ABSTRACT
Purpose: To limit corneal damage and potential loss of vision, bacterial keratitis must be treated aggressively. Innovation in antimicrobials is required due to the need for empirical treatment and the rapid emergence of bacterial resistance. Designed host defense peptides (dHDPs) are synthetic analogues of naturally occurring HDPs, which provide defense against invading pathogens. This study investigates the use of novel dHDPs for the treatment of bacterial keratitis. Methods: The minimum inhibitory concentrations (MICs) were determined for dHDPs on both Gram-positive and -negative bacteria. The minimum biofilm eradication concentrations (MBEC) and in vitro time-kill assays were determined. The most active dHDP, RP444, was evaluated for propensity to induce drug resistance and therapeutic benefit in a murine Pseudomonas aeruginosa keratitis model. Results: Designed HDPs were bactericidal with MICs ranging from 2 to >64 µg/mL and MBEC ranging from 6 to 750 µg/mL. In time-kill assays, dHDPs were able to rapidly reduce bacterial counts upon contact with as little as 2 µg/mL. RP444 did not induce resistance after repeated exposure of P. aeruginosa to subinhibitory concentrations. RP444 demonstrated significant efficacy in a murine model of bacterial keratitis as evidenced by a significant dose-dependent decrease in ocular clinical scores, a significantly reduced bacterial load, and substantially decreased inflammatory cell infiltrates. Conclusions: Innovative dHDPs demonstrated potent antimicrobial activity, possess a limited potential for development of resistance, and reduced the severity of murine P. aeruginosa keratitis. These studies demonstrate that a novel dHDP may have potential to treat patients with sight-threatening bacterial keratitis.
Subject(s)
Biofilms/drug effects , Cornea/microbiology , Eye Infections, Bacterial/drug therapy , Keratitis/drug therapy , Organotechnetium Compounds/administration & dosage , Peptides, Cyclic/administration & dosage , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/isolation & purification , Animals , Culture Media, Serum-Free , Disease Models, Animal , Dose-Response Relationship, Drug , Eye Infections, Bacterial/microbiology , Keratitis/microbiology , Mice, Inbred C57BL , Microbial Sensitivity Tests , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effectsABSTRACT
In this work perfluorinated substrates fabricated from SiO2 glass slides are modified with oligo(ethylene glycol) (OEG) units for long-term resistance of cell adhesion purposes, based on fluorous interactions and click chemistry. Specifically, fluorous substrates, prepared by treatment of glass slides with 1H, 1H, 2H, 2H-perfluorodecyltrimethoxysilane (FAS17), were coated with ethynyl-OEG-C8F17, followed by covalent attachment of an azido-OEG via copper-catalyzed azide-alkyne cycloaddition (CuAAC) "click" reaction. We demonstrate that the resultant surface avoid fibrinogen adsorption and resisted cell adhesion for over 14days. X-ray photoemission spectroscopy (XPS) analysis and contact angle goniometry measurements confirm the presence of the OEG molecules on the fluorous substrates. Bright field optical images show total absence of 3T3 fibroblast cells on the OEG modified fluorinated substrate for 1 and 5days, and a remarkably decrease of cell adhesion at 14days.
Subject(s)
Click Chemistry , Ethylene Glycol/chemistry , Fluorine/chemistry , Cell Adhesion , Glass/chemistry , Molecular Structure , Silicon Dioxide/chemistryABSTRACT
We report a practical method for biofunctionalization of fluoropolymers based on noncovalent, fluorous interactions and click chemistry that allows incorporation of biomolecules under physiological solutions. We demonstrate the method by immobilization of an antimicrobial peptide (AMP) on fluorous thin films and fluorosilicone contact lens. The fluorous surfaces were dip-coated with fluorous-tagged oligo(ethylene) chain terminated with a reactive group, such as an alkynyl group. This simple step generates a "clickable" surface. The noncovalent fluorous interaction was strong enough to allow subsequent covalent attachment of IG-25, a truncated version of the most extensively studied human AMP LL-37. The attachment was through copper-catalyzed click reaction between the alkynyl group on the surface and the azido-OEG tag at the N-terminus of IG-25. In comparison to surfaces presenting IG-25 randomly bound via carbodiimide chemistry, the surfaces presenting IG-25 tethering to the surface at the N-terminus via click chemistry displayed higher antibacterial activities against an ocular pathogen Pseudomonas aeruginosa (strain PA-O1).
Subject(s)
Antimicrobial Cationic Peptides/chemistry , Click Chemistry , Immobilized Proteins/chemistry , Polymers/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Antimicrobial Cationic Peptides/metabolism , Contact Lenses/microbiology , Fluorescein/chemistry , Humans , Immobilized Proteins/metabolism , Pseudomonas aeruginosa/drug effects , Silicones/chemistryABSTRACT
The eye and its associated tissues including the lacrimal system and lids have evolved several defence mechanisms to prevent microbial invasion. Included among this armory are several host-defence peptides. These multifunctional molecules are being studied not only for their endogenous antimicrobial properties but also for their potential therapeutic effects. Here the current knowledge of host-defence peptide expression in the eye will be summarised. The role of these peptides in eye disease will be discussed with the primary focus being on infectious keratitis, inflammatory conditions including dry eye and wound healing. Finally the potential of using host-defence peptides and their mimetics/derivatives for the treatment and prevention of eye diseases is addressed.
Subject(s)
Antimicrobial Cationic Peptides/immunology , Eye Infections/immunology , Eye/immunology , Antimicrobial Cationic Peptides/metabolism , Eye/metabolism , Eye/microbiology , Eye Infections/metabolism , Eye Infections/microbiology , HumansABSTRACT
Antimicrobial peptide IG-25 (a truncated version of LL-37 of the cathelicidin family) tethering an azido-capped poly(ethylene glycol) chain at the N-terminus was site-specifically attached to alkynyl-terminated polymerized liposomes using copper catalyzed "click" reaction, leading to an 18 fold enhancement in efficacy against Pseudomonas aeruginosa when compared to LL-37 without any increase in cytotoxicity to human corneal epithelial cells.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Liposomes/chemistry , Polyethylene Glycols/chemistry , Pseudomonas aeruginosa/drug effects , Anti-Bacterial Agents/chemistry , Antimicrobial Cationic Peptides/chemistry , Catalysis , Click Chemistry , Copper/chemistry , Cornea/cytology , Cornea/drug effects , Epithelial Cells/cytology , Epithelial Cells/drug effects , Humans , Liposomes/chemical synthesis , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity Relationship , CathelicidinsABSTRACT
The biological properties of polyunsaturated fatty acid (PUFA) classes have been the source of much contention. For example, n-3 PUFA are chemoprotective, whereas n-6 PUFA may promote tumor development. Since dietary components can have combinatorial effects, we further examined the apoptotic properties of n-3 or n-6 fatty acids when combined with different fiber sources. Mice were fed diets supplemented with either fish oil (FO; enriched in n-3 PUFA) or corn oil (CO; enriched in n-6 PUFA) and nonfermentable (cellulose) or fermentable (pectin) fiber sources. In complementary experiments, immortalized young adult mouse colonic (YAMC) cells were treated with docosahexaenoic acid (DHA; 22:6n-3) or linoleic acid (LA; 18:2n-6) with or without butyrate. Mice fed a FO and pectin diet had significantly (p < 0.05) increased levels of apoptosis in colonocytes compared to all other diets. Similarly, apoptosis was highly induced in DHA and butyrate cotreated YAMC cells. In contrast, in both YAMC and mouse models, LA/CO with butyrate/pectin treatment reduced apoptosis and enhanced expression of bcl-2. The LA and butyrate induced antiapoptotic phenotype was reversed by knocking down bcl-2 using targeted siRNA. In comparison, overexpression of bcl-2 blocked the proapoptotic effect of DHA and butyrate. These data provide new mechanistic insights into the regulation of apoptosis by dietary PUFA and fiber.
