High-throughput screening method for the identification of biomarkers, therapeutic targets or therapeutic agents

Abstract

The present invention relates to a method for screening a molecule of interest by means of nanoparticles comprising a candidate molecule, a tracer, and a single DNA tag specific to said molecule. The present invention also relates to a method for screening biomarkers of a disease and/or of a phenotype feature, more particularly of a cancer or an infection, by means of said nanoparticles. The invention finally relates to the nanoparticles as such as well as to a library of said nanoparticles.

Claims

1. A method for screening a molecule of interest comprising: (a) transfecting cells with a library of synthetic nanoparticles comprising a plurality of candidate molecules, a tracer, and a plurality of different unique DNA tags that are specific to the plurality of candidate molecules, wherein each synthetic nanoparticle of the library of synthetic nanoparticles is a droplet of an oil-in-water emulsion, each synthetic nanoparticle of the library of synthetic nanoparticles comprising: (1) the tracer, wherein tracer is selected from the group consisting of a magnetic tracer, a radioactive tracer, and a fluoropolymer, (2) one of the candidate molecules of the plurality of candidate molecules, said one of the candidate molecules being localized at the surface of each nanoparticle, (3) a single unique DNA tag of the plurality of different single unique DNA tags, said single unique DNA tag comprising a unique sequence of at least 10 nucleotides which is specific to said one of the candidate molecules of the plurality of candidate molecules, such that said one of the candidate molecules of the plurality of candidate molecules are identified by virtue of this unique sequence comprised within the unique DNA tag, (4) at least 5% molar of an amphiphilic lipid, (5) from 15 to 70% molar of at least one cationic surfactant selected from the group consisting of: N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride, 1,2-dioleyl-3-trimethylamonium-propane, N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy-1-propananium), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium, and dioctadecylamidoglycylspermine, and (6) from 10% to 55% molar of a co-surfactant comprising at least one poly(ethylene oxide) chain comprising between 25 to 500 ethylene oxide units, (7) a solubilizing lipid, and (8) optionally a fusogenic lipid, wherein the molar percentages of amphiphilic lipid, cationic surfactant and co-surfactant are relative to the total molar amounts of amphiphilic lipid, cationic surfactant, co-surfactant, and optional fusogenic lipid under conditions allowing said cells to be transfected by at least one of said synthetic nanoparticles of the library of synthetic nanoparticle whereby the tracer is integrated within the cells; (b) selecting the cells having integrated the tracer and having a phenotype of interest; and (c) identifying as a molecule of interest, one of the candidate molecules of the plurality of candidate molecules that has been integrated into the cells selected in (b) by identifying the unique sequence of at least 10 nucleotides of said single unique DNA tag of the plurality of different unique DNA tags.

2. The method for screening method according to claim 1, wherein the tracer is a magnetic tracer and selecting cells that have integrated said magnetic tracer is performed by magnetic cell sorting, or the tracer is a fluorophore and selecting cells that have integrated said fluorophore is performed by flow cytometry.

3. The method for screening method according to claim 1, wherein each synthetic nanoparticle is a particle of less than 1 micron in diameter.

4. The method for screening method according to claim 1, wherein said synthetic nanoparticles further comprise a biological ligand for targeting a cell and/or an organ.

5. The method for screening method according to claim 1, wherein in said synthetic nanoparticle is an oil-in-water nanoemulsion; said fusogenic lipid is 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine; and said amphiphilic lipid is a phospholipid.

6. The method for screening method according to claim 4 wherein, in said synthetic nanoparticle, said co-surfactant is grafted with said biological ligand for targeting a cell and/or an organ.

7. The method for screening according to claim 1, wherein said candidate molecule is a nucleotide sequence which modulates RNA interference mechanisms.

8. The method for screening method according to claim 7, wherein said nucleotide sequence which modulates RNA interference mechanisms is selected from the group consisting of: (a) a small interfering RNA (siRNA), (b) a locked nucleic acid (LNA), (c) a microRNA (miRNA), and (d) a long double-stranded RNA (dsRNA).

9. The method for screening method according to claim 1, wherein said single unique DNA tag consists in a sequence of DNA with at least 50 nucleotides or base pairs (bp) selected from the group consisting of, on one strand and in the 5-3 direction: (a) a first sequence of at least 20 nucleotides common to all the single DNA tags of the library, (b) a single sequence of at least 10 nucleotides specific to said molecule, and (c) a second sequence of at least 20 nucleotides common to all the single DNA tags of the library.

10. The method for screening according to claim 9, wherein said single unique DNA tag comprises double-stranded DNA.

Description

FIGURES

(1) FIG. 1 illustrates a diagram illustrating the core/crown structure of a droplet of a formulation in the form of a nano-emulsion, comprising a continuous aqueous phase and at least one dispersed phase, as defined in the invention, and comprising a nucleotide sequence which may modulate interfering RNA endogenous mechanisms, a tracer and a single DNA tag. 1 represents the continuous aqueous phase, 2 the crown (partly) of the droplet and 3 the core (partly) of the droplet. In the crown 2, are illustrated: the amphiphilic lipid (for example a neutral phospholipid such as lecithin), the cationic surfactant (cationic phospholipid DOTAP for example), the co-surfactant for which the poly(ethylene oxide) chain which folds back, is represented in the aqueous phase, the double-strand nucleotide sequence which may modulate the interfering RNA endogenous mechanisms (siRNA for example) and the single DNA tag.

(2) FIG. 2 illustrates an electrophoresis gel revealed under UV made by complexation of siRNA with the formulation B10 with concentrations specified in table 8. The scale and the siRNAs (controls) are on the left.

(3) FIG. 3 gives the results of the amount of SiRNA (ng) obtained by processing the data of the electrophoresis on the software package ImageJ of the electrophoresis gel revealed under UV of FIG. 2.

(4) FIG. 4 illustrates the intensity obtained on a ZetaSizer Malvern apparatus versus the hydrodynamic diameter in nm for free siRNA (comparison) and for three formulations according to the invention obtained by complexation with an N/P ratio: amount of positive charges due to the cationic surfactant in the premix formulation (due to charged nitrogen, whence the N of N/P), over the amount of negative charges provided by the siRNAs (due to the phosphorus of siRNA, whence the P of N/P) of 4/1, 8/1 or 16/1.

(5) FIG. 5 represents an electrophoresis gel revealed under UV with from left to right: the scale, the free siRNA (control)
and then the migrations obtained by complexation of siRNA: with formulation B1 (comparative example) (no complexation, the siRNAs are free), with the formulation B6 with a ratio of the amount of positive charges due to the cationic surfactant in the formulation over the amount of negative charges brought by the siRNAs, of 8/1 (quantitative complexation), with the formulation B6 with a ratio of the amount of positive charges due to the cationic surfactant in the formulation over the amount of negative charge brought by the siRNAs, of 8/1 (quantitative complexation), with the formulation B10 with a ratio of the amount of positive charges due to the cationic surfactant in the formulation over the amount of negative charge brought by the siRNAs, of 8/1 (quantitative complexation), with lipofectamine (comparative example) (incomplete complexation).

(6) FIG. 6 illustrates an electrophoresis gel revealed under UV of an siRNA/formulation B10 complex just after complexation (T0) and then 15 min, 45 min, 1 h 30 min, 3 h and 6 h after complexation. The scale and the siRNAs (controls) are on the left.

(7) FIG. 7 illustrates the decrease of the fluorescence intensity FITC in % by transfecting the cell lines with: Lipofectamine RNAimax (commercial agent), the siRNA/formulation B1 complex, with the siRNA/formulation B6 complex, the formulation having been kept for 7 months at room temperature before complexation, with the siRNA/formulation B6 complex, the formulation having been kept for 12 months at room temperature before complexation, with the siRNA/formulation B10 complex, the formulation having been kept for 12 months at room temperature before complexation, with an siRNA/formulation of cationic liposomes complex comprising DOTAP (58% wt), DOPE (18% wt), cholesterol (2% wt) and DSPE-PEG3000 (22% wt)) (comparative).

(8) FIG. 8 illustrates the fluorescence intensity (FITC) for 3 cell lines experimentally over expressing the fluorescent protein GFP (green fluorescent protein): U2OS, PC3 and Hela for the siRNA/formulation B10 complex, the siRNA called siGFP being targeted against the mRNA coding for the GFP protein, and for the negative control (cells incubated with a complexed formulation B10 with an siRNA, negative control, i.e., inert without any effect on the transcriptome of the cell called siAllStar).

(9) FIG. 9 illustrates an electrophoresis gel revealing the nanoparticles A3/DNA tag/siRNA complexes. The nucleic acids are revealed by means of the reagent, GelRed Nucleic Acid Gel Stain (Interchim, Ref. BY1740)

(10) FIG. 10 illustrates the quantification of the averages of fluorescences for GFP after FACS analysis of the HeLa cells and incubated for 72 h with nanoparticles A3/DNA tag/siGFP complexes.

(11) FIG. 11 illustrates the quantification of averages of fluorescences for the encapsulated DID fluorophore after FACS analysis of the HeLa cells and incubated for 72 h with nanoparticles A3/DNA tag/siGFP complexes.

(12) FIG. 12 illustrates the FACS analysis of the FITC signal corresponding to expression of GFP by the HeLa cells. The sorting of these cells was accomplished according to their criterion of expression of GFP and allows identification of two populations: a population which strongly expresses GFP and a population which weakly expresses GFP.

(13) FIG. 13 illustrates an electrophoresis gel of the DNA tags after extraction and amplification with PCR on HeLa cells sorted beforehand.

EXAMPLES

Example 1

Design and Preparation of Fluorescent Nanoparticles Containing a DNA Tag and an siRNA

(14) 1.1. Preparation and Composition of Formulations in the Form of a Nano-Emulsion, Comprising a Continuous Aqueous Phase and at Least One Dispersed Phase

(15) The aqueous phase used is a buffer solution PBS 1.

(16) The suppliers of the compounds are the following: Lipoid S75-3: Lipoid Lipoid S75: Lipoid Lipoid S100-3: Lipoid DOTAP: Avanti Polar DOPE: Avanti Polar MyrjS40: Croda Suppocire NB: Gattefoss Soya oil: Croda

(17) The hydrodynamic diameter of the droplets of the formulations as well as their zeta potential were measured by quasi-elastic scattering of light with an apparatus of the ZetaSizer type from Malvern. The hydrodynamic diameter of the droplets was measured in a solution of PBS 0.1, the zeta potential in an aqueous solution of NaCl 0.15 mM.

(18) Sixteen different formulations were made, the compositions of which are indicated in tables 1 to 5.

(19) TABLE-US-00002 TABLE 1 A1 (comp.) A2 A3 CROWN Amphiphilic lipid: Lipoid S75-3 S75-3 S75-3 % wt Lipoid/droplet 35.29 8.83 3.53 % wt Lipoid/crown without PEG 100 25 10 % wt Lipoid/crown 46.15 11.54 4.62 % mol. Lipoid/crown 70.02 16.86 6.74 Cationic surfactant DOTAP X DOTAP DOTAP % wt DOTAP/droplet 0 26.47 26.47 % wt DOTAP/crown without PEG 0 75 75 % wt DOTAP/crown 0 34.61 34.61 % mol. DOTAP/crown 0 54.28 54.24 Co-surfactant Myrj S40 Myrj S40 Myrj S40 % wt co-surfactant droplet 41.17 41.17 41.17 % wt (co-surfactant) crown 53.85 53.85 53.85 % mol. (co-surfactant)/crown 29.98 28.86 28.84 Fusogenic ipid DOPE X X DOPE % wt DOPE/droplet 0 0 5.29 % wt DOPE/crown without PEG 0 0 15 % wt DOPE/crown 0 0 6.92 % mol. DOPE/crown 0 0 10.18 CORE Solubilizing Lipid Suppocire NB Suppocire NB Suppocire NB % wt solubilizing lipid/core 75 75 75 % wt solubilizing lipid/droplet 17.65 17.65 17.65 Oil Soya oil Soya oil Soya oil % wt oil/core 25 25 25 % wt oil/droplet 5.88 5.88 5.88 Formulation (number of repetitions) 3 4 3 RESULT Hydrodynamic diameter (nm) - average 124.9 49.1 44.48 ZP (mV)_in NaCl 0.15 mM 26 25.38 30.87 Stability ok ok Ok Complexation (%) 0 100 100 Extinction efficiency (%) 0 72.88 75.06

(20) TABLE-US-00003 TABLE 2 B1 (comp.) B2 B3 CROWN Amphiphilic lipid: Lipoid S75-3 S75 S100-3 % wt Lipoid/droplet 8.75 4.25 4.25 % wt Lipoid/crown without PEG 100 50 50 % wt Lipoid/crown 15.98 7.99 7.99 % mol. Lipoid/crown 34.14 17.89 17.89 Cationic surfactant DOTAP X DOTAP DOTAP % wt DOTAP/droplet 0 4.25 425 % wt DOTAP/crown without PEG 0 50 50 % wt DOTAP/crown 0 7.99 7.99 % mol. DOTAP/crown 0 17.85 17.85 Co-surfactant Myrj S40 Myrj S40 Myrj S40 % wt co-surfactant/droplet 46 46 46 % wt (co-surfactant)/crown 84.02 84.02 84.02 % mol. (co-surfactant)/crown 65.86 64.27 64.27 Fusogenic lipid DOPE X X X % wt DOPE/droplet 0 0 0 % wt DOPE/crown without PEG 0 0 0 % wt DOPE/crown 0 0 0 % mol. DOPE/crown 0 0 0 CORE Solubilizing lipid Suppocire NB Suppocire NB Suppocire NB % wt solubilizing lipid/core 75 75 75 % wt solubilizing lipid/droplet 33.94 33.94 33.94 Oil Soya oil Soya oil Soya oil % wt oil/core 25 25 25 % wt oil/droplet 11.31 11.31 11.31 Formulation (number of repetitions) 6 2 2 RESULT Hydrodynamic diameter (nm) - average 59.51 42.11 61.43 ZP (mV)_in NaCl 0.15 mM 21.8 21.4 6.72 Stability ok ok ok Complexation (%) 0 ND ND Extinction efficiency (%) 0 0 0

(21) TABLE-US-00004 TABLE 3 B4 (comp.) B5 B6 CROWN Amphiphilic lipid: Lipoid S75-3 S75-3 S75-3 % wt Lipoid/droplet 6.58 2.19 2.84 % wt Lipoid/crown without PEG 100 25 25 % wt Lipoid/crown 14.29 4 6.9 % mol. Lipoid/crown 31.24 8.39 12.39 Cationic surfactant DOTAP X DOTAP DOTAP % wt DOTAP/droplet 0 6.56 8.52 % wt DOTAP/crown without PEG 0 75 75 % wt DOTAP/crown 0 11.99 20.67 % mol. DOTAP/crown 0 26.99 39.87 Co-surfactant Myrj S40 Myrj S40 Myrj S40 % wt co-surfactant/droplet 39.48 46 29.87 % wt (co-surfactant)/crown 85.71 84.02 72.43 % mol. (co-surfactant)/crown 68.76 64.63 47.74 Fusogenic lipid DOPE X X X % wt DOPE/droplet 0 0 0 % wt DOPE/crown without PEG 0 0 0 % wt DOPE/crown 0 0 0 % mol. DOPE/crown 0 0 0 CORE Solubilizing lipid Suppocire NB Suppocire NB Suppocire NB % wt solubilizing lipid/core 75 75 75 % wt solubilizing lipid/droplet 40.46 33.94 44.07 Oil Soya oil Soya oil Soya oil % wt oil/core 25 25 25 % wt oil/droplet 13.49 11.31 14.69 Formulation (number of repetitions) 3 4 6 RESULT Hydrodynamic diameter (nm) - average 84.88 56.68 86.77 ZP (mV)_in NaCl 0.15 mM 18.89 26.51 36.38 Stability Ok ok ok Complexation (%) 0 100 100 Extinction efficiency (%) 0 0 42.81

(22) TABLE-US-00005 TABLE 4 B9 (Comp.) B10 CROWN Amphiphilic lipid: Lipoid S75-3 S75-3 % wt Lipoid/droplet 0 1.71 % wt Lipoid/crown without PEG 0 15 % wt Lipoid/crown 0 4.14 % mol. Lipoid/crown 0 7.43 Cationic surfactant DOTAP DOTAP DOTAP % wt DOTAP/droplet 8.25 8.25 % wt DOTAP/crown without PEG 75 75 % wt DOTAP/crown 20.67 20.67 % mol. DOTAP/crown 39.87 39.87 Co-surfactant Myrj S40 Myrj S40 % wt co-surfactant/droplet 29.87 29.87 % wt (co-surfactant)/crown 72.43 72.43 % mol. (co-surfactant)/crown 47.74 47.74 Fusogenic lipid DOPE DOPE DOPE % wt DOPE/droplet 2.84 1.71 % wt DOPE/crown without PEG 25 10 % wt DOPE/crown 6.9 2.76 % mol. DOPE/crown 10.18 4.99 CORE Solubilizing lipid Suppocire NB Suppocire NB % wt solubilizing lipid/core 75 75 % wt solubilizing lipid/droplet 44.07 44.07 Oil Soya oil Soya oil % wt oil/core 25 25 % wt oil/droplet 14.69 14.69 Formulation (number of repetitions) 1 poor 5 formulation RESULT Hydrodynamic diameter (nm) - average ND 88.64 ZP (mV)_in NaCl 0.15 mM ND 36.9 Stability ND Ok Complexation (%) ND 100 Extinction efficiency (%) ND 49.34

(23) TABLE-US-00006 TABLE 5 C1 (comp.) C2 C3 CROWN Amphiphilic lipid: Lipoid S75-3 S75-3 S75-3 % wt Lipoid/droplet 28.44 7.11 4.27 % wt Lipoid/crown without PEG 100 25 15 % wt Lipoid/crown 70.24 17.56 10.54 % mol. Lipoid/crown 86.55 20.65 12.38 Cationic surfactant DOTAP X DOTAP DOTAP % wt DOTAP/droplet 0 21.33 21.33 % wt DOTAP/crown without PEG 0 75 75 % wt DOTAP/crown 0 52.68 52.68 % mol. DOTAP/crown 0 66.51 66.47 Co-surfactant Myrj S40 Myrj S40 Myrj S40 % wt co-surfactant/droplet 12.05 12.05 12.05 % wt (co-surfactant)/crown 29.76 29.76 29.76 % mol. (co-surfactant)/crown 13.45 12.84 12.83 Fusogenic lipid DOPE X X DOPE % wt DOPE/droplet 0 0 2.84 % wt DOPE/crown without PEG 0 0 10 % wt DOPE/crown 0 0 7.02 % mol. DOPE/crown 0 0 8.32 CORE Solubilizing lipid Suppocire NB Suppocire NB Suppocire NB % wt solubilizing lipid/core 75 75 75 % wt solubilizing lipid/droplet 44.63 44.63 44.63 Oil Soya oil Soya oil Soya oil % wt oil/core 25 25 25 % wt oil/droplet 14.88 14.88 14.88 Formulation (number of repetitions) 4 4 3 RESULT Hydrodynamic diameter (nm) - average 153.03 162.2 168.9 ZP (mV)_in NaCl 0.15 mM 37.71 53.7 51.83 Stability Ok Ok Ok Complexation (%) 0 100 100 Extinction efficiency (%) 0 80.51 81.72

(24) In tables 1 to 5: % wt corresponds to a mass percent. % mol. Corresponds to a molar percentage. ND (not determined) means that the experiment was not conducted. The percentages /droplet represents percentages relatively to the (Lipoid/DOTAP/Myrj S40/optional DOPE/Suppocire NB/Soys oil) assembly. The percentages /crown represent percentages relatively to the (Lipoid/DOTAP/Myrj S40/optional DOPE) assembly. The percentages /crown without PEG represent percentages relatively to the (Lipoid/DOTAP/optional DOPE) assembly. The percentages /core represent percentages relatively to the (Suppocire NB/Soya oil) assembly. Lipoid S75-3 comprises 65-75% of phosphatidylcholine. The aliphatic chains of the phospholipids are in majority saturated (average composition: 12-16% of C16:0, 80-85% of C18:0, <5% of C18:1, <2% of C18:2). Lipoid S75 comprises 65-75% of phosphatidylcholine. The aliphatic chains of the phospholipids are in majority unsaturated (average composition: 17-20% of C16:0, 2-5% of C18:0, 8-12% of 018:1, 58-65% of C18:2, 4-6% of C18:3). Lipoid S100-3 comprises >94% of phosphatidylcholine. The aliphatic chains of the phospholipids are in majority saturated (average composition: 12-16% of C16:0, 85-88% of C18:0, <2% of C18:1, <1% of C18:2).

(25) The formulations A1, B1, B4 and C1 are comparative examples since they do not comprise any cationic co-surfactant.

(26) Their zeta potentials are negative.

(27) Complexation of siRNA does not occur at the surface of the droplets, according to what was expected.

(28) The formulation B9 is a comparative example since it does not comprise any amphiphilic lipid. The emulsion was not able to be prepared.

(29) The following preparation method was followed:

(30) (i) Preparation of the oily phase: Soya oil, suppocire NC, amphiphilic lipid, DOTAP, optional DOPE, were weighed and then mixed with dichloromethane before being heated to 60 C. in order to obtain a homogenous viscous solution. The dichloromethane gives the possibility of promoting solubilization. The solvents are then evaporated in vacuo.

(31) (ii) Preparation of the aqueous phase: During the phase for evaporating ethanol, the aqueous phase was prepared. In an Eppendorf of 5 ml, the co-surfactant, glycerol and the aqueous solution of PBS (NaCl 154 mM, pH 7.4) were mixed and then dissolved in a bath at 75 C.

(32) (iii) Mixing both phases: The oily phase was at about 40 C. (in viscous form) and the aqueous phase at about 70 C. (upon leaving the bath). The aqueous phase was poured into the oily phase.

(33) (iv) Emulsification: The flask containing both phases was attached in the sonication enclosure a sonicator AV505 (Sonics, Newton, USA). The procedure consisted in producing sonication cycles (10 seconds of activity every 30 seconds) at a power of 100 W over a period of 40 minutes.

(34) (v) Purification: The thereby produced droplets were then purified by dialysis (cut off threshold: 12 kDa, against NaCl 154 mM, overnight) in order to remove the lipid components not integrated to the LNPs. Finally, the formulation was sterilised by filtration on a cellulose membrane.

(35) Size of the Droplets of the Formulations and Zeta Potential

(36) Influence of the Composition of the Formulation

(37) The results of table 1 to 5 show that a decrease in the proportion of co-surfactant (Myrj S40) leads to an increase in the diameter of the droplets.

(38) Time Dependent Change

(39) The time dependent change in the size of the droplets (Table 6) and of the zeta potential (table 7) of the formulations were measured at 40 C. (accelerated stability). The formulations were kept at 40 C. between two measurements.

(40) TABLE-US-00007 TABLE 6 Time dependent change of the size of the droplets and of the polydispersity index (PDI) as measured by quasi-elastic scattering of light versus time Days B1 B4 B5 B6 B10 C1 C2 0 58.27 98.87 60.03 87.93 91.53 157.43 171.4 7 60.52 97.12 58.96 87.1 90.51 156.17 172.63 14 62.59 98.36 58.28 86.25 89.87 156.46 170.15 21 59.41 97.81 59.23 85.78 90.3 153.03 162.2 28 61 97.15 60.36 87.61 90.96 153.9 161.47

(41) TABLE-US-00008 TABLE 7 Time dependent change of the zeta potential of the formulations versus time. Days B1 B4 B5 B6 B10 C1 C2 0 21.3 21.16 24.03 34.13 34.97 40.27 57.33 7 25.6 21.17 28.23 31.77 34.73 43.13 56.77 14 24.73 21.03 29.45 28.4 33.47 42.33 55.33 21 21.8 20.47 28.63 34.3 33.07 38.1 53.9 28 18.93 19.83 27.5 33 31.23 39.03 51.6

(42) It was observed that the size of the droplets and the zeta potential of formulations according to the invention kept at 40 C. for 300 days do not change.

(43) These results show that the formulations according to the invention are stable over time.

(44) 1.2. Complexation with siRNAs

(45) The following general procedure was followed:

(46) The complexation consists in a simple mixture of the formulations prepared above and of an siRNA solution, the whole in a buffer. The selection of the buffer depends on the envisioned application: for a study in vitro, the optimized culture medium for the transfection steps, OptiMEM, was used. For a complexation study, buffer Hepes 5 mM was used.

(47) An amount of 0.5 g of siRNA (GFP-22 siRNA rhodamine (catalogue no. 1022176) (Qiagen) or siGFP (Sigma)) was used (25 g/mL in 20 L).

(48) The mixture was stirred for 30 minutes at 600 rpm, this at room temperature (about 25 C.).

(49) The complexation was viewed through two tools: Agarose gel electrophoresis which allows observation of the migration of the siRNAs. If there is good complexation, then the droplets comprising the complexed siRNAs will be heavier than the free siRNAs and will be viewed in the wells. If the complexation is less significant, free siRNAs migrate to another position. In DLS, by observing the impact of the complexation on the hydrodynamic diameter, the more efficient is complexation, the more the profile is oriented towards a single mode distribution.

(50) The amount of formulation required for having a quantitative complexation yield of the siRNAs was optimized.

(51) In practice, the negative charges brought by the siRNAs are compensated by the positive charges of the formulation (i.e., the positive charges of the cationic surfactant DOTAP).

(52) Typically, when the sole cationic surfactant of the formulation is DOTAP (which only comprises a single positive charge), a quantitative complexation yield is obtained when the ratio of the amount of positive charges due to the cationic surfactant in the premix formulation over the amount of negative charge brought by the siRNAs is greater than 8/1, as illustrated in FIGS. 2 and 3.

(53) FIG. 2 illustrates an electrophoresis gel revealed under UVs produced after complexation of siRNA with the formulation B10 with concentrations specified in table 8, by mixing an siRNA solution and the formulation in a Hepes buffer 5 mM. After deposition on 1.5% agarose gel, 2 L of charged buffer were added to the tests. After 1 h 30 min of electrophoresis at 100 V, the gel was immersed in GelRed 3. Finally, development with UVs was carried out.

(54) TABLE-US-00009 TABLE 8 Ratio of the amount of positive charges due to the cationic surfactant in the premix formulation over the amount of negative charge provided by the siRNAs 1/1 2/1 4/1 6/1 8/1 10/1 12/1 16/1 siRNA 25 concentration (g/mL) DOTAP 0 25 50 100 150 200 250 300 400 concentration (g/mL)

(55) FIG. 3 gives the results obtained by processing the data from the electrophoresis on the software package ImageJ of the same experiments.

(56) FIGS. 2 and 3 show that for a ratio of the amount of positive charges due to the cationic surfactant in the premix formulation over the amount of negative charge provided by the siRNAs, greater than 8/1, there is no longer any free siRNA in the medium and the siRNA has been completely complexed.

(57) FIG. 4 illustrates the intensity obtained on a ZetaSizer Malvern apparatus according to the hydrodynamic diameter in nm for free siRNA (comparison) and for three formulations according to the invention obtained by complexation with a ratio of the amount of positive charges due to the cationic surfactant in the premix formulation over the amount of negative charge brought by the siRNAs of 4/1, 8/1 or 16/1.

(58) The diameter is larger for free siRNA (comparison).

(59) With a ratio of the amount of positive charges due to the cationic surfactant in the premix formulation over the amount of negative charge brought by the siRNAs, of 4/1, 2 populations are observed: an siRNA/droplet complex around 100 nm and a population of a larger size representing the siRNAs in free form (arrow).

(60) With the ratios of the amount of positive charges due to the cationic surfactant in the premix formulation over the amount of negative charge provided by the siRNAs, of 8/1 and 16/1, only a single population representing the siRNA/droplet complex was observed.

(61) These results also show that quantitative complexation (100%) of the siRNAs on the droplets is allowed.

(62) The complexation step was carried out with diverse formulations.

(63) As a comparison, a complexation test was conducted with a formulation free of cationic surfactant: the formulation B1 described above. As expected, complexation of the siRNA did not occur and the siRNA remained in free form.

(64) FIG. 5 illustrates an electrophoresis gel revealed with UV with, from left to right: the scale, the free siRNA (control)
and then migrations obtained by complexation of siRNA: with the formulation B1 (comparative example) (no complexation, the siRNAs are free), with the formulation B6 (a premix formulated 6 months before) with a ratio of the amount of positive charges due to the cationic surfactant in the premix formulation over the amount of negative charge brought by the siRNAs, of 8/1 (quantitative complexation), with the formulation B6 (a premix formulated 12 months before) with a ratio of the amount of positive charges due to the cationic surfactant in the premix formulation over the amount of negative charge provided by the siRNAs, of 8/1, the formulation having been kept for 7 months at room temperature before complexation (quantitative complexation), with the formulation B10 with a ratio of the amount of positive charges due to the cationic surfactant in the premix formulation over the amount of negative charge brought by the siRNAs, of 8/1 (quantitative complexation), with lipofectamine (comparative example) (incomplete complexation).

(65) The complexation of siRNA is quantitative when the formulations B6 or B10 were used. The storage of the formulation at room temperature before its complexation with siRNA has no influence on the complexation yield, which remains quantitative, which shows the stability of the formulations used.

(66) The yield is quantitative regardless of whether the formulation used comprises or not DOPE.

(67) With the commercial transfection agent Lipofectamine RNAimax, more than 60% of the siRNAs were found in free form. This implies that the formulations of nanoemulsions used in the invention provide a better complexation yield than Lipofectamine.

(68) Finally, siRNA sorting-out kinetics of the siRNA/formulation B10 complex prepared above were effected in order to observe the time dependent change of the complexation and is illustrated in FIG. 6. Up to 3 hours after complexation, the siRNAs were not found in free form. At 6 hours, the siRNAs begin to be sorted out. The complex is therefore stable for at least three hours.

(69) 1.3. Transfection in vitro

(70) Preliminary transfection tests of the nanoparticles defined in example 1.1. and complexed according to Example 1.2. with an siRNA specifically inhibiting a sequence of messenger RNA of GFP (GFP-22 siRNA rhodamine (catalogue no. 1022176) (Qiagen)) were conducted on different cell lines over expressing the Green Fluorescent Protein (GFP).

(71) The transfection was carried out with a final siRNA concentration of 100 nM.

(72) Thus, cells expressing GFP were sown in 12-well plates (25,000 cells/well) and then treated with siRNA/Formulations (B1, B6, B6 7 months after its preparation or B10) complexes obtained above. The cells are then incubated for 72 hours at 37 C., and then recovered for analysing the fluorescence intensity with the flow cytometer in order to determine the efficiency of the formulation as a transfection agent. The active delivery of siRNA specifically inhibiting the expression of the GFP protein induces a decrease in the fluorescence brought by this protein.

(73) The commercial transfection agent, Lipofectamine RNAimax was used as a comparison.

(74) FIG. 7 illustrates the decrease in the fluorescence intensity FITC in % upon transfecting cell lines with: Lipofectamine RNAimax, siRNA/formulation B1 complex, with the siRNA/formulation B6 complex, the formulation having been kept for 7 months at room temperature before complexation, with the siRNA/formulation B6 complex, the formulation having been kept for 12 months at room temperature before complexation, with the siRNA/formulation B10 complex, with an siRNA/formulation of cationic liposomes comprising DOTAP (58% wt), DOPE (18% wt), cholesterol (2% wt) and DSPE-PEG3000 (22% wt)) (comparative) complex.

(75) Thus, a decrease of the fluorescence by 33 to 50% is observed with the formulations according to the invention which were tested. The formulations according to the invention therefore allow active delivery of siRNA inducing relative extinction of the expression of the gene of GFP.

(76) Further, by incorporating DOPE into the formulations, a more substantial extinction of fluorescence is visible, DOPE promoting endosomal escape.

(77) No decrease in the fluorescence was observed with the siRNA/formulation of cationic liposomes complex, which may for example be explained by poor stability of the liposomes in the culture medium, a poor complexation yield and/or a poor retention of the siRNAs by the liposomes after complexation.

(78) Finally, such results on the active delivery of siRNA mediated by the formulations according to the invention were reproduced on 3 cell lines expressing GFP: U2OS, PC3 and Hela, as illustrated in FIG. 8.

(79) 1.4. Complexation with Single DNA Tags

(80) Complexation consists in a simple mixing of the formulations prepared above and of an siRNA solution, the whole in a buffer. The selection of the buffer depends on the envisioned application: for a study in vitro, the optimized culture medium for transfection steps, OptiMEM, was used. For a complexation study, Hepes buffer 5 mM was used. The mixture is stirred for at least 30 minutes at 600 rpm, this at room temperature (about 25 C.).

(81) 1.5. Encapsulation of the Fluorophore

(82) The encapsulation of the fluorophore is achieved according to the method defined in application WO2008104717. More specifically, the fluorescent nanoparticles are obtained by sonication of the oily phase in the aqueous phase. The oily phase comprises a mixture of soya oil and of Suppocire NC as well as lecithin and fluorophores (for solubility reasons). The aqueous phase as for it comprises the pegylated surfactant, the aqueous solution (NaCl or PBS) and optionally glycerol in order to increase the viscosity of the mixture. The fluorescent nanoparticles are produced by batches of 2 or 5 mL. Briefly, the oily phase is prepared by mixing the soya oil, Suppocire NC and lecithin (dispersed phase+lecithin). An organic solvent (dichloromethane) is added in order to facilitate dissolution of lecithin. Once all the compounds are dissolved, the solvent is evaporated in vacuo at a temperature above the boiling point of the wax. The fluorescent molecules are then added into the oily phase. In order to facilitate their dispersion, the fluorophores are dissolved beforehand in an organic solvent (ethanol). The solution is homogenized and the organic solvent is removed by evaporation in vacuo. The aqueous phase is prepared by hot mixing of glycerol, of the pegylated surfactant and of the aqueous phase. Both phases maintained beforehand at about 50 C. are mixed and then homogenized with ultrasound, so as to form nanoparticles trapping in their cores the lipophilic fluorophores. The solutions of fluorescent nanoparticles obtained are then purified by dialysis so as to remove the molecules which possibly have not been encapsulated.

(83) 1.6. Preparation of the Library of Nanoparticles

(84) The preparation of 1000 fluorescent nanoparticles is carried out by using siRNAs from the collection of 1292 siRNAs from Qiagen targeting 646 kinases (Human Kinase siRNA set V1.0; Ref. 1027091), or the collection of 2375 siRNAs from Qiagen targeting 1183 genes involved in cancers (Human Cancer siRNA set V2.0), or the collection of 278 siRNAs targeting 139 genes involved in cancers (Human Cancer siRNA set V1.0; Ref. 1022171), or the collection of 91,800 siRNAs from Qiagen targeting 22,950 human genes (Human Genome Wide siRNA set), or by using LNAs from the collection of 982 LNAs from Exiqon targeting all the known human miRNAs (miRCURY LNA Human microRNA Inhibitor Library; Ref. 190102-2).

(85) The single DNA tag contains two sequences of 50 bp in 5 and in 3 allowing amplification of the tag flanking a single sequence of 10 bp containing synthetic bases. Each siRNA or each LNA of the collections is associated in silico with a single sequence of 10 bp.

Example 2

2D and 3D Cultivation of Prostate Cells

(86) The following prostate cells are used: PTN1 and RWPE1 cells, which are immortalized normal epithelial cells of the prostate, the cells WPE1-NA22, WPE1-NB14, WPE1-NB11 and WPE1-NB26, which are derived from RWPE1 cells and which mimic different tumorigenesis stages after exposure to N-methyl-N-nitrosourea, the cell line 22Rv1, which is a line of human prostate carcinoma epithelial cells responding to a deficiency of androgens, the cell lines VcaP and LNCaP, which are metastatic prostate cancer cell lines in vitro and in vivo sensitive to androgens, the cell lines PC3 and DU145, which are metastatic prostate cancer cell lines which no longer respond to androgen deficiency, primary prostate cells from healthy subjects.

(87) The 3D structure production in the form of acini from these prostate cells is also achieved.

(88) The transfection efficiency and the toxicity of the nanoparticles comprising a fluorophore, an siRNA and a single DNA tag are evaluated on each of these prostate cell cultures.

Example 3

High Throughput Phenotype Analysis by Flow Cytometry

(89) The cells are screened according to two different phenotypes: cell proliferation, cell death. The proliferation is evaluated by marking with propidium iodide, EdU, or Hoechst 33342. Cell death is evaluated by marking with Annexine V-FITC or by so called TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labelling) marking or by the analysis of activation of caspases. For each of these phenotypes, the optimal conditions for marking and selecting the cells are evaluated, in order to select suitable positive and negative controls.

(90) Once the conditions are determined for marking and selecting the cells according to their phenotype, primary screenings on about 10 prostate cell lines having different types of response to hormonal treatment or on prostate primary cells are carried out.

Example 4

Deconvolution of the Single DNA Tag, Analysis of the Data and Validation

(91) A small aliquot of cells selected after flow cytometry is used for identifying the single DNA tag. The DNA is extracted from fluorescent cells, subject to PCR with universal primers, and sequenced with a second generation sequencer (Illumina or Roche 454 or ABI solid). The identification of the sequence of 10 bp of the single DNA tag is then correlated with the siRNA or the LNA which has been associated with it in silico. A list of genes for which inhibition by siRNA or LNA induces the phenotype of interest is then established. The screening method also allows the establishment of sets of functional genomic data for each of the cell lines tested according to their sensitivity to hormonal therapies, lists of genes coding for proteins or miRNAs which are potential markers of sensitivity to hormones.

(92) A validation in vitro on the remaining cells of the occurrence of the phenotype and of the inhibition of the gene is achieved by quantitative real time PCR and by Western-blot.

Example 5

Validations in vivo and Clinical Validations

(93) A validation strategy in two phases is achieved: for validation in vivo, control human prostate cancer cells and prostate cancer cells transfected with the nanoparticles according to the invention (e.g. containing siRNAs or LNAs) are implanted in nude athymic cells. The volume of tumours is monitored and after euthanasia, the tumours are excised and fixed for immunohistological analysis of the proliferation or apoptosis in cells transformed with the nanoparticles according to the invention. for clinical validation, commercial microchips of prostate tissues containing several hundred prostate cancer tissues of different grades are used.

(94) This allows validation of biomarkers for prostate cancer, new therapeutic targets and siRNA as new therapeutic agents.

Example 6

Co-delivery of siRNA of Interest and of a DNA Tag (Bar Code) by Fluorescent Nanoparticles, Sorting of the Cells Having Incorporated the Nanoparticles According to a Phenotype of Interest and a Posteriori Identification of the Bar Code of the DNA Tag by Extracting DNA and Specific Amplification from the Sorted Cells

(95) 6.1. Complexation Between Different Nucleic Acids, Tag DNA and siRNA, with a Formulation of Fluorescent Nanoparticles

(96) In this example, co-transfection of the target cells is carried out with an siRNA of interest and a specific DNA tag of the siRNA by resorting to dispersion of fluorescent nanoparticles.

(97) Gel Delay Experiment (FIG. 9):

(98) The formulations of lipid nanoparticles used in this example were achieved according to the same manufacturing method as the one described in Example 1 and corresponds to the formulation A3. The general complexation procedure is the same as the one followed for example 1. Briefly, complexation consists in simply mixing a formulation A3 comprising a lipophilic fluorophore encapsulated in the core (DiD, Invitrogen, Ref. D7757) and of a siRNA and DNA tag solution with nanoparticles, the whole in a buffer. In this study, the buffer used is HEPES (5 mM, pH 7.2). For the well 1, the nanoparticles A3 were complexed with a solution containing 11 ng of siRNA (siAllStar Negative Control siRNA, Qiagen, Ref. 1027280) and 20 ng of DNA tag (Eurogentec). For the following wells (wells numbered from 2 to 7), this siRNA/DNA tag solution was diluted by a factor two before complexation with the nanoparticles, according to the cascade dilution technique. The N/P ratios used here (N=positive charge brought by the ammonium group of the nitrogen of the cationic lipids making up the crown of the lipid particle; P=negative charge brought by the phosphate group of the nucleic acids) are 12/1, 24/1, 48/1, 96/1, 192/1, 384/1, 768/1 for the wells 1 to 7 respectively. The mixture was stirred for 30 minutes at 600 rpm at room temperature (about 25 C.). An electrophoresis on agarose gel (gel with 1.5% of agarose with Agarose ultrapure 1000, Invitrogen, Ref. 16550100; buffer TBE 10X, Ref. 15581044; ultrapure water, Ref. 10977035) gives the possibility of demonstrating that both nucleic acids are actually complexed at the fluorescent lipid droplet, as illustrated in FIG. 9. Indeed, if the nanoparticles/siRNA/DNA tag complexes are stable, all the nucleic acids (siRNA and DNA tag) are retained in the wells (wells 1 to 7) and cannot migrate within the gel. Conversely, a free siRNA (not complexed with the nanoparticles) will be visible in the form of a band migrating towards 21 bp (well 8). Also, a free DNA tag (non-complexed with the nanoparticles) will migrate in the form of a visible band towards 129 bp (well 9). This experiment shows the efficiency of the lipid nanoparticles, of the type Lipidot of formulation A3, to be simultaneously and in a stable way for complexing the siRNAs and the DNA tags.

(99) 6.2. Co-transfection of Two Nucleic Acids, siRNA and DNA Tag, by Fluorescent Nanoparticles

(100) A Transfection Experiment in vitro of HeLa Over Expressing GFP with Formulation A3/DNA Tag/siRNA Complexes:

(101) Simultaneous delivery, in target cells in vitro, of the different molecules transported by the lipid nanoparticle (formulation A3) containing a fluorophore was evaluated.

(102) To do this, a study was conducted on HeLa cells, i.e., cells stemming from uterine cervix cancer (ATCC, Ref. HeLa-CCL2), which were modified in order to over express the GFP protein. Active delivery of siRNA is validated by extinction of the GFP protein (decrease in the FITC signal) by specific screening of its mRNA by an siGFP. In parallel, the observation of the interaction of the fluorescent nanoparticles with the cell is made possible by studying the time dependent change of the fluorescent signal brought by the encapsulated fluorophore, DID (followed by the APC signal). This first step shows a very strong decrease in the FITC signal and therefore of the expression of GFP in cells treated with the nanoparticle A3/DNA tag/siGFP complex as well as an increase in the APC signal indicating the interaction of the fluorescent nanoparticles (bearing the fluorophore DID) with the cells (FIGS. 10 and 11).

(103) These results show that the presence of tag DNA does not perturb the delivery of siRNA in the cells and that the functionality of the latter is preserved, i.e., the interaction with the mRNA coding for GFP and inhibition of the expression of this protein by an RNA interfering mechanism. The efficiency of extinction of FITC fluorescence corresponding to the expression of GFP is of the order of 70% in these experiments following incubation of the cells with the fluorescent nanoparticle A3/DNA tag/siRNA targeted against mRNA of GFP complexes (FIG. 10).

(104) Identification of the DNA Tag in the Sorted Cells on the Basis of GFP Extinction After Transfection by A3/DNA Tag/siRNA Complexes:

(105) In this experiment, the cell populations strongly or weakly expressed in GFP were sorted by flow cytometry (Cytomation, MoFlo). In practice, 200,000 HeLa-GFP cells were sown in wells of 6-well plates. After 24 hours of cultivation, these cells were transfected with fluorescent nanoparticles A3/DNA tag/siAllStar complexes or fluorescent nanoparticles A3/DNA tag/siGFP complexes. Seventy two hours after transfection, the cells are detached from the wells by adding trypsin. The cells contained in three wells for a same transfection condition are added and sorted into two distinct populations, according to their strong or weak expression level of GFP (FIG. 12). The DNA content of the thereby recovered cells is extracted via the QiaAmp DNA mini kit (Qiagen, Ref. 51304) and then a PCR is carried out by using the specific primers of the DNA tag. The primers used for this amplification are the pGEX 3 and 5 primers (Eurogentec, Ref. UN-PR130-005 and UN-PR135-005). The PCR is carried out with 35 amplification cycles (Qiagen, HotStarTaq Master Mix, Ref. 203443). The thereby amplified nucleic acids are then deposited in the wells of a 2% agarose gel prepared beforehand. In FIG. 13, we may observe that the cells transfected with the fluorescent nanoparticles A3/DNA tag/siGFP complexes have a very strong decrease in the expression of GFP, and also bear the DNA tag which is detected by PCR. This result shows the capability of the fluorescent nanoparticles of formulation A3 of simultaneously delivering within the cells, a DNA tag and a functional siRNA.

(106) These results demonstrate: that the nanoparticles according to the invention allow simultaneous delivery in target cells of two nucleic acids (siRNA and DNA tag), the cells transfected by siRNA being therefore fluorescent. the feasibility of the sorting of the cells having incorporated the fluorescent nanoparticles and having a phenotype of interest, the feasibility of a posteriori identification of the siRNA of interest by analysing the bar code of the DNA tag specifically associated with this siRNA, after extraction of cell DNA and amplification of the tag DNA.