Understanding the Role of Polymer Surface Nanoscale Topography

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Understanding the Role of Polymer Surface Nanoscale Topography on Inhibiting Bacteria Adhesion and Growth Luting Liu, Linlin Sun, Batur Ercan, Katherine S. Ziemer, and Thomas J. Webster ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00431 • Publication Date (Web): 19 Nov 2015 Downloaded from http://pubs.acs.org on November 24, 2015

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ACS Biomaterials Science & Engineering

Understanding the Role of Polymer Surface Nanoscale Topography on Inhibiting Bacteria Adhesion and Growth Luting Liu1, Batur Ercan1, 2, Linlin Sun3, 5, Katherine S. Ziemer1 and Thomas J. Webster1, 4, 5* 1

2

Department of Chemical Engineering, Northeastern University, Boston, MA, 02115, USA

Department of Metallurgical and Materials Engineering, Middle East Technical University, Ankara, 06800, Turkey 3

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Department of Bioengineering, Northeastern University, Boston, MA, 02115, USA

Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, 21589, Saudi Arabia

5

Wenzhou Institute of Biomaterials and Engineering, Wenzhou Medical University, Wenzhou, China

*Address correspondence to [email protected]

ABSTRACT: Catheter-associated infections, most of which are caused by microbial biofilms, are still a

serious issue in healthcare and are associated with significant morbidity, mortality, and excessive medical costs. Currently, the use of nanostructured materials, especially materials with nanofeatured topographies, which have more surface area, altered surface energy, enhanced select protein adsorption, and selectively increased desirable cell functions while simultaneously decreasing competitive cell functions, seem to be among the most promising ways for reducing initial bacteria attachment, biofilm formation and infections. In this study, polydimethylsiloxane (PDMS), a commonly used polymeric catheter material, was formulated to mimic the nano-patterned topography of natural tissue by using a template method with nanotubular anodized titanium. Results showed that increased PDMS surface nanoscale roughness alone can inhibit both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria adhesion and growth for up to two days, the time length of the current study. Additionally, increased fibroblast and endothelial cell adhesion on nano-PDMS indicated that this ACS Paragon Plus Environment

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nanoscale topography had no toxic effects towards mammalian cells. Mechanistically, this study also developed a model for the first time to correlate bacteria responses to nanoscale roughness with initial protein and biomolecule adsorption (specifically, casein protein and glucose, which are unique biomolecules that mediate bacteria functions). Data revealed that the increase in nanoscale roughness and associated energy contributed to greater select casein adsorption during the first several minutes of culture, which is critical for decreasing bacteria attachment and growth. In contrast, no significant differences for glucose adsorption between samples before and after nanofabrication were identified. These results together indicated that the present biomimetic nano-patterned PDMS surface without any chemical or antibiotic modification has the potential to combat catheter-associated infections and should be further investigated. KEYWORDS: catheter-associated infections, nanotechnology, cell adhesion, antibacterial, protein adsorption INTRODUCTION The insertion or implantation of foreign materials has been widely used for biomedical applications, however, infections associated with medical devices are becoming the leading causes of preventable deaths in the hospital and result in a substantial increase in healthcare costs every year.1 Among these, catheter-associated infections account for a large proportion of these infections.2 For example, approximately 2/3 of infections occur in three main areas of the body: the urinary tract, the blood stream, and the respiratory tract, while almost all of the infections follow catheterization. Intravascular and urinary catheters are two of the most common sources of infections, due to their extensive use in the hospital.3-5 According to the US CDC, more than 30 million urinary catheters are inserted every year with a 10%-30% rate of infection, of particular concern is up to 5% of these infections result in death. In addition, an estimated 80, 000 catheter-associated bloodstream infections are caused by central venous catheters at a cost of $25, 000 per incident annually.2 The longer a catheter is left in place, the greater the patient’s risk of infection. Microorganisms such as the Staphylococcus ACS Paragon Plus Environment

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species, Gram-positive bacteria, are the leading causes of vascular catheter-associated bloodstream infections.4 On the other hand, Gram-negative bacteria, such as Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), have been identified as the predominant pathogens responsible for urinary tract infections.5 Microbial biofilms, which are often formed by antimicrobial-resistant organisms, are responsible for most of these infections.6, 7 However, biofilms are resistant to host defense mechanisms and antibiotic agents, making the treatment of catheter-associated infections more challenging.

Thus, to substantially reduce the extent of bacterial colonization on

catheter surfaces and related infections, the development of effective strategies is urgently needed here. The application of surface coatings is one of the most frequently used methods to implement antibacterial properties to combat catheter-associated infections.2,

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For example, coating catheter

surfaces with antimicrobial agents, such as silver, has been exploited and more recently, silver and silver ions have been used in medical devices since they demonstrate antimicrobial activity at low concentrations.11,

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Despite proving to be efficacious at reducing catheter microbial colonization,

antibiotic-coated catheters have been associated with some considerable drawbacks, such as the possibility of the emergence of drug-resistant pathogens, toxicity to mammalian cells, and delamination.13 Most recently, another proposed method to decrease infection is the use of nanotechnology, especially nanomaterials or creating a nanofeatured topography on the surface of catheters to reduce initial bacteria attachment.14 Many researchers have confirmed that nanofeatured surface topographies are a potent tool for selectively promoting desirable cell functions while simultaneously decreasing competitive cell functions.15-17 For example, Wu et al. found that both the vertical and lateral surface feature dimensions could be modified to not only optimize bone-implant interactions but also to simultaneously minimize implant-bacteria interactions by using four clinically relevant titanium surface finishes: polished, satin, grit-blasted and plasma-sprayed.18 Also, natural surfaces, such as nano-patterned cicada wing surfaces, appear to be bactericidal to P. aeruginosa, which was thought to ACS Paragon Plus Environment

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be due to the surface nanostructure of the wing rather than a chemical effect.10, 19 Yee’s group used industrial nanostructuring techniques (nanoimprint lithography) to generate biomimetic antibacterial nanostructures on the surfaces of poly(methylmethacrylate) (PMMA), a polymer commonly used in medical applications. They found that E. coli appeared to deflate and die as they stretch over nanopillars.20 Motivated by these findings, the aim of this work was to create an antibacterial nanostructure on the raw surface of a catheter composed of polydimethylsiloxane (PDMS) for the prevention of bacterial attachment and growth.21 One of the methods for polymer nanofabrication that has drawn a lot of attention recently is the template method, in which a material with a special structure is used as a template to imprint its structure onto another material.22-24 The template method with its simple fabrication procedure, low production cost and limited facility requirement, has found widespread application in the preparation of nanomaterials. Among materials that can be used as a template, anodized nanotubular titanium has attracted more attention because of its highly ordered nanotubular structures, low cost of manufacturing and easy preparation method.15,

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Furthermore, the diameter of such nanotubes can be easily

controlled by altering experimental conditions (such as electrolyte composition, electrolyte concentration, anodization time and working temperature). With this in mind, the present in vitro study aimed to modify the PDMS surface to possess nano-patterned structures by using a highly ordered anodized nanotubular titanium substrate as the template. The designed nano-patterned PDMS samples were tested for surface morphology, wettability and roughness, chemical composition, cytotoxicity, and antibacterial properties. To study the mechanism of these nanostructured features towards inhibiting bacteria adhesion and growth, the adsorption of biomolecules from tryptic soy broth (TSB) that influence bacteria functions (such as glucose and casein protein) were determined.27-29 MATERIALS AND METHODS Materials. ACS Paragon Plus Environment

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99.5% titanium (Ti) foils (0.25 mm thick, annealed) and platinum (Pt) meshes were purchased from Alfa Aesar. A Sylgard 184 silicone elastomer kit (Base and Curing agent) to produce PDMS was purchased from Dow Corning Co. A CSI3645A DC power supply was purchased from Circuit Specialists Inc. All the chemicals used were purchased from Sigma-Aldrich and Fisher Scientific. Sample Preparation. Ti samples were cut into 2.5 cm × 2.5 cm squares and were cleaned with acetone, 70% ethanol, and deionized water (Milli-Q water) separately, each for 15 minutes. Then, the cleaned samples were etched for 1 minute with a 1.5% by weight nitric acid (HNO3) and 1.5% by weight hydrofluoric acid (HF) solution to remove the naturally occurring oxide layer. The electrochemical anodization process, which consisted of a two-electrode configuration with a Pt mesh serving as the cathode and a Ti specimen as the anode, is schematically shown in Figure 1. One side of both the Ti and Pt samples was immersed in an electrolyte solution consisting of 1% HF, another side was connected to the DC power supply through copper wires for 10 minutes at voltages ranging from 5V to 20 V to obtain the optimized surface structure for the replica mold process. After anodization, Ti samples were rinsed with large amounts of deionized water immediately and dried in an oven at 100 °C for 30 minutes. The nano-patterned PDMS template was prepared using a replica mold process as shown in Figure 2. For this, the PDMS base and curing agent at a weight ratio of 10:1 were thoroughly mixed and stirred for 15 minutes and de-aired in a vacuum chamber for 1 hour to remove air bubbles in the mixture. The PDMS mixture was poured onto the nanotubular anodized Ti master and then was placed into the vacuum chamber for another hour to remove the bubbles between the patterned interface and the PDMS slurry. The fabricated PDMS together with the master was cured at 60 °C for 2 hours and then was allowed to cool down to room temperature. Afterwards, it was soaked in cold water for 10 minutes to easily peel the PDMS off from the Ti template. The plain PDMS substrates were prepared by easily casting the PDMS slurry on empty petri dishes following the same procedure as described above.

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For the cell and bacteria experiments, all the PDMS samples were cut into identical size pieces (1 cm × 1 cm) and were sterilized by ultrasonication in 70% ethanol followed by UV irradiation. Surface Characterization. Scanning electron microscopy (SEM). All the samples were thoroughly rinsed with deionized water and then dried at room temperature. The surface morphology of Ti before and after anodization, plain PDMS as well as nano-patterned PDMS was characterized by scanning electron microscopy (SEM, Hitachi S-4800). Before scanning the surface of PDMS samples under SEM, the PDMS samples were coated with 3 nm of a palladium layer using a sputter coater (Cressington Sputter Coater 208HR) to make them conductive. Atomic force microscopy (AFM). For surface roughness measurements, an atomic force microscope (Parks Scientific XE-7 AFM) was used to scan both Ti and PDMS samples. Each sample was analyzed in ambient air under non-contact mode using a silicone ultrasharp cantilever (probe tip radius of 10µm; MikroMasch); 1.25 µm × 1.25 µm AFM fields were analyzed and the scan rate was chosen as 0.4 Hz. Image analysis software (XEI) was used to generate micrographs and to quantitatively compare the root-mean-square roughness of the samples. At least three different spots on each sample were measured for statistical purposes (n=3). Contact angle analysis. Water contact angles were determined using a drop shape analysis system (SEO Phoenix 300) at room temperature. A 7 µL droplet of DI water was added to the sample surface by controlling injection. Contact angles were measured (5 seconds after being dropped on the surface) at four different positions for each test sample (n=4) at room temperature. Chemical analysis. For chemical analysis of the top surface layer of the PDMS samples, X-ray photoelectron spectroscopy (XPS) and peak curve fitting software (Escalab250, Thermo Electron) were employed. The XPS system consisted of a dual source, non-monochromated X-ray source (Phi model

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04-548) and a hemispherical analyzer (Phi model 10-360). The two X-ray options were MgKα (1253.6 eV) and Al Kα (1486.6 eV) operated at 300 W. Bacterial Assays. Microorganisms such as the Staphylococcus aureus (S. aureus), Gram-positive bacteria, are the leading causes of vascular catheter-associated bloodstream infections.4 On the other hand, Gram-negative bacteria, such as E. coli, P. aeruginosa, and Klebsiella pneumoniae have been identified as the predominant pathogens responsible for urinary tract infections.5 For bacterial experiments, bacteria cell lines of S. aureus and E. coli (ATCC 25923 and 25922, respectively) were used in this study as a model system. All sterile PDMS samples were transferred into 24-well non-tissue culture plates, treated at the prepared bacterial solutions with a concentration of 106 bacteria/mL in tryptic soy broth (TSB, 0.03 g/mL) and cultured for either 24 or 48 hours in an incubator (37 °C, humidified, 5% CO2). For those samples that were cultured for 48 hours, the medium was changed with 1 mL of sterile and fresh TSB after 24 hours. Afterwards, the samples were rinsed twice with phosphate-buffered saline (PBS) and were transferred into 15 mL tubes with 4 mL of PBS. Following this, the samples were sonicated for 10 minutes to release the bacteria attached on the sample surface into the solution. Then, a series of diluted solutions with bacteria were spread onto agar plates (100 µL/agar) and bacteria colonies were counted after 18 hours of incubation. Cell Adhesion Assays. Human fibroblasts (ATCC, CCL-110) and endothelial cells (Life Technologies) at population numbers less than ten were used for all cell experiments. Fibroblasts and endothelial cells were cultured respectively in Eagle’s Minimum Essential Medium (EMEM; ATCC) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 1% penicillin/streptomycin (P/S; Gibco) and Dulbecco’s Modified Eagle Medium (DMEM; ATCC) supplemented with 10% FBS and 1% P/S. The cells were seeded onto the samples at a density of 5000 cells/cm2 per substrate and were allowed to adhere for 4 hours in a 37 °C, humidified 5% CO2 atmosphere. Then, the samples were washed twice with PBS and ACS Paragon Plus Environment

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were transferred to fresh 24-well tissue culture plates. Next, 150 µL of a dye solution (MTT; Promega) was added to each well and the plates were cultured for another 4 hours, then 1 ml of a stop solution (MTT; Promega) was added to each well and the plates were incubated overnight. A plate reader (Molecular Devices, SpectraMax M3, 570 nm) was used to determine cell density. Mechanism of Anti-bacterial Properties Variation in Protein Adsorption as a Function of Incubation Time. A Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Scientific) was used to quantify the total amount of protein and target protein (casein) adsorbed onto the sample surfaces. To determine the variation of total protein adsorption, samples were incubated with TSB medium for 15 minutes, 30 minutes, 1 hour, 2 hours, 16 hours and 24 hours. After the prescribed time points, samples were removed from the medium and re-suspended in PBS with RIPA buffer (Sigma-Aldrich) to solubilize the adsorbed protein. Afterwards, the amount of total proteins in the supernatant was then tested according to the BCA assay protocol. Casein protein has been shown to be a key protein in TSB medium (17 g casein/L), thus, to quantify casein protein adsorption, samples were incubated with prepared 1.7% by weight casein solution (Sigma-Aldrich) and then the same procedures described above to determine total protein adsorption were used.30 Effects of Casein Protein on Bacterial Attachment. To further investigate the effects of casein protein adsorption on different surfaces, samples were soaked with 1.7% and 17% casein solutions and were incubated for 1 hour (since the maximum adsorbed protein occurred at an incubation time of approximately 1 hour). Then, the same procedures described above were used to perform S. aureus and E. coli assays on all the samples. Variation in Glucose Adsorption as a Function of Incubation Time. Glucose functions as the carbon energy source in tryptic soy broth (2.5 g/L). To determine the variation of glucose adsorption on PDMS samples with different surface topographies, samples were incubated with a glucose solution (Sigma-Aldrich) at a concentration of 120 µg/mL (a diluted glucose ACS Paragon Plus Environment

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solution was used here for more-refined analysis) for 2 hours and 24 hours, respectively. Then, the Glucose Assay Kit (Sigma-Aldrich) was used to quantify the glucose concentration in solution.31 Statistical Analysis. All cell, bacteria and mechanism studies were conducted in triplicate and repeated at least three times. Data were collected and the significant differences were assessed with the probability associated with one-tailed Student’s t-tests. Statistical significance was considered at p

Understanding the Role of Polymer Surface Nanoscale Topography

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