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cells, differentiated by retinoic acid and BDNF (Kou et al., 2008). The role of
the omega-3 fatty acid in the presence of BDNF has been explained for its
effects on the synaptic plasticity and cognition (Kaplan et al., 1993; Matsumoto
et al., 1995). Polyunsaturated fatty acids have shown effi ciency in inhibiting
neuronal apoptosis (Kim et al., 2000).
In the neuronal phenotype, differences have been discovered between two
subclones of the SH-SY5Y cells induced by differentiation: (i) the SH-SY5Y-E
cells, which differentiate upon alternative treatment by retinoic acid and
BDNF, and (ii) SH-SY5Y-A cells, which differentiate easier via the formation
of a neuronal network after 5 days of treatment with retinoic acid. An increased
neuronal phenotype has been found when a sequential treatment with retinoic
acid and BDNF has been conducted over 3 days (Nishida et al., 2008).
It should be emphasized that the treatment of the human neuroblastoma
cell line SH-SY5Y by retinoic acid allows its differentiation by slowing down
or stopping the cellular proliferation mechanism. As a result, the characteristic
properties of the human neuronal cells, when they are affected by neurode-
generation, are reproduced in the chosen cellular model. BDNF has been
evidenced to stimulate the neuronal growth of differentiated human neuro-
blastoma cells. Thus, the SH-SY5Y cell line, treated with retinoic acid, repre-
sents an appropriate cellular model to evaluate in vitro the activity of
neurotrophic factors.
11.3
EXPERIMENTAL SECTION
11.3.1
Preparation of Lipid Nanoparticle Dispersions
Toward the objective to conceive anionic, multicompartment liquid crystalline
type of nanocarriers, constituted by the hydrated monoglyceride MO as the
main cubic-phase forming lipid, we employed the self-assembly method.
Oleic acid (OA) and EPA, which is an omega-3 polyunsaturated fatty acid
(20 : 5), were added to MO, in various proportions, in order to functionalize
the cubic-phase nanovector. A PEGylated phospholipid was also included in
the self-assembled lipid mixture in an attempt to generate sterically stabilized
multicompartment nanoparticles.
Monoolein (1 - oleyl -
rac
-glycerol), oleic acid (OA),
cis
- 5,8,11,14,17 eicosap-
entaenoic acid (20 : 5, EPA), dimyristoylphosphatidylcholine (DMPC), and
retinoic acid were purchased from Sigma-Aldrich (St. Quentin, France).
The 1,2 - dioleyl -
sn
- glycero - 3 - phosphoethanolamine -
N
- [methoxy (polyethyl-
ene glycol) - 2000] (DOPE - PEG
2000
) was a product of Avanti Polar Lipids.
Recombinant human BDNF and a primary monoclonal antihuman TrkB
antibody (MAB397) were purchased from R&D Systems (Lille, France).
The affi nity - purifi ed fl uorescent secondary antibody Alexa Fluor 633 goat
antimouse IgG
2b
(A21146) (2 mg/ml) was purchased from Invitrogen (Cergy
Pontoise, France). Mono- and dibasic sodium phosphate salts were dissolved
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