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S. Kolar, A. Kumar, C. Cai, A. M. McDermott; Antimicrobial Activity of a Cathelicidin Derivative Conjugated to Polymerized-Liposomes. Invest. Ophthalmol. Vis. Sci. 2010;51(13):431.
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Human antimicrobial-peptides (AMPs) are novel bioactive-molecules that are effective despite the emergence of multidrug-resistant pathogens. Therefore, they serve as potential alternatives to traditional-antibiotics. However, very high doses are needed for in-vivo activity which can be associated with cytotoxicity. In an attempt to improve their efficacy and minimize toxicity, a polyvalent presentation tethering AMPs to the surface of polymerized-liposomes was tested.
Cathelicidin derivative LL-25 was conjugated onto polymerized-liposomes (10,12- pentacosadiynoic-acid) via covalent bonding using copper-assisted click reaction or cabodiimide-coupling reaction or via non-covalent immobilization through electrostatic-interactions on anionic surfaces. Pseudomonas aeruginosa (PA01) were incubated with 0 µg/ml - 1 mg/ml of LL-25 conjugated-liposomes or 50 µg/ml LL-25 as control for 2h at 37°C. Samples were plated and colony-forming-units counted after overnight incubation. The amount of LL-25 incorporated into the liposomes was quantified by measuring the amino-groups using Fluorescamine.
The amount of LL-25 incorporated per mg/ml of liposome was 310, 80 and 20 µg/ml using the click, electrostatic and carbodiimide methods respectively. Unconjugated-liposomes did not cause bacterial killing whereas all three forms of the LL-25 modified liposomes caused concentration dependent killing ranging from 4% to 100%. When expressed relative to the antibacterial activity of LL-25 alone, click-modified conjugates demonstrated 72 fold greater activity whereas that for electrostatic and carbodiimide conjugates was 137 and 1304 fold lower respectively (n= 2-3 experiments). However, as conjugation via carbodiimide-coupling involves reaction of amine with carboxylic-acid, the amount incorporated by carbodiimide-coupling is an underestimate thus the activity relative to LL-25 is also artificially low.
This study demonstrates that click-reaction provides for high-efficiency conjugation of AMPs onto polymerized-liposomes which results in a high local AMP concentration and enhanced antimicrobial efficacy. Thus, AMPs have significant potential as novel antibiotics for clinical use.
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