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Physical and Functional Characterization of PLGA Nanoparticles Containing the Antimicrobial Peptide SAAP-148.

ABSTRACT: Synthetic antimicrobial and antibiofilm peptide (SAAP-148) commits significant antimicrobial activities against antimicrobial resistant (AMR) planktonic bacteria and biofilms. However, SAAP-148 is limited by its low selectivity index, i.e., ratio between cytotoxicity and antimicrobial activity, as well as its bioavailability at infection sites. We hypothesized that formulation of SAAP-148 in PLGA nanoparticles (SAAP-148 NPs) improves the selectivity index due to the sustained local release of the peptide. The aim of this study was to investigate the physical and functional characteristics of SAAP-148 NPs and to compare the selectivity index of the formulated peptide with that of the peptide in solution. SAAP-148 NPs displayed favorable physiochemical properties [size = 94.1 ± 23 nm, polydispersity index (PDI) = 0.08 ± 0.1, surface charge = 1.65 ± 0.1 mV, and encapsulation efficiency (EE) = 86.7 ± 0.3%] and sustained release of peptide for up to 21 days in PBS at 37 °C. The antibacterial and cytotoxicity studies showed that the selectivity index for SAAP-148 NPs was drastically increased, by 10-fold, regarding AMR Staphylococcus aureus and 20-fold regarding AMR Acinetobacter baumannii after 4 h. Interestingly, the antibiofilm activity of SAAP-148 NPs against AMR S. aureus and A. baumannii gradually increased overtime, suggesting a dose-effect relationship based on the peptide's in vitro release profile. Using 3D human skin equivalents (HSEs), dual drug SAAP-148 NPs and the novel antibiotic halicin NPs provided a stronger antibacterial response against planktonic and cell-associated bacteria than SAAP-148 NPs but not halicin NPs after 24 h. Confocal laser scanning microscopy revealed the presence of SAAP-148 NPs on the top layers of the skin models in close proximity to AMR S. aureus at 24 h. Overall, SAAP-148 NPs present a promising yet challenging approach for further development as treatment against bacterial infections.

SUBMITTER: Ali M

PROVIDER: S-EPMC9918011 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Physical and Functional Characterization of PLGA Nanoparticles Containing the Antimicrobial Peptide SAAP-148.

Ali Muhanad M van Gent Miriam E ME de Waal Amy M AM van Doodewaerd Bjorn R BR Bos Erik E Koning Roman I RI Cordfunke Robert A RA Drijfhout Jan Wouter JW Nibbering Peter H PH

International journal of molecular sciences 20230202 3

Synthetic antimicrobial and antibiofilm peptide (SAAP-148) commits significant antimicrobial activities against antimicrobial resistant (AMR) planktonic bacteria and biofilms. However, SAAP-148 is limited by its low selectivity index, i.e., ratio between cytotoxicity and antimicrobial activity, as well as its bioavailability at infection sites. We hypothesized that formulation of SAAP-148 in PLGA nanoparticles (SAAP-148 NPs) improves the selectivity index due to the sustained local release of th ...[more]

PMID: 36769188

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Project description:BackgroundWe investigated the efficacy of a synthetic antimicrobial peptide SAAP-148, which was shown to be effective against Methicillin-resistant Staphylococcus aureus (MRSA) on tape-stripped mice skin. Unexpectedly, SAAP-148 was not effective against MRSA in our pilot study using rats with excision wounds. Therefore, we investigated factors that might have contributed to the poor efficacy of SAAP-148. Subsequently, we optimised the protocol and assessed the efficacy of SAAP-148 in an adapted rat study.MethodsWe incubated 100 µL of SAAP-148 with 1 cm2 of a wound dressing for 1 h and determined the unabsorbed volume of peptide solution. Furthermore, 105 colony forming units (CFU)/mL MRSA were exposed to increasing dosages of SAAP-148 in 50% (v/v) human plasma, eschar- or skin extract or PBS. After 30 min incubation, the number of viable bacteria was determined. Next, ex vivo skin models were inoculated with MRSA for 1 h and exposed to SAAP-148. Finally, excision wounds on the back of rats were inoculated with 107 CFU MRSA overnight and treated with SAAP-148 for 4 h or 24 h. Subsequently, the number of viable bacteria was determined.ResultsContrary to Cuticell, Parafilm and Tegaderm film, < 20% of peptide solution was recovered after incubation with gauze, Mepilex border and Opsite Post-op. Furthermore, in plasma, eschar- or skin extract > 20-fold higher dosages of SAAP-148 were required to achieve a 2-log reduction (LR) of MRSA versus SAAP-148 in PBS. Exposure of ex vivo models to SAAP-148 for 24 h resulted in a 4-fold lower LR than a 1 h or 4 h exposure period. Additionally, SAAP-148 caused a 1.3-fold lower mean LR at a load of 107 CFU compared to 105 CFU MRSA. Moreover, exposure of ex vivo excision wound models to SAAP-148 resulted in a 1.5-fold lower LR than for tape-stripped skin. Finally, SAAP-148 failed to reduce the bacterial counts in an adapted rat study.ConclusionsSeveral factors, such as absorption of SAAP-148 by wound dressings, components within wound exudates, re-colonisation during the exposure of SAAP-148, and a high bacterial load may contribute to the poor antimicrobial effect of SAAP-148 against MRSA in the rat model.

Project description: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.

Project description:The interaction of the periodontal pathogen Porphyromonas gingivalis (Pg) with commensal streptococci promotes Pg colonization of the oral cavity. Previously, we demonstrated that a peptide (BAR) derived from Streptococcus gordonii (Sg) potently inhibited adherence of Pg to streptococci and reduced Pg virulence in a mouse model of periodontitis. Thus, BAR may represent a novel therapeutic to control periodontitis by preventing Pg colonization of the oral cavity. However, while BAR inhibited the initial formation of Pg/Sg biofilms, much higher concentrations of peptide were required to disrupt an established Pg/Sg biofilm. To improve the activity of the peptide, poly(lactic-co-glycolic acid) (PLGA) nanoparticles were surface-modified with BAR and shown to more potently disrupt Pg/Sg biofilms relative to an equimolar amount of free peptide. The goal of this work was to determine the in vivo efficacy of BAR-modified NPs (BNPs) and to assess the toxicity of BNPs against human gingival epithelial cells. In vivo efficacy of BNPs was assessed using a murine model of periodontitis by measuring alveolar bone resorption and gingival IL-17 expression as outcomes of Pg-induced inflammation. Infection of mice with Pg and Sg resulted in a significant increase in alveolar bone loss and gingival IL-17 expression over sham-infected animals. Treatment of Pg/Sg infected mice with BNPs reduced bone loss and IL-17 expression almost to the levels of sham-infected mice and to a greater extent than treatment with an equimolar amount of free BAR. The cytotoxicity of the maximum concentration of BNPs and free BAR used in in vitro and in vivo studies (1.3 and 3.4 μM), was evaluated in telomerase immortalized gingival keratinocytes (TIGKs) by measuring cell viability, cell lysis and apoptosis. BNPs were also tested for hemolytic activity against sheep erythrocytes. TIGKs treated with BNPs or free BAR demonstrated >90% viability and no significant lysis or apoptosis relative to untreated cells. In addition, neither BNPs nor free BAR exhibited hemolytic activity. In summary, BNPs were non-toxic within the evaluated concentration range of 1.3-3.4 μM and provided more efficacious protection against Pg-induced inflammation in vivo, highlighting the potential of BNPs as a biocompatible platform for translatable oral biofilm applications.

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Physical and Functional Characterization of PLGA Nanoparticles Containing the Antimicrobial Peptide SAAP-148.

Publications

Physical and Functional Characterization of PLGA Nanoparticles Containing the Antimicrobial Peptide SAAP-148.

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