Inherent antibacterial activity of a peptide-based beta-hairpin hydrogel

Abstract

Among several important considerations for implantation of a biomaterial, a main concern is the introduction of infection. We have designed a hydrogel scaffold from the self-assembling peptide, MAX1, for tissue regeneration applications whose surface exhibits inherent antibacterial activity. In experiments where MAX1 gels are challenged with bacterial solutions ranging in concentrations from 2 x 10(3) colony forming units (CFUs)/dm2 to 2 x 10(9) CFUs/dm2, gel surfaces exhibit broad-spectrum antibacterial activity. Results show that the hydrogel surface is active against Gram-positive (Staphylococcus epidermidis, Staphylococcus aureus, and Streptococcus pyogenes) and Gram-negative (Klebsiella pneumoniae and Escherichia coli) bacteria, all prevalent in hospital settings. Live-dead assays employing laser scanning confocal microscopy show that bacteria are killed when they engage the surface. In addition, the surface of MAX1 hydrogels was shown to cause inner and outer membrane disruption in experiments that monitor the release of beta-galactosidase from the cytoplasm of lactose permease-deficient E. coli ML-35. These data suggest a mechanism of antibacterial action that involves membrane disruption that leads to cell death upon cellular contact with the gel surface. Although the hydrogel surface exhibits bactericidal activity, co-culture experiments indicate hydrogel surfaces show selective toxicity to bacterial versus mammalian cells. Additionally, gel surfaces are nonhemolytic toward human erythrocytes, which maintain healthy morphologies when in contact with the surface. These material attributes make MAX1 gels attractive candidates for use in tissue regeneration, even in nonsterile environments.

Figure 1

(A) Mechanism of folding and self-assembly for MAX1 hydrogel formation. Resulting gels are self-supporting as image at far right shows. (B) Sequence of MAX1 β-hairpin.

Figure 2

TCTP control surface (○) and 2 wt % MAX1 hydrogel surface (■) challenged with an increasing number of CFUs of Gram-negative bacteria. (A) E. coli; 24 h. (B) E. coli; 48 h. (C) K. pneumoniae; 48 h. N = 3.

Figure 3

TCTP control surface (○) and 2 wt % MAX1 hydrogel surface (■) challenged with an increasing number of CFUs of Gram-positive bacteria for 48 h. (A) S. aureus. (B) S. epidermidis. (C) S. pyogenes. N = 3.

Figure 4

LSCM xy projections taken of 2.5 × 10³ CFUs/dm² E. coli incubated on a borosilicate control surface (A) and 2 wt % MAX1 hydrogels (B) after 24 h. Gel is viewed parallel to the z-axis. Green fluorescence denotes live cells, and red fluorescence denotes dead cells with compromised membranes. (C) LSCM xy projections taken of 2.5 × 10⁹ CFUs/dm² E. coli incubated on a 2 wt % MAX1 hydrogel surface viewed perpendicular to the z-axis. Arrows denote the gel–bacterial interface.

Figure 5

Membrane permeabilization assay monitoring the activity of cytoplasmic β-galactosidase released from E. coli ML-35 incubated on a TCTP control surface, □; TCTP control surface after cell sonication, ■; 2 wt % MAX1 hydrogel surface, ×. N = 3.

Figure 6

Proliferation of 2 × 10⁶ CFUs/dm² E. coli and S. aureus on a TCTP control surface in the absence (checkered bars), in the presence of 100 μM soluble MAX1 (white bars), and in the presence of 38.7 mM TFA (black bars). N = 3.

Figure 7

Co-culture of NIH 3T3 murine fibroblasts, A. xylosoxidans (xylosoxidans) and S. maltophilia, on a TCTP control surface (A) and on a 2 wt % MAX1 hydrogel surface (B) after 32 h. (Scale bar = 100 μm)

Figure 8

(A) Hemolytic activity of a TCTP control surface and a 2 wt % MAX1 hydrogel surface toward 6.9 × 10⁵ and 1.4 × 10⁶ hRBC’s under shear flow conditions. TCTP control surface (white bars); TCTP control surface + 1% Triton-X-100 (black bars); and 2 wt % MAX1 hydrogel surface (checkered bars). (B) Image of hRBC’s resting on a 2 wt % MAX1 hydrogel surface after 5 h of incubation at 37 °C. N = 3.