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. 2024 Aug 7;16(31):40628-40640.
doi: 10.1021/acsami.4c07413. Epub 2024 Jul 27.

Development and Evaluation of Microencapsulated Oregano Essential Oil as an Alternative Treatment for Candida albicans Infections

Liliana Fernandes  1 , Ainara Barco-Tejada  2   3 , Elena Blázquez  2   3 , Daniela Araújo  1   4   5 , Artur Ribeiro  1   5 , Sónia Silva  1   4   5 , Lorena Cussó  2   6   7 , Sofia Costa-de-Oliveira  8 , M Elisa Rodrigues  1   5 , Mariana Henriques  1   5
Affiliations

Development and Evaluation of Microencapsulated Oregano Essential Oil as an Alternative Treatment for Candida albicans Infections

Liliana Fernandes et al. ACS Appl Mater Interfaces. .

Abstract

Vulvovaginal candidiasis (VVC) is characterized as a very common fungal infection that significantly affects women's health worldwide. Essential oils (EOs) are currently being evaluated as an alternative therapy. The development of efficient techniques such as micro- or nanoencapsulation for protecting and controlling release is essential to overcome the limitations of EO applications. Therefore, the aim of this study was to develop and characterize oregano EO-loaded keratin microparticles (OEO-KMPs) as a potential treatment for VVC. OEO-KMPs were produced using high-intensity ultrasonic cycles and characterized in terms of morphological and physicochemical parameters. In vitro evaluation included assessing the toxicity of the OEO-KMPs and their effect against Candida albicans using microdilution and agar diffusion, while the activity against biofilm was quantified using colony forming units (CFU). The efficacy of the OEO-KMPs in an in vivo VVC mouse model was also studied. Female BALB/c mice were intravaginally infected with C. albicans, 24 h postinfection animals were treated intravaginally with 15 μL of OEO-KMPs and 24 h later vaginal fluid was analyzed for C. albicans and Lactobacillus growth (CFU mL-1). The results showed the stability of the OEO-KMPs over time, with high encapsulation efficiency and controlled release. This nanoparticle size facilitated penetration and completely inhibited the planktonic growth of C. albicans. In addition, an in vitro application of 2.5% of the OEO-KMPs eradicated mature C. albicans biofilms while preserving Lactobacillus species. In in vivo, a single intravaginal application of OEO-KMPs induced a reduction in C. albicans growth, while maintaining Lactobacillus species. In conclusion, this therapeutic approach with OEO-KMPs is promising as a potential alternative or complementary therapy for VVC while preserving vaginal microflora.

Keywords: Biofilm treatment; Candida albicans; Keratin microcapsules; Phytotherapeutic applications; Vaginal microflora; Vulvovaginal candidiasis.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Characterization of keratin nanoparticles prepared in PBS encapsulating oregano oil (OEO-KMPs) for 5 months (155 days). (A) particle size (Z-average) and polydispersity (PDI); (B) surface charge (zeta-potential). * indicates statistical difference in particle size when compared to the results obtained at time 0 (initial conditions) (*p < 0.1, **p < 0.01, ****p < 0.0001).
Figure 2
Figure 2
In vitro release profiles of oregano essential oil from keratin-based particles in simulated vaginal and sweat fluids, over 72 h. * indicates statistical difference compared to the results obtained at time 0 h (*p < 0.1, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Figure 3
Figure 3
Keratin microparticles encapsulating oregano oil (OEO-KMPs) cytotoxicity measured in Galleria mellonella model. (A) Survival curves of G. mellonella larvae in contact with OEO-KMPs and respective controls (without exposure to OEO-KMPs and keratin solution) during 72 h; and (B) G. mellonella larvae health index after 24, 48, and 72 h in contact with OEO-KMPs and respective controls.
Figure 4
Figure 4
Effect of the keratin microparticles encapsulating oregano oil (OEO-KMPs) on single and mixed infection of Candida albicans with Lactobacillus gasseri. (A) Single infection of C. albicans; (B) Mixed infection (C. albicans); (C) Mixed infection (L. gasseri). * indicates statistical reduction of biofilms cell cultivability in comparison with the respective control (**p < 0.01, *** p < 0.001, **** p < 0.0001).
Figure 5
Figure 5
Effect of OEO-KMPs on mice model of vaginal candidiasis. (A) Timeline of Candida albicans infection and OEO-KMPs’ treatment of mice; (B) Mice weight according to the timeline of infection; (C) C. albicans and Lactobacillus cells detected in mice vaginal lavage fluid (Log CFU mL–1) represented is the median with 95% confidence interval (*p < 0.05); (D) Histological images of vaginal tissue stained with periodic acid-Schiff (PAS), increasing magnification along the column: I. Mice uninfected (negative control); II. Mice infected with C. albicans, untreated (positive control); III. Mice infected with C. albicans, treated with OEO-KMPs for 24 h after infection. (a) Layer of superficial squamous epithelial cells, (b) basal membrane, (c) lamina propria (d) vaginal lumen, (e) and (f) C. albicans cells and (g) neutrophils. The results were registered under 10× and 20× magnification. The data presented in this study are representative of at least two independent experiments.
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