Protective Liquid Crystal Nanoparticles for Targeted Delivery of PslG: A Biofilm Dispersing Enzyme

Abstract

The glycoside hydrolase, PslG, attacks and degrades the dominant Psl polysaccharide in the exopolymeric substance (EPS) matrix of Pseudomonas aeruginosa biofilms and is a promising therapy to potentiate the effect of antibiotics. However, the need for coadministration with an antibiotic and the potential susceptibility of PslG to proteolysis highlights the need for an effective delivery system. Here, we compared liposomes versus lipid liquid crystal nanoparticles (LCNPs) loaded with PslG and tobramycin as potential formulation approaches to (1) protect PslG from proteolysis, (2) trigger the enzyme's release in the presence of bacteria, and (3) improve the total antimicrobial effect in vitro and in vivo in a Caenorhabditis elegans infection model. LCNPs were an effective formulation strategy for PslG and tobramycin that better protected the enzyme against proteolysis, triggered and sustained the release of PslG, improved the antimicrobial effect by 10−100-fold, and increased the survival of C. elegans infected with P. aeruginosa. Digestible LCNPs had the advantage of triggering the enzyme's release in the presence of bacteria. However, compared to nondigestible LCNPs, negligible differences arose between the LCNPs' ability to protect PslG from proteolysis and potentiate the antimicrobial activity in combination with tobramycin. In C. elegans, the improved antimicrobial efficacy was comparable to tobramycin-LCNPs, although the PslG + tobramycin-LCNPs achieved a greater than 10-fold reduction in bacteria compared to the unformulated combination. Herewith, LCNPs are showcased as a promising protective delivery system for novel biofilm dispersing enzymes combined with antibiotics, enabling infection-directed therapy and improved performance.

Liquid Crystal Nanoparticles Enhance Tobramycin Efficacy in a Murine Model of Pseudomonas aeruginosa Biofilm Wound Infection

“…Importantly, this approach resulted in significant improvements in wound healing outcomes (Figure 4). The in vivo data presented here was consistent with our previous in vitro and ex vivo reports on tobramycin-LCNPs 21,27 now providing further preclinical data on the potential of tobramycin-LCNP formulation as an important next-generation therapy approach to combat P. aeruginosa biofilms. Tobramycin-LCNPs have been previously demonstrated to form a patch over in vitro biofilms, increasing the penetration of tobramycin directly into the bacteria, increasing the total antimicrobial effect.…”

“…Consistently, a dose-dependent effect was observed in vitro and in vivo in C. elegans P. aeruginosa infection models. 21,27 At 15 μg dose used in the present study, there was a 1000-fold improvement in antimicrobial efficacy, which was similar to the 15 μg dose used against a human pulmonary bronchial epithelium infected with P. aeruginosa biofilm 21 and C. elegans infection models. 27 A reasonable correlation was further demonstrated between the P. aeruginosa infection load following various treatments in both in vitro and ex vivo models to the in vivo data presented here.…”

Advances in Nanostructures for Antimicrobial Therapy

“…LCNPs [ 61 ] have shown great potential for clinical applications in antimicrobial therapy due to their ability to overcome numerous biological, chemical and physical barriers in bacteria [ 172 ]. Pronounced differences in the uptake mechanism of cubosomes (self-assembled lipid NCs of cubic symmetry) into Gram-positive and Gram-negative bacteria are observed.…”

“…Rapid internalization of NCs by the Gram-negative bacteria via the fusion uptake mechanism helps to overcome the outer bacterial membrane, ensuring an enhanced toughness to these bacteria, which results in a considerable reduction of the required dose of antibiotics [ 173 ]. LCNPs loaded with tobramycin and glycoside hydrolase (PslG), which attacks and degrades the dominant Psl polysaccharide in the exopolymeric substance matrix of P. aeruginosa biofilms, protected PslG against proteolysis, showed sustained release of PslG and ameliorated the antimicrobial effect by one till two orders; such NPs enable infection-directed therapy with improved efficiency [ 172 ].…”

Recent Advances in Antimicrobial Nano-Drug Delivery Systems

“…However, PSLG is sensitive to proteolysis. To stabilize and protect PSLG from degradation by proteases, Chelsea R. Thorn et al [ 35 ] have used LCNPs as delivery systems for PSLG and tobramycin. The results indicated the prepared LCNPs could protect the enzyme from proteolysis, trigger and maintain the release of PSLG, increase the antibacterial effect by 10–100 times.…”