Lentisk (Pistacia lentiscus) Oil Nanoemulsions Loaded with Levofloxacin: Phytochemical Profiles and Antibiofilm Activity against Staphylococcus spp

Abstract

Most clinical isolates of both Staphylococcus aureus and Staphylococcus epidermidis show the capacity to adhere to abiotic surfaces and to develop biofilms resulting in a contribution to chronic human skin infections. Antibiotic resistance and poor biofilm penetration are the main causes of ineffective therapeutic treatment in killing bacteria within biofilms. A possible strategy could be represented by drug delivery systems, such as nanoemulsions (composed of bioactive oil, surfactant and water phase), which are useful for enhancing the drug permeation of a loaded drug inside the biofilm and its activity. Phytochemical characterization of Pistacia lentiscus oil (LO) by direct infusion Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) allowed the identification of bioactive compounds with antimicrobial properties, including fatty acids and phenolic compounds. Several monoterpenes and sesquiterpenes have been also detected and confirmed by gas chromatography-mass spectrometric (GC-MS) analysis, together providing a complete metabolomic profiling of LO. In the present study, a nanoemulsion composed of LO has been employed for improving Levofloxacin water solubility. A deep physical-chemical characterization of the nanoemulsion including hydrodynamic diameter, ζ-potential, morphology, entrapment efficiency, stability release and permeation studies was performed. Additionally, the antimicrobial/antibiofilm activity of these preparations was evaluated against reference and clinical Staphylococcus spp. strains. In comparison to the free-form antibiotic, the loaded NE nanocarriers exhibited enhanced antimicrobial activity against the sessile forms of Staphylococcus spp. strains.

Keywords: Pistacia lentiscus L.; Staphylococcus spp.; antibiofilm activity; bioactive oil; mass spectrometry; nanoemulsion.

Figure 1

(A) Van Krevelen plot gained from the molecular formulas obtained by ESI FT-ICR MS analysis of LO. (B) Van Krevelen diagram displays homology series (dashed lines) related to the following chemical reactions: A lines (green) stand for (de)hydrogenation; B lines (red) stand for oxidation or reduction; C lines (light blue) stands for (de)hydration and condensation processes; D lines (purple) stand for (de)methylation.

Figure 2

Donut chart regarding the elemental composition of LO sample. The chart shows the relative frequency of the CHO, CHNO, CHNOS, CHNOP, CHOS, CHN, CHOP, CHNOPS population.

Figure 3

Histograms of the relative abundance distribution within specific classes of FA: saturated (A); C18 series (B) obtained by ESI(−) FT-ICR MS analyses of LO sample.

Figure 4

Percentage areas of the most concentrated monoterpenes detected through static HS (red bars) and HS-SPME-GC-MS (blue bars) analyses.

Figure 5

Percentage areas of the sesquiterpenes detected through HS-SPME-GC-MS analyses.

Figure 6

Ternary phase diagram of LO, Brij O10, and Hepes Buffer. The resulting phases were the homogeneous phase (light blue area) and the not homogeneous phase (dark blue area). NEs composition has been placed into the homogeneous region.

Figure 7

Representative images of the transmission electron microscopy observations of selected empty NEs (A) and NEsL (B) samples.

Figure 8

Stability study over time of NEs and NEsL at room temperature (A,C) and 4 °C (B,D).

Figure 9

LVX release profile from NEsL by cellulose dialysis tubing.

Figure 10

Permeated amount of LVX from NEsL through Strat-M^® in a Franz cell system.