Background: Glycoside hydrolases have emerged as potent, novel therapeutics that can disrupt biofilms, thereby increasing the susceptibility of the residing bacteria to co-administered antibiotics [1]. The broader clinical use of glycoside hydrolases such as alginate lyase (AL) is limited due to challenges in maintaining enzyme stability, adequate delivery and release of the enzyme at the site of infection. Herein, we present a nanostructured Trojan Horse carrier for AL using environment-sensing lyotropic liquid crystalline gels (LLC) [2].

Aim: To design a LLC-gel carrier based on the lipid glycerol monooleate to protect, deliver and release AL in combination with the antibiotic gentamicin (GENT) as a novel anti-biofilm strategy.

Methods: The effect of Pseudomonas lipase on the release of AL/GENT from LLC-gels was evaluated and the efficacy of the gel was determined over 1 week in vitro against biofilms formed by alginate producing P. aeruginosa (clinical isolate) and compared to an unformulated simple drug solution. Finally, the stability of AL after fabrication of the LLC gel was assessed.

Results: GENT and AL were released at different rates and extent from the LLC-gels (10% AL over 9 days; 60% GENT over 2 days, respectively). Addition of Pseudomonas lipase increased AL release >2-fold (20-30% within 2 days). The LLC-gel demonstrated similar anti-biofilm activity (2.5 log reduction in CFU) compared to unformulated solution, confirming preservation of AL activity in the LLC-gels. Interestingly the antimicrobial effect could not be sustained over extended period (>2 days) which was attributed to a gradual loss of AL activity from prolonged exposure to 37^oC during the assay, rather than short exposure to higher temperatures (60^oC) during LLC-gel fabrication.

Summary: LLC gels present a promising Trojan Horse strategy to conceal and protect biologicals such as glycoside hydrolase. The ability of infection-triggered release provides potential as a future site-specific anti-biofilm therapeutic system.