FR2850388A1 - New cationic (co)polymers obtained from ethylenic monomer(s) containing a terminal aminoalkyl group, useful as vectors for negatively charged biomolecules, e.g. in nucleic acid transfection - Google Patents

Abstract

Cationic polymers or copolymers (A) formed from ethylenically unsaturated monomer(s) (I) containing an amide, amine or ester linkage, optionally a spacer group and a terminal alkyl group substituted by at least one primary, secondary or tertiary amino function, optionally protected or in protonated form, are new. New cationic polymers or copolymers (A) are formed from one or more monomers of formula H2C=C(R1)-(CH2)n-X-Sp-Y-R2 (I). R1 = H or 1-4C alkyl; n = 0 or 1; X = OC(O), C(O)O, NH, C(O)NH or NHC(O); Sp = spacer group (such as (CH2)m or (CH2)mO(CH2)l) or direct bond; m, l = 1-4; Y = OC(O), C(O)O, NH, C(O)NH, NHC(O) or direct bond; R2 = 1-6C alkyl substituted by at least one primary, secondary or tertiary amino function, optionally protected or in protonated form.

Description

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 The invention relates to novel cationic polymers and copolymers and the intermediates necessary for their synthesis as well as the use of cationic polymers and copolymers for the vectorization of natural or synthetic biomolecules, complexes comprising said cationic polymers and / or copolymers and said biomolecules.

 The use of genetic material in a therapeutic or prophylactic strategy seems to be a promising avenue for the last ten years. However, this genetic material, whether DNA, RNA and / or their fragments, is rather fragile, in vivo (degradation by nucleases), during direct administrations by conventional parenteral routes or known invasives in the pharmaceutical and biotechnology industries. Satisfactory transfection levels are obtained only when the administration of the naked genetic material is carried out near the target cells (for example injection of naked DNA directly into the muscle tissues in contact with the myoblasts). To this observation, it must be added that these molecules, negatively charged, have difficulties in crossing the various cell membranes themselves being negatively charged. All these obstacles currently lead to a reduced effectiveness of these natural or synthetic biomolecules and it has thus been possible to demonstrate that a very small proportion of the total mass of the material administered actually reaches the targeted cell compartment. and to exercise the role assigned to it.

 In order to overcome these problems, the use of vectors is an original method of choice. These vectors are mainly divided into two categories: (i) viral vectors, which were first tested, and (ii) synthetic vectors.

 Among the viral vectors, mention may be made of retroviruses, adenoviruses and viruses associated with adenoviruses (AAV). While many trials are underway with viral vectors, this strategy still raises doubts about their safety (MD Brown et al., Word Markets Series, Business Briefing, Pharmatech 2001, Word Markets Research Center Ltd. Ed., 158-164). Indeed, although capable of ensuring a high transfection rate, certain viral vectors can cause strong inflammatory and immune reactions (activation of oncogenes), the repeated administrations of doses then becoming impossible. In addition, these vectors can only transfer DNA fragments of limited size, which prohibits the transfection of large genes.

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In addition, difficulties of production, quality control and storage are encountered with such vectors.

 As an alternative to virus-derived vectors, researchers have turned to other purely synthetic vectors, which can be classified into two families: cationic lipids and cationic polymers. These positively charged compounds associate with negatively charged genetic material through electrostatic interactions to form so-called "lipoplex" or "polyplex" complexes depending on the nature of the vector. The formation of a complex with excess positive charges promotes interaction with the various membranes of the cells.

 There is a wide variety of cationic lipids leading to variable transfection efficiencies (A. Aissaoui et al., Curr Drug Targets 2002, 3.1-16). One of the major drawbacks of these compounds is undoubtedly their higher or lower toxicity.

 Similarly, the transfecting power of many cationic polymers has been evaluated (S.C. De Smedt et al., Pharm.Research, 2000, 17, 113-126;

Garnett, Critical Reviews in Therapeutic Drug Carrier Systems, 1999, 16, 147-207). Among them, there may be mentioned polylysine, polyethyleneimine and certain cationic patterned dendrimers. These synthetic vectors are more stable than cationic lipids and benefit from a certain ease of production.

 In this context, the aim of the present invention is to solve the technical problem consisting in the provision of new synthetic polymers or copolymers able to form polyplex-type structures, endowed with specific properties allowing, in vitro and in vivo, an efficient vectorization of natural or synthetic biomolecules such as DNA, RNA and / or fragments thereof.

dans laquelle : - R1 est H ou un C1-C4 alkyle ; - n est zéro ou un ; The invention thus relates, according to a first aspect, to a cationic polymer or copolymer essentially formed from at least one monomer of formula (I)

wherein: - R1 is H or C1-C4 alkyl; - n is zero or one;

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 X is -O-C (O) -, -C (O) -O-, -NH-, -C (O) -NH- or NH-C (O) -; - Sp is a spacer group such that - (CH2) m- or - (CH2) m -O- (CH2) I- or does not exist; m and I, identical or different, represent an integer of 1 to 4; Y is -O-C (O) -, -C (O) -O-, -NH-, -C (O) -NH- or NH-C (O) - or does not exist; R2 is a linear or branched (C1-C6) alkyl group substituted with at least one primary, secondary or tertiary amine function, optionally protected by a protecting group or in protonated form.

 The expression "essentially formed" used in the context of the present description means that the repeating units constituting the polymers or copolymers according to the invention are derived exclusively from monomers of formula (I), the resulting polymers or copolymers possibly containing in very small proportions, some impurities generated during synthesis.

 The originality of the monomers of formula (I) lies in the fact that they comprise at least one primary, secondary or tertiary amine function protonable, that is to say capable of being made cationic by adding a proton.

 According to a mechanism hypothesis, said monomer containing at least one protonatable amine function is capable of ensuring the interaction with natural or synthetic biomolecules such as DNA, RNA and / or their fragments, thus forming complexes, via positive charges associated with the negative charges of biological material. Said complexes also represent an object of the invention.

 Being formed exclusively from monomer (s) at least one protonable function, the cationic polymers or copolymers according to the invention exhibit water solubility properties at physiological pH.

 Preferred protonated monomers having at least one amine function of formula (I) are those wherein - R 1 is H or methyl, preferably methyl; n is zero or one, preferably zero; X is -C (O) -O- or -C (O) -NH-; - Sp is a spacer group selected from -CH2- and - (CH2) 2-, or does not exist; - Y is -O-C (O) - or does not exist;

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 R2 is a group -CH2-NH2, -CH (CH3) -NH2, -CH (CH2-NH2) 2 or CH (NH2) - (CH2) 3 -NH2, the amine functions of which are optionally protected by a protective group such as the tert-butyloxycarbonyl group.

Other monomers of formula (I) that are advantageous for the purposes of the invention are the compounds of formulas (II), (III) and (IV) below:

These monomers of formula (II), (III), (IV) may be used: either to form homopolymers; or to form copolymers consisting exclusively of recurring units of formula (II), (III) and / or (IV); or to form copolymers consisting of repeating units of formula (II) and / or (III) and / or (IV) and of formula (I).

 In general, the monomers of formula (I) are prepared by methods known to those skilled in the art by coupling an appropriately functionalized methacrylate, vinyl or allylic derivative with a molecule also functionalized carrying one or more functions (s). ) protected amine (s) or not.

 For example, an amino acid residue whose terminal amine function is protected by a protecting group with a hydroxyethylmethacrylate or allyl alcohol derivative may be condensed according to known methods,

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 the presence of, for example, dicyclohexylcarbodiimide as a coupling agent and 4-dimethylaminopyridine as a catalyst in a solvent such as dichloromethane.

 The monomers of formula (I) may comprise one or more protected amine function (s) or unprotected (s).

When they contain one or more protected function (s), that is (are) preferably deprotected (s) only after the synthesis of the polymer or copolymer to allow the use of different types of polymerization processes and to ensure better reactivity of the monomers in a wider variety of solvents.

 When the amine function (s) of the group R2 is (are) protected by a protective group, it is chosen from the groups usually used for this purpose, such as, for example, the tertiary group. butyloxycarbonyl.

 The invention also relates to the use of cationic polymers and copolymers as defined above for the vectorization of natural or synthetic biomolecules which are preferably negatively charged, in particular of biological material, in particular of genetic material, such as DNA, RNA and / or fragments thereof (anti-sense oligonucleotides, aptamers, "small interfering" RNA, etc.); of proteins, natural or synthetic peptides.

 By "biomolecule" is meant here generally any molecule having a prophylactic or therapeutic biological activity, especially an anti-infectious agent, in particular an antiseptic agent, antibiotic, antiviral, antiparasitic or antimitotic, especially anticancer.

 The invention also relates to complexes formed from said cationic polymers or copolymers according to the invention and to said biomolecules, in particular DNA, RNA and / or their corresponding fragments.

 Said complexes are obtained by contacting said cationic polymers or copolymers and said biomolecules in solution.

 The examples below illustrate, without limitation, the invention.

AIBN : a,a'-Azoisobutyronitrile BCA : Bicinchoninic Acid Solution

t-Boc : tertioutyloxycarbonyle DCC : dicyclohexylcarbodiimide DMAP : 4-diméthylaminopyridine In these examples, the following abbreviations are used:

AIBN: α, α'-Azoisobutyronitrile BCA: Bicinchoninic Acid Solution

t-Boc: tert-butyloxycarbonyl DCC: dicyclohexylcarbodiimide DMAP: 4-dimethylaminopyridine

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MTT : (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;
Thiazolyl blue)
Mw : masse molaire moyenne en poids n.d : non déterminé
Réf. : référence
THF : tétrahydrofurane. DMSO: dimethylsulfoxide OD: optical density ELISA: Enzyme-Linked Immunosorbent Assay HEMA: hydroxyethyl methacrylate Ip: polymolecularity index LB: Luria Bertani Me: methyl

MTT: (3- [4,5-Dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide;
Thiazolyl blue)
Mw: average molar mass in weight nd: not determined
Ref. : reference
THF: tetrahydrofuran.

1 - Synthesis of Monomers Comprising a Protected Amine Function
The amino acid derivative (Neosystem) and the other reagents (Aldrich, Merck, Fluka) were used as they were unless specified to indicate their purification mode. The solvents were used without prior purification except where expressly meant.

 The monomers are characterized by thin layer chromatography (TLC) performed with plates coated with 60F254 silica gel as a stationary phase.

 The tlc plates were successively exposed under UV illumination at a wavelength of 254 nm and then heated to 100 ° C. after spraying with a 1% ninhydrin ethyl solution. Depending on the case, the purifications of the monomers were carried out by precipitation, crystallization or flash chromatography.

 The analytical purity of the prepared compounds was controlled by obtaining a single spot in TLC for two different eluent systems.

The following eluent systems were used: A (ethyl acetate / petroleum ether: 1/1 (v / v)) and B (methanol / dichloromethane: 5/95 (v / v)).

 The characterization was carried out by spectroscopic analysis (proton NMR at 250 MHz) and the NMR data are expressed as follows: chemical shift (8) given in ppm; s singlet; d doublet; t triplet;

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Exemple 1 : tert-butyloxyca rbonylami noéthyl méthacrylate (t-Boc-AEMA)

Composé de formule (I) dans laquelle R1 = CH3 ; n = 0 ; X = -C(O)-O- ; Sp = CH2 ; Y n'existe pas ; R2 = CH2-NH-t-Boc a/ Synthèse du précurseur tert-butyloxycarbonylaminoéthanol (t-BocNH-CH2-CH2-OH)

q quadruplet; m multiplet; the position (the domain) and the number of protons are specified for each resonance line.

Example 1: tert-butyloxycarbonylamiethylethyl methacrylate (t-Boc-AEMA)

A compound of formula (I) wherein R1 = CH3; n = 0; X = -C (O) -O-; Sp = CH2; It does not exist; R2 = CH2-NH-t-Boc a / Synthesis of the precursor tert-butyloxycarbonylaminoethanol (t-BocNH-CH2-CH2-OH)

Une solution de 10 mmoles de di-tert butyldicarbonate (t-BOC20) dans 30 ml de dioxane est ajoutée sur une période de 2 h à une solution de 30 mmoles d'aminoéthanol dissous dans 30 ml de dioxane. Le mélange réactionnel est laissé sous agitation durant 22 h à température ambiante puis concentré. On ajoute alors 50 ml d'eau et la phase aqueuse est ensuite extraite par 3 x 50 ml de dichlorométhane. La phase organique est séchée sur MgS04, filtrée, puis concentrée sous pression réduite. Le mélange réactionnel est purifié par chromatographie éclair sur gel de silice dans un mélange dichlorométhane/méthanol 95/5 (v/v).

A solution of 10 mmol of di-tert-butyldicarbonate (t-BOC20) in 30 ml of dioxane is added over a period of 2 hours to a solution of 30 mmol of aminoethanol dissolved in 30 ml of dioxane. The reaction mixture is stirred for 22 h at room temperature and then concentrated. 50 ml of water are then added and the aqueous phase is then extracted with 3 × 50 ml of dichloromethane. The organic phase is dried over MgSO4, filtered and then concentrated under reduced pressure. The reaction mixture is purified by flash chromatography on silica gel in a 95/5 (v / v) dichloromethane / methanol mixture.

Yield: 88%; oil ; Rf (A): 0.30, Rf (B): 0.55; 1H-NMR (250 MHz; CDCl3): δ 5.29 (m, 1H, NH); (dd, 2H, J1 = 5.00 Hz, J2 = 11.70 Hz, CH2); 3.52 (m, 1H, OH); 3.17 (dd, 2H, J1 = 5.00 Hz, J2 = 10.50 Hz, CH2); 1.36 (s, 9H, t-Boc).

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b/ Synthèse du monomère onomere Me han a
A une solution de 10 mmoles d'aminoéthanol monoprotégé dissous dans 50 ml de dichlorométhane sont ajoutées 12 mmoles d'acide méthacrylique. Après solubilisation complète, 2 mmoles de DMAP sont ajoutées puis 11 mmoles de DCC dissous dans 20 ml de dichlorométhane sont alors introduites au goutte à goutte dans le mélange réactionnel. Après une nuit sous agitation à température ambiante, le mélange réactionnel est filtré puis la phase organique est lavée successivement par 50 ml d'une solution 1M de KHS04 et par 50 ml d'une solution saturée de NaCI. La phase organique est séchée sur MgS04, filtrée, puis concentrée sous pression réduite. Le mélange réactionnel est purifié par chromatographie éclair sur gel de silice dans un mélange acétate d'éthyle/éther de pétrole 1/4 (v/v).

b / Synthesis of the onomeric monomer Me han a
To a solution of 10 mmol of monoprotected aminoethanol dissolved in 50 ml of dichloromethane are added 12 mmol of methacrylic acid. After complete solubilization, 2 mmol of DMAP are added and then 11 mmol of DCC dissolved in 20 ml of dichloromethane are then introduced dropwise into the reaction mixture. After stirring overnight at room temperature, the reaction mixture is filtered and the organic phase is washed successively with 50 ml of a 1M solution of KHSO 4 and with 50 ml of a saturated solution of NaCl. The organic phase is dried over MgSO4, filtered and then concentrated under reduced pressure. The reaction mixture is purified by flash chromatography on silica gel in ethyl acetate / petroleum ether 1/4 (v / v).

Yield: 74%; solid; Rf (A): 0.71, Rf (B): 0.89; 1H-NMR (250MHz, CDCl3): # 6.10 (s, 1H, CH); 5.57 (s, 1H, CH); 4.81 (m, 1H, NH); 4.18 (t, J = 5.25 Hz, 2H, CH 2); 3.42 (m, 2H, CH 2); 1.42 (s, 9H, t-Boc).

Composé de formule (I) dans laquelle Ri = CH3 ; n = 0 ; = -C(0)-0- ; Sp = - (CH2)2- ; Y = -O-C(O)- ; R2 = -CH(CH3)-NH-t-Boc
A une solution de 10 mmoles de tert-butyloxycarbonylalanine (t-BocAlaOH) dissous dans 50 ml de dichlorométhane sont ajoutées 10 mmoles de HEMA. Après solubilisation complète, 2 mmoles de DMAP sont ajoutées puis EXAMPLE 2 Tetra-Butyloxycarbonylalanine Hydroxyethyl Methacrylate Ester
Boc AlaEMA)

A compound of formula (I) wherein R1 = CH3; n = 0; = -C (O) -O-; Sp = - (CH2) 2-; Y = -OC (O) -; R2 = -CH (CH3) -NH-t-Boc
To a solution of 10 mmol of tert-butyloxycarbonylalanine (t-BocAlaOH) dissolved in 50 ml of dichloromethane is added 10 mmol of HEMA. After complete solubilization, 2 mmoles of DMAP are added then

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 11 mmol of DCC dissolved in 20 ml of dichloromethane are then introduced dropwise into the reaction mixture. After stirring overnight at room temperature, the reaction mixture is filtered and the organic phase is washed successively with 50 ml of a 1M solution of KHSO 4 and with 50 ml of a saturated solution of NaCl. The organic phase is dried over MgSO4, filtered and then concentrated under reduced pressure. The reaction mixture is purified by flash chromatography on silica gel in ethyl acetate / petroleum ether 1/4 (v / v).

Yield: 85%; white solid; Rf (A): 0.70, Rf (B): 0.90; 1 H-NMR (250 MHz, CDCl 3): # 6.07 (m, 1H, CH); (m, 1H, CH); (d, 1H, J = 6.70 Hz, NH); 4.37 (m, 1H, CH?); 4.30 (m, 4H, CH 2); 1.88 (m, 3H, CH 3); 1.38 (s, 9H, t = Boc).

Composé de formule (I) dans laquelle Ri = CH3 ; n = 0 ; X = -C(O)-NH- ; Sp = CH2 ; Y = n'existe pas ; R2 = -CH2-NH-t-Boc

a/ Synthèse du précurseur tert-buylorcarbonyldiaminoéthane (t-Boc- NH-CH2-CH2-NH2)

Example 3 tert-butyloxycarbonylaminoethylmethacrylamide (t-Boc-AEMAA)

A compound of formula (I) wherein R1 = CH3; n = 0; X = -C (O) -NH-; Sp = CH2; Y = does not exist; R2 = -CH2-NH-t-Boc

a / Synthesis of the tert -benzylcarbonylaminoethane precursor (t-Boc-NH-CH 2 -CH 2 -NH 2)

87 mmol of diaminoethane dissolved in 40 ml of dioxane are added dropwise through a dropping funnel to a solution of 11 mmol of di-tert-butyldicarbonate (t-Boc2O) dissolved in 40 ml of dioxane. with stirring. The addition is done over a period of about 45 minutes. The reaction medium is stirred for 15 hours at room temperature. The organic phase is then concentrated under vacuum and taken up in 50 ml of water. The

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 reaction product is extracted from the aqueous phase by 3 successive washes with, each time, 25 ml of dichloromethane. The combined organic phases are then dried over MgSO 4 and concentrated under reduced pressure. The product is dried completely before analysis.

Yield: 72%; solid; Rf (A): 0.11, Rf (B): 0.27; 1H-NMR (250 MHz; CDCl3): δ 4.90 (s, 1H, NH); 3.15 (m, 2H, CH 2); 2.75 (m, 2H, CH 2); 1.45 (s, 9H, t-Boc); 1.25 (s, 2H, NH2) - b / Synthesis of the monomer

To a solution of 13 mmol of tert-butyloxycarbonyl diaminoethane (tBoc-NH-CH 2 -CH 2 -NH 2) dissolved in 30 ml of dichloromethane is added 16 mmol of methacrylic acid. After complete solubilization, 4 mmol of DMAP are added. 16 mmol of DCC dissolved in 30 ml of dichloromethane are then added dropwise to the reaction medium. After stirring overnight at room temperature, the reaction mixture is filtered and the organic phase is washed successively with 50 ml of a 1M solution of KHSO 4 and with 50 ml of a saturated solution of NaCl. The organic phase is dried over MgSO4, filtered and then concentrated under reduced pressure. The crude reaction product is then purified by flash chromatography on silica gel in a 9/1 (v / v) dichloromethane / isopropanol mixture.

Yield: 54%; solid; Rf (A): 0.37, Rf (B): 0.71; 1 H-NMR (250 MHz; CDCl 3): 8.65 (s, 1H, NH); 5.75 (s, 1H, CH); 5.30 (m, 1H, J = 5.30 Hz, CH); 4.90 (s, 1H, NH); (m, 4H, CH 2); 1.95 (m, 3H, CH 3); 1.45 (s, 9H, t-Boc).

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II - Synthesis of Monomers Containing Two Protected Amine Functions
The purification of the monomers and the methods of characterization of the latter are carried out under the conditions mentioned in point I above for the monomers comprising a single amine function.

Composé de formule (I) dans laquelle R1 = CH3 ; n = 0 ; X = -C(O)-O- ; Sp n'existe pas ; Y n'existe pas ; R2 = -CH(CH2-NH-t-Boc)2

a/ Synthèse du précurseur 1,3-di-tertbuyloxycarbonylamino 2- hydroxypropane

Example 4a: 1,3-di-tert-butyloxycarbonylamino-2-hydroxypropane methacrylate (di-t-Boc-APMA)

A compound of formula (I) wherein R1 = CH3; n = 0; X = -C (O) -O-; Sp does not exist; It does not exist; R2 = -CH (CH2-NH-t-Boc) 2

a / Synthesis of the precursor 1,3-di-tertbuyloxycarbonylamino 2-hydroxypropane

solution sont additionnées goutte à goutte 80 mmoles de di-teri-butyldicarbonate (t-Boc2O) solubilisées dans 50 ml de dioxane. Après une nuit sous agitation à température ambiante, le mélange réactionnel est évaporé sous pression réduite puis extrait par 3 x 50 ml de dichlorométhane. La phase organique est séchée sur MgS04, filtrée et concentrée sous pression réduite. Le mélange réactionnel est purifié par chromatographie éclair sur gel de silice dans un mélange acétate d'éthyle/éther de pétrole 1/1 (v/v). 27 mmol of 1,3-diamino-2-hydroxypropane are dissolved in a mixture consisting of 50 ml of dioxane and 30 ml of water. At this

80 mmol of di-tert-butyldicarbonate (t-Boc 2 O) solubilized in 50 ml of dioxane are added dropwise. After stirring overnight at room temperature, the reaction mixture is evaporated under reduced pressure and then extracted with dichloromethane (3 × 50 ml). The organic phase is dried over MgSO4, filtered and concentrated under reduced pressure. The reaction mixture is purified by flash chromatography on silica gel in ethyl acetate / petroleum ether 1/1 (v / v).

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bl Synthèse du monomère }--o HN )v \O >-<0 HNrl- /-0
A une solution de 10 mmoles de 1,3-di-tert-butyloxycarbonylamino 2hydroxypropane dissous dans 50 ml de dichlorométhane sont ajoutées 10 mmoles d'acide méthacrylique. Après solubilisation complète, 2 mmoles de DMAP sont ajoutées et 11 mmoles de DCC dissous dans 30 ml de dichlorométhane sont alors introduites au goutte à goutte dans le mélange réactionnel. Après une nuit sous agitation, le mélange réactionnel est filtré, puis la phase organique est lavée successivement par 50 ml d'une solution 1M de KHS04 et par 50 ml d'une solution saturée de NaCI. La phase organique est séchée sur MgS04, filtrée, puis concentrée sous pression réduite. Le mélange réactionnel est purifié par chromatographie éclair sur gel de silice dans un mélange acétate d'éthyle/éther de pétrole 3/7 (v/v). Yield: 78%; solid; Rf (A): 0.50, Rf (B): 0.50; 1H-NMR (250 MHz; CDCl3): # 5.10 (m, 2H, NH); (m, 1H, CH); (m, 1H, OH); (m, 4H, CH 2); 1.43 (s, 9H, t-Boc).

bl Synthesis of the monomer} - o HN) v \ O> - <0 HNrl- / -0
To a solution of 10 mmol of 1,3-di-tert-butyloxycarbonylamino-2-hydroxypropane dissolved in 50 ml of dichloromethane is added 10 mmol of methacrylic acid. After complete solubilization, 2 mmol of DMAP are added and 11 mmol of DCC dissolved in 30 ml of dichloromethane are then introduced dropwise into the reaction mixture. After stirring overnight, the reaction mixture is filtered and the organic phase is washed successively with 50 ml of a 1M solution of KHSO 4 and with 50 ml of a saturated solution of NaCl. The organic phase is dried over MgSO4, filtered and then concentrated under reduced pressure. The reaction mixture is purified by flash chromatography on silica gel in a mixture of ethyl acetate / petroleum ether 3/7 (v / v).

Yield: 90%; solid; Rf (A): 0.47, Rf (B): 0.61; 1H-NMR (250 MHz; CDCl3): δ 6.23 (m, 1H, CH); 5.82 (m, 1H, CH); 5.00 (m, 2H, NH); (t, 1H, J = 5.20 Hz, CH); (m, 4H, CH 2); 1.93 (s, 3H, CH3); 1.30 (s, 18H, t-Boc).

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Exemple 4b : Acrylate de 1,3-di-fe/t-butyloxycarbonylamino 2-hydroxypropane (di-t-Boc-APA)

Composé de formule (I) dans laquelle R1 = H ; n = 0 ; X = -C(0)-0- ; Sp n'existe pas ; Y n'existe pas ; R2 = -CH(CH2-NH-t-Boc)2

a/ Synthèse du précurseur 13-di-terbutrfoxycarbony(amino 2- hydroxypropane

Cette synthèse est identique à celle décrite dans l'exemple 4a ci-dessus. b/ Synthèse du monomère

Example 4b 1,3-Di-Fe / t-Butyloxycarbonylamino-2-Hydroxypropane Acrylate (di-t-Boc-APA)

A compound of formula (I) wherein R1 = H; n = 0; X = -C (O) -O-; Sp does not exist; It does not exist; R2 = -CH (CH2-NH-t-Boc) 2

a / Synthesis of the precursor 13-di-terbutrfoxycarbonyl (amino-2-hydroxypropane

This synthesis is identical to that described in Example 4a above. b / Synthesis of the monomer

To a solution of 10 mmol of 1,3-di-tert-butyloxycarbonylamino-2-hydroxypropane dissolved in 50 ml of dichloromethane is added 10 mmol of acrylic acid. After complete solubilization, 2 mmol of DMAP are then added and then 11 mmol of DCC dissolved in 30 ml of dichloromethane are introduced dropwise into the reaction mixture.

After stirring overnight, the reaction mixture is filtered and the organic phase is washed successively with 50 ml of a 1M solution of KHSO4 and with 50 ml of a saturated solution of NaCl. The organic phase is dried over MgSO 4,

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 filtered, then concentrated under reduced pressure. The reaction mixture is purified by flash chromatography on silica gel in a mixture of ethyl acetate / petroleum ether 3/7 (v / v).

Yield: 90%; solid; Rf (A): 0.71, Rf (B): 0.86; 1 H-NMR (250 MHz; CDCl 3): # 6.33 (m, 1H, CH); 6.02 (m, 1H, CH); 5.72 (m, 1H, CH); 5.00 (m, 2H, NH); 4.84 (t, 1H, J = 5.20 Hz, CH); (m, 4H, CH 2); 1.30 (s, 18H, t-Boc).

Composé de formule (I) dans laquelle Ri = CH3 ; n = 0 ; X = -C(O)-O- ;

Sp = (CH2)2 ; Y = -O-C(O)- ; R2 = CH(NH-t Boc)-(CHZ)3-NH-t Boc

a/ Synthèse du précurseur Adt-/butyfoxycarbonytomithtne o o ho # y- nh y # o V- (CH2)3 / ni 7(o-ç
20 mmoles de chlorhydrate de L(+)-Ornithine sont mises en solution dans un mélange constitué de 40 ml de dioxane, 20 ml d'eau et 20 ml d'une solution molaire de soude. La solution est placée sous agitation à 0 C et 44 mmoles de di-tert-butyldicarbonate (t-BOC20) sont ajoutées. Après 30 min, le mélange réactionnel est évaporé sous pression réduite puis est repris par 50 ml d'éther éthylique et par 50 ml d'eau. Après lavage de la phase aqueuse, 50 ml d'acétate d'éthyle sont ajoutés, puis le mélange est acidifié à l'aide d'une solution EXAMPLE 5 Adi-6-butyloxycarbonyl ornithine hydroxyethyl methacrylate ester (t Boc-Orn (t Boc) EMA)

A compound of formula (I) wherein R1 = CH3; n = 0; X = -C (O) -O-;

Sp = (CH2) 2; Y = -OC (O) -; R2 = CH (NH-t Boc) - (CH2) 3-NH-t Boc

a / Synthesis of the precursor Adt- / butyfoxycarbonyltomithene oo ho # y-nh y # o V- (CH2) 3 / ni 7 (o-ç
20 mmol of L (+) - Ornithine hydrochloride are dissolved in a mixture consisting of 40 ml of dioxane, 20 ml of water and 20 ml of a molar solution of sodium hydroxide. The solution is stirred at 0 ° C. and 44 mmol of di-tert-butyldicarbonate (t-BOC20) are added. After 30 min, the reaction mixture is evaporated under reduced pressure and then taken up in 50 ml of ethyl ether and 50 ml of water. After washing the aqueous phase, 50 ml of ethyl acetate are added and the mixture is acidified with a solution of

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 1M KHSO4 to pH = 2-3. The organic phase is then isolated and led, after drying over MgSO 4, filtration and evaporation, to obtain a solid which is characterized in the following eluent system: chloroform / methanol / acetic acid in the proportions 85/10 / 5 (v / v / v).

b/ Synthèse du monomère 0 Me 0 >-O =<M' r -t:}-o =\~ NH A" O (CH2)3 // \\a 0 NH 7( -{H \\0

A une solution de 10 mmoles de di-tertutyloxycarbonylornithine (t- Boc-Orn (Boc)OH) dissous dans 50 ml de dichlorométhane sont ajoutées 10 mmoles d'hydroxyéthylméthacrylate. Après solubilisation complète, 2 mmoles de DMAP sont ajoutées puis 11 mmoles de DCC dissous dans 50 ml de dichlorométhane sont introduites au goutte à goutte dans le mélange réactionnel. Yield: 20%; solid; Rf (A): 0.32, Rf (B): 0.44; 1H-NMR (250MHz, CDCl3): # 7.88 (s, 1H, OH); 5.26 (d, 1H, NH); 4.82 (d, 1H, NH); 4.11 (m, 1H, CH); 3.11 (m, 2H, CH 2); 1.68 (m, 2H, CH 2); 1.50 (m, 2H, CH 2); 1.42 (s, 18H, CH3).

b / Synthesis of the monomer: ## STR1 ##

10 mmol of hydroxyethyl methacrylate are added to a solution of 10 mmol of di-tert-butyloxycarbonylornithine (t-Boc-Orn (Boc) OH) dissolved in 50 ml of dichloromethane. After complete solubilization, 2 mmol of DMAP are added and then 11 mmol of DCC dissolved in 50 ml of dichloromethane are introduced dropwise into the reaction mixture.

After stirring overnight, the reaction mixture is filtered and the organic phase is washed successively with 150 ml of a 1M solution of KHSO 4 and with 50 ml of saturated NaCl solution. The organic phase is dried over MgSO4, filtered and then concentrated under reduced pressure. The reaction mixture is purified by flash chromatography on silica gel in a mixture of ethyl acetate / petroleum ether 3/7 (v / v).

Yield: 73%; oil ; Rf (A): 0.74, Rf (B): 0.91; 1H-NMR (250 MHz; CDCl3): δ6.12 (m, 1H, CH); 5.59 (m, 1H, CH); 5.05 (d, 1H, J = 6.50 Hz, NH); 4.58 (m, 1H, NH); 4.45 - 4.26 (m, 4H, CH 2); 3.86 (m, 1H, CH?); 3.12 (m, 2H, CH 2); 1.86 - 1.45 (m, 6H, CH 2); 1.42 (s, 18H, t-Boc).

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Exemple 6 : Vinyl acétate de 1,3 di-tertbutyloxycarbonylamino 2- hydroxypropane

Composé de formule (I) dans laquelle Rl = H ; n = 1 ; X = -C(O)-O- ; Sp n'existe pas ; Y n'existe pas ; R2 = -CH(CH2-NH-t-Boc)2

a/ Synthèse du précurseur 1,3-di-tert-butyloxycarbonylamino 2- hvdroxypropane

Cette synthèse est décrite dans l'exemple 4a.

b/ Synthèse du monomère 0)-0 HN /V O HN 0 HN /-0

Example 6 Vinyl acetate of 1,3 di-tertbutyloxycarbonylamino-2-hydroxypropane

A compound of formula (I) wherein R1 = H; n = 1; X = -C (O) -O-; Sp does not exist; It does not exist; R2 = -CH (CH2-NH-t-Boc) 2

a / Synthesis of the precursor 1,3-di-tert-butyloxycarbonylamino-2-hydroxypropane

This synthesis is described in Example 4a.

b / Synthesis of the monomer 0) -0 HN / VO HN 0 HN / -0

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A une solution de 10 mmoles de 1,3-di-tert=butyloxycarbonylamino 2- hydroxypropane dissous dans 50 ml de dichlorométhane sont ajoutées 10 mmoles d'acide acétique vinylique. Après solubilisation complète, 2 mmoles de DMAP sont ajoutées puis 11 mmoles de DCC dissous dans 50 ml de dichlorométhane sont introduites au goutte à goutte dans le mélange réactionnel.

To a solution of 10 mmol of 1,3-di-tert-butyloxycarbonylamino-2-hydroxypropane dissolved in 50 ml of dichloromethane is added 10 mmol of vinyl acetic acid. After complete solubilization, 2 mmol of DMAP are added and then 11 mmol of DCC dissolved in 50 ml of dichloromethane are introduced dropwise into the reaction mixture.

After stirring overnight, the reaction mixture is filtered and the organic phase is washed successively with 150 ml of a 1M solution of KHSO 4 and with 50 ml of a saturated solution of NaCl. The organic phase is dried over MgSO4, filtered and then concentrated under reduced pressure. The reaction mixture is purified by flash chromatography on silica gel in ethyl acetate / petroleum ether 4/6 (v / v).

Yield: 65%; solid; Rf (A): 0.80, Rf (B): 0.94; 1H-NMR (250MHz; CDCl3) δ5.90 (m, 1H, CH); 5.14 (m, 2H, CH 2); 5.03 (m, 2H, NH); 4.82 (t, 1H, J = 5.00 Hz, CH); (m, 4H, CH 2); 3.06 (m, 2H, CH 2); 1.41 (s, 18H, Boc).

III - Preparation of cationic (co) polymers
The (co) polymers are characterized by: proton NMR (1H-NMR)
An NMR analysis before deprotection in CDCl3 makes it possible to observe the presence of a characteristic peak of the t-Boc protecting group at 8 = 1.45 (s, 9H, 3CH 3) as well as the possible presence of residual monomer (s) ( s) (no peak corresponding to the protons of the double bond at around 6 ppm). After deprotection, the disappearance of the characteristic singlet of the t-Boc protecting group is observed in D20, demonstrating that the deprotection step is complete.

 Assuming that copolymers are prepared from several amino monomers, NMR analysis gives access to the average composition of the polymer chains. The final average composition of the copolymer can then be given by the ratio between the intensity of the characteristic peaks of each of the comonomers.

- Size exclusion chromatography (SEC)
This analysis technique carried out on the (co) polymers soluble in THF, that is to say before their deprotection, generally highlights the

<Desc / Clms Page number 18>

 the presence of a monomodal peak having, however, a fairly wide distribution in molar mass, but sometimes also the presence of residual oligomers which can be removed by dialysis or fractionation. The molar masses indicated in the following examples were determined using a refractometric detector and a calibration made from a polystyrene range of low polymolecularity index.

 The (co) polymers according to the invention were prepared by radical (co) polymerization.

 Radical (co) polymerizations were carried out in test tubes ("batch" type process). The temperature of the tubes is regulated by means of a thermostatically controlled heating bath and the stirring is of magnetic type. After introducing into the tube the monomer (s) (generally between 4 and 6 mmol) and the polymerization solvent, most often bidistilled THF, the reaction mixture is degassed by bubbling nitrogen, with vigorous stirring. for 5 min. The contents of the tube are then gradually brought to the decomposition temperature of the initiator and the latter is added rapidly, most often in the form of a solution in the polymerization solvent. The polymerization is continued for 15 to 24 hours and the solvent is then evaporated off under reduced pressure. The (co) polymer is precipitated in an aqueous phase to remove residual monomer (s) possibly present.

 Hydrochloric acid gas was chosen both as a deprotection agent allowing the removal of the protective group t-Boc and as protonating agent of the amine function thus released. It is obtained by adding a solution of hydrochloric acid (12M) on concentrated sulfuric acid. The (co) polymers containing the protected amine functions are solubilized in ethyl acetate (30 ml) and are placed under a current of HCl gas.

After a few minutes, the copolymers precipitate in ethyl acetate. This phenomenon is characteristic of the loss of the t-Boc groups which until now provided the (co) polymers their solubility in most organic solvents (THF, ethers, etc.). The final product is then isolated by filtration, solubilized in a small volume of distilled water (15 ml) and then lyophilized.

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Example 7 Preparation of Polymer P (HCl, AEMA)
The procedure described above is used starting from the monomer of Example 1.

Table 1

<Tb>
<tb> Reference <SEP> Mass <SEP> total <SEP> of the <SEP>% <SEP> mass <SEP> Mw <SEP> (g / mol)
<tb> monomer <SEP> (g) <SEP> of initiator <SEP> (Ip)
<tb> 72. <SEP> 000
<tb> 7a <SEP> 0.5 <SEP> 1.0 <SEP> (2,6)
<Tb>

The polymerization is conducted in THF using cyclohexyl percarbonate as an initiator at 40 ° C. for 24 hours. The monomer is dissolved in 3 ml of solvent and the initiator is introduced into the polymerization medium as a solution in 1 ml of solvent.

Example 8 Preparation of P (HCI, AlaEMA)
The procedure described above is used starting from the monomer of Example 2.

Table 2

<Tb>
<tb> Reference <SEP> Mass <SEP> total <SEP> of the <SEP>% <SEP> mass <SEP> Mw <SEP> (g / mol)
<tb> monomer <SEP> (g) <SEP> of initiator <SEP> (Ip)
<tb> 32. <SEP> 600
<tb> 8a <SEP> 1.0 <SEP> 1.0 <SEP> (3.9)
<Tb>

The polymerization is carried out in THF using cyclohexyl percarbonate as an initiator at 40 ° C. for 24 hours. The monomer is dissolved in 3 ml of THF and the initiator is introduced into the polymerization medium in the form of a 1 ml solution of THF.

Example 9: Preparation of P (HCI, AEMAA)
The procedure described above is used starting from the monomer of Example 3.

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Table 3

<Tb>
<tb> Reference <SEP> Mass <SEP> total <SEP> of the <SEP>% <SEP> mass <SEP> Mw <SEP> (g / mol)
<tb> monomer <SEP> (g) <SEP> of initiator <SEP> (Ip)
<tb> 8. <SEP> 100
<tb> 9a <SEP> 1.3 <SEP> 1.0 <SEP> (2.5)
<Tb>

The polymerization is carried out in THF using AIBN as initiator, at 60 ° C., for 24 hours. The monomer is dissolved in 3 ml of THF and the initiator is introduced into the polymerization medium in the form of a 1 ml solution of THF.

Example 10 Preparation of P (HCI, diAPMA)
The procedure described above is used starting from the monomer of Example 4a.

Table 4

<Tb>
<tb> Reference <SEP> Mass <SEP> total <SEP> of the <SEP>% <SEP> mass <SEP> Mw <SEP> (g / mol)
<tb> monomer <SEP> (g) <SEP> of initiator <SEP> (Ip)
<tb> 43. <SEP> 000
<tb> 10a <SEP> 1.0 <SEP> 1.0 <SEP> (3,3)
<Tb>

The polymerization is carried out in THF using cyclohexyl percarbonate as an initiator at 40 ° C. for 24 hours. The monomer is dissolved in 3 ml of THF and the initiator is introduced into the polymerization medium in the form of a 1 ml solution of THF.

Example 11 Preparation of P (HCI, OrnEMA)
The procedure described above is used starting from the monomer of Example 5.

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Table 5

<Tb>
<tb> Reference <SEP> Mass <SEP> total <SEP> of the <SEP>% <SEP> mass <SEP> Mw <SEP> (g / mol)
<tb> monomer <SEP> (g) <SEP> of initiator <SEP> (Ip)
<tb> 0.5 <SEP> 1.0 <SEP> 27,800
<tb> 11a <SEP> 0.5 <SEP> 1.0 <SEP> (3.1)
<Tb>

The polymerization is carried out in THF using AIBN as initiator, at 60 ° C., for 20 hours. The monomer is dissolved in 3 ml of THF and the initiator is introduced into the polymerization medium in the form of a 1 ml solution of THF.

Example 12: Preparation of P (HCl, OrnEMA-r-HCl, AEMA)
The procedure described above is used starting from the monomers of Examples 1 and 5.

Table 6

<Tb>
<tb>% <SEP> molar
<tb>, <SEP>, <SEP> Mass <SEP> total <SEP> of <SEP>% <SEP> mass <SEP> Mw <SEP> (g / mol)
<tb> Reference <SEP> HCI, OrnEMA /
<tb> monomers <SEP> (g) <SEP> HCI, <SEP> AEMA <SEP> of initiator <SEP> (Ip)
<tb> 48,900
<tb> 12a <SEP> 0.7 <SEP> 20/80 <SEP> 0.5
<tb> (2,4)
<tb> 42. <SEP> 900
<tb> 12b <SEP> 0.7 <SEP> 50/50 <SEP> 0.5 <SEP> (2.4)
<tb> 36. <SEP> 900
<tb> 12c <SEP> 0.7 <SEP> 80/20 <SEP> 0.5
<Tb>

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~')~~~
La polymérisation est conduite dans le THF en utilisant l'AIBN comme amorceur, à 60 C, pendant 20 h. Les monomères sont dissous dans 1 ml de THF et l'amorceur est introduit dans le milieu de polymérisation sous la forme d'une solution de 1 ml de THF.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ') ~~~
The polymerization is carried out in THF using AIBN as initiator, at 60 ° C., for 20 hours. The monomers are dissolved in 1 ml of THF and the initiator is introduced into the polymerization medium in the form of a solution of 1 ml of THF.

Example 13 Preparation of P (HCl, OrnEMA-r-HCl, AEMAA)
The procedure described above is used starting from the monomers of Examples 3 and 5.

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Table 7

<Tb>
<tb> Mass <SEP> total <SEP> of <SEP>% <SEP> molar <SEP>% <SEP> mass <SEP> Mw <SEP> (g / mol)
<tb> Reference <SEP> HCI, OrnEMA /
<tb> monomers <SEP> (g) <SEP> $ HCl, AEMAA <SEP> of initiator <SEP> (Ip)
<tb> 77. <SEP> 800
<tb> 12a <SEP> 0.7 <SEP> 20/80 <SEP> 0.5
<tb> (3,5)
<tb> 66. <SEP> 400
<tb> 12b <SEP> 0.7 <SEP> 50/50 <SEP> 0.5
<Tb>

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\,-3j~~~

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \ - 3d ~~~

<Tb>
<tb> 54. <SEP> 800
<tb> 12c <SEP> 0.7 <SEP> 80/20 <SEP> 0.5
<tb> (2,7)
<Tb>

The polymerization is carried out in THF using AIBN as initiator, at 60 ° C., for 20 hours. The monomers are dissolved in 1 ml of THF and the initiator is introduced into the polymerization medium in the form of a solution of 1 ml of THF.

Example 14: Preparation of P (HCl, AlaEMA-r-HCl, AEMA)
The procedure described above is used starting from the monomers of Examples 1 and 2.

Table 8

<Tb>
<tb>% <SEP> molar
<tb> Mass <SEP> total <SEP> of <SEP>% <SEP> mass <SEP> Mw <SEP> (g / mol)
<tb> Reference <SEP> HCl, AlaEMA /
<tb> monomers <SEP> (g) <SEP> HCI <SEP> AEMA <SEP> d <SEP> initiator <SEP> (Ip)
<tb> 55. <SEP> 500
<tb> 12a <SEP> 0.7 <SEP> 20/80 <SEP> 0.5
<tb> 68. <SEP> 300
<tb> 12b <SEP> 0.7 <SEP> 50/50 <SEP> 0.5 <SEP> (2.6)
<tb> 72. <SEP> 600
<tb> 12c <SEP> 0.7 <SEP> 80/20 <SEP> 0.5 <SEP> (2.8)
<Tb>

The polymerization is carried out in THF using AIBN as initiator, at 60 ° C., for 20 hours. The monomers are dissolved in 1 ml of THF and the initiator is introduced into the polymerization medium in the form of a solution of 1 ml of THF.

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IV - In Vitro Transfections in the Presence of Cationic Polymers
Several cationic polymers according to the invention have been tested as transfection agents. The tests were carried out by transfection of BHK-21 cells (fibroblasts) and / or SRDC (murine splenic dendritic cell line whose production is detailed in paragraph b / below) with the plasmid pCMV-Luc (7, 1 kb), containing the gene coding for the luciferase protein.

The transfection experiments are carried out with varying N / P molar ratio complexes (N, nitrogen from the copolymer and P, phosphate from DNA). For all these reports, the estimation of the transfection efficiency is directly performed by measuring the luciferase activity. An estimate of cell viability is jointly conducted. a / Production of plasmid pCMV-Luc
The plasmid pCMV-Luc, not commercially available, required a molecular construction step. Thus, it was obtained by insertion of the Luc gene into the plasmid pCMV (Invitrogen), according to a protocol conventionally used in the literature (J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, 1987, Chapter 1). . In practice, the ligation of the vector and the insert was first carried out, then competent bacteria were prepared and the transformation was carried out. the bacteria were subjected to a thermal shock).

 E.coli DH5a recombinant bacteria clones, carrying the plasmid pCMV-Luc, resistant to ampicillin (previously spread on Petri dishes containing LB agar medium and ampicillin) are incubated for approximately 8 h at 37 ° C. with shaking. in LB medium containing ampicillin. This preculture is then incubated overnight at 37 ° C. with stirring under the same conditions as those described previously. After centrifugation of the cultures, the bacterial pellets containing the plasmids are treated according to the protocol described by Qiagen, supplier of the ion exchange columns (EndoFree Plasmid Giga, Qiagen GmbH, Germany) necessary for the purification of the plasmids.

 The integrity of the plasmid pCMV-Luc produced was analyzed on a 1% agarose gel (1X Tris-Acetate-EDTA) before and after digestion with the restriction enzyme EcoRI (linearization of the plasmids). The quality of the DNA (protein contamination ...) is analyzed by the evaluation of the D026o / D028o ratio and their quantity is also determined by UV spectrophotometry (260 nm).

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b / Cell culture bl / Lineage BHK-21
The line BHK-21, derived from Syrian hamster kidney (ATCC, CCL-10) is cultured in BHK-21 medium (Gibco BRL) containing 2% tryptose phosphate, 1% penicillin / streptomycin and 5% serum. of fetal calf (FCV).

24 h before the transfection experiments, the cells are distributed in wells of 24-well plates under 1 ml of culture medium, at a rate of 7.5 × 10 4 cells / well. The cells are cultured at 37 C in an oven at 5% CO2 up to 60-70% confluence. b.2 / SRDC dendritic cell line
Dendritic cells are obtained from female rats of 8 weeks old CBA / J (H2k) mice. They are purified and cultured according to the procedure described by Iwasaki and Kelsall (J. Exp. Med., 190 (2), 229-239, 1999) applied to dendritic cells of Peyer's patches.

 After 1 month of culture, some cells representing a very small cell proportion survive and begin to proliferate.

(Gibco BRL), 50 pm 3-mercaptoéthanol (SIGMA), 100 unités/ml pénicilline et 100 pg/ml de streptomycine (SIGMA). These spontaneously immortalized cells are cultured in complete IMDM medium: IMDM (Gibco BRL) supplemented with 5% fetal calf serum decomplemented and filtered (0.22 m porosity filter), 2 mM L-glutamine

(Gibco BRL), 50 μM 3-mercaptoethanol (SIGMA), 100 units / ml penicillin and 100 μg / ml streptomycin (SIGMA).

 These immortalized cells are incubated at 4 ° C. for 30 min with magnetic beads coated with anti-CD11c antibody (clone N418) (MILTENYL BIOTECH), a marker specific for dendritic cells. The cells expressing the CD11c antigen at the surface are selected on MACS columns (MILTENYL BIOTECH) using magnets and cultured in complete IMDM medium, and cloned by two successive dilutions at 0.1 cell / well in 96-well culture plates (CORNING). Under these conditions, 15 clones were obtained. The clones were analyzed by flow cytometry to verify the expression of the CD11c marker and to look for the expression of other markers, such as Mac-1, Mac-3, DEC205 (specific for dendritic cells) ... The results of this analysis resulted in the selection of a clone, hereinafter referred to as SRDC.

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24 h before the transfection experiments, the SRDC cells are distributed in the wells of 6-well plates in 2 ml of culture medium, at a rate of 7.5 × 10 5 cells / well. The cells are cultured at 37 ° C. in an oven containing 5% CO 2. c / Polymer preparation
The various freeze-dried polymers are dissolved in HBS buffer (Hepes Buffered Saline, containing 150 mM NaCl, 20 mM Hepes, pH 7.4) at a concentration of 2 mg / ml. In some cases, a sonication of 3 to 5 min (300 UltraSonik, NEY) may be necessary for complete dissolution. The solutions are then sterilized by filtration on 0.22 m porosity filters. d / Transfection protocol d.1 / BHK-21 cells
The transfection medium is Optimem 1 medium (Gibco BRL). The DNA / cationic polymer complexes are prepared by adding different amounts of polymers to 1 g of plasmid. Depending on the case considered, stock solutions of polymers are diluted in HBS to obtain solutions of appropriate concentrations.

 The complexes are formed by mixing and incubating at room temperature for 30 min, 125 l of polymer solution at different concentrations and 125 l of plasmid solution (20 g / ml) to obtain N / P ratios. variables. Then, 100 l of the complex solution are distributed on the cells cultured the day before and previously rinsed with Optimem 1 medium, and then incubated with 150 l of Optimem 1.

The incubation is continued for 4 h at 37 ° C. (in an oven containing 5% CO 2). The transfection medium is then removed and replaced with 1 ml of complete culture medium. The cells are again cultured for 24 hours. All transfection experiments are performed in 2 identical series on 2 plates of 24 different wells, one allowing the estimation of the expression of the reporter gene, the other being reserved for the determination of the cytotoxic effects of the complexes. On each plate, a cell well is reserved for the control. They are BHK-21 cells that do not undergo transfection, they are only in contact with Optimem 1 medium and complete culture medium.

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d.2 / SRDC cells
The transfection medium is Optimem 1 medium (Gibco BRL). The DNA / cationic polymer complexes are prepared by adding different amounts of polymers to 1.5 g of plasmid pCMV-Luc. Transfection assays with commercial transfection agents Lipofectamine (Gibco BRL) and DOTAP (Boehringer Mannheim) were performed in parallel following the recommendations of the supplier. In the case of DOTAP, the formation of the complex is carried out in HBS medium instead of Optimem 1 medium.

Formation of the DNA / cationic polymer complexes is carried out by mixing and incubating at room temperature for 30 min, 100 l of polymer solution and 100 l of plasmid solution. The mixture (200 l) is distributed on the cells cultured the day before and rinsed with 1 ml of Optimem 1 medium, and then incubated with 800 l of Optimem 1. The incubation is continued for 4 h at 37 ° C. (in an oven containing 5% of 002). The transfection medium is then removed and replaced with 2 ml of complete culture medium. The SRDC cells are cultured under these conditions for 36 h. e / Evaluation of cytotoxicity of cationic polymer / DNA complexes and quantitative determination of luciferase activity
To determine the cytotoxic effect of the cationic polymer / DNA complexes, the number of living cells is evaluated using an MTT colorimetric test (T. Mosmann, J. Immunol Meth, 1983, 65, 55-63). . On the other hand, the GeneLux kit (PerkinElmer, Ref L002-100) is used for the assay of luciferase activity. el / MTT colorimetric test
24 h after transfection, the culture medium is replaced by DMEM medium (without FBS) (500 l per well). 50 l of MTT of concentration 5 mg / ml (in 1X PBS), sterile (filter filtration with a porosity of 0.22 m) are added to each well to reach a final concentration of 0.5 mg MTT / m.
The plate is incubated for 2 h at 37 ° C (5% CO 2). The "MTT medium" is then gently removed and 200 l per well of sterile DMSO (ACROS ORGANICS) are deposited on the cells in order to dissolve the formed formazan crystals. The plate is shaken gently for a few minutes

<Desc / Clms Page number 27>

 to ensure the complete dissolution of the formazan crystals. 50 l per well are taken and deposited in the wells of a 96-well plate. The reading of the OD is then carried out at 540 nm (use of an ELISA plate reader, Wallac VICTOR). Dilution of the samples in DMSO or in distilled water (1/4 or 1/8) is often necessary in order to observe OD values close to 1.

The percentage of living cells is calculated according to the formula:
Live cells (%) = (OD 540 (sample) / OD 540 (control)) X 100 where OD 540 (sample) represents the measurement of cells transfected with the different cationic polymer / DNA complexes and OD 540 (control) represents the measurement untransfected cells having only been in contact with the transfection medium (Optimem 1) and the complete culture medium. The OD read is directly proportional to the number of living cells. e. 2 / Determination of luciferase activity
Case of BHK-21 cells
24 h after transfection, the culture medium is removed. 100 l of lysis buffer (1 X) are added to the cells. After incubation for 5 min at room temperature, 20 μl of sample are transferred to a 96-well black white-bottomed plate (PerkinElmer, Ref 1450-581). In each well, 50 l of luciferin reagent and then 50 l of ATP reagent are added. The emission of emitted light is measured using a luminometer (Wallac VICTOR), the duration of a measurement being fixed at 10 s. The results are expressed in RLU ("Relative Light Unit") for a test portion of 20 l of cell lysate.

 In order to express these results with respect to the amount of protein extracted, a BCA assay (SIGMA) is performed.

Case of SRDC cells
36 hours after transfection, the culture supernatant is removed. The adherent dendritic cells are recovered by scraping, washed in 1 ml of PBS and then centrifuged for 5 min at 400 g. After removal of the supernatants, the cell pellet is taken up in 150 l of lysis buffer supplemented with 150 l of water. After incubation for 15 minutes at room temperature, 20 μl of sample are transferred to a 96-well black white-bottom plate (PerkinElmer, Ref 1450-581). In each well, 50 l of luciferin reagent then 50 l of ATP reagent

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are added. The emission of emitted light is measured using a luminometer (Wallac VICTOR), the duration of a measurement being fixed at 10 s. The results are expressed in RLU for a test portion of 20 l of cell lysate. The results are compared with a negative control consisting of SRDC cells incubated in the presence of 1.5 g of plasmid pCMV-Luc. f / Protein Assay (BCA)
For the determination of the amount of protein present in the lysed BHK-21 cells, previously transfected with various cationic polymer complexes / plasmid pCMV-Luc, the "Bicinchoninic acid protein assay" kit (SIGMA, BCA-1) was used. . Sampling of samples was done according to the supplier's instructions. g / Results gl / BHK-21 cells
For all transfection assays of BHK-21 cells, a positive transfection control is systematically introduced. The latter consists of cells transfected with a PEI / DNA complex, the PEI being the 25 kDa branched polyethyleneimine (Aldrich). This cationic polymer is perfectly described in the literature for its high transfection power (W.

T. Godbey et al., J. Control. Release 1999.60, 149-160; A. Kichler et al., J. Gene Med 2001,3, 135-144). Preliminary transfection experiments had shown that with a N / P ratio of high transfection efficiency was obtained.

The following two tables represent the luciferase activity and the percentage of living cells at different N / P ratios for the AEMA and AEMAA cationic polymers of Examples 7 and 9.

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Table 9

<Tb>
<tb> Agent <SEP> of <SEP> N / P <SEP> Quantity <SEP> of agent <SEP> RLU / mg <SEP> of <SEP> Living <SEP> cells
<tb> transfection <SEP> of <SEP> transfection <SEP> proteins <SEP> (%)
<tb> (g / well)
<tb> 7a <SEP> 1 <SEP> 1.24 <SEP> 2.0.104 <SEP> 100
<tb> 7a <SEP> 2 <SEP> 2.48 <SEP> 6.3.105 <SEP> 100
<tb> 7a <SEP> 3 <SEP> 3.72 <SEP> 8.5.105 <SEP> 100
<tb> 7a <SEP> 4 <SEP> 4.96 <SEP> 4.3.105 <SEP> 100
<tb> 7a <SEP> 5 <SEP> 6.20 <SEP> 3.4.105 <SEP> 100
<tb> 7a <SEP> 6 <SEP> 7.45 <SEP> 5.1.105 <SEP> 86
<tb> PEI <SEP> 10 <SEP> 1.30 <SEP> 3.2.107 <SEP> 55
<Tb>
Table 10

<Tb>
<tb> Agent <SEP> of <SEP> N / P <SEP> Quantity <SEP> of agent <SEP> RLU / mg <SEP> of <SEP> Living <SEP> cells
<tb> transfection <SEP> of <SEP> transfection <SEP> proteins <SEP> (%)
<tb> (g / well)
<tb> 9a <SEP> 1 <SEP> 1.23 <SEP> 1.8.104 <SEP> 100
<tb> 9a <SEP> 2 <SEP> 2.47 <SEP> 2.3.106 <SEP> 100
<tb> 9a <SEP> 3 <SEP> 3.70 <SEP> 2.5.106 <SEP> 100
<tb> 9a <SEP> 4 <SEP> 4.93 <SEP> 3.0.106 <SEP> 100
<tb> 9a <SEP> 5 <SEP> 6 <SEP> 17 <SEP> 8.2.106 <SEP> 80
<tb> 9a <SEP> 6 <SEP> 7.40 <SEP> 1.4.10 '<SEP> 100
<tb> 9a <SEP> 7 <SEP> 8.63 <SEP> 1.8.107 <SEP> 80
<tb> PEI <SEP> 10 <SEP> 1.30 <SEP> 5.6.107 <SEP> 65
<Tb>

In view of the results obtained, it is clear that the cationic polymers according to the invention have properties of transfectants. They indeed make it possible to efficiently transfect BHK-21 cells and moreover are very little toxic towards cells with the most favorable N / P ratios in terms of transfection. On the other hand, the positive control PEI, although being a very good transfection agent, has the disadvantage of being highly toxic towards the cells. At equal toxicity (90 - 100% of living cells), it has been demonstrated (results not reported) that the transfection power of polymers

<Desc / Clms Page number 30>

cationic compounds according to the invention could be up to 100 times higher than that of PEI (in particular for the polymer composed of AEMAA). g.2 / SRDC cells
Table 11 summarizes the transfection conditions implemented.

Table 11

<Tb>
<tb> Agent <SEP> of <SEP><SEP> Agent Quantity <SEP> of
<tb> N / P <SEP> transfection
<tb> transfection
<tb> (g / well)
<tb> 9a <SEP> 2.5 <SEP> 2.5
<tb> 8a <SEP> 45.0 <SEP> 48.0
<tb> Lipofectamine <SEP> 11.3 <SEP> 20.0
<tb> DOTAP <SEP> 1.4 <SEP> 5.0
<Tb>

The transfection results are shown in FIG. 1. They show that, unlike the commercial transfection agents Lipofectamine and DOTAP, the two tested polymers are capable of transfecting dendritic cells, in particular the AEMAA-based polymer 9a.

Claims (8)

Polymer or cationic copolymer, characterized in that it is essentially formed from one or more monomers corresponding to the following formula (I):

 wherein: - Ri is H or a C1-C4 alkyl; - n is zero or one; X is -O-C (O) -, -C (O) -O-, -NH-, -C (O) -NH- or -NH-C (O) -; - Sp is a spacer group such that - (CH2) m- or - (CH2) m -O- (CH2) I-; or does not exist; m and I, identical or different, represent an integer of 1 to 4; Y is -O-C (O) -, -C (O) -O-, -NH-, -C (O) -NH- or -NH-C (O) - or does not exist; - R2 is a linear or branched (C1-C6) alkyl group substituted by at least one primary, secondary or tertiary amine function, protected or not by a protecting group or in protonated form.  2. Polymer or cationic copolymer according to claim 1, characterized in that it is essentially formed from one or more monomers corresponding to the above formula (I) in which: R 1 is H or methyl, preferably methyl; n is zero or one, preferably zero; X is -C (O) -O- or -C (O) -NH-; - Sp is a spacer group selected from -CH2- and - (CH2) 2-, or does not exist; - Y is -O-C (O) - or does not exist; R2 is a group -CH2-NH2, -CH (CH3) -NH2, -CH (CH2-NH2) 2 or CH (NH2) - (CH2) 3 -NH2, whose amine functions are protected or not by a grouping protector such as the tert-butyloxycarbonyl group.  3. Polymer or cationic copolymer according to claim 2, characterized in that it consists essentially of one or more monomers corresponding to one of the following formulas: <Desc / Clms Page number 32>

4. Polymer or cationic copolymer according to one of claims 1 to 3, characterized in that it is essentially formed from one or more monomers of formula (I) corresponding to one of formulas (II), ( III) and (IV)

 following: O Me O / ## Hic) 'H2N 00 O H2N O

5. Use of a polymer or copolymer according to any one of claims 1 to 4 for the vectorization of at least one natural or synthetic biomolecule, preferably negatively charged. <Desc / Clms Page number 33>  6. Use according to claim 5, characterized in that said (said) biomolecule (s) is (are) constituted (s) of genetic material such as DNA, RNA and / or their fragments (oligonucleotides) ), proteins, natural or synthetic peptides.  7. Complex comprising at least one polymer or copolymer according to any one of claims 1 to 4 and at least one natural or synthetic biomolecule, preferably negatively charged.  8. Complex according to claim 7, characterized in that said biomolecule (s) is (are) constituted (s) of genetic material such as DNA, RNA and / or their fragments (oligonucleotides), of proteins, natural or synthetic peptides.