YRGDS Fibronectin Fragment

Poly(ethylene glycol):poly(acrylate) PEG-g-PA co-polymers were made that inhibited nonspecific protein and cellular adhesion. PEG-g-PA co-polymers were then covalently modified with either cell adhesion peptides or fragments of antibodies to monocyte/macrophage integrin receptors (anti-VLA4, anti-beta(1), anti-beta(2), and anti-CD64) known to enhance macrophage adhesion and, perhaps, modulate their activation. Peptides were either directly conjugated to the base material or linked by way of PEO-star tethers. Fragments of the antibody region containing the antigen-binding site (Fab' fragments) were coupled to other PEG-g-PA samples using the sulhydryl end groups on Fab' fragments to amine-bearing PEO stars. Macrophage adhesion rates, phagocytic response (oxidative burst), and cytokine expression were determined for each PEG-g-PA material. Luminol-enhanced chemiluminescence was used as a semiquantitative indication of monocyte-macrophage phagocytic activation (oxidative burst). Macrophage cytokine expression in response to control, base, and modified materials was determined by ELISAs for TNF-alpha, IL-1 beta, IL-6, and IL-8. Tissue culture poly(styrene) (TCPS)-mediated the greatest number of adherent monocyte/macrophage cells relative to PEG-g-PA materials. Both YRGDS and YEILDV peptides, whether directly or indirectly (via StarPEO) conjugated to PEG-g-PA, increased adhesion versus controls. Fab' fragments of all four antibodies also promoted enhanced adhesion versus controls. Fab'StarPEO materials presented two orders of magnitude fewer ligands per surface unit area than peptide star materials (10(8) vs. 10(10)), but were able to adhere similar numbers of cells. For surfaces presenting Fab'(VLA-4) or YEILDV, both of which may both bind to a cell's VLA-4 receptor, the Star:VLA4 surface showed a greater number of adherent monocyte/macrophages. This result suggests that the Fab' had a higher affinity to the cell receptor than a corresponding minimal peptide binding sequence. All materials exhibited low oxidative burst (luminescence counts per minute, LCPM) per cell DNA without the addition of exogenous stimuli (LCPM/DNA < 100). Directly conjugated peptide materials, poly(propylene) (PP), and TCPS showed the lowest levels of LCPM/DNA without the addition of exogenous stimulus (LCPM/DNA < 20). There was no correlation between LCPM/DNA ratios, with and without added LPS stimulus, versus the individual substrates. Monocyte/macrophages adherent to TCPS substrata showed the overall highest stimulatory potential in cytokine expression response to exogenous LPS, followed by PP > PEG-g-PA > StarPEO. Cells adherent to peptide-modified materials and Fab'-modified materials were overall less stimulated. The method of presenting the peptides (i.e., directly or via Star PEO) influenced the level of cytokine secreted by the adherent macrophage.

The ability of various surface modifications of poly(ethylene glycol)-graft-polyacrylate (PEG-g-PA) copolymers (tethered adhesion peptides and fragments of monoclonal antibodies) to modulate monocyte-macrophage cell interactions with surface colonizing bacteria is reported. The PEG-g-PA copolymers were made to inhibit nonspecific protein and cellular adhesion. The copolymers were then covalently modified with either cell adhesion peptides (YRGDS, YEILDV, or YRGES) or fragments of antibodies to monocyte-macrophage integrin receptors (anti-VLA4, anti-beta(1), anti-beta(2), and anti-CD64), which are known to enhance macrophage adhesion and perhaps modulate their activation. Cytokine expression and phagocytosis response by surface adherent monocyte-macrophages to Staphylococcus epidermidis and Pseudomonas aeruginosa bacteria were quantified. The cytokine expression (interleukins 6 and 1 beta) of adherent macrophages in response to the modified polymers only and to bacterial challenges were quantified by dynamic ELISA assays. The adherent macrophage phagocytic response (oxidative burst) to various materials is compared to oxidative responses to both opsonized and nonopsonized S. epidermidis and P. aeruginosa bacteria. The efficiency of adherent macrophages to ingest and kill both species was determined using radiolabeled and fluorescent labeled bacterial cell ingestion studies as a function of the PEG-g-PA surface modification. Materials modified with adhesion peptides marginally enhanced (2x) macrophage attachment versus controls but, upon bacterial challenges, these materials predisposed adherent macrophages to overexpress proinflammatory cytokines and to exhibit a significant phagocytic response. Conversely, PEG-g-PA materials modified by fragments of monoclonal antibodies significantly enhanced (7x) macrophage adhesion but, upon bacterial challenge, "per cell" cytokine expression levels were reduced compared to peptide modified materials. Macrophages adhering to antibody fragment modified surfaces also exhibited sustained enhanced phagocytic response and higher bacterial killing efficiencies when compared with peptide modified materials.

Polymer brushes of poly(ethylene glycol) have long been considered the gold standard for antifouling surfaces that resist adsorption of biomolecules and attachment of microorganisms. However, despite displaying excellent resistance to protein adsorption, the polymer brush coatings cannot entirely avoid colonization by bacteria. Here we investigate and identify which non-proteinaceous bacterial adhesins challenge the antifouling properties of polymer brush coatings and how these challenges might be overcome. We quantified biofilm formation on a well-known polymer brush coating of poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) on titanium. The coating successfully resisted colonization by Staphylococcus aureus and Pseudomonas aeruginosa, but not Staphylococcus epidermidis. This colonization pattern was also reflected on the adhesion forces measured on single bacterial cells. The biofilm produced from S. epidermidis on PLL-g-PEG were found to be rich in polysaccharides and extracellular DNA, and quantification of DNA, polysaccharides and proteins on PLL-g-PEG surfaces revealed that although the coating almost fully resisted protein adsorption, polysaccharides could adsorb, and exposure to DNA led to desorption of the polymer from the titanium surface. We hypothesized that this problem could be overcome by increasing the polymer brush density to better resist the penetration of DNA and polysaccharides into the polymer layer. Indeed, high density PLL-g-PEG brushes prepared by the recently discovered temperature-induced polyelectrolyte (TIP) grafting method resisted the interaction with DNA and polysaccharides, and therefore also the colonization by S. epidermidis. The TIP grafting is a simple improvement of PLL-g-PEG brush formation, and our results suggest that it provides an important advancement to the bacterial resistance by polymer brush coatings. Statement of significance: The antifouling properties of poly(ethylene glycol) brush coatings against protein adsorption are well documented, but it is not well understood why these coatings do not perform as well against bacterial colonization when tested against a wide range of species and over periods of days. Here we investigated bacterial colonization on poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) grafted on Ti, and revealed that bacteria relying mostly on polysaccharides and extracellular DNA for adhesion and biofilm formation could successfully colonize PLL-g-PEG coated surfaces. The coatings could not resist adsorption of polysaccharides, and DNA could even desorb the coatings from the Ti surface. Fortunately, the shortcomings of conventional PLL-g-PEG could be overcome by increasing the graft density, using the recently discovered and very simple grafting method, 'temperature-induced polyelectrolyte (TIP) grafting'. Our study highlights that it is of utmost importance to develop coatings which resist adsoprtion of non-proteinaceous bacterial adhesins such as polysaccharides and DNA, and we demonstrated that TIP grafted high density PLL-g-PEG coatings are promising materials to achieve diverse bacterial resistance.