Silica microparticles made of diatomaceous earth have become particularly attractive materials for designing drug delivery systems. In order to investigate the use of natural diatoms as drug scaffolds for carbon monoxide releasing molecules (CORMs), we evaluated the chemisorption of the cis-[Re(CO)2Br4]2− complex (ReCORM-2) and its vitamin B12 derivative (B12-ReCORM-2) on Coscinodiscus frustules by 3D FT-IR spectroscopic imaging, and the drugs’ neovascularization effects in vivo in the zebrafish (Danio rerio) model. By mapping the symmetric Re-C≡O υ(CO) stretching vibration of the CORMs in the 2000 cm−1 region, we found that the drugs are mostly localized at the girdle band of the diatom frustule. Both ReCORM-2 and B12-ReCORM-2 retain their CO-releasing ability when chemisorbed on the diatoms. When applied in vivo at doses ≥25 µM, the molecules markedly reduced intersegmental and subintestinal vessels development in zebrafish, revealing high anti-angiogenic potential. In addition, diatom frustules did not provoke any toxic in vivo response in the zebrafish embryos, including inflammation. Overall, our results indicate that: (1) CORMs chemisorbed on diatom frustules retain their CO-releasing abilities; (2) both CO-releasing molecules show a concentration-dependent effect on the neovascularization in developing zebrafish; (3) silicate frustules are not toxic and could be used as CORMs drug carriers.
Bacterial infections have become increasingly difficult to treat due to the occurrence of antibiotic-resistant strains. A promising strategy to increase the efficacy of therapy is to combine antibacterials with agents that decrease pathogen virulence via the modulation of the quorum sensing (QS). Lactonases inhibit acylated homoserine lactone (AHL)-mediated QS in Gram-negative bacteria, including the leading nosocomial pathogen Pseudomonas aeruginosa. Here we describe the characteristics of heterologously expressed YtnP lactonase from Bacillus paralicheniformis ZP1 (YtnP-ZP1) isolated from agricultural soil using the culture enrichment method. Purified YtnP-ZP1 hydrolyzed different AHLs with preference to substrates with long acyl residues as evaluated in assays with biosensors and HPLC. The enzyme showed good thermostability and activity in a wide temperature range. YtnP-ZP1 in 50 μg mL–1 concentration reduced the amount of P. aeruginosa-produced long-chain AHLs by 85%, while it hydrolyzed 50% of short-chain AHLs. Incubation of P. aeruginosa PAO1 with YtnP-ZP1 reduced its swarming motility and elastolytic activity without bactericidal effect. YtnP-ZP1 caused the inhibition of biofilm formation and disintegration of mature biofilms in P. aeruginosa PAO1 and multiresistant clinical strain BR5H that was visualized by crystal violet staining. The treatment with YtnP-ZP1 in concentrations higher than 25 μg mL–1 improved the survival of P. aeruginosa PAO1-infected zebrafish (Danio rerio), rescuing 80% of embryos, while in combination with tobramycin or gentamicin survival rate increased to 100%. The treatment of P. aeruginosa PAO1 biofilms on infected zebrafish tail wounds with 50 μg mL–1 YtnP-ZP1 and 2 × MIC tobramycin led to infection clearing in 2 days. The extensive toxicity studies proved YtnP-ZP1 was non-toxic to human cells and zebrafish. In conclusion, novel YtnP-ZP1 lactonase with its effective anti-virulence activity could be used to increase the efficacy of clinically approved antibiotics in clearing both systemic and biofilm-associated P. aeruginosa infections.
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