Aqueous synthesis and rapid in situ screening of amphiphilic polymers (Machine-translation by Google

专利摘要:
The present invention relates to a novel screening method for transfection using novel amphiphilic polymers based on acryloyl. (Machine-translation by Google Translate, not legally binding)
公开号:ES2618373A1
申请号:ES201531831
申请日:2015-12-17
公开日:2017-06-21
发明作者:Javier MONTENEGRO GARCÍA；Francisco FERNÁNDEZ-TRILLO
申请人:Universidade de Santiago de Compostela；University of Birmingham；
IPC主号:

专利说明:

  QUICK SYNTHESIS AND QUICK IN-SITE SCREENING OF AMPHIFIED POLYMERS Description Field of the Invention The present invention relates to screening methods using new amphiphilic polymers conjugated to biomolecules such as DNA, RNA or siRNA.  The new methods allow faster and more flexible screening of suitable transfection agents and release of biomolecules of interest.  The application also describes novel transfection agents identified following the screening method.   BACKGROUND OF THE INVENTION The pharmaceutical industry is continuously looking for more and more efficient drug identification methods.  One of the main challenges facing the industry in recent years is to provide compositions or devices capable of overcoming the cell membrane barrier and yielding the active pharmaceutical ingredient in the target cell.  This problem is critical in the case of gene therapy, where relatively large and charged molecules, and therefore lipophobic, such as nucleic acids (DNA, RNA or siRNA), have to overcome the lipophilic cell membrane.  For many years, the 15 researchers have been looking for amphiphilic molecules that can be conjugated with such hydrophobic molecules and at the same time pass through cell membranes.  The first success was obtained with positively charged lipid molecules, such as those described in WO 94/05624 (Invitrogen).  Each lipid has to be synthesized separately and then screened, and therefore they are not suitable for high performance screening.  Recently, the Siegwart group has described a method for the preparation of lipocathionic polyester libraries by ring opening polymerization of valerolactones.  The high efficiency of the polymerization conditions allowed direct screening of the resulting polymers for the transfer of siRNA.  However, polymerization requires the use of specialized equipment, such as a dry box to prevent the presence of moisture, which is detrimental to the polymerization process.  In addition, the resulting polymers have to be combined with various additives, such as (PEGylated lipids, cholesterol and DSCP lipids) in order to obtain efficient vehicles for assignment.  Hao, Jing, Petra Kos, Kejin Zhou, Jason B Miller, Lian Xue, Yunfeng Yan, Hu Xiong, Sussana Elkassih, and Daniel J Siegwart.  “Rapid Synthesis of a Lipocationic Polyester Library via Ring-Opening Polymerization of Functional Valerolactones for Efficacious siRNA Delivery. "J.  A.M.  Chem  Soc.  2015, 137, 9206-9209.  30 In fact, as far as we know, there are no examples in the public domain of technologies for the synthesis and in-situ evaluation of polymeric agents for the release of genetic material.  Anderson et al.  (US 8,557,231, US 8,287,849, US 7,427,394 or J.  J.  Green, G.  T.  Zugates, N.  C.  Tedford, Y.  H.  Huang, L.  G.  Griffith, D.  TO.  Lauffenburger, J.  TO.  Sawicki, R.  Langer, D.  G.  Anderson, Adv.  Mater.  2007, 19, 2836-2842) have developed a remarkable volume of research based on amphiphiles generated by the condensation of 35 diacrylates and amines.  The length of the polymer and the molecular weight distribution of the products obtained are, however, intrinsically different, since each polymer is derived from a "unique" polymerization, which makes systematic screening and the identification of structure-activity relationships difficult. .  Organic solvents are used, so in-situ screening is not possible.  Klibanov et al.  (M.  Thomas, J.  J.  Lu, C.  Zhang, J.  Chen, A.  M.  Klibanov, Pharm.  Beef.  2007, 24, 1564–1571) 40 applied a similar strategy but using PEI (polyethyleneimine).  Again, in-situ screening is not possible since organic solvents are used and the polymers need to be purified before conjugation and screening.  Yu et al.  (L.  Gan, J.  L.  Olson, C.  W.  Ragsdale, L.  Yu, Chem.  Commun.  2008, 573-575; T.  Potta, Z.  Zhen, T.  S.  P.  Grandhi, M.  D.  Christensen, J.  Ramos, C.  M.  Breneman, K.  Rege, Biomaterials 2014, 35, 1977–1988) use disulfonamides instead of acrylates.  Again, organic solvents are used and the polymers must be purified.  Rege et al.  (S.  Barua, A.  Joshi, A.  Banerjee, D.  Matthews, S.  T.  Sharfstein, S.  M.  Cramer, R.  S.  Kane, K.  Rege, Mol.  Pharmaceutics 2008, 6, 86–97) use diepoxides instead of acrylates.  A pseudo in situ screening is possible since net starting materials are used, which are then diluted in the buffer used for the formation of nucleic acid complex (Polyplex).  The molecular weight of the products obtained is, however, difficult to control and each polymer is derived from a "unique" polymerization, which makes systematic screening and identification of structure-activity relationships difficult.  The solubility cannot be easily modulated, and the compounds are synthesized first and then the solubility is checked.  In Merkel et al.  (V.  Nadithe, R.  Liu, B.  TO.  Killinger, S.  Movassaghian, N.  H.  Kim, A.  B.  Moszczynska, K.  S.  Masters, S.  H.  Gellman, O.  M.  Merkel, Mol.  Pharmaceutics 2015, 12, 362–374) a library was prepared by co-polymerization of protected functional monomers.  Even though polymers with similar molecular weight can be synthesized (for example table 1 entries P G2A and G3) the number of monomer units are intrinsically different, depending on the polymerization efficiency of each monomer.  The polymerizations are made using protected monomers and organic solvents, again requiring deprotection and purification.  Schubert et al.  (WO2015 / 048940) they prepared poly (alkene) polymers, which are functionalized with thiols.  The solubility of poly (alkene) under aqueous conditions is limited, compromising their potential for in situ screening.  The functionalization is done in methanol, which is toxic.  Bertozzi R. , C.  et al.  (K.  Godula, C.  R.  Bertozzi, J.  A.M.  Chem  Soc.  2010, 132, 9963-9965) describes poly (acroyl hydrazides) of 174 units, which however are conjugated with reducing (hydrophilic) sugars and thus are not suitable for transfection.  The synthesis also requires the generation of a hydrazide residue using hydrazine, which is a toxic and explosive reagent.  In Matile et al.  (C.  Gehin, J.  Montenegro, E. -K.  Bang, A.  Cajaraville, S.  Takayama, H.  Hirose, S.  Futaki, S.  Matile, H.  Riezman, J.  A.M.  Chem  Soc.  2013, 135, 9295-9298) small amphiphilic molecules containing hydrazone bonds for transfection are described.  Matile's strategy is to fix the cationic fragment to the molecular skeleton and evaluate to this cationic part different hydrophobic groups through hydrazone bonds.  This system is thus limited by the amount of cationic residues that can be incorporated, (two cationic charges and four hydrophobic tails in the examples described), which is essential to increase the stability of the conjugates with polyanionic biomolecules such as DNA, RNA and XNA. (Niidome, T. , Takaji, K. , Urakawa, M. Ohmori, N. , Wada, A. , Hirayama, T. , and Aoyagi, H.  "Chain length of 15 cationic R-helical peptide sufficient for gene delivery into cells" Bioconjugate Chem.  1999, 10, 773-780; Ward, C. M. , Read, M.  L.  and Seymour, L. W “Systemic circulation of poly (L-lysine) / DNA vectors is influenced by polycation molecular weight and type of DNA: differential circulation in mice and rats and the implications for human gene therapy” Blood, 2001, 97, 2221–2229).  There is therefore a need to provide flexible and more efficient methods for screening multivalent polymers suitable for the transfection of cells with active pharmaceutical ingredients.   BRIEF DESCRIPTION OF THE INVENTION The inventors have solved the problems of the previous screening methods for new polymers with potential in the transfection of nucleic acids by providing a novel polymeric scaffolding and demonstrating that said scaffolding can be easily functionalized with lipophilic and cationic moieties. easily affordable to provide amphiphilic polymers.  The arrangement of said amphiphilic polymers (and their precursors) and screening methods will significantly improve the current situation as discussed below.  Thus, a first aspect of the invention is a polymer of formula (I), salts and stereoisomers thereof, R3X1X2OR0R3n (I) where n is the average number of monomer units and is a number equal to or greater than 10; R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl each R3 is independently selected from the group consisting of hydrogen and a C1-C3 alkyl group; X1 is a group that is selected from the group consisting of –N (H) -, -O-, -N (H) -Alkyl- and –O-Alkyl-; X2 is independently selected in each unit of the group consisting of –NH2, -N = C (H) R1 and 40 –N = C (H) R2; where R1 is a lipophilic moiety and R2 is a cationic moiety; and where the percentage of lipophilic moieties present in the polymer with respect to the total number of groups X2 is between 1 and 99%; where the percentage of cationic residues present in the polymer with respect to the total number of groups X2 is between 1 and 99%; and where the sum of the percentage of lipophilic moieties and cationic moieties is between 2 and 100%.  The inventors have confirmed that these polymers are surprisingly efficient in the transfection of 5 pharmaceutical active ingredients that would otherwise be unable to overcome the lipid bilayer.  Such pharmaceutical active ingredients are negatively charged compounds, such as large polymeric biomolecules (for example DNA, RNA or siRNA).  A further aspect of the invention is then a composition comprising the polymer, salts and stereoisomers thereof, of formula (I), and a negatively charged compound.  Due to the amphiphilic nature of the polymer of formula (I), the composition described above can be easily prepared and directly used in screening tests.  Thus another aspect of the invention is a screening method comprising the step of contacting the composition described above and a lipophilic membrane.  The consistency of the amphiphilic polymer conjugates was validated by the reproducibility of all transfection experiments.  The polymer of formula (I) and the composition resulting from its association with negatively charged molecules (eg nucleic acids) can be used in the preparation of medicaments (or pharmaceutical compositions), and thus additional aspects of the invention are: polymer of formula (I), salts and stereoisomers thereof, for use as a medicine.  - The polymer of formula (I), salts and stereoisomers thereof, for use in the transfection of 20 cells.  - The composition for use as a medicine.  - The composition for use in cell transfection.  - Pharmaceutical compositions comprising the composition of the invention.  The inventors have devised a method and reagents that allow the preparation of amphiphilic polymer molecules suitable for transfection of polynucleotides.  All steps can be performed in aqueous medium.  From a novel polymeric scaffolding of easy and reliable preparation it is possible to introduce a great diversity of lipophilic and cationic moieties to modulate the properties of the resulting amphiphilic polymer and, without further purifications, mix it with a pharmaceutical active ingredient of interest, and test in situ the transfection properties of the resulting composition.  It is also possible to store the existence of solutions (stock) of the different intermediate compounds in order to use them at any time.  The result is unprecedented flexibility and efficiency in the screening of molecules, and new methods and precursors for transfection.  Additional aspects of the invention are thus the precursors of the polymer of formula (I) and synthetic methods thereof.  Accordingly, a further aspect of the invention is a process for the preparation of the polymer of formula (I), the salts and stereoisomers thereof, which comprises the step of contacting a polymer of formula (II), salts and stereoisomers thereof R3X1NH2OR0R3n (II) 40 with an aldehyde of formula O = C (H) R1 and an aldehyde of formula O = C (H) R2; where n, X1, R0, R1, R2 and R3 are as defined above.  A further aspect of the invention is a polymer of formula (II), salts and stereoisomers thereof. R3X1NH2OR0R3n (II) where n is the average number of monomer units that is a number between 10 and 150; R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl each R3 is independently selected from the group consisting of hydrogen and a C1-C3 alkyl group; and X1 is a group that is selected from the group consisting of –N (H) -, -O-, -N (H) -Alkyl- and –O-Alkyl-.  A further aspect of the invention is a process for the preparation of the polymer of formula (II), salts and stereoisomers thereof, which comprises the step of contacting a polymer of formula (III), salts and stereoisomers thereof, with an acidic medium R3X1NHOR8R0R3n (III) where n, X1, R0 and R3 are as defined above; and R8 is a labile group in acidic medium.  A further aspect of the invention is thus a polymer of formula (III), salts and stereoisomers thereof R3X1NHOR8R0R3n (III) where 20 n is the average number of monomer units which is a number equal to or greater than 10; R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl each R3 is independently selected from the group consisting of hydrogen and a C1-C3 alkyl group; X1 is a group selected from the group consisting of –N (H) -, -O-, -N (H) -Alkyl- and –O-Alkyl-; 5 and R8 is a labile group in acidic medium.  A further aspect of the invention is a process for the preparation of a polymer of formula (III), salts and stereoisomers thereof, which comprises polymerizing a compound of formula (IV), salts and stereoisomers thereof, preferably, in the presence of a radical initiator 10 R3X1NHOR8R0R3 (IV) where X1 is a group that is selected from the group consisting of -N (H) -, -O-, -N (H) -Alkyl- and -O-Alkyl-; R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl each R3 is independently selected from the group consisting of hydrogen and a C1-C3 alkyl group; and R8 is a labile group in acidic medium.  A further aspect of the invention is a compound of formula (IV), salts and stereisomers thereof, R3X1NHOR8R0R3 (IV) where X1 is a group selected from the group consisting of -N (H) -, -O-, -N (H) -Alkyl- and -O-Alkyl-; R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl each R3 is independently selected from the group consisting of hydrogen and a C1-C3 alkyl group; and R8 is a labile group in acidic medium.   A further aspect of the invention is a process for the preparation of a compound of formula (IV), salts and stereoisomers thereof, which comprises the step of contacting a compound of formula (V), salts and stereoisomers thereof, with a protecting group - R8 or with a compound of the formula –N (H2) -N (H) - R8, -ON (H) - R8, -N (H2) -Alkyl-N (H) - R8 and –O- Alkyl-N (H) - R8 R3X3OR0R3 5 (V) where R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl each R3 is independently selected from group consisting of hydrogen and a C1-C3 alkyl group; and X3 is selected from the group consisting of –OH, halogen, O-Alkyl, -N (H) -N (H2), -ON (H2), -N (H) -Alkyl-N (H2) and –O -Alkyl-N (H2).  The above procedure provides polymers consistent in terms of molecular weight and size.  Thus, the present invention provides amphiphilic molecules with excellent transfection activity by means of a flexible and efficient screening method, where the process, from the polymers of formula (II) to transfection assays, including amphiphilic functionalization, and conjugation and screening can be done in aqueous medium without intermediate purification.  Brief description of the figures Figure 1: Changes in the normalized emission intensity / (t) (Figure 1) and dose-response curve (Figure 20 1B) for the transport of Herring DNA (125 M) in EYPC-LUVs ⊃ HPTS / DPX with increased concentrations of the amphiphilic polymer prepared using 15% benzaldehyde and 85% guanidinium aldehyde of example 5 (GA-5) as ligands.  Amphiphilic polymer (AP) concentrations: 75 µM (), 50 µM (), 37. 5 µM (), 25 µM (), 12. 25 µM (), 6 µM (), 1. 5 µM (), 0. 6 µM (), 0. 06 µM ().  Figure 2: Transfection efficiency in HeLa EGFP at a constant concentration of amphiphilic polymer 25 (12. 25 µM, 15% iso-valeraldehyde and 85% GA-5) and increased concentrations of siEGFP.  Figure 1: Figure 3A: Transfection efficiency in HeLa GFI-EGFP at a constant concentration of siRNA (14 nM) and increased concentrations of amphiphilic polymers (e. g.  15% iso-valeraldehyde and 85% GA-5).  Figure 3B: Transfection efficiency in HeLa EGFP at a constant concentration of siRNA (14 nM) and fixed concentrations of amphiphilic polymers (12. 25 µM) prepared from different percentages of iso-valeraldehyde.  Percentage of GA-5 = 100% - percentage of iso-valeraldehyde.  Figure 4: Cell viability obtained from the decrease in fluorescence and cytotoxicity assay with HeLa-EGFP at constant concentration of siRNA (14 nM) and increase in concentrations of amphiphilic polymers (15% iso-valeraldehyde and 85% GA-5) (Figure 4A), at a fixed concentration of the amphiphilic polymer (12. 25 µM) and with different percentages of iso-valeraldehyde (Percentage of GA-5 = 100% - 35 percent of iso-valeraldehyde) (Figure 4B).  Figure 5: General screening method.  A polymer (1) is mixed with the required amounts of aldehydes (3) of formula O = C (H) R1 and aldehydes (2) of formula O = C (H) R2.  The resulting polymer (4) is then conjugated with a negatively charged compound (5), such as DNA, RNA or siRNA.  The resulting composition (6) is then tested in the transfection of model membranes or cell membranes (7).  Detailed Description of the Invention Definitions Many of the compounds described herein can form salts.  For example, polymers can include nitrogen atoms that can be protonated to form a positive charge, and / or carboxylic acids or thiol moieties that can be deprotonated and have a negative charge, depending on the pH of the medium.   All these variations are readily available to the person skilled in the art in view of the present description, and are part of the invention.  Such salts are preferably pharmaceutically acceptable salts.  Non-limiting examples are halides; sulfates; hydrohalide salts; phosphates; short chain alkane sulfonates; arylsulfonates; salts of mono-, di- or tribasic C1-C20 aliphatic acids that may contain one or more double bonds, an aryl core or other functional groups such as hydroxy, amino or ketone; 5 salts of aromatic acids in which the aromatic core may or may not be substituted with groups such as hydroxyl, short chain alkoxy, amino, mono- or di-alkylamino sulfonamido short chain.  Also included within the scope of the invention are quaternary salts of tertiary nitrogen atoms with haloalkyl or short chain alkyl sulfates, and oxygenated derivatives of tertiary nitrogen atoms, such as N-oxides.  The compounds of the present invention can also form salts with different inorganic acids or bases, such as hydrochloric acid, phosphoric acid or sodium hydroxide, all included within the scope of the present invention.  A "stereoisomer" in the present description refers to compounds formed by the same atoms linked by the same sequence of bonds but having different three-dimensional structures that are not interchangeable.  "Alkyl" refers to a linear or branched hydrocarbon chain consisting of carbon and hydrogen atoms, which does not contain unsaturations, which has the number of carbon atoms indicated in each case, which is attached to the rest of the molecule by a simple link  If a carbon number is not given in a specific case, it is understood to be an alkyl group having between 1 and 12 carbon atoms, preferably between 1 and 6, preferably between 1 and 3 carbon atoms.  Examples of alkyl groups may be methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, or even larger, depending on the size required.  "Cycloalkyl" refers to a saturated carbocyclic ring having the number of carbon atoms indicated in each case.  Suitable cycloalkyl groups include, but are not limited to cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.  "Alkenyl" refers to a linear or branched hydrocarbon chain consisting of carbon atoms and hydrogen, which contains at least one unsaturation, which has the number of carbon atoms indicated in each case, and which binds to the rest of the molecule through a single bond.  Examples of alkenyl groups may be allyl, butenyl (for example, 1-butenyl, 2-butenyl, 3-butenyl), or pentenyl (for example, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl).  "Cycloalkenyl" refers to a carbocyclic ring having the number of carbon atoms indicated in each case, and at least one unsaturation.  Suitable cycloalkenyl groups include, but are not limited to cycloalkenyl groups, such as 1-cyclobutenyl, 2-cyclobutenyl, 1-cyclopentenyl, 2-cyclopentenyl or 3-cyclopentenyl.  "Alkynyl" refers to a linear or branched hydrocarbon chain consisting of carbon atoms and hydrogen, containing at least one carbon-carbon triple bond, conjugated or not, having the number of carbon atoms indicated in each case, and that it is attached to the rest of the molecule by a single bond, such as -CCH, -CH2CCH, -CCCH3, -CH2CCCH3.  "Cycloalkynyl" refers to a carbocyclic ring having the number of carbon atoms indicated in each case, and at least one triple bond.  Suitable cycloalkynyl groups include, but are not limited to cyclooctinyl, cyclononinyl or cyclododecinyl.  "Alkylcarboxy acid" refers to a group having the number of carbon atoms indicated in each case, and comprises (i) an alkyl group attached to the rest of the molecule by a single bond; and (ii) a carboxy derivative selected from esters and amides attached to said alkyl group.  "Aryl" refers to an aromatic hydrocarbon radical having the number of carbon atoms indicated in each case, such as phenyl or naphthyl.  "Aralkyl" refers to an aryl group attached to the rest of the molecule by an alkyl group such as benzyl and phenethyl.  "Heterocyclyl" refers to a stable ring having the number of carbon atoms indicated in each case, consisting of carbon atoms and one to five heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, preferably a ring 4-8 members with one or more heteroatoms, more preferably a 5 or 6 member ring with one or more heteroatoms.  For the purposes of this invention, the heterocycle may be a monocyclic, bicyclic or tricyclic ring system, which may include a fused ring system; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may optionally be quaternized; and the heterocyclyl radical may be partially or completely saturated or aromatic.   Examples of such heterocycles include, but are not limited to azepines, benzimidazole, benzothiazole, isothiazole, imidazole, indole, piperidine, piperazine, purine, quinoline, thiadiazole, tetrahydrofuran.  "Heteroaryl" refers to a heterocyclic group where at least one of the rings is an aromatic ring.  Any of the above mentioned groups may be optionally substituted with 1 to 10 selected groups of halogens, such as fluorine or ether linkages.  5 Amphiphilic polymers and compositions The polymers of formula (I), as previously defined, are conjugated with aldehydes, that is, positively charged modulator (for example GA-5) and different hydrophobic modulators (for example iso-valeraldehyde).  The resulting amphiphilic polymers are combined with negatively charged biomolecules 10 to provide the compositions of the invention, which are suitable as transfection agents and capable of generating a large library of screening candidates in a direct method.  The inventors have also confirmed that the polymers mentioned above and the compositions provide positive results in the transfection of different model membranes.  Different variants of the polymers of formula (I), as defined above, will be readily recognized by one skilled in the art.  For example, a representative example of polymers according to the present description is a polymer of formula (VII), salts and stereoisomers thereof R5R4R3X1X2OR0n (VII) where 20 n is the average number of monomer units that is an equal or greater number that 10; R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl each R3 is independently selected from the group consisting of hydrogen and a C1-C3 alkyl group; R4 is selected from the group consisting of -SH, -S-Alkyl, -O-Alkyl, -OH and -NH2, preferably, -SH, -S-Alkyl, -O-Alkyl; R5 is a C2-C12 alkylcarboxy acid or a C2-C12 alkylcarboxy acid derivative; X1 is a group that is selected from the group consisting of –N (H) -, -O-, -N (H) -Alkyl- and –O-Alkyl-, preferably where X1 is –N (H) -; 30 X2 is independently selected in each unit from the group consisting of –NH2, -N = C (H) R1 and -N = C (H) R2; where R1 is a lipophilic moiety and R2 is a cationic moiety; and where the percentage of lipophilic moieties present in the polymer with respect to the total number of groups X2 is between 1 and 99%; where the percentage of cationic residues present in the polymer with respect to the total number of groups X2 is between 1 and 99%; and where the sum of the percentage of lipophilic moieties and cationic moieties is between 2 and 100%.  The size of the polymers of the present description is not of particular relevance since they maintain their amphiphilic properties.  Regarding this issue, the choice of the inventors of a polymer scaffolding that has multiple groups -X1-X2 available for functionalization is an additional advantage.  In addition to the flexibility that has already been mentioned, the use of these polymers provides greater functionalization with less synthetic effort and allows rapid identification of efficient transfection reagents without toxicity in cellular models as shown by the MTT viability test (Fig.  4A and 4B).  Therefore, n the average number of monomer units may be a number between 10 and 300, for example 45 equal to or less than 150, such as between 20 and 120, for example between 30 and 100.  The term "amphiphilic" in the This description corresponds to the generally accepted meaning for the person skilled in the art, and therefore refers to a molecule that combines hydrophilic and lipophilic (hydrophobic) properties.  The polymers of the present description are typically based on acryloyl derivatives and thus R0 is generally selected from the group consisting of hydrogen, a C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl.  R3 is typically for all hydrogen or methyl units, preferably hydrogen.  The person skilled in the art can recognize that the methodology described in the present invention can be applied to different derivatives of (meth) acryloyl following the same principles.  The polymerization may be a radical polymerization, but other polymerization methods are also available to the person skilled in the art.  As explained below, the polymerization reaction is typically carried out in the presence of a chain transfer agent (CTA), and thus the polymers of the invention can terminate at both terminations with moieties derived from such CTAs.  Other polymerization methods are possible, and those moieties are not of particular relevance to the function of the composition of the invention and the person skilled in the art can select from a wide variety of commercial CTAs (or any other available polymerization method), for example, derivatives that combine a (((ethylthio) carbonothioyl) thio) moiety and a carboxylic acid, which would give rise to a polymer of formula (VII) where R4 is -SH and R5 is a C2-C12 alkylcarboxy acid, for example , which has the formula - (R6) (R7) CC (= O) OH, where R6 and R7 are each independently selected from a C1-C3 alkyl or hydrogen group, preferably where R6 and R7 are both methyl.  Additional details are provided in the synthesis section below.  However, the structure of R4 and R5 may vary depending on the polymerization method used and the particular reagents in each case.  The use of atomic transfer polymerization (ATRP) or radical polymerization by nitroxide mediation (NMP) will result in polymers having the same units described herein, but with different terminations R4 and R5.  ATRP generally employs a transition metal complex as a catalyst with an alkyl halide as an initiator, giving the possibility that R4 is a halogen atom.  Other possible mechanisms can be recognized by the person skilled in the art and can be found in book references such as 1) Matyjaszewski, K. , and Möller, M.  (Eds. ).  Polymer Science: A Comprehensive Reference.  Elsevier B. V.  vol 3 Chain Polymerization of Vinyl Monomers; or (2) Tsarevsky, N.  V. , and Sumerlin, B.  S.  (Eds. ).  (2013) Fundamentals of Controlled / Living Radical Polymerization.  Royal Society of Chemistry, Cambridge.  In addition, the R4 or R5 terminations can be functionalized to include other molecules that can aid in screening, such as chromophores or targeting agents.  Group X1 is a bridge between the polymeric skeleton and the nitrogen atom to which the lipophilic and cationic moieties will adhere.  It is preferred that X1 and X2 together form a group (-N (H) -N (H) -) or a group (-N (H) -N =), since the inventors have discovered that protected carbazones can be made Easily react with the acryloyl monomers and then polymerize and readily deprotect (see below).  Therefore, it is preferred that in the polymers of the present description X1 be -N (H) -.  Other polymers in which X1 is -O-, -N (H) -Alkyl- or -O-Alkyl are also suitable and readily available following the synthesis procedures shown herein (see below).  One of the key aspects of the polymers and methods of the present description is the possibility of functionalizing the polymeric skeleton with a wide range of lipophilic and cationic residues with unprecedented flexibility and simplicity.  As a result of the polymers described herein and the corresponding compositions, a wide variety of lipophilic moieties and cationic (hydrophilic) moieties can be included.  The terms "lipophilic" or "hydrophobic", as used herein, are given their normal meaning in the state of the art, and refers to substances that have a higher lipid solubility than in aqueous media.  When lipophilic residues are considered in the field of chemistry, the person skilled in the art has in his hand a wide variety of possibilities to select and it is of general understanding which substances will impart lipophilicity and which will not.  Such groups are widely described in literature, for example in C.  Gehin, J.  Montenegro, E. -K.  Bang, A.  Cajaraville, S.  Takayama, H.  Hirose, S.  Futaki, S.  Matile, H.  50 Riezman, J.  A.M.  Chem  Soc.  2013, 135, 9295–9298, already discussed in the background.  Preferably, the term "lipophilic" refers to moieties having a logKow value greater than 1. 0, more preferably a logKow value greater than 2. 0, where the value of logKow is measured by the behavior of the rest distribution in a biphasic system such as in the octanol / water partition test.  This test involves measuring the equilibrium concentration of a substance dissolved in a two-phase octanol and water system 55 also as a chromatographic method and is described in ASTM E1147.  Generally, lipophilicity will be achieved by organic molecules, such as hydrocarbons (for example alkyl, alkenyl, aryl and the like).  These molecules are generally nonpolar, although they may contain a small relative amount of polar groups or groups capable of forming hydrogen bonds.  Examples of R1 groups are those in which the corresponding aldehyde is readily available 60 (commercial or easy to synthesize).  Thus, R1 can be selected from the group consisting of C1-C40 alkyl, C2-C40 alkenyl, C2-C40 alkynyl, C3-C40 cycloalkyl, C4-C40 cycloalkenyl, C5-C40 cycloalkynyl, C6-C40 aryl, C7-C40 alkylaryl, C3-C40 heterocyclyl and C5-C40 heteroaryl optionally substituted with 1 to 10 selected groups of halogens such as fluorine or ether bonds.  Preferably, R1 is selected from the group consisting of C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl, C4-C40 cycloalkenyl, C5-C10 cycloalkynyl, C7-C15 alkynyl and C5-C15 heteroaryl optionally substituted with 1 to 5 groups 5 selected from halogen such as fluorine or ether bonds.  Preferably, R1 is selected from the group consisting of C1-C10 alkyl, C3-C10 cycloalkyl, C7-C15 alkylaryl and C5-C15 heteroaryl, optionally substituted with 1 to 5 selected halogen groups such as fluorine or ether bonds.  Examples of the R1 groups are those corresponding to available aldehydes (commercial or easy to synthesize).  Examples of aryl and alkylaryl groups are phenyl, naphthyl, phenyl substituted with C7-C12 alkyl (for example methylphenyl), or biphenyl.  Examples of alkyl and cycloalkyl groups have 3 to 7 carbon atoms and can be cyclopentyl, cyclohexyl, butyl, tert-butyl, propyl, isopropyl, neopentyl, neobutyl (iso-varyl), pentyl, hexyl.  Excellent transfection activity has been achieved when R1 is selected from the group consisting of C1-C7 branched alkyl.  Examples of heteroaryl groups are imidazoyl, furyl or thiophenyl.  R2 is a cationic moiety that imparts hydrophilicity to the polymers and compositions of the present description.  R2 typically comprises a cationic group that has a positively charged heteroatom.  Such a cationic group is positively charged to the pH to which it is exposed for the medical test or application, typically when protonated it has a pka (pkaH) greater than 4, or a pka over 7, for example a group that will protonate at the pH of the medium. , for example physiological pH.  Non-limiting examples of cationic groups are benzimidazolium (pKaH approximately above 5. 6), imidazolium (pKaH 20 approximately above 7. 0), morpholinium (pKaH approximately above 8. 76), piperacinium (pKaH approximately above 9. 8), azepanium (pKaH approximately above 11. 07), piperidinium (pKaH approximately above 11. 22), pyrrolidinium (pKaH approximately above 11. 27), indolinium (pKaH approximately above 16. 2), ammonia (pKaH approximately above 9. 25), phosphonium (pKaH approximately above 9) or guanidinium groups (pKaH approximately 25 above 13), for example, ammonium (pKaH approximately above 9. 25), phosphonium (pKaH approximately above 9) or guanidinium groups (pKaH approximately above 13).   The cationic groups may be a residue of the formula -L-G, where L is a linker and G is a positively charged group.  The specific nature of the L or G connector group is not critical.  Simple and commercially available compounds of formula O = C (H) -R2 that can be used in the present invention are compounds of formula (IX) O = C (H) -ZG, where Z is a group comprising 1 to 40 carbon atoms, and G is a positively charged group.  The group Z may be an alkyl group (for example a C1-C20 alkyl group), a C1-C20 cycloalkyl group, which contains or not the group G in the molecular skeleton ring, may comprise an aromatic ring or a heterocyclic group or heteroaryl, which contains or does not contain a group G in the ring of the molecular skeleton, all of them may be optionally substituted.  G can be positively charged ammonium, phosphonium or guanidinium.  For example, the compound of formula O = C (H) -R2 may be a compound of formula (X) O = C (H) -YG, where Y is selected from the group consisting of C1-C12 alkyl, optionally including as part of the alkyl chain of 1 to 3 amido groups or ester groups, C6-C16 aryl, C4-C16 heterocyclyl and C4-C16 heteroaryl, all optionally substituted, and G is a positively charged ammonium, phosphonium or guanidinium group.  Thus, for example, Y may be a chain of alkyl- (CH2) r- having 1 to 12 carbon atoms (r = 1-12) or a phenyl group.  Examples of molecules that can be used as cationic moieties are betain aldehyde, 4- (trimethylamino) butyraldehyde, 4- (dimethylamino) benzaldehyde, 1- (3-formyl-4-hydroxyphenyl) guanidino hydrochloride (Chemical and Pharmaceutical Bulletin, 2003, vol.  51, # 6 p.  625), 1- (4-formylphenyl) guanidino (SU172307), 1- (5-formyl-4-methylthiazol-2-yl) guanidino (US6521643), piperidine-4-carbaldehyde hydrochloride (piperidin-4-45 hydrochloride carbaldehyde), 1- (4-oxobutyl) guanidino (Biochemical Journal, 2015, vol.  468, # 1 p.  109).  A further example of a group R2 is a residue of formula (XI) NH OGab (XI) where a is a number between 1 and 6, for example 1, 2, 3, 4, 5 or 6, for example between 1 and 3 ; b is a number between 50 1 and 6, for example 1, 2, 3, 4, 5 or 6, for example between 1 and 3; and G is a positively charged ammonium, phosphonium or guanidinium.  Ammonium groups are typically of formula -N + H3, but other possibilities can be recognized by one skilled in the art, such as a group of formula (XII) N + R12R12 R12 (XII) where each R12 group is independently selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C4-C6 cycloalkenyl, C5-C7 cycloalkynyl, aryl C6-C8, C7-C10 alkylaryl, C3-C10 heterocyclyl and C5-C10 heteroaryl.  Guanidinium groups are typically of the formula - [N (H) -C (NH2) = NH2] +, but other possibilities may be recognized by one skilled in the art, such as a group of formula (XIII) NHN + N R12R12R12R12 ( XIII) where each R12 group is independently selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C4-C6 cycloalkenyl, C5-C7 cycloalkynyl, C6-C8 aryl, C7-C10 alkylaryl, C3-C10 heterocyclyl and C5-C10 heteroaryl.  Phosphonium groups are typically of formula –P + H3, but other possibilities can be recognized by the person skilled in the art, such as a group of formula (XIV) P + R12R12 R12 15 (XIV) where each R12 group is independently selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C4-C6 cycloalkenyl, C5-C7 cycloalkynyl, C6-C8 aryl, C7-C10 alkylaryl, C3-C10 heterocyclyl and C5 heteroaryl -C10.  The corresponding aldehydes that carry a remainder of formula (XI), where b is between 1 and 4 are new 20 according to our knowledge.  Thus, a further aspect of the invention is a compound of formula O = C (H) -R11, where R11 is a residue of formula (XI) NH OGab (XI) where a is a number between 1 and 6; b is a number between 1 and 4; and G is positively charged ammonium, phosphonium or guanidinium.  The polymers of the invention allow extraordinarily flexible screening, and the amphiphilicity of the polymers and compositions can be modulated by the nature of the lipophilic and cationic moieties, as well as by the proportion of each.  Thus, the percentage of lipophilic moieties present in the polymer with respect to the total number of groups X2 may be between 5 and 70%, preferably between 7 and 60%, preferably between 7 and 20%, more preferably between 10 and 20% .  Also, it can be readily understood that different mixtures of cationic moieties can be made and thus provide a polymer of formula (I) where more than one type of cationic molecule has been incorporated.  Also, it can be easily understood that different mixtures of lipophilic moieties can be made and thus provide a polymer of formula (I) where more than one type of lipophilic molecule has been incorporated.  The percentage of cationic moieties present in the polymer with respect to the total number of groups X2 may be between 10 and 99%, preferably between 40 and 95%, preferably between 60 and 90%, more preferably between 65 and 85%.  At the same time, the sum of the percentage of lipophilic residues and remains Cationic can be between 40 and 80%.  The percentage of lipophilic residues R1 (% of R1) is one hundred times the result of dividing the average number of groups R1 (number of R1) by the total average number of available positions X2 (positions X2), for example% of R1 = 100x (number of R1) / (positions X2).  The percentage of lipophilic residues R2 (% of R2) is one hundred times the result of dividing the average number of groups R2 (number of R2) by the total average value of available positions X2 (positions X2), for example% of R2 = 100x (R2 number) / (positions X2).   In view of the foregoing and the specific examples provided, the person skilled in the art can devise different combinations of the above parameters and substituents, all of them included in the present description.  For example, the polymer of the invention may be a polymer of formula (VIIa), salts 10 and stereoisomers thereof R5R4R3NHX2OR0n (VIIa) where n is the average number of monomer units that is a number between 10 and 70; R0 is selected from the group consisting of hydrogen, C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl R3 is hydrogen or methyl; R4 is selected from the group consisting of -SH, -S-Alkyl, -O-Alkyl, -OH and -NH2, preferably, -SH, -S-Alkyl, -O-Alkyl; R5 is a C2-C12 alkylcarboxy acid or a C2-C12 alkylcarboxy acid derivative; X2 is independently selected in each unit from the group consisting of –NH2, -N = C (H) R1 and -N = C (H) R2; where R1 is a lipophilic moiety and R2 is a cationic moiety; and where the percentage of lipophilic moieties present in the polymer with respect to the total number of groups X2 is between 5 and 30%; Where the percentage of cationic residues present in the polymer with respect to the total number of groups X2 is between 60 and 90%; and where the sum of the percentage of lipophilic moieties and cationic moieties is between 5 and 95%.  A further example may be a polymer of formula (VIIb), salts and stereoisomers thereof 30 nSHR3NHX2OR6R7OOHR0 (VIIb) where n is the average number of monomer units that is a number between 10 and 70; R0 is selected from the group consisting of hydrogen, C1-C3 alkyl group and CN, for example, where R035 is hydrogen or methyl R3 is hydrogen or methyl; R6 and R7 are independently selected from hydrogen and C1-C3 alkyl; X2 is independently selected in each unit from the group consisting of –NH2, -N = C (H) R1 and -N = C (H) R2; where R1 is a lipophilic moiety and R2 is a cationic moiety; and where the percentage of lipophilic moieties present in the polymer with respect to the total number of groups X2 is between 5 and 30%; where the percentage of cationic residues present in the polymer with respect to the total number of groups X2 is between 60 and 90%; and where the sum of the percentage of lipophilic moieties and cationic moieties is between 5 and 95%.   Preparation of Polymers 10 The polymers and compositions described herein can be synthesized from readily available materials.  The synthesis begins with the preparation of the appropriate monomer of formula (IV), salts and stereoisomers thereof, R3X1NHOR8R0R3 15 (IV) where X1 is a group selected from the group consisting of –N (H) -, -O-, -N (H) -Alkyl- and -O-Alkyl-, preferably -N (H) -; R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl each R3 is independently selected from the group consisting of hydrogen and a C1-C3 alkyl group; and R8 is a labile group in acidic medium.   comprising the step of contacting a compound of formula (V), salts and stereoisomers thereof, with a protective group -R8, preferably labile in acidic medium, or with a compound of formula -N (H2) -N ( H) -R8, -ON (H) -R8, -N (H) -Alkyl-N (H) -R8 and -O-Alkyl-N (H) -R8 R3X3OR0R3 (V) 30 where R0 is selected from the group consisting of hydrogen, a C1-C3 and CN alkyl group, for example, where R0 is hydrogen or methyl; each R3 is independently selected from the group consisting of hydrogen and a C1-C3 alkyl group; and X3 is selected from the group consisting of –OH, halogen, O-alkyl, –N (H) -N (H2), -ON (H2), -N (H) -Alkyl-N (H2) and –O -Alkyl-N (H2).  The reaction typically takes place in the presence of an appropriate solvent, preferably an aqueous based solvent, and a suitable base.  The compounds of the formula -N (H2) -N (H) -R8 are carbazates.  Different carbazates are available to the person skilled in the art, for example t-butyl carbazato, benzyl carbazato, ethyl carbazato, methyl carbazato or mixtures thereof.  The compounds of the formula -O-N (H) -R8 are protected hydroxy amines, many of which are commercially available, such as N-Boc-hydroxylamine or N- (Benzyloxycarbonyl) hydroxylamine.  In order to prepare the compounds of formula (IV) where X1 is -N (H) -Alkyl- or -O-Alkyl-, the person skilled in the art can react a compound of formula (V) where X3 is - OH, halogen or -OR, with a compound of the formula -N (H2) -Alkyl-N (H) -R8 or -O-Alkyl-N (H) -R8.  Alternatively, it is possible to directly protect with the protecting group -R8 a compound of formula (V) where X3 is -N (H) -Alkyl-N (H2) or -O-Alkyl-N (H2); such compounds of formula (V) are commercially available, for example 2-Aminoethyl methacrylate hydrochloride or N- (3-15 Aminopropyl) methacrylamide hydrochloride (available from Aldrich®).  Since the residue R8 acts as an amino protecting group during polymerization, it is designed to be labile, preferably under acidic conditions, in order to cleave it once the polymerization is complete.  "Protective group" in the present invention refers to a group that blocks an organic functional group and can be removed under controlled conditions.  Protective groups, their relative reactivities 20 and conditions under which they remain inert are known to the person skilled in the art.  "Amino protecting group" refers to a group that blocks the -NH2 or -N (H) -NH2 function for other reactions and can be removed under controlled conditions.  The amino protecting groups are well known in the art, representative protecting groups are -amides of formula -C (= O) R9, such as acetate amide, benzoate amide; pivalate amide; 25 methoxyacetate amide; Chloroacetate Amide; levulinate amide; - carbamates of the formula -C (= O) -O-R9, such as benzyl carbamate, p-nitrobenzyl carbamate, tert-butyl carbamate, ethyl carbamate, 2,2,2-trichloroethyl carbamate, 2- (trimethylsilyl) ethyl carbamate.   In all of the above formulas R9 represents a group selected from the group consisting of C1-C6 alkyl, C6-C15 aryl or aralkyl.  Carbamates of the formula - (O =) C-O-R9, where R9 is a C1-C12 alkyl group, are preferred.  Additional examples of amino protecting groups can be found in reference books such as Greene and Wuts' "Protective Groups in Organic Synthesis", John Wiley & Sons, Inc. , New York, 4th Ed. , 2007.  The next step in the synthesis is the preparation of the corresponding polymer of formula (III), salts and stereoisomers thereof by polymerization of a compound of formula (IV), preferably, in the presence of a radical initiator R3X1NHOR8R0R3n (III) where n is the average number of monomer units that is a number equal to or greater than 10; R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl each R3 is independently selected from the group consisting of hydrogen and a C1-C3 alkyl group; X1 is a group that is selected from the group consisting of –N (H) -, -O-, -N (H) -Alkyl- and –O-Alkyl-; 45 and R8 is a protective group, preferably labile in acidic medium.   Typically the polymerization takes place in aqueous medium.  When a radical polymerization is used different radical initiators are available in the art and include both peroxide compounds and azo compounds.  Examples of available free radical initiators are peroxide catalysts such as dibenzoyl peroxide, lauroyl peroxide, t-amylperoxy-2-ethylhexanoate, di-t-butyl peroxide, diisopropyl peroxide carbonate, t-butyl peroxy-2-ethylhexanoate, t-butylperpivalate, t -butylperneo-decanoate, t-butylperbenzoate, t-butyl percrotonate, t-butyl perisobutyrate, t-butylperoxy-l-methylpropanoate, t-butylperoxy-2-ethylpentanoate, t-butylperoxioctanoate and di-t-butylperphthalate.  Examples of azo compounds are azobis-isobutyronitrile (AIBN), 4,4’-azobis (4-cianovaleric) (ACVA) and azobis- (2-methylbutanenitrile).  The amount of initiator may vary from 0.01 percent by weight to 5 percent by weight based on the total weight of monomer (s).   The polymerization typically takes place in the presence of a Chain Transfer Agent (CTA), also known as "Reversible Addition Fragmentation Chain Transfer" (RAFT), which is known to the person skilled in the art.  Non-limiting examples are those described in US2015024488 or in US2012128743.  The CTA may be a compound of formula (XV) or salts thereof SZ1SZ2 (XV) where Z1 is a hydrophobic group and Z2 is a hydrophilic group.  This group of CTAs is widely known in the art and described, for example, in US2012128743.  Preferably, Z1 is a thiol such as an R10-S- group, where R10 is a C1-C20 alkyl, C1-C20 aryl or a C1-C20 arylalkyl, preferably one such that the Z1-C moiety (= S) - it is labile under acidic conditions in order to be cleaved at the same time as group 20 R8.  Preferably, Z2 is a C2-C12 alkylcarboxy acid or a C2-C12 alkylcarboxy acid derivative, such as -alkyl-CO2H, -alkyl-C (= O) (NH2), -alkyl-SO3.  The CTA is preferably 2 - (((ethylthio) carbonothioyl) thio) -2-methylpropanoic acid.  The person skilled in the art can recognize that any other method for the polymerization of (meth) acryloyl derivatives is available for the purposes of the present invention while the result of the reaction is the formation of polymers of formula (III).  Other methods for polymerizing (meth) acryloyl derivatives include but are not limited to Atomic Transfer Radical Polymerization (ATRP), Nitroxide Mediated Polymerization (NMP), Degenerative Transfer with Alkyl Iodide, Cobalt Catalyzed Chain Transfer Polymerization, Polymerization Organometallic-mediated radical (OMRP), Anionic Polymerization, Cationic Polymerization, Alkene Polymerization 30 Metallocene, or Live polymerization of alkenes catalyzed by transition metals.  Additional examples of polymerization methods can be found in reference books such as Matyjaszewski, K. , and Möller, M.  (Eds. ).  Polymer Science: A Comprehensive Reference.  Elsevier B. V.  Thus, a preferred polymer of formula (III) is a compound of formula (IIIa), salts and stereoisomers thereof R3X1NHOR8R5R4R0n (IIIa) where n is the average number of monomer units that is a number equal to or greater than 10; R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl R3 is selected from the group consisting of hydrogen and a C1-C3 alkyl group; R4 is selected from the group consisting of -SH, -S-Alkyl, -O-Alkyl, -OH and -NH2, preferably, -SH, -S-Alkyl, -O-Alkyl; R5 is a C2-C12 alkylcarboxy acid or a C2-C12 alkylcarboxy acid derivative; X1 is a group selected from the group consisting of –N (H) -, -O-, -N (H) -Alkyl- and –O-Alkyl-, preferably –N (H) -; and R8 is a labile group in acidic medium.  Additional examples of chain transfer agents include mercapto compounds, such as thioglycolic acid, thiomalic acid, thiosalicylic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, 3-mercaptobutyric acid, N- (2-mercaptopropionyl) glycine, 2 -mercaptonicotinic, 3- [N- (2-mercaptoethyl) carbamoyl] propionic acid, 3- [N- (2-mercaptoethyl) amino] propionic acid, N- (3-mercaptopropionyl) alanine, 2-mercaptoethanesulfonic acid, 3- acid mercaptopropanesulfonic acid, 4-mercaptobutanesulfonic acid, dodecyl (4-methylthio) phenyl ether, 2-mercaptoethanol, 3-mercapto-1,2-propanediol, 1-10 mercapto-2-propanol, 3-mercapto-2-butanol, mercaptophenol, 2 -mercaptoethylamine, 2-mercaptoimidazole, 2-mercapto-3-pyridinol, 2-mercaptobenzothiazole, mercaptoacetic acid, trimethylolpropane tris (3-mercaptopropionate), and pentaerythritol tetrakis (3-mercaptopropionate); disulfide compounds obtained by oxidation of the mercapto compounds mentioned; and alkyl iodine compounds, such as iodoacetic acid, iodopropionic acid, 2-iodoethanol, 2-iodoethanesulfonic acid, and 3-15 iodopropanesulfonic acid.  Once the protected polymer is obtained, deprotection takes place under acidic conditions in order to provide the corresponding polymer of formula (II), salts and stereoisomers thereof R3X1NH2OR0R3n (II) 20 where n is the average number of monomer units which is a number between 10 and 150; R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl each R3 is independently selected from the group consisting of hydrogen and a C1-C3 alkyl group; and X1 is a group that is selected from the group consisting of –N (H) -, -O-, -N (H) -Alkyl- and -O-Alkyl-, preferably –N (H) -.  A preferred polymer of formula (II) is a polymer of formula (IIa), salts and stereoisomers thereof R3X1NH2OR5R4R0n 30 (IIa) where n is the average number of monomer units that is a number between 10 and 150; R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group and CN, for example, where R0 is hydrogen or methyl R3 is selected from the group consisting of hydrogen and a C1-C3 alkyl group; R4 is selected from the group consisting of -SH, -S-Alkyl, -O-Alkyl, -OH and -NH2, preferably, -SH, -S-Alkyl, -O-Alkyl; R5 is a C2-C12 alkylcarboxy acid or a C2-C12 alkylcarboxy acid derivative; and X1 is a group that is selected from the group consisting of –N (H) -, -O-, -N (H) -Alkyl- and -O-Alkyl-, preferably –N (H) -.  The final stage in the preparation of the polymers of formula (I) is the functionalization of the available nitrogen atoms with the aldehydes of the corresponding groups R1 (lipophilic) and R2 (cationic), for example with aldehydes of formula O = C (H) -R1 and O = C (H) -R2, where R1 and R2 are as defined elsewhere in this description.  The reaction comprises contacting the aldehydes of formula O = C (H) -R1 and O = C (H) -R2 with the polymer in an appropriate medium, preferably aqueous medium, and proceeds smoothly.  Both aldehydes of formula O = C (H) -R1 and O = C (H) -R2 can be simultaneously or sequentially brought into contact with the polymer.  As already discussed, the method allows to easily change the proportion or the total amount in which the aldehydes are incorporated into the polymer.  The added equivalents are not particularly relevant and the invention works in a wide range of aldehyde charges.  The person skilled in the art can determine which amounts and proportions are the most suitable for each particular case and typical conditions are those in which said aldehyde of formula O = C (H) R1 and said aldehyde of formula O = C (H) R2 are added in amounts, for example, between 0. 001 and 10 equivalents with respect to the total amount of NH2 residues available, for example between 0. 01 and 4 or between 0. 01 and 3, typically between 0. 1 and 2. 5 equivalents   20 Screening Methods The general screening method of the present description is shown in Figure 5.  A polymer (1) of formula (II), salts and stereoisomers thereof, is mixed with selected amounts of said aldehydes (3) of formula O = C (H) R1 and said aldehydes (2) of formula O = C (H ) R2, in order to provide a polymer (4) of formula (I), salts and stereoisomers thereof, which is then conjugated with a negatively charged compound (5), typically a nucleic acid, such as DNA, RNA or siRNA.  The resulting composition (6) is then transfected into cells, membranes or membrane models (7).  Preferably, a polymer (1) of formula (IIa), salts or stereoisomers thereof, is mixed with selected amounts of said aldehydes (3) of formula O = C (H) R1 and said aldehydes (2) of formula 30 O = C (H) R2, in order to provide a polymer (4) of formula (VII), salts and stereoisomers thereof, which is then conjugated with a negatively charged compound (5), typically a nucleic acid, such as DNA , RNA or siRNA.  The resulting composition (6) is then transfected to a membrane or membrane model (7).  Additional examples of nucleic acids are possible and may comprise, for example, one or more than 35 DNA plasmids (pDNA), cosmids, double stranded RNA (dsRNA), interference RNA (siRNA), endogenous microRNA (miRNA), RNA short hairpin (shRNA), oligodeoxynucleotides (ODN), primary RNA transcriptionists (pri-miRNA).  These and others are described in bibliography, such as (1) Deng, Y. , Wang, C.  C. Choy, K.  W. , Du, Q. , Chen, J. , Wang, Q. , Li, L. , Chung, T.  K.  H. , and Tang, T.  (2014) “Therapeutic potentials of gene silencing by RNA interference: Principles, challenges, and new strategies” Gene 538, 217-227; (2) Li, 40 Z. , and Rana, T.  M.  (2014) “Therapeutic targeting of microRNAs: current status and future challenges” Nat.  Rev.  Drug Discovery 13, 622-638; or (3) Alexander, C. , and Fernandez-Trillo, F.  (2013) “Bioresponsive Polyplexes and Micelleplexes, in Smart Materials for Drug Delivery” (Alvarez-Lorenzo, C. , and Concheiro, A. , Eds. ) 1st ed. , pp 256-282.  Royal Society of Chemistry.  Artificial nucleic acids such as XNAs or PNAs can also be used.  Examples thereof can be found in (1) Turner, J.  J. , Jones, S. , Fabani, M.  M. , Ivanova, G. , Arzumanov, A.  TO. , and Gait, M.  J.  (2007) "RNA targeting with peptide conjugates of oligonucleotides, siRNA and PNA" Blood Cells Mol.  Dis.  38, 1–7); (2) Pinheiro, V.  B. , and Holliger, P.  (2012) “The XNA world: progress towards replication and evolution of synthetic genetic polymers” Curr.  Opin.  Chem  Biol  16, 245-252; (3) Pinheiro, V.  B. , and Holliger, P.  (2014) “Towards XNA nanotechnology: new materials from synthetic genetic polymers” Trends Biotechnol.  32, 321–50 328.   One of the key advantages of the present screening method is that all steps from the polymer (1) to the transfection test of the compound (6) can be carried out in aqueous medium without purification of the intermediates.  For example, solutions of the polymer of formula (II), salts and stereoisomers thereof can be made to store and later mix them with different aldehydes in parallel and / or automated experiments, each resulting polymer conjugated to a molecule of interest being negatively loaded, and the resulting composition subjected to transfection.  Thus, a further aspect of the present invention is a kit comprising a polymer of formula (II), salts and stereoisomers thereof.   A further aspect of the present invention is a kit comprising a polymer of formula (I), salts and stereoisomers thereof.  A further aspect of the present invention is a kit comprising the composition of the invention comprising a polymer of formula (I), salts and stereoisomers thereof and a negatively charged molecule.  The compositions resulting from the screening method of the invention can thus be used as a medicament, specifically in the transfer of biologically active molecules that have a negative charge which otherwise are unable to pass through the lipid membrane.  The present invention thus includes a pharmaceutical composition comprising the compositions of the invention and pharmaceutically acceptable carriers and / or other auxiliary substances.  The medicament or pharmaceutical composition according to the present invention may be in any form suitable for application to humans and / or animals, preferably humans including infants, children and adults and may be produced by standard procedures known to those skilled in the art. .  The medicament can be produced by methods known to the person skilled in the art, for example from the table of contents of "Pharmaceutics: The Science of Dosage Forms", Second Edition, 15 Aulton, M. AND.  (ED.  Churchill Livingstone, Edinburgh (2002); "Encyclopedia of Pharmaceutical Technology", Second Edition, Swarbrick, J.  and Boylan J. C.  (Eds. ), Marcel Dekker, Inc.  New York (2002); "Modern Pharmaceutics", Fourth Edition, Banker G. S.  and Rhodes C. T.  (Eds. ) Marcel Dekker, Inc.  New York 2002 and "The Theory and Practice of Industrial Pharmacy", Lachman L. , Lieberman H.  And Kanig J.  (Eds. ), Lea & Febiger, Philadelphia (1986).  The composition of the medicament may vary depending on the route of administration, and generally comprises mixing the compositions of the invention with suitable carriers and / or other auxiliary substances.  The vehicles and auxiliary substances necessary to manufacture the desired dosage form of administration of the pharmaceutical composition of the invention will depend, among other factors, on the dosage form of administration chosen.  Said pharmaceutical forms of administration of the pharmaceutical composition will be manufactured according to conventional methods known to those skilled in the art.  A review of methods of administration of different active ingredients, excipients used and processes to produce them can be found in "Galency Pharmacy Treaty", C.  Faulí i Trillo, Luzán 5, S. TO.  of Editions, 1993.  Non-limiting examples are preparations for oral administration, for example, tablets, capsules, syrups or suspensions.  Also, the pharmaceutical compositions of the invention may include topical compositions, for example creams, ointments and pastes, or transdermal preparations such as patches or plasters.  The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and typically do not produce allergic reactions or similar adverse reactions, such as gastric discomfort, dizziness and the like, when administered to a human being.  Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the US Pharmacopoeia. UU.  or other pharmacopoeia generally recognized for use in animals, and more particularly in humans.   Examples Example 1: Materials and Methods 40 Nuclear Magnetic Resonance (NMR) spectra were recorded on a Brucker Avance III 300 MHz, Bruker Avance III 400 MHz, Varian Mercury 300 MHz or a Varian Inova 500 MHz spectrometer.  Chemical shifts are expressed in ppm (δ units) with respect to the following reference solvent signals: DMSO-d6 δH 2. 50, D2O δH 4. 79 and CDCl3, δH 7. 26.  The average degree of polymerization (DP) (for example the ratio between monomer units and terminal groups) in polymers of formula (II) was calculated by 1H-NMR spectrum by comparison of the integration of methyl substituents into the terminal groups ( 0. 95 and 1. 01 ppm, 6 H) with the integration of the aliphatic region in the polymer backbone (1. 59-2. 08 ppm).  Electrospray ionization mass spectroscopy (ESI-MS) for the characterization of the new compounds was carried out on a Finnigan MAT SSQ 7000 instrument or an ESI API 150EX and was recorded as the ratio of mass to charge m / z (intensity in%, [allocation]).  The 50 precise mass determinations (HR-MS) using ESI-MS were carried out on a Sciex QSTAR Pulsar mass spectrometer.  The infrared (IR) spectra were recorded on a Perkin Elmer Spectrum Two FT-IR spectrometer.  Ultraviolet-visible (UV-vis) spectra were recorded on a Campsec M550 Double Beam Scanning UV-vis Spectrophotometer.  DP in the polymer of formula (III) was calculated by measuring the absorbance at 300, 305 and 310 nm and comparing them against a calibration curve using CTA.  The amount (mg · mL-1) of CTA in polymers of formula (III) was obtained in this way and the DP was calculated.  Fluorescence measurements were carried out with a FluoroMax-2 spectrofluorimeter (Jobin-Yvon Spex) equipped with a stirrer and a temperature controller.  The size exclusion chromatography (SEC) spectra were recorded on a Shimadzu Prominence LC-20A equipped with a Thermo Fisher Refractomax 521.  The polymers of formula (III) were analyzed using 0. 05 M LiBr in DMF at 60 ° C as eluent and a flow of 1 mL · min-1.  The instrument was equipped with a Polymer Labs PolarGel pre-column (50 × 7. 5 mm, 5 µm) followed by two PLGel columns PL1110-6540 (300 × 7. 5 mm, 5 µm).  Molecular weights were calculated based on the standard calibration method using polymethylmethacrylate (see (1) Pasch, H.  Chromatography, in Polymer Science: A Comprehensive Reference (Matyjaszewski, K. , and Möller, M. , Eds. ), 5 pp 33–64.  Elsevier B. V. ).  For analytical HPLC we use a reverse phase C18 HPLC column [Nucleosil 100-7 C18, H2O (0.1% TFA) / CH3CN (0.1% TFA) 95: 5 (0 → 5 min), 100: 0 → 25:75 (5 → 35 min), 0: 100 (> 35 min)] with a binary gradient of Solvent A and Solvent B, the collected fractions were lyophilized and stored at -20 ° C.  2 - (((ethylthio) carbonothioyl) thio) -2-methylpropanoic acid (CTA) (J.  Skey, R.  K.  O'Reily, Chem.  Commun.  2008, 10 4183) was synthesized according to the protocols described in the literature.  The trisodium salt of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) was purchased from Sigma-Aldrich® and p-xylene-bis-pyridinium bromide (DPX) was purchased from InvitrogenTM.  Egg yolk L-α-phosphatidylcholine (EYPC) was purchased from Avanti Polar Lipids, Inc.  All other reagents were purchased from Sigma-Aldrich®, Scharlau, Panreac Química SLU, Fisher Scientific® or Acros® and used without further purifications.  All solvents were grade 15 HPLC, purchased from Sigma-Aldrich® or Fisher Scientific®, and used without further purification.  Example 2: Synthesis of the tert-Butyl-2-acryloylhydrazine-1-carboxylate monomer (compound of formula (IV)) Acrylic acid (3. 81 mL, 54. 95 mmol) and tert-butyl carbazate (8. 89 g, 65. 95 mmol) in an H2O / THF mixture (2: 1, 180 mL) at room temperature.  N- (3-Dimethylaminopropyl) -N'-20 ethylcarbodiimide hydrochloride (EDC) (11. 75 g, 61. 29 mmol) in portions to the solution for 15 minutes and kept under stirring for 3 h.  The reaction crude was extracted with ethyl acetate (3 x 75 mL) and the organic phase was washed with 0. 1 M HCl (3 x 75 mL), H2O (50 mL) and a saturated sodium chloride solution (2 x 50 mL).  The organic phase was dried with anhydrous Na2SO4 and the solvent was removed under reduced pressure to obtain the crude product as a white solid.  The crude product was purified by recrystallization from ethyl acetate (70 ° C 25 to rt) to obtain 5. 05 g of a white crystalline powder (50%).  Rf = 0. 87 (100% ethyl acetate); IR (net) νmax 3311m sh (N-H), 3221m sh (N-H), 2981w sh (C-H), 1715s sh (C = O), 1668s sh (C = O) cm-1.  1H-NMR (300 MHz, DMSO-d6) δ (ppm) 9. 79 (s, 1H, C4 (O) NH), 8. 84 (s, 1H, C3 (O) NH), 6. 17-6. 20 (m, 2H, CHCH2), 5. 69 (dd, 3JH, H = 7. 8, 4. 5 Hz, 1H, CH2CH), 1. 40 (s, 9H, C (CH3) 3.  13C-NMR (100 MHz, DMSO-d6) δ (ppm) 164. 3 (C4), 155. 3 (C3), 129. 4 (C5), 126. 2 (C6), 79. 2 (C2), 28. 1 (C1).  Example 3: Synthesis of poly (tert-butyl-2-acryloyl) hydrazine-1-carboxylate A precursor of the polymer (polymer of formula (III)) A solution of 4,4'-azobis (4-cyanovaleric acid) (ACVA ) (18. 4 mg, 0. 064 mmol) in DMSO (1. 5 mL) and a CTA solution (72. 3 mg, 0. 322 mmol; 2 - (((ethylthio) carbonothioyl) thio) -2-methylpropanoic acid) in DMSO (1. 5 mL) were added sequentially to a solution of tert-butyl-2-acryloylhydrazine-1-carboxylate (3. 00 g, 35 16. 095 mmol) in DMSO (14. 88 mL).  A 50 µL aliquot of this solution was taken at this stage to be used in the conversion calculation.  The reaction mixture was then sealed and deoxygenated with Argon for 30 min.  The deoxygenated solution was maintained at 70 ° C for 7 h.  The reaction was cooled to room temperature and exposed to air.  A 50 µL aliquot of this solution was taken at this stage to be used in the conversion calculation.  The polymer was purified by dialysis against water.  Water was removed by lyophilization and drying in a desiccator with P2O5 to obtain 2. 2 g of poly (tert-butyl-2-acryloyl) hydrazine-1-carboxylate A (a polymer of formula (III)) as an off-white powder (73% yield).  UV (DMSO) λmax 300 nm.  1H-NMR (300 MHz, DMSO-d6) δ (ppm) 9. 22 (br, 1H, NH), 8. 60 (br, 1H, NH), 2. 03 (br, 1H, CH2CH), 1. 41 (br, 11H, 9H in C (CH3) 3, 2H in CHCH2).  40% conversion.  The average number of molecular mass Mn 10270, dispersion in molecular mass ĐM 1. 39 (as defined in Pure 45 Appl.  Chem  2009, Vol.  81, No.  2, pp.  351–353).  DP (UV-vis) 45.  Example 4: Synthesis of a reactive polymer scaffolding - synthesis of poly (acrylohydrazide) (polymer of formula (II)) Trifluoroacetic acid (TFA) (15 mL) was added dropwise to poly (tert-butyl-2-acryloyl) hydrazine -1-carboxylate) A (1. 5 g) (a polymer of formula (III)) obtained in example 3 and the yellow solution was stirred at room temperature for 2 h.  Excess TFA was removed by insufflating a constant stream of Argon and the resulting oil was diluted in water (15 mL).  The TFA salt of the polymer was neutralized by adding NaHCO3 until no foam formation was observed.  The colorless solution was kept stirring overnight.  The crude polymer was purified by dialysis against water.  The water was removed by lyophilization and drying in a desiccator with P2O5 to obtain 650 mg of poly (acrylohydrazide) B (a polymer of formula (II)) as a white powder (92%).  IR (net) νmax 3254w br (N-H), 1609m br (C = O), 1428s sh cm-1.  1H-NMR (300 MHz, D2O) δ (ppm) 1. 59-2. 08 (br, (3 x DP) H, CHCH2), 1. 01 (s, 3H, HOOCCH3b), 0. 95 (s, 3H, HOOCCH3a).  13C-NMR (100 MHz, D2O) δ (ppm) 174. 9 (CONH), 40. 2-40. 5 (CH), 34. 4-35. 7 (CH2).  Degree of polymerization (1H-NMR) 40.  Example 5: Synthesis of 3-guanidino-N- (3-oxopropyl) propanamide (GA-5) (an aldehyde of formula O = C (H) -R1) A solution of 3- (2,3-bis ( tert-butoxycarbonyl) guanidino) propanoic acid (520 mg, 1. 57 mmol) in dichloromethane (30 mL) was treated with N, N, N ′, N′-Tetramethyl-O- (benzotriazol-1-yl) uronium tetrafluoroborate (TBTU) (519. 67 mg, 1. 57 mmol), 2- (1,3-dioxolan-2-yl) ethanamine (316 µl, 2. 83 mmol) and N, N-Diisopropylethylamine (DIPEA) (1 mL, 6. 28 mmol, added dropwise).  The reaction mixture was stirred at room temperature under Argon atmosphere for 1 hour.  The reaction crude was washed with aqueous HCl (5%, 3 x 20 mL) and saturated aqueous NaHCO3 solution (2 x 20 mL).  The organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuo.  The residue was purified by flash chromatography (gradient MeOH / Dichloromethane 1-5 10%, Rf = 0. 76) to get 542. 6 mg of 2- (1,3-dioxolan-2-yl) ethyl-3- (2,3-bis (tert-butoxycarbonyl) guanidino) propanamide (80%).  1H-NMR (300 MHz, CDCl3) δ (ppm) 11. 4 (s, 1H), 8. 7 (t, 3JH, H = 4. 9 Hz, 1H), 6. 66-6. 57 (m, 1 H), 4. 8 (td, 3JH, H = 4. 3, 0. 7 Hz, 1H), 3. 95-3. 91 (2H, m), 3. 85-3 81 (2H, m), 3. 70-3 59 (2H, m), 3. 4-3. 3 (2H, m), 2. 41 (t, 3JH, H = 6. 2 Hz, 2H, m), 1. 86-1 84 (2H, m), 1. 4 (18H, s).  13C-NMR (500 MHz, CDCl3) δ (ppm) 170. 79 (s), 163. 40 (s), 156. 25 (s), 152. 84 (s), 103. 89 (d), 83. 09 (s), 79. 28 (s), 64. 89 (t), 10 36. 05 (t), 35. 40 (t), 34. 71 (t), 32. 80 (t), 28. 28 (q).  ESI-MS (H2O / CH3CN) m / z 431 (100, [M + H] +), 453 (20, [M + Na] +).  A solution of 2- (1,3-dioxolan-2-yl) ethyl-3- (2,3-bis (tert-butoxycarbonyl) guanidino) propanamide (0. 69 mmol, 300 mg) in water was treated with an aqueous solution of HCl (3M, 10 mL).  The reaction mixture was stirred at 60 ° C for 1 h.  Then the solvent was evaporated in vacuo.  The reaction crude was dissolved in H2O and purified by RP-HPLC.  The fractions collected (tR 4. 0 min) were lyophilized and stored at -20 ° C to obtain 90 mg of 3-guanidino-N- (3-oxopropyl) propanamide (GA-5) (70%).   RP-HPLC [Nucleosil 100-7 C18 H2O (0. 1% TFA) / CH3CN (0. 1% TFA) 100: 0 → 75: 35 (10 → 35 min), 0: 100 (> 35 min)].  Purity and characterization were confirmed by analytical RP-HPLC, 1H-NMR and ESI-MS.  1H-NMR (300 MHz, D2O) δ (ppm) 9. 53 (s, 1 H), 5. 00-4. 77 (m, 1 H), 3. 40-3 25 (m, 2H), 3. 24-3 04 (m, 2H), 2. 65-2. 60 (m, 2H), 2. 52-2. 29 (m, 20 2H), 1. 94-1. 73 (m, 1 H), 1. 66 (dd, 3JH, H = 12. 7, 6. 9 Hz, 2H).  ESI-MS (H2O / CH3CN) m / z 187 (100, [M + H] +, 205 (30, [M + H2O] +).  HR-MS (MS): Calcd for C7H15N4O2: 187. 1185; Found: 187. 1190  Example 6: General conditions for the synthesis of amphiphilic polymers In a standard experiment, 25 µl of a solution of a polymer of formula (II) (100 mM) in acetate buffer (100 mM, pH 4) was mixed. 5), 3. 8 µl of a solution of a hydrophobic aldehyde (200 mM) of formula 25 O = C (H) -R2 in dry DMSO and 21. 2 µl of a solution of hydrophilic aldehyde (200 mM) of formula O = C (H) -R1 in dry DMSO to obtain a final concentration of amphiphilic polymer of 50 mM.  This mixture was stirred at 60 ° C for 2 h.   Example 7: Specific examples of synthesis of amphiphilic polymers Poly (acrylohydrazide) (B) was reacted in acetate buffer (100 mM, pH 3. 0) with 2 equivalents of 30 different molar fractions of GA-5 and different lipophilic aldehydes of formula O = C (H) -R1.  In a type experiment with mixtures of modulators, 25 µl of a solution of poly (acrylohydrazide) (B) (100 mM) in acetate buffer (100 mM, pH 3) was mixed. 0), 3. 8 µl of a hydrophobic aldehyde solution of formula O = C (H) -R1 (200 mM) (Table 1) in dry DMSO and 21. 2 µl of a solution of GA-5 aldehyde (200 mM) in dry DMSO to obtain a final concentration of amphiphilic polymers of 50 mM.  This mixture was stirred at 60 ° C for 2 h.  Amphiphilic polymers were used without further purification in vesicle transport experiments and in HeLa cell transfection experiments.  Example 8: General conditions for the evaluation of transport through model nucleic acid membranes - Experiments with vesicles.  Stock solutions of unilamellar long vesicles (LUVs) (5 µl) were diluted with buffer (10 mM Tris, 107 mM NaCl, pH 7. 4), in a fluorescence cuvette with temperature control (25 ° C) and with sufficient agitation (total volume ~ 2000 µl; final lipid concentration ~ 13 µM).  HPTS efflux was monitored at a wavelength λ 511 nm (λex 413 nm) as a function of time after the addition of the amphiphilic polymer (20 µl in DMSO / AcOH buffer, t = 25 s), nucleic acid (NA, 20 µl stock solution in buffer, t = 50 s) and aqueous triton X-100 (1. 2%, 40 µl, 370 µM final concentration, t = 225 s).  Total time of the experiment = 45 250 s.  The fluorescent emission intensity was normalized to the fractional emission intensity I (t) using Equation Equation S1.  Equation S1: It = I-I / I-I where I0 = It to the addition of nucleic acid, I∞ = It to saturation after rupture.  Effective concentration of amphiphilic polymer or nucleic acid - EC50 - and the Hill coefficient - n - were determined by 50 the representation of fractional activity Y (= I (t) at saturation just before rupture, t = 200 s) in function of the concentration of amphiphilic polymer or nucleic acid [Analyte] and adjusting this data to the Hill equation (Equation S2).  Equation S2: = + - / 1+ [] where Y0 is Y without nucleic acid (or amphiphilic polymer), Ymax is Y saturated in the presence of an excess of amphiphilic polymer (or nucleic acid), EC50 is the acid concentration nucleic (or amphiphilic polymer) needed to reach 50% activity and n is the Hill coefficient.  The results are shown in Table 1.    Aldehyde EC50 (µM) Ymax (%) n 1 12. 54 ± 2. 25 56. 00 ± 5. 04 2. 94 ± 1. 51 2 17. 27 ± 6. 88 35. 72 ± 7. 73 1. 50 ± 0. 63 3 8. 38 ± 0. 53 23. 12 ± 1. 31 4. 82 ± 1. 34 4 2. 40 ± 0. 20 53. 58 ± 3. 32 3. 71 ± 1. 02 5 1. 32 ± 0. 16 50. 9 ± 2. 60 2. 88 ± 0. 96 6 3. 49 ± 0. 19 48. 48 ± 1. 90 5. 28 ± 1. 42 7 9. 17 ± 7. 60 28. 34 ± 9. 07 1. 02 ± 0. 57 8 2. 97 ± 0. 52 26. 71 ± 1. 51 2. 36 ± 0. 89 9 9. 19 ± 1. 97 17. 84 ± 1. 79 1. 61 ± 0. 50 10 2. 10 ± 0. 51 11. 51 ± 0. 57 1. 70 ± 0. 67 11 16. 28 ± 13. 17 18. 27 ± 5. 05 0. 92 ± 0. 39 Table 1: EC50 (µM), YMAX (%) and n for the transport of Herring DNA (125 µM) in EYPC-LUVs⊃HPTS / DPX with increasing concentrations of amphiphilic polymer prepared from a 15% percentage of hydrophobic modulator and 85% of the GA-5 modulator (4).  All experiments were carried out in triplicate.  5 Figure 1 shows the normalized emission intensity I (t) (A) and the dose-response curves (B).  Example 9: In Vitro Screening of SiRNA Assignment with Amphiphilic Polymers HeLa Cells Stably Expressing Enhanced Fluorescent Green Protein (EGFP) were maintained in Dulbecco's Modified Eagle's Medium of Life TechnologiesTM (DMEM, High Glucose, GlutaMAXTM, Pyruvate) supplemented with 10% (v / v) fetal bovine serum (FBS) of HycloneTM (Thermo Fisher Scientific Inc) and 500 10 µg · mL-1 of Geneticin® (Life TechnologiesTM).  HeLa-EGFP transfection was carried out in the same medium, free of antibiotics.  Cell incubations were carried out in a 37 ° C / 5% CO2 water jacket incubator.  HeLa-EGFP cells were transfected with Ambion® Silencer® GFP (EGFP) siRNA (siEGFP) from Life Technologies ™ or RNA (siMOCK, All Star Negative Control) from Qiagen.  After 72 h after transfection of siRNA, the cell supernatant was removed and EGFP expression was measured by fluorimetry (λex 489nm; λem 509nm).  The percentage of EGFP knockdown was calculated as the percentage decrease in fluorescence observed in cells transfected with siEGFP compared to transfection with siMOCK with the same reagents under the same conditions.  The percentage of cell viability was calculated as the percentage of fluorescence remaining in samples transfected with siMOCK compared to cells not transfected in DMEM, high glucose, GlutaMAX ™ and pyruvate, supplemented with 0. 125% (v / v) DMSO.   Lipofectamine® 2000 was used as a positive control of the transfection of siRNA in in vitro screening of amphiphilic polymers in HeLa-EGFP.  The quality of the transfection experiments was evaluated by calculating the Z factor using Equation S3, Equation S3: Z – factor = 1-- 25 With the mean and standard deviation of relative unit (RFU) of both the positive (p = cells transfected with mixture of siEGFP and amphiphilic polymers or Lipofectamine® 2000) and negative (n = cells not transfected in medium supplemented with 0. 125% (v / v) DMSO) controls (µp, σp, and µn, σn).  Where µ is the average value and σ is the standard deviation.  A Z factor between 0. 5 and 1. 0 indicates an excellent trial, 0. 5 is equivalent to separation of 12 standard deviations between µp and µn.  As shown in Figure 2, the compositions of the present invention are as effective as Lipofectamine® 2000 but at 10 times lower concentrations.  Example 10: General conditions for In Vitro screening of amphiphilic polymers in the transfer of siRNA The stock solutions of the freshly prepared amphiphilic polymers were prepared in DMSO / buffer (v / v) 10 as described above.  The siRNA solutions / amphiphilic polymer compositions were prepared just before the transfection experiments.  10 µl of the siRNA solution (1 µM in DMEM) and 8 µl of amphiphilic polymer solution in DMEM, high glucose, GlutaMAXTM, pyruvate, 10% (v / v) DMSO, supplemented with 10% (v / v) FBS, were added to 190 µl DMEM, high glucose, GlutaMAXTM, pyruvate, supplemented with 10% (v / v) FBS and the mixture was homogenized by pipetting.  Then, the cell medium was aspirated from the 96-well plate and 50 µl of the mixture was added in each well.  The final concentration of DMSO in each well was 0.125% (v / v).  All experiments were performed in triplicate and the results are shown in Figure 3.  Example 11: General conditions for the evaluation of cell viability: MTT assay The cell viability of HeLa-EGFP cells was initially measured as the percentage decrease in fluorescence in samples transfected with siMOCK / amphiphilic polymer complexes and compared. with untreated cells in medium supplemented with 0. 125% (v / v) of DMSO.  The results are shown in Figure 4.     

权利要求:
Claims (1)
[1]
Claims 1. A polymer, salts and stereoisomers thereof, of formula (I) R3X1X2OR0R3n (I) where n is the average number of monomer units that is a number equal to or greater than 10; R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group, and CN; each R3 is independently selected from the group consisting of hydrogen and a C1-C3 alkyl group; X1 is a group selected from the group consisting of -N (H) -, -O-, -N (H) -Alkyl-, and -O-Alkyl-; X2 is independently selected on each unit from the group consisting of -NH2, -N = C (H) R1, and -N = C (H) R2; where R1 is a lipophilic residue and R2 is a cationic residue; and where the percentage of lipophilic residues present in the polymer with respect to the total number of X2 groups is comprised between 1 and 99%; where the percentage of cationic residues present in the polymer with respect to the total number of X2 groups is comprised between 1 and 99%; and the sum of the percentage of lipophilic residues and of cationic residues is comprised between 2 and 100%. 2. The polymer according to claim 1, where n is a number between 10 and 300, preferably equal to or less than 150, preferably between 20 and 120, more preferably between 30 and 100. 3. The polymer according to any of the claims above, where X1 is –N (H) -. 4. The polymer according to any of the preceding claims, wherein R1 is selected from the group consisting of C1-C40 alkyl, C2-C40 alkenyl, C2-C40 alkynyl, C3-C40 cycloalkyl, C4-C40 cycloalkenyl, C5-C40 cycloalkynyl , C6-C40 aryl, C7-C40 alkylaryl, C3-C40 heterocyclyl and C5-C40 heteroaryl, optionally substituted with 1 to 10 groups selected from halogens such as fluorine or ether linkages. 5. The polymer according to any of the preceding claims, wherein R1 is selected from the group consisting of C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl, C4-C40 cycloalkenyl, C5-C10 cycloalkynyl , C7-C15 alkylaryl and C5-C15 heteroaryl, optionally substituted with 1 to 5 groups selected from halogen such as fluorine or ether linkages. 6. The polymer according to any of the preceding claims, wherein R1 is selected from the group consisting of C1-C7 branched alkyl. 7. The polymer according to any of the preceding claims, wherein R2 comprises a positively charged heteroatom. 8. The polymer according to any of the preceding claims, wherein R2 comprises a cationic group having a pKa greater than 4 when protonated. 9. The polymer according to any of the preceding claims, where R2 is a residue of formula -L-G, where L is a connecting group comprising an organic residue, and G is a positively charged group. The polymer according to claim 10, wherein the remainder of formula -L-G has the formula (XI) NH OGab (XI) where a is a number between 1 and 6 ,; b is a number between 1 and 6; and G is a positively charged ammonium, phosphonium, or guanidinium. 11. A polymer according to any of the preceding claims, wherein the percentage of lipophilic residues present in the polymer with respect to the total number of X2 groups is between 5 and 70%, preferably between 7 and 60%, preferably between 7 and 20 %, more preferably between 10 and 20%. 12. A polymer according to any of the preceding claims, wherein the percentage of cationic moieties present in the polymer with respect to the total number of X2 groups is comprised between 10 10 and 99%, preferably between 40 and 95%, preferably between 60 and 90 %, more preferably between 65 and 85%. 13. A polymer according to any of the preceding claims, wherein the sum of the percentage of lipophilic residues and the percentage of cationic residues is between 40 and 80%. 14. A composition comprising the polymer defined in any of claims 1 to 13 and a negatively charged compound. The composition according to claim 14, wherein said negatively charged compound is a nucleic acid or poly (nucleotide). 16. A screening method comprising the step of contacting the composition defined in any of claims 14 or 15, and a lipophilic membrane. 17. A polymer, salts and stereoisomers thereof, as defined in any one of claims 1 to 13 for use as a medicine. 18. A polymer, salts and stereoisomers thereof, as defined in any one of claims 1 to 13 for use in transfection into cells. 19. A composition as defined in any of claims 14 or 15, for use as a medicament. 20. A composition as defined in any of claims 14 or 15, for use in transfection into cells. 21. A pharmaceutical composition comprising the composition defined in any of claims 14 or 15. 22. A process for the preparation of the polymer salts and stereoisomers thereof as defined in any of claims 1 to 13, comprising the step of contacting a polymer of formula (II), salts and stereoisomers thereof R3X1NH2OR0R3n (II) with an aldehyde of formula O = C (H) R1 and an aldehyde of formula O = C (H) R2; where n, X1, R0, R1, R2 and R3 are as defined in any of claims 1 to 13. 23. The process according to claim 22, wherein said aldehyde of formula O = C (H) R1 and said aldehyde of formula O = C (H) R2 are mixed together simultaneously with the polymer of formula (II). 24. The process according to claim 22, wherein said aldehyde of formula O = C (H) R1 and said aldehyde of formula O = C (H) R2 are mixed together consecutively with the polymer of formula (II). 25. The process according to any of claims 22 to 24, wherein said aldehyde of formula O = C (H) R1 and said aldehyde of formula O = C (H) R2 are added in amounts between 0.001 and 10 equivalents with respect to total number of NH2 moieties available. 26. The process according to any of claims 22 to 25, wherein the preparation of the polymer of formula (II), salts and stereoisomers thereof, comprises the step of eliminating the R8 group of a polymer of formula (III), salts and stereoisomers thereof, R3X1NHOR8R0R3n (III) where n, X1, R0 and R3 are as defined in any one of claims 1 to 13; and R8 is a protecting group. 27. The process according to claim 26, wherein R8 is a protecting group of formula - (O =) C-O-R9, where R9 is a C1-C12 alkyl group. 28. The process according to any of claims 26 or 27, wherein the reaction takes place in aqueous medium. 29. The process according to any of claims 26 to 28, wherein the preparation of the polymer of formula (III), salts and stereoisomers thereof, comprises polymerizing a compound of formula (IV), salts and stereoisomers thereof, R3X1NHOR8R0R3 (IV ) where X1 is a group selected from the group consisting of -N (H) -, -O-, -N (H) -Alkyl- and -O-Alkyl-15; R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group, and CN; each R3 is independently selected from the group consisting of hydrogen and a C1-C3 alkyl group; and R8 is a protecting group. 30. The process according to claim 29, wherein the preparation of a compound of formula (IV), salts and stereoisomers thereof, comprises the step of contacting a compound of formula (V), salts and stereoisomers thereof, with a protecting group - R8 or with a compound of formula -N (H2) -N (H) -R8, -ON (H) -R8, -N (H2) -Alkyl-N (H) -R8 and -O- Alkyl-N (H) -R8 R3X3OR0R3 (V) where R0 is selected from the group consisting of hydrogen, a C1-C3 alkyl group, and CN; each R3 is independently selected from the group consisting of hydrogen and a C1-C3 alkyl group; and X3 is selected from the group consisting of -OH, halogen, O-alkyl, -N (H) -N (H2), -ON (H2), -N (H) -Alkyl-N (H2) and - O-Alkyl-N (H2). 31. The process according to claim 30, wherein said carbazate is selected from the group consisting of t-butyl carbazate, benzyl carbazate, ethyl carbazate, methyl carbazate, and mixtures thereof.

类似技术:

---

公开号 | 公开日 | 专利标题

---

Blasco et al.2017|50th anniversary perspective: polymer functionalization

---

Danial et al.2013|Janus cyclic peptide–polymer nanotubes

---

Fitzgerald et al.2016|A novel, anisamide-targeted cyclodextrin nanoformulation for siRNA delivery to prostate cancer cells expressing the sigma-1 receptor

---

Danial et al.2014|Thermal gating in lipid membranes using thermoresponsive cyclic peptide–polymer conjugates

---

ES2759112T3|2020-05-07|Cucurbituril-based hydrogels

---

Xu et al.2009|Synthesis of dendritic carbohydrate end‐functional polymers via RAFT: Versatile multi‐functional precursors for bioconjugations

---

BRPI0014652B1|2016-08-09|manufacture of therapeutic agent-polyglutamate conjugates

---

WO2007048190A1|2007-05-03|Macromolecular compounds having controlled stoichiometry

---

KR20100119491A|2010-11-09|Oligocarbonate molecular transporters

---

Sánchez-Nieves et al.2014|Amphiphilic cationic carbosilane–PEG dendrimers: synthesis and applications in gene therapy

---

ES2797026T3|2020-12-01|Amphiphilic Polymer Systems

---

Bartolami et al.2015|Multivalent DNA recognition by self-assembled clusters: deciphering structural effects by fragments screening and evaluation as siRNA vectors

---

KR20140120316A|2014-10-13|Chitosan covalently linked with small molecule integrin antagonist for targeted delivery

---

Tschiche et al.2016|Crosslinked Redox‐Responsive Micelles Based on Lipoic Acid‐Derived Amphiphiles for Enhanced siRNA Delivery

---

ES2618373B1|2018-04-09|Aqueous synthesis and rapid in situ screening of amphiphilic polymers

---

Su et al.2020|Cationic dynamic covalent polymers for gene transfection

---

KR20140127234A|2014-11-03|Integrin antagonist conjugates for targeted delivery to cells expressing vla-4

---

WO2011056645A2|2011-05-12|Poly| and derivatives thereof

---

ES2705068T3|2019-03-21|Chemical synthesis and screening of bicyclic peptide libraries

---

Bang et al.2018|Amphiphilic small peptides for delivery of plasmid DNAs and siRNAs

---

DK2806897T3|2017-10-16|INTEGRIN ANAGONIST CONJUGATES FOR TARGETED RELEASE TO LFA-1 EXPRESSING CELLS

---

US10562901B2|2020-02-18|Temozolomide compounds, polymers prepared therefrom, and method of treating a disease

---

Abel2016|Addressing the Issues of Drug Delivery via Advanced Macromolecular Design

---

Yadav et al.2017|Dendrimeric amide-and carbamate-linked lysine-based efficient molecular transporters

---

Simpson2018|Synthesis, Characterization, and Assessment of Cationic Polypeptoids Toward Gene Delivery and Development of Air Stable N-Substituted N-Thiocarboxyanhydrides

同族专利:

---

公开号 | 公开日

---

EP3390471A1|2018-10-24|

---

WO2017102894A1|2017-06-22|

---

ES2618373B1|2018-04-09|

---

US20180371138A1|2018-12-27|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

US3395134A|1960-05-12|1968-07-30|Gaetano F D'alelio|Chelating polymers and method of preparation|

---

US4782900A|1987-04-24|1988-11-08|Pfizer Inc.|Aminoalkylated polyacrylamide aldehyde gels, their preparation and use in oil recovery|

---

DE60307133T2|2002-11-29|2007-04-12|Ciba Speciality Chemicals Holding Inc.|AQUEOUS COMPOSITIONS WITH HOMO AND / OR COPOLYMERS|

---

US20060228406A1|2005-03-17|2006-10-12|Invitrogen Corporation|Transfection reagent for non-adherent suspension cells|

---

CN101778873B|2007-06-15|2012-10-31|巴科曼实验室国际公司|High solids glyoxalated polyacrylamide|

---

US20100034748A1|2008-08-07|2010-02-11|Guizhi Li|Molecular imaging probes based on loaded reactive nano-scale latex|

---

AU2012377385A1|2012-04-18|2014-01-23|Arrowhead Research Corporation|Poly polymers for in vivo nucleic acid delivery|

---

法律状态:
2018-04-09| FG2A| Definitive protection|Ref document number: 2618373 Country of ref document: ES Kind code of ref document: B1 Effective date: 20180409 |

优先权:

---

申请号 | 申请日 | 专利标题

---

ES201531831A|ES2618373B1|2015-12-17|2015-12-17|Aqueous synthesis and rapid in situ screening of amphiphilic polymers|ES201531831A| ES2618373B1|2015-12-17|2015-12-17|Aqueous synthesis and rapid in situ screening of amphiphilic polymers|

---

EP16822404.6A| EP3390471A1|2015-12-17|2016-12-14|Aqueous synthesis and in-situ rapid screening of amphiphilic polymers|

---

PCT/EP2016/081085| WO2017102894A1|2015-12-17|2016-12-14|Aqueous synthesis and in-situ rapid screening of amphiphilic polymers|

---

US16/062,536| US20180371138A1|2015-12-17|2016-12-14|Aqueous synthesis and in-situ rapid screening of amphiphilic polymers|