Antibacterial Plasma Polymer Films Conjugated with Phospholipid

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Antibacterial plasma polymer films conjugated with phospholipid encapsulated silver nanoparticles Shima Taheri, Alex Cavallaro, Susan Christo, Peter J. Majewski, Mary Barton, John Dominic Hayball, and Krasimir Vasilev ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00338 • Publication Date (Web): 02 Nov 2015 Downloaded from http://pubs.acs.org on November 3, 2015

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ACS Biomaterials Science & Engineering

Antibacterial plasma polymer films conjugated with phospholipid encapsulated silver nanoparticles Shima Taheri1, Alex Cavallaro1, Susan N. Christo2, Peter Majewski1, Mary Barton3, John D. Haybal2,3 and Krasimir Vasilev1,* 1

School of Engineering, University of South Australia, Mawson Lakes, SA 5095 Australia

2

Sansom Institute, University of South Australia, Adelaide, SA 5000, Australia

3

School of Pharmacy and Medical Sciences, University of South Australia, SA 5000, Australia

KEYWORDS: silver nanoparticles, phospholipid bilayer, inflammatory response, antibacterial coating

ABSTRACT

Medical device associated infections are a persistent medical problem which has not found a comprehensive solution yet. Over the last decades, there have been intense research efforts towards developing antibacterial coatings that could potentially improve medical outcomes. Silver nanoparticles have attracted a great deal of attention as a potent alternative to conventional antibiotics. Herein, we present a biologically inspired approach to synthesize phospholipid encapsulated silver nanoparticles and their surface immobilization to a functional plasma polymer interlayer to generate antibacterial coatings. The antibacterial efficacy of the coatings

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was evaluated against three medically-relevant pathogens including the Gram-positive Staphylococcus aureus and Staphylococcus epidermidis, and the Gram-negative Pseudomonas aeruginosa. The innate immune response to the coatings was assessed in vitro using primary bone marrow derived macrophages (BMDM). Any potential cytotoxicity was studied with primary human dermal fibroblasts (HDF). Overall, the coatings had excellent inhibition of bacterial growth. We also observed reduced expression of pro-inflammatory cytokines from BMDM which suggests a reduced inflammatory response. The combined properties of coatings developed in this study may make them a good candidate for application on medical devices such as catheters and wound dressings.

Introduction The battle between humans and pathogenic bacteria is a dynamic relationship which requires adoption of new tactics and strategies from each side. For example, the discovery and use of antibiotics by humans has led to a relatively quick evolution of resistance in bacteria. Some infections leading to high mortality, morbidity and costs are those associated with medical devices such as intravascular and urinary catheters and orthopedic implants.1,

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Following

adhesion to the surface of the device, the pathogens establish colonies and produce biofilms which are complex structures of bacteria and extracellular matrix polymers, such as proteins and sugars, which provide new communication routes and protect bacteria from the environment. Bacteria in biofilms are notoriously difficult to eradicate or treat with commonly used antibiotics.3-5 Biofilms are also capable of avoiding immune surveillance and facilitate development of antibiotic resistance. Since the formation of biofilms begins with the attachment


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of individual viable bacterial cells to the surface, preventing the initial stages of adhesion has been considered to be of paramount importance and has become the focus of intensive research over the last two decades. It has been demonstrated that incorporating nanoscale material into surface coatings can play a pivotal role in controlling bacterial adhesion and biofilm formation on indwelling medical devices.6, 7 Such coatings are even considered by some in the field as a viable alternative to systemic application of antibiotics.8,

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Amongst other nanomaterials, the

application of silver nanoparticles (AgNPs) based coatings on medical device surfaces is an approach that carries a significant potential to protect medical device surfaces from bacterial colonization device-associated infections. However, biocompatibility,8,

10-13

long term activity

and possible inflammatory responses caused by silver remain controversial.14, 15 Nevertheless, in some applications such as burns, where the probability for severe infections is very high, the benefits of the application of various formulations of antibacterial silver arguably outweighs any possible negative consequences. Lipids have often been employed for encapsulation of nano- and micro-particles as well as pharmaceuticals for applications such as imaging,16 drug delivery, 16-18 and biosensing.19, 20 As a natural constituent of the cell membrane, lipid coatings are expected to bring benefits to medical formulations and therapies by improving biocompatibility and reducing the potential of undesirable inflammatory responses. Silver nanoparticles have been encapsulated in lipid coatings previously.21-23 However, to be used in antibacterial coating these nanoparticles need to be immobilized on solid carriers. Herein, we report a method for the simultaneous synthesis of silver nanoparticles and their encapsulation into a phospholipid bilayer. The composition of the lipid bilayer was rationally designed to provide a negative surface charge of the resultant nanoparticles in order to facilitate


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immobilization to positively charged substrates. In order to make the coatings applicable to the various materials that medical devices are manufactured of, we used an interlayer deposited by plasma polymerization. These types of films are known to adhere well to most substrate materials.24 The antibacterial efficacy of the coatings was assessed via in vitro biofilm susceptibility assays against medically relevant pathogens including the Gram-positive Staphylococcus aureus and Staphylococcus epidermidis and the Gram-negative Pseudomonas aeruginosa. Essential for medical devices, we also interrogated the biocompatibility of the coatings developed in this work. The innate immune response was studied in culture of primary bone marrow-derived macrophages and the potential cytotoxicity was assessed in culture of primary human fibroblasts.

Experimental Section Materials All reagents were used as received without further purification.

1-palmitoyl-2-oleoyl-sn-

glycero-3-phospho-L-serine (sodium salt), (POPS, >99% 16:0-18:1 PS) was supplied in powder form from Avanti polar. Allylamine (AA, 98%), silver nitrate (AgNO3, 99.99 %), sodium borohydride (NaBH4), and nitric acid (70%) were supplied by Aldrich (Australia). Hydrochloric acid (36%) by Ajax Finechem Pty. Ltd (Australia), Safranin O, acetic acid glacial and sodium hydroxide pellet were purchased from Chem Supply (Australia). All solution preparation and glassware cleaning procedures were performed using ultrapure (Milli-Q) water (resistivity 18.2 Ω). All glassware and magnet stirrer were soaked in aqua regia solution (3:1 conc.HCl: conc.HNO3) and then rinsed thoroughly with the Milli-Q water before nanoparticle synthesis.


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Microorganisms: S. epidermidis strain ATCC 35984, S. aureus ATCC 43300 (Methicillinresistant Staphylococcus aureus (MRSA)), P. aeruginosa ATCC 27853 were cultured in tryptone soya broth (TSB, Oxoid, UK) in an incubator at 37°C and 60% humidity. Fibroblasts cells: Human dermal fibroblasts were harvested and grown as described previously25 from split thickness skin grafts (STSG´s) obtained from scavenged tissue specimens following routine breast reductions and abdominoplasties. All patients gave informed consent for skin to be used for research through a protocol approved by the Ethical Committee at the Queen Elisabeth Hospital and the University of South Australia Human Ethics Committee. Briefly, fibroblasts were grown in fibroblast culture medium (FCM) consisting of Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Gibco, Life Technologies, Australia), 10% v/v fetal calf serum (FCS, Ausgenex, Australia), 0.625 µg/mL amphotericin B (Sigma-Aldrich, Australia), 100 IU/mL penicillin and 100 mg/mL streptomycin (Gibco, Life Technologies, Australia) in an incubator at 37 °C, 5% CO2 in a humidified atmosphere. The medium was changed every 3–4 days until the cells were 80% confluent. HDFs between passages 3 and 9 were used. Cells were stained with the Phalloatoxin “Alexa Fluor 488® phalloidin” (a filamentous actin probe from Life) and DAPI- dilactate were purchased from Life Technologies, Invitrogen. Macrophages: Roswell Park Memorial Institute (RPMI; Sigma Aldrich) medium or DMEM (Sigma Aldrich) was supplemented with 10% foetal calf serum, penicillin (100 U/ml), gentamicin (100 µg/ml), β-mercaptoethanol (2 mM), L-glutamine (2 mM), and HEPES (10 mM) to produce complete RPMI (cRPMI) or complete DMEM (cDMEM), respectively. The L-929 conditioned media was prepared by culturing L-929 cells in culture flasks to >95% confluency and cultured in cDMEM until the media was exhausted. The conditioned media containing the


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required macrophage colony stimulating factor was removed, sterilely filtered and aliquoted for storage at -20˚C until use.

Instrumental UV–visible spectra were recorded on a Cary 5 UV–vis spectrometer (Varian Australia Pty Ltd) from 250 nm to 700 nm. Milli-Q water was used as blank and 1 ml of sample was diluted in a ratio 3:1 with Milli-Q water before UV-vis analysis. Dynamic light scattering (DLS) was used to measure the average size and the size distribution using a PSS Nicomp Particle Sizer 380 (Nicomp Particle Sizing Systems, USA). In order to study the morphology of the polymer particles, samples were sputter coated with 5 nm of Cr using a Quorum Q150T sputter coating system. Transmission Electron Microscope (TEM) Images were acquired using a Philips CM200 transmission electron microscope. The zeta potential of the nanoparticles was measured in a 10−3 M potassium chloride (KCl) solution (100 µl of sample in 900 µl KCl) with a Zeta Nanosizer (Malvern Instruments, U.K.) at 25°C. Topographical images were provided by Atomic force microscopy (AFM) using an NT-MDT NTEGRA SPM in non-contact mode with silicon nitride non-contact tips coated with gold on the reflective side (NT-MDT, NSG03) (resonance frequencies between 65 and 100 kHz). The scan rate was 0.5 Hz with the maximum scanner range of 100 µm. The scanner was calibrated in the x, y, and z directions using 1.5 µm grids with a height of 22 nm. X-ray Photoelectron Spectroscopy (XPS) spectra were recorded on a SPECS SAGE spectrometer. Processing and component fitting of high resolution spectra were performed with CasaXPS software. Thickness measurements of deposited films on silicon wafers were performed using an imaging ellipsometer (Beaglehole Instruments, New Zealand). The


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measurements were carried out at a constant wavelength of ∼600 nm as the angles of incidence and reflected light detection were varied between 40° and 85°.

Synthesis of silver nanoparticles: Silver nanoparticles were synthesized via a wet chemistry route as follows: lipid assemblies were prepared by the solvent evaporation of a mixture consisting 20 ml chloroform (CHCl3) and 0.357 mM 1-palmitoyl-2-oleoyl-sn-glycero-3phospho-L-serine (sodium salt) (POPS) in a 100 ml round-bottom flask with a long extension neck, using a rotary evaporator at 25°C under reduced pressure. The system was then purged with nitrogen and the dry lipid films were hydrated in 50 ml Milli-Q water by agitation in a rotary evaporator for 45 minutes. Then a 10 ml aqueous solution of 1 mM silver nitrate (AgNO3) was added and the resulting homogeneous vesicle suspension was sonicated briefly for 3 min using a digital ultrasonic cleaner (model CD-7810(A), Richsea Development Co., Ltd.). The mixture was then cooled in an ice bath and 10 ml aqueous solution of 5 mM NaBH4 was added dropwise under vigorous stirring. This resulted in a homogeneous dark brown solution. The solution was than kept in dark and cold conditions (4-8°C) and were stable over 6 months. Preparation of allylamine plasma polymer film: Plasma polymerization was carried out in a custom-built reactor described elsewhere26 using a 13.56 MHz plasma generator (Advanced Energy, USA) and a matching network (Advanced Energy, USA). The 13 mm Thermanox cover slips substrates (Thermo Fisher Scientific, USA) were cleaned with ethanol and Milli-Q water before being loaded in the reactor chamber. Then, they were cleaned by oxygen plasma for 2 min at pressure of 2.5x10-2 mbar using a power of 20 W. The deposition of the allylamine (AA) plasma polymer was carried out at a monomer pressure of 2×10-3 mbar. The input radiofrequency (RF) power of 10 W and the monomer flow rate of 10 sccm. The time of


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deposition was for 5 min. These conditions resulted in nitrogen-rich films with a thickness of about 24 nm. Allylamine plasma polymer (AApp) substrates were kept sealed overnight at ambient temperature before undergoing any further modification, in order to let the polymer surface to find its stable structure with minimum interfacial energy. The allylamine was plasma polymerized on silicon wafers for SEM analysis, and on glass and Thermanox coverslips for bacterial and cell culture studies. Immobilization of nanoparticles on surfaces: AApp coated substrates were immersed in the solution of POPS encapsulated AgNPs for 24 h at room temperature (ca 23°C). The samples were rinsed thoroughly with Milli-Q water, dried with nitrogen and kept sealed. Determination of antibacterial activity in vitro Bacteria were inoculated onto blood agar plates and incubated overnight at 37°C. Individual bacterial colonies were isolated and incubated overnight at 37 °C in 10 ml Tryptic Soya Broth (TSB). 1ml of solution was diluted in 9 ml of fresh TSB and incubated for 2h at 37°C. 1 × 106 cfu ml−1 of bacteria was prepared using fresh TSB. Control and modified substrates were placed in 24 well plates. Each well was filled with 400 µl of diluted culture of bacteria (1 × 106 cfu ml−1) in TSB. Two sets of AApp coverslips covered with 400 µl TSB were used as positive and negative control. The sample box was incubated in a container with moist foam for 4 h at 37 °C, the broth in each well was replaced with 400 µl fresh TSB and incubated in similar fashion for 24 h at 37°C. After the second incubation period, the TSB was removed and wells were rinsed twice with 400 µl of sterile saline (0.9% sterile solution of sodium chloride in water) to clean any loose biofilm formed on the surface. The biofilm formed on the other side of each cover slip was removed with a swab immersed in ethanol (EtOH, 70%). 400 µl of Safranin–O (0.1%, Ajax Chemical) solution was added to each well to stain the biofilm adhered to the surface. After 30


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min Safranin solution was removed and wells were rinsed twice with 400 µl of Milli-Q water to remove excess Safranin and allowed to dry at ambient temperature. 400 µl of acetic acid (AcOH, 33%) was introduced into each well to lyse the bacterial cells and release the trapped dye. 100 µl of the content of each well was transferred into a 96 well plate and the optical density (OD) was measured by an ELISA plate reader at 490 nm. Cell viability and morphology Cell viability: Samples were placed in 24 well tissue culture plates. Human dermal fibroblasts were seeded at a density of 1x104 cells per well in FCM. After 2 days cell viability was measured using the resazurin assay. A stock solution of 110 µg/ml resazurin was prepared in phosphate buffered saline and filter sterilized using a 0.2 µm filter. This was then diluted 1:10 in fibroblast culture medium. 500 µl of the resazurin solution was added to each sample. After 4 hours 200 µl of the reduced solution was removed from each well and placed into a well of a 96 well plate and the fluorescent intensity read using a plate reader (λex =544 nm and λem =590 nm). Cell morphology: The samples were fixed in 4% paraformaldehyde in PBS for immunocytochemistry staining of the cytoskeleton to assess cell morphology. Fixed cells were incubated at room temperature with 0.1% (v/v) Triton X-100 for 5 minutes to permeablize the membranes. Phallotoxin Alexa – 488 and DAPI to final concentrations of 0.165 µM and 0.1 µg/ml respectively in 200 µL of PBS was added to each sample and samples incubated further in the dark. Excess dye was washed from the samples and samples were imaged with a Nikon A1-R Confocal laser scanning microscope running NIS elements A R software. Immune response: In vitro activation of bone marrow-derived macrophages


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Bone marrow derived macrophage cells were generated by flushing bone marrow cells from the femurs and tibia of 8-week old C57Bl/6 mice in cRPMI supplemented with 20% L-929 conditioned media. On day 4 of culture, non-adherent cells were removed, and media replenished before being seeded onto control and modified coverslips. On day 7, the in vitro generated BMDMs were stimulated with lipopolysaccharide (LPS) (100 ng/ml; Sigma Aldrich) or left unstimulated for 4 h, and supernatant collected for the analysis of tumor necrosis factor-alpha (TNF-α), and interleukin (IL-6). BMDMs were then stimulated with ATP (5 mM; Sigma Aldrich) for 1 h, and supernatant collected for IL-1β analysis using standard ELISA protocols. Transmission images were collected on day 7 using an Olympus IX51 Fluorescence Microscope and the CellSens program. To determine cell viability, adherent BMDMs were gently scraped off the coverslips and stained with the nuclear counterstain DAPI (1 µg/ml) for 10 mins and analyzed on the FACSCantoII immediately. Viable cells were gated as side scatter (SSC) and DAPI low. Data was analyzed using the FlowJo (Treestar) software. Statistical analysis method All statistical analysis was performed on GraphPad Prism 5 and a one-way ANOVA test was performed on data. The posttest performed on antibacterial results was Tukey’s posttest and on fibroblast cell results a Dunnett’s posttest. In antibacterial and fibroblast cell studies AApp and glass surfaces were the chosen, respectively, as controls in which data were compared against.

Results and discussion The process of encapsulation of silver nanoparticles in phospholipid bilayer is schematically depicted in Figure 1. The first phase involves the generation of lipid vesicles based on the Bangham’s method.27, 28 Lipids (POPS in this instance) are dissolved in chloroform followed by solvent evaporation on a rotary evaporator. Liposomes are then spontaneously formed when the


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dried phospholipids were hydrated in aqueous medium. Sonication was then carried out to reduce the size of the lipid vesicles.28 During sonication, a given amount of AgNO3 was added to the dispensed lipid solution and mixed thoroughly. This was followed by dropwise addition of NaBH4. After the addition of NaBH4 the color of the suspension turned gradually to dark red /brownish which is indicative for the presence of silver nanoparticles. UV-Visible spectroscopy characterization revealed a peak at 404 nm (Figure 2a) consistent with the plasmon resonance absorption band of silver nanoparticles. The solution was stable over six months without visible aggregation and precipitation. The surface potential of the nanoparticles was determined to be 70 mV. This high negative value is a result of the carboxyl acid groups in the hydrophilic section of the lipid molecules which in aqueous medium point towards the outer surface of the bilayer. POPS was selected for this preparation for the purpose of electrostatic immobilization of the lipid-encapsulated silver nanoparticles on positively charges surfaces such as those deposited from plasma of allylamine.


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Figure 1. Schematic representing the incorporation of silver nanoparticle into a POPS lipid bilayer (the image is not to scale). (a) Lipid molecules are dissolved in chloroform and (b) dried forming a thin lipid film on the glass wall. (c) Hydration in Milli-Q water leads to the formation of lipid vesicles of various sizes. (d) Reactants are added dropwise and the solution sonicated to form smaller lipid vesicles with AgNPs. (e) final lipid coated AgNPs. TEM measurements were carried out to gain insight of the structure, size and morphology of the nanoparticles. The nanoparticles had a spherical morphology with an average size of 30 ± 10 nm. The size of the AgNPs was further confirmed by dynamic light scattering (DLS) studies which showed that the majority of the particles had size of 35 nm (75.5%). However, there were some smaller nanoparticles present of less than 10 nm (24.5%) in diameter. The structure of the nanoparticles was revealed by the higher magnification image shown in Figure 2c. The dark appearance of the core is consistent with a material of high electron density such as silver. The light-grey area around the nanoparticles is attributed to the lipid encapsulation. The thickness of this region is in the rage of 4±1 nm which is consistent of the thickness of a lipid bilayer.

Figure 2. (a) UV-Visible spectrum of colloidal solution of AgNPs; (b) A photograph showing the resultant colloidal solution of POPS encapsulated AgNPs; (c) TEM image of a silver nanoparticle encapsulated in a 4 nm thin layer of POPS (scale bar is showing 10 nm)


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To generate antibacterial coatings, the POPS encapsulated AgNPs were immobilized on model substrata modified with a thin interlayer of allylamine plasma polymerized (AApp). This type of plasma polymer film contains a population of amine groups which acquire a positive charge in aqueous medium at pH below 8.29 The positive charge of the surface facilitates electrostatic binding of the POPS capped silver nanoparticles which are negatively charged (surface charge of -70mV). The nanoparticles surface immobilization was evaluated using XPS analysis. Figure 3a shows a typical survey spectrum after nanoparticles immobilization for 24 h. Five distinct peaks were detected on the surface corresponding to the O1s, N1s, C1s, Ag3d and P2p binding energies. The presence of silver and phosphorus can be attributed to the silver nanoparticles that are capped with a phospholipid. Quantification of the surface chemical composition based on the peak area results in 8.9 At% oxygen, 14.2 At% nitrogen, 70.4 At% carbon, 5.7 At% silver and 0.8 At% phosphorus. UV-visible spectroscopy characterization (Figure 3b) of the surface after immobilization of the nanoparticles shows a peak at about 404 nm corresponding to the plasmon resonance absorption band of silver nanoparticles. The position of the peak is consistent to that measured in solution (Figure 2a). A photograph of a Thermanox coverslip before and after nanoparticle immobilization is shown in Figure 3c. After nanoparticle immobilization the substrate acquires the characteristic light brown color. AFM characterization of the surface after nanoparticles immobilization was also carried out.30 The immobilization process results in a fairly dense coating as shown in Figure 3d and Figure 3e. The nanoparticles adhere as well separated entities without apparent aggregations.


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Figure 3.(a) XPS survey spectrum of POPS encapsulated AgNPs immobilized on AApp film for 24 h.(b) UV-Visible spectrum of a glass slide POPS encapsulated AgNPs immobilized on AApp film for 24 h. (c) A photograph showing the visible color change of Thermanox substrate coated with AApp after AgNPs immobilization. (d) A representative AFM topography image of a surface with POPS encapsulated AgNPs immobilized on AApp film for 24 h. (e) Topography histogram analysis of the surface coated with AgNPs. (f) A 3D representation of the image shown in d.

The antibacterial efficacy of the coatings was evaluated against three pathogenic bacteria including S. aureus, S. epidermidis as Gram-positive species, and P. aeruginosa as a


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representative of Gram-negative species (Figure 4). These bacteria were chosen because of their relevance to medical device-associated infections.31-33 The capacity of the coatings to inhibit bacterial colonization and biofilm formation was assessed via safranin-O staining after 24 hours of incubation. Figure 4 shows the relative level of surface bonded bacteria compared to AApp control. Surface colonization by S. epidermidis was almost completely inhibited while there was 70% reduction of S. aureus attachment and 80% reduction of P. aeruginosa growth. This result is consistent with published studies where S. aureus was found to have higher tolerance to silver nanoparticles.34 Although the inhibition of bacterial growth is not complete in the case of S. aureus and P. aeruginosa the level of reduction is substantial and potentially sufficient to assist the immune system in handling the bacterial invasion. We should also note that the tests are conducted with a very high initial concentration of bacteria (106 cfu/ml). Such high level of bacterial contamination in a hospital environment is rather unlikely.


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Figure 4. Bacterial colonization of AgNPs modified surface relative to AApp control by Staphylococcus

aureus

(ATCC

4330),

Staphylococcus

epidermidis

(ATCC

35984),

Pseudomonas aeruginosa (ATCC 27853) after 24 h of incubation evaluated by Safranin-O staining. Values are expressed as mean ± SEM, with a one way ANOVA statistical analysis being performed using a Tukey’s posttest (AApp comparison group).*** P0.05; * p

Antibacterial Plasma Polymer Films Conjugated with Phospholipid

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