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Responsable : Montier Tristan • Responsable Tech. : Benvegnu Thierry (20%, ENSC Rennes) Jaffres Paul (15%, Brest) Loyer Pascal (30%, Rennes)
Adresse : Plateforme SynNanoVect, Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, 22 rue Camille Desmoulins, CS 93837
29238 BREST Cedex 3 - France



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Presentation de la plate-forme

SynNanoVect provides several products and services to its customers. The chemistry part of the platform based on UMR CNRS 6521 (UBO) and UMR CNRS 6226 (ENSCR) are highly specialised into the synthesis and the characterisation of synthetic carriers. Considering the evaluation of the formulations associating synthetic vectors from the platform (or not) and nucleic acids constructs or biomolecules provided by the partners, the biology part of the platform (INSERM U1078 - Brest and NuMeCam ex-U991 - Rennes) disposes of numerous in vitro and in vivo models as well as various equipments to measure the efficiency and the toxicity / inflammation of the nanocarrier formulations.

I. Synthesis and formulation of nanocarriers
A- Design and production of synthetic reagents for the delivery of nucleic acids constructs and/or biomolecules
The platform is specialised in the organic synthesis of lipid molecules within a context of vectorisation. Three types of original lipid structures are available and synthesised in the platform (Cationic lipids, Neutral lipids and Targeting lipids) which would permit to fulfil numerous vectorisation requirements. Cationic lipids include glycine betaine-type (quaternary ammonium) and lipophosphoramidate-type lipids. These lipids efficiently complex anionic nucleic acid constructs and would be also useful for the vectorisation of other anionic biomolecules. These cationic lipids have been widely and successfully used for in vitro transfection assays. Some of these vectors (in particular the lipophosphoramide with an arsonium polar head) proved to be efficient for in vivo DNA delivery as well. Research is still in progress to produce new innovative cationic lipids. Neutral lipids include archaeal-type (diether or tetraether) and lipophosphoramidate (imidazole) lipids. The tetraether lipids are known to improve stabilities of liposomes, while the diether lipids have fusogenic properties. Regarding the neutral lipophosphoramidate characterized by an imidazole polar head, it has been recently shown that this type of vector has a stabilizing effect of the liposome at physiological pH while at lower pH it exhibits fusogenic properties. These neutral lipids will be included into the formulations required by the external demands. Targeting lipids include archaeal lipids bearing targeting ligands such as folic acid or trisaccharidic clusters (mannose, lactose) through a pegylated spacer. Such lipids are also formulated with cationic or neutral lipids (few percents) in the aim to deliver nucleic acid constructs or biomolecules selectively to a specific cell/tissue type. The cationic lipids and neutral co-lipids have been design to carry DNA into cells. These formulations have also the potential to carry other type of bioactives molecules including for instance RNA.

B- Preparation of nanoparticles, encapsulation and characterisation of supramolecular assembly properties
The efficiency of a drug delivery system depends on the physico-chemical behaviour of the complexes resulting from the association of the chemical carrier and the bio-active molecule. A better understanding of the physical properties of both the carrier and the complex is essential for a rational design of new carriers or formulations. Several techniques are available in the platform for the production of nanoparticles, i.e. lipid film hydration followed by sonication or extrusion through polycarbonate membrane, nanoprecipitation or ethanolic injection. In addition, we have the knowledge required for the encapsulation of various biological active molecules (hydrophilic, hydrophobic, neutral or charged). To use such nanocarriers as drug delivery systems and for gene therapy, it is of great importance to well characterize these supramolecular assemblies containing or not the therapeutic agent. For that purpose, several techniques are available on our platform and are listed thereafter: 1) Zetasizer based on Dynamic Light Scattering (DLS) for average diameter and size distribution analysis as well as zeta potential measurement ; 2) Small Angle X-Ray Scattering (SAXS) for internal molecular structure of the nanocarriers; 3) UV-Vis spectrometry for the quantification of drug encapsulation; 4). Electrophoretic mobility for DNA-binding ability; 5) Transmission Electron Microscopy (TEM) and cryo-TEM to analyze nanocarrier morphologies (this apparatus will be available at the beginning of the year 2011); 6) Isothermal Titration Calorimetry (ITC) to characterize biomolecular interactions (ligand/membrane receptor, availability of targeting agents on nanoparticle surface, this apparatus will be available in 2009); 7) A Varian Cary Eclipse spectrofluorimeter and a YAG Laser coupled with an optical parametric oscillator and an ICCD camera. These equipments are used to achieve FRET experiments and anisotropy of fluorescence measurement. These analyses are used to evaluate the fusogenic properties of the synthetic vectors.

II. Biological evaluation and conception
C- In vitro and in vivo efficacy of complexes
Firstly, the efficiency of the formulations is evaluated on relevant cell lines and/or on primary cellular models (airway epithelium, melanocytes, hepatocytes HepaRG cells...). Although the transfection efficiency of a given DNA / biomolecules associated with a nanocarrier with cell lines does not completely predict its in vivo efficiency, the proposed in vitro evaluation allow us to get a better insight into the relationships between the in vitro and in vivo transfection efficiency of the formulations studied herein. The efficiency of the formulation is evaluated directly in living animals (mice) by using an in vivo bioluminescence imaging system. The INSERM U1078 team is equipped with a such recent highly sensitive imaging system. Here, the luminescent photons are detected and quantified without euthanazing the animals. Thus, in addition to a simple global evaluation of the efficacy of the different formulations, such an imaging system allows to study, in standardized groups of living animals, the kinetics of transgene or labelled molecule (peak, duration), and it also allows to evaluate the efficacy of repeated dosing (re-administration). Obviously, different routes of administration can be evaluated (systemic, aerosol, or HLV) and different types of mice can be studied (normal or transgenic). We could also insure the localisation of the transgene by immunohistochemistry with specific antibodies. This allows the precise identification of the transfected cells and the quantification of the number of treated cells. For our partner, we could also provide a housing mice service, especially if the experiment requires the use of specific rodent models.

D- Electroporation facilities
Although lipids synthesized by the chemists from the platform SynNanoVect exhibit high levels of transfection in several cell types in vitro and in vivo, for non dividing cells and/or non adherent cells (hematopoietic cells), cationic lipids and polymers either generated by us or commercially available remain poorly efficient. To address the need of academic investigators and private companies for efficient transfection procedures to deliver nucleic acids in these cell types “hard to transfect”, we set up a core facility dedicated to electroporation using two devices: 1) the Microporator MP100 (engineered by Digital Bio and originally distributed by LabTech France; now commercially available from Invitrogen and sold under the name of NeonTM system). This equipment was purchased in 2007, 2) the last 4D-Nucleofector station from Amaxa/Lonza including the core unit, the X unit (electroporation of single cuvettes or nucleocuvette strips) and the Shutlle device for electroporation of 96-well plates purchased in December 2010. This electroporation devices purchased with financial supports from the GIS IBiSA, Région Bretagne and Inserm U991 federal fundings, are hosted by the Inserm UMR991 (Rennes) in a tissue culture room dedicated to the SynNanoVect platform, which also contains a culture hood, a CO2 incubator and all the small equipment for cell culture. Since the electroporation devices are very easy to manipulate, the experiments are performed by the users themselves without the requirement for man power coming from the SynNanoVect platform. The transfection efficiencies are evaluated by the users too, either by a molecular biology approach, microscopy or flow cytometry using the other core facilities available on the site.
The core facility also performs R&D by optimizing electroporation protocols on mammalian cells and by developing brand new procedures of electroporation on non-mammalian cells including in fish cells.

E- Pre-clinical safety evaluation and biodistribution studies
As the accumulation of cationic lipids and polymers could lead to disturbances in cells affecting survival potential and the rate of proliferation, we also propose to investigate stress responses in cells of interest following siRNA or plasmid transfection by measuring oxidative stress, mitochondrial and endoplasmic reticulum, proteasome activities (using commercial standard kits or our own cytotoxic tests). We also could measure the proliferation activity to determine if molecular vectors and transfected nucleic acids inhibit proliferation with downstream decrease in nuclear translocation of expression DNA plasmids or gene knockdown by siRNAs. Evaluation of hepatotoxicity of synthetic vectors will include the in vitro studies of toxicity using several hepatic cell lines especially the highly differentiated human hepatocarcinoma cell lines HepaRG which currently represents the most relevant in vitro cell system for drug screening.
It is obviously of crucial importance that the most efficient formulations are also safe, especially when considering their clinical application. Thus, we propose to perform several measurements to assess their in vivo toxicity by using clinical assays (adapted for small volumes of blood and urines) in living animals; this is performed in addition to the classical histopathological techniques. Serum samples will be obtained and the concentration of alkaline phosphatase, AST and ALT are measured. CPK and Alcaline phosphatase are also quantified and the ionogram will be in a short time also established. Cytokine levels in mouse serum are measured using specific immunoassays kits. The urine pH is measured and glucose, bilirubine, ketones and protein concentrations are also determined by colorimetric measurements.

III. Experiment conception service and tailor made experiments
An increasing number of investigators in academic and biotechnology research need to vehicle DNA, siRNA or biomolecules in order to investigate genomic, metabolic or pharmacologic effects of the delivered molecules in a large variety of cells from yeast to human but also from plants. The ultimate goal of our platform is to provide to poorly experimented investigators a large set of synthetic vectors and also electroporation technologies to transfect their cell(s) of interest with a high efficiency. To reach that ambitious goal, the strategy is the following:
- To request a detailed description of molecule(s) to be delivered and targeted cell system(s) for each project in order to rapidly evaluate the feasibility of the project. At this stage, the committee may decide to turn down some “out of scoop” projects. Accordingly to the ISO9001 normes, all the submitted projects are considered as confidential.
- To propose to project leaders the protocols already available on our platform or tailor-made protocols according to specific requirements linked to the molecules to be delivered or the target cell system. Milestones to conduct the project will be proposed. According to geographic location of the project leader and depending on the specificity of the project, the experiments will be conducted either in Brest and/or Rennes. The development of the project may include man training of people belonging to the group submitting the project.
- To analyse with the project leaders the initial results in order to write down a finalized protocol or to conduct further experiments to improve the delivery.

Moyens et equipements

SynNanoVect platform presents a catalog with more than twenty transfection molecules for the transfer of nucleic acids constructs (DNA, siRNA, ODN....) or biomolecules (peptides, therapeutic compounds). It offers a large number of equipment for the synthesis and the characterization of nanovectors (UMR CNRS 6521-Brest and UMR CNRS 6226-Rennes) as well as their biological evaluation in term of efficacy and toxicity (NuMeCam - ex-Inserm UMR991-Rennes and Inserm U1078-Brest).

- Quantitative determination of encapsulation efficiencies of drugs : LC-MS2020 Schimadzu with an ESI detector (Liquid chromatography coupled with mass spectrometry)
- Characterization of the formulations (Zeta potential, sizes...): Dynamic light scattering using a DelsaTM Nano Beckman Coulter apparatus and a zetasizer (Malvern instrument)
- Analysis of the lipid components: NMR spectroscopy using A Brucker ARX 400 Instrument
- Optical microscopy under polarized light Leika DMLS with a 350 Dig. heating plate : determination of the lamellar phases formed by the liposomes.
- Diffusion/diffraction of X-ray small and medium angles (SAXS) : characterisation of nanovectors with or without therapeutic agents.
- Spectrofluorimeter : Delivery kinetic of therapeutic molecules
- Spectrometer UV-visible : determination of the encapsulation rate and salting out of active compounds
- Exclusion chromatography: molecular weight analysis of polymers
- TEM and cryo-TEM: morphologies of the nanovectors
- Isothermal titration calorimetry (ITC): molecular interactions (ligand/membrane receptors)
- Förster resonance energy transfer FRET : measure distance and detect molecular interactions

- Extrusion and sonication systems : preparation of liposomes
- Bioluminescence and biofluorescence imaging systems : in vivo transfection evaluation and tracking complexes - successive adaptation with close collaboration with Berthold Technologies.
- Cellular and molecular devices (Levels 1 and 2)
- Biochemistry machine, haematology machine and Ionogram : measurements of parameters and biological constances from small animal.
- Electroporation devices: Microporateur MP100 (Invitrogen - NeonTM system), dedicated to non adherent cells ; Nucleofecteur 4D d'Amaxa/Lonza for the electroporation of adherent and non adherent cells

Close collaboration with the platform IMPACCELL (Rennes)