“Click” Immobilization on Alkylated Silicon Substrates: Model for the Study of Surface Bound Antimicrobial Peptides

Abstract

We describe an effective approach for the covalent immobilization of antimicrobial peptides (AMPs) to bioinert substrates via CuI-catalyzed azide–alkyne cycloaddition (CuAAC). The bioinert substrates were prepared by surface hydrosilylation of oligo-(ethylene glycol) (OEG) terminated alkenes on hydrogen-terminated silicon surfaces. To render the OEG monolayers “clickable”, mixed monolayers were prepared using OEG-alkenes with and without a terminal alkyne protected by a trimethylgermanyl (TMG) group. The mixed monolayers were characterized by X-ray photoelectron spectroscopy (XPS), elliposometry and contact angle measurement. The TMG protecting group can be readily removed to yield a free terminal alkyne by catalytic amounts of CuI in an aqueous media. This step can then be combined with the subsequent CuAAC reaction. Thus, the immobilization of an azide modified AMP (N3-IG-25) was achieved in a one-pot de-protection/coupling reaction. Varying the ratio of the two alkenes in the deposition mixture allowed for control over the density of the alkynyl groups in the mixed monolayer, and subsequently the coverage of the AMPs on the monolayer. These samples allowed for study of the dependence of antimicrobial activities on the AMP density. The results show that a relative low coverage of AMPs (~1.6×1013 molecule per cm2) is sufficient to significantly suppress the viability of Pseudomonas aeruginosa, while the surface presenting the highest density of AMPs (~2.8×1013 molecule per cm2) is still cyto-compatible. The remarkable antibacterial activity is attributed to the long and flexible linker and the site-specific “click” immobilization, which may facilitate the covalently attached peptides to interact with and disrupt the bacterial membranes.

Immobilization and orientation‐dependent activity of a naturally occurring antimicrobial peptide

“…In contrast, immobilized peptide on a planar solid surface may collectively act on the bacteria with only other neighboring, localized peptide molecules. Another reason may relate to peptide density, 1.5–2× greater for the beads than the planar surface, which is in concurrence with previous studies demonstrating a relationship between peptide densities and activity . Although less potent, the ability to derive efficacy in the planar glass immobilized systems indicates the substrate shape/size does not appear to play a significant role other than limiting potency.…”

“…The reduction was attributed to tethering parameters such as peptide density, peptide accessibility and spacer length as well as adverse influence on parameters that are critical for lytic behavior such as diffusivity, flexibility and membrane permeation. Moreover, Li et al suggests a relationship between peptide densities and activity, while also identifying surface distribution, particularly effective local concentrations, as important for efficacy . The concentration and density of immobilized SMAP_c and c_SMAP on the superparamagnetic beads were variable to a certain degree and likely influenced the degree of potency compared with the soluble forms.…”

CAP37 Activation of PKC Promotes Human Corneal Epithelial Cell Chemotaxis

“…Quadrisected corneas were incubated overnight at 48C on ice in Hank's balanced salt solution (Gibco) containing dispase (25 caseinolytic U/mL; Becton Dickinson) and 5 lg/mL gentamicin (A.G. Scientific, Inc., San Diego, CA). 20 Corneal epithelial cells were then detached from the stroma by gently scraping the corneal surface with a scalpel. The removed cells were digested for 5 minutes in 0.25% trypsin-EDTA (Gibco) at 378C followed by the addition of an equal volume of heat inactivated fetal bovine serum (FBS; Gibco) and were centrifuged at 450g for 5 minutes.…”

Immobilization of Antimicrobial Peptide IG-25 onto Fluoropolymers via Fluorous Interactions and Click Chemistry

“…13 We have previously shown that the fluorously immobilized films were stable against desorption during the subsequent covalent modification in aquous solutions. 27, 28 The alkynyl surface A allows site-specific attachment of the peptide with an OEG-azido tag ( N 3 -EG 12 -IG-25 ) via click reaction to provide the surface B . Random grafting of the unmodified peptide IG-25 onto the carboxylic acid surfaces C was performed using a procedure similar to a reported one.…”