Dual nanocarrier of chlorhexidine and fluconazole: Physicochemical characterization and effects on microcosm biofilms and oral keratinocytes.

OBJECTIVES: This study assembled and characterized a dual nanocarrier of chlorhexidine (CHX) and fluconazole (FLZ), and evaluated its antibiofilm and cytotoxic effects. METHODS: CHX and FLZ were added to iron oxide nanoparticles (IONPs) previously coated by chitosan (CS) and characterized by physical-chemical analyses. Biofilms from human saliva supplemented with Candida species were grown (72 h) on glass discs and treated (24 h) with IONPs-CS carrying CHX (at 39, 78, or 156 µg/mL) and FLZ (at 156, 312, or 624 µg/mL) in three growing associations. IONPs and CS alone, and 156 µg/mL CHX + 624 µg/mL FLZ (CHX156-FLZ624) were tested as controls. Next, microbiological analyses were performed. The viability of human oral keratinocytes (NOKsi lineage) was also determined (MTT reduction assay). Data were submitted to ANOVA or Kruskal-Wallis, followed by Fisher's LSD or Tukey's tests (α=0.05). RESULTS: Nanocarriers with spherical-like shape and diameter around 6 nm were assembled, without compromising the crystalline property and stability of IONPs. Nanocarrier at the highest concentrations was the most effective in reducing colony-forming units of Streptococcus mutans, Lactobacillus spp., Candida albicans, and Candida glabrata. The other carriers and CHX156-FLZ624 showed similar antibiofilm effects, and significantly reduced lactic acid production (p<0.001). Also, a dose-dependent cytotoxic effect against oral keratinocytes was observed for the dual nanocarrier. IONPs-CS-CHX-FLZ and CHX-FLZ significantly reduced keratinocyte viability at CHX and FLZ concentrations ≥7.8 and 31.25 µg/mL, respectively (p<0.05). CONCLUSION: The nanotherapy developed outperformed the effect of the combination CHX-FLZ on microcosm biofilms, without increasing the cytotoxic effect of the antimicrobials administered. CLINICAL SIGNIFICANCE: The dual nanocarrier is a promising topically-applied therapy for the management of oral candidiasis considering that its higher antibiofilm effects allow the use of lower concentrations of antimicrobials than those found in commercial products.

Efeitos antibiofilme e citotóxico de um novo nanosistema carreador de clorexidina e fluconazol / Antibiofilm and cytotoxic effects of a new nanocarrier of chlorhexidine and fluconazole

Os objetivos do presente estudo foram montar e caracterizar um novo nanocarreador dual de clorexidina (CLX) e fluconazol (FLZ), bem como avaliar seu efeito sobre biofilmes microcosmos e sua citotoxicidade sobre queratinócitos orais. Para montar o nanocarreador dual, CLX e FLZ foram adicionados a nanopartículas de óxido de ferro (NPsOF) previamente revestidas por quitosana (QTS), seguido de um processo de solubilização sob agitação magnética. O nanocarreador foi, então, caracterizado por microscopia eletrônica de transmissão, difração de raios X, espectroscopia no infravermelho por transformada de Fourier e análise termogravimétrica. A suscetibilidade de Candida albicans e Candida glabrata no estado planctônico ao nanocarreador dual foi determinada pelos valores de concentração inibitória mínima, utilizando o método da microdiluição em caldo. Um pool de saliva de 2 doadores saudáveis suplementado com espécies de Candida foi usado como inóculo para a formação de biofilmes microcosmos. Os biofilmes foram cultivados (72 h) sobre discos de vidro posicionados no Amsterdam Active Attachment model e tratados (24 h) com NPsOF-QTS carreando 39 µg/mL de CLX + 156 µg/mL de FLZ (NPsOF-QTS-CLX39-FLZ156), 78 µg/mL de CLX + 312 µg/mL de FLZ (NPsOF-QTS-CLX78-FLZ312) e 156 µg/mL de CLX + 624 µg/mL de FLZ (NPsOF-QTS-CLX156-FLZ624). NPsOF (218,5 µg/mL), QTS (218,5 µg/mL) e 156 µg/mL de CLX + 624 µg/mL de FLZ (CLX156-FLZ624) foram testados como controles. Posteriormente, foram realizadas as análises de quantificação das unidades formadoras de colônias (UFCs), produção de ácido lático (LA), composição da matriz extracelular (ME) e viabilidade celular por microscopia confocal de varredura a laser (MCVL). Para o ensaio de citotoxicidade, queratinócitos orais humanos (linhagem NOKsi) foram expostos a diferentes concentrações do nanocarreador dual, por 24 ou 48 h, e a viabilidade celular foi determinada pelo ensaio de redução de MTT. Os dados foram analisados por ANOVA ou teste de Kruskal-Wallis, seguidos dos testes de Fisher LSD ou Tukey (α = 0,05). Os testes de caracterização físico-química mostraram que um nanocarreador dual com dimensões em torno de 6 nm foi obtido, sem comprometer a propriedade cristalina e a estabilidade de NPsOF. Os compostos que formam o nanocarreador estabeleceram uma interação sinérgica em relação ao efeito sobre células planctônicas de Candida. Para os ensaios de biofilme, NPsOF-QTS-CLX156-FLZ624 foi o composto mais eficaz na redução de UFCs de Streptococcus mutans, Lactobacillus spp., C. albicans e C. glabrata, diferindo significativamente dos outros grupos, e esses achados foram confirmados por MCVL. NPsOF-QTS-CLX39-FLZ156, NPsOF-QTS-CLX78-FLZ312 e CLX156- FLZ624 mostraram efeitos antibiofilme similares. O nanocarreador dual também reduziu significativamente a produção de AL e a quantidade de carboidratos e ácidos nucleicos da ME. Um efeito citotóxico dose-dependente sobre queratinócitos orais foi observado para o nanocarreador dual, independentemente do período de exposição testado (24 ou 48 h). NPsOF-QTS-CLX-FLZ e CLX-FLZ reduziram significativamente a viabilidade dos queratinócitos em concentrações de CLX e FLZ iguais ou superiores a 7,8 e 31,25 µg/mL, respectivamente. Por fim, a nanoterapia testada no presente estudo é promissora e constitui um grande avanço dentro dos métodos alternativos aos antimicrobianos tradicionais para o controle da candidíase oral(AU)

The objectives of the present study were to assemble and characterize a new dual nanocarrier of chlorhexidine (CHX) and fluconazole (FLZ), and evaluate its effect on microcosm biofilms and its cytotoxicity against oral keratinocytes. To assemble the dual nanocarrier, CHX and FLZ were added to iron oxide nanoparticles (IONPs) previously coated by chitosan (CS), followed by a solubilization process under magnetic stirring. The nanocarrier was then characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The susceptibility of Candida albicans and Candida glabrata in the planktonic state to the dual nanocarrier was determined by the minimum inhibitory concentration values, using the broth microdilution method. A saliva pool from 2 healthy donors supplemented with Candida species was used as an inoculum for microcosm biofilm formation. Biofilms were grown (72 h) on glass discs positioned in the Amsterdam Active Attachment model and treated (24 h) with IONPs-CS carrying 39 µg/mL CHX + 156 µg/mL FLZ (IONPsCS-CHX39-FLZ156), 78 µg/mL CHX + 312 µg/mL FLZ (IONPs-CS-CHX78-FLZ312), and 156 µg/mL CHX + 624 µg/mL FLZ (IONPs-CS-CHX156-FLZ624). IONPs at 218.5 µg/mL, 218.5 µg/mL CS, and 156 µg/mL CHX + 624 µg/mL FLZ (CHX156-FLZ624) were tested as controls. Next, analyses of the quantification of colony-forming units (CFUs), lactic acid production (LA), composition of the extracellular matrix (EM), and viability by confocal laser scanning microscopy (CLSM) were performed. For the cytotoxicity assay, human oral keratinocytes (NOKsi lineage) were exposed to different concentrations of the dual nanocarrier, for 24 or 48 h, and cell viability was determined by the MTT reduction assay. Data were analyzed by ANOVA or Kruskal-Wallis test, followed by Fisher LSD or Tukey tests (α = 0.05). The physico-chemical characterization tests showed that a dual nanocarrier with dimensions around 6 nm was assembled, without compromising the crystalline property and stability of IONPs. The compounds that form the nanocarrier established a synergistic interaction in relation to the effect on Candida planktonic cells. Regarding biofilm assays, IONPs-CS-CHX156-FLZ624 was the most effective compound in reducing CFUs from Streptococcus mutans, Lactobacillus spp., C. albicans, and C. glabrata, differing significantly from the other groups, and these findings were confirmed by CLSM. IONPs-CS-CHX39-FLZ156, IONPs-CS-CHX78-FLZ312, and CHX156-FLZ624 showed similar antibiofilm effects. The dual nanocarrier also significantly reduced LA production and the amount of carbohydrates and nucleic acids from the EM. A dose-dependent cytotoxic effect against oral keratinocytes was observed for the dual nanocarrier, regardless of the exposure period tested (24 or 48 h). IONPs-CSCHX-FLZ and CHX-FLZ significantly reduced keratinocyte viability at CHX and FLZ concentrations equal to or greater than 7.8 and 31.25 µg/mL, respectively. In conclusion, the nanotherapy tested in the current study is promising and constitutes a major advance in alternative methods to traditional antimicrobials for oral candidiasis control(AU)

Effects of Antifungal Carriers Based on Chitosan-Coated Iron Oxide Nanoparticles on Microcosm Biofilms.

Resistance of Candida species to conventional therapies has motivated the development of antifungal nanocarriers based on iron oxide nanoparticles (IONPs) coated with chitosan (CS). This study evaluates the effects of IONPs-CS as carriers of miconazole (MCZ) or fluconazole (FLZ) on microcosm biofilms. Pooled saliva from two healthy volunteers supplemented with C. albicans and C. glabrata was the inoculum for biofilm formation. Biofilms were formed for 96 h on coverslips using the Amsterdam Active Attachment model, followed by 24 h treatment with nanocarriers containing different concentrations of each antifungal (78 and 156 µg/mL). MCZ or FLZ (156 µg/mL), and untreated biofilms were considered as controls. Anti-biofilm effects were evaluated by enumeration of colony-forming units (CFUs), composition of the extracellular matrix, lactic acid production, and structure and live/dead biofilm cells (confocal laser scanning microscopy-CLSM). Data were analyzed by one-way ANOVA and Fisher LSD's test (α = 0.05). IONPs-CS carrying MCZ or FLZ were the most effective treatments in reducing CFUs compared to either an antifungal agent alone for C. albicans and MCZ for C. glabrata. Significant reductions in mutans streptococci and Lactobacillus spp. were shown, though mainly for the MCZ nanocarrier. Antifungals and their nanocarriers also showed significantly higher proportions of dead cells compared to untreated biofilm by CLSM (p < 0.001), and promoted significant reductions in lactic acid, while simultaneously showing increases in some components of the extracellular matrix. These findings reinforce the use of nanocarriers as effective alternatives to fight oral fungal infections.