Convenient Preparation of Bactericidal Hydrogels by Covalent Attachment of Stabilized Antimicrobial Peptides Using Thiol–ene Click Chemistry

Abstract

This report describes the design and synthesis of a bactericidal poly(ethylene glycol)-based (PEG) hydrogel coating with covalently attached antimicrobial peptides (AMP) stabilized against proteolytic degradation. As such, mimics of the highly active AMP HHC10 (H-KRWWKWIRW-NH2) were designed for optimal stability in human serum while retaining strong antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermidis, the major causative agents of biomaterial associated infection. In order to investigate the selectivity of the AMPs, their hemolytic activity was determined. A N-terminal cysteine facilitated thiol–ene chemistry for a fast, single-step immobilization/photopolymerization strategy. The antimicrobial activity of the resulting thin layer hydrogel coating on a PET surface was established using the Japanese Industrial Standard (JIS) Z2801 assay, showing complete killing (>99.9%) of inocula of S. aureus ATCC 49230, S. epidermidis ATCC 35984, and E. coli ATCC 8739.

Highly potent antimicrobial peptide derivatives of bovine cateslytin

“…A wide range of haemolytic tendencies in studied antimicrobial peptides has been observed such as the highly toxic non-selective peptides mastoparan B (96% haemolysis at 30 µM) and melittin (100% haemolysis at 10 µM) or less toxic peptides that are more selective to bacterial cells like magainin 2 (50% haemolysis at 428 µM) and HHC10 (only 0.4% haemolysis at 15.6 µg/mL). [34][35][36][37] The haemolytic activity of peptides 1-7 was determined using a haemolytic assay developed in our group with minor modifications (Table 4). 37 In this assay sheep erythrocytes were used as a convenient source of red blood cells.…”

“…[34][35][36][37] The haemolytic activity of peptides 1-7 was determined using a haemolytic assay developed in our group with minor modifications (Table 4). 37 In this assay sheep erythrocytes were used as a convenient source of red blood cells.…”

Characterization and Activity of an Immobilized Antimicrobial Peptide Containing Bactericidal PEG-Hydrogel

“…30 A quantitative method to determine the amount of antimicrobial peptide in the hydrogel was found in a modified Lowry assay. The use of the Lowry assay for short peptides was described before.…”

“…Peptides were synthesized by solid phase peptide synthesis on Rink Amide resin (0.24 mmol/g) (Rapp Polymere GmbH, Tübingen, Germany) on a 0.25 mmol scale. The peptide was assembled using an automatic ABI 433A Peptide Synthesizer, equipped with an UVmonitoring system as described before 30. …”

Triple-targeting Gram-negative selective antimicrobial peptides capable of disrupting the cell membrane and lipid A biosynthesis

“…Therefore, the cytotoxicity of peptides 2 and 3 was determined in a haemolysis assay using sheep erythrocytes. 20 In this assay the extent of haemolysis was determined by measuring the concentration of released haemoglobin into solution compared to 100% haemolysis (1% Triton X-100) after 1h at 37 °C. The percentage of haemolysis at 64 µM is presented as this is at least 10 times higher than the MIC concentrations for peptide 3 in Gram-negative bacteria (Table 3).…”