Biology Reference
In-Depth Information
Lipid Isolation
Lipids were extracted by the method of Bligh and Dyer as modified for extreme halo-
philes [60], from frozen and thawed biomass of each colony with Cl 3 CH:CH 3 OH:H 2 O
(1:2:0.9; v:v), and the TPL fraction was collected by precipitation from cold acetone.
Phospholipids of the TPL were quantified by Bötcher [61] and Stewart [62] methods.
ESI-MS Analysis
The ESI-MS analysis was performed using a Thermo Finnigan LCQ Ion Max mass
spectrometer (Thermo Finnigan MAT, San Jose, CA, USA) equipped with a electro-
spray ionization source. Analyses were carried out in the loop injection mode with
dried lipid extracts dissolved in chloroform-methanol (1:1 vol/vol). Samples (5 μl)
injected via a 10 μl loop were transferred to an MS electrospray interface (ESI) at flow
rate of 10 μl/min. Interface conditions were as follows: nebulizer gas (air) 12 l/min,
curtain gas (nitrogen), 1.2 l/min needle voltage -5.0 kV (negative ions), mass range
50-2,000 amu.
ARC Preparation and Physicochemical Characterization
Two different types of ARC were prepared from TPL isolated from each colony: ARC-
GC and ARC- BM . Briefly, 20 mg of TPL from CHCl 3 : CH 3 OH (9:1, v/v) solution was
rotary evaporated at 40°C in round bottom flask until organic solvent elimination.
The thin lipid film was flushed with N 2 and hydrated at 40°C with 1 ml of 10 mM
Tris-HCl buffer plus 0.9% w/v NaCl, pH 7.4 (Tris buffer) (empty ARC) or with 1 ml
of 10 mg/ml BSA solution in Tris buffer (ARC-CG/ BM -BSA). The resultant suspen-
sions were sonicated (20 min in a bath type sonicator 80 W, 40 KHz) and submitted
to five cycles of freeze/thaw between -80 and 37°C. Free BSA was removed by cen-
trifugation and washing with Tris buffer (10,000 × g for 20 min). Liposomes made of
HSPC:cholesterol (1:1 M:M) were prepared in the same way.
The BSA/phospholipid weight ratio from each preparation was determined by
phospholipid and protein quantitation. Phospholipids were quantifi ed by Bötcher [61]
whereas BSA content was measured by Bradford [63].
Mean ARC size was determined by dynamic light scattering with a 90 Plus Par-
ticle size analyzer (Brookhaven Instruments) and zeta-potential was determined with
a Zetasizer 4 (Malvern). Electron microscopy images of ARC upon phosphotungstic
acid negative staining were obtained with a TEM Jeon 1210, 120 Kv, equipped with
EDS analyzer LINK QX 2000.
CYTOTOXICITY
Upon incubation with ARC, cell viability was measured as mitochondrial dehydroge-
nase activity employing a tetrazolium salt (XTT) on Vero cells and the murine macro-
phage-like cell line J-774 [64].
Cells maintained at 37°C with 5% CO 2 , in RPMI 1640 medium supplemented
with 10% heat-inactivated FBS, 2 mM glutamine, 100 UI/ml penicillin and 100 μg/
ml streptomycin, were seeded at a density of 5 × 10 4 cells/well in 96-well fl at bottom
microplates. Culture medium of nearly confl uent cell layers was replaced by 100 μl
 
Search WWH ::




Custom Search