Transcript Présentation PowerPoint

New Lipid Nanoparticles Formulations for Imaging
and Drug delivery purposes
Thomas Delmas1, Fabrice P. Navarro1, Isabelle Texier1, Jérôme Bibette2, Françoise Vinet1, Anne Claude Couffin1
1. DTBS/LETI-Minatec, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble – France ([email protected])
2. ESPCI, Laboratoire Colloïdes et matériaux divisés, UMR 7612, 10 rue Vauquelin, 75005 Paris - France
1.Introduction
Nanomedicine is the use of nanotechnology for medical applications. In this field, drug delivery and in vivo imaging
show great promises through the use of nanoparticulate systems as nanocarriers for the protection and targeting of active
pharmaceutical ingredients (API) and/or molecular contrast agents. Among a wide variety of nanocargos, Lipid
NanoParticles (LNP) present numerous advantages over other formulations. These nanocarriers are biocompatible,
biodegradable, allow controlled release and can easily be produced by versatile and up-scalable processes. In this work, we
investigated the physicochemistry of LNP to propose optimised formulations for API and/or dye encapsulation. The size will
be critical when considering biological interactions [1] and targeting (EPR effect) [2], but may also affect release profiles
[3]. LNP core composition should also impact the release properties [4]. Meanwhile, the size distribution will affect LNP
physical stability: the more monodisperse the dispersion, the highest the physical stability [5].
Oil + Wax
Hydrophobic surfactant
Hydrophilic surfactant
Vector molecule
Fluorophore
and/or Drug
Figure 1 : LNP definition
2. LNP Formulation
Model:
The model was constructed on a quadratic design with main effects. The
relevance of the diameter model is illustrated in Fig. 3. The model diameter
is thus reliable for LNP of sizes ranging from 20 to 120nm. The model
regression coefficients are higher than 0.85 (R²=0.90 / R²adjusted = 0.88).
Design definition:
An experimental design was used to model the physicochemical behaviour
of the LNP system. Its definition is summarized in Fig. 2.
Limits
(%w/w)
Hydrophilic
surfactant (Myrj52)
5
5
Quantitative:
(DLS)
95
Size (nm)
Polydispersity
(UA)
30
Qualitative:
(comparative
scale)
30
75
y = 0.9849x
R2 = 0.8268
600
82 trials
Homogeneity
Transparency
90
80
70
50
30
20
10
5
1
120
y = 1.0604x
R2 = 0.4656
95
500
Dp experimental (nm)
5
Dispersion for dp model ≤ 120nm
140
99
Dp experimental (nm)
50
Total dispersion (all dp model)
700
High
Normal % Probability
Low
Normal % probability
Input variables:
Aqueous phase
(PBS 1X)
Lipid mixture
(25%oil+75%wax)
Lipophilic surfactant
(Lipoid S75)
Normal
plot
of residuals
Normal
Plot
of Residuals
Output parameters:
400
experimental design
trials
test points
300
200
formulations reference
100
Linéaire (experimental
design trials)
Viscosity
-1.03
0.01
1.04
experimental design
trials
test points
60
40
formulations reference
Linéaire (experimental
design trials)
0
0
2.08
80
20
0
-2.07
100
100
Externally studentised residuals
200
300
400
500
600
700
0
20
Dp predicted (nm)
40
60
80
100
120
140
Dp predicted (nm)
Internally Studentized Residuals
Figure 3 : Relevance of the diameter model: Normal plot and dispersion analysis
Figure 2 : Variables defining the experimental design approach
100
Size (nm)
Polydispersity (AU)
FXX
61
dp model
dp exp (n=5)
PDI model
PDI exp (n=5)
Zeta Potential
(mV)
41.5
F30
27.2
30.6 ± 1.8
0.075
0.177 ± 0.034
ND*
22
F50
49.7
55.7± 2,4
0.187
0.168 ± 0,030
-5.1 ± 2.5
C (0.278)
A (0.500)
B (0.050)
B (0.328)
C (0.000)
F100
84.2
94.6 ± 2.8
0.194
0.127 ± 0.017
-6.9 ± 1.1
F120
115.1
122.2 ± 2.5
0.186
0.106 ± 0.012
-8.4 ± 0.4
A (0.777)
[ Mean ± standard deviation (n=5) / * ND=Not determined ]
Figure 4 : F50 standard
formulation
Figure 5 : Standard formulations FXX: physicochemical properties
3. Effect of LNP core composition
Varying
LNP
internal
composition may be the way
to tune the encapsulation and
the release behavior of the
nanocarrier. Increasing the
internal weight fraction of
wax lead to smaller particles
(Fig. 6).
4. Cytotoxicity
The LNP cytotoxicity was
evaluated by a cell viability assay
(ViaCount®). Murine fibroblasts
(NIH-3T3
cell
line)
were
incubated in presence of LNP for
24h at 37°C (Fig. 7). LNP
displayed no cytotoxicity for
concentrations up to 1000 µg/ml.
[ Mean ± standard deviation (n=3)]
Figure 6 : Effect of core composition on
LNP size
100
Percent viability
D ia m e tre
80.5
Diametre (nm)
Optimisation:
The model was then used to propose
formulations
giving
monodisperse
populations of specific size. Standard
formulations with different sizes (30, 50,
80, 100, 120 nm) were isolated to study
the effect of particle size on biological
interactions,
biodistribution
and
encapsulation/release properties (Fig. 5).
Results
200 displayed thereafter focused on
the 50nm formulation, called F50 (Fig.
7
4).
95
90
85
80
75
CTRL
LNP
[ Mean ± standard deviation (n=3)]
Figure 7 : Cell viability assay
(Abbreviation: CTRL, control)
5. Conclusion & Perspectives
To conclude, we designed and characterised ready made lipid nanoparticles.
Being able to incorporate a wide range of lipophilic molecules, LNP could be
foreseen as a nanoplatform for imaging and drug delivery purposes through
fluorophore and/or API encapsulation. The large flexibility of LNP formulation
should allow their characteristics to be tuned to obtain optimised encapsulation
and/or release properties.
References: [1] Lundqvist, M. et al. PNAS 105 (2008) 14265-14270 [2] Tbata, Y. et al. J. Control Release 50
(1998) 123-133 [3] zur Mühlen, A. et al. Eur. J. Pharm. Biopharm. 454 (1998) 149-155 [4] Müller, R. H. et al.
Int. J. Pharm. 242 (2002) 121-128 [5] Uner, M. Pharmazie 61 (2006) 5
Further studies will be envisioned:
- LNP biodistribution recording using fluorescence imaging
- Surface chemistry modification for improving particle targeting and
internalisation
- Correlation between the internal physical state of LNP and their
encapsulation/release properties
Acknowledgments: We thank Dr F. De Crecy and Pr P. Ozil for their help for
the experimental design
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