Protective and antifungal properties of Nanodisk-Amphotericin B over commercially available Amphotericin B

Objective:Amphotericin B (AMB),a potent antifungal agent,has been employed as topical and systemic therapy for sinonasal fungal infections.A novel formulation of nanodisc (ND) containing super aggregated AMB (ND-AMB) for the treatment of fungal infections has been recently developed to provide greater protection from AMB toxicity than current,clinically approved lipid-based formulations.The objective of the current study was to evaluate the safety and potency of ND-AMB for sinonasal delivery using an in vitro model.Methods:Human sinonasal tissue was harvested during endoscopic sinus surgery and grown at air-liquid interface until well-differentiated.Cultures were exposed to ND-AMB vs AMB and changes in K+ permeability and resistance were measured and recorded via Ussing chamber assay.Ciliary beat frequency (CBF) was analyzed in parallel as well as cytotoxic assay.Potency was assessed using real-time PCR measurement of the Aspergillus fumigatus 18S rRNA.Results:Ussing chamber studies revealed K+ currents that increased rapidly within 30 s of adding AMB (10 μg/mL) to the apical side,indicating apical membranes had become permeable to K+ ions.In contrast,negligible induction of K+ current was obtained following addition of NDAMB [AMB =(107.7 ± 15.9) μA/cm2 AMB vs ND-AMB =(2.3 ± 0.7) μA/cm2 ND-AMB;P =0.005].ND-AMB also protected nasal epithelial cells from cytotoxicity of AMB (P ＜ 0.05).There was no difference in ciliary beat frequency between the two groups (P =0.96).The expression of A.fumigatus 18S rRNA with exposure of lower dose of ND-AMB was significantly lower compared to that with AMB (P ＜ 0.05).Conclusions:Data from the present study suggests ND-AMB protects human nasal epithelia membranes from AMB toxicity by protecting against apical cell K+ permeability while maintaining uncompromised antifungal property compared to AMB.ND-AMB could provide a novel topical therapy for sinonasal fungal diseases.

Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro

The purpose of this study was to compare cell growth characteristics,ciliated cell differentiation,and function of human nasal epithelial cells established as explant outgrowth cultures or dissociated tissue cultures.Human nasal mucosa of the uncinate process was obtained by endoscopy and epithelial cell cultures were established by explant outgrowth or dissociated tissue culture methods.Epithelial cell growth characteristics were observed by inverted phase contrast microscopy.Ciliated cell differentiation was detected byβ-tubulin IV and ZO-1 immunocytochemistry.Basal and ATP-stimulated ciliary beat frequency(CBF)was measured using a high-speed digital microscopic imaging system.Both the explant and dissociated tissue cultures established as monolayers with tight junctions and differentiated cell composition,with both types of cultures comprising ciliated and non-ciliated epithelial cells.Fibroblasts were also frequently found in explant cultures but rarely seen in dissociated tissue cultures.In both culture systems,the highest ciliated cell density appeared at 7th–10th culture day and declined with time,with the lifespan of ciliated cells ranging from 14 to 21 days.Overall,10%of the cells in explant cultures and 20%of the cells in the dissociated tissue cultures were ciliated.These two cultures demonstrated similar ciliary beat frequency values at baseline(7.78±1.99 Hz and 7.91±2.52 Hz,respectively)and reacted equivalently following stimulation with 100μM ATP.The results of this study indicate that both the explant outgrowth and dissociated tissue culture techniques are suitable for growing well-differentiated nasal ciliated and non-ciliated cells,which have growth characteristics and ciliary activity similar to those of nasal epithelial cells in vivo.