The goal of this study was to determine the effect of apolipoprotiens on Amphotericin B lipid complex (ABLC). We report that incubation of ABLC with recombinant human apolipoprotein A-I (apoA-I) induces solubilization of ABLC by transforming the micron sized phospholipid/AMB assemblies into discrete nanoscale disk-shaped complexes termed nanodisks (ND). ApoA-I induced changes in ABLC solubility and morphology were monitored by spectroscopy and electron microscopy. AMB efficacy was evaluated in yeast and pathogenic fungi growth inhibition assays and the effect of AMB formulation on cell toxicity was assessed in cultured Hep3B cells. AMB associated with ND were more efficiently nebulized than AMB associated with ABLC. Thus, transformation of ABLC into ND preserves the potent biological activity of AMB as well as the reduced toxicity of the ABLC formulation. ABLC derived AMB-ND offer advantages over conventional ABLC in terms of stability, storage, nebulization efficiency and provides an intrinsic "handle" for tissue specific targeting via genetic engineering of its protein component.

Nanometer scale apolipoprotein A-I stabilized phospholipid disk complexes (nanodisks; ND) have been formulated with the polyene antibiotic amphotericin B (AMB). The present studies were designed to evaluate if a peptide can substitute for the function of the apolipoprotein component of ND with respect to particle formation and stability. An 18-residue synthetic amphipathic α-helical, solubilized vesicles comprised of egg phosphatidylcholine (egg PC), dipentadecanoyl PC or dimyristoylphosphatidylcholine (DMPC) at rates greater than or equal to solubilization rates observed with human apolipoprotein A-I (apoA-I; 243 amino acids). Characterization studies revealed that interaction with DMPC induced a near doubling of 4F tryptophan fluorescence emission quantum yield (excitation 280 nm) and a ~7 nm blue shift in emission wavelength maximum. Inclusion of AMB in the vesicle substrate resulted in formation of 4F AMB-ND. Spectra of AMB containing particles revealed the antibiotic is a highly effective quencher of 4F tryptophan fluorescence emission, giving rise to a Ksv = 7.7 × 10 4 . Negative stain electron microscopy revealed that AMB-ND prepared with 4F possessed a disk shaped morphology similar to ND prepared without AMB or prepared with apoA-I. In yeast and pathogenic fungi growth inhibition assays, 4F AMB-ND was as effective as apoA-I AMB-ND. The data indicate that AMB-ND generated using an amphipathic peptide in lieu of apoA-I form a discrete population of particles that possess potent biological activity. Given their intrinsic versatility, peptides may be preferred for scale up and clinical application of AMB-ND.

BACKGROUND Macrolide polyene antibiotics possess potent broad-spectrum antifungal properties. Use of these agents in the field or in controlled environments is impeded by their poor water solubility and susceptibility to oxidation- and/or light-induced degradation. While typically used for human disease therapy, there is potential to expand the utility of polyene macrolide antibiotics, such as amphotericin B, for control of fungal disease infestation in agricultural settings. Thus, the susceptibility of this antibiotic to exposure-induced activity loss was evaluated. RESULTS Incubation of the prototype polyene amphotericin B (AMB) with phospholipid vesicles and apolipoprotein A–I results in the formation of nanoscale complexes, termed nanodisks (NDs), capable of solubilizing significant quantities of AMB. To evaluate whether AMB incorporation into NDs conferred protection against light- or oxidation-induced damage, yeast growth inhibition assays were conducted. Compared with AMB solubilized in detergent micelles, AMB incorporated into NDs was protected from damage caused by exposure to UV light as well as by KMnO4-induced oxidation. Furthermore, AMB-NDs inhibited growth of the turfgrass fungus Marasmius oreades Fr. CONCLUSION Results suggest that this water-soluble formulation of a natural, biodegradable, antifungal agent represents a potential cost-effective, non-toxic and environmentally friendly substitute for chemical agents currently employed to control a range of fungal infestations.

Objective: Twenty to 30 percent of children in cross-sectional studies have significant bedtime problems or night waking. Melatonin, a synthetic form of the hormone produced by the pineal gland as a biomarker of the circadian system, is a commonly used nonprescription pharmacologic treatment for sleep disorders in children. Many studies have demonstrated the effect of melatonin supplementation on sleep duration and sleep quality, which can improve overall systemic health and disease prevention. However, despite the growing number of studies demonstrating the effects of melatonin to improve disordered sleep, no available studies have evaluated the effects of melatonin on normal oral tissues. Based upon this lack of knowledge, the primary objective of this study is to evaluate any potential effects of melatonin on normal oral cells and tissues within the physiologically relevant (supplementation) range. Methods: Normal oral keratinocytes (OKF4) and human gingival fibroblasts (HGF-1) were obtained and cultured for this study. Melatonin was administered in 96-well growth assays at supplement-equivalent physiologic concentrations at the low, mid and high range (1, 5 and 10 ug/uL) to determine any effects on cellular growth and proliferation. Changes in cellular viability and expression of cell cycle and apoptosis-related pathways were also evaluated. Results: Curvilinear U-shaped dose responses were observed in OKF cells under melatonin administration, ranging from -11.4% (low), to a maximum of -13.6% (mid) and  -5.0% (high) compared with non-treated controls, p=0.029. Dose-responses among HGF-1 cells ranged from +12.1% (low), +17.4% (mid), and +5.0% (high), p=0.021. No changes in cellular viability were observed between control and experimental cells. However, qPCR screening of total RNA revealed significant changes in cell cycle related pathways, including c-myc, GAPDH and P53 but no changes in any apoptosis-related pathways, including Bcl-2, Bax, caspase-3, caspase-8 and caspase-9. Conclusions: This study demonstrated that melatonin does affect growth but not viability among these cell lines, which was found to be dose-dependent. These results suggest that melatonin may have some limited effects on oral tissues that may influence wound healing and repair but may not affect normal physiologic function or other cellular pathways. In agreement with other pediatric literature supporting the safety of melatonin use, this pilot study does not reveal any deleterious effects that would caution against its use in children or adults.