Chitosan nanoparticles loaded with the antimicrobial peptide temporin B exert a long-term antibacterial activity in vitro against clinical isolates of Staphylococcus epidermidis

Abstract

Nowadays, the alarming rise in multidrug-resistant microorganisms urgently demands for suitable alternatives to current antibiotics. In this regard, antimicrobial peptides (AMPs) have received growing interest due to their broad spectrum of activities, potent antimicrobial properties, unique mechanisms of action, and low tendency to induce resistance. However, their pharmaceutical development is hampered by potential toxicity, relatively low stability and manufacturing costs. In the present study, we tested the hypothesis that the encapsulation of the frog-skin derived AMP temporin B (TB) into chitosan nanoparticles (CS-NPs) could increase peptide's antibacterial activity, while reducing its toxic potential. TB-loaded CS-NPs with good dimensional features were prepared, based on the ionotropic gelation between CS and sodium tripolyphosphate. The encapsulation efficiency of TB in the formulation was up to 75%. Release kinetic studies highlighted a linear release of the peptide from the nanocarrier, in the adopted experimental conditions. Interestingly, the encapsulation of TB in CS-NPs demonstrated to reduce significantly the peptide's cytotoxicity against mammalian cells. Additionally, the nanocarrier evidenced a sustained antibacterial action against various strains of Staphylococcus epidermidis for at least 4 days, with up to 4-log reduction in the number of viable bacteria compared to plain CS-NPs at the end of the observational period. Of note, the antimicrobial evaluation tests demonstrated that while the intrinsic antimicrobial activity of CS ensured a "burst" effect, the gradual release of TB further reduced the viable bacterial count, preventing the regrowth of the residual cells and ensuring a long-lasting antibacterial effect. The developed nanocarrier is eligible for the administration of several AMPs of therapeutic interest with physical-chemical characteristics analog to those of TB.

Keywords: Staphylococcus epidermidis; antimicrobial peptides; chitosan; delivery system; nanoparticles; temporin B.

FIGURE 1

Release profile of temporin B from TB-CS-NPs in SPB pH 7.4, 37°C. After a short lag time, the system displayed a progressive linear release of the loaded active agent.

FIGURE 2

Cytocompatibility evaluation of free TB (A) and of TB loaded into CS-NPs (B) in mouse embryo fibroblast cell line Balb 3T3/Clone A31. (A) Determination of free TB cytotoxicity. Results show that free TB displays an IC₅₀ value of ∼119 μg/ml after 24 h of incubation; (B) Comparison of cytotoxicity of different concentrations of free TB (116.5, 233, and 466 μg/ml) with the same amount loaded into CS-NPs (TB-CS-NPs), corresponding to TB-CS-NPs concentrations of 2.5, 5, and 10 mg/ml, respectively. Results show a statistically significant reduction of TB toxicity upon its loading into nanoparticles (^∗p < 0.05, Student’s t-test).

FIGURE 3

Kinetics of the antibacterial activity of the developed nanoparticle-based AMP-delivery system against Staphylococcus epidermidis ATCC35984. Bacterial cells were exposed for different time intervals to free TB, CS-NPs, or TB-CS-NPs. At the end of the exposure time aliquots of the cultures were serially diluted, plated on solid medium, and incubated at 37°C for 48 h before assessing the CFU count. Results are the mean values ± SEM of three independent experiments. CTRL, control bacteria incubated in the assay medium only. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 as compared to TB-CS-NPs, Student’s t-test.

FIGURE 4

Antibacterial activity of TB-CS-NPs as compared to the plain nanocarrier against clinical isolates of S. epidermidis after 4 days of incubation. Bacterial cells were exposed for 4 days to plain CS-NPs or TB-CS-NPs. At the end of the exposure time aliquots of the cultures were serially diluted, plated on solid medium, and incubated at 37°C for 48 h before assessing the CFU count. Results are the mean values ± SEM of two independent experiments performed in duplicate. CTRL, control bacteria incubated in the assay medium only. ^∗p < 0.05; ^∗∗p < 0.01; as compared to TB-CS-NPs, Student’s t-test.