Effect of Poly(Oxanorbonene)- and Poly(Methacrylate)-Based Polyzwitterionic Surface Coatings on Cell Adhesion and Gene Expression of Human Keratinocytes.

Polyzwitterions are generally known for their anti-adhesive properties, including resistance to protein and cell adhesion, and overall high bio-inertness. Yet there are a few polyzwitterions to which mammalian cells do adhere. To understand the structural features of this behavior, a panel of polyzwitterions with different functional groups and overall degrees of hydrophobicity is analyzed here, and their physical and biological properties are correlated to these structural differences. Cell adhesion is focused on, which is the basic requirement for cell viability, proliferation, and growth. With the here presented polyzwitterion panel, three different types of cell-surface interactions are observed: adhesion, slight attachment, and cell repellency. Using immunofluorescence methods, it is found that human keratinocytes (HaCaT) form focal adhesions on the cell-adhesive polyzwitterions, but not on the sample that has only slight cell attachment. Gene expression analysis indicates that HaCaT cells cultivated in the presence of a non-adhesive polyzwitterion have up-regulated inflammatory and apoptosis-related cell signaling pathways, while the gene expression of HaCaT cells grown on a cell-adhesive polyzwitterion does not differ from the gene expression of the growth control, and thus can be defined as fully cell-compatible.

Stimulus-Responsive Polyelectrolyte Surfaces: Switching Surface Properties from Polycationic/Antimicrobial to Polyzwitterionic/Protein-Repellent.

Surfaces coated with polyzwitterions are most well-known for their ability to resist protein adsorption. In this article, a surface-attached hydrophobically modified poly(carboxybetaine) is presented. When protonated by changes of the pH of the surrounding medium, this protein-repellent polyzwitterion switches to a polycationic state in which it is antimicrobially active and protein-adhesive. The pH range in which these two states exist are recorded by zeta potential measurements. Adsorption studies at different pH values (monitored by surface plasmon resonance spectroscopy) confirm that the adhesion of protein is pH dependent and reversible, that is, protein can be released upon a pH change from pH 3 to pH 7.4. At physiological pH, the poly(carboxyzwitterion) is antimicrobially active, presumably because it becomes protonated by bacterial metabolites during the antimicrobial activity assay. Stability studies confirm that the here presented material is storage-stable, yet hydrolyses after longer incubation in aqueous media.

Bifunctional Bioactive Polymer Surfaces with Micrometer and Submicrometer-sized Structure: The Effects of Structure Spacing and Elastic Modulus on Bioactivity.

This study presents a comparison of two types of bifunctional structured surface that were made from the same polymer -- an antimicrobial polycation (a synthetic mimic of an antimicrobial peptide, SMAMP) and a protein-repellent polyzwitterion (poly(sulfobetaines), PSB). The first type of bifunctional surface was fabricated by a colloidal lithography (CL) based process where the two polymers were immobilized sequentially onto pre-structured surfaces with a chemical contrast (gold on silicon). This enabled site-selective covalent attachment. The CL materials had a spacing ranging from 200 nm to 2 µm. The second type of structured surface (spacing: 1 - 8.5 µm) was fabricated using a microcontact printing (µCP) process where SMAMP patches were printed onto a PSB network, so that 3D surface features were obtained. The thus obtained materials were studied by quantitative nanomechanical measurements using atomic force microscopy (QNM-AFM). The different architectures led to different local elastic moduli at the polymer-air interface, where the CL surfaces were much stiffer (Derjaguin-Muller-Toporov (DMT) modulus = 20 ± 0.8 GPa) compared to the structured 3D networks obtained by µCP (DMT modulus = 42 ± 1.1 MPa). The effects of the surface topology and stiffness on the antimicrobial activity against Escherichia coli, the protein repellency (using fibrinogen), and the compatibility with human gingival mucosal keratinocytes were investigated. The softer 3D µCP surfaces had simultaneous antimicrobial activity, protein repellency, and cell compatibility at all spacings. For the stiffer CL surfaces, quantitative simultaneous antimicrobial activity and protein repellency was not obtained. However, the cell compatibility could be maintained at all spacings. The optimum spacing for the CL materials was in the range of 500 nm-1 µm, with significantly reduced antimicrobial activity at 2 µm spacing. Thus, the soft polymer network obtained by µCP could be more easily optimized than the stiff CL surface, and had a broader topology range of optimal or near-optimal bioactivity.

Synthesis and Bioactivity of Polymer-Based Synthetic Mimics of Antimicrobial Peptides (SMAMPs) Made from Asymmetrically Disubstituted Itaconates.

A series of asymmetrically disubstituted diitaconate monomers is presented. Starting from itaconic anhydride, functional groups could be placed selectively at the two nonequivalent carbonyl groups. By using 2D NMR spectroscopy, it was shown that the first functionalization step occurred at the carbonyl group in the ß position to the double bond. These monomers were copolymerized with N,N-dimethylacrylamide (DMAA) to yield polymer-based synthetic mimics of antimicrobial peptides (SMAMPs). They were obtained by free radical polymerization, a metal-free process, and still maintained facial amphiphilicity at the repeat unit level. This eliminates the need for laborious metal removal and is advantageous from a regulatory and product safety perspective. The poly(diitaconate-co-DMAA) copolymers obtained were statistical to alternating, and the monomer feed ratio roughly matched that of the repeat unit content of the copolymers. Investigations of varied R group hydrophobicity, repeat unit ratio, and molecular mass on antimicrobial activity against Escherichia coli and on compatibility with human keratinocytes showed that the polymers with the longest R groups and lowest DMAA content were the most antimicrobial and hemolytic. This is in agreement with the biological activity of previously reported SMAMPs. Thus, the design concept of facial amphiphilicity has successfully been transferred, but the selectivity of these polymers for bacteria over mammalian cells still needs to be optimized.

Simultaneously Antimicrobial, Protein-repellent and Cell-compatible Polyzwitterion Networks: More Insight on Bioactivity and Physical Properties.

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 pH-responsive 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 protein-repellency, 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.

A Simultaneously Antimicrobial, Protein-Repellent, and Cell-Compatible Polyzwitterion Network.

A simultaneously antimicrobial, protein-repellent, and cell-compatible surface-attached polymer network is reported, which reduces the growth of bacterial biofilms on surfaces through its multifunctionality. The coating was made from a poly(oxonorbornene)-based zwitterion (PZI), which was surface-attached and cross-linked in one step by simultaneous UV-activated CH insertion and thiol-ene reaction. The process was applicable to both laboratory surfaces like silicon, glass, and gold and real-life surfaces like polyurethane foam wound dressings. The chemical structure and physical properties of the PZI surface and the two reference surfaces SMAMP ("synthetic mimic of an antimicrobial peptide"), an antimicrobial but protein-adhesive polymer coating, and PSB (poly(sulfobetaine)), a protein-repellent but not antimicrobial polyzwitterion coating were characterized by Fourier transform infrared spectroscopy, ellipsometry, contact angle measurements, photoelectron spectroscopy, swellability measurements (using surface plasmon resonance spectroscopy, SPR), zeta potential measurements, and atomic force microscopy. The time-dependent antimicrobial activity assay (time-kill assay) confirmed the high antimicrobial activity of the PZI; SPR was used to demonstrate that it was also highly protein-repellent. Biofilm formation studies showed that the material effectively reduced the growth of Escherichia coli and Staphylococcus aureus biofilms. Additionally, it was shown that the PZI was highly compatible with immortalized human mucosal gingiva keratinocytes and human red blood cells using the Alamar Blue assay, the live-dead stain, and the hemolysis assay. PZI thus may be an attractive coating for biomedical applications, particularly for the fight against bacterial biofilms on medical devices and in other applications.