1. © 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 200 7 www.leti.fr New Lipid Nanoparticles Formulations for Imaging and Drug delivery purposes and Drug delivery purposes Thomas Delmas 1, Fabrice P. Navarro 1, Isabelle Texier 1, Jérôme Bibette 2, Françoise Vinet 1, Anne Claude Couffin 1 1. DTBS/LETI-Minatec, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble – France (thomas.delmas@cea.fr) 2. ESPCI, Laboratoire Colloïdes et matériaux divisés, UMR 7612, 10 rue Vauquelin, 75005 Paris - France 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 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]. Experimental design: An experimental design was used to model the physicochemical behaviour of the LNP system. Its definition is summarized in Fig. 2. 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. Figure 2 : Variables defining the experimental design approach 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). Limits (%w/w) Input variables: LowHigh Aqueous phase (PBS 1X) 5095 Lipid mixture (25%oil+75%wax) 530 Lipophilic surfactant (Lipoid S75) 530 Hydrophilic surfactant (Myrj52) 575 Output parameters: Quantitative: (DLS) Size (nm) Polydispersity (UA) Qualitative: (comparative scale) Homogeneity Transparency Viscosity 82 trials Acknowledgments: We thank Dr F. De Crecy and Pr P. Ozil for their help for the experimental design Hydrophilic surfactant Hydrophobic surfactant Fluorophore and/or Drug Oil + Wax Figure 1 : LNP definition Figure 3 : Relevance of the diameter model: Dispersion analysis Vector molecule 2. LNP Formulation 1.Introduction 5. Conclusion & Perspectives 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 purposes (Fig. 8). Figure 8 : Examples of encapsulated species: From right to left: LNP, LNP(Nile Red), LNP(ICG), LNP(DiD),LNP(Paclitaxel) et LNP(mTHPC) Figure 7 : Cell viability assay 5. Fluorophore and/or API encapsulation4. Toxicity 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). 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). Figure 4 : Standard formulations FXX: physicochemical properties 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). [ Mean ± standard deviation (n=5) / dp : LNP diameter] FXX Size (nm)Polydispersity (AU) Zeta Potential (mV) dp modeldp exp (n=5)PDI modelPDI exp (n=5) F3027.2 30.6 ± 1.8 0.075 0.177 ± 0.034-4.9 ± 1.8 F5049.7 55.7± 2.4 0.187 0.168 ± 0.030-5.1 ± 2.5 F10084.2 94.6 ± 2.8 0.194 0.127 ± 0.017-6.9 ± 1.1 F120115.1 122.2 ± 2.5 0.186 0.106 ± 0.012-8.4 ± 0.4 Figure 6 : Accelerated stability (40°C): effect of size and internal composition Figure 5 : LNP cryoTEM imaging E. Neumann, IBS NCXX = % wax (w/w) FXX = size XX (nm)