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
Experimental design:
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)
50
95
5
30
5
30
5
Output parameters:
700
experimental design trials
140
High
Quantitative:
(DLS)
Qualitative:
(comparative
scale)
82 trials
75
Size (nm)
Polydispersity
(UA)
Homogeneity
Transparency
600
test points
500
standard formulations
y = 0.9849x
R² = 0.8268
120
400
dp experimental (nm)
Low
dp experimental (nm)
Input variables:
Aqueous phase
(PBS 1X)
Lipid mixture
(25%oil+75%wax)
Lipophilic surfactant
(Lipoid S75)
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).
y = 1.0604x
R² = 0.4656
300
200
100
80
60
model points
40
test points
100
standard formulations
20
Linéaire (model points)
Viscosity
0
0
0
100
200
300
400
500
600
700
0
20
40
dp predicted (nm)
Figure 2 : Variables defining the experimental design approach
60
80
100
120
140
dp predicted (nm)
Figure 3 : Relevance of the diameter model: Dispersion analysis
Optimisation
The model was then used to propose formulations, giving monodisperse
populations of specific sizes. Standard formulations with different sizes (30,
50, 100, 120 nm) were isolated to study the effect of particle size on
biological interactions, biodistribution and encapsulation/release properties
(Fig. 4).
Stability:
All the sizes and compositions were shown to be stable at all
temperatures (4°C, Troom and 40°C) for more than 6 months (see 40°C:
Fig. 6). Increasing wax content in LNP core leads to decrease in particle
diameter (Fig. 6).
80
140
70
F30
F50
27.2
200
F100
7
F120
dp exp (n=5)
30.6 ± 1.8
PDI model
0.075
PDI exp (n=5)
0.177 ± 0.034 -4.9 ± 1.8
49.7
55.7± 2.4
0.187
0.168 ± 0.030 -5.1 ± 2.5
84.2
94.6 ± 2.8
0.194
0.127 ± 0.017 -6.9 ± 1.1
115.1
122.2 ± 2.5
0.186
0.106 ± 0.012 -8.4 ± 0.4
NC0
F120
100
60
F100
dp (nm)
dp model
120
Zeta
Potential
(mV)
dp (nm)
Polydispersity (AU)
E. Neumann, IBS
Size (nm)
FXX
F80
80
F50
60
NC25
NC50
50
NC75
NC100
40
40
Figure 5 : LNP
cryoTEM imaging
30
20
0
20
0
50
100
150
time (days)
FXX = size XX (nm)
[ Mean ± standard deviation (n=5) / dp : LNP diameter]
200
250
0
50
100
150
200
250
time (days)
NCXX = % wax (w/w)
Figure 6 : Accelerated stability (40°C): effect of size and internal composition
Figure 4 : Standard formulations FXX: physicochemical properties
4. Toxicity
5. Fluorophore and/or API encapsulation
125
115
NIH3T3 Fibroblasts viability (%CTRL)
LNP toxicity has been assessed both
in vitro and in vivo, with promising
results. Murine fibroblasts (NIH-3T3
cell line) present a viability > 95% after
24h incubation in the presence of high
LNP doses (1mg/mL) (Fig. 9).
Meanwhile, high LNP doses (150
mg/kg) were well tolerated after in vivo
systemic injection in rat (100% survival
after 5 weeks, n=6).
105
95
85
75
65
55
45
35
25
CTRL
H2O2 10
mM
150
500
1000
LNP (µg/mL)
Different lipophilic molecules
can be encapsulated in LNP.
Some fluorophores (ICG, Nile
Red, DiD, DiR, DiL…) and some
therapeutics (Paclitaxel, mTHPC,
mTHPP…) have already been
successfully encapsulated for both
imaging and drug delivery
Figure 8 : Examples of encapsulated species:
purposes (Fig. 8).
From right to left: LNP, LNP(Nile Red), LNP(ICG),
LNP(DiD),LNP(Paclitaxel) et LNP(mTHPC)
Figure 7 : Cell viability assay
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
www.leti.fr
© CEA 2008. Tous droits réservés.
Toute reproduction totale ou partielle sur quelque support que ce soit ou utilisation du
contenu de ce document est interdite sans l’autorisation écrite préalable du CEA
All rights reserved. Any reproduction in whole or in part on any medium or use of the
information contained herein is prohibited without the prior written consent of CEA