CATIONIC POLOXAMERS AND THEIR USE IN TRANSDUCTION

Abstract

Disclosed is a method for the enhancement of the transduction of a target cells by a viral vector using a cationic block-copolymer introduced as an additive alone or formulated with nanoparticles. The method includes a step of contacting a target cells with viruses and a cationic block co-polymer. The structure of this additive incorporates both hydrophilic and hydrophobic regions which represents different areas in the backbone of the polymer. This polymeric construction is ended by cationic chemical functions which contribute to further enhance the viral transduction. Also disclosed are new cationic poloxamers that can be used in the disclosed method. Furthermore, another embodiment is the colloidal stabilization of iron-based nanoparticles using these polymers and their use in increasing transduction efficiency.

Claims

1) Method for transducing target cells with a viral vector, comprising a step of contacting the target cells with the viral vector and a cationic poloxamer.

2) The method according to claim 1, wherein said cationic poloxamers is used alone or formulated with nanoparticles.

3) The method according to claim 1, wherein said cationic poloxamers is used alone or with any composition comprising said cationic poloxamers.

4) (canceled)

5) The method according to claim 41, wherein said target cells are contacted with the cationic poloxamer prior, at the same time or after contacting with the viral vector.

6) The method according to claim 41, wherein the cationic poloxamer and the viral vector are added at the same time onto the target cells as mixture, or sequentially.

7) The method according to claim 1, wherein the time of contact between the viral vector, the cationic poloxamer and the target cells ranges from 5 seconds to 3 months.

8) The method according to claim 1, wherein said cationic poloxamer is provided at a stock concentration of 0.01 to 500 mg/ml.

9) The method according to claim 2, wherein when said cationic poloxamer is used formulated with nanoparticles, said nanoparticles being magnetic nanoparticles.

10) The method according to claim 2, wherein it comprises a further step of applying a magnetic field when the viral vector, the target cells and the adjuvant have been put in contact for a time ranging from 10 seconds to 96 h.

11) The method of claim 1 comprising a further step of spinoculation or centrifugation prior to or after contacting said target cells with the viral vector and the cationic poloxamer.

12) Novel linear or branched cationic poloxamer according to formula I or formula II: ##STR00023## wherein P is according to Formula III a or b: ##STR00024## or P is according to Formula IV a or b: ##STR00025## and wherein “a” represents the number of hydrophilic units repeated in the polymeric backbone P, and is an integer that ranges from 2 to 10000; “a−1” is “a” described before in which 1 unit has been subtracted and is an integer that ranges from 2 to 10000; “b” is the number of hydrophobic unit repeated in the polymeric backbone P and is an integer that ranges from 2 to 1000; “b−1” is the number “b” described before, in which 1 unit has been subtracted and is an integer that ranges from 2 to 1000; X.sub.1, X.sub.2, X.sub.3 and X.sub.4, same or different, refer to heteroatoms and are covalently bonded to respectively R.sub.1, R.sub.2, R.sub.3 and R.sub.4; n is an integer comprised between 1 to 20; R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are simultaneously or independently non polymeric chemical entities to be chosen from: 1 to 6 hydrogen atoms. 1 to 8 heteroatoms; 1 to 24 linear, branched and/or cyclic, saturated or unsaturated hydrocarbon group comprising from 1 to 24 carbon atoms, incorporating or not one or more heteroatoms; 1 to 6 amino acid residues, natural or not; Any combination of these definitions; A.sub.1, A.sub.2, A.sub.3 and A.sub.4” represent the counter ions identical or different selected from one or several of the organic groups consisting of: Halogen-based anions; Organic groups bearing a negative charge, centred or not on a carbon atom; Inorganic anions; Inorganic, non-coordinating inorganic anions; Boron-centered organic anions based on tetrakis[3,5-bis(trifluoromethyl)phenyl]borate backbone; with the exception of F108 backbone, wherein a is an integer comprised between 130 and 135, and b is an integer comprised between 48 and 52, a−1 is an integer comprised between 129 and 134, and X.sub.1R.sub.1═X.sub.2R.sub.2═NH.sub.2, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—S—S—C.sub.5H.sub.4N, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—NH.sub.2; F127 backbone, wherein a is an integer comprised between 98 and 103, and b is an integer comprised between 52 and 58, a−1 is an integer comprised between 97 and 102, and X.sub.1R.sub.1═X.sub.2R.sub.2═NH.sub.2, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—NH.sub.2, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—NH—C.sub.19—H.sub.18—N.sub.7—O.sub.5, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—(CH.sub.2).sub.2—NH—(CH.sub.2).sub.3—NH—(CH.sub.2).sub.4—NH—(CH.sub.2).sub.3—NH.sub.2, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—CH(NH.sub.2)—CH—(CH.sub.3).sub.2, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—NH—(CH.sub.2).sub.2—NH—(CH.sub.2).sub.2—NH.sub.2, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—CH(NH.sub.2)—CH.sub.2—SH; F68 backbone wherein a is an integer comprised between 72 and 78, and b is an integer comprised between 25 and 32, a−1 is an integer comprised between 71 and 77, and X.sub.1R.sub.1═X.sub.2R.sub.2═NH.sub.2, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—S—S—C.sub.5H.sub.4N, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—NH.sub.2; P123 backbone wherein a is an integer comprised between 17 and 23, and b is an integer comprised between 67 and 73, a−1 is an integer comprised between 16 and 22, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—NH.sub.2; L121 backbone wherein a is an integer comprised between 8 and 12, and b is an integer comprised between 64 and 72, a−1 is an integer comprised between 7 and 11, and X.sub.1R.sub.1═X.sub.2R.sub.2═—CH═N—(CH.sub.2).sub.2—NH.sub.2 or X.sub.1R.sub.1═X.sub.2R.sub.2═—CH═N—(CH.sub.2).sub.4—NH.sub.2 or X.sub.1R.sub.1═X.sub.2R.sub.2═—CH═N—(CH.sub.2).sub.2—O—(CH.sub.2).sub.2—O—(CH.sub.2).sub.2—NH.sub.2; P85 backbone wherein a is an integer comprised between 24 and 28, and b is an integer comprised between 38 and 42, a−1 is an integer comprised between 23 and 27, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.3—NH.sub.2; P105 backbone wherein a is an integer comprised between 34 and 39, and b is an integer comprised between 52 and 60, a−1 is an integer comprised between 33 and 38, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—S—S—C.sub.5H.sub.4N, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—NH.sub.2; F88 backbone wherein a is an integer comprised between 94 and 100, and b is an integer comprised between 34 and 42, a−1 is an integer comprised between 93 and 99, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—S—S—C.sub.5H.sub.4N, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—NH.sub.2; P124 backbone wherein a is an integer comprised between 8 and 14, and b is an integer comprised between 16 and 24, a−1 is an integer comprised between 7 and 13, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—CH.sub.2—C.sub.12H.sub.20N.sub.3O.sub.8, or X.sub.1R.sub.1═X.sub.2R.sub.2═N((CH.sub.2).sub.3—NH.sub.2)—(CH.sub.2).sub.4)—NH.sub.2; P104 backbone wherein a is an integer comprised between 24 and 30, and b is an integer comprised between 56 and 64, a−1 is an integer comprised between 23 and 29, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—(CH.sub.2).sub.2—NH—(CH.sub.2).sub.3—NH—(CH.sub.2).sub.4—NH—(CH.sub.2).sub.3—NH.sub.2; P103 backbone wherein a is an integer comprised between 14 and 20, and b is an integer comprised between 56 and 64, a−1 is an integer comprised between 13 and 19, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—(CH.sub.2).sub.2—NH—(CH.sub.2).sub.3—NH—(CH.sub.2).sub.4—NH—(CH.sub.2).sub.3—NH.sub.2; L64 backbone wherein a is an integer comprised between 10 and 16, and b is an integer comprised between 26 and 34, a−1 is an integer comprised between 9 and 15, and X.sub.1R.sub.1═N.sub.3 and X.sub.2R.sub.2═—C.sub.17H.sub.15N.sub.2O.sub.2; T908 backbone wherein a is an integer comprised between 116 and 122, and b is an integer comprised between 15 and 20, a−1 is an integer comprised between 115 and 121, and X.sub.1R.sub.1═X.sub.2R.sub.2═X.sub.3R.sub.3═X.sub.4R.sub.4═NH.sub.2.

13) Novel linear or branched cationic poloxamer according to claim 12, wherein “—X.sub.1—R.sub.1”, “—X.sub.2—R.sub.2”, “—X.sub.3—R.sub.3” and “—X.sub.4—R.sub.4” are non-polymeric entities selected from a group consisting of: Primary, secondary or tertiary amines cationic moiety and amines derivates; Organic quaternary phosphonium moieties; Quaternary ammonium salts based on a nitrogen atom covalently linked to 4 carbon moieties; Organic tertiary sulfonium salts based on a sulphur atom covalently linked to 3 carbon moieties; Organic heterocycles bearing a net positive charge, delocalized or not on the cycle, incorporating at least 1 to 6 similar or different heteroatoms and including at least one unsaturation providing them an aromatic character; Basic amino-acids residue as a source of cationic charges; Natural non-polymeric polyamines.

14) Cationic poloxamer according to claim 13, wherein said organic heterocycles bearing a net positive charge are selected from a group consisting of: pyridine, and its cationic counterpart pyridinium; imidazole and its cationic counterpart imidazolium; triazole and its cationic counterpart triazolium; piperidine and its cationic counterpart piperidinium; morpholine and its cationic counterpart morpholinium.

15) Cationic poloxamers according to claim 13, chosen from the compounds of formula: ##STR00026## ##STR00027##

16) Composition comprising a cationic poloxamer of formula I or II as defined in claim 12.

17) A composition comprising a cationic poloxamer and a viral vector to transduce a target cell.

18) A kit comprising a cationic poloxamer alone or formulated as defined in claim 12, further comprising a viral vector.

19) Transduced cells obtained by the method of claim 1.

20) A method of treating a subject in need of a treatment with cell gene therapy, said method comprising administering to said subject an effective amount of the transduced cells of claim 19.

21) A method for transducing cells, comprising contacting the cells with the cationic poloxamer according to claim 12 and a viral vector.

22) Method according to claim 1, wherein the cationic poloxamer is a novel linear or branched cationic poloxamer according to formula I or formula II: ##STR00028## wherein P is according to Formula III a or b: ##STR00029## or P is according to Formula IV a or b: ##STR00030## and wherein “a” represents the number of hydrophilic units repeated in the polymeric backbone P, and is an integer that ranges from 2 to 10000; “a−1” is “a” described before in which 1 unit has been subtracted and is an integer that ranges from 2 to 10000; “b” is the number of hydrophobic unit repeated in the polymeric backbone P and is an integer that ranges from 2 to 1000; “b−1” is the number “b” described before, in which 1 unit has been subtracted and is an integer that ranges from 2 to 1000; X.sub.1, X.sub.2, X.sub.3 and X.sub.4, same or different, refer to heteroatoms; n is an integer comprised between 1 to 20; R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are simultaneously or independently non polymeric chemical entities to be chosen from: 1 to 6 hydrogen atoms. 1 to 8 heteroatoms; 1 to 24 linear, branched and/or cyclic, saturated or unsaturated hydrocarbon group comprising from 1 to 24 carbon atoms, incorporating or not one or more heteroatoms; 1 to 6 amino acid residues, natural or not; Any combination of these definitions; A.sub.1, A.sub.2, A.sub.3 and A.sub.4” represent the counter ions identical or different selected from one or several of the organic groups consisting of Halogen-based anions; Organic groups bearing a negative charge, centred or not on a carbon atom; Inorganic anions; Inorganic, non-coordinating inorganic anions; Boron-centered organic anions based on tetrakis[3,5-bis(trifluoromethyl)phenyl]borate backbone; with the exception of F108 backbone, wherein a is an integer comprised between 130 and 135, and b is an integer comprised between 48 and 52, a−1 is an integer comprised between 129 and 134, and X.sub.1R.sub.1═X.sub.2R.sub.2═NH.sub.2, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—S—S—C.sub.5H.sub.4N, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—NH.sub.2; F127 backbone, wherein a is an integer comprised between 98 and 103, and b is an integer comprised between 52 and 58, a−1 is an integer comprised between 97 and 102, and X.sub.1R.sub.1═X.sub.2R.sub.2═NH.sub.2, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—NH.sub.2, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—NH—C.sub.19—H.sub.18—N.sub.7—O.sub.5, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—(CH.sub.2).sub.2—NH—(CH.sub.2).sub.3—NH—(CH.sub.2).sub.4—NH—(CH.sub.2).sub.3—NH.sub.2, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—CH(NH.sub.2)—CH—(CH.sub.3).sub.2, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—NH—(CH.sub.2).sub.2—NH—(CH.sub.2).sub.2—NH.sub.2, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—CH(NH.sub.2)—CH.sub.2—SH: F68 backbone wherein a is an integer comprised between 72 and 78, and b is an integer comprised between 25 and 32, a−1 is an integer comprised between 71 and 77, and X.sub.1R.sub.1═X.sub.2R.sub.2═NH.sub.2, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—S—S—C.sub.5H.sub.4N, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—NH.sub.2; P123 backbone wherein a is an integer comprised between 17 and 23, and b is an integer comprised between 67 and 73, a−1 is an integer comprised between 16 and 22, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—NH.sub.2; L121 backbone wherein a is an integer comprised between 8 and 12, and b is an integer comprised between 64 and 72, a−1 is an integer comprised between 7 and 11, and X.sub.1R.sub.1═X.sub.2R.sub.2═—CH═N—(CH.sub.2).sub.2—NH.sub.2 or X.sub.1R.sub.1═X.sub.2R.sub.2═—CH═N—(CH.sub.2).sub.4—NH.sub.2 or X.sub.1R.sub.1═X.sub.2R.sub.2═—CH═N—(CH.sub.2).sub.2—O—(CH.sub.2).sub.2—O—(CH.sub.2).sub.2—NH.sub.2; P85 backbone wherein a is an integer comprised between 24 and 28, and b is an integer comprised between 38 and 42, a−1 is an integer comprised between 23 and 27, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.3—NH.sub.2; P105 backbone wherein a is an integer comprised between 34 and 39, and b is an integer comprised between 52 and 60, a−1 is an integer comprised between 33 and 38, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—S—S—C.sub.5H.sub.4N, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—NH.sub.2; F88 backbone wherein a is an integer comprised between 94 and 100, and b is an integer comprised between 34 and 42, a−1 is an integer comprised between 93 and 99, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—(CH.sub.2).sub.2—S—S—C.sub.5H.sub.4N, or X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—NH—NH.sub.2; P124 backbone wherein a is an integer comprised between 8 and 14, and b is an integer comprised between 16 and 24, a−1 is an integer comprised between 7 and 13, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—CH.sub.2—C.sub.12H.sub.20N.sub.3O, or X.sub.1R.sub.1═X.sub.2R.sub.2═N((CH.sub.2).sub.3—NH.sub.2)—(CH.sub.2).sub.4)—NH.sub.2; P104 backbone wherein a is an integer comprised between 24 and 30, and b is an integer comprised between 56 and 64, a−1 is an integer comprised between 23 and 29, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—(CH.sub.2).sub.2—NH—(CH.sub.2).sub.3—NH—(CH.sub.2).sub.4—NH—(CH.sub.2).sub.3.NH.sub.2; P103 backbone wherein a is an integer comprised between 14 and 20, and b is an integer comprised between 56 and 64, a−1 is an integer comprised between 13 and 19, and X.sub.1R.sub.1═X.sub.2R.sub.2═—O—C(O)—(CH.sub.2).sub.2—NH—(CH.sub.2).sub.3—NH—(CH.sub.2).sub.4—NH—(CH.sub.2).sub.3—NH.sub.2; L64 backbone wherein a is an integer comprised between 10 and 16, and b is an integer comprised between 26 and 34, a−1 is an integer comprised between 9 and 15, and X.sub.1R.sub.1═N.sub.3 and X.sub.2R.sub.2═—C.sub.17H.sub.15N.sub.2O.sub.2; T908 backbone wherein a is an integer comprised between 116 and 122, and b is an integer comprised between 15 and 20, a−1 is an integer comprised between 115 and 121, and X.sub.1R.sub.1═X.sub.2R.sub.2═X.sub.3R.sub.3═X.sub.4R.sub.4═NH.sub.2.

Description

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Examples

[0179] a) Materials

[0180] Most of the solvents and reagents have been obtained from VWR Prolabo (Briare, France), Sigma-Aldrich SA (St Quentin-Fallavier, France), TCI Europe N V (Zwijndrecht, Belgium), and Bachem Biochimie SARL (Voisin-le-Bretonneux, France). Poloxamers used as starting materials are from BASF Performance polymers Europe (Rudolstadt, Germany) and from CRODA Industrial specialties Europe (Gouda, The Netherlands). All reagents purchased with reagent grade have been used without any further purification procedure, unless otherwise stated. Amino-acids used in this work all derived from natural one (i.e. L-isomer), unless otherwise stated.

[0181] b) Methods

[0182] Thin layer chromatographies (TLC) are carried out on aluminum plates covered with silica gel 60 Fasa (Merck). The compounds are developed under UV light (254 nm), with iodine, by immersion in a ninhydrin developer (0.2% in butanol) followed by a stage of heating at 150° C. In the case of the compounds possessing a primary amine function, or by immersion in a cerium/concentrated molybdate (HO/HSO.sub.4/(NH.sub.4)Mo.O.4H.sub.2O/Ce(SO).3HO developer: 90/10/15/1) followed by a stage of heating at 110° C. in the case of the Sulphur-containing compounds.

[0183] The synthesized products are purified on chromatography columns on silica. The flash chromatography separations are carried out on silica gel 60 (230-400 mesh ASTM) (Merck).

[0184] Unless otherwise stated, polymers purifications have been processed through dialysis using membranes of different cutoffs sold by Sigma Aldrich or Spectrum Labs Europe BV (Breda, Netherlands). Dialyses have been processed against 4 liters of double distilled water. The medium was changed after 2 hours, 8 hours, 24 hours, 36 hours and 48 hours.

[0185] Freeze-drying has been carried out using an Heto PowerDry 3000 freeze dryer (ThermoFisher) on samples kept frozen 12 h at −80° C.

[0186] Size Exclusion Chromatography (SEC)

[0187] SEC Experiments have been processed following the Gel-permeation chromatography technique using an Agilent HPLC 1260 Chain (Agilent, Les Ulis France). This apparatus includes: an Agilent 1260 infinity Quaternary pump with manual injection valve, a refractive index detector RID Agilent 1260 Infinity, A UV detector with multiple wavelength Agilent infinity 1260, and a thermostated column's oven Agilent 1260 infinity. Data were collected and processed using Agilent Open Lab LC Chemstation software and its GPC extension. The stationary phase used in these experiments is an Agilent Biosec 3 column (100 Angstroms, 7.8*300 mm) thermostated at 40° C. Mobile phase is constituted by 0.5M acetate buffer, whose pH is corrected to 4.75. The mobile phase has been extensively degassed by sonication and filtered twice prior to use. Flow was adjusted at 1 mL/min to reach a working pressure ranging around 105 bars.

[0188] For analysis of the molecular mass properties of the polymer, a molecular weight calibration curve has been built using polyethylene oxide standards (Mw ranging from 20400 to 136 Da) provided by Agilent. Sample to be analyzed have been dissolved in double distilled water at a concentration of 1 mg/mL, and have been filtered before injection (syringe filter, 0.22 μm, Merck). The dilution water used for dissolving samples contained tetrahydrofuran (THF, 10 μL/100 mL of water) that was used as elution marker for all measurements.

[0189] Particle Size and Zeta Potential Measurements.

[0190] The mean hydrodynamic particle size and charge measurements for cationic poloxamers were performed using Dynamic Light Scattering (DLS) and Laser Doppler Velocimetry (LDV), respectively, using Malvern Nano ZS instrument and DTS software (Malvern Instruments, UK) in a water of Grade 2. Various amounts of cationic poloxamers were diluted in 100 μl of water and mixed with an equal volume of the same water containing DNA. The measurements were carried out in automatic mode and the results are presented as mean+/−SEM, n=3. Each mean represents the average value of 30 measurements.

[0191] Examples of Synthesized Molecules

[0192] A large ensemble of modified cationic poloxamers has been synthesized according to the method described in the present invention. All the molecules obtained in these examples derived from commercially available unmodified “hydrophilic” linear and regular poloxamer, which have a variety of value of HLB preferentially greater or close from 20 (see before for complete definition). The main structural features of the commercially available polymers used as starting material in this study are compiled in Table 1 below:

TABLE-US-00001 TABLE 1 Physical properties of the commercially available unmodified poloxamers used as template in this study. Poloxamer Property F108 F127 F87 F68 F88 F98 MW 14600 12600 7700 8400 11400 13000 “a” 132.7 100.22 61.25 76.4 103.7 118.2 “b” 50.3 65.2 39.83 28.97 39.3 44.8 HLB 27 22 24 29 28 28 Molecular weights (MW) are expressed in Daltons (Da) and are provided by the manufacturers. “a” and “b” value have been defined earlier in the text and are calculated according the MW. HLB have been calculated by the manufacturers.

[0193] A large panel of cationic modifications is presented in the following examples, with different strategies applied to the different commercially available unmodified poloxamers described before. This led to different cationic polymers belonging to the present invention. Each of these polymers has been named after numbers (Tag), which are depicted in Table 2 hereafter:

TABLE-US-00002 TABLE 2 Attributed numbers (tags) to the cationic poloxamers synthesized for this study: each polymer is identified as a combination as an unmodified poloxamer backbone from commercial sources, with a modification of the endgroups using cationic chemical functions. Polymer Template Cationic/anionic End-groups F87 F108 F127 F68 F88 F98 [00006] I VIII XV XXII XXXIX XLII [00007]  1b  2b  5b 16b 23b 12b [00008] XXIX II IX XVI XXIII XL [00009] XXXV XXX III X XVII XXIV [00010] 25b  4b 31b 36b 11b 18b [00011] XXV IV XXXI XXXVI XI XVIII [00012] 19c 26c  5c 32c 37c 12c [00013] XIX XXVI V XXXII XXXVII XII [00014] XIII XX VI XXVII XXXIII XXXVIII [00015] XLI XIV VII XXI XXVIII XXXIV

[0194] With the aim to keep the examples as simple as possible, only one example of each synthetic strategy will be described in its entirety. However, all these synthetic procedures have been applied with similar efficiencies for the obtaining of the compound described in Table 2.

[0195] Each synthetic step will thus be described completely for the chosen cationic poloxamer, while the results using the other polymeric backbones will be each time resumed in a table following the main procedure.

Example 1: Synthesis of Bis-Spermine-Cationic Poloxamer Derivative

[0196] Example 1 deals with the introduction of spermine derivatives as endgroups on different hydrophilic backbones of the unmodified poloxamers. The chemical strategy will lead to compounds bearing the following general structure:

##STR00016##

[0197] The physico-chemical features of the related compounds are dependent of the polymeric backbone chosen. They are condensed in the following Table 3.

TABLE-US-00003 TABLE 3 Main physico-chemical features of the spermine-grafted cationic poloxamer derivatives described in this study, with S.d.: Spermine derivative; Tfa: trifluoroacetates; XR-A: Cationic endgroups (XR)-counteranion(A) as described before; Tag: Number attributed to the obtained polymer; a;b: Number of the PEO (a) and PPO (b) blocks as defined before. HLB refers to hydrophilic/lipophilic balance as described before. Backbone Features F87 F108 F127 F68 F88 F98 XR; A S.d.; Tfa S.d.; Tfa S.d.; Tfa S.d.; Tfa S.d.; Tfa S.d.; Tfa Tag I VIII XV XXII XXXIX XLII a; b 61.25; 132.7; 100.22; 76.4; 103.7; 118.2; 39.83 50.3 65.2 28.97 39.3 44.8 HLB >24 >27 >22 >29 >28 >28

[0198] To illustrate the synthetic strategy, the detailed procedure for the obtaining of compound I is developed below. It started by the synthesis of compound 1b, described in Figure II:

Step 1: Synthesis of Bis-Tosylated F87 1a

[0199] Prior to the synthesis, 10 g of F87 (Croda) are dissolved in 100 mL of MilliQ double distilled water and put in a 500 mL flask. The mixture is then frozen at −80′C overnight, before being freeze-dried.

[0200] Then, 3.36 g (0.436 mmoles) are dissolved under argon in 86 mL of a mixture of dry dichloromethane with pyridine (volume ratio 73/13). The mixture is stirred vigorously before adding a catalytic amount of DMAP (TCI), and para-toluenesulfonic acid chloride (Sigma-Aldrich, 4.36 mmoles, 891 mg) portion wise over 45 minutes. The reaction is subsequently stirred for 48 h at room temperature. The whole mixture is then concentrated under vacuum and the resulting yellowish gum is re-dissolved in DCM (50 mL). The organic extract is then washed twice with an aqueous saturated solution of NH.sub.4Cl (2×100 mL) and put aside. The combined washings are then extracted extensively with dichloromethane (5*50 mL), before combining the organic extracts and washing them twice with saturated NH.sub.4Cl, twice with MilliQ water, and once with brine (each 150 mL). The obtained organic phase is then dried on magnesium sulphate, filtered and concentrated under vacuum to provide the title compound with has been used without further purification (3.17 g).

[0201] Following Table 4 presents number and quantities of the starting material and name and quantity of the bis-tosylated product, in which m represents the mass of the compound while n the number of moles.

TABLE-US-00004 TABLE 4 Backbone Material F87 F108 F127 F68 F88 F98 Starting F87 F108 F127 F68 F88 F98 material (3.36 g; (16.8 g; (11.7 g; (5.2 g; (2.2 g; (3.8 g; (m; n) 0.436 1.15 mmol) 0.929 mmol) 6.19 mmol) 0.193 mmol) 0.292 mmol) mmol) Isolated 1a 2a 5a 16a 23a 12a Product (3.17 g; (15.8 g; (11.3 g; (4.45 g; (2.1 g; (2.7 g; (m; n) 0.397 mmol) 1.06 mmol) 0.870 mmol) 0.512 mmol) 0.180 mmol) 0.203 mmol)

Step 2: Synthesis of Bis-Amino F87 1b

[0202] Compound 1b (1.66 g, 0.208 mmoles) is dissolved in 30 mL of a solution of methanol containing 2M of ammonia (TCI) under vigorous stirring. The solution is degassed with argon and then heated to reflux for 24 h. Ammonia in methanol is then added (15 mL) and the mixture is refluxed for another 6 h before being concentrated under vacuum. The resulting syrup is then dissolved in 20 mL of double distilled water, kept at 4° C. for one night and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product is then recovered after lyophilisation (1.48 g). This compound is of cationic nature. It thus belongs to the present invention.

[0203] Following Table 5 shows Tosyl displacement by ammonia step assessment: this table presents number and quantities of the starting material and name and quantity of the bis-amino product in which m represents the mass of the compound while n the number of moles.

TABLE-US-00005 TABLE 5 Backbone Material F87 F108 F127 F68 F88 F98 Starting 1a 2a 5a 16a 23a 12a material (1.66 g; (4.65 g; (3 g; (1.5 g; (1.5 g; (1.2 g; (m; n) 0.208 mmol) 0.312 mmol) 0.233 mmol) 0.173 mmol) 0.128 mmol) 0.090 mmol) Isolated 1b 2b 5b 16b 23a 12b Product (1.48 g; (3.38 g; (2.2 g; (1.30 g; (1.08 g; (1.04 g; (m; n) 0.192 mmol) 0.232 mmol) 0.174 mmol) 0.155 mmol) 0.095 mmol) 0.080 mmol.

[0204] The achievement of the title compounds requires then the synthesis of ornithin-derivative 1e.

Synthesis of Compound 1e, Intermediate of I is Summarized in Figure III

Step 3: Synthesis of 2,5-bis(2-cyanoethylamino)pentanoic acid 1c

[0205] Sodium hydroxide (1.78 g, 44.5 mmoles, VWR) is finely grounded in a mortar and suspended in 150 mL of methanol in a round-bottom flask. L-Ornithine monohydrochloride (5 g, 29.7 mmoles, TCI) is then introduced portion wise and stirred intensively for 1 h30. The suspension is then filtered on a short pad of Celite (Sigma-Aldrich). The remaining clear filtrate is reintroduced in a flask and stirred, before 4.29 mL of acrylonitrile are added dropwise over 1 h. After complete addition, the mixture is stirred at room temperature for 36 h in the dark full until completion shown by TLC. When over, 12 mL of 37% HCl (VWR) are added carefully to the mixture creating a turbid precipitate upon stirring. The solid is collected by filtration and dried under vacuum (S1, 2.51 g). A few drops of 30% concentrated sodium hydroxide in water (VWR) are then added, resulting in a second solid formation, which is collected and dried the same way (S2, 1.13 g). Analysis by TLC proved that S1 and S2 are the pure target product while remaining filtrate does not contain any targeted material. Global yield is 46% (3.64 g, 13.7 mmoles).

Step 4: 5-amino-2-(3-aminopropylamino)pentanoic acid 1d

[0206] 2,5-bis(2-cyanoethylamino)pentanoic acid 1c (2.51 g, 9.44 mmoles) is suspended in 40 mL of absolute ethanol. The pH of the mixture is slightly increased with sodium hydroxide to help solubilization, before being transferred in a 50 mL steel lab autoclave (BüchiGlass; Uster Switzerland). A catalytic amount of Raney Nickel (Sigma Aldrich) is introduced and the autoclave is closed and put under 8 bars pressure of dihydrogen. The mixture is stirred that way for 12 h. The gas is then evacuated and the mixture filtered on a short pad of Celite. After removal of the ethanol the resulting oil is used without further purification in the next step.

Step 5: Synthesis of 2,5-bis[tert-butoxycarbonyl-[3-(tert-butoxycarbonylamino)propyl]amino]pentanoic acid 1e

[0207] The crude product of step 4 is put in a three-necks round bottom flask containing 15 mL of a solution composed of tetrahydrofuran and water (Volume ratio of 5/1). The mixture is then cooled to 0° C. with an ice/water bath. Under stirring, 4M NaOH in water (9.12 mL) and Boc.sub.2O (11.4 g, 57 mmoles) dissolved in THE (12 mL), are added in 4 times each alternatively. The whole addition process takes 30 minutes to be completed, and then, the reaction is let stirred 12 hours at room temperature. The reaction is then concentrated under vacuum, re-dispersed in 100 mL of 6M HCl, and extracted with diethyl ether (6×35 mL). The organic extracts are then dried on magnesium sulphate, filtered and concentrated. The product is purified on silica gel, using DCM/MeOH as mobile phase (gradient from 2% to 12% MeOH in DCM). After concentration, the target compound is isolated with 48% yield (2 steps, 2.91 g, 4.53 mmoles).

[0208] This compound has been synthesized in high quantities and used associated with each bis-amino poloxamer following the strategy depicted in Figure IV.

Step 6: Coupling Between 1b and 1e

[0209] Compound 1e (32 mg, 4.8.Math.10.sup.−2 mmoles) is dissolved in 2 mL of dry DMF. DIC (9 μL, 5.6.Math.10.sup.−2 mmoles) and HOBt (8 mg, 5.6.Math.10.sup.−2 mmoles) are added successively to the mixture and stirred for 2 h30. Poloxamer 1b (127 mg) is then added directly in the stirring flask, and the reaction is pursued for 24 h at room temperature, before being concentrated under high vacuum. The resulting syrup is then dissolved in 20 mL of double distilled water, kept at 4° C. for one night and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product is then recovered after lyophilisation (123 mg).

[0210] Following Table 6 shows the Coupling with compound 1e step assessment: this table presents number and quantities of the starting material and name and quantity of the bis-protected product in which m represents the mass of the compound while n the number of moles.

TABLE-US-00006 TABLE 6 Backbone Material F87 F108 F127 F68 F88 F98 Starting 1b 2b 5b 16b 23b 12b material (127 mg; (292 mg; (252 mg; (168 mg; (228 mg; (260 mg; (m; n) 0.0165 0.020 mmol) 0.02 mmol) 0.02 mmol) 0.02 mmol) 0.02 mmol) mmol) Isolated 1f 2f 5f 22f 39f 42f Product (123 mg; (254 mg; (236 mg; (106 mg; (165 mg; (214 mg; (m; n) 0.0137 mmol) 0.016 mmol) 0.017 mmol) 0.011 mmol) 0.013 mmol) 0.015 mmol)

Step 7: Synthesis of Bis-Substituted F87 Derivative 1

[0211] Compound if (123 mg) is dissolved in 4 mL of a solution of dichloromethane. TFA is then introduced dropwise (VWR, 2 mL). The solution is degassed with argon and then stirred for 2 h. Mixture is then concentrated under vacuum, and co-evaporated twice with DCM, three times with diethyl ether and dried one night under vacuum. The remaining residue is then resuspended in MilliQ water and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product is then recovered after lyophilisation (124 mg). [0212] Following Table 7 shows Boc-deprotection step assessment: this table presents number and quantities of the starting material and name and quantity of the bis-spermine product in which m represents the mass of the compound while n the number of moles.

TABLE-US-00007 TABLE 7 Backbone Material F87 F108 F127 F68 F88 F98 Starting 1f 2f 5f 22f 39f 42f material (123 mg; (254 mg; (236 mg; (106 mg; (165 mg; (214 mg; (m; n) 0.0137 mmol) 0.016 mmol) 0.017 mmol) 0.011 mmol) 0.013 mmol) 0.015 mmol. Isolated I VIII XV XXII XXXIX XLII Product (124 mg; (256 mg; (238 mg; (108 mg; (166 mg; (216 mg; (m; n) 0.0137 mmol 0.016 mmol) 0.017 mmol) 0.011 mmol) 0.013 mmol) 0.015 mmol)

Example 2: Synthesis of Bis-Trimethylammonium-Cationic Poloxamer Derivative

[0213] Example 2 deals with the introduction of trimethyl ammonium moieties as endgroups on different hydrophilic unmodified poloxamers. The chemical strategy will lead to compounds bearing the general structure depicted below:

##STR00017## [0214] Structure of trimethylammonium grafter cationic poloxamer derivatives

[0215] The physico-chemical features of the related compounds are dependent of the polymeric backbone chosen. They are condensed in the following Table 8.

TABLE-US-00008 TABLE 8 Main physico-chemical features of the trimethylammonium-grafted cationic poloxamer derivatives described in this study. Backbone Features F87 F108 F127 F68 F88 F98 XR; A Trimethyl- Trimethyl- Trimethyl- Trimethyl- Trimethyl- Trimethyl- ammonium; ammonium; ammonium; ammonium; ammonium; ammonium; iodide iodide iodide iodide iodide iodide Tag XXIX II IX XVI XXIII XL a; b 61.25; 132.7; 50.3 100.22; 76.4; 28.97 103.7; 39.3 118.2; 44.8 39.83 65.2 HLB >24 >27 >22 >29 >28 >28 XR; A: Cationic endgroups (XR); counteranion (A) as described before; Tag: number attributed to the obtained polymer; a; b: Number of the PEO (a) and PPO (b) blocks as defined before. HLB refers to hydrophilic/lipophilic balance as described before.

[0216] The synthetic strategy, the detailed procedure for the achievement of compound II is developed in Figure V.

Step 1: Synthesis of Bis-Tosylated F108 2a

[0217] Prior to the synthesis, 20 g of F108 (Croda) are dissolved in 200 mL of MilliQ double distilled water and put in a 1000 mL flask. The mixture is then frozen at −80′C overnight, before being freeze-dried.

[0218] Compound 2a is obtained following the strategy and proportions used in the synthesis of compound 1a, starting from 16.8 g of F108 to get 15.8 g of 2a. See Table 4 for detailed results.

Step 2: Synthesis of Bis-Amino F108 2b

[0219] Compound 2b is obtained following the strategy and proportions used in the synthesis of compound 1b, starting from 4.65 g of 2a to get 3.38 g of 2b. This compound is of cationic nature. It thus belongs to the present invention and will be evaluated further in the document.

[0220] See Table 5 for detailed results.

Step 3: Synthesis of bis-trimethyl-ammonium F108 Derivative II

[0221] In a round-bottom flask under argon are introduced 300 mg (2.06.Math.10.sup.−2 mmoles) of compound 2b, in 8 mL of dry DMF. After complete dissolution within a few minutes are added DIPEA (Sigma-Aldrich, 60 μL, 3.44.Math.10.sup.−1 mmoles) and methyl iodide (Sigma-Aldrich, 75 μL, 1.21 mmoles) and the resulting mixture is stirred for 48 h at room temperature. After removal of the solvent, the remaining residue is then resuspended in MilliQ water and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product is then recovered after lyophilisation (290 mg).

[0222] Following the Table 9 shows Trimethylation step assessment: this table presents number and quantities of the starting material and name and quantity of the bis-trimethylated product in which m represents the mass of the compound while n the number of moles.

TABLE-US-00009 TABLE 9 Backbone Material F87 F108 F127 F68 F88 F98 Starting 1b 2b 5b 16b 23a 12b material (154 mg; (300 mg; (252 mg; (168 mg; (229 mg; (260 mg; (m; n) 0.02 mmol) 0.0206 mmol) 0.020 mmol) 0.02 mmol) 0.02 mmol) 0.02 mmol). Isolated XXIX II IX XVI XXIII XL Product (119 mg; (290 mg; (234 mg; (123 mg; (212 mg; (187 mg; (m; n) 0.015 mmol) 0.019 mmol) 0.018 mmol) 0.014 mmol) 0.018 mmol) 0.014 mmol)

Example 3: Synthesis of TriphenylPhosphonium-Cationic Poloxamer Derivatives

[0223] Example 3 deals with the introduction of triphenylphosphonium moieties as endgroups on different hydrophilic unmodified poloxamers. The chemical strategy will lead to compounds bearing the general structure depicted below:

##STR00018## [0224] General structure of triphenylphosphonium grafted cationic poloxamers derivatives

[0225] The physico-chemical features of the related compounds are dependent of the polymeric backbone chosen. They are condensed in the following Table 10.

TABLE-US-00010 TABLE 10 Main physico-chemical features of the triphenylphosphonium-grafted cationic poloxamer derivatives described in this study Backbone Features F87 F108 F127 F68 F88 F98 XR; A T; b T; b T; b T; b T; b T; b Tag XXXV XXX III X XVII XXIV a; b 61.25; 132.7; 100.22; 76.4; 28.97 103.7; 39.3 118.2; 44.8 39.83 50.3 65.2 HLB >24 >27 >22 >29 >28 >28 (T; b: Triphenylphosphonium; bromide) XR; A: Cationic endgroups (XR); counteranion (A) as described before; Tag: Number attributed to the obtained polymer; a; b: Number of the PEO (a) and PPO (b) blocks as defined before. HLB refers to hydrophilic/lipophilic balance as described before.

[0226] To illustrate the synthetic strategy, the detailed procedure for the achievement of compound III is developed in Figure VI.

Step 1: Synthesis of Bis-Brominated F127 3a

[0227] Prior to the synthesis, 20 g of F127 (Croda) are dissolved in 200 mL of MilliQ double distilled water and put in a 1000 mL flask. The mixture is then frozen at −80′C overnight, before being freeze-dried.

[0228] In a 100 mL round-bottom flask, are dissolved 5.45 g of F127 (0.432 mmoles) under an argon atmosphere. Carbon tetrabromide (Sigma, 2.46 g, 7.42 mmoles) and triphenyl phosphine (Sigma, 2.13 g, 8.14 mmoles) are added simultaneously in three portions over a 1-hour period. The mixture is vigorously stirred during 48 h at room temperature before removing dichloromethane under vacuum. The resulting solid is then resuspended in 50 mL of MilliQ water and kept at 4° C. for 12 hours. After removal of the solid by centrifugation, the title product is isolated without further purification by lyophilization (1.62 g).

[0229] Following Table 11 shows bromination step assessment: this table presents number and quantities of the starting material and name and quantity of the bis-brominated product in which m represents the mass of the compound while n the number of moles.

TABLE-US-00011 TABLE 11 Backbone Material F87 F108 F127 F68 F88 F98 Starting F87 F108 F127 F68 F88 F98 material (3.85 g; (6.13 g; (5.45 g; (2.94 g; (3.99 g; (4.55 g; (m; n) 0.5 mmol) 0.42 mmol) 0.432 mmol) 0.35 mmol) 0.35 mmol) 0.35 mmol) Isolated 35a 30a 3a 10a 17a 24a Product (1.89 g; (1.55 g; (1.62 g; (1.88 g; (1.85 g; (1.25 g; (m; n) 0.24 mmol) 0.105 mmol) 0.127 mmol) 0.22 mmol) 0.16 mmol) 0.095 mmol)

Step 2: Synthesis of Bis-Phosphonium F127 Derivative III

[0230] 1 g (0.078 mmoles) of compound 3a is dissolved in 30 mL of dry DMF. PPh.sub.3 (1.6 mmoles 420 mg) is subsequently added under argon and the reaction is heated to 120° C. in an oil bath while keeping an argon atmosphere. After 36 h of stirring, the reaction is cooled down to RT, and concentrated under vacuum. The resulting syrup is washed extensively with several portions of hexane giving a solid which is then dried 12 h under high vacuum. The remaining residue is then resuspended in MilliQ water and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product is then recovered after lyophilisation (654 mg). [0231] Following Table 12 shows Phosphonium introduction step assessment: this table presents number and quantities of the starting material and name and quantity of the bis-phosphonium product in which m represents the mass of the compound while n the number of moles.

TABLE-US-00012 TABLE 12 Backbone Material F87 F108 F127 F68 F88 F98 Starting 35a 30a 3a 10a 17a 24a material (1 g; 0.127 (1 g; 0.068 (1 g; 0.078 (1 g; 0.117 (1 g; 0.0865 (1 g; 0.076 (m; n) mmol) mmol) mmol) mmol) mmol) mmol) Isolated XXXV XXX III X XVII XXIV Product (679 mg; (550 mg; (654 mg; (799 mg; (833 mg; (602 mg; (m; n) 0.081 0.036 mmol) 0.049 mmol) 0.088 mmol) 0.069 mmol) 0.044 mmol) mmol)

Example 4: Synthesis of Bis-1-methyl-5-[(trimethyl-lambda5-azanyl)methyl]-1lambda5,2,3-triazacyclopenta-1,4-diene (MTAMTD)—Cationic Poloxamer Derivatives

[0232] Example 4 deals with the introduction of 1-methyl-5-[(trimethyl-lambda5-azanyl)methyl]-1 lambda5,2,3-triazacyclopenta-1,4-diene (MTAMTD) moieties as endgroups on different hydrophilic unmodified poloxamers. The chemical strategy will lead to compounds bearing the following general structure:

##STR00019## [0233] General structure of MTAMTD-grafted cationic poloxamer derivatives

[0234] The physico-chemical features of the related compounds are dependent of the polymeric backbone chosen. They are condensed in the following Table 13.

TABLE-US-00013 TABLE 13 Main physico-chemical features of the MTAMTD-grafted cationic poloxamer derivatives described in this study. Backbone Features F87 F108 F127 F68 F88 F98 XR; A MTAMTD; MTAMTD; MTAMTD; MTAMTD; MTAMTD; MTAMTD; iodide iodide iodide iodide iodide iodide Tag XXV IV XXXI XXXVI XI XVIII a; b 61.25; 39.83 132.7; 50.3 100.22; 65.2 76.4; 28.97 103.7; 39.3 118.2; 44.8 HLB >24 >27 >22 >29 >28 >28 XR; A: Cationic endgroups (XR); counteranion (A) as described before; Tag: number attributed to the obtained polymer; a; b: Number of the PEO (a) and PPO (b) blocks as defined before. HLB refers to hydrophilic/lipophilic balance as described before.)

[0235] To illustrate the synthetic strategy, the detailed procedure for the achievement of compound IV is developed in Figure VII.

Step 2: Synthesis of Bis-Azido F108 4a

[0236] Compound 2a (7.44 g, 0.5 mmoles) described earlier in this document, is dissolved under argon in 30 mL of dry DMF. Sodium azide (6 mmoles, 390 mg) is then introduced and the mixture is stirred at 90° C. for 3 days. After cooling down, DMF is removed using a rotary evaporator. The remaining residue is then resuspended in MilliQ water and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product is then recovered after lyophilisation (6.18 g). [0237] Following Table 14 shows Azido introduction step assessment: this table presents number and quantities of the starting material and name and quantity of the bis-azido product in which m represents the mass of the compound while n the number of moles.

TABLE-US-00014 TABLE 14 Backbone Material F87 F108 F127 F68 F88 F98 Starting  1a 2a  5a 16a 23a 12a (1.5 g; (3 g; 0.376 (7.44 g; 0.5 (3 g; 0.233 (1.5 g; 0.173 (1.5 g; 0.128 0.113 material (m; mmol) mmol) mmol) mmol) mmol) mmol) n) Isolated 25a 4a 31a 36a 11a 18a Product (m; (2.31 g; (6.18 g; (2.35 g; (1.11 g; (1.10g; (693mg; n) 0.297 mmol) 0.421 mmol) 0.185 mmol) 0.131 mmol) 0.096 mmol) 0.053 mmol)

Step 2: Synthesis of bis-1H-triazol-4-ylmethanamine F108 4b

[0238] Copper sulphate heptahydrate (VWR, 0.012 mmoles, 3 mg), ascorbic acid (Sigma, 0.06 mmoles, 12 mg) and triphenylphosphine (0.012 mmoles 3 mg) are mixed together under stirring in 1 mL of dry DMSO. While this solution is stirring (15 minutes), compound 4a (2 g, 0.136 mmoles) is dissolved in 4 mL of a 3/1 mixture of water/DMSO. After complete dissolution of the polymer the copper-based yellowish solution is added to the reaction flask and stirred with the starting material for 12 h at room temperature. The reaction is then diluted in 10 mL of water and extracted with 4×.sub.20 mL of DCM. The organic extracts are then combined, dried on magnesium sulphate, filtered and concentrated under vacuum. The remaining residue is then resuspended in MilliQ water and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product is then recovered after lyophilisation (1.72 g). This compound is of cationic nature. It thus belongs to the present invention. [0239] Following Table 15 shows Huysgen-click reaction step assessment: this table presents number and quantities of the starting material and name and quantity of the products bearing triazole rings in which m represents the mass of the compound while n the number of moles.

TABLE-US-00015 TABLE 15 Backbone Material F87 F108 F127 F68 F88 F98 Starting 25a 4a 31a 36a 11a 18a material (2 g; (2 g; (2 g; (1.11 g; (1.10 g; (693 mg; (m; n) 0.257 0.136 0.158 0.131 0.096 0.053 mmol) mmol) mmol) mmol) mmol) mmol) Isolated 25b 4b 31b 36b 11b 18b Product (1.66 g; (1.72 g; (1.59 g; (902 mg; (893 mg; (515 mg; (m; n) 0.21 0.116 0.124 0.105 0.077 0.039 mmol) mmol) mmol) mmol) mmol) mmol)

Step 3: Synthesis of bis-1-methyl-5-[(trimethyl-lambda5-azanyl)methyl]-1 lambda5,2,3-triazacyclopenta-1,4-diene F108 Derivative IV

[0240] In a round-bottom flask under argon are introduced 600 mg (4.05.Math.10.sup.−2 mmoles) of compound 4b, in 18 mL of dry DMF. After complete dissolution within a few minutes are added DIPEA (Sigma-Aldrich, 120 μL, 6.88.Math.10.sup.−1 mmoles) and methyl iodide (Sigma-Aldrich, 150 μL, 2.42 mmoles) and the resulting mixture is stirred for 48 h at room temperature. After removal of the solvent, the remaining residue is then resuspended in MilliQ water and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product is then recovered after lyophilisation (501 mg).

[0241] Following Table 16 shows methylation reaction step assessment: this table presents number and quantities of the starting material and name and quantity of the products bearing methylated triazolium rings in which m represents the mass of the compound while n the number of moles.

TABLE-US-00016 TABLE 16 Backbone Material F87 F108 F127 F68 F88 F98 Starting 25b 4b 31b 36b 11b 18b material (660 (600 (590 mg; (402 mg; (493 mg; (315 mg; (m; n) mg; mg; 0.0461 0.0468 0.0425 0.0239 0.0839 0.0405 mmol) mmol) mmol) mmol) mmol) mmol) Isolated XXV IV XXXI XXXVI XI XVIII Product (477 (501 (416 mg; (203 mg; (354 mg; (194 mg; (m; n) mg; mg; 0.031 0.022 0.029 0.014 0.056 0.0325 mmol) mmol) mmol) mmol) mmol) mmol)

Example 5: Synthesis of Bis-3,4-Dimethyl-1H-Imidazolium Cationic Poloxamer Derivatives

[0242] Example 5 deals with the introduction of 3,4-dimethyl-1H-imidazolium moieties as endgroups on different hydrophilic unmodified poloxamers. The chemical strategy will lead to compounds bearing the general structure depicted below:

##STR00020## [0243] General structure of 3,4-dimethyl-1H-imidazolium-grafted cationic poloxamer derivatives

[0244] The physico-chemical features of the related compounds are dependent of the polymeric backbone chosen. They are condensed in the following Table 17.

TABLE-US-00017 TABLE 17 Main physico-chemical features of the 3,4-dimethyl-1H-imidazolium- grafted cationic poloxamer derivatives described in this study Backbone Feature F87 F108 F127 F68 F88 F98 XR; A 3,4- 3,4- 3,4- 3,4- 3,4- 3,4- dm1Hiz; dm1Hiz; dm1Hiz; dm1Hiz; dm1Hiz; dm1Hiz; ms ms ms Ms ms ms Tag XIX XXVI V XXXII XXXVII XII a; b 61.25; 132.7; 100.22; 76.4; 103.7; 39.3 118.2; 44.8 39.83 50.3 65.2 28.97 HLB >24 >27 >22 >29 >28 >28 XR; A: Cationic endgroups (XR); counteranion (A) as described before. Tag: number attributed to the obtained polymer; a; b: Number of the PEO (a) and PPO (b) blocks as defined before. HLB refers to hydrophilic/lipophilic balance as described before. 3,4-dm1Hiz: 3,4-dimethyl-1H-imidazolium; ms: methyl-sulfate.

[0245] To illustrate the synthetic strategy, the detailed procedure for the achievement of compound V is developed in Figure VIII.

Step 1: Synthesis of Bis-Tosylated F127 5a

[0246] Prior to the synthesis, 20 g of F127 (Croda) are dissolved in 200 mL of MilliQ double distilled water and put in a 1000 mL flask. The mixture is then frozen at −80′C overnight, before being freeze-dried.

[0247] Compound 5a is obtained following the strategy and proportions used the synthesis of compound 1a, starting from 11.7 g of F127 to get 11.3 g of 5a.

[0248] See Table 4 for detailed results.

Step 2: Synthesis of Bis-Amino F127 5b

[0249] Compound 5b is obtained following the strategy and proportions used the synthesis of compound 1b, starting from 3 g of 5a to get 2.2 g of 5b. This compound is of cationic nature. It thus belongs to the present invention.

[0250] See Table 5 for detailed results.

Step 3: Synthesis of Bis-Methylimidazole F127 5c

[0251] Compound 5b (1 g, 7.93.Math.10.sup.−2 mmoles) is dissolved in 10 mL of dry DMF under an argon atmosphere. After complete dissolution, acetaldehyde (TCI, 1.6 mmoles, 100 μL) is introduced in the mixture, which is consequently stirred for 3 hours at room temperature. Potassium carbonate (VWR, 0.47 mmoles, 65 mg) and para-toluenesulfonic isocyanide (Sigma, 0.47 mmoles, 91 mg) are added subsequently and the reaction is kept under stirring for 24 hours at room temperature. At the end of the reaction the solvent is evaporated under vacuum, and then co-evaporated several times with ethyl acetate. The remaining residue is then resuspended in MilliQ water and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product is then recovered after lyophilisation (831 mg). This compound is of cationic nature. It thus belongs to the present invention. [0252] Following Table 18 shows VanLeusen cyclisation step assessment: this table presents number and quantities of the starting material and name and quantity of the products bearing imidazoles rings in which m represents the mass of the compound while n the number of moles.

TABLE-US-00018 TABLE 18 Backbone Material F87 F108 F127 F68 F88 F98 Starting 1b 2b 5b 16b 23b 12b material (770 mg; (1.46 g; (1 g; (840 mg; (1.14 g; (1.3 g; (m; n) 0.1 mmol) 0.1 mmol) 0.079 mmol) 0.1 mmol) 0.1 mmol) 0.1 mmol). Isolated 19c 26c 5c 32c 37c 12c Product (574 mg; (1.27 g; (831 mg; (591 mg; (925 mg; (763 mg; (m; n) 0.073 mmol) 0.086 mmol) 0.065 mmol) 0.069 mmol) 0.080 mmol) 0.058 mmol)

Step 4: Synthesis of Bis-Methylimidazolium Salt F127 V

[0253] Compound 5c (412 mg) is dissolved in 20 mL of dry toluene under argon. The flask is then cooled to 0° C. using an ice-bath. While stirring intensively, dimethyl sulfate (Sigma, 6.3 mmoles, 600 μL) is introduced in the mixture carefully so that temperature inside the flask does not rise above 40° C. After complete addition, the reaction is stirred at room temperature for two more days, concentrated under vacuum and then co-evaporated several times with diethyl ether and ethyl acetate. The remaining residue is then resuspended in MilliQ water and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product is then recovered after lyophilisation (274 mg).

[0254] Following Table 19 shows Imidazole methylation step assessment. this table presents number and quantities of starting materials and name and quantity of the products bearing methylated imidazolium rings in which m represents the mass of the compound while n the number of moles.

TABLE-US-00019 TABLE 19 Backbone Material F87 F108 F127 F68 F88 F98 Starting material 19c 26c 5c 32c 37c 12c (m; n) (287 mg; (635 mg; (412 mg; (297 mg; (925 mg; (381 mg; 0.037 0.043 0.032 0.035 0.040 0.029 mmol) mmol) mmol) mmol) mmol) mmol) Isolated Product XIX XXVI V XXXII XXXVII XII (m; n) (237 mg; (497 mg; (274 mg; (124 mg; (356 mg; (148 mg; 0.029 0.033 0.021 0.014 0.030 0.011 mmol) mmol) mmol) mmol) mmol) mmol)

Example 6: Synthesis of Bis-Arginine-Based Cationic Poloxamers Derivatives

[0255] Example 6 deals with the introduction of arginine moieties as endgroups on different hydrophilic unmodified poloxamers. The chemical strategy will lead to compounds bearing the general structure depicted below:

##STR00021## [0256] General structure of cationic poloxamers derivatives bearing arginine moieties

[0257] The physico-chemical features of the related compounds are dependent of the polymeric backbone chosen. They are condensed in the following Table 20.

TABLE-US-00020 TABLE 20 Main physico-chemical features of the arginine-grafted cationic poloxamers derivatives described in this study (A; TFA: Arginine/Trifluoroacetate) XR; A: Cationic endgroups (XR); counteranion (A) as described before; Tag: number attributed to the obtained polymer; a; b: number of the PEO (a) and PPO (b) blocks as defined before. HLB refers to hydrophilic/lipophilic balance as described before. Backbone Features F87 F108 F127 F68 F88 F98 XR; A A; TFA A; TFA A; TFA A; TFA A; TFA A; TFA Tag XIII XX VI XXVII XXXIII XXXVIII a; b 61.25; 132.7; 100.22; 76.4; 103.7; 118.2; 39.83 50.3 65.2 28.97 39.3 44.8 HLB >24 >27 >22 >29 >28 >28

[0258] To illustrate the synthetic strategy, the detailed procedure for the achievement of compound VI is developed in Figure IX.

Step 1: Coupling Between 5b and Boc.SUB.3.Arg

[0259] Boc.sub.3Arg (Bachem, 30 mg, 6.4.Math.10.sup.−2 mmoles) is dissolved in 2 mL of dry DMF. DIC (15 μL, 9.6.Math.10.sup.−2 mmoles) and HOBt (17 mg, 13.Math.10.sup.−1 mmoles) are added successively to the mixture and stirred for 2 h30. Cationic poloxamer 5b (200 mg) is then added directly in the stirring flask, with triethylamine (TCI, 4.Math.10.sup.−2 mmoles, 6 μL) and the reaction is pursued for 24 h at room temperature, before being concentrated under high vacuum. The resulting syrup is then dissolved in 20 mL of double distilled water, kept at 4° C. for one night and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product 6a is then recovered after lyophilisation (198 mg). [0260] Following Table 21 shows Coupling with protected Arginine step assessment: this table presents number and quantities of starting materials and name and quantity of the products bearing protected arginine moieties in which m represents the mass of the compound while n the number of moles.

TABLE-US-00021 TABLE 21 Backbone Material F87 F108 F127 F68 F88 F98 Starting material 1b 2b 5b 16b 23b 12b (m; n) (116 mg; (219 mg; (200 mg; (134 mg; (182 mg; (208 mg; 0.015 0.015 0.016 0.016 0.016 0.016 mmol) mmol) mmol) mmol) mmol) mmol). Isolated Product 13a 20a 6a 27a 33a 38a (m; n) (103 mg; (186 mg; (198 mg; (84 mg; (99 mg; (139 mg; 0.012 0.012 0.0147 0.009 0.008 0.010 mmol) mmol) mmol) mmol) mmol) mmol)

Step 2: Synthesis of Bis-Arginine Substituted F127 Derivative VI

[0261] Compound 6a (198 mg) is dissolved in 4 mL of a solution of dichloromethane. TFA is then introduced dropwise (VWR, 1 mL). The solution is degassed with argon and then stirred for 2 h. Mixture is then concentrated under vacuum, and co-evaporated twice with DCM, three times with diethyl ether and dried one night under vacuum. The remaining residue is then resuspended in MilliQ water and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product is then recovered after lyophilisation (194 mg).

[0262] Following Table 22 shows deprotection of protected Arginine step assessment: this table presents number and quantities of starting materials and name and quantity of the products bearing unprotected arginine moieties in which m represents the mass of the compound while n the number of moles.

TABLE-US-00022 TABLE 22 Backbone Material F87 F108 F127 F68 F88 F98 Starting 13a 20a 6a 27a 33a 38a material (103 mg; (186 mg; (198 mg; (84 mg; (99 mg; (139 mg; (m; n) 0.012 0.012 0.0147 0.009 0.008 0.010 mmol) mmol) mmol) mmol) mmol) mmol) Isolated XIII XX VI XXVII XXXIII XXXVIII Product (104 mg; (187 mg; (163 mg; (85 mg; (99 mg; (140 mg; (m; n) 0.012 0.012 0.0147 0.009 0.008 0.010 mmol) mmol) mmol) mmol) mmol) mmol)

Example 7: Synthesis of Bis-Histidine-Based Cationic Poloxamers Derivatives

[0263] Example 7 deals with the introduction of histidine moieties as endgroups on different hydrophilic unmodified poloxamers. The chemical strategy will lead to compounds bearing the general structure depicted below:

##STR00022## [0264] General structure of histidine-based cationic poloxamers derivatives

[0265] The physico-chemical features of the related compounds are dependent of the polymeric backbone chosen. They are condensed in the following Table 23.

TABLE-US-00023 TABLE 23 Main physico-chemical features of the histidine-grafted cationic poloxamers derivatives described in this study. XR; A: Cationic endgroups (XR); counteranion (A) as described before. Tag: number attributed to the obtained polymer. a; b: number of the PEO (a) and PPO (b) blocks as defined before. HLB refers to hydrophilic/lipophilic balance as described before. Backbone Features F87 F108 F127 F68 F88 F98 XR; A histidine; histidine; histidine; histidine; histidine; histidine; trifluoro- trifluoro- trifluoro- trifluoro- trifluoro- trifluoro- acetate acetate acetate acetate acetate acetate Tag XLI XIV VII XXI XXVIII XXXIV a; b 61.25; 132.7; 100.22; 76.4; 103.7; 118.2; 39.83 50.3 65.2 28.97 39.3 44.8 HLB >24 >27 >22 >29 >28 >28

[0266] To illustrate the synthetic strategy, the detailed procedure for the achievement of compound VII is developed in Figure X.

Step 1: Coupling Between 5b and Boc.SUB.2.Hist

[0267] Di-Boc-Histidine dicyclohexylammonium salt (Sigma, 34 mg, 6.4.Math.10.sup.−2 mmoles) is dissolved in 2 mL of dry DMF. DIC (15 μL, 9.6.Math.10.sup.−2 mmoles) and HOBt (17 mg, 13.Math.10.sup.−1 mmoles) are added successively to the mixture and stirred for 2 h30. Cationic poloxamer 5b (200 mg) is then added directly in the stirring flask, with triethylamine (TCI, 4.Math.10.sup.−2 mmoles, 6 μL) and the reaction is pursued for 24 h at room temperature, before being concentrated under high vacuum. The resulting syrup is then dissolved in 20 mL of double distilled water, kept at 4° C. for one night and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product 6a is then recovered after lyophilisation (198 mg). [0268] Following Table 24 shows coupling with protected histidine step assessment: this table presents number and quantities of starting materials and name and quantity of the products bearing protected histidine moieties in which m represents the mass of the compound while n the number of moles.

TABLE-US-00024 TABLE 24 Backbone Material F87 F108 F127 F68 F88 F98 Starting material 1b 2b 5b 16b 23b 12b (m; n) (116 mg; (219 mg; (200 g; (134 mg; (182 mg; (208 mg; 0.015 0.015 0.016 0.016 0.016 0.016 mmol) mmol) mmol) mmol) mmol) mmol). Isolated Product 41a 14a 7a 21a 38a 34a (m; n) (75 mg; (153 mg; (159 mg; (91 mg; (133 mg; (164 mg; 0.009 0.010 0.012 0.010 0.011 0.012 mmol) mmol) mmol) mmol) mmol) mmol)

Step 2: Synthesis of Bis-Histidine Substituted F127 Derivative VII

[0269] Compound 7a (159 mg) is dissolved in 4 mL of a solution of dichloromethane. TFA is then introduced dropwise (VWR, 1 mL). The solution is degassed with argon and then stirred for 2 h. Mixture is then concentrated under vacuum, and co-evaporated twice with DCM, three times with diethyl ether and dried one night under vacuum. The remaining residue is then resuspended in MilliQ water and introduced in a dialysis bag of 3.5 kDa cutoff (Spectrum Labs Europe). The dialysis is performed against 4 liters of double-distilled water with 5 medium-changes (2 hours, 8 hours, 24 hours, 36 hours and 48 hours). Final product is then recovered after lyophilisation (160 mg). [0270] Following Table 25: deprotection of histidine step assessment: this table presents number and quantities of starting materials and name and quantity of the products bearing deprotected histidine moieties in which m represents the mass of the compound while n the number of moles.

TABLE-US-00025 TABLE 25 Backbone Material F87 F108 F127 F68 F88 F98 Starting 41a 14a 7a 21a 38a 34a material (75 mg; (153 mg; (159 mg; (91 mg; (133 mg; (164 mg; (m; n) 0.009 0.010 0.012 0.010 0.011 0.012 mmol) mmol) mmol) mmol) mmol) mmol) Isolated XLI XIV VII XXI XXXVIII XXXIV Product (76 mg; (153 mg; (160 mg; (91 mg; (133 mg; (165 mg; (m; n) 0.009 0.010 0.012 0.010 0.011 0.012 mmol) mmol) mmol) mmol) mmol) mmol)

Example 8: Synthesis of Cationic Poloxamer-Coated Magnetic Nanoparticles

[0271] Some of the cationic poloxamers derivatives described in this invention have been used to coat negatively or positively charged iron-based magnetic nanoparticles. Thus after the initial NPs synthesis several coating strategies have been envisioned. All of them involved the resuspension of a magnetic fluid (aqueous or ethylic suspension of NPs) in an aqueous polymeric suspension. We observed during initial trials huge differences in stabilizing behavior between the cationic poloxamers, depending on three mains factors: [0272] Cationic end group selection [0273] Nature of the polymeric backbone [0274] Concentration of the cationic poloxamer solution.

[0275] Based on these observations, several experimental strategies have been attempted in parallel to figure out what is the best way to get colloidally stable magnetic NPs coated with cationic poloxamers. So after the synthesis of the magnetic core by a well described and known in the art coprecipitation strategy (any other synthesis procedure can also be considered), the polymeric coating has been introduced using different pathways, depending on the concentration of cationic poloxamer solution used. We can sort these pathways in four categories as “diluted” “moderate concentration” “concentrated” and “highly concentrated” that corresponds to aqueous cationic poloxamer coating solutions being equal to respectively at 0.25, 1, 5 and 10% w/v, while the iron content is fixed each time at 0.5 mg of iron per mL of polymeric aqueous solution.

Step 1: Synthesis of Hydrophobically Coated Iron-Based Magnetic Core

[0276] The iron based magnetic core is obtained via a classical co-precipitation strategy (Lu, A. H. et al. Angew. Chem. Int. Ed. 2007, 46:1222-44; Huber, D. L. Small 2005, 1: 482-501). Using Iron (III) chloride hexahydrate (Sigma) and Iron (II) chloride tetrahydrate (Sigma) both dissolved in MilliQ water. After filtration, precipitation is carried out under an argon atmosphere upon mechanical stirring. NH.sub.4OH (TCI) has been used to precipitate iron oxide. The hydrophobic precoating is then introduced at a temperature of 80° C., while stirring is maintained for 2 hours. After cooling to RT, the reaction is then transferred in an Erlenmeyer and is magnetically decanted over 12 hours. The supernatant is removed and the remaining solid is washed extensively with absolute ethanol (4 portions of 30 mL), and use as it in the next step, suspended in 20 mL of ethanol noted Solution A.

Step 2: Coating of the Magnetic Core Using an Aqueous Diluted Solution of Cationic Poloxamer (0.25% w/v)

[0277] An aqueous solution of the cationic poloxamer of interest is prepared by suspending the polymer in MilliQ water at a concentration of 100 mg/mL (10% w/v). To help dissolution and prevent gelation, the polymer once suspended is kept at 4° C. for 12 hours while being stirred regularly. After complete dissolution, the solution is filtered on a 0.22 μm membrane, providing what we will call after the mother solution.

Step 3.1: Coating of the Magnetic Core Using an Aqueous Diluted Solution of Cationic Poloxamer (0.25% w/v)

[0278] To work using the “diluted” conditions, 1 mL of the mother solution is diluted with 37 mL of MilliQ water to give a Solution noted B. For the coating procedure, solution A is titrated using a standard colorimetric method (Jiang C et al.; J Magn Magn Mater. 2017, 439:126-134) providing the total iron concentration of the Oleic-acid coated magnetic core. Then a volume of solution A containing 0.5 mg of iron is magnetically decanted for 12 hours on magnetic rack (OZ Biosciences, Marseille, France). After removal of the supernatant, the remaining solid is then resuspended in 1 mL of solution B and vortexed intensively for 5 minutes. The resulting suspension is then sonicated (Branson apparatus, Power output 3, 70% duty cycles, 10 minutes sonication) before being characterized by DLS.

Step 3.2: Coating of the Magnetic Core Using a Moderately Concentrated Solution of Cationic Poloxamer (1% w/v)

[0279] Previous steps 1 & 2 are repeated, and solution A is titrated as before. Meanwhile 100 μL of polymeric aqueous mother solution is diluted in 900 μL of MilliQ water providing solution C (in which cationic poloxamer concentration equals 1% w/v). Then a volume of solution A containing 0.5 mg of iron is magnetically decanted for 12 hours on magnetic rack (OZ Biosciences, Marseille, France). After removal of the supernatant, the remaining solid is then resuspended in 1 mL of solution C and vortexed intensively for 5 minutes. The resulting suspension is then sonicated (Branson apparatus, Power output 3, 70% duty cycles, 10 minutes sonication) before being characterized by DLS.

Step 3.3: Coating of the Magnetic Core Using a Concentrated Solution of Cationic Poloxamer (5% w/v)

[0280] Previous steps 1, 2 and 3.2 are repeated, and solution A is titrated as before. Meanwhile 500 μL of polymeric aqueous mother solution is diluted in 500 μL of MilliQ water providing solution D (in which cationic poloxamer concentration equals 5% w/v). Then the NPs resuspended in a 1% w/v cationic poloxamer (see Step 3.2) are again magnetically decanted for 12 hours on a magnetic rack. After removal of the supernatant, the remaining solid is then resuspended in 1 mL of solution D and vortexed intensively for 5 minutes. The resulting suspension is then sonicated (Branson apparatus, power output 4, 80% duty cycles, 10 minutes sonication). After cooling to room temperature, a second sonication cycle is performed before characterizing the NPs by DLS.

Step 3.4: Coating of the Magnetic Core Using a Highly Concentrated Solution of Cationic Poloxamer (10% w/v)

[0281] Previous steps 1, 2, 3.2 and 3.3 are repeated, and solution A is titrated as before. Then the NPs resuspended in a 5% w/v cationic poloxamer (see Step 3.3) are again magnetically decanted for 12 hours on a magnetic rack. After removal of the supernatant, the remaining solid is then resuspended in 1 mL of cationic poloxamer mother solution and vortexed intensively for 5 minutes. The resulting suspension is then sonicated (Branson apparatus, power output 4, 80% duty cycles, 10 minutes sonication). After cooling to room temperature a second sonication cycle is performed, then a third one, before characterizing the NPs by DLS.

[0282] Biological Evaluation of the Modified Cationic Poloxamers

[0283] Materials & Methods for the Transduction Experiments

[0284] Cationic Poloxamer Formulation in Water

[0285] An aqueous solution of the cationic poloxamer of interest is prepared by suspending the polymer in MilliQ water at a concentration of 100 mg/mL (10% w/v). To help dissolution and prevent gelation, the polymer once suspended is kept at 4° C. for 12 hours while being stirred regularly. After complete dissolution, the solution is filtered on a 0.22 μm membrane, and is ready to be used in transduction.

[0286] For adenoviruses or AAV-mediated experiments, 250 mg/mL (25% w/v) cationic poloxamers solutions have been prepared, following the same general guidelines to preserve a liquid form of the mixture.

[0287] Cells and Virus

[0288] Human cervical carcinoma (HeLa), Human Embryonic Kidney (HEK-293T), Adult Mouse Hypothalamic cell line (CLU-500), Mouse fibroblasts (NIH-3T3), immortalized murine microglial cell line (BV2), and Rat glioma (C6) cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM, Lonza, Walkersville, Md., USA) supplemented with 10% Foetal Bovine Serum (FBD, St. Louis, Mo., USA), 2 mM final L-Glutamine, 100 units/ml penicillin and 100 μ/ml streptomycin (Lonza, Walkersville, Md., USA). Human immortalized Jurkat T-cells were cultured in Roswell Park Memorial Institute medium (RPMI, Lonza, Walkersville, Md., USA) supplemented with 10% Foetal Bovine Serum (FBD, St. Louis, Mo., USA), 2 mM final L-Glutamine, 100 units/ml penicillin and 100 μ/ml streptomycin (Lonza, Walkersville, Md., USA). CD34+ KG-1a cell line was cultured Iscove's Modified Dulbecco's Medium (IMDM, Lonza, Walkersville, Md., USA) supplemented with 20% Foetal Bovine Serum. Primary CD34+ stem cells isolated from cord blood and were cultured in P6-well plates and all cell lines were cultured in 75 cm.sup.2 flasks (Sarstedt AG & Co., Germany) at 37° C. in a humidified incubator (Sanyo, Tokyo, Japan) with 5% CO2 atmosphere, and trypsinized at 80% confluency in order to maintain an exponential division rate before transduction assays. Transduction experiments were performed in 24 well-plate (Sarstedt AG & Co., Germany).

[0289] Preparation of the Lentivirus/Cationic Poloxamers Solution for Infection

[0290] HIV-1-derived vector coding for the green fluorescent protein (GFP) reporter gene under the control of a SFFV promoter (HIV-SFFV-GFP) was produced at the facility of SFR Biosciences (UMS3444/CNRS, US8/INSERM, ENS de Lyon, UCBL, France). Viral suspensions were collected from cell supernatants and aliquoted without any concentration step before conservation at −150° C. (Sanyo, Tokyo, Japan). Previous to each experiment, virus stock was thawed and diluted at the desired Multiplicity of Infection (MOI) in 50 μL of complete medium. After addition of the cationic poloxamer, suspension was mixed by tube inversion and directly added in a dropwise manner onto cells. Cells were then incubated for 72H under classic condition until evaluation of experiment.

[0291] Adenovirus and Adeno Associated Virus Infection

[0292] Type 5 replication-deficient Adenovirus encoding GFP (Ad-GFP) and AAV serotype A virus, both under the control of a cytomegalovirus (CMV) promoter were purchased from Vector Biolabs. Ad-GFP aliquots of 1×10.sup.10 IFU/ml viral stock corresponding to 5×10.sup.11 VP/ml were diluted extemporaneously in medium without any supplement in order to reach the desired MOI. AAV-1 were diluted in medium without supplement for a final MOI of 100.000 genome copies per cell. When needed, cationic poloxamers mixed with viral particles and added in a dropwise manner onto cells. Cells were then incubated for 72H under classic condition until evaluation of experiment.

[0293] Flow Cytometry.

[0294] Cells were washed two times with phosphate buffered saline without calcium and magnesium (PBS, Lonza, Walkersville, Md., USA) and adherent cells were detached with a Trypsin/EDTA 0.2% solution (Lonza, Walkersville, Md., USA) before being fixed in 200 μL of 4% PFA (Sigma Aldrich). Cells were then analysed for % of GFP positive cells and fluorescence intensity by flow cytometry using a CytoFlex Flow Cytometer (Beckman Coulter, Miami, Fla.).

[0295] Evaluation in Transduction Experiments of the Use of Modified Cationic Poloxamers as Chemical Transduction Enhancers Using Lentiviruses.

[0296] As disclosed herein, the main purpose of this invention is to develop a new method with chemical agents able to enhance efficiently and reliably the infective behavior of viruses. The first part of the results focuses on the use of lentivirus as viral vector, as it has been described as potent candidate for cell therapy clinical studies. For this evaluation we used mouse fibroblast cell lines, NIH-3T3; the cationic poloxamers described in the examples have been associated with HIV-1-derived vector coding for the green fluorescent protein (GFP) reporter gene. This first evaluation described modified cationic poloxamers derived from F87, F127 and F108.

[0297] NIH-3T3 cell line was infected with Lentivirus (MOI 1) in presence or not of various doses of cationic poloxamers. After 72 h incubation, % of GFP positive cells (A) and mean intensity (B) of transduced cells were evaluated by flow cytometry. Results are described in Figure XI.

[0298] The biological evaluation of cationic poloxamers as transduction enhancers highlighted well their propensity to act positively in such experiments. Compounds I, 2b, II &VI, show a net increase in the number of cells infected compared with the viral vector used without adjuvant at the same MOI, with a maximum 6-fold increase when using 10 μL of compound II. This increase is also visible to a lesser degree when comparing the mean intensities of the different experiments.

[0299] For the compound showing a positive effect, clear dose dependence is noticeable, with a dramatic increase of the numbers of infected cells related to an increasing amount of cationic poloxamer. This dose effect is less visible when considering the mean of intensity, a clear increase is observed only when compound 2b & VI are chosen.

[0300] On the other hand, compound III showed no positive effect on the transduction while used in the same experimental conditions. The number of infected cells remains at the same level than with the lentivirus alone, whatever the cationic poloxamer dose employed. It is however important to note that compound III does not inhibit the behavior of the virus, and does not affect its infecting skills.

[0301] The difference of behavior between compounds III and VI, both based on F127 backbone, is a first proof of the tremendous importance of the ending group nature on the transduction efficiency.

[0302] A second set of potent adjuvants has been evaluated following the same experimental plan than previously. Results are summarized in Figure XII.

[0303] This evaluation of the different cationic poloxamers candidates gives similar results to the previous one. It indeed seems that some of the modifications have clearly a positive impact on the transduction efficiency while others just do not lead to any improvement.

[0304] In case of tetrazole-based compounds 4b and IV, only the IV leads to a net improvement of the transduction efficiency, both in terms of number of infected cells and of mean of GFP intensity. The dose response is also clearly visible, highlighting the positive impact of the adjuvant. In the same time, the use of a non-substituted tetrazole ending groups to modify the cationic poloxamer did not bring the same level of enhancement. Indeed, when 4b is used as transduction enhancer, the number of cells transduced remained really close to the level reached with the lentivirus alone.

[0305] Imidazole-based compounds 5c & V both show a positive effect in transduction associated with a lentivirus. In both cases the number of cells infected by the virus is dependent to the amount of modified cationic poloxamer used. However, the improvement is much more pronounced with compound V, that shows around four times more transduced cells than the virus alone. In this case also, the methylation of the heteroatomic ring lead to a better behavior as adjuvant, with compound V being more efficient than its counterpart 5c.

[0306] This screening has been finally completed by a last run of transduction experiments, involving this time the use of lentivirus to infect HEK-293 and NIH-3T3 cells. Here again the results evidenced the difference of behavior among the different poloxamers, as shown in table 26.

TABLE-US-00026 TABLE 26 Cationic poloxamers differential transduction effect depending on the MOI and cell type. NIH-3T3 and HEK293 cell lines were infected with Lentivirus encoding for GFP protein at MOI of 0.5 and 1 in presence or not of 5 or 10 μL of cationic poloxamer. 72 hours after infection, % of GFP positive cells was analyzed by flow cytometry. NIH-3T3 HEK-293 MOI 0.5 MOI 1 MOI 0.5 MOI 1 cationic cationic cationic cationic polox. polox. polox. polox. — 5 μL 10 μL — 5 μL 10 μL — 5 μL 10 μL — 5 μL 10 μL LV 2.50% — — 4.01% — — 23.60% — — 42.90% — — alone 5b —  5.97%  5.00% —  6.25% 15.13% — 29.29% 34.98% — 45.90% 52.87% XXIX — 13.32% 11.52% — 12.70% 50.17% — 34.75% 37.18% — 46.75% 50.26% IX —  4.69%  8.26% —  7.52% 24.14% — 27.80% 33.63% — 41.27% 54.61% VI —  3.44%  8.85% —  7.95% 25.88% — 26.57% 34.36% — 39.28% 56.09%

[0307] For example, in the case of NIH-3T3, all the cationic poloxamers presented in this table induce a positive effect on the quantity of transduced cells. Compound XXIX, based on a F87 backbone, is clearly efficient, enabling to multiply by 6 the number of infected cells at a lentivirus MOI of 0.5 (13.32% of infected cells vs 2.5% for the lentivirus used alone) while at a MOI of 1 the number of infected cells raises by more than a factor 10 when compared with the lentivirus control. Furthermore, a clear dose effect is visible using these conditions, as the number of transduced cells goes from 12.7 to more than 50% when doubling the amount of poloxamer used using the same quantity of virus.

[0308] This poloxamer exhibits a similar behavior on HEK-293 cells, even if the effect is less visible, due to the greater permissivity of those cells to the lentiviral infection. At a MOI of 0.5, compound XXIX allowed to observe of the number of infected cells going from 23% to 37% when 10 μL of adjuvant are added in the experiment. Increasing the amount of virus does not change the positive tendency brought by the use of this compound. At a MOI of 1, adding compound XXIX put the number of infected cells from 42 to 50%. These results are a clear illustration of one of the main benefits of this invention, that is using less virus to obtain a similar quantity of positively transduced cells.

[0309] Compounds 5b, VI and IX, all based on a F127 backbone have also been included in this round of experiments, the three of them showing positive results on both cell lines, when associated with various quantity of lentivirus. However, their efficiency stands below what has been observed with compound XXIX used simultaneously. Indeed, the maximum enhancement is observed when using compound VI on NIH-3T3 cells, with a 5 times enhancement at MOI 1 when using 10 μL of poloxamer versus the virus alone. On HEK-293, the three compounds offer similar enhancement factor, offering a benefit of about 10% to 15% of cellule transduced compared with the lentivirus used alone, whatever the MOI used.

[0310] An interesting observation can be made when comparing the results observed with compounds XXIX and IX that have both been modified to include quaternary trimethylammonium salts as ending groups, but that are based respectively on F87 and F127 backbones.

[0311] In each of these experiments, compound XXIX presents a transduction enhancing behavior almost always superior to the one observed with compound IX, this difference peaking when NIH-3T3 are used as cells, with a lentivirus used at a MOI of 0.5 (6 times enhancement with compound XXIX vs 3 times with compound IX). This observation correlated with the other results observed in the present demand, emphasizes well on the importance to find a good balance between the choice of the polymeric backbone and the choice of the cationic endgroup to reach optimal efficiency. It is also clear observing these data that no predictable behavior can be adopted, neither for choosing the best backbone nor the best ending groups to reach the perfect balance.

[0312] The figure XIII A-F highlighted this point by presenting the results of several transduction experiments combining the use of a lentivirus as viral vectors on HEK-293T cells, using a broad range of modified poloxamers as transduction adjuvants.

[0313] The poloxamers used in this figure have been grouped by the poloxamer backbone used for the modification.

[0314] Consequently, figure XIII A & B present the results of experiments using lentiviral infection on HEK-293T involving cationic poloxamers based on the F87, namely compounds 1b & XXIX, that includes respectively primary amines and quaternary trimethyl ammonium as endgroups. Both of them display a positive effect on the number of cells infected, with a 10% benefit when compared with the conditions using the virus alone.

[0315] The figure XIII C & D focused on the behavior of poloxamers based on F108 backbone. in this case a broad array of modifications has been compared. All the compounds tested induced a positive effect on the rate of infection, with compounds 2b and VIII giving the best results. In these conditions almost 100% of the cells were transduced versus only 75% of the cells infected when the lentivirus is used alone. Compounds II, XX and XIV also displayed a positive effect globally. When considering the mean of fluorescence intensity, a tendency clearly appears, with compounds 2b, II and VIII providing the best infection enhancement. Compound 2b allows the fluorescence intensity to reach 150% of the value observed with the virus alone, while in the case of II, the benefit is of 120%. On the other hand, poloxamers XX & XIV bearing respectively arginine and histidine as endgroups, while promising when considering the number of infected cells, did not lead to high level of fluorescence intensity. This is a supplementary proof of the tremendous importance of the choice of the cationic modifications, and how several readouts must be considered in parallel to efficiently evaluate the potency of the cationic modification to enhance the transduction procedure.

[0316] The figure XIII E&F features the same experiment involving poloxamers based on a F127 backbone bearing different cationic modifications. On figure XIIIE, the number of transduced cells obtained with each poloxamers used as adjuvant has been compared with the one obtained with the lentiviral vector alone. Here again the results are positive, with the compounds 5b, XV, IX, VII showing a clear benefit when used to enhance the infection rate. Compounds XV, that includes spermine residues as end-groups, looks as the more efficient candidate, allowing the rate of transduced cell to reach 90% at the end of experiment. A similar beneficial effect is observed with compound VII which bears cationic histidines.

The observation of the mean of fluorescence intensity for this experiment allowed us to discriminate easily between the evaluated polymers. Compounds XV and VII, that where the most promising adjuvant when looking at the number of infected cells when combined with the lentivirus, remained the most efficient enhancers, as they both allowed to observe a mean of intensity higher than the one observed with the virus alone. But while both provided similar enhancement when dealing with the number of infected cells, compound XV is clearly the most efficient globally, as its association with the viral vector provide a gain in intensity of 40%, versus only 3% for compound VII. Also, we can observe that compounds 5b and IX that were promising in respect of the number of infected cells, failed to be as efficient when we looking at the mean of intensity of fluorescence.

[0317] This whole set of experiments provides interesting insights relative to the choice of the best cationic poloxamer to efficiently enhance a transduction experiment: [0318] first, the choice of the polymeric backbone which will serve as modification platform is clearly of importance, as different poloxamers provides different response in transduction. However, as no study provides comparison between all the available poloxamer as transduction enhancer, their influence is unpredictable. [0319] second, all the experiments should be looked through several readouts, to finely screen between all the possible candidates. [0320] foremost, the cationic modifications display a crucial role on the global efficiency of the experiment. However, the figure XIII clearly highlights the fact that a certain cationic function won't provide the same results in transduction when associated with different poloxamers' skeleton, hampering the scientist to predict any enhancement capacity of a polymer of this invention, only based on the choice of its cationic endgroups. It is seeable when comparing adjuvants 1b, 2b, 5b, based on poloxamer F87, F108 and F127 respectively, and which all bears primary amine functions as endgroups. From figure XIII, it is clear that primary amines endgroups are beneficial to the transduction efficiency when they are associated with a F108 backbone. On the other hand, the positive effect observed with compound XV witnesses the benefit of linking a spermine residue to a F127 backbone, while the lower efficiency of the compound VIII, that also bears spermine endgroups, exclude this cationic modification for F108-based polymers.

[0321] All in all, only a careful screening of a maximum of the modifications provided in this demand would allow a skilled scientist to find the most efficient adjuvant candidate for his experiment. This should combine several poloxamer backbone with several cationic modifications provided in this demand.

[0322] Importance of the Modification: Comparison Between Unmodified Poloxamer and Cationic Poloxamer as Adjuvant in Lentivirus-Mediated Transduction Experiment

[0323] The innovative feature of this invention stands on the cationic modification of the ending groups of poloxamers to enhance their adjuvant skills in transduction experiments. The biological activity of the modified cationic poloxamers with their non-modified counterpart were then compared. Accordingly, we used compounds 2b & II as modified cationic poloxamers and compared them with Pluronic F108, associated or not with polybrene, following a well-described procedure.

[0324] Two cell lines were tested, namely Human Embryonic Kidney (HEK-293T) and immortalized murine microglial cell line (BV2). The cationic poloxamers have been associated with HIV-1-derived vector coding for the green fluorescent protein (GFP) reporter gene. Results are depicted in Figure XIV.

[0325] Results pointed out that the three cationic poloxamers efficiently enhance the transduction in both cell lines. F108 in combination with the cationic polymer, polybrene doubles the number of BV2 cells infected by the virus compared with the virus alone. More interestingly, the introduction of cationic endgroups into the F108 backbone has a net positive effect on the number of positive GFP cells, which goes from 18% when F108+polybrene is used as adjuvant to 26% for compounds 2b & II. In another hand, the mean of fluorescence intensity remained unaffected whatever the polymer used. In the case of HEK-293T cells, the positive effect of the use of cationic poloxamer on the transduction is also clearly noticeable. The unmodified and modified cationic poloxamers provide the same enhancement for the number of infected cells. This number goes from around 40% when the LV is used alone to around 60% when a cationic poloxamer is used as enhancer. However, the mean of fluorescence intensity is clearly enhanced by the introduction of cationic endgroups on the adjuvant. Indeed, with both 2b and II, this value is higher compared with the F108, gaining almost one third after the cationic modifications. In the end, these results clearly demonstrate the benefits of cationic modification of the poloxamer backbone in different parameters related to global efficiency of the procedure.

[0326] Moreover, it is clear from the previous evaluations, that the nature of the cationic function introduced has a vital importance for the success of the transduction experiment enhancement. Consequently, we have compared the transduction efficiency of different cationic poloxamers based on a F108 backbone, but modified with different polymeric functions. Compounds, 2b, 4b and IV were evaluated in parallel on human Jurkat T-cells. The cationic poloxamers have been associated with HIV-1-derived vector coding for the green fluorescent protein (GFP) reporter gene. As before the F108 has been used as control. Results are depicted in Figure XV.

[0327] The result confirmed that compound 2b once again provides better results than its non-modified counterpart F108. Indeed, while both cationic poloxamers induces 20% more cells to be infected by the viral vector, the cationic modification provokes an increase of the mean of intensity of 20% versus the F108. On the other hand, results with compounds 4b & IV are less efficient. On Jurkat cells, they both induce a slight increase in the number of transduced cells compared with the viral vector used alone and no significant effect in comparison to virus alone, were observed in terms of mean of intensity. More importantly, the results observed with the compounds 4b and IV were lower than with the non-modified cationic poloxamer highlighting the critical needs to design a suitable cationic modification.

[0328] By confirming that the behavior of the cationic poloxamer in transduction experiment is dependent on the nature of the cationic endgroup introduced, this experiment supports the fact that the careful choice of the modification is the key of the success in such procedure. Indeed, introducing a cationic group is not sufficient to get transduction improvement over the virus alone and furthermore against the same reaction adjuvanted by a non-modified poloxamer.

[0329] Transduction Optimization: Cell Screening and Dose Responses

[0330] In order to achieve optimal transduction efficiency, we have monitored various parameters such as cationic poloxamer quantity, virus titer, type of cells etc. The cationic poloxamer dose response study has been carried out using KG-1a cell line, which are immortalized human bone marrow acute myelogenous leukaemia cells, known to be very difficult to infect using classical lentiviruses-mediated transduction procedures. Various doses of cationic poloxamers have been associated with HIV-1-derived vector coding for the green fluorescent protein (GFP) reporter gene. Results are depicted in Figure XVI.

[0331] The results highlight again the positive effect of the use of compounds 2b & II in this kind of transduction procedure. Indeed, while transduction mediated with the lentivirus alone fails to give more than 2% of infected cells at MOI 2 after 72 hours of incubation, the use of cationic poloxamers increases this level to values close to 15% at the same MOI. Results are really close between 2b & II, with in both cases a clear dose response effect while increasing the amount of adjuvant added.

[0332] This positive effect is confirmed by the analysis of the mean intensity of fluorescence. With both compounds this value increases spectacularly compared with the use of the viral vector alone. The best observed result is obtained when 10 μL of compound 2b is used as additive, with a 9-fold gain compared to the control. Dose response profile are slightly different between 2b & II, as the first reached its maximum at a value of 10 μL whereas compound II induces a constant increase of the mean intensity between 2 μL and 20 μL, not reaching a dose plateau like 2b.

[0333] In conclusion both cationic poloxamers showed a dose-dependent positive effect on the transduction mediated by lentiviruses. Despite their structural similarity, compounds 2b & II present slight differences in behavior depending on the dose added, which emphasizes again the importance of the choice of the cationic function on the global efficiency of the transduction procedure.

[0334] The next experiment had for objective to demonstrate the ability of cationic poloxamer to enhance the efficiency of lentivirus-mediated transduction procedure using a large variety of cell types, and thus to demonstrate the broad-range character of the present invention.

[0335] Compound 2b has been tested on 6 different cell lines which are Human cervical carcinoma (HeLa), Adult Mouse Hypothalamic cell line (CLU-500), immortalized murine microglial cell line (BV2), Rat glioma (C6) cell lines, human immortalized Jurkat T-cells and CD34+KG-1a human bone marrow acute myelogenous leukaemia cell line. HIV-1-derived vector coding for the green fluorescent protein (GFP) reporter gene has been used as viral vector, optimized MOI for each cell line has been selected to emphasize on the cationic poloxamer effect. Results are depicted in Figure XVII.

[0336] This study on a variety of cells clearly demonstrates the consistency of the cationic poloxamer used as adjuvant in lentivirus-mediated transduction experiment. Indeed, whatever the cell line tested, introduction of compound 2b always lead to a clear increase of the number of cells infected. This gain varies depending on the cell line used, ranging from about 10% increase of number of infected cells compared with virus alone (Jurkat) to 650% benefit with KG1-a. The homogeneity of these impressive results can also be observed when looking at the mean of the fluorescence intensity. With this readout as well, the use of the cationic poloxamers as adjuvant enhances notably the results obtained with the lentivirus alone in all cell lines tested, the enhancement being highly cell-dependent. Whereas the benefit of intensity is only of a factor 1.5 when considering BV-2 cell line, transduction of KG1-a using compound 2b is 2.5-fold more intense compared with the virus alone. In conclusion of these series of experiments, the benefits of the cationic modification of the poloxamer backbone as enhancer for transduction experiments mediated by lentivirus was clearly demonstrated. Cationic modification of the poloxamers evidently influence their adjuvant properties. It gives rise to a panel of adjuvants well-suited to the requirements meet in gene therapy applications.

[0337] Finally, the cationic poloxamers' efficiency to enhance lentiviral induced transduction was confirmed on primary human CD34+ stem cells issued from cord blood. A dose response study assessed the capacity of cationic poloxamers to infect primary human CD34+ cells with HIV-1-derived vector coding for the green fluorescent protein (GFP) reporter gene. Results are depicted in Figure XVIII and show the positive effect of the use of compounds 2b & II to raise transduction efficiency to 35% at MOI 5 after 72 hours incubation compared to the lentivirus alone that gives about 7% of infected cells at the same MOI of 5 after 72 hours of incubation.

[0338] As observed previously cationic poloxamers also succeeded in dramatically improving the mean intensity of fluorescence when compared with the use of the viral vector alone. The best observed result is obtained when 20 μL of compound 2b is used as additive, with a 5-fold gain compared to the control.

[0339] In conclusion these results demonstrate the capacity of the cationic poloxamers to enhance the transduction of primary CD34+ stem cells mediated by lentiviruses in a dose-dependent manner.

[0340] Use of Cationic Modified Poloxamers as Adjuvant in Adenoviruses-Mediated Transduction Experiments.

[0341] To extend the scope of the current invention, the effect of the cationic poloxamers as efficient adjuvant in adenovirus-mediated transduction experiment was also investigated. As described in the introduction, non-modified poloxamers have been used as adjuvant in adenovirus-mediated transduction experiments but required very high concentration. In these conditions, the polymer acts as a “pseudo-gel” agent that reduces the diffusion of the viral particles in the biological medium. The main consequence is that as the viruses diffuses less and it is more likely to stay longer in contact with the cells to infect. This prolonged contact increases mechanically virus uptake and thus the efficiency of the whole procedure. It was possible to formulate cationic poloxamer 2b at a 25% w/v concentration which is near its gelation point, demonstrating that the cationic modifications did not inhibit the “gel” behavior observed with the corresponding non modified poloxamer. The main goal of this experiment is to check the pseudo-gel efficacy of the modified cationic poloxamers and also, to monitor if the cationic modifications bring a supplement of efficiency for the adenoviral-mediated transduction procedure, as it was the case with lentiviruses.

[0342] To do so, C6 and HEK-293 cell lines were transduced with adenovirus encoding for GFP (AdGFP) with a MOI of 5 in presence or not of commercially available F108 and its cationic counterpart 2b at a 7.5% w/v final concentration. The results of this experiment are depicted in Figure XIX.

[0343] This experiment clearly demonstrates the benefit of using a modified cationic poloxamer as transduction adjuvant associated with adenoviruses. Indeed, on either HEK-293 or C6 cells, the adenovirus used alone failed to infect 5% of the cells. On the other hand, the cationic modifications lead to a significant enhancement of the adenovirus transduction efficiency. Indeed, when compound 2b is used against its unmodified counterpart, the number of infected cells raises from 46% to 55% on C6 cells and from 32 to 46% on HEK293 cells. The positive effect of the cationic modification is even more impressive when considering the mean of fluorescence intensity with almost a 2-fold increase with compound 2b on both cell lines. This experiment demonstrates that cationic modification of poloxamer opens a wide-range of applications for virus-mediated transduction experiment and that the observed improvement is not limited to lentiviruses.

[0344] To confirm these positive results, this experiment was further extended using several other poloxamers based both on F108 or F127 backbones. The main results are depicted on the following tables:

TABLE-US-00027 TABLE 27A & B Cationic poloxamers differences in adenoviral infection and transduction enhancement. C6 and HEK293 cell lines were infected with Adenovirus encoding for GFP protein at MOI of 4 in presence or not of cationic poloxamer (final concentration 7.5%). 72 hours after infection, % of GFP positive cells (upper table) and mean intensity (lower table) were analysed by flow cytometry Table A: Transduction percent, F108 derivatives AdGFP alone XXVI XXX 2b F108 % S.D. % S.D. % S.D. % S.D. % S.D. C6 1.20% 0.65% 13.98% 3.61% 11.02% 1.84% 55.07% 3.52% 46.19% 2.17% HEK293 1.13% 1.56% 14.12% 2.30%  8.34% 3.01% 45.74% 4.89% 31.79% 1.98% Table B: Mean of intensity, F127 derivatives AdGFP alone III 5b F127 RFU S.D. RFU S.D. RFU S.D. RFU S.D. C6 4158 1207 3850 987 7484 1541 4142 1127 HEK293 1519 564 1652 987 5505.7 1308 3229.4 378

[0345] In the case of F108 derivatives we monitored the number of transduced cells versus the virus alone. On C6 cells, all tested poloxamers clearly show a benefit in term of infected cells ranging from a 10-fold increase with compound XXX to a 50-fold increase for compound 2b. This one, exhibits also a better adjuvating effect than the non-modified poloxamer F108, that is 10% less efficient.

[0346] The results observed on HEK-293 are similar, with all the compounds showing a clear benefit when compared with the virus alone. Here again, the poloxamer 2b, displays the best enhancing capacity, proving one more time to be more efficient than its non-modified counterpart. These results are another proof of the positive influence brought by the cationic modifications depicted in the present invention.

[0347] The F127 derivatives were evaluated through the monitoring of the mean of fluorescence intensity, keeping the adenovirus alone as control. Between the two chosen compounds III & 5b, only the latter one display a gain in fluorescence intensity on the two chosen cell lines, versus the adenovirus alone, with an almost two-fold gain. Furthermore, the non-modified F127, tested in parallel in the experiment, show no positive effect when associated with the adenovirus, validating once again our cationic modification strategy. Finally, it is interesting to note that compound 5b, that was not optimally efficient when associated with lentiviruses, proved to be a powerful adjuvant when associated with adenoviruses.

[0348] This round of experiment has been completed by a study of the influence of the use of serum in association with a poloxamer in an adenovirus-mediated transduction procedure. NIH-3T3 cells were transduced using an adenovirus and the poloxamer II at different concentrations, with or without serum. Results are depicted in the figure XX.

[0349] The results underline that the addition of serum potentializes the positive effect of cationic poloxamer on the efficiency of the transduction mediated by adenovirus. Indeed, when the poloxamer is used at a 12.5% concentration, the presence of serum allows a threefold increase in the efficiency of the transduction, while the effect is less visible without. At a 6.5% concentration of compound II, the effect of serum is still visible, going from a two-fold increase without to a three-fold when serum is used,

These results, witnesses the perfect compatibility between the use of serum and the compounds of the present invention, making them consistent with the procedure used routinely in cell biology processes.

[0350] Use of Cationic Poloxamers as Adjuvant in AAV-Mediated Transduction Experiments.

[0351] To extend further the scope of this invention, the effect of the cationic poloxamers were also tested as efficient adjuvant in AAV-mediated transduction experiment. Compound II at a final concentration of 15% w/v has been associated with AAV(GFP) and tested on HEK-293 cells. The results of this experiment are resumed in Figure XXI:

[0352] In this procedure again, the use of modified cationic poloxamer proved to be a real source of enhancement of the efficiency of the transduction procedure. The addition of 15% w/v of compound II allows AAV to infect 57% of cells while AAV used alone infects only 12%. The improvement is less important when considering the mean of intensity, with nonetheless a net gain of 5% compared with virus alone. In the end, this experiment demonstrated that the cationic poloxamers are also valuable and efficient as adjuvant in AAV-mediated transduction experiment.

[0353] Poloxamers 5b and 2b have also been tested in these experimental conditions. They have been combined at different concentrations with AAVs to infect HEK-293. Results are depicted in the figure XXII.

[0354] The compound 5b show a similar adjuvant behavior compared with compound II, with a net increase of the number of transduced cells to almost 60%, when the poloxamer is used at 15% concentration. It is interesting to note that the compound II show its maximal enhancing factor at a concentration of 7.5%, highlighting clear differences of adjuvant behavior. The effect brought by compound 2b, based on F108 like compound II, is less impressive, but remains very positive when compared with the virus alone, with more than 40% of cells transduced at the end of the experiment.

[0355] When considering the mean of fluorescence intensity, compounds 2b and 5b, were less efficient that compound II. In the end, this set of experiments clearly confirmed the potency of the cationic poloxamers to efficiently enhance AAVs-based transduction experiment.

[0356] Furthermore, the polyvalence of the method described here has been clearly illustrated by the use of several types of virus in association with a broad array of different cationic poloxamers. The cationic modification of poloxamer is without any doubt a step further towards an increased efficiency in transduction procedures. Some compounds proved to be particularly efficient when associated with one kind of viral vectors, whereas some polymers looked more like all-around enhancers

[0357] Cationic Poloxamers and Magnetic Nanoparticles Formulation: DLS Characterization of the Cationic Poloxamer-Coated MNPs

[0358] As mentioned previously, magnetic nanoparticles (MNPs) have been demonstrated as powerful to boost, accelerate and synchronize viral infection. Consequently, it could have been foreseen that the combination of magnetic nanoparticles and unmodified poloxamer lead a strong synergistic effect. Unfortunately, the non-ionic nature of unmodified poloxamer lead to nanoparticles destabilization or precipitation which hampered their use as coating or formulating agent of nanoparticles.

[0359] To investigate the benefits of the poloxamer cationic modification on the magnetic nanoparticle's formulation and stabilization, we focused first on DLS (Dynamic Light Scattering) to measure the size and the charge of the obtained nanoparticles. Size and net charge (zeta potential) measurements are nowadays key parameters to check when focusing on MNPs synthesis and characterization. Indeed, a stable colloidal suspension is mainly characterized by its propensity to maintain the magnetic objects in a non-aggregated state. In other words, it is critically important that the nanoparticles present in solution remain of nanometric sizes. This is even more important when this colloidal suspension is intended to have biological applications, as aggregated objects have proved in the past to have disastrous consequences on the cell's viability. The zeta potential is a direct electric consequence of the presence of charged chemical functions at the surface of the nanometric object. Thus, a suspension made of charged nanoparticles (either positively or negatively) is less likely to witness aggregation of its components, because of the electrostatic repulsions which limit the propensity of magnetic cores to stack into micrometric objects. Furthermore, a net positive charge will allow these beads to form stable complexes with negatively charged viruses (such as many Lentivirus or adenovirus), which appears to be a favorable step in a magnetically-driven transduction experiment.

[0360] The lack of chemically charged functions in the native unmodified poloxamer backbone forbids any repulsive interactions when associated with magnetic beads and leads to destabilization and/or aggregation of those nanoparticles. On the of goal of this present invention was to demonstrate that the cationic modified polymers are able to stabilize magnetic MNPs by providing to the nanometric objects synthesized, enough positive charges on its surface to create electrostatic repulsive interactions, to be able to maintain the colloidal suspension and to allow achieving a viral transduction enhancement. As a consequence, the resulting MNPs will cumulate several features useful for an efficient magnetic NPs-driven transduction experiment: [0361] ability to complex virus at the surface of the NPs, following Magnetofection™ requirements [0362] presence of cationic poloxamer in the complex to act as adjuvants during transduction [0363] stabilization of the magnetic NPs to avoid the formation of micrometric objects, detrimental for cell viability.

[0364] Following these guidelines several magnetic nanoparticles have been synthesized using a well-described co-precipitation strategy. The obtained iron core is mainly made of magnetite crystals, conferring the object strong magnetic properties. The strategy involved the use of a pre-coating molecule prior to the cationic poloxamer introduction. This pre-coating molecule must be charged, negatively or positively, in order to stabilize the magnetic core, and may also include hydrophobic areas, that will be useful to associate the cationic poloxamer to the surface of the NP through hydrophobic interactions. Examples of precoating include a wide array of well-known molecules such as dextran, starch, Zonyl FSA, Linoleic acid, oleic acid, silicium oxide polymer, polyethyleneimine, chitosan, Tween, Span. In the following study, it was chosen to use an oleic acid-based pre-coating.

[0365] Different unmodified or cationically modified poloxamers solutions have been used to resuspend this MNPs, while the quantity of iron has been kept constant.

[0366] Coating of the Magnetic Core Using Aqueous Diluted and Concentrated Solutions of Cationic Poloxamer.

[0367] The MNPs were first resuspended with 0.25% w/v of cationic polymer solution, then after another decantation, the MNPs were formulated within the corresponding 1% w/v cationic poloxamer solution. This intermediate dilution/decantation step appeared to be a reliable solution to avoid direct aggregation of the resulting MNPs. Only the NPs suspended in the 1% w/v solution were evaluated by DLS.

[0368] Abroad array of modified polymers was tested to figure out if other interactions such as (hydrophobic, pi/pi . . . ) brought by the cationic endgroups could be advantageous over short (5 days) and long incubation time (1 month). The size measurements results are presented in the Table 28 below:

TABLE-US-00028 TABLE 28 Size measurements of magnetic NPs resuspended with various 1% w/v cationic poloxamer solution. The sizes are expressed in nm and have been recorded att wo different time points (“meas time” of 5 days (d), 1 month) after resuspension in the cationic poloxamer solution. The “Agg”” tag means that no traces of nanometric objects could have been measured, meaning a likely aggregation of the beads. Poloxamers Naked Meas time NPS F108 2b II 4b XXX XIV F127 III VII IX XV Size (nm) 178.3 Agg 307 677 208 720 201.2 Agg 1103 204.5 817 326.1 after 5 d Size (nm) 180.5 Agg 204.7 Agg 198.7 198 199 Agg Agg 192 Agg Agg after 1 month

[0369] This experiment highlights the impossibility to stably formulate or coat magnetic NPs with a diluted solution of the native (non-ionic) or non-modified poloxamer. Indeed, it leads to the quick, systematic and irreversible aggregation of the beads, whatever the polymer chosen, either F108 or F127.

[0370] In the case of MNPs coated or formulated with cationic F108-derivatives (2b, II, 4b, XXX and XIV) all cationic poloxamers gave, after the resuspension, colloidally stable nanoparticles after 5 days. Amines (2b), triazole derivative (4b) and histidine (XIV) look the most promising candidates as stabilizing agents for MNPs leading to MNPs having sizes in the perfect range for transduction applications, i.e. between 200 and 300 nm. This result was confirmed after 1 month of production When compared with non-coated NPs, the introduction of cationic polymers at the surface of the NPs induces a small increase in the global size of the construct, with a distribution peak that remains thin after the redispersion/sonication procedure, highlighting the association between the cationic polymer and the bead surface.

[0371] However, and despite their cationic modifications, some cationic poloxamers such as the II presented a characteristic behavior of aggregation, which was not completed after 5 days (size of 677 nm), but was after one month. The MNPs formulated with various cationic poloxamer based on the F127 backbone, showed also potential as stabilizing agents for magnetic nanoparticles (see Table 29); one cationic poloxamers (VII) led to a stable MNPs formulation, even after 1 month.

[0372] In summary, while it was not possible to formulate stable MNPs coated with commercially available non-modified and non-ionic poloxamers, it was proved that the cationic functions introduced on the poloxamer endgroups is a necessary and mandatory condition for the stability of the corresponding coated MNPs. Moreover, it appears that depending on the property of the cationic endgroups, the stability of the MNPs is variable. Secondary interactions such as hydrogen bonding (compounds 2b), pi/pi stacking (compounds XXX) or both (compounds VII & 4b) might be involved as well. In parallel of these measurements Zeta potential has been evaluated with the same compounds at the same time points, results are depicted in the following Table 29:

TABLE-US-00029 TABLE 29 Zeta potential (Zp) measurements of magnetic NPs resuspended with various 1% w/v non-ionic and cationic poloxamer solution. The potentials are expressed in mV and have been recorded at two different time points (Meas times of 5 days & 1 month) after resuspension in the non-ionic or cationic poloxamer solution. The “Agg”” tag means that no traces of nanometric objects could have been measured, meaning a likely aggregation of the beads. Cationic poloxamer Naked Meas time NPs F108 2b II 4b XXX XIV F127 III VII IX XV Zp (mV) −14.87 Agg 8.68 3.36 4.18 4.64 8.75 Agg 11.3 9.08 3.13 4.78 after 5 d Zp (mV) −15.09 Agg 13.2 Agg 7.99 17.8 10.2 Agg Agg 9.60 Agg Agg after 1 month [0373] The results presented in this table correlate extremely well with the previous size measurements. Commercially available native and non-ionic poloxamer F108 and F127 when used as coating agents, mask some of the negative charges of the original MNPs, decrease their stabilization through electrostatic interactions and favor their aggregation. On the other hand, cationic poloxamers, all provide to the resulting nanoparticles a positive surface charge which clearly helps the colloidal stabilization over time, highlighted by the great behavior of compounds 2b, 4b, XIV, VII even one month after the resuspension procedure. These experiments validate the fact that the introduction of cationic endgroups into the structure of poloxamers allowed to stabilize NPs coated with these modified polymers, contrary to their native counterparts.

[0374] Using this strategy, we further extend the coating and formulation of MNPs with higher concentration of cationic poloxamer (5% and 10% w/v). We selected for this study compounds 2b, 4b and XXX derived from the F108 backbone, and compound VII derived from the backbone of the F127. To accomplish this, a resuspension procedure which involves intermediate stage of suspension/decantation with less concentrated cationic poloxamer solution has been used.

TABLE-US-00030 TABLE 30 Size measurements of magnetic NPs resuspended with various 5% w/v non-ionic and cationic poloxamer solution. The sizes are expressed in nm and have been recorded at two different time points (“Meas time” of 5 days (d), 1 month) after resuspension in the non-ionic and cationic poloxamer solution. The “Agg”” tag means that no traces of nanometric objects could have been measured, meaning a likely aggregation of the beads. Poloxamer Naked Meas time NPs F108 2b 4b XXX F127 VII Size (nm) after 5 d 178.3 Agg 206 198 236 Agg 186.7 Size (nm) after 1 month 180.5 Agg 210.3 199.4 518 Agg 201

[0375] As before, the unmodified and neutral poloxamers (F108 and F127) have led to an instantaneous aggregation of the MNPs whereas the cationic poloxamers were able to coat in a stable manner MNPs at this higher concentration. On the other hand, the four selected compounds led to the achievement of stable beads after 5 days, having size ranging around 200 nm suitable with for transduction applications and minimized cell-toxicity. The nature of the chosen compounds does not seem to have an influence on the recorded size, with the exception of beads derived from compounds XXX, which looked slightly bigger than its relative. The 1-month stability study displayed no clear differences; most of the NPs keeping equivalent sizes compared with the sizes recorded 5 days after the resuspension. Here again, the beads derived from compound XXX showed a difference, with size ranging above 500 nm. This net increase in size might be a sign of preliminary stacking leading to aggregation, even if no sign of such phenomena is evident at this time point. As a conclusion, here again, the electrostatic repulsive interactions embedded by the cationic modifications of these poloxamers allowed the formation of stable suspension of magnetic NPs, even if the concentration of the cationic poloxamer solution used for resuspension is increased. The corresponding zeta potential values recorded at the same time points are presented in the Table 31 below.

TABLE-US-00031 Poloxamer Naked Meas time NPs F108 2b 4b XXX F127 VII Zp (mV) −14.87 Agg 17.6 12.8 14.6 Agg 16 after 5 d Zp (mV) −15.09 Agg 18.5 12.5 9.8 Agg 16.9 after 1 month Table 31 shows Zeta potential (Zp) measurements of magnetic NPs resuspended with various 5% w/v non-modified and cationic poloxamer solution. The potentials are expressed in mV and have been recorded at two different timepoints (5 days (d) & 1 month) after resuspension in the neutral or cationic poloxamer solution. The “Agg”” tag means that no traces of nanometric objects could have been measured, meaning a likely aggregation of the beads.

[0376] Zeta potential measurements correlates perfectly with the results observed for sizes. All the nanoparticles exhibit high positive charges after formulation with the cationic poloxamers, without any significant changes after 5 days or one month. Only the beads resulting from polymer XXX showed a slight decrease of the Zeta potential, possibly due to the increase in size. These data confirmed that concentrated solutions of cationic poloxamers might be suitable for the resuspension of decanted magnetic NPs. The cationic modifications at this concentration still allowed electrostatic repulsive interactions able to stabilize the colloidal nanostructure.

[0377] The last part of this detailed study, concerned the resuspension of the magnetic cores into 10% w/v cationic poloxamers solutions. This concentration is the preferred one to get the best results in lentivirus-mediated transduction experiments using cationic poloxamers as adjuvant. Having beads stably suspended at such concentrations might emphasize the adjuvant effect of the cationic poloxamer. We selected the same compounds (2b, 4b, VII, XXX) than previously to resuspend the beads. Here again a sequential decantation/resuspension procedure has been carried out to avoid direct aggregation of the beads. This time, the only chosen time point is 14 days. The size results are summarized into the Table 32 below:

TABLE-US-00032 TABLE 32 Size measurements of magnetic NPs resuspended with various 10% w/v unmodified or cationic poloxamer solution. The sizes are expressed in nm and have been recorded at one time point (14 days (d) after resuspension in the unmodified or cationic poloxamer solution. The “Agg”” tag means that no traces of nanometric objects could have been observed, meaning a really likely aggregation of the beads. Poloxamer Naked Meas time NPs F108 2b 4b XXX F127 VII Size (nm) after 14 d 179 Agg 287 453 1030 Agg 190.7

[0378] At this concentration, once again commercially available unmodified poloxamer 10% w/v solutions have not been able to resuspend efficiently the magnetic nanoparticles; in contrast the cationic modifications succeeded in stabilizing the MNPs. The compounds 2b and VII still impressively stabilize the colloid with only a slight increase in size of the beads coated by 2b and VII and without any sign of aggregation at this time. Those 2b and VII cationic poloxamers are clearly the best candidates for resuspension at this concentration, that is emphasized by the analysis of the Zeta potentials recorded in the same conditions. Nonetheless, some cationic poloxamers such as compound XXX did not allowed the achievement of stable nanometric NPs solution and induced stacking of the beads probably due to stronger Pi/pi interactions at this concentration. The results with compound 4b showed that a size of 453 nm that clearly indicates the beginning of an aggregating behavior, which is not completed after 14 days.

TABLE-US-00033 TABLE 33 Zeta potential measurements of magnetic NPs resuspended with various 10% w/v neutral or cationic poloxamer solution. The potentials are expressed in mV and have been recorded at one timepoint (14 days) after resuspension in the neutral or cationic poloxamer solution. The “Agg”” tag means that no traces of nanometric objects could have been measured, meaning a likely aggregation of the beads. Poloxamer Naked Meas time NPs F108 2b 4b XXX F127 VII Zp (mV) after −14.8 Agg 8.88 0.6 7.12 Agg 9.36 14 d

[0379] This last set of data is in total accordance with the previous ones. The compounds able to maintain a positive charge at the surface of the particles (2b, VII) are less likely to induce aggregation during the resuspension step. In contrary, the beads coated with 4b became almost neutral with time. Compound XXX looks like an exception from this aspect, as its positive charges seem to not counterbalance the aggregation probably caused by Pi stacking of the triphenylphosphonium moieties. This highlights the fact that secondary interactions might have positive impact on the stabilization of the particles if they are correctly balanced and do not become a factor of stacking or aggregation.

[0380] As a conclusion, we clearly demonstrated the positive impact of the cationic modification of the poloxamers on the stabilization of resuspended magnetic nanoparticles. In opposition to commercially available neutral poloxamer, some modified cationic poloxamers create positive charge on the surface of the beads to ensure a colloidal stabilization through electrostatic repulsive interactions. With a careful choice of the chemical function and an optimized decantation/suspension strategy, MNPs with appropriated iron/cationic poloxamer content have been stably obtained. They have been tested in a magnetically driven transduction experiment mediated by lentivirus.

[0381] Biological Evaluation of the Modified Magnetic Nanoparticles Coated with Cationic Poloxamers

[0382] This invention clearly highlighted the reliable benefits of using cationic poloxamers to ensure a colloidal stabilization of magnetic nanoparticles. Next, the purpose was to check if such MNPs are able to associate the positive adjuvant effect described earlier herein with the propensity of cationic nanoparticles to enhance the efficiency of lentivirus-mediated transduction experiment. We have thus compared the transduction efficiency on KG1a cell line, of a lentiviral vector associated with either: [0383] ViroMag nanoparticles, from OZ Biosciences (Marseille, France) which are a longtime reference in enhancing lentivirus-mediated transduction, [0384] Cationic poloxamer 2b, which has proved earlier in this document to be efficient as adjuvant in transduction experiment, [0385] ViroMag nanoparticles coated with cationic poloxamer 2b, following the strategy developed in the present document.

[0386] KG1a cells were transduced with Lentivirus encoding for GFP with a MOI of 5 associated with ViroMag magnetic nanoparticles (VM), cationic poloxamer 2b or ViroMag NPs coated with a 5% w/v solution of cationic poloxamer 2b (VM+2b). After 72 h incubation, % of GFP positive cells (A) and mean intensity (B) of genetically modified cells were evaluated by flow cytometry. The results are depicted in Figure XXIII:

[0387] The results showed the benefits of the cationic poloxamer. The ViroMag MNPS were successful at enhancing the transduction as expected and published, as it allows to double the number of cells infected by the lentivirus. Compound 2b, as described earlier allowed also to boost transduction efficiency, as the number of cells infected when it is used as adjuvant reached almost 40%, with a mean of intensity 1.5-fold higher than the LV alone. The combination of cationic poloxamer 2b with ViroMag led the highest efficiency. The synergistic effect brought by the cationic poloxamer and the MNPs is demonstrated. Indeed, VM+2b potentialized the effect of the adjuvant 2b with the benefits of magnetically driven transduction experiment leading to higher number of cells transduced and higher mean of intensity. This experiment not only confirmed the benefits of introducing cationic functions on the endgroups of poloxamers to enhance their adjuvant behavior, but also proved that this cationic modification also allows to associate the intrinsic enhancing features of modified cationic poloxamers with the benefits of magnetically driven lentivirus transduction in only one formulation. This is of a critical importance to face today's emerging challenges of the use of virus mediated gene therapies or the use if virus to generate cell therapy product for clinical trials.