Effects of efflux pumps on antifungal activity of chitosan against Candida albicans.

Background: Antifungal resistance is a major problem, commonly caused by drug-efflux pump overexpression. To evaluate if chitosan could be effective in drug-resistant Candida infections, we investigated the effects of efflux pumps on antifungal activity of chitosan. Materials and Methods: The minimal fungicidal concentration (MFC) of oligomer (7-9 kD) and polymer (900-1,000 kD) chitosan against Saccharomyces cerevisiae and Candida albicans were evaluated by broth and agar dilution methods. The MFCs of S. cerevisiae with single deletion of efflux pump genes, with deletion of seven efflux pumps (AD∆), and AD∆ overexpressing C. albicans efflux pump genes (CDR1, CDR2 and MDR1) were determined. C. albicans with homozygous deletions of CDR1 and of CDR2 were generated using CRISPR-Cas9 system and tested for chitosan susceptibility. Results: While deleting any individual efflux pump genes had no effect on chitosan susceptibility, simultaneous deletion of multiple pumps (in AD∆) increased sensitivity to both types of chitosan. Interestingly, the overexpression of CDR1, CDR2 or MDR1 in AD∆ barely affected its sensitivity. Moreover, C. albicans with homozygous deletions of CDR1 and/or CDR2 showed similar sensitivity to wildtype. Conclusion: Thus, C. albicans susceptibility to chitosan was not affected by drug-efflux pumps. Chitosan may be a promising antifungal agent against pump-overexpressing azole-resistant C. albicans.

1. Neither deletion of efflux pump genes, nor overexpression of major C. albicans efflux pumps in pump-deficient S. cerevisiae, nor deletion of major efflux pumps in C. albicans affects yeast susceptibility to chitosan. 2. Chitosan may be an effective antifungal agent against drug-resistant C. albicans.

The Evaluation of Anti-Osteoclastic Activity of the Novel Calcium Hydroxide Biodegradable Nanoparticles as an Intracanal Medicament.

INTRODUCTION: The aim of this study was to evaluate the anti-osteoclastic activity of calcium hydroxide-loaded poly(lactic-co-glycolic acid) nanoparticles [Ca(OH)2-loaded PLGA NPs] in comparison to calcium hydroxide nanoparticles [Ca(OH)2 NPs]. METHODS: RAW 264.7 cell lines (third-fifth passage) were cultured and incubated with soluble receptor activator of nuclear factor kappa B ligand in triplicate. Subsequently, Ca(OH)2-loaded PLGA NPs and Ca(OH)2 NPs were added for 7 days to evaluate their effects on receptor activator of nuclear factor kappa B ligand-induced osteoclast differentiation of RAW 264.7 cells by tartrate-resistant acid phosphatase activity. Additionally, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted to confirm the cytotoxicity of treatments to cells. RESULTS: Tartrate-resistant acid phosphatase staining showed a significant reduction in the osteoclast number when treated with Ca(OH)2-loaded PLGA NPs compared with Ca(OH)2 NPs (P < .01). In comparison to the control, the number of osteoclasts significantly reduced upon treatment with Ca(OH)2-loaded PLGA NPs (P < .05), but there was no significant difference in Ca(OH)2 NPs. Furthermore, osteoclast morphology in both treatment groups exhibited smaller sizes than the control group. Neither Ca(OH)2-loaded PLGA NPs nor Ca(OH)2 NPs demonstrated cytotoxic effects on RAW264.7 cells. CONCLUSIONS: Both Ca(OH)2 NPs with and without poly(lactic-co-glycolic acid) have the ability to inhibit osteoclast differentiation. However, Ca(OH)2-loaded PLGA NPs exhibit greater potential than Ca(OH)2 NPs, making them a promising intracanal medicament for cases of root resorption.

The Effects of Calcium Hydroxide-loaded Poly(Lactic-co-glycolic Acid) Biodegradable Nanoparticles in the ex vivo External Inflammatory Root Resorption Model.

AIM: To evaluate the calcium ions (Ca2+) diffusion of calcium hydroxide-loaded poly(lactic-co-glycolic acid) biodegradable nanoparticles [Ca(OH)2-loaded PLGA NPs] compared with conventional Ca(OH)2 in a simulated external root resorption ex vivo model using inductively coupled plasma mass spectrometry (ICP-MS). MATERIALS AND METHODS: Thirty human mandibular premolars were prepared by sectioning the root segments to create roots measuring 10 mm from the anatomical apex. The root canals were instrumented and irrigated. The external root surface cavities were created. The specimens were randomly divided into the following three groups: Poly(lactic-co-glycolic acid) (PLGA; control group, n = 10), conventional calcium hydroxide [Ca(OH)2] (Metapaste, n = 10), and Ca(OH)2-loaded PLGA NPs [15% Ca(OH)2, n = 10]. The intracanal materials were placed in the root canals, and the teeth were stored in phosphate-buffered saline at 37°C. The release of Ca2+ was measured at 7, 30, and 60 days using ICP-MS. RESULTS: Both Ca(OH)2-loaded PLGA NPs and Metapaste groups exhibited higher levels of Ca2+ release compared to the PLGA group at all time points. During the initial 7-day period, the Ca(OH)2-loaded PLGA NPs exhibited a significantly greater release of Ca2+ compared to Metapaste. From day 7 to day 30, Metapaste displayed a significantly higher release of Ca2+ than the Ca(OH)2-loaded PLGA NPs, but it experienced a subsequent decline in Ca2+ release after the 30-day period. After the 30-day mark, the Ca(OH)2-loaded PLGA NPs once again exhibited a significantly higher release of Ca2+ compared to Metapaste. CONCLUSION: The Ca(OH)2-loaded PLGA NPs exhibited sustained release of Ca2+ that exceeded conventional Ca(OH)2, particularly during the first week, demonstrating a greater amount of Ca2+ release. CLINICAL SIGNIFICANCE: The utilization of Ca(OH)2-loaded PLGA NPs as an intracanal medication for external inflammatory root resorption provided sustained release and had the potential to enhance the efficacy of inhibiting root resorption more effectively than conventional Ca(OH)2.