Simultaneously Antimicrobial, Protein-repellent and Cell-compatible

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Simultaneously Antimicrobial, Protein-repellent and Cell-compatible Polyzwitterion Networks: More Insight on Bioactivity and Physical Properties Monika Kurowska, Alice Eickenscheidt, Ali Al-Ahmad, and Karen Lienkamp ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00100 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 29, 2018

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ACS Applied Bio Materials

Simultaneously Antimicrobial, Protein-repellent and Cell-compatible Polyzwitterion Networks: More Insight on Bioactivity and Physical Properties Monika Kurowska,1 Alice Eickenscheidt,1 Ali Al-Ahmad,2 and Karen Lienkamp1,*

AUTHOR ADDRESS 1

Bioactive Polymer Synthesis and Surface Engineering Group, Department of Microsystems Engineering (IMTEK) and Freiburg Centre for Interactive Materials and Bioinspired Technologies (FIT), Georges-Köhler-Allee 105, 79110 Freiburg, Germany

2

Department of Operative Dentistry and Periodontology, Medical Centre of the University of Freiburg, Faculty of Medicine, University of Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany.

* [email protected]

KEYWORDS Antimicrobial polymer, antibiofouling polymer, hydrogel, polynorbornene, polyzwitterion, protein-repellent polymer, surface plasmon resonance spectroscopy.

1

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ABSTRACT A poly(oxanorbornene)-based polyzwitterion with primary ammonium and carboxylate groups (PZI) has been reported previously as the first simultaneously antimicrobial and protein-repellent polyzwitterion. Here, additional physical and biological properties of three poly(oxanorbornene)based polyzwitterions with different functional groups (PZI, the polycarboxybetaine PCB, and the polysulfobetaine PSB) are compared to understand the molecular origins of this unusual bioactivity. Additionally, the three polyzwitterions and the antimicrobial, polycationic SMAMP are exposed to proteins, bacteria suspensions, human plasma and serum. These interactions are investigated by surface plasmon resonance spectroscopy. In protein adhesion studies, neither fibrinogen nor lysozyme adhere irreversibly to PZI, yet reversible interaction with lysozyme is observed at pH 7 and 8. In the presence of bivalent cations, reversible fibrinogen adhesion on PZI and PSB is observed, but not on PCB. This might explain why mammalian cells grow on PZI and PSB, but not on PCB. PZI does not show human plasma adhesion, while PCB and PSB have 0.27 and 0.48 ng mm-2 adhered plasma, and SMAMP even 6.3 ng mm-2. Both PZI and SMAMP show strong serum adhesion, while no serum adhered to PCB, and only little to PSB. This could be related to the pH difference between serum and plasma, to which the pHresponsive primary ammonium groups are susceptible, while the permanently charged NR4+ groups are unaffected. Both PZI and PCB showed none or only little bacterial adhesion. PCB is also intrinsically antimicrobial against E. coli and S. aureus bacteria and thus is also simultaneously protein-repellent and antimicrobially active. Thus, while the carboxylate groups of PZI and PCB seems to be a prerequisite for the dual antimicrobial activity and proteinrepellency, the pH-responsiveness of the primary ammonium group seems to make the PZI molecule vulnerable for protein adhesion in fluids that are slightly out of the physiological range. 2


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MANUSCRIPT Introduction. Motivation. According to the "The Review on Antimicrobial Resistance" which was commissioned for the UK government in 2014, resistant bacteria will be causing 10 million deaths annually worldwide by 2050 if current trends continue, thus exceeding even the yearly death toll to cancer.1 The reports of the World Heath Organization,2 the Center for Disease Control3 and the European Antimicrobial Resistance Surveillance Network4 on the spreading of antimicrobial resistance are no less worrying. In the context of medical devices, one important infection pathway is biofilm formation on the surfaces of medical devices.5-8 These can act as a gateway of bacteria into the human body. Besides clinical measures (increased hygiene, faster diagnostics, reduction of antibiotics use and patient screenings prior to surgery),1 new drugs and materials that can help contain bacterial infections are thus direly needed. Significant progress on polymer-based biofilm-suppressing materials has been made in the previous years, for which we refer to the literature.9-14 In short, these materials typically either have an active (antimicrobial) or a passive (protein-repellent) component, or contain several active and/or passive components, so that the final material is either protein-repellent or antimicrobially active, or both. Since protein adhesion to the material surface is the first step of biofilm formation, passive coatings slow down biofilm growth by preventing the formation of a protein conditioning layer onto which bacteria would adhere. Active components, which are either leaching or covalently attached, kill adherent and/or planktonic bacteria and thereby slow down surface colonization by these organisms. Materials that significantly slow down protein-adhesion and are at the same time strongly antimicrobially active are rare, and often complicated to fabricate.10, 15, 16,

12, 13, 17-20

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State of the Art: Polyzwitterions. We recently reported a material consisting of a poly(oxanorbornene)-based polyzwitterion21 (PZI, Figure 1) that was strongly protein-repellent, highly antimicrobially active, cell-compatible and consisted of a single component only. Additionally, it could be used to coat both laboratory model materials (glass, silicon wafers, gold) and real-life surfaces (PDMS tubes and polyurethane wound dressings). This was surprising because polyzwitterions are typically considered as passive, i.e. just protein-repellent materials without intrinsic antimicrobial activity. They consist of an equal number of positive and negative charges that are placed regularly along a polymer backbone, so that polymer-bound ion pairs are formed. This makes them overall charge neutral. Due to the strong Coulomb interactions between these ion pairs, polyzwitterions are highly hydrophilic and thus strongly hydrated in aqueous saltcontaining media.22 This is also the origin of their strongly protein-repellency, since the interfacial energy with water at their surfaces is so low that there is no enthalpy gain when proteins adhere.23 It was recently shown that the biocompatibility of a surface correlates with its swellability.24, 25 It is thus little surprising that strongly hydrated polyzwitterions are also highly biocompatible.22 Another interesting property of polyzwitterions is their peculiar solubility. While polyelectrolytes shrink in size with increasing ionic strength of the solvent (because their identical charges are increasing shielded, "polyelectrolyte effect"), polyzwitterions have larger molecular dimensions at high salt conditions than at low salt conditions, and they become increasingly insoluble when the salt concentration of the solvent is reduced ("anti-polyelectrolyte effect").22 The presence of salt breaks the ion pairs and thus solubilizes the polyzwitterion. Even though their are notoriously difficult to handle in the lab because they are only soluble in salt solutions and harsh organic solvents (trifluoroethanol, hexafluoroisopropanol), and also challenging to analyze, polyzwitterions have found a number of interesting applications: they 4


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have been used as lubricants, cell membrane mimetics, protein-repellent polymer coatings that slow down biofilm formation and improve hemocompatibility, drug delivery agents, and as passivating layer for sensor applications.22, 26-31 In particular, Jiang and coworkers demonstrated that carboxybetaine-based polyzwitterions reduced long-term biofilm formation (> 100 h) by various bacterial species by 95 % under flow conditions.32 Previous work on the polyzwitterion PZI.21 In our recent report on the polyzwitterion PZI (Figure 1),21 we described the synthesis of surface-attached polymer networks from that polymer, and compared its bioactivity to a structurally similar, strongly antimicrobial and protein-adhesive polycation (SMAMP, Figure 1), as well as to a non-antimicrobial, protein-repellent sulfobetainbased polyzwitterion (PSB, Figure 1). With a number of experiments on these three polymers, including the full physical characterization of the materials, an antimicrobial assay, a biofilm formation assay, a toxicity assay and a protein adhesion assay at 7, we demonstrated that PZI was indeed antimicrobially active like SMAMP, and not just passively protein-repellent like PSB. Additionally, it was non-toxic to mammalian cells. Since technical substrates could be coated with PZI, this makes it a promising material for medical applications.21 However, based on that data, we could not fully understand the molecular origins of the bioactivity of PZI. In particular, it was unclear which role the carboxylate group played for its bioactivity, and whether the primary ammonium group was prerequisite or not. This was to be clarified in this follow-up study. Study Design. In this study, we had three aims. First, we included the polycarboxybetaine PCB (Figure 1) carrying a carboxylate and a quaternary ammonium group as an additional reference in our investigation, which was not present to our previous study.21 Comparing PCB with the 5



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polysulfobetaine PSB would allow us to differentiate between effects caused by the carboxylate and the sulfonate groups, respectively. A comparison of PCB with PZI, on the other hand, would enable us to compare the effect of a primary and a quaternary ammonium group. Thus, PCB is a key molecule to isolate certain structural motifs in these molecules, and correlate them to their bioactivity. We here present the physical characterization and bioactivity (antimicrobial activity, cell toxicity and protein adhesively) of PCB in comparison to the bioactivity of the previously reported polymers PZI, SMAMP and PSB. Second, we needed to understand the physical properties of PZI in more detail to explain its bioactivity, particularly its interaction with proteins and mammalian cells. As mentioned above, polyzwitterion solubility and swelling is salt-concentration dependent. For polyzwitterions with -responsive ionic groups, it might also be -dependent. Thus, to appreciate whether PZI is well-solubilized under physiological conditions, we here investigated its swelling at different ionic strengths. We also measured the swellability of PZI at the values 3, 7 and 8, and compared it to that of PCB and PSB at these values. Since real-life biological fluids contain bivalent cations that might affect the charge situation at the polyzwitterions' surface, and thus protein adhesion on polyzwitterions, we also investigated the effect of the presence of Mg2+ when exposing PZI, PCB and PSB to proteins. Last but not least, we exposed the entire sample set of this study (PZI, PCB, PSB and SMAMP networks, respectively) to real-life biological fluids, and studied the interaction of the materials with these fluids by surface plasmon resonance spectroscopy (SPR). These fluids were suspensions of Escherichia coli and Staphylococcus aureus bacteria, human blood plasma and human blood serum. The results of these experiments would enable us to predict which of the 6


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polymer surfaces would be suitable for different biomedical applications. Thus, correlating the additional data obtained in the there parts of this study and connecting it with the previous results for PZI gives a more detailed picture on the molecular origin of the physical properties and bioactivity of these materials, as detailed below.

Figure 1: Structure of the polymers used in this study: the simultaneously antimicrobial and protein-repellent polyzwitterion PZI, the carboxybetaine-based polyzwitterion PCB, the sulfobetaine-based polyzwitterion PSB, and the polycationic SMAMP. They were simultaneously cross-linked and surface-attached by a UV-activated thiol-ene reaction (with the tetrathiol crosslinker) and a C,H insertion reaction (with the benzophenone anchor group on the surface).

Experimental

General.

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All chemicals were obtained as reagent grade from typical sources like Sigma-Aldrich, Fluka, Acros, or Carl Roth, and were used as received unless otherwise indicated. Gel-permeation chromatography (GPC) was measured in chloroform or tetrahydrofuran (THF) on a PSS SDV column (PSS, Mainz, Germany) and calibrated with PMMA standards. For the polybetaines, GPC was measured in 2,2,2-trifluoroethanol TFE with addition of sodium trifluoroacetate NaTFA salt (at 0.1M NaTFA for PCB and at 0.25M NaTFA for PSB). Solvents for GPC were HPLC quality and obtained from Carl Roth. The thickness of the dry polymer layers on silicon wafers was measured with the auto-nulling imaging ellipsometer Nanofilm EP3 (Nanofilm Technologie GmbH, Göttingen, Germany), which was equipped with a 532 nm solid-state laser. The contact angle system OCA 20 (Dataphysics GmbH, Filderstadt, Germany) was used to measure the static, advancing and receding contact angles of the PCB network. The FTIR spectra were recorded from 4000 to 400 cm-1 with a Bio-Rad Excalibur spectrometer (Bio-Rad, München, Germany), using a spectrum of the blank double side polished silicon wafer as background. The topography of the PCB surface was imaged with a Dimension FastScan and Icon from Bruker. Commercial FastScan-A cantilevers (length: 27 µm; width: 33 µm; spring constant: 18 Nm-1; resonance frequency: 1400 kHz) and ScanAsyst Air cantilevers (length: 115 µm; width: 25 µm; spring constant: 0.4 Nm-1; resonance frequency: 70 kHz) were used. All AFM images were recorded in Peak Force Tapping mode in air and ScanAsyst in air, respectively. The obtained images were analyzed and processed with the software ‘Nanoscope Analysis 9.1’. The streaming current measurements were performed with an electrokinetic analyzer with integrated titration unit (SurPASS, Anton Paar GmbH, Austria). The analyzer was equipped with an adjustable gap cell. Ag/AgCl electrodes were used to detect the streaming current. The respective polymers were spin-cast on fused silica substrates (MaTeC, 20 x 10 x 1 mm lp, Ch.Nr. 8


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13112704) and put into the measuring cell. XPS measurement was performed under ambient condition on a PerkinElmer PHI 5600 ESCA system (PerkinElmer, Waltham, MA, USA). The Xray source was a Mg anode with an energy of 1253.6 eV. The aperture size was 400 µm, the angle was 45°. Optical micrographs of the keratinocytes grown on PCB and the untreated glass slides (growth control) were taken using a phase contrast objective (10x magnification) on a Leica DMIL microscope with a Leica D-LUX-3 CCD camera (10x magnification). Synthesis. Molecules for surface attachment and functionalization of model surfaces with surface functionalization agents. The molecules used for surface attachment of the polymer network (3EBP for all surfaces except gold, and LS-BP for gold surfaces) were synthesized as described in the literature.17, 33 Silicon wafers, glass slides, quartz glass slides, and gold surfaces for SPR measurements were prepared as described previously.21 Synthesis of polyzwitterions and polyzwitterion networks. The monomers for PZI,24 PCB34 and PSB35 were synthesized using literature procedures. Each monomer was dissolved in the appropriate solvent, and was polymerized by ring-opening metathesis polymerization (ROMP) using Grubbs 3rd generation catalyst. They were then characterized by gel permeation chromatography (GPC). Full details of the reagents amounts used for the polymers studied in this paper as well as the GPC results are given in Table 1. PZI was initially obtained in its protected form carrying a Boc protective group; PCB and PSB were directly obtained in their polyzwitterionic form.

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Table 1: Experimental parameters for the synthesis of polyzwitterions PZI, PSB and PCB: monomer and catalyst amounts (in mg and mmol). GPC results of the polyzwitterions obtained: molecular weight Mn and polydispersity PDI. Polymer

Monomer

Catalyst

Mn / g mol-1

PDI

m / mg

n / mmol

m / mg

n / mmol

PZI

500

1.53

3.7

0.005

70 000

1.6

PSB

500

1.34

7.3

0.01

34 000

1.9

PCB

500

1.62

7.3

0.01

36 000

1.2

The PZI and PSB networks were synthesized as described previously,21 the PCB network was synthesized analogously: First, the PCB polymer was dissolved in TFE at a concentration of 25 mg mL-1. The tetrafunctional cross-linker pentaerythritol-tetrakis-(3-mercaptopropionate) (20 mg dissolved in TFE) was added, and the mixture was spin-coated onto 3EBP-functionalized silicon wafers pieces (1.5 x 1.5 cm²), glass slides or quartz glass slides (1 x 2 cm2), or onto LS-BP functionalized gold substrates (2.5 x 2.5 cm²). The thus coated substrates were crosslinked by UV irradiation (254 nm, 30 min). The PSB and PCB network was already obtained as a polyzwitterion and did not have to be further modified. To remove the Boc groups from the protected PZI network, it was immersed into HCl in dioxane for 12 h. All networks were finally rinsed with ethanol (PZI) or TFE (PSB and PCB), and dried under nitrogen flow.

Polymer network characterization. Measurements by ellipsometry, Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), photoelectron spectroscopy (XPS), and of the contact angles were performed as reported previously.21 The zeta potential was determined by electrokinetic measurements using 10


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an electrokinetic analyzer with integrated titration unit (SurPASS, Anton Paar GmbH, Austria; with adjustable gap cell and Ag/AgCl electrodes).21 For these measurements, the respective polymers were spin-cast on fused silica substrates (MaTeC, 20 x 10 x 1 mm lp, Ch.Nr. 13112704) and mounted into the measuring cell. Titration started with electrolyte (1 mM KCl) at pH 3.5; after each measurement 0.1 M NaOH was added incrementally until pH 10.5. was reached. Representative titration curve for PCB network (zeta potential vs. pH) is shown in Figure S3 in the supporting information. The data was fitted separated into two parts at the central plateau; each part was fitted with the Hill equation, a standard fit for sigmoidal curve shapes: ζ = ζ + (ζ − ζ )

(with ζ = extrapolated start value of the first

plateau; ζ = the extrapolated end value of the second plateau; k = the point of inflection; n = factor adjusting the width and steepness of the sigmoidal curve). The isoelectric point (IEP, pH were the zeta potential is zero) was determined from the curve. The acid constant value of

each surface bound acid-base pair was not calculated from = ζ + 0.4343

ζ !

as

has been done previously21 because that expression is only strictly valid if ζ is less than 25 mV.36 Instead, has been estimated graphically from the titration curve with ζ" ≈ ζ −

ζ $ζ%

(where ζ is the zeta potential at the point of inflection & of the titration curve) in

analogy to the definition of the half-titration point of conventional volumetric acid-base titrations. is then the pH value measured at ζ" . The values thus (re)calculated for PZI, PCB, PSB and SMAMP are included in Table 2. Ethics statement. Gingival mucosal keratinocytes and blood for the plasma and serum studies were obtained from human volunteers who had previously given their written consent according 11



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to the Helsinki declaration. This was approved by the ethics vote number 381/15 of the Ethics Board of the Albert-Ludwigs University, Freiburg, Germany. Swelling and adhesion studies, including protein adhesion, bacterial adhesion, serum adhesion and plasma adhesion. General. Surface plasmon resonance spectroscopy (SPR) experiments were performed on a RT2005 RES-TEC device in Kretschmann configuration from Res-Tec, Framersheim, Germany. Plasmons were excited with a He-Ne-Laser with λ= 632.8 nm, as reported previously. SPR substrates were homemade (LaSFN9 glass from Hellma GmbH, Müllheim, Germany; coated with 1 nm Cr and 50 nm Au at the Clean Room Service-Center (RSC) of the Department of Microsystems Engineering, University of Freiburg, using the device CS 730 S (Von Ardenne, Dresden, Germany). Swelling study. To study the swelling of the PCB polymer network in water, full SPR angular reflectivity curves (reflectivity vs. angle of incidence) were measured against nitrogen and water as described previously.21 The thickness of layer of the material was calculated by simulations of the reflectivity curves based on the Fresnel equations, which were performed with the software ‘Winspall’. The following permittivities ε’ and ε’’ were used: LaSFN9 (ε’ = 3.4036; ε’’ = 0), Cr (ε’ = –6.3; ε’’ = 20), Au (ε’ = –11.85; ε’’ = 1.5), PCB (ε’ = 2.36; ε’’ = 0), water (ε’ = 1.77; ε’’ = 0), nitrogen (ε’ = 1; ε’’ = 0). For the surface-attached PZI network, the swelling behavior in NaCl solutions with different concentration in the range from cNaCl = 0.001M to cNaCl = 1M was studied. First, the angular reflectivity curve of the dry polymer surface was measured. Then, the surface was covered with deionized water (MilliQ, Merck KGaA, Darmstadt, Germany) for a short time, and 0.001 M NaCl 12


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solution was injected into the SPR flow cell (10 µL, Teflon, Res-Tec, Mainz, Germany). The salt solution was supplied to the SPR flow cell with flow rate 30 µL min-1 using peristaltic pump (Ismatec Reglo ICC, Idex, Wertheim, Germany). The swelling process was completed when the polymer layer had reached equilibrium (At that point, the reflectivity signal in the kinetic mode did not change, or the SPR reflectivity curves measured against the salt solution every 10-15 min remained the same). After that, the NaCl solution with the next higher concentration was injected. Each time the polymer layer was allowed to swell for about 90 min. After the complete swelling, SPR reflectivity curves were recorded in swollen state. The swelling behavior of surface-attached PZI, PCB and PSB networks was studied using NaCl solutions (cNaCl = 0.15M) with different pH values. For the PZI surface, swelling was measured in the range from pH 3 to pH 8. For PCB and PSB surfaces, measurements were taken at pH 3 and pH 7 only. The procedure for the pH-dependent experiments was the same as for the salt solution series described above. Briefly, angular reflectivity scan measurements of the dry polymer surface were performed. Then, the surface was hydrated with Milli-Q water for a short time. After that, salt solutions with increasing pH were injected into the SPR flow cell. Each time after complete swelling (about 90 min), angular reflectivity curves against salt solution with respective pH were measured. The layer thickness of the material was calculated by simulations of the reflectivity curves based on the Fresnel equations, which were performed with the software ‘Winspall’. For the swelling study in NaCl solutions with different concentrations, the following permittivities ε’ and ε’’ were used: LaSFN9 (ε’ = 3.4036; ε’’ = 0), Cr (ε’ = –6.3; ε’’ = 20), Au (ε’ = –12.15; ε’’ = 1.2), LS-BP (ε’ = 2.25; ε’’ = 0), PZI (ε’ = 2.434; ε’’ = 0), 0.001 M and 0.01 M NaCl (ε’ = 1.777; ε’’ = 0), 0.1 13



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M NaCl (ε’ = 1.779; ε’’ = 0), 0.6 M NaCl (ε’ = 1.793; ε’’ = 0), 1 M NaCl (ε’ = 1.802; ε’’ = 0), water (ε’ = 1.776; ε’’ = 0), nitrogen (ε’ = 1; ε’’ = 0). For the swelling study in NaCl solutions with different pH values, the following permittivities ε’ and ε’’ were used: LaSFN9 (ε’ = 3.4036; ε’’ = 0), Cr (ε’ = –6.3; ε’’ = 20); Au (-12.05≤ ε’ ≤ -11.55; 1.5 ≥ ε’’ ≥ 1.2); LS-BP (ε’ = 2.25; ε’’ = 0); PZI (ε’ = 2.434; ε’’ = 0); PCB (ε’ = 2.38; ε’’ = 0); PSB (ε’ = 2.427; ε’’ = 0); 0.15 mol·L-1 NaCl (ε’ = 1.781; ε’’ = 0); water (ε’ = 1.776; ε’’ = 0), nitrogen (ε’ = 1; ε’’ = 0). For the swelling experiments, thick samples (200 nm or more) were used, which had plasmon and waveguide peaks. SPR reflectivity curves of the dry polymer networks were recorded first, followed by measurements on the fully swollen network. The sample thickness d and the real part of the permittivity (ε’) of the polymer network were obtained by fitting the minima of the wave guide modes and plasmons, and are shown in Table S5. The swelling ratio of the polymer network was calculated as ' =

()*+,-. (/01

. SPR curves of the swollen and non-swollen material are

shown in Figure S9. Protein adhesion study. Protein adsorption was studied using SPR in the kinetics mode using thinner samples, as reported previously.21 These only had plasmon signals but no waveguides modes. Full angular reflectivity scans of the dry samples were taken as described above. The angle was set to 23 = 2 − 1 (on the left flank of the plasmon peak), and the signal intensity at that angle was monitored vs. time (kinetics mode). The baseline intensity against buffer was recorded for about 20 min. Next, fibrinogen or lysozyme solution was injected (concentration 1 mg mL-1, in HEPES buffer, flow rate 50 µL min-1) until the adsorption reached equilibrium (plateau in the curve). Next, buffer was injected to remove any loosely adhering protein. To quantitatively determine the average thickness of the adsorbed protein layer after the kinetics 14


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experiment, the surfaces were rinsed with MilliQ water for 15 min to remove residual salt, and dried under nitrogen flow, and another full angular reflectivity curve was measured. This curve was also modeled based on the Fresnel equations using the Winspall software. The amount of adsorbed protein on the surface was calculated by: Γ =

5

=

5∙∆(

∙

5∙∆( 5

= 8 ∙ ∆9 (Γ = average

protein layer thickness (in ng mm-2), m = mass of adsorbed protein, A = surface area, ∆d = protein layer thickness, ρ = density of the protein. The literature value for the density of the fibrinogen is 1.085 g cm-3, that of lysozyme is 1.185 g cm-3.35 Human blood serum and plasma adhesion study. For the adhesion study of the blood components, first serum and plasma were isolated from freshly drawn human blood. To recover the plasma, the blood was collected in EDTA-coated plastic tubes (S-Monovette® KaliumEDTA, Sarstedt AG & Co, Nümbrech, Germany) and centrifuged at 3000 rpm (relative centrifugal force (rcf) 775 g, radius of the centrifuge rotor r = 7.7 cm) for 5 min in 2 mL Eppendorf tubes (Eppendorf AG, Hamburg, Germany). The yellow supernatant, i.e. the pure plasma was used for the adhesion study. To recovery the serum, the whole human blood was collected in a plastic tube containing granulated material coated with silicate (S-Monovette® Serum-Gel, Sarstedt AG & Co, Nümbrech, Germany) and allowed kept upright under ambient conditions until the blood clotting process was finished (about 60 min). The yellow supernatant thus obtained was transferred into 2 mL Eppendorf tubes (Eppendorf AG, Hamburg, Germany) and centrifuged at 3000 rpm (rcf 775 g) for 5 min. The yellow liquid thus obtained was 100% serum. It was used for the adhesion study as pure serum an in a 1:9 dilution (10% (v/v) serum). To obtain 10% (v/v) serum, the sample was diluted by adding HEPES buffer (1 mL, 10 mM, 154 mM NaCl, pH 7.4 Merck KGaA, Darmstadt, Germany). To study the kinetics of blood 15



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plasma or serum adhesion on the model surfaces, surface-attached PZI, PCB, PSB or SMAMP networks were mounted in the SPR instrument. First, the angular reflectivity curve of the dry sample was measured. It was then exposed to a HEPES buffer solution (6 mL, 10 mM, 154 mM NaCl, pH 7.4 Merck KGaA, Darmstadt, Germany) for 15 min under ambient conditions. Then, either plasma or serum was injected into the SPR flow cell. After about 15 – 20 min, an adsorption equilibrium was typically reached, and the surface was rinsed with HEPES to remove reversible adsorbed plasma or serum components. After a short rinse with deionized water to remove salt from the HEPES buffer, the surface were dried by a nitrogen flow, and scan measurements of the dry polymer surface was performed to estimate the total surface coverage with blood proteins (compared to the initial measurement). The change of reflectivity monitored during exposure of the surface-attached PZI, PCB, PSB and SMAMP network to human blood is shown in Figure 5. The baseline for each polymer surface was adjusted (lowered or raised) to a common level for easier comparison of the results. Bacterial adhesion study. For the bacterial adhesion study, suspensions of Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 29523) bacteria at concentrations of 106 and 108 bacteria per mL were used. For the experiment, the bacterial suspensions were freshly prepared. Overnight cultures were prepared in tryptic soy broth (TSB, Merck, Darmstadt, Germany). Log-phase cultures were prepared from the respective overnight-culture by dilution a certain volume into fresh TSB culture medium and incubating for 3–4 h. The optical density of these cultures was measured using a Smart-Spec plus spectrophotometer (Bio-Rad, Life Science Group, Hercules, USA) at 595 nm. A bacterial solution with a concentration of ca. 106 and ca. 108 colony forming units (CFUs) per ml were prepared for each bacterial strain by dilution in 0.9% saline solution. To study the kinetics of bacterial adsorption, the surface-attached PZI, 16


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PCB, PSB or SMAMP networks were mounted in the SPR instrument. First, the angular reflectivity curve of the dry sample was measured. It was then were exposed to 0.9% NaCl solution (physiological saline) for about 15 min under ambient conditions. Then, the respective bacterial suspension was injected into the SPR flow cell. After about 20 – 30 min, the surface was rinsed with NaCl solution to remove reversible adsorbed bacteria. After short rinsing with deionized water, angular reflectivity scans of the dry polymer surface was performed and compared to the initial scan measurement to estimate the surface coverage with bacteria.

Bioactivity Studies. Antimicrobial Activity. The antimicrobial activity of the PZI, SMAMP, PSB, and PCB networks was tested as reported previously.37 S. aureus (ATCC29523) and E. coli (ATCC25922) were used. In short, the test surfaces were sprayed with a suspension of bacteria and incubated for a defined contact time. The surviving bacteria where then transferred onto agar plates and further incubated to grow colonies. These colony forming units (CFUs) were counted and reported as % Growth relative to the negative and positive control. The negative control (growth control) was an uncoated silicon wafer piece; the positive control was a silicon wafer that had been incubated with chlorohexidine digluconate (CHX). The growth percentage was calculated as % ;?ℎ =

ABC)D+- $ ABCD*). E*.0*+

ABC-F. E*.0*+ $ ABCD*). E*.0*+

∙ 100. The data obtained is shown in Figure S8.

Cell compatibility. Cell compatibility was tested with the Alamar blue assays using previously reported procedures21 (with human gingival mucosal keratinocyte cells, approved by vote 381/15 of the Ethics Board of the Albert-Ludwigs-University, Freiburg, Germany). Optical micrographs of the keratinocytes grown on PZI, PSB, SMAMP and PCB and the untreated glass slides 17



Page 18 of 51

(growth control) were taken using a phase contrast objective (10x magnification) on a Leica DMIL microscope with a Leica D-LUX-3 CCD camera (10x magnification). Live-Dead staining of keratinocytes grown on polymer networks was also performed as reported previously using a Keyence BZ-9000E fluorescence microscope.21 Green fluorescence was measured at about 490 nm excitation; red fluorescence at about 536 nm. The image contrast was adjusted to better visualise the staining.

Results. Part 1: PCB - an additional reference molecule. In the first part of this study, the importance of the carboxylate and primary ammonium groups of PZI for its antimicrobial activity was to be investigated. This was not possible with the reference molecules of our previous study21 (which were the polycationic, antimicrobial SMAMP, and the protein-repellent, not intrinsically antimicrobial polysulfobetaine PSB) since none of them carried a carboxylate group. Therefore, we included the polycarboxybetaine PCB as an additional reference molecule in this study (Figure 1). Thus, the sample set consisted of the four molecules shown in Figure 1: PZI carries a primary ammonium and a carboxylate on each repeat unit, which are both -responsive. PCB also has a -responsive carboxylate group, but a permanently charged quaternary ammonium group. PSB, the other reference polymer, has a -inert sulfonate group in addition to its permanently charged quaternary ammonium group. Thus, by a comparison between PCB and PSB, it should be possible to relate differences in physical or biological activity to the two different anion moieties and thus clarify the role of the carboxylate group. Additionally, a comparison of the properties of PZI with PCB should allow differentiating between the impact 18


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of a primary and a quaternary ammonium group. The synthesis of surface-attached PCB networks was analogous to the previously reported procedure for the other three polymers:17, 21, 25 In short, PCB polymer was dissolved in trifluoroethanol, mixed with the tetrafunctional UVcrosslinker pentaerythritol-tetrakis-(3-mercaptopropionate) (=tetrathiol) and spin-coated onto substrates that had been pre-functionalized with a benzophenone-containing anchor groups.21, 25 These polymer layers were UV-irradiated, which initiated thiol-ene reactions between the tetrathiol and the polymer double bonds, and also triggered the CH-insertion reactions between the substrate-attached benzophenone groups and C-H groups of nearby polymer chains (Figure 1). Full details of the procedure have been reported elsewhere.17,

21, 25

The basic physical and

biological characterization of the additional reference molecule PCB can be found in the supporting information of this paper (FTIR spectrum: Figure S1; XPS data: Table S1, AFM images: Figure S2; zeta potential measurement Figure S3, swelling studied by SPR: Table S2; Fibrinogen adhesion studied by SPR: Table S3 and Table S4). A summary of the data thus obtained for PCB is included in Table 2. The physical characterization and the first set of biological experiments on PZI, PSB and SMAMP were already included in our previous report.21 For the readers convenience, we also summed up the key results on PZI, SMAMP and PSB in Table 2, and present them along with our new data on PCB in the text below.

19



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Table 2: Physical characterization data of the surface-attached PZI, PCB, PSB and SMAMP networks. The layer thickness was determined by ellipsometry, the rms roughness was obtained by atomic force microscopy. CA = contact angle. Fibrinogen adhesion was determined by surface plasmon resonance spectroscopy. The maximal surface charge in acidic medium (ζmax), the surface charge at physiological (ζphys), the isoelectric point IEP and the acid constant of the acid base pairs of the polymers were determined by electrokinetic measurements. a) Data obtained by extrapolation of the curve.

Polymer

Thickness / nm

PZI

86 ± 1

PCB

89 ± 3

PSB

71 ± 3

SMAMP

152 ± 1

CA / ° Static Adv. Rec. 21 ± 2 37 ± 1 14 ± 1 36 ± 2 55 ± 3 21 ± 3 37 ± 2 56 ± 2 22 ± 2 70 ± 3 68 ± 3 17 ± 2

FibrinoRough- Swellgen ability ness adhesion / nm in H2O / ng mm-2

ζmax

IEP

/ mV

ζphys / mV

GH

antimicro. activity E.coli S. aureus

cell viability

5.0 ± 0.3

1.9

0

51 ± 5

6.5 ± 0.1

-29 ± 5

3.5 ± 0.2 5.9 ± 0.2

high high

good

17 ± 1.0

1.9

0.1

50 ± 7

5.4 ± 0.1

-28 ± 5

3.0 ± 0.2 6.5 ± 0.2

good good

medium

19 ± 2.0

1.6

0

0 a)

2.4a)

-34 ± 5

n/d

low low

good

2.0 ± 0.2

1.2

8±2

86 ± 5

7.4 ± 0.1

-2 ± 5

6.5 ± 0.2

high high

lower

For all polymers, the network thickness was determined by ellipsometry, and similar thicknesses were aimed at for the three polyzwitterions in order to have comparable results. The relative hydrophilicity of each compound was estimated by contact angle measurements. The contact angles of PCB and PSB were almost identical, indicating that these molecules were almost equally hydrophilic. PZI was more hydrophilic than the two, while SMAMP was more hydrophobic since it only has a single charge per repeat unit, and an additional hydrophobic propyl group. The swellability (= swollen layer thickness/dry layer thickness) and the fibrinogen adhesion on the networks was studied by surface resonance spectrometry. The swellability ratios of PZI and PCB were almost equal, and significantly larger than that of PSB, even though the contact angles of PCB and PSB were identical. This apparent contradiction can be explained by 20


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the much higher surface roughness of both PCB and PSB, which could cause pinning effects and thus lead to larger contact angles, and thus a larger apparent hydrophobicity. A possible explanation for the stronger swelling of PCB and PZI, compared to PSB, is that both have carboxylate groups, which are not permanently charged at a neutral . PSB, on the other hand, has two permanently charged groups, and thus more electrostatic interactions between its oppositely charged ion pairs at neutral . This could lower the swellability in spite of a comparable hydrophilicity. The pH-dependent zeta potential of the polymers was measured by electrokinetic measurements; the thus obtained titration curve for PCB, and the fit to this curve (compared to the other three previously reported polymers) is shown in Figure 2.

Figure 2. Zeta potential titration curves (ζ versus H) obtained by electrokinetic measurements for PZI, PCB, PSB and SMAMP networks, together with the respective data fits. (Note: the second of PCB is probably ring opening under basic conditions). The titration curves (ζ potential versus H, Figure 2) of all four samples are markedly different. Expectedly, the polycationic SMAMP has the highest ζmax in acidic conditions. PZI has a higher

ζ potential than PCB for most parts of the titration curve, particularly at lower . Consequently, 21



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the isoelectric point of PCB is slightly lower than that of PZI. Interestingly, the ζ potential of PCB under physiological conditions is very similar to that of PZI. PCB has two values: one at pH 3.0 (corresponding to the protonation - deprotonation equilibrium of the carboxylate), the other at pH 6.5 (ring opening of the imide). We also report two values for the PZI here, the first one at pH 3.5 (corresponding to the protonation - deprotonation equilibrium of the carboxylate), the other at pH 5.9 (corresponding to the protonation - deprotonation equilibrium of the primary ammonium group). In our previous report,21 had overlooked that second equilibrium, and indeed it is difficult to exactly locate these values based on graphical determination from the titration curves. However, in the light of the overall experimental data, these graphically determined values are more plausible than the previously reported, calculated values.21 It has been previously reported that the of the COO/COOH equilibrium in polycarboxybetaines depends on the spacing between the charges: the longer the spacing, the higher the .38 In the case of the PZI, the two charges are not connected by a spacer. However, due to their preorganization in the exo-exo-configured oxanorbornene repeat unit, and the flexibility of the ethylene spacer to which the primary ammonium group is attached, one can imagine that these two charges can have a quite short distance, which would explain the relatively low value of the COO/COOH equilibrium in PZI. The zeta potential titration curve of PSB indicates that PSB is a "true" polyzwitterion, i.e. that its charge is almost unaffected by changes over a large range,22 unlike PCB and PZI, whose surface charge is affected by protonation-deprotonation equilibria. Interestingly, PSB, PCB and PZI have all negative zeta potentials at physiological , and these values differ only by 6 mV. This is consistent with the finding that all three polyzwitterions have very low protein adhesion when exposed to fibrinogen solution (Table 2): At these negative surface potentials, the also negatively charged protein fibrinogen will not gain 22


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sufficient adhesion energy to irreversibly attach to the hydrophilic polyzwitterion surfaces. Similarly low protein adhesion for polyzwitterions has been previously reported by other groups.35, 39 Toxicological studies of PZI, SMAMP and PSB have been previously reported.21 The additional toxicity studies of PCB can be found in the supporting information of this paper (Figure S4: Alamar Blue toxicity test; Figure S5: optical micrographs of cell growth on PCB; Figure S6: life dead stain; all experiments were performed with human keratinocytes). The results are summarized in Table 2. All data indicate that PCB is not particularly conducive to keratinocyte growth. While the live-dead stain indicated that PCB is not toxic, the amount of cells present in the optical experiments showed that the cells cannot proliferate on the PCB surface, presumably due to problems to adhere. In contrast, cells grew significantly better on PSB and PZI. While the cell growth was reduced on PCB due to reduced cell adhesion, the SMAMP surface had, due to its higher positive charge, a higher cell toxicity than the three polyzwitterions.21 The data of the antimicrobial activity assays on PCB using S. aureus and E. coli bacteria are shown in Figure S7. These results are also included in a comparison of the antimicrobial activity of all polymer networks in Figure 3. The data show that like PZI, PCB shows intrinsic antimicrobial activity and not just protein-repellency. The antimicrobial activity of PCB, particularly against S. aureus, is less pronounced than that of PZI, yet it is clearly observable, especially in comparison to the antimicrobial inactivity of PSB (Figure 3). Thus, PCB is another antimicrobial polyzwitterion, only the second one so far reported.

23



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Figure 3: Summary of the antimicrobial activity of the four polymer networks: time-dependent killing of S. aureus (a) and E. coli (b) by PZI, SMAMP, PSB, and PCB (% bacterial growth, expressed as percentage of colony forming units vs. time; percentage bacterial growth was normalized to an uncoated silicon wafer as growth control (100% growth); a silicon wafer treated with chlorhexidine digluconate was used as positive control (0% growth)).

Swelling of PZI networks at different ionic strengths. As discussed above, polyzwitterions have a better solubility in salt solutions than in pure water. This should reflect in their swelling behavior. Using surface plasmon resonance spectroscopy (SPR), we studied the swelling behavior of the surface-attached PZI network. For this, PZI was swollen in aqueous sodium chloride solutions (with concentrations from 0.01 to 1 mol L-1) until equilibrium was reached, i.e. no further 24


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swelling occurred. This point was typically reached after 90 min. Full angular reflectivity curves of the sample in equilibrium were taken, and the sample was then rinsed with the salt solution with the next higher ionic strength. The reflectivity curves thus obtained are shown in Figure S8a in the supporting information. The data shows a systematic shift of the reflectivity minima with increasing ionic strength. The curves were fitted to determine the layer thickness of the swollen layers, and related to the thickness of the dry layer. The results thus obtained are summarized in Table 3a and Figure 4a (swellability = swollen layer thickness / dry layer thickness). The data indicates that the swelling increased systematically with increasing ionic strength of the solvent; however, the strongest effect of additional salt was seen at low salt concentration (up to 0.1 mol L-1). At that concentration, the ion pairs of PZI seem to be sufficiently separated so that the swelling at 0.1 mol L-1 NaCl is significantly stronger than in pure water. Thus, under physiological conditions (0.154 mol L-1), the PZI networks are swollen and their functional groups are thus bioavailable. Table 3: Swellability of surface-attached PZI, PCB and PSB networks in NaCl solutions a. with different NaCl concentrations; and b. at different pH values but constant ionic strength (0.15 mol L-1). Swellability = swollen layer thickness / dry layer thickness. a. Swellability PZI

NaCl solution / mol L-1 0.001

0.01

0.1

0.6

1M

1.63

1.66

1.73

1.74

1.78

b. Swellability

NaCl solution pH 3

pH 4

pH 6

pH 7

pH 8

PZI

1.92

1.99

2.06

2.06

2.11

PCB

1.76

-

-

1.94

-

PSB

1.66

-

-

1.82

25



Page 26 of 51

Figure 4. a. Swellability (= swollen layer thickness/dry layer thickness) of surface-attached PZI networks as a function of the salt concentration cNaCl at pH = 5. The dry thickness of the PZI layer was 1052 nm. b. Swellability of surface-attached PZI (●), PCB (▲) and PSB (♦) networks as a function of pH (cNaCl = 0.15 mol L-1). The dry thickness of the PZI, PCB and PSB layer was 1072 nm, 230 nm and 683 nm, respectively.

2.2 - Swelling of polyzwitterion networks at different pH values. To investigate the effect of increasing on swellability, PZI swelling was monitored for different values at constant ionic strength (c = 0.15 mol L-1 NaCl, Figure 4b; full reflectivity curves are given in Figure S8b). A slight increase of swelling (about 20%) with increasing was also observed in this series of experiments. The same trend was observed for the PCB and PSB networks, whose swelling was determined at 3 and 7, respectively - both networks were also more swollen at higher . (In line with theory, the swelling of all networks at 0.15 M NaCl and 7.4 was also stronger than their swelling in pure water; Table 2.) Overall, PZI was more strongly swollen than PCB and PSB at 3 and 7. 26


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2.3 - -dependent adsorption of proteins on PZI networks. In a previous report, we had only studied the adhesion of fibrinogen on PZI under near-physiological conditions.21 To get more detailed insight into the robustness of the observed protein-repellency of PZI, we studied the adsorption of the two proteins fibrinogen and lysozyme on the PZI network at different values. Fibrinogen was chosen as a test protein because it is a large protein with a relatively high molecular weight (M = 341 kg mol-1) and has an isoelectric point (IEP) of around 6. It thus has a net negative charge under physiological conditions. Lysozyme, on the other hand, is relatively small (M = 14.4 kg mol-1) and has an IEP at 10.9.35 It is thus positively charged under physiological conditions. The protein adsorption studies were monitored by SPR in the kinetics mode to get a qualitative impression of the amount of protein adsorbed and of the timedependency of the adhesion process. Additional full reflectivity scans on the dry materials before and after protein adhesion were used to quantify the mass of adsorbed protein. In the kinetics experiments, first the baseline value of the reflectivity at constant angle was recorded. Then, protein solution was flown over the PZI surfaces (black arrows in Figure 4). When equilibrium was reached, the surfaces were washed with buffer to remove reversibly adhered protein (open arrows in Figure 5). The data thus obtained for fibrinogen and lysozyme adsorption at 4, 7 and 8 is shown in Figure 5, where the change in reflectivity is plotted versus time. The full SPR reflectivity curves of the dry surfaces before and after protein adhesion are shown Tables S6 and S7 in the supporting information. These were modelled using the Fresnel equations to determine the thickness of any adhered protein. This data shows that zero irreversible protein adhesion was measured for both proteins at all values on PZI. For fibrinogen, the kinetics experiment also shows that also no time-dependent, reversible protein adhesion occurred: After the protein solution was injected (at 15-20 min, black arrows in Figures 5a), the reflectivity value remained 27



Page 28 of 51

unaffected for the duration of the experiment (about 30 min) and also did not alter upon rinsing with buffer (at 35-50 min, open arrows in Figures 5a). Thus, both data sets are consistent and show neither reversible nor irreversible adhesion of fibrinogen on PZI at all three values. For lysozyme, the kinetics experiments show that this protein adheres reversibly to PZI at 7 and 8: first, the reflectivity value increased when the protein was injected (black arrows in Figures 5b), while the curve of the measurement at 4 remained flat. However, when injecting buffer (open arrows in Figures 5b), the adhered lysozyme was quantitatively removed also at high , as the signal returns to the baseline level. Thus, as also observed from the full reflectivity scans, there is indeed no irreversible lysozyme adhesion on PZI. Yet the kinetics data shows that reversible adhesion of lysozyme (and thus of other positively charged proteins) under physiological conditions is possible. This may or may not be relevant in the context of biofilm formation. The difference between fibrinogen and lysozyme can be understood as follows: since the isoelectric point of fibrinogen is at 6, and that of the PZI network is at 6.5, both should have a similar zeta potential over the range investigated: positive at 4, slightly negative at 7, and negative at 8. Thus, they would repel each other at all three values, and therefore no fibrinogen adhesion was observed. Lysozyme, on the other hand, with its IEP at 10.9, is positively charged at all three values. Thus, at 4, when PZI is also positively charged, no lysozyme adhesion is observed. At 7, there is an attractive interaction between the slightly negatively charged PZI and lysozyme, and even more so at 8, when PZI is more strongly negative, but lysozyme still positively charged. Yet overall, this electrostatic interaction is still reversible, possibly due to a low overall number of charges at the contact area between PZI and the protein. Nevertheless, the data demonstrates that the zeta potential of both the PZI surface and the protein strongly impact the outcome of the protein adsorption studies. 28


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Figure 5: Time-dependent adsorption and desorption of fibrinogen (a.) and lysozyme (b.) on PZI networks at different values, studied by SPR kinetics measurements. At the time points marked by the black arrows, protein solution was flown over the PZI surfaces. About 30 min later (open arrows), the surface was washed with buffer to remove reversibly adhered protein. Upon exposing PZI to fibrinogen, only little change in reflectivity was detected, whereas the reflectivity increased strongly upon contact to lysozyme solution at higher .

2.4 - Adsorption of proteins on PZI, PCB and PSB networks in the presence of bivalent counterions. In real life, protein adhesion on surfaces occurs in the presence of many other species, among them multivalent cations. These could enhance the adsorption of negatively charged proteins like fibrinogen. To investigate this effect in a well-defined model system, a solution of fibrinogen and magnesium chloride in PBS buffer (at physiological ionic strength and pH) was prepared and flown over PZI, PCB and PSB networks. Again, the protein adhesion was 29



Page 30 of 51

monitored by SPR in the kinetics mode (Figure 6). For PZI, minimal reversible adsorption was observed (Figure 6a), indicating that protein adsorption was minimally enhanced in the presence of Mg2+. For PCB, the kinetics curve remained smooth, indicating no detectable adsorption of the protein, just like in the absence of magnesium ions (Table S3). On PSB, on the other hand, a measurable hump in the curve was observed, which indicates significant reversible adhesion on that surface. However, all curves return to baseline after flushing the surfaces with buffer, indicating that fibrinogen adhesion was fully reversible in all cases. This result was confirmed by the full reflectivity scans on the dry surfaces before and after protein adhesion, which indicated that there was no irreversible protein adhesion (Tables S8 and S9, respectively). The reversible protein adhesion in the presence of Mg2+ can be understood with reference to the dependency of the zeta potential (Figure 2). PZI, with an isoelectric point at 6.5 in the absence of Mg2+, will be slightly negatively charged at physiological due to the adsorption of anions from the medium. Thus, a small fraction of Mg2+ could adhere to the PZI surface, thereby generating a slight net positive charge excess. This charge imbalance could then enable the adhesion of a small amount of fibrinogen, which is also negatively charged and thus would not adhere in the absence of Mg2+. Since the fibrinogen adhesion was low and fully reversible, such a charge imbalance can only be small. However, in real-life samples, e.g. when mammalian cells attempt to adhere to PZI, this charge imbalance in the presence of bivalent cations might be sufficient to induce cell attachment. Interestingly, not even reversible protein adhesion was observed for PCB, even though PCB is also net negatively charged at physiological value and has a zeta potential similar to PZI. Both PCB and PZI have carboxylate ions, which are known to bind Mg2+. However, as known for example from the titration of Mg2+ with EDTA (a common lab class experiment), the ability to of carboxylates to bind Mg2+ is -dependent and is most 30


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effective at basic . Thus, neither PCB nor PZI bind Mg2+ irreversibly under physiological conditions through the carboxylate groups, and the differences observed for PZI and PCB should be related to the permanently charged ammonium group of PCB. Overall, Mg2+ does not seem to bind to PCB, presumably because of local electrostatic repulsion by the NR4+ groups, and thus there is no fibrinogen adhesion, just like in the absence of Mg2+. PSB, on the other hand, consists of two permanent charges, RSO3- and NR4+, and has a negative zeta potential in the entire range investigated in the zeta potential titration curves (Figure 2). Thus, the negatively charged sulfonate groups are available for Mg2+ binding. This could lead to a more permanent positive charge excess on PSB than on PZI, which thus becomes more adhesive for fibrinogen in the presence of bivalent ions. However, as the data indicates, the interaction with the magnesium ions still remains reversible. Nevertheless, we observed that PCB, with not even reversible protein adhesion in the presence of Mg2+, was strongly repellent for mammalian cells in the cell studies here reported (Figures S5 and S6), while these cells grew well on both PZI and PSB, which showed reversible protein adhesion when Mg2+ was present. This might be related to the response of the two materials to the presence of bivalent cations.

31



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Figure 6: Kinetics of fibrinogen adsorption in the presence of divalent cation Mg2+ studied by SPR kinetic measurements: time dependent adsorption and desorption fibrinogen (in PBS buffer, 1 mM MgCl2) on PZI (a.), PCB (b.) and PSB (c.). The black arrows indicate injection of fibrinogen, the open arrows mark injection of buffer. Upon exposing PZI, PCB and PSB to fibrinogen in the presence of Mg2+, only little change in reflectivity was detected. After washing with buffer, the reflectivity signal returned back to start values.

Part 3: Interaction with Biological Fluids. 3.1 - Adsorption of human plasma and serum on PZI, PCB, PSB and SMAMP networks. The interaction of biomaterials with real biological fluids like plasma and serum is crucial for any biomedical applications. To that end, we studied the adhesion of plasma and serum obtained from 32


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fresh human blood on the PZI, PCB, PSB and SMAMP surfaces using SPR kinetics studies and full angular reflectivity measurements. As is well known, plasma is obtained from fresh blood by adding EDTA as an anti-coagulant. It is then centrifuged, so that all blood cells precipitate, and the supernatant plasma can be recovered. Plasma proteins cannot coagulate further, however plasma still contains all coagulation factors including fibrin and fibrinogen. The main components of plasma are proteins (60-80 g L-1, corresponding to 6-8 mass%), lipids (4.5-8.5 g L-1), inorganic ions, and a small amount of organic acids. Isolated plasma has a of around 7.95.40 The kinetics of plasma adsorption on PZI is shown in Figure 7a. While the plasma proteins adhere to a much larger extent to PZI than pure fibrinogen (black arrow in Figure 7a), this adhesion and is fully reversible, as all proteins are washed off upon buffer injection (open arrow in Figure 7a). This was also confirmed with full reflectivity scans of the dry material before and after plasma adhesion (Table S10 and S11 in the supporting information), which indicate that 0 ng mm-2 plasma protein (within the accuracy limit of the method) were adsorbed. The kinetics of plasma adhesion on PCB and PSB (Figure 7a) were qualitatively similar, however the dry scans before and after plasma treatment (Table S12 and S13 in the supporting information) indicate that 0.28 ng mm-2 of irreversibly adhered plasma protein remained on PCB, while 0.47 ng mm-2 was adsorbed on PSB. For the SMAMP surfaces, it is already apparent from the kinetics experiment (Figure 7a) that the adsorption of plasma proteins on SMAMP would be irreversible. When plasma is injected, the adsorption increase was significant (about 8 a.u.s), and the curve did not return to baseline level when washed with buffer. Full reflectivity scans before and after plasma treatment (Table S14 and S15 in the supporting information) confirm this observation: overall, 6.3 ng mm-2 of plasma protein were adsorbed.

33



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Figure 7: Kinetics of human blood plasma and serum adsorption on PZI, PCB, PSB and SMAMP networks, studied by SPR kinetic measurements: time dependent adsorption and desorption of a. human plasma and b. human serum at physiological and ionic strength. After exposure of the network to plasma or serum (black arrows), the surfaces were rinsed with buffer (open arrows).

The same experiments were repeated with human serum. Unlike plasma, serum is obtained from a coagulated blood sample, the blood cells of which have been removed by centrifugation. Thus, serum does not contain anticoagulants, yet it also contains less coagulation factors, notably less fibrinogen. Otherwise, the composition of serum is similar to that of plasma; i.e. the protein content (about 7 mass%) mainly consists of albumin and different globulins, and a lower amount 34


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of fibrinogen. Serum has a slightly higher than plasma, around 8.18.40 The kinetics of serum adsorption on PZI is shown in Figure 7b. Interestingly, the serum adsorption on PZI is a two stage process: first, one species is adsorbed and the curve gradient decreases, then another species is adsorbed which apparently displaces the first one. This has been previously reported in the literature for other materials and is reported as the Vroman effect.41 While the plasma adhesion was fully reversible, the adhesion of proteins from 10% serum was partially irreversible (0.1 ng mm-2 of plasma protein was adsorbed, see Tables S10 and S11 in the supporting information). When full reflectivity scans were taken on dry PZI before and after exposure to undiluted serum proteins, it was found that 7.53 ng mm-2 of serum protein was irreversibly adsorbed on PZI (Table S10 and S11). When comparing that data to the serum protein adhesion on PCB and PSB, it is found that on these materials, no Vroman effect was observed (Figure 7b). The serum adhesion on PCB and PSB, according to full reflectivity scans on the dry materials (Tables S12 and S13 in the supporting information)), was 0 ng mm-2 and 0.27 ng mm-2, respectively. The highest irreversible serum adhesion was observed for the SMAMP network (Figure 6b, Tables S14 and S15 in the supporting information), with 8.35 ng mm-2. The reason why only PZI and none of the other polymer hydrogels show the Vroman effect when exposed to serum is not yet understood. The general difference in plasma and serum adhesion of the four polymers can either be related to the difference of the protein constituents of serum and plasma, respectively, or to the different of these two fluids. The common structural feature of the two serum-adhesive materials, PZI and SMAMP, is their -responsive primary ammonium group. The common feature of the two serum-repellent materials, PCB and PSB, is that they have permanently charged NR4+ groups. Since serum is more basic than plasma, both PZI and SMAMP will be

35



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more strongly deprotonated in serum than in plasma. This could facilitate the interaction with the bivalent cations in the fluid, and thus trigger irreversible protein adhesion on PZI and SMAMP. The isoelectric point of albumin, the most abundant serum protein, is at 4.6. It is thus negatively charged under physiological conditions and thus not surprisingly would adhere to the SMAMP network. Apparently, fluctuating charges on the PZI surface, which presumably also make it antimicrobial,21 could be the reason why albumin adheres to the PZI. Yet fibrinogen, which has a similar isoelectric point, adheres reversibly, while some albumin and/or the other serum proteins remain on the surface after flushing with buffer. 3.2 - Adsorption of E. coli and S. aureus bacteria on PZI, PCB, PSB and SMAMP networks. In our previous report on PZI,21 we conducted time-kill experiments and biofilm formation experiments with E. coli and S. aureus bacteria on PZI, SMAMP and PSB. These indicated that PZI and SMAMP were indeed antimicrobial (Figure 3) while PSB was only protein-repellent and passively reduced biofilm formation. Our new data on PCB (Figures S7 and Figure 3) also indicated antimicrobial activity of this polyzwitterion. To look more closely into the interaction of bacteria with these networks, we also studied the in-situ bacterial adhesion on them by surface plasmon resonance spectroscopy. The SPR kinetics curves of E. coli and S. aureus adsorption on PZI at a bacterial density of 106 and 108 bacteria per cm3, respectively, are shown in Figure 8a and b. The respective full reflectivity scans on the dry material are shown in Table S16 and S17 in the supporting information. The data indicates that a small amount of reversible adhesion is observed, and that in all four experiments, the bacteria were fully removed after rinsing with buffer (corresponding to a bacterial load of 0 ng mm-2). Since the bacterial interaction with PZI was fully reversible even at 108 bacteria per cm3 (corresponding to the bacterial density in the 36


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human small intestine!), the experiments on PCB, PSB and SMAMP were only performed with this high bacterial density. The kinetics data of the experiments on PCB and PSB are shown in Figure 8c and d, respectively. The corresponding full reflectivity scans on the dry materials are shown in Tables S18-S21 in the supporting information. For PCB, 0.02 ng cm-2 E. coli were irreversibly adsorbed, while no S. aureus adsorption was observed. For PSB, the trend was opposite, and 0.00 ng cm-2 E. coli were irreversibly adsorbed, while a significant amount of S. aureus (1.80 ng cm-2) adhered irreversibly. It is interesting to note that PSB, the permanently charged polyzwitterion, was the least bacteria-repelling of the polyzwitterions. PZI and PSB, on the other hand, with a -responsive carboxylate group, did not enable the bacteria to adhere significantly. Since bacteria are negatively charged, they would need permanent positive charges on the surface to adhere. This is not found in PZI, but in PCB and PSB. Apparently, the responsive PCB stays sufficiently polyzwitterionic even in the presence of bacteria to prevent a large amount of adhesion, whereas PSB gets contaminated. In a final experiment involving bacteria, the bacterial adhesion of the SMAMP surface with E. coli and S. aureus was tested (Figure 8e, full reflectivity scans on the dry material are shown in Tables S22-23 in the supporting information). The data shows that 1.6 ng mm-2 of E. coli and 26.9 ng mm-2 of S. aureus were adsorbed on SMAMP, which is thus, as expected, highly protein adhesive.

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Figure 8: Kinetics of Escherichia coli and Staphylococcus aureus bacteria adsorption on different surfaces at physiological pH in saline solution, studied by SPR kinetics measurements. a. PZI / 106 bacteria mL-1, b. PZI / 108 bacteria mL-1. In both cases, no irreversible bacteria adsorption was detected. c. PCB / 108 bacteria mL-1, d. PSB / 108 bacteria mL-1 (a dotted line indicates a baseline), e. SMAMP / 108 bacteria mL - 1.

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Table 3: SPR results of protein adsorption (fibrinogen and lysozyme), adsorption of human blood plasma and serum, and bacterial adsorption (E. coli and S. aureus) measured for surface-attached PZI, PCB, PSB and SMAMP networks. Adsorption / ng mm-2 Conditions

Fibrinogen

pH 4, HEPES

0

-

-

-

pH 7, HEPES

0

-

-

-

pH8, HEPES

0

-

-

-

0

0

0

-

pH 4, HEPES

0

-

-

-

pH 7, HEPES

0

-

-

-

pH 8, HEPES

0

-

-

-

0

0.28

0.47

6.3

100% serum

7.53

0

0.27

8.35

10% serum

0.1

-

-

-

106 bacteria per cm3

0

-

-

-


0

0.02

0

1.6


0

-

-

-


0

0

1.80

26.9

1mM MgCl2 + PBS, pH 7.4

Lysozyme

PZI PCB PSB SMAMP

100% Human blood plasma

Human blood serum

E. coli

S. aureus

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The results of all SPR investigations have been summarized in Table 3. Overall, PZI resisted to irreversible adhesion of all fluids, except serum. PCB had no serum adhesion, but showed slight adhesion of plasma proteins and of E. coli bacteria. PSB, on the other hand, had adhesion of S. aureus but not of E. coli, and also had a low amount of plasma and serum adhesion. In literature reports on a poly(methacrylate)-based poly(carboxybetaine), it was reported that protein adhesion was -sensitive. Protein adhered at < 5, but not above, corresponding to a protonation of the carboxylate group at low , which shifted the charge of the material to polycationic.39 On the other hand, the protein adhesion of a poly(methacrylate)-based poly(sulfobetaine) was insensitive of .42 Our data on PZI also shows a -dependency of protein adhesion, but a very different one. For a protein with a zeta potential and isoelectric point similar to PZI, no adhesion was observed; for proteins with markedly a higher isoelectric point than PZI, reversible protein adhesion occurred for values larger than 5. Thus, the responsiveness of PZI is different to poly(carboxybetaines), presumably due to its primary ammonium group which can be deprotonated at higher values. For polyzwitterions in contact with plasma and serum, Jiang and coworkers had previously reported that polycarboxybetaines had less plasma adhesion than polysulfobetaines, and that the same trend was also observed for serum.43 This was also observed here when comparing PCB and PSB. This finding was explained by the observation that carboxybetaine units are more strongly hydrated than sulfobetaine units,44 and that the charge densities of the respective anions and cations in a polycarboxybetaine are more dissimilar on the corresponding polysulfobetaine.44 For the difference in plasma and serum adhesion (plasma adsorbed less than serum on polycarboxybetaine and polysulfobetaine,43, 45 analogously to the results for PCB and PSB here 40


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reported), Jiang proposed that the anticoagulants present in plasma help keep proteins in their native state, while they denature in serum more easily and thus adhere to the surface.45 In addition to that explanation, the difference between plasma and serum, which is 0.2 units, might account for the different protein adhesion behavior of polyzwitterions in these two fluids. Particularly PZI, with its responsive primary ammonium group, will lose its zwitterionic characteristics at higher , which might explain the high serum adhesion of PZI. To fully understand this behavior, additional experiments with plasma proteins at different will be needed.

Conclusion. In this study, we conducted a number of comparative experiments with the surface-attached polymer networks PZI, PCB, PSB and SMAMP with the aim to understand why the PZI network can be simultaneously protein-repellent and antimicrobially active. For this, we first characterized the additional reference polymer PCB and found that it was also intrinsically antimicrobial against E. coli and S. aureus bacteria. Since both PZI and PCB carry carboxylate groups in addition to their respective ammonium groups, we postulate that the carboxylate functionality is critical for the antimicrobial activity found. We speculate that these carboxylates are (partially) protonated in the presence of bacteria (maybe due to acidic metabolites secreted by the bacteria), so that the PZI and PCB surfaces become net positively charged and thus antimicrobial. So far, however, we do not have experimental evidence for such a "bacteria responsiveness" of these two polymer hydrogels.

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We also studied the -dependent protein adhesion on PZI in detail and found that neither fibrinogen nor lysozyme adhered irreversibly to PZI over the entire range investigated, yet some reversible interaction with lysozyme was observed, a consequence of the opposite the zeta potentials of PZI and lysozyme at 7 and 8. In the presence of bivalent cations, some reversible fibrinogen adhesion on PZI and PSB was observed, while no interaction with PCB was found. Since mammalian cells also did not grow well on PCB, but on PZI and PSB, we speculate that the ability of the substrate to interact with such cations is prerequisite for mammalian cell adhesion. PZI was the only polymer in the sample set that did not show human plasma adhesion, while PCB and PSB had slight plasma adhesion (0.27 and 0.48 ng mm-2), and SMAMP even more so (6.3 ng mm-2). However, both PZI and SMAMP showed strong serum adhesion, while no serum adhered to PCB, and only little serum adhered to PSB. We speculate that the fact that both PZI and SMAMP have a -responsive primary ammonium group makes them more susceptible to protein adhesion in serum than in plasma. PCB and PSB on the other hand, with their permanently charged NR4+ groups, are insensitive to the change between plasma and serum, and only show little protein adhesion in either medium. From a practical perspective, plasma adhesion is more relevant in in-dwelling medical devices such as venous catheters. Thus, PZI might be a promising material for these devices. Interestingly, both PZI and PCB showed none or only little bacterial adhesion in the SPR studies. Such studies under flow conditions are an interesting model experiment for urinary catheters. Thus, both PZI and PCB could be interesting materials for such devices. In summary, we have performed a number of additional experiments on PZI and its reference molecules. While the carboxylate group seems to be a prerequisite for the dual antimicrobial activity and protein-repellency, the -responsiveness of the primary ammonium group seems to make the molecule vulnerable for protein adhesion in 42


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fluids that are slightly out of the physiological range. These results are of relevance for the design of the next generation antimicrobial and protein-repellent materials. In spite of the observed protein adhesion when exposed to serum, PZI should be studied in more detail, particularly in model experiments that are even more relevant for clinical applications.

Acknowledgement

Funding for this project by the Emmy-Noether-program of the German Research Foundation (DFG, grant ID: LI1714/5-1) and the VIP+ Program of the German Federal Ministry of Education and Research (BMBF, grant ID AntiBug) is gratefully acknowledged. Many thanks to Diana Lorena Guevara Solarte for help with the microbiological experiments.

Supporting Information Physical and biological characterization of polycarboxybetaine (PCB): FTIR spectra, XPS data, AFM images, swellability in water (SPR), fibrinogen adsorption (SPR),

cell toxicity (Alamar

Blue Assay), antimicrobial activity against E.coli and S.aureus. Swellability of PZI in NaCl solutions and at different pH values (SPR); protein adhesion on PZI at different pH values (SPR). Protein adhesion on PZI, PCB and PSB in the presence of Mg²+ (SPR). Human blood plasma, human serum and bacteria adhesion on PZI, PCB, PSB and SMAMP (SPR).

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Simultaneously Antimicrobial, Protein-repellent and Cell-compatible

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