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. 2019 May 13;11(5):231.
doi: 10.3390/pharmaceutics11050231.

Clotrimazole-Loaded Mediterranean Essential Oils NLC: A Synergic Treatment of Candida Skin Infections

Claudia Carbone  1   2 , Maria do Céu Teixeira  3 , Maria do Céu Sousa  4   5 , Carlos Martins-Gomes  6   7 , Amelia M Silva  8   9 , Eliana Maria Barbosa Souto  10   11 , Teresa Musumeci  12
Affiliations

Clotrimazole-Loaded Mediterranean Essential Oils NLC: A Synergic Treatment of Candida Skin Infections

Claudia Carbone et al. Pharmaceutics. .

Abstract

The increasing development of resistance of Candida species to traditional drugs represents a great challenge to the medical field for the treatment of skin infections. Essential oils were recently proposed to increase drug effectiveness. Herein, we developed and optimized (23 full factorial design) Mediterranean essential oil (Rosmarinus officinalis, Lavandula x intermedia "Sumian", Origanum vulgare subsp. hirtum) lipid nanoparticles for clotrimazole delivery, exploring the potential synergistic effects against Candida spp. Small sized nanoparticles (<100 nm) with a very broad size distribution (PDI < 0.15) and long-term stability were successfully prepared. Results of the in vitro biosafety on HaCaT (normal cell line) and A431 (tumoral cell line), allowed us to select Lavandula and Rosmarinus as anti-proliferative agents with the potential to be used as co-adjuvants in the treatment of non-tumoral proliferative dermal diseases. Results of calorimetric studies on biomembrane models, confirmed the potential antimicrobial activity of the selected oils due to their interaction with membrane permeabilization. Nanoparticles provided a prolonged in vitro release of clotrimazole. In vitro studies against Candida albicans, Candida krusei and Candida parapsilosis, showed an increase of the antifungal activity of clotrimazole-loaded nanoparticles prepared with Lavandula or Rosmarinus, thus confirming nanostructured lipid carriers (NLC) containing Mediterranean essential oils represent a promising strategy to improve drug effectiveness against topical candidiasis.

Keywords: DSC; Lavandula x intermedia “Sumian”; Lumisizer; MIC; Origanum vulgare subsp. hirtum; Rosmarinus officinalis L.; Turbiscan; cytotoxicity; factorial design; stability.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Design of Experiment (DoE) results: observed vs predicted values for Zave (a) and PDI (d), respectively; Response surface plots for Zave (b) and PDI (e); Pareto Chart of standardized effects for Zave (c) and PDI (f).
Figure 2
Figure 2
Cell viability results for A-431 (at the top) and for HaCaT (at the bottom) cells exposed with various concentrations of essential oils from Lavandula, Origanum and Rosmarinus (as denoted). Results are presented as mean ± SD (n = 3 independent experiments). * Significance for p < 0.05.
Figure 3
Figure 3
Cell viability results for A-431 (at the top) and for HaCaT (at the bottom) cells treated with NLC-L, NLC-O and NLC-R. Results are presented as mean ± SD (n = 3 independent experiments). * Significance for p < 0.05.
Figure 4
Figure 4
Fourier Transform-Infrared (FT-IR) spectra of pure clotrimazole (CLZ), cryoprotectant (Trehalose), CLZ-loaded NLC prepared with Lavandula and Rosmarinus EO (CLZ-LNLC, CLZ-RNLC).
Figure 5
Figure 5
Speed of creaming at normal gravity obtained by LUMiSizer® for unloaded and CLZ-loaded NLC prepared with Lavandula and Rosmarinus EO.
Figure 6
Figure 6
Creaming velocity prediction of CLZ-RNLC. (a) Evolution of transmission profiles at 3000 RCF. (b) Volume based size distribution of CLZ-RNLC suspension. (c) Transient variation of the front position in panel (a). The fitter straight line is determined as the creaming velocity. (d) Comparison of the measured creaming velocities and the theoretical values from Stoke’s law at different RCFs.
Figure 7
Figure 7
Amount (µg/mL) of CLZ released in vitro from NLC prepared with Lavandula EO and Rosmarinus EO.
Figure 8
Figure 8
Thermograms of MLV at different dilutions C1, C2, C3 respectively 0.05%, 0.1%, 0.2% (v/v) (A), MLV with Lavandula (B) and Rosmarinus (C). EOs were tested at different dilutions: D1, D2, D3, D4 corresponding to 0.01%, 0.05%, 0.1%, 0.2% v/v respectively.
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