Preparation, Characterization, and Biological Evaluation of a Hydrophilic Peptide Loaded on PEG-PLGA Nanoparticles

The encapsulation of peptides and proteins in nanosystems has been extensively investigated for masking unfavorable biopharmaceutical properties, including short half-life and poor permeation through biological membranes. Therefore, the aim of this work was to encapsulate a small antimicrobial hydrophilic peptide (H-Ser-Pro-Trp-Thr-NH2, FS10) in PEG-PLGA (polyethylene glycol-poly lactic acid-co-glycolic acid) nanoparticles (Nps) and thereby overcome the common limitations of hydrophilic drugs, which because they facilitate water absorption suffer from rapid degradation. FS10 is structurally related to the well-known RNAIII inhibiting peptide (RIP) and inhibits S. aureus biofilm formation. Various parameters, including different method (double emulsion and nanoprecipitation), pH of the aqueous phase and polymeric composition, were investigated to load FS10 into PEG-PLGA nanoparticles. The combination of different strategies resulted in an encapsulation efficiency of around 25% for both the double emulsion and the nanoprecipitation method. It was found that the most influential parameters were the pH—which tailors the peptides charge—and the polymeric composition. FS10-PEG-PLGA nanoparticles, obtained under optimized parameters, showed size lower than 180 nm with zeta potential values ranging from −11 to −21 mV. In vitro release studies showed that the Nps had an initial burst release of 48–63%, followed by a continuous drug release up to 21 h, probably caused by the porous character of the Nps. Furthermore, transmission electron microscopy (TEM) analysis revealed particles with a spherical morphology and size of around 100 nm. Antimicrobial assay showed that the minimum inhibitory concentration (MIC) of the FS10-loaded Nps, against S. aureus strains, was lower (>128 µg/mL) than that of the free FS10 (>256 µg/mL). The main goal of this work was to develop polymeric drug delivery systems aiming at protecting the peptide from a fast degradation, thus improving its accumulation in the target site and increasing the drug-bacterial membrane interactions

Helicobacter pylori ATCC 43629/NCTC 11639 Outer Membrane Vesicles (OMVs) from biofilm and planktonic phase associated with extracellular DNA (eDNA)

Helicobacter pylori persistence is associated with its capacity to develop biofilms as a response to changing environmental conditions and stress. Extracellular DNA (eDNA) is a component of H. pylori biofilm matrix but the lack of DNase I activity supports the hypothesis that eDNA might be protected by other extracellular polymeric substances (EPS) and/or Outer Membrane Vesicles (OMVs), which bleb from the bacteria surface during growth. The aim of the present study was to both identify the eDNA presence on OMVs segregated from H. pylori ATCC 43629/NCTC 11639 biofilm (bOMVs) and its planktonic phase (pOMVs) and to characterize the physical-chemical properties of the OMVs. The presence of eDNA in bOMVs and pOMVs was initially carried out using DNase I-gold complex labeling and Transmission Electron Microscope analysis (TEM). bOMVs and pOMVs were further isolated and physical-chemical characterization carried out using dynamic light scattering (DLS) analysis. eDNA associated with OMVs was detected and quantified using a PicoGreen spectrophotometer assay, while its extraction was performed with a DNA Kit. TEM images showed that eDNA was mainly associated with the OMV membrane surfaces; while PicoGreen staining showed a four-fold increase of dsDNA in bOMVs compared with pOMVs. The eDNA extracted from OMVs was visualized using gel electrophoresis. DLS analysis indicated that both planktonic and biofilm H. pylori phenotypes generated vesicles, with a broad distribution of sizes on the nanometer scale. The DLS aggregation assay suggested that eDNA may play a role in the aggregation of OMVs, in the biofilm phenotype. Moreover, the eDNA associated with vesicle membrane may impede DNase I activity on H. pylori biofilms. These results suggest that OMVs derived from the H. pylori biofilm phenotype may play a structural role by preventing eDNA degradation by nucleases, providing a bridging function between eDNA strands on OMV surfaces and promoting aggregation

Biofilm Formation by Stenotrophomonas maltophilia: Modulation by Quinolones, Trimethoprim-Sulfamethoxazole, and Ceftazidime

We investigated the in vitro effects of seven fluoroquinolones (ciprofloxacin, grepafloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, and rufloxacin), compared to those of trimethoprim-sulfamethoxazole (SXT) and ceftazidime on total biomass and cell viability of Stenotrophomonas maltophilia biofilm. S. maltophilia attached rapidly to polystyrene, within 2 h of incubation, and then biofilm formation increased over time, reaching maximum growth at 24 h. In the presence of fluoroquinolones at one-half and one-fourth the MIC, biofilm biomass was significantly (P < 0.01) reduced to 55 to 70% and 66 to 76% of original mass, respectively. Ceftazidime and SXT did not exert any activity. Biofilm bacterial viability was significantly reduced by all antibiotics tested at one-half the MIC. At one-fourth the MIC all antibiotics, except levofloxacin, significantly reduced viability. Treatment of preformed biofilms with bactericidal concentrations (500, 100, and 50 μg/ml) of all fluoroquinolones caused, except for norfloxacin, significant reduction of biofilm biomass to 29.5 to 78.8, 64.1 to 83.6, and 70.5 to 82.8% of original mass, respectively. SXT exerted significant activity at 500 μg/ml only. Ceftazidime was completely inactive. Rufloxacin exhibited the highest activity on preformed biofilm viability, significantly decreasing viable counts by 0.6, 5.4, and 17.1% at 500, 100, and 50 μg/ml, respectively. Our results show that (i) subinhibitory (one-half and one-fourth the MIC) concentrations of fluoroquinolones inhibit adherence of S. maltophilia to polystyrene and (ii) clinically achievable concentrations (50 and 100 μg/ml) of rufloxacin are able to eradicate preformed S. maltophilia biofilm