Chitosan microparticles loaded with essential oils inhibit duo-biofilms of Candida albicans and Streptococcus mutans

Abstract

Objective: Oral candidiasis is a common fungal infection that affects the oral mucosa, and happens when Candida albicans interacts with bacteria in the oral microbiota, such as Streptococcus mutans, causing severe early childhood caries. C. albicans and S. mutans mixed biofilms are challenging to treat with conventional antimicrobial therapies, thus, new anti-infective drugs are required. This study aimed to test a drug delivery system based on chitosan microparticles loaded with geranium and lemongrass essential oils to inhibit C. albicans and S. mutans mixed biofilms.

Methodology: Chitosan microparticles loaded with essential oils (CM-EOs) were obtained by spray-drying. Susceptibility of planktonic were performed according CLSI at 4 to 2,048 µg/mL. Mixed biofilms were incubated at 37ºC for 48 h and exposed to CM-EOs at 256 to 4,096 µg/mL. The antimicrobial effect was evaluated using the MTT assay, with biofilm architectural changes analyzed by scanning electron microscopy. RAW 264.7 cell was used to evaluate compound cytotoxicity.

Results: CM-EOs had better planktonic activity against C. albicans than S. mutans. All samples reduced the metabolic activity of mixed C. albicans and S. mutans biofilms, with encapsulated oils showing better activity than raw chitosan or oils. The microparticles reduced the biofilm on the slides. The essential oils showed cytotoxic effects against RAW 264.7 cells, but encapsulation into chitosan microparticles decreased their toxicity.

Conclusion: This study demonstrates that chitosan loaded with essential oils may provide an alternative method for treating diseases caused by C. albicans and S. mutans mixed biofilm, such as dental caries.

Figure 1. Inhibitory effects of geranium essential oil (GEO), lemongrass essential oil (LEO), chitosan (C), chitosan microparticle (CM) chitosan microparticle loaded with lemongrass essential oil (CMLEO), and chitosan microparticle loaded with geranium essential oil (CMGEO) on the metabolic activity of the mature C. albicans and S mutans mixed biofilm, compared to mixed biofilms not exposed to the test samples (control). *, P˂0.05, the obtained test group values are given as the percentages of biofilm formation in relation to the control group. Results are shown as means ± SD

Figure 2. Scanning electron microscopy of two C. albicans - S.mutans species’ biofilms treated with CM-EOs. Untreated C. albicans- S. mutans mixed biofilm (A and B), C. albicans - S. mutans mixed biofilm treated with lemongrass essential oil (C), C. albicans - S. mutans mixed biofilm treated with 4,096 µg/mL of geranium essential oil (D), C. albicans - S. mutans mixed biofilm treated with 4,096 µg/mL of chitosan (E), C. albicans - S. mutans mixed biofilm treated with 4,096 µg/mL of chitosan microparticle (F), C. albicans - S. mutans mixed biofilm treated with 4,096 µg/mL of chitosan microparticle incorporated with lemongrass essential oil (CMLEO) (G), C. albicans - S. mutans mixed biofilm treated with 4,096 µg/mL of chitosan microparticle incorporated with geranium essential oil (CMGEO) (H). Yellow arrows indicate S. mutans cells and red arrows indicate C. albicans cells

Figure 3. Toxic effects of lemongrass essential oil (A), geranium essential oil (B), chitosan microparticle (C), chitosan microparticle incorporated with lemongrass essential oil (D) and chitosan microparticle incorporated with geranium essential oil (E) on the viability of RAW 264.7 cells. Values were expressed as means ± SEM of three independent determinations (n=8). Vehicle (positive control), Dx = 50% DMSO in medium (negative control). *p<0.05; **p<0.01; ***p<0.001 vs. control (ANOVA, followed by the Student-Newman-Keuls test)