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in MilliQ water (Millipore Co., Molsheim, France) to prepare the aqueous
buffer phase, which was sterilized.
Chloroform solutions of MO, OA, EPA, and DOPE-PEG
2000
were prepared
at desired concentrations by weighing the lipid powders or oils; the addition
of solvent (chloroform) was done using a Hamilton syringe. After mixing, the
solvent was evaporated from the MO/DOPE-PEG
2000
/OA/EPA systems under
a gentle stream of nitrogen gas to form fi ne and homogeneous lipid fi lms,
which were lyophilized overnight and stored at 4°C. The hydration of the
mixed lipid layers was performed by adding a phosphate buffer. Nanoparticu-
late dispersions were obtained by vortexing and ultrasonic irradiation in an
ice bath for less than 20 min (Branson 2510 ultrasonic bath, “set sonics” mode,
power 100 W). Filtration through a 0.22-
m fi lter (Minisart High Flow, Sarto-
rius, Palaiseau, France) was then carried out under a laminar fl ow hood in
order to sterilize the samples, which were stored in glass vials at room tem-
perature before the physicochemical and cellular tests.
μ
11.3.2
Characterization of Lipid Nanocarriers
11.3.2.1 Quasi-Elastic Light Scattering (QELS) and Optical Density
Measurements
The hydrodynamic diameter of the generated lipid nanoob-
jects was determined using a Nanosizer apparatus (Nano-ZS90, Malvern,
Orsay, France) at a scattering angle of 90° and at 25°C. The average hydrody-
namic diameter,
d
h
, was calculated considering the mean translational diffu-
sion coeffi cient,
D
, of the particles in accordance with the Stokes-Einstein law
for spherical particles in the absence of interactions:
d
h
=
k
B
T
/3
ηπ
D
, where
k
B
is the Boltzmann constant,
T
is temperature, and
η
is the viscosity of the
aqueous medium.
The size measurements were performed after dilution of 150
l of a lipid
NP sample in 1 ml of phosphate buffer or 0.2 ml of sample in 2 ml of cell
culture medium. The results were analyzed using the MALVERN Zetasizer
software (version 6.11). The particle distributions were expressed via the
“volume” analysis mode by averaging three measurements with the same
sample. Optical density (OD) measurements were conducted on a double-
beam ultraviolet (UV)/visible spectrophotometer PerkinElmer Lambda 2
(PerkinElmer, Courtaboeuf, France) at 25°C.
μ
11.3.2.2 Small-Angle X-ray Scattering (SAXS)
The small - angle X - ray
scattering measurements with the MO/DOPE-PEG
2000
/EPA (83/2/15 mol %)
and MO/DOPE - PEG
2000
(98/2 mol %) samples were performed at the ID22
beam line of the Diamond Light Source (Didcot, UK). The investigated lipid
dispersions were liquid solutions of nanoparticles that were fi lled in borosili-
cate glass capillaries (diameter 1.5 mm, 10 -
m wall thickness). The samples
were mounted in a holder in air and at room temperature (21°C), without a
special vacuum chamber. The detector at the I22 station was a gas wire RAPID
2D detector (www.diamond.ac.uk/Home/Beamlines/I22/tech/detectors.html),
μ
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