Materials and Methods
Murine C1498, 32D-p210-GFP, and 32D-FLT3-ITD cells, and human HL-60/vcr cells
were maintained at 37°C, and 5% CO2, in RPMI-1640 supplemented with 10% Fetal Bovine
Serum (FBS) and 1% penicillin/streptomycin. Human KG-1 cells were likewise maintained,
in IMDM supplemented with 20% FBS and 1% penicillin/streptomycin.
Nano liposomes were prepared by the Penn State College of
Medicine Drug Discovery Core following previously established
methods. All lipids were obtained from Avanti Polar Lipids (Alabaster,
AL, USA). Ghost nanoliposomes (Lip-Ghost) and Lip-C6 were
prepared as previously described [19, 21]. Briefly, lipids dissolved
in chloroform, or other organic solvents, were combined in specific
molar ratios, dried to a film under a stream of nitrogen, and then
hydrated by addition of 0.9% NaCl. Solutions were sealed, heated at
60°C (60 min), and subjected to vortex mixing and sonicated until
light no longer diffracted through the suspension. The lipid vesiclecontaining
solution was quickly extruded at 60°C by passing the
solution 10 times through 100 nm polycarbonate filters in an Avanti
Mini-Extruder. Nanoliposomal size and integrity was determined
using a Malvern Zetasizer Nano ZS at 25°C. Nanoliposome
formulations were stored at room temperature until use.
In Vitro assays
Cellular viability assays were performed as previously described
using a Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay
according to the manufacturer’s instructions (Promega, Madison,
WI) [11,19,21]. N-SMase assays were performed as previously
described using a Sphingomyelinase Inhibitor Screening kit from
Cayman Chemical (Ann Arbor, MI) according to the manufacturer’s
instructions . An ELISA assay was performed using previously
established methods adapted using a specific cysteine sulfenic acid
monoclonal antibody from Millipore (Billerica, MA) .
Blueberry extraction and fractionation
Solvents and reagents were obtained from VWR (Radnor, PA)
and Sigma (St. Louis, MO). Vaccinium uliginosum was harvested in
the interior of Alaska for extraction as previously described [14,15].
Briefly, whole berries were lyophilized, crushed to powder, and a
crude extract was prepared by extracting with aqueous acetone
(70/30 acetone/water), and dried by rotary- evaporation and
lyophilization. For fractionation, crude extracts were separated
by silica gel chromatography. Fractions were collected by elution
with 80/20 dichloromethane/methanol, assessed individually
by Thin Layer Chromatography (TLC), and dried by rotary
evaporation. Fraction 1 was further separated by silica gel flash
column chromatography, eluted using 92/8 dichloromethane/
methanol followed by pure methanol, and TLC was used to assess
fractions (Figure 1A-B). The N-SMase assay was used through the
fractionation process to define inhibitory bioactive fractions using
an inflammation-stimulated SH-SY5Y neuroblastoma cell model
(unpublished). Fraction 1,28 underwent a final clean-up purification by silica gel flash column chromatography, where elution with 85/15
dichloromethane/methanol yielded a relatively pure compound that
was characterized by LC-MS (ESI, Q-TOF, in positive ion mode) and
1H-NMR and identified as quercetin-3-O-arabinoside (Figure 1C).
Figure 1:Crude blueberry extract was separated by repeated silica column fractionation and a N-SMase assay was used to determine fractions that inhibited enzyme activity.
(a)Representative thin layer chromatography of the initial fractionation using dichloromethane/methanol as the
solvent (92/8) with fraction #1 selected for further separation.
(b)Representative thin layer chromatography of the second fractionation using dichloromethane/methanol as the
solvent (85/15) with fraction #28 selected for further separation.
(c)Following a clean-up fractionation, quercetin-3-Oarabinoside was identified by LC-MS (ESI, Q-TOF, in positive
ion mode) and 1H- NMR (inset) as the primary component of fraction 1,28.
CalcuSyn Software (Biosoft, Cambridge, UK) was used to
determine combinatorial effects of treatments . Cellular viability
data was used for this analysis, and a Combination Index (CI) less
than or equal to 0.9 was considered synergistic. CI values greater
than or equal to 1.1 were considered antagonistic, whereas CI values
between 0.9 and 1.1 were considered additive.