was observed (212 nm: 1.25 × 103 - 1.51 × 103 cells per mm2), (475nm: 1.5 × 103 – 2.7 × 103 cells per mm2), and (610 nm: 0.8 × 103 – 3.4 × 103 cells per mm2). Changes in cell adhesion to bSi and silane-coated bSi may be attributed only to changes in surface chemistry rather than changes to physical surface characteristics, due to the bSi topography being preserved despite the addition of a self-assembled monolayer. Indeed, it was reported previously that nanostructured surfaces were less desirable for bacterial colonization due to a decrease in the contact area between the bacterial cell and the substratum 21, 33. It was also reported that nanoscale topography affects the attachment behaviour of bacterial cells in terms of their orientation, their expression of attachment organelles (fimbriae), and preference for the substratum 34. For example, rates of adhesion and bacterial killing have been attributed to surface nanoarchitecture structural dimensions and material stiffness 18, 21.



Staphylococcus aureus Cell attachment ×103

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Figure 3. The antibacterial effects of hydrophobic and hydrophilic bSi surfaces of varying surface geometries. Control surfaces were silicon wafer coated with PFTS and as-received silicon wafer. The graphs showing bacterial cell density plot both total cell attachment of live and dead cells and just specifically stained (dead) cells reveal that most attached cells for both P. aeruginosa and S. aureus are non-viable. ** P < 0.01. There is no statistical significance in the differences in the percentage of non-viable cells for S. aureus (P > 0.05). Error bars are standard deviation values. The addition of a self-assembled monolayer of silane to bSi surfaces, however, did not seem to increase the bactericidal efficacy of the hydrophilic surfaces to a significant extent, resulting in the confirmation that, although the attachment patterns changed dramatically, surface hydrophobicity did not impact upon the mechanobactericidal effect. The most active bactericidal pattern was the bSi substratum with 212 nm nanopillars (Figure 3). Hydrophilic


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bSi achieved approximately 84% killing efficiency against P. aeruginosa and silane-coated 212 nm bSi substrate (WCA ~160 º) achieved 98% inactivation of cells. Similar killing efficiencies were observed for S. aureus (approximately 90% bSi and approximately 97% bSi + PFTS), the difference was found to be statistically insignificant (P > 0.05). Therefore, the adhesion affinity of bacterial cells to a nanostructured surface did not play a pivotal part in in the mechanistic killing of bacterial cells. In this study, EPS staining using Concanavalin A 633 conjugate did not show production of extracellular matrix, which would indicate the formation of a biofilm, regardless of a high number of cells observed on the surface (Figure S3). This finding indicated that bacteria are dying within a short time period as they come into contact with the surface by means of mechanical stress placed upon the membrane and this process does not allow any time to secrete EPS. In fact it was shown that bacterial rupturing upon contact with cicada wing surfaces takes between 3 to 5 minutes 12. The killing mechanism of nanopillared surfaces has, in recent years, been understood to be a purely biophysical action involving the adherence of the bacterial cell to the nanostructured surface, stretching of the membrane in the areas suspended between attachment points and finally rupturing of the membrane as mechanical stress overcomes the elasticity of the membrane 35. The in-depth biophysical model developed for mechanobactericidal action of cicada wings proposed that, if the cell rigidity is decreased, or the initial cell membrane stretching is increased, cells that were initially resistant to mechanical killing by the natural nanopillared surface would be susceptible to the mechanobactericidal action of the wings 38. The bactericidal efficacy of topographies found in nature have since been optimised with the fabrication of synthetic analogues where surface geometries have been manipulated to increase killing efficiency for bacterium with a greater stretching modulus, i.e., Gram positive bacteria and spores 12, 17. Here, the surface geometrical parameters have been adjusted so that 11 ACS Paragon Plus Environment


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cells possessing greater natural rigidity, i.e., S. aureus, are also susceptible to mechanical cell rupturing via interaction with nanopillared surfaces. Both bSi surfaces and dragonfly wings have been shown to possess comparative randomness in size, shape and spatial distribution12 (Figure S4). In comparison to the dragonfly wing pillar clusters, which possess a sigmoidal population distribution within 90 nm, bSi surfaces exhibit a multi-modal pillar cluster distribution pattern (with a broader spatial distribution), with inter-pillar spacing increasing as the pillars increase in size and clustering decreases accordingly. Dragonfly pillars have been observed to be approximately 240 nm in height, although both short and tall nanopillars are evident. The bSi pillars in this study range from 212 nm – 610 nm in height. Previously, both the dragonfly wing nanoarchitecture and bSi surfaces have been found to be effective against Gram-negative, Gram-positive bacteria and

Bacillus subtilis spores and additionally, the killing efficiency of both these surfaces were found to be comparable over a 3 h period for both P. aeruginosa and S. aureus cells12. Therefore, black silicon is a successful synthetic analogue of dragonfly wing surfaces. Recently it has been proposed that cell-surface affinity plays a role in increasing the bactericidal efficiency of nanostructured surfaces and that the killing efficiency is dependent on the adhesion strength of the cell to the surface 36. Additionally, Bandara et al has proposed an alternative killing mechanism for the nanostructured surfaces of dragonfly wings whereby

E. coli cells are adhered strongly to the dragonfly wing surfaces via production of EPS. As the result of such high affinity, membrane damage and loss of cellular morphology was proposed to be due to the tearing of the bacterial membrane as the motile cell attempts to manoeuvre on the nanostructured substratum 20. FIB milling of S. aureus and P. aeruginosa cells incubated for 30 m on both hydrophilic and hydrophobic bSi surfaces was used to reveal the initial stretching of the bacterial membrane. Samples were fixed with 2.5% glutaraldehyde and subjected to ethanol dehydration prior to 12 ACS Paragon Plus Environment

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FIB milling. An FEI Nanolab 200 dual-beam FIB system was used for cross-sectioning of bacterial cells. Before FIB milling the bSi surfaces incubated with bacteria were coated with a Pt protection layer using an e-beam Pt deposition process. Figure 4 shows the interface between the nanopillars and the cell, stretching the membrane so it envelopes the nanopillar, but not piercing the cell. This event is most obvious for S. aureus but is also evident for P.

aeruginosa and E. coli cells (Figure 4, Figure S5). FIB milling of both bacterial species attached onto hydrophobic and hydrophilic bSi surfaces revealed similar occurrences where the nanopillar contact points with the membrane are clearly observed to be stretching the membrane inwards. These findings contrast with the suggested alternative killing mechanism



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a)a)

b)

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Figure 4. SEM images of focused ion beam milling of P. aeruginosa and S. aureus attached onto bSi surface. (a) P. aeruginosa hydrophilic, (b) P. aeruginosa hydrophobic, (c) S. aureus hydrophilic and (d) S. aureus hydrophobic. Milling was completed following 30 m of incubation on the black silicon surfaces revealing the initial stretching of the membrane as the nanospike needles contact with the cell. Scale bars are 400 nm.

that nanotextured surface-induced damage occurs without direct contact between the nanopillars and the bacterial cell membrane 20. Therefore, the results of this study are in agreement with previously reported mechanisms of bactericidal activity 35, 37-38, and provide new evidence that high rates of cell death on mechanobactericidal surfaces could not be achieved before the cells are adhered to the surface by means of enhanced secretion of extracellular polymeric substances. In conclusion, we have designed and fabricated superhydrophobic and superhydrophilic bSi surfaces with varying spatial geometries, and heights. All types of the nanopatterned surfaces exhibited similar killing efficiencies against P. aeruginosa and S. aureus bacterial cells. It 14 ACS Paragon Plus Environment

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was found that cellular affinity for a surface or motility of bacterial cells does not determine the bactericidal efficacy of the surface and that EPS does not play a role in the mechanobactericidal action of nanopillared surfaces. ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Fabrication and characterization of black silicon surfaces, XPS analysis, bacterial strains growth condition and cell surface characterizations, characterization of EPS production using CLSM, comparative characterization of black silicon and dragonfly wing surfaces, FIB/SEM of E.coli on black silicon surfaces (PDF). AUTHOR INFORMATION

Corresponding author Email: [email protected]

ORCID Elena Ivanova: 0000-0002-5509-8071

Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS The authors would like to acknowledge Bio21 Advanced Microscopy Facility for assistance with FIB milling. Denver Linklater is supported by a SUPRA scholarship.

REFERENCES



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