Silver is widely used as a biocidal agent in ointments and wound dressings. However, it has also been associated with tissue toxicity and impaired healing. In vitro characterization has also revealed that typical loadings of silver employed in ointments and dressings (∼ 100 μg/cm2) lead to cytotoxicity. In this paper, we report the results of an initial study that sought to determine if localization of carefully controlled loadings of silver nanoparticles within molecularly thin films immobilized on surfaces can lead to antimicrobial activity without inducing cytotoxicity. Polymeric thin films of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were prepared by layer-by-layer deposition and loaded with ∼0.4 μg/cm2 to ∼23.6 μg/cm2 of silver nanoparticles. Bacterial killing efficiencies of the silver-loaded films were investigated against Staphylococcus epidermidis, a gram-positive bacterium, and it was determined that as little as ∼0.4 μg/cm2 of silver in the polymeric films caused a reduction of 6 log10 CFU/mL (99.9999%) bacteria in suspensions incubated in contact with the films (water-borne assays). Significantly, whereas the antibacterial films containing high loadings of silver were found to be toxic to a murine fibroblast cell line (NIH-3T3), the polymeric films containing ∼0.4 μg/cm2 of silver were not toxic and allowed attachment, and growth of the mammalian cells. Thus, the results of this study go beyond prior reports by identifying silver-impregnated, polymeric thin films that are compatible with in vitro mammalian cell culture yet exhibit antibacterial activity. These results support the hypothesis that localization of carefully controlled loadings of silver nanoparticles within molecularly thin polymeric films can lead to antimicrobial activity without cytotoxicity. More broadly, this strategy of modifying surfaces with minimal loadings of bioactive molecules indicates the basis of approaches that may permit management of microbial burden in wound beds without impairment of wound healing.

ABSTRACTHuman and bovine neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of extracellular trapping and killing of pathogens. Recently, we reported that bovine neutrophils release NETs in response to the important respiratory pathogenMannheimia haemolyticaand its leukotoxin (LKT). Here, we demonstrate macrophage extracellular trap (MET) formation by bovine monocyte-derived macrophages exposed toM. haemolyticaor its LKT. Both native fully active LKT and noncytolytic pro-LKT (produced by anlktCmutant ofM. haemolytica) stimulated MET formation. Confocal and scanning electron microscopy revealed a network of DNA fibrils with colocalized histones in extracellular traps released from bovine macrophages. Formation of METs required NADPH oxidase activity, as previously demonstrated for NET formation. METs formed in response to LKT trapped and killed a portion of theM. haemolyticacells. Bovine alveolar macrophages, but not peripheral blood monocytes, also formed METs in response toM. haemolyticacells. MET formation was not restricted to bovine macrophages. We also observed MET formation by the mouse macrophage cell line RAW 264.7 and by human THP-1 cell-derived macrophages, in response toEscherichia colihemolysin. The latter is a member of the repeats-in-toxin (RTX) toxin family related to theM. haemolyticaleukotoxin. This study demonstrates that macrophages, like neutrophils, can form extracellular traps in response to bacterial pathogens and their exotoxins.

Mannheimia haemolytica is an important member of the bovine respiratory disease complex, which is characterized by abundant neutrophil infiltration into the alveoli and fibrin deposition. Recently several authors have reported that human neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of trapping and killing pathogens. Here, we demonstrate that the leukotoxin (

Acquired resistance to the facultative intracellular bacterium Listeria monocytogenes is thought to require immunologically activated macrophages. Using peritoneal exudate cells from nonimmunized mice in a suspension bactericidal assay, however, we found that peritoneal neutrophils obtained early during the inflammatory process (4 hr after elicitation) and macrophages obtained later during inflammation (maximal listericidal activity at 48 hr after elicitation) were able to kill Listeria in vitro. The kinetics of expression of bactericidal activity by inflammatory neutrophils and macrophages against both L monocytogenes and E coli were similar. Although intraperitoneal immunization or intravenous hyperimmunization markedly enhanced resistance of mice to Listeria in vivo, immunization did not increase the ability of inflammatory peritoneal phagocytes to kill Listeria in vitro. However, in response to intraperitoneal injection of proteose-peptone or dead Listeria, immunized mice mobilized more neutrophils and monocytes into the inflamed peritoneum. These data suggest that, rather than systemic activation of mononuclear phagocyte bactericidal activity, increased mobilization of neutrophils and mononuclear phagocytes into sites of infection may be of prime importance in resistance to listeriosis.

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We report the design of polyelectrolyte multilayers (PEMs) that can be prefabricated on an elastomeric stamp and mechanically transferred onto biomedically-relevant soft materials, including medical-grade silicone elastomers (E′~450–1500 kPa; E′-elastic modulus) and the dermis of cadaver-skin (E′~200–600 kPa). Whereas initial attempts to stamp PEMs formed from poly(allylamine hydrochloride) and poly(acrylic acid) resulted in minimal transfer onto soft materials, we report that integration of micrometer-sized beads into the PEMs (thicknesses of 6–160 nm) led to their quantitative transfer within 30 seconds of contact at a pressure of ~196 kPa. To demonstrate the utility of this approach, PEMs were impregnated with a range of loadings of silver-nanoparticles and stamped onto the dermis of human cadaver-skin (a wound-simulant) that was subsequently incubated with bacterial cultures. Skin-dermis stamped with PEMs that released 0.25±0.01 μg cm−2 of silver ions caused a 6 log10 reduction in colony forming units of Staphylococcus epidermidis and Pseudomonas aeruginosa within 12 h. Significantly, this level of silver release is below that which is cytotoxic to NIH 3T3 mouse fibroblast cells. Overall, this study describes a general and facile approach for the functionalization of biomaterial surfaces without subjecting them to potentially deleterious processing conditions.

f Biofilm formation by Pseudomonas aeruginosa has been implicated in the pathology of chronic wounds. Both the D and L isoforms of tryptophan inhibited P. aeruginosa biofilm formation on tissue culture plates, with an equimolar ratio of D and L isoforms producing the greatest inhibitory effect. Addition of D-/L-tryptophan to existing biofilms inhibited further biofilm growth and caused partial biofilm disassembly. Tryptophan significantly increased swimming motility, which may be responsible in part for diminished biofilm formation by P. aeruginosa.

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