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Impact of Bioinspired Nanotopography on the Antibacterial and Antibiofilm Efficacy of Chitosan Abinash Tripathy, Suman Pahal, Rajeev J. Mudakavi, Ashok M Raichur, Manoj M. Varma, and Prosenjit Sen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00200 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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Impact of Bioinspired Nanotopography on the Antibacterial and Antibiofilm Efficacy of Chitosan Abinash Tripathy#§, Suman Pahal#§, Rajeev J. Mudakavi#‡, Ashok M. Raichur‡, Manoj M. Varma§ and Prosenjit Sen*§ §

Centre for Nano Science and Engineering, Indian Institute of Science, Bangalore, 560012

‡

Department of Materials Engineering, Indian Institute of Science, Bangalore, 560012

Corresponding Author: [email protected]

Abstract Chitosan derived from chitin is one of the most abundant naturally occurring biocompatible polymers obtained from fungi and arthropods. In this work, we report the enhancement in the bactericidal efficacy of CHI in the presence of a sharp nanotopography. High-aspect ratio nanostructured surface (NSS) was fabricated using a single-step deep reactive ion etching technique (DRIE). Post fabrication, CHI coating was carried out using a layer-by-layer (LBL) dip coating process on the flat and nanostructured surfaces. Antibacterial efficacy of the flat silicon surface coated with CHI (Si_CHI) and NSS coated with CHI (NSS_CHI) was tested against both Gram-negative (G-ve) bacteria E. coli and Gram-positive (G+ve) bacteria S. aureus. NSS_CHI exhibited superior antibacterial property against G-ve and G+ve microbes as compared to Si_CHI and NSS substrates. Scanning electron microscopy (SEM) and fluorescence microscopy were used to study the morphology and viability of the bacteria on all the surfaces. Also, biofilm quantification was carried out on all the engineered surfaces for both E. coli and S. aureus using crystal violet (CV) staining. NSS_CHI was found to have the

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minimum biofilm formation on its surface exhibiting its superior antibacterial property. This study shows that the antibacterial and antibiofilm efficiency of CHI can be augmented by combining it with a sharp nanotopography. Keywords: Chitosan, DRIE, antibacterial, NSS, NSS_CHI, nanotopography

1. Introduction Antibacterial resistance (AMR) has become a global threat to the present world1,2. AMR infections currently claim ~0.7 million lives every year and researchers are predicting this figure will reach 10 million by 2050 if not stopped3. The emergence of antimicrobial resistance has been attributed to the misuse of antibiotics and the rapid evolution of drug resistant bacteria4,5. In nature, bacteria proliferate and colonise on surfaces where nutrients are available. The colonization of bacteria on such surfaces and interfaces results in biofilm formation6–8. Biofilm mode of growth allows them to proliferate even in hostile environment such as low pH, high chlorine content, environment with thermal variations and exposure to antibiotics9. It is therefore wise to design surfaces with antibacterial properties by inhibiting initial bacterial attachment and growth to stop biofilm formation. Different parameters such as surface chemistry10–17, surface topography18–23, other physicochemical properties of the surface24–29 etc. play crucial role in the antibacterial/anti-biofouling performance of the surface. Due to the emergence of antimicrobial resistance, antibiotics have become less effective in eliminating bacteria. Apart from antibiotics certain biopolymers such as chitosan also exhibit antibacterial property. There have been several studies reporting the antibacterial performance of CHI against different kinds of bacteria30–34. Chitosan’s biocompatible35 property is very desirable in designing antibacterial surfaces for applications like water purification, biomedical devices, food packaging industry etc.


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Contrary to bacterial killing using antibiotics or surface chemistry, recent mechanisms exploring physico-mechanical concept of killing have gained attention. One of the first studies by Ivanova et al. showed the bactericidal behaviour of Cicada wing against P. aeruginosa by physical killing mechanism36. Cicada wing is composed of nanostructures on its wing surface. When the bacterium encounters such nanostructured surface, it gets stretched and deformed as it tries to settle on such rough surface and during this action cell lysis takes place. Following the same line of work there have been many studies on killing bacteria using the physico-mechanical technique where bacteria are killed by such sharp nanostructures/nanospear by physically rupturing the cell wall37–50. In this work we have studied the impact of sharp nanotopography on the bactericidal efficacy of CHI against G-ve and G+ve bacteria. Also, biofilm quantification has been carried out to examine the efficiency of CHI coated nanostructured surface to inhibit biofilm formation. SEM, fluorescence microscopy and CV staining were used to study the bacterial morphology, viability and biofilm quantification respectively.

2. Materials and Methods 2.1 NSS Fabrication orientation silicon (Si) wafers (purchased from SVM Technology, USA) were used as base substrates for fabricating the nanostructures. Silicon wafers were cleaned in piranha solution (H2SO4 and H2O2 (3:1)) for 10 minutes at 90°C, followed by a dip in HF for 30 seconds. The wafers were rinsed in deionized water, dried using N2 purge and then baked on a hot plate at 250°C for 10 minutes. An optimized deep reactive ion etching (DRIE) recipe51 (Modified Bosch Process) was used to etch the silicon substrate. Etching and passivation steps were carried out for 30 cycles (6:43 minutes) to get the high aspect ratio nanostructured



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surface (NSS). Octafluorocyclobutane (C4F8) gas was used for passivation and for etching Sulphur hexafluoride (SF6) and oxygen (O2) gases were used.

2.2 Antibacterial Coating Materials Polymers Polyethylenimine (PEI, Molecular Weight (MW) ≈ 25 kDa), hyaluronic acid sodium salt (HA, from Streptococcus equi sp, MW ~ 1.5-1.8 x 106 Da), chitosan (CHI, MW ≈ 150 kDa), were purchased from Sigma-Aldrich. Sodium chloride (NaCl), sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from Merck. Glacial acetic acid ((CH3COOH) was purchased from Rankem, RFLC Limited (Bangalore, India). All solutions were prepared by using ultrapure water from Millipore with a resistivity of 18.2 MΩ. All chemicals were used without any further purification.

2.3 Polyelectrolyte solutions Polyelectrolyte solutions of PEI and HA were prepared by dissolving the respective polymer in Milli-Q water at concentrations of 1% (w/v) by using 0.15 M NaCl solution. CHI (1% (w/v)) was dissolved by drop-wise addition of 100 mM glacial acetic acid solution. All polyelectrolyte solutions were stirred for 12 hrs and maintained at pH 4 by using 0.1 M HCl and 0.1 M NaOH solutions.

2.4 Polyelectrolyte multilayer assembly Polyelectrolyte multilayer films were deposited on the flat and Nanostructured substrates by dip assisted layer-by-layer (LbL) self-assembly process, where multilayer assembly takes place due to electrostatic interactions. PEI is deposited on the Si and the NSS surfaces by dipping the surfaces for 30 min. PEI acts as an adhesion promoting layer. Then, HA and CHI were deposited by alternate dipping into solutions of polyanions and polycations. The dip time in each polyelectrolyte solution was 10 min, followed by three wash cycles in water with


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agitation and drying in pure nitrogen. Two bilayers (BL) of HA/CHI were deposited by repeating the LbL cycle.

2.5 Contact Angle Measurement Contact angle measurement on all the fabricated surfaces was carried out using a custommade Goniometer setup. For this, 8 µL water droplet was placed gently on the substrate and image of the droplet was captured by a CMOS camera (Thor Labs). ImageJ©52 software was used to measure the contact angle from the captured images. To ensure reproducibility, all the measurements were repeated three times.

2.6 Scanning Electron Microscopy (SEM) High-resolution images of all the substrates used in our study were captured at different magnifications using a Carl-Zeiss field emission SEM (FE-SEM). Bacterial samples were first fixed with 2.5% of glutaraldehyde in phosphate buffered saline (PBS) followed by dehydration in serially diluted Ethanol (40%, 50%, 60%, 70%, 80%, 90% and 100%). Next, the substrates were dried in room temperature and then kept in vacuum prior to doing SEM. Quorum sputter coater was used to coat a thin layer of gold (15 nm) on the samples to avoid sample charging during SEM.

2.7 Atomic Force Microscopy (AFM) Thickness measurements of HA/CHI coatings were carried at the step edge using Bruker AFM in tapping mode. The imaging was performed by scanning an area of 10 µm X 10 µm with TESPA- V2 tip having a resonant frequency of ∼320 kHz and spring constant of ∼42N/m. Tapping mode was chosen to achieve high resolution data.

2.8 Fluorescence Live/Dead Assay Sample substrates (Si, Si-CHI, NSS, NSS-CHI) in triplicates were placed in a 24 well plate and inoculated with E. coli and S. aureus for 6 hours in PBS. Another Si wafer sample treated



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with isopropyl alcohol for 15 minutes served as positive control. The Live/Dead staining was carried out using BacLight bacterial viability kit (L7012, ThermoFisher Inc., USA) and imaged using Fluorescence microscope (Olympus BX51M upright Fluorescence microscope with DP 71 CCD camera, magnification - 50X, exposure time – 200 ms). A mixture of SYTO 9 and propidium iodide (PI) fluorescent dyes (1:1) was used for staining. SYTO 9 binds to the nucleic acid by penetrating both intact and damaged cell wall membranes and fluoresces green when excited by 485 nm wavelength (λ). In contrary, PI enters cell wall which is damaged significantly (non-viable cells) and binds to the nucleic acid with higher affinity than SYTO 9. PI fluoresces red when excited by λ=535 nm.

2.9 Biofilm Quantification The sample wafers (Si, Si-CHI, NSS, NSS-CHI) in triplicates were placed in a 24 well plate and inoculated with E. coli and S. aureus in LB broth medium and incubated at 25°±2°C. After 4 days the plates were retrieved and stained with crystal violet (CV) solution for 10 minutes. The CV solution was completely removed, and the plates were washed thrice with PBS to remove excess stain and dried in air for 15 minutes. To the dried samples 2-3 mL of 70 % Ethanol solution was added and agitated lightly for 30 minutes. 100 µL of the ethanol solution was aliquoted from each sample well and placed into 96 well plate. The intensity of the blue coloured solution was read at 600 nm using the microplate reader (TECAN, infinite pro series). The quantity of biomass adhered to the surface is directly proportional to the intensity of the colour developed. Similarly, samples without CV staining were processed for biofilm SEM imaging.


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3. Results and Discussion 3.1 Fabricated Nanostructured Surface Figure 1(a) shows the photograph of the fabricated nanostructured surface (NSS) on silicon. EDS analysis confirmed the presence of the elements on the NSS (Figure 1(b)). The nanostructures possess random size (average nanoposts diameter ~350 ) and spatial distribution (average spacing between nanostructures ~300 800 ) similar to a dragon fly wing37 (Figure 1(c)&(d), Figure S1(a),(b)&(c)). Height of the nanostructures formed are ~8 (aspect ratio ~1: 23) (Figure S1(d)).

Figure 1: Details of NSS surface. (a) photograph of NSS. (b) EDS spectra of the NSS surface showing the elements present on the surface. (c) SEM and (d) AFM images of the NSS surface showing the random nanostructures.

3.2 HA/CHI Bi-layer on Si and NSS Coating of HA/CHI was carried using simple LBL assisted dip coating procedure as explained in materials and methods section. SEM (Figure S2(b)&(d)), EDS (Figure



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S2(b)&(d)) and AFM (Figure S3) confirmed the coating of the CHI layer on the flat and the NSS. There was no morphological change of the nanostructures before and after the CHI coating. The thickness of the HA/CHI bi-layer was measured to be ~13.7 (Figure S4). To test the durability of the CHI film, flat Si_CHI was kept in PBS for 2 days. Post 2 days of dipping, thickness of the CHI layer was measured using AFM. There was no decrease in the thickness (post exposure thickness ~15.1 ) of the CHI layer showing its stability against a long exposure to wet medium (Figure S5).

Figure 2: Process flow for HA/CHI bilayer coating on flat and nanostructured silicon surfaces.

3.2 Bacterial Morphology SEM was used to observe the morphology of the bacteria on the flat and nanostructured surfaces with and without chitosan coating. Figure 3 shows the SEM images of the G-ve and G+ve bacteria on the Si and Si_CHI surfaces. Both E. coli and S. aureus cell walls look 8 ACS Paragon Plus Environment

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smooth and cells appear healthy on the Si surface by retaining their rod and spherical shapes respectively (Figure 3(a)&(c). In contrary, the cell wall morphology of both the G-ve and G+ve microbes appear compromised and visibly rough on the Si_CHI surface. Also, the exact shapes were not retained by both the bacteria. This can be attributed to the presence of the CHI coating which initiates an electrostatic interaction between the NH3+ ion of CHI with the phosphoryl group present in the phospholipid components of the cell membrane. When this interaction crosses a threshold, cell wall is damaged and the cellular contents are released leading to cell lysis53.

Figure 3: Representative FESEM images of E. coli and S. aureus on the Si and Si_CHI surfaces. Bacterial cell wall looks smooth on the flat silicon surface, while the cell wall looks rough on the chitosan coated silicon samples for both the bacteria. Figure 4 shows the morphology of E. coli and S. aureus on the NSS and NSS_CHI surfaces. As observed from the SEM images, E. coli cells try to anchor to multiple nanostructures to 9 ACS Paragon Plus Environment


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adhere on the available surface and in doing so the cell wall gets stretched and when the threshold is reached, cell lysis takes place. E. coli cell wall was stretched and deformed on the NSS and NSS_CHI surfaces as observed in the SEM images (Figure 4(a)&(b)). The degree of stretching was found to be more on the NSS as compared to the NSS_CHI (see Figure S7 and Table S1). This can be because of the presence of the additional CHI layer where the microbes are killed at a reduced degree of stretching. On the contrary S. aureus cells were found to settle on top and in between the nanostructures on both the NSS and NSS_CHI surfaces (Figure 4(c)&(d)). This is due to the spherical shape and smaller size (~0.5 μm) of the Gram-positive microbe which allows it to settle in between the nanostructures which was not observed in case of E. coli. At some places a change in the morphology of S. aureus was observed on the NSS and NSS_CHI surfaces. There was a transition from the perfect spherical shape to elliptical shape due to trapping of the G+ve cells in between the nanostructures (Figure 4(c)&(d)). However, the bacteria on top of the nanostructures retained the spherical shape.


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Figure 4: FESEM images of Gram-negative E. coli and Gram-positive S. aureus on the NSS and NSS_CHI surfaces.

3.3 Surface Bactericidal Activity using Fluorescent Staining To examine the bactericidal efficacy of the fabricated surfaces, fluorescent staining was performed, and images were captured using a fluorescent microscope after 6 hours of incubation. Most of the cells (both G-ve and G+ve) on the flat silicon surface appeared green which was used as control in our experiments (Figure 5(a)&(b)). A mixture of red and green cells was observed on the Si_CHI surface for both E. coli and S. aureus confirming the reported antibacterial nature of CHI coating (Figure 5(a)&(b)). NSS and NSS_CHI surfaces were found to kill E. coli very efficiently as seen in Figure 5(a). However, in case of S. aureus few green cells were observed on the NSS. The unstretched S. aureus cells between the nanostructures could be the reason for these green signals (see Figure 4(c)). All the S.



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aureus cells were found dead on the NSS_CHI surface showing its superior bactericidal efficiency over the Si, Si_CHI and NSS surfaces. NSS surface is not able to kill the S. aureus bacteria trapped between the nanostructures as the bacteria does not interact with the sharp tips of the nanostructures. However, in case of the NSS_CHI surface the additional CHI layer on the nanostructures interacts with those trapped bacteria and eventually kills them.

Figure 5: Fluorescence microscopy images of E. coli and S. aureus attached for 6 hours on the Si, Si_CHI, NSS and NSS_CHI surfaces (Scale bar - 20 µm). To quantify the number of viable bacteria on each surface, live cell counting was performed. ImageJ52 was used to count the number of live bacteria for all the substrates from the fluorescent images. Only live cells counting was performed as it was difficult to distinguish between the dead cells on the nanostructured surface due to stretching and merging of the bacterial cell wall. At least 20 images for each case were scanned and cells were counted and plotted (Figure 6(a)&(b)). The scanning area for each image was 220 X 170 µm2. For E. coli there was clear statistical significance in the number of live bacteria between the Si & NSS, Si & NSS_CHI and Si_CHI & NSS_CHI pairs. However, no statistical significance was found between the NSS & NSS_CHI pair. Similarly, for S. aureus, statistical significance was observed between the Si & NSS, Si & NSS_CHI and Si_CHI & NSS_CHI pairs in the 12 ACS Paragon Plus Environment

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enumeration of the live cells. In contrary to E. coli, a statistical significance was also observed between the NSS & NSS_CHI surfaces for S. aureus in terms of the number of live bacteria on the surface. S. aureus cells which are present on top of the nanostructures in case of NSS come under higher stress at the sharp contact points and the sharp nanotips cause a localised high stress to the cell membrane leading the cell lysis37. However, the NSS cannot kill the S. aureus cells which are trapped in between the nanostructures. This is not true in case of the NSS_CHI surface as the additional CHI layer coating on the nanostructures interact with the trapped S. aureus cells in between the nanostructures and kill them.

Figure 6: Quantification of live cells on all the engineered surfaces using Live/Dead kit. ImageJ was used to calculate the number of live cells in each image [area of each image (220 X 170) µm2]. At least 20 images were used for quantification for each surface. Statistical significance was performed using Student’s t-test [∗∗ 0.01,∗∗∗ 0.001,∗∗∗∗ 0.0001].

3.4 Biofilm Quantification Bacterial biofilm is a community of bacteria in which the microbes are embedded in an extracellular polymeric substances (EPS)9. The main challenge in designing any antibacterial surface is to prevent/minimize the formation of the bacterial biofilm. Crystal violet (CV) stain was used to quantify the bacterial biofilm on all the engineered surfaces for E. coli and S. aureus. The bacterial biofilm mass was quantified by measuring the intensity of the blue 13 ACS Paragon Plus Environment


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colour of CV stain retained on the biofilm using the microplate reader. Figure 7(a)&(c) show the intensity of the blue colour of the stained CV obtained from all the fabricated surfaces expressed in percentage for the E. coli and S. aureus biofilms respectively (see Figure S8 for intensity of the blue colour of the stained CV obtained from all the fabricated surfaces expressed in terms of optical density (O.D.) at 600 nm). NSS_CHI was found to have the minimum biofilm formation for both the G-ve and G+ve bacteria (Figure 7(a)&(c)). SEM images of the bacterial biofilm of E. coli and S. aureus on the NSS_CHI surface has been shown in Figure 7(b)&(d) and Figure S9. The presence of both the nanostructures and CHI coating helps to prohibit the formation of the biofilms more effectively as compared other test surfaces (Si, Si_CHI & NSS) used in our study.

Figure 7: Bio-film quantification of (a) E. coli and (c) S. aureus on all the engineered surfaces. Representative SEM images of the (b) G-ve and (d) G+ve biofilms on the NSS_CHI substrates.


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4. Conclusion In this study impact of nanotopography on the bactericidal efficacy of CHI has been studied. CHI was found to have enhanced bactericidal performance in the presence of sharp nanotopography against both G-ve and G+ve bacteria. The NSS_CHI surface is also found to be the more efficient in preventing the formation of bacterial biofilm. For E. coli, NSS_CHI was found to have 25 %, 9 % and 1% less biomass on its surface as compared to Si, Si_CHI and NSS respectively. For S. aureus, NSS_CHI was found to have 38 %, 16 % and 10 % less biomass on its surface as compared to Si, Si_CHI and NSS respectively. This concept can be further used to design structured surfaces with CHI coating for several practical applications such as water purification, food packaging, biomedical devices etc.

Associated Content Supplementary Information FESEM images of bacteria, ImageJ analysis to find size distribution, CHI thickness measurements using AFM, EDAX spectra, surface area of E. coli, wettability of the engineered surfaces, SEM images of bacterial biofilms. (PDF) Video showing the live bacteria on the Si_CHI and NSS_CHI surfaces. (AVI)

Author Information Corresponding Author Prosenjit Sen, [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given # approval to the final version of the manuscript. AT, SP and RM contributed equally.



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ORCID Abinash Tripathy – 0000-0003-3546-2806 Suman Pahal – 0000-0002-8135-0402 Rajeev M. Mudakavi – 0000-0001-8686-8561 Ashok M. Raichur – 0000-0001-5042-3122 Prosenjit Sen – 0000-0001-6519-1707

Conflict of Interest The authors declare no competing financial interest.

Acknowledgement AT would like to acknowledge the financial support from Meity, Government of India. Authors would also like to thank Department of Science and Technology (DST), Government of India for funding the research work.

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