EP4720137A1 - Polymers for delivery of active agents, compositions comprising said polymers, and uses thereof - Google Patents

Abstract

The present invention relates to a polymer for delivery of active agents. Said polymers comprising amino acid derivatives, such as cationic polymers comprising amino acid derivatives, wherein the polymer according to the invention may also be referred to as PiPOx-XXX, wherein XXX refers to the amino acid derivative. Furthermore, the invention also relates to a composition comprising said polymer and use thereof.

Description

POLYMERS FOR DELIVERY OF ACTIVE AGENTS, COMPOSITIONS COMPRISING SAID POLYMERS, AND USES THEREOF

FIELD OF THE INVENTION

The present invention pertains to the field of smart biomaterials. More in particular, the present invention pertains to polymers comprising amino acid derivatives, such as cationic polymers comprising amino acid derivatives. Furthermore, the invention also relates to a composition comprising said polymers and uses thereof.

BACKGROUND TO THE INVENTION

Gene therapy has great potential in the treatment of genetically based diseases, such as cancer. A major problem in exploiting the full potential of, for example, cancer gene therapy is the lack of safe and efficient delivery systems for nucleic acids used therein. Other examples which may lack safe and efficient delivery systems may be protein replacement therapy and nucleic acidbased vaccination. Despite having a good transfection efficiency, viral vectors raise concerns about their safety, due to risk of immunogenicity and insertional mutagenesis. In this context, non-viral vectors represent a safer alternative for gene delivery. The non-viral transfection of genetic material (e.g. nucleic acids, such as DNA, RNA or proteins, such as ribonucleoproteins complexes, etc.), into cells also known as gene delivery, was simplified over the years by the development of synthetic vectors. Synthetic vectors are materials that bind electrostatically and condense nucleic acids into nanoparticles, having a size of tens to several hundreds of nm, thereby protecting them against degradation and mediating their entry into cells. Both, cationic lipids and polymers can be used to complex nucleic acids, thereby creating lipoplexes, also known as lipid nanoparticles (LNPs) and polyplexes, respectively. Even though LNPs have rapidly entered the market as mRNA vaccines for COVID-19, their production is cumbersome and expensive.

The development of cationic polymers plays an important role in delivery of genetic material, due to the large possibility to modulate their structural and functional parameters. One of the most commonly used non-viral polymeric vectors for nucleic acid transfection is linear polyethylenimine (PEI). However, high molecular weight PEI (with 500 repeat units) showed high in vitro and in vivo cytotoxicity, while low molecular weight PEI (with less than 250 repeat units), on the other hand, has demonstrated low toxicity in cell culture studies but also leads to lower transfection efficiency. Alternatives such as poly(P-amino esterjs, poly(lysine)s, cyclodextrin derivatives, dendrimers, poly(amidoamine)s, PEGylated polymers, methacrylate/methacrylamide polymers, poly(allylamine)s, and poly(2-oxazoline)s (PiPOx) have been developed gaining ground on efficacy and reducing toxicity, albeit without meeting satisfaction. Therefore, it can be concluded that successful gene delivery is a complex multi-step process requiring nucleic acid complexation, nanoparticle formation, diffusion through biological barriers, cellular uptake, endosomal escape, and nucleic acid release. Up to now, efficiently tackling all these steps still represent a big challenge for synthetic carriers, therefore the search for novel polymers for efficient transfection remains a current goal.

Polymer-based transfection remains of interest owing to the ease of polyplex formation by simply mixing the polymer and the nucleic acids as well as the chemical diversity of polymers to access linear, branched, or dendritic polymeric structures with multiple functions to optimize the transfection efficiency. It is known that the structural and functional parameters of polymers such as length, dispersity, composition, linkage, sequences, as well as capacity to further selfassemble into higher-order assemblies (e.g. micelles, polymersomes) determine the binding efficacy, binding selectivity, cell penetration, and toxicity towards transfected cells.

The present invention aims at obviating or at least reducing the aforementioned problems and to enable efficient and effective transfection/ delivery.

It is an objective of the invention to provide a (cationic) polymer for efficient transfection of active agents, in particular nucleic acids.

It was surprisingly found that a (cationic) polymer comprising amino acid derivatives provides efficient and effective transfection.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a polymer comprising m monomers according to formula (I), or a stereoisomer, a tautomer, a racemic, a salt, a hydrate, a N-oxide or solvate thereof, wherein: m is an integer in the range of 10 to 1000; n is an integer in the range of 0 to 50;

R¹ in combination with the adjacent carboxy group may be for each monomer individually an amino acid derivative, wherein the polymer comprises at least two different amino acid derivatives; and

R² may be for each monomer individually selected from hydrogen or methyl.

It is noted that R¹ is individually selected for each monomer. Therefore, different amino acid derivatives may be present in the polymer according to the invention.

In a further presently preferred embodiment according to the invention, each amino acid derivative may be independently selected from the group of tryptophan derivative, tyrosine derivative, arginine derivative, glycine derivative, alanine derivative, valine derivative, proline derivative, lysine derivative, phenylalanine derivative, histidine derivative, isoleucine derivative, leucine derivative, methionine derivative, threonine derivative, cysteine derivative, glutamine derivative, aspartic acid derivative, asparagine derivative, glutamic acid derivative, serine derivative, selenocysteine derivative, pyrrolysine derivative.

In a further presently preferred embodiment according to the invention, each amino acid derivative may be independently selected from the sub-group of aromatic amino acid derivatives, carboxylic amino acid derivatives, charged amino acid derivatives, hydrophobic amino acid derivatives, hydrophilic amino acid derivatives, and polar uncharged amino acid derivative. It is noted that amino acid derivatives may be present in multiple sub-groups. Preferably, the charged amino acid derivative comprises a positive charge, more preferably comprises a positively charged and/or protonatable group.

The aromatic amino acid derivatives may include histidine derivative, phenylalanine derivative, tyrosine derivative, and tryptophan derivative. The charged amino acid derivatives may include arginine derivative, glycine derivative, histidine derivative, lysine derivative, proline derivative, and selenocysteine derivative. The hydrophobic amino acid derivatives may include alanine derivative, valine derivative, isoleucine derivative, leucine derivative, methionine derivative, phenylalanine derivative, tyrosine derivative, and tryptophan derivative. The hydrophilic amino acid derivatives may include arginine derivative, lysine derivative, asparagine derivative, histidine derivative, and proline derivative. The polar uncharged amino acid derivative may include serine derivative, threonine derivative, asparagine derivative, and glutamine derivative.

In a further presently preferred embodiment according to the invention, each amino acid derivative may be independently selected from the group of tryptophan derivative, tyrosine derivative, arginine derivative, glycine derivative, alanine derivative, valine derivative, proline derivative, lysine derivative, phenylalanine derivative, and histidine derivative. Preferably, each amino acid derivative may be independently selected from the group of tryptophan derivative, tyrosine derivative, and arginine derivative.

In a further presently preferred embodiment according to the invention, the at least two different amino acid derivatives comprises tryptophan, more preferably the at least two different amino acid derivatives are phenylalanine derivatives or tryptophan derivatives and valine derivatives or proline derivatives. In other words, the two different amino acid derivatives are phenylalanine derivatives and valine derivatives, or phenylalanine derivatives and proline derivatives, or tryptophan derivatives and valine derivatives, or tryptophan derivatives and proline derivatives. It is noted that the polymer according to the invention preferably comprises tryptophan derivatives and valine derivatives, or tryptophan derivatives and proline derivatives.

In a further presently preferred embodiment according to the invention, one or more amino acid derivatives comprise an aromatic moiety.

In a further presently preferred embodiment according to the invention, the amino acid derivatives are coupled via the carboxylic acid on the C-terminus.

In a further presently preferred embodiment according to the invention, the amino acid derivative comprises a hydrophobic amino acid derivative. Preferably, at least 20 mol% of the amino acid derivatives present in the polymer is hydrophobic, more preferably at least 25 mol% of the amino acid derivatives present in the polymer is hydrophobic, even more preferably at least 30 mol% of the amino acid derivatives present in the polymer is hydrophobic, even more preferably at least 35 mol% of the amino acid derivatives present in the polymer is hydrophobic, most preferably at least 40 mol% of the amino acid derivatives present in the polymer is hydrophobic.

In a further presently preferred embodiment according to the invention, at most 100 mol% of the amino acid derivatives present in the polymer is hydrophobic, preferably, at most 95 mol% of the amino acid derivatives present in the polymer is hydrophobic, more preferably at most 90 mol% of the amino acid derivatives present in the polymer is hydrophobic, most preferably at most 85 mol% of the amino acid derivatives present in the polymer is hydrophobic.

In a further presently preferred embodiment according to the invention, between 20 mol% and 100 mol% of the amino acid derivatives present in the polymer may be hydrophobic, preferably between 20 mol% and 95 mol% of the amino acid derivatives present in the polymer may be hydrophobic, more preferably between 25 mol% and 95 mol% of the amino acid derivatives present in the polymer may be hydrophobic, even more preferably between 30 mol% and 95 mol% of the amino acid derivatives present in the polymer may be hydrophobic, even more preferably between 30 mol% and 90 mol% of the amino acid derivatives present in the polymer may be hydrophobic, even more preferably between 35 mol% and 90 mol% of the amino acid derivatives present in the polymer may be hydrophobic, most preferably between 40 mol% and 85 mol% of the amino acid derivatives present in the polymer may be hydrophobic.

In a further presently preferred embodiment according to the invention, m may be an integer in the range of 50 to 1000, preferably in the range of 100 to 500, more preferably in the range of 100 to 250.

In a further presently preferred embodiment according to the invention, n may be an integer in the range of 1 to 40, more preferably n may be an integer in the range of 1 to 30, even more preferably n may be an integer in the range of 1 to 20, even more preferably n may be an integer in the range of 1 to 10, most preferably n may be an integer in the range of 1 to 5.

In a further presently preferred embodiment according to the invention, n is I .

In a further presently preferred embodiment according to the invention, R² may be methyl.

In a further presently preferred embodiment according to the invention, the polymer further comprises k monomers individually selected from the group of ethylene glycol, acrylate, and methacrylate, wherein k is an integer in the range of 1 to 1000. Preferably, the k monomers are statistically or randomly distributed in the polymer or form blocks.

In a further presently preferred embodiment according to the invention, one or more amino acid derivatives comprises a Boc group, trifluoroacetic acid counterion group, acetate counterion group.

In a second aspect, the present invention provides a composition comprising a polymer according to the invention, wherein the polymer is a delivery agent.

In a further presently preferred embodiment according to the invention, the composition further comprises an active agent.

In a further presently preferred embodiment according to the invention, the active agent comprises nucleic acids.

In a further presently preferred embodiment according to the invention, the active agent may be one or more selected from the group of RNA, siRNA, mRNA, self-amplifying mRNA, circular RNA, tRNA, rRNA, viral cRNA, miRNA, IncRNA, antisense oligonucleotides of DNA, antisense oligonucleotides of RNA, guide RNA, DNA, plasmid DNA, ribonucleoproteins. Preferably, the active agent may be one or more selected from the group of RNA, siRNA, mRNA, DNA, plasmid DNA, more preferably the active agent is siRNA.

In a third aspect, the present invention provides a polymer according to the invention, or composition according to the invention, for use as a human or veterinary medicine, or for use in field crop protection.

In a fourth aspect, the present invention provides a use of a polymer according to the invention, or a composition according to the invention, as a delivery agent of an active agent, preferably as a delivery agent of siRNA; or as a delivery agent of plant protection agents, such as siRNA molecules used in plant protection applications.

In a further presently preferred embodiment according to the invention, the active agent is a medicament.

In a further presently preferred embodiment according to the invention, the polymers or compositions of the invention are for use in the prevention and/or treatment of at least one disease or disorder selected from the group comprising oncological diseases, infectious diseases, genetic disorders. The invention also provides the use of the polymers or compositions of the invention in the prevention and/or treatment of at least one disease or disorder selected from the group comprising oncological diseases, infectious diseases, genetic disorders, or crop diseases.

In a fifth aspect, the present invention provides a method for the prevention and/or treatment of at least one disease or disorder selected from the group comprising oncological diseases, infectious diseases, genetic disorders; said method comprising administering to a subject in need thereof a therapeutic effective amount of a compound according to the invention, or a composition according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

Fig. 1 : Reaction route for (a) the synthesis of fully modified PiPOx homopolymers with N-(tert- Butoxycarbonyl)-amino acids (NBAA), (b) the synthesis of fully modified PiPOx copolymers with NBAA, and (c) for the preparation of the final cationic polymethacrylamide (co)polymers. Fig. 2 : Flow cytometric quantification of silencing efficiency in H1299-eGFP cells of polymers that showed to complex siRNA. Transfection was performed at a siRNA concentration of 50 nM for each sample and the silencing is expressed as mean ± standard deviation for three technical replicates (a); Representative histograms of eGFP silencing induced by PiPOx-Pro Trp complexed with eGFP targeting siRNA (sieGFP) compared to complexation with non-targeting siRNA (siCTRL) (b).

Fig. 3: Quantification of the percentage of H1299-eGFP cells in which Cy5-labeled siRNA (siCy5) could be detected (a); Mean fluorescence intensity (MFI) of Cy5 -positive cell populations (b); Representative dot-plots of cells transfected with the indicated polyplexes, loaded with Cy5-labeled siRNA (c). Data are represented as mean ± standard deviation for three technical replicates.

Fig. 4: Efficiency of eGFP silencing of PiPOx-AA copolymers in comparison to JetPRIME® commercial transfection reagent. Data is presented as mean ± standard deviation.

Fig. 5: Cell viability of H1299-eGFP cell treated for 4h with mounting concentrations of the indicated PiPOx polymers. The concentrations used for cell transfection experiments corresponded to < 0.01 mg/ml, maintaining a cell viability above 90% for both polymers.

Fig. 6: Flow cytometric quantification of silencing efficiency in H1299-eGFP cells of PiPOx- Val Trp and PiPOx-Pro Trp copolymers with different chain length: a) details of studied polymers; b) Mean fluorescence intensity for transfection at a siRNA concentration of 50 nM for each sample and the silencing is expressed as mean ± standard deviation for three technical replicates. siCTRL samples were transfected with non-eGFP silencing siRNA, siEGFP samples wree transfected with EGFP silencing siRNA and NTC is the non-transfected control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The term “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/- 10 % or less, preferably +/- 5 % or less, more preferably +/- I % or less, and still more preferably +/- 0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

When describing the compounds of the invention, the terms used are to be construed in accordance with the following definitions, unless a context dictates otherwise:

Whenever the term “substituted” is used in the present invention, it is meant to indicate that one or more hydrogens on the atom indicated in the expression using “substituted” is replaced with a selection from the indicated group, provided that the indicated atom’s normal valency is not exceeded, and that the substitution results in a chemically stable compound, i.e. a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into a therapeutic agent.

As described herein, some of the compounds of the invention may contain one or more asymmetric carbon atoms that serve as a chiral center, which may lead to different optical forms (e.g. enantiomers or diastereoisomers). The invention comprises all such optical forms in all possible configurations, as well as mixtures thereof.

More generally, from the above, it will be clear to the skilled person that the compounds of the invention may exist in the form of different isomers and/or tautomers, including but not limited to geometrical isomers, conformational isomers, E/Z-isomers, stereochemical isomers (i.e. enantiomers and diastereoisomers) and isomers that correspond to the presence of the same substituents on different positions of the rings present in the compounds of the invention. All such possible isomers, tautomers and mixtures thereof are included within the scope of the invention.

As already detailed herein above, the present invention provides a polymer comprising m monomers according to formula (I), or a stereoisomer, a tautomer, a racemic, metabolite, pro- or predrug, a salt, a hydrate, a N-oxide or solvate thereof, wherein: m is an integer in the range of 10 to 1000; n is an integer in the range of 0 to 50;

R¹ in combination with the adjacent carboxy group is for each monomer individually an amino acid derivative, wherein the polymer comprises at least two different amino acid derivatives; and

R² is for each monomer individually selected from the group of hydrogen or methyl.

It is noted that the monomers, such as monomers m, in a coupled state form a polymer, and that the weight of the polymer is (partly) defined by the amount of monomers connected to each other.

It is also noted that the carboxy group of formula I may also be referred to as ester. Thus, R¹ in combination with the adjacent carboxy group may be referred to as R¹ in combination with the adjacent ester is for each monomer individually an amino acid derivative.

It is also noted that R¹ in combination with the adjacent carboxy group forms an amino acid derivative. Throughout this application an amino acid derivative refers to a chemical compound or chemical group that is structurally derived from a parent compound through one or several steps. For example, the reaction of a (protected, for example Boc protected) amino acid or salt thereof with a starting polymer provides a polymer comprising amino acid derivatives.

In other words, in spite of this specific determination of amino acids or derivatives thereof, it is obvious that derivatives of these amino acids resembling alike structural and physico-chemical properties, lead to functional analogs with similar biological activity, and therefore still form part of the gist of the present invention. Thus, the amino acid derivative refers to the part of a molecule, for example monomer unit, originating from an amino acid which is covalently coupled with another part of a molecule.

It is also noted that R¹ is individually selected for each monomer. Therefore, different amino acid derivatives are present in the polymer according to the invention.

It is also noted that throughout this application the polymer according to the invention may also be referred to as PiPOx-XXX, wherein XXX refers to the amino acid derivative or may be replaced by the letter code, for example three letter code, of an amino acid derivative. In addition, a polymer comprising two different amino acid derivatives may be referred to as PiPOx-

XXX XXX. For example, PiPOx-Pro refers to PiPOx with proline derivatives. Nonetheless, the same polymers can be accessed through polymerization (radical or anionic) of the corresponding

(meth)acrylamide monomers that comprise a (protected) amino acid side chain.

It is noted that the following three letter codes apply for the amino acid derivatives:

Tryptophan Trp Valine Vai Isoleucine He

Tyrosine Tyr Proline Pro Leucine Leu

Arginine Arg Lysine Lys Methionine Met

Glycine Gly Phenylalanine Phe Threonine Thr

Alanine Ala Histidine His Selenocysteine Sec

Glutamine Gin Serine Ser Asparagine Asn

Pyrrolysine Pyl

An advantage of the polymers according to the invention is that polymers may be non-viral vectors for transfection. As a result, stable complexes with further molecules such as active agents, for example active agents comprising nucleotides and/or nucleic acids, may be formed. Furthermore, it was found that the polymers according to the invention showed efficient and effective transfection ability of > 90% of the cells and potent gene silencing with IC50 values down to 5.5 nM. In addition, the polymers according to the invention provide higher gene silencing compared to commercial polycationic transfection reagents, such as JetPRIME®, without reducing cell viability (figure 4).

In addition, the toxicity of the polymers according to the invention was lower compared to conventional polymers and/or delivery agents when used in humans, animals, or plants (figure 5).

A further advantage of the polymers according to the invention is that said polymers exhibited good thermal stability and preserved their optical activity. As a result, the polymers according to the invention may be applicable in the development of chiral materials for applications in asymmetric catalysis, enantiomer selective release, and chiral resolution.

It was found that amino acid derivatives provide efficient and effective transfection of molecular structures comprising nucleic acids.

In a further preferred embodiment, each amino acid derivative may be independently selected from the group of tryptophan derivative, tyrosine derivative, arginine derivative, glycine derivative, alanine derivative, valine derivative, proline derivative, lysine derivative, phenylalanine derivative, histidine derivative, isoleucine derivative, leucine derivative, methionine derivative, threonine derivative, cysteine derivative, glutamine derivative, aspastic acid derivative, asparagine derivative, glutamic acid derivative, serine derivative, selenocysteine derivative, pyrrolysine derivative. Preferably, each amino acid derivative may be independently selected from the group of tryptophan derivative, tyrosine derivative, arginine derivative, glycine derivative, alanine derivative, valine derivative, proline derivative, lysine derivative, phenylalanine derivative, and histidine derivative. More preferably, each amino acid derivative may be independently selected from the group of tryptophan derivative, tyrosine derivative, and arginine derivative, even more preferably the amino acid derivative is a tryptophan derivative.

In a further preferred embodiment, one or more amino acid derivatives may be a hydrophobic amino acid, preferably, the hydrophobic amino acid is one or more independently selected from the group of alanine derivative, valine derivative, leucine derivative, isoleucine derivative, proline derivative, phenylalanine derivative.

In a further preferred embodiment, the amino acid derivatives are a combination of a valine derivative and a tryptophan derivative, or a proline derivative and a tryptophan derivative.

An advantage of amino acid derivatives such as tryptophan derivative, tyrosine derivative, lysine derivative, histidine derivative, and arginine derivative, preferably tryptophan derivative, tyrosine derivative, and arginine derivative, is that a positive charge can be efficiently distributed. As a result, efficient and effective delivery of molecules comprising nucleotides to desired tissue is achieved.

A further advantage of said amino acids and/or all the aforementioned amino acids is that polymer-based transfection is achieved. Furthermore, said amino acid derivatives do not influence the chemical diversity of the polymers. Therefore, chemical diversity of the polymers according to the invention, such as access to linear, branched, hyperbranched, and/or dendritic polymeric structures with multiple functions to optimize the transfection efficiency, is achieved.

It was found that said amino acids influence the structural and functional parameters of the polymers according to the invention and that said parameters could be adapted to the desired properties. For example, it was found that the length, dispersity, composition, linkage, sequences, as well as capacity to further self-assemble into higher-order assemblies (e.g. micelles, polymersomes) determine the binding efficacy, binding selectivity, cell penetration, and toxicity towards transfected cells.

In a further preferred embodiment, the polymer may comprise monomers according to formula

II, or a stereoisomer, a tautomer, a racemic, a salt, a hydrate, a N-oxide or solvate thereof, wherein:

R³ is for each monomer individually selected from the group of hydrogen and methyl. Preferably, R³ is methyl.

It is noted that a monomer according to formula II may be a starting material to form the monomer according to formula I.

It was found that the monomer according to formula II did not influence the properties of the polymers according to the invention when present for less than 15% of the total monomers (monomeric units) in the polymers according to the invention, preferably less than 10% of the total monomers (monomeric units) in the polymers according to the invention, more preferably less than 5% of the total monomers (monomeric units) in the polymers according to the invention.

In a further presently preferred embodiment according to the invention, one or more amino acid derivatives comprise one or more of an aromatic moiety, and/or a positive charge, a carboxylic group, one or more primary amino group in a side chain of the monomer. Preferably, the one or more amino acid derivative comprising a positive charge in said side chain comprises a positively charged and/or protonatable group.

It is noted that, without to being bound to theory, the aromatic units may interact with the bases of the nucleic acid. Amine moieties may provide a positive charge an may interact with negatively charged nucleic acid units such that solubility, in for example water, is achieved.

Preferably, the polymer according to the invention is a cationic polymer and/or comprises one or more amino acid derivative comprising an aromatic group and/or one or more primary amino group.

An advantage of a cationic polymer according to the invention is the possibility of said polymers to modulate the structural and functional parameters.

It was found that cationic polymers with amino acid moieties in the side chain with exposed primary amino groups provide efficient transfection and show low cytotoxicity and strong binding ability with molecules such as nucleic acids. These effects were even stronger for polymers comprising tryptophan derivatives. Said polymers showed low water solubility and induced aggregation.

Furthermore, it was found that incorporation of the tryptophan derivative (indole unit) into a polymeric sequence improves the formation of polyplexes with increased cellular uptake and transfection efficiency. In addition, the tryptophan derivatives enable membrane interaction of cell penetrating peptides, determining increase in cellular internalization. Thus, partial incorporation of amino acid derivatives comprising aromatic moieties in polymers as side chain groups provide more effective delivery of molecules comprising nucleotides, such as genes, by enabling facilitating the uptake and endosomal escape of the payload.

In a further presently preferred embodiment, the amino acid derivatives are coupled via the carboxylic acid on the C-terminus.

An advantage of the amino acid derivatives which are coupled via the carboxylic acid on the C- terminus is that the polymer according to the invention is positively charged in water.

In a further preferred embodiment, the polymer according to the invention is soluble in an aqueous solvent. Preferably, the solvent comprises at least 25 vol% of water, more preferably the solvent comprises at least 30 vol% of water, even more preferably the solvent comprises at least 35 vol% of water, even more preferably the solvent comprises at least 40 vol% of water, even more preferably the solvent comprises at least 45 vol% of water, most preferably the solvent comprises at least 50 vol% of water.

In a further preferred embodiment, the solvent comprises at most 100 vol% of water, preferably the solvent comprises at most 90 vol% of water, even more preferably the solvent comprises at most 80 vol% of water, most preferably the solvent comprises at most 70 vol% of water.

In a further presently preferred embodiment according to the invention, the amino acid derivative comprises a hydrophobic amino acid derivative. Preferably, at least 20 mol% of the amino acid derivatives present in the polymer is hydrophobic, more preferably at least 25 mol% of the amino acid derivatives present in the polymer is hydrophobic, even more preferably at least 30 mol% of the amino acid derivatives present in the polymer is hydrophobic, even more preferably at least 35 mol% of the amino acid derivatives present in the polymer is hydrophobic, most preferably at least 40 mol% of the amino acid derivatives present in the polymer is hydrophobic.

In a further presently preferred embodiment according to the invention, at most 100 mol% of the amino acid derivatives present in the polymer is hydrophobic, preferably, at most 95 mol% of the amino acid derivatives present in the polymer is hydrophobic, more preferably at most 90 mol% of the amino acid derivatives present in the polymer is hydrophobic, most preferably at most 85 mol% of the amino acid derivatives present in the polymer is hydrophobic.

Providing a polymer comprising one or more hydrophobic amino acids enables an efficient and effective transfection and enables an efficient and effective delivery agent of molecules comprising nucleotides. The amount of hydrophobic amino acid derivative is determined using standard 'H-NMR. In a further preferred embodiment according to the invention, m may be an integer in the range of 50 to 1000, preferably in the range of 100 to 500, more preferably in the range of 100 to 250.

A value of m being an integer in the range of 50 to 1000, preferably in the range of 50 to 500, more preferably in the range of 60 to 400; more preferably in the range of 75 to 350; more preferably in the range of 100 to 250, enables efficient and effective transfection.

In a further preferred embodiment according to the invention, R² may be methyl. Preferably, R² being methyl is in combination with the polymer according to the invention being cationic.

An advantage of R² being methyl is that said group increases the stability of the polymer according to the invention.

In a further preferred embodiment according to the invention, the polymer further comprises k monomers individually selected from the group of ethylene glycol, acrylate, and methacrylate, wherein k is an integer in the range of 1 to 1000. Preferably, the k monomers are statistically or randomly distributed in the polymer or form blocks.

An advantage of including a k monomers in the polymer according to the invention is that the properties of the polymer according to the invention as delivery agent may be further adapted to the needs of a user. Therefore, a wider range of active agents may be delivered with the polymer according to the invention, including proteins.

In a further preferred embodiment according to the invention, one or more amino acid derivatives comprise a Boc group, trifluoroacetic acid counterion group, acetate counterion group.

It is noted that one or more amino acid derivatives comprising a Boc group, trifluoroacetic acid counterion group, or acetate counterion group may not have been deprotected or are introduced to increase the transfection or lower toxicity.

As already detailed herein above, the present invention provides a composition comprising a polymer according to the invention, wherein the polymer is a delivery agent.

The compositions provide the same effects and advantages as those described for the polymers according to the invention.

An advantage of the compositions according to the invention is that the polymers are less toxic, efficient and effective delivery agents compared to conventional delivery agents.

In a preferred embodiment according to the invention, the compositions further comprise an active agent. Preferably, the active agent comprises nucleic acids.

In a preferred embodiment, the invention provides a composition comprising one or more of the polymers of the invention complexed with nucleic acids, in particular having an N/P ratio between 1 and 40, preferably between 1 and 30, more preferably between 1 and 25, most preferably between 1 and 20.

It was found that the polymers according to the invention and the active agent comprising nucleic acids provide an efficient composition for transfection.

In a further preferred embodiment according to the invention, the active agent may be one or more selected from the group of RNA, siRNA, mRNA, circular RNA, self-amplifying mRNA, tRNA, rRNA, miRNA, IncRNA, antisense oligonucleotides (DNA or RNA), guide RNA, viral cRNA, DNA, plasmid DNA, ribonucleoproteins. Preferably, the active agent may be one or more selected from the group of RNA, siRNA, mRNA, DNA, plasmid DNA, proteins, more preferably the active agent is siRNA.

It was found that the polymer according to the invention provides an efficient and effective composition with one or more active agent selected from the group of RNA, siRNA, mRNA, tRNA, rRNA, viral cRNA, DNA, plasmid DNA, proteins. In particular, the polymer according to the invention provides an effective and efficient composition with siRNA.

As already detailed herein above, the present invention provides a polymer according to the invention, or composition according to the invention, for use as a human or veterinary medicine.

The use as a human or veterinary medicine, provides the same effects and advantages as those described for the polymers according to the invention and compositions according to the invention.

In a preferred embodiment according to the invention, the use of the polymers or compositions is in the prevention and/or treatment of at least one disease or disorder selected from the group comprising oncological diseases, infectious diseases, genetic disorders.

As already detailed herein above, the present invention provides a use of a polymer according to the invention, or a composition according to the invention, as a delivery agent of an active agent, preferably as a delivery agent of siRNA; or as a delivery agent of plant protection agents. Preferably, the active agent is a medicament.

The use as a delivery agent of an active agent, for example being a medicament, preferably as a delivery agent of siRNA; or as a delivery agent of plant protection agents provides the same effects and advantages as those described for the polymers according to the invention, compositions according to the invention, and uses as a human or veterinary medicine, or use in field crop protection according to the invention.

Furthermore, the active agent enables the use as prophylactic agent. As already detailed herein above, the present invention provides a method for the prevention and/or treatment of at least one disease or disorder selected from the group comprising oncological diseases, infectious diseases, genetic disorders; said method comprising administering to a subject in need thereof a therapeutic effective amount of a compound according to the invention, or a composition according to the invention.

The method for the prevention and/or treatment according to the invention provides the same effects and advantages as those described for the polymers according to the invention, compositions according to the invention, uses as a human or veterinary medicine, or uses in field crop protection according to the invention, and uses as a delivery agent of an active agent, for example a medicament, preferably as a delivery agent of siRNA according to the invention.

The compounds of the present invention can be prepared according to the reaction schemes provided in the examples hereinafter, but those skilled in the art will appreciate that these are only illustrative for the invention and that the compounds of this invention can be prepared by any of several standard synthetic processes commonly used by those skilled in the art of organic chemistry.

In a preferred embodiment, the polymer according to formula I may be prepared through radical or anionic polymerization of the corresponding acrylamide or methacrylamide monomers with amino acid side chains.

The invention will now be illustrated by means of the following synthetic and biological examples, which do not limit the scope of the invention in any way.

EXAMPLES

The A'-Boc-amino acids (NBAA) with a high grade of purity (A'-Boc-glycine, X-Boc-L-alanine, X-Boc-L-valine, X-Boc-L-lysine, X-Boc-L-proline, X-Boc-L-phenylalanine, X-Boc-L- tryptophan, X-Boc-L-histidine), the solvents (X,X'-dimcthylformamidc (DMF), methanol, dichloromethane (DCM), diethyl ether), and trifluoracetic acid (TFA) were purchased from Sigma Aldrich and used as such, unless stated otherwise. 2-isopropenyl-2-oxazoline (Sigma- Aldrich, 98%, iPOx) was distilled over CalL under reduced pressure before use. Tetrahydro furan (Sigma-Aldrich, THF) was freshly distilled over Na/benzophenone under Ar flow before use. X-Butyllithium solution 2.5 mol L"¹ in hexane (Sigma-Aldrich, zi-BuLi) was used as received.

The 21 -nucleotide siRNA duplexes targeting the enhanced green fluorescent protein (siEGFP) and the negative control siRNA (siCTRL) were purchased from Eurogentec (Seraing, Belgium). The siCTRL sequence presents no homology with any known eukaryotic gene. Sequences of siEGFP: sense strand = 5'-CAAGCUGACCCUGAAGUUCtt-3'; antisense strand = 5'- GAACUUCAGGGUCAGCUUGtt-3'. Sequence of siCTRL: sense strand = 5'- UGCGCUACGAUCGACGAUGtt-3'; antisense strand = 5'-

CAUCGUCGAUCGUAGCGCAtt-3'. Capital letters represent ribonucleotides, lower case letters represent 2’-deoxyribonucleotides. Fluorescently labeled siRNA used for cell uptake experiments consists of the siCTRL sequence modified with Cy5® dye at the 5’ end of the sense strand (siCy5). Labeling and quality control was performed by Eurogentec (Seraing, Belgium). The siRNA was dissolved in nuclease-free water (Ambion-Life Technologies, Ghent, Belgium) and stored at -80 °C. The concentration of the siRNA stock was calculated by absorption measurements at 260 nm (1 OD260 = 40 pg/mL) with a NanoDrop 2000c UV-Vis spectrophotometer (Thermo Fisher Scientific, MA, USA).

The human non-small cell lung cancer cell line that stably expresses eGFP (H1299-eGFP) was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS, HycloneTM, GE Healthcare, Machelen, Belgium), 2 mM L-Glutamine and 100 U mF¹ penicillin/ streptomycin (i.e. ‘complete cell culture medium’ or CCM). The cell line was maintained in a humidified atmosphere containing 5% CO2 at 37 °C and culture medium was renewed every other day. When 80% to 90% confluence level was reached, cells were split using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA). Cells were regularly tested and found negative for mycoplasma.

’ll NMR spectra were recorded at 25 °C on a Bruker instrument operating at 300 MHz. Chemical shifts (5) are referenced to CDCL (5 7.26 ppm) or D2O (5 4.79 ppm). Infrared spectra were measured on a Bruker Vertex 70 spectrometer fitted with a Harrick MVP2 diamond ATR device and were reported in wavenumber (cm^-1). Polarimetry measurements were performed on a Perkin Elmer 241 Polarimeter with solutions of 20 mg/ 2 mL in MeOH.

Size exclusion chromatography (SEC) for the Boc-protected polymers was performed using an Agilent 1260-HPLC system equipped with a 1260 refractive index detector (RID), a 1260 online degasser, a 1260 isocratic pump, a 1260 automatic liquid sampler, a thermostatted column compartment at 50 °C equipped with two PSS Novema Max linear M columns (8 mm x 300 mm) and a guard column in series. The eluent used as mobile phase was DMAc containing 50 mM ofLiCl at a flow rate of 0.5 mL/min. The spectra are analysed using the Agilent Chemstation software with the GPC add on. Molar mass values and dispersity values are calculated against PMMA standards.

Size exclusion chromatography (SEC) for the cationic charged polymers was performed using an Agilent 1260 Infinity II HPLC system equipped with a 1260 refractive index detector (RID), a 1260 online degasser, a 1260 isocratic pump, a 1260 automatic liquid sampler, a thermostatted column compartment at 30 °C with one Phenomenex PolySep-P3000 (7.8 mm x 300 mm) and a guard column in series. The eluent used as mobile phase was methanol (MeOH) and sodium acetate buffer (0.1 M, pH 6.5) mixture (80:20 vol.%) at a flow rate of 0.5 mL min'¹. The spectra are analysed using the OpenLab CDS software with the GPC add on.

Light scattering (LS) measurements were performed on a Wyatt Heleos II Multi Angle Light Scattering detector (MALS), from Wyatt Technology. The detector is coupled on-line to an Agilent 1260 Infinity II HPLC system (MeOH-sodium acetate-SEC) and used to determine absolute molar mass of the analysed polymer samples. The measurements are performed at ambient temperature, i.e., no temperature control unit is supplied/installed with the above- mentioned LS detector. The refractive index (RI) increment (dn/dc) values determined via online size-exclusion chromatography (SEC) equipped with an RI detector, which measures the RI increase for a 1 To 10 mg mL'¹ concentration series of the herein reported polymers. The LS results were analysed with the Astra 7 software (Wyatt Technology).

The thermal analysis and glass transition temperatures (T_g) (simultaneous TGA-DSC, MS hyphenated) was performed on a NETZSCH STA 449C Jupiter system, coupled to an Aeolos II MS detector. Degradations were performed in the scanning mode for all samples, from ambient temperature up to 750 °C, at a heating rate of 10 °C min'¹ under helium flow (25 mL min'¹). Differential scanning calorimetry (DSC) was performed on a Setaram DSC 131 instrument with a heating/ cooling rate of 10 °C min¹ under nitrogen flow.

PiPOx, for example PiPOx with an average molecular weight of 14,300 Da, was prepared via living anionic polymerization as disclosed below. In a typical run, the anionic polymerization of iPOx and all manipulations were performed in clean/dry glassware and under Ar flow. A 1.6 mol L'¹ solution of 5 mL (47.7 mmol) of iPOx in 25 mL of THF was cooled down to -40 °C. Then, 146.8 pL of zi-BuLi (0.367 mmol) in hexane was injected into the reaction flask. The polymerization was kept for 10 min at this temperature, then, the reaction mixture was allowed to warm to room temperature. The polymerization was terminated by injecting 5 mL of methanol. The polymer was precipitated from diethyl ether and dried in a vacuum oven at 55 °C. The PiPOx polymer was obtained with a 96% yield as a fine white powder. The absolute number average molecular weight (Mn) of the analysed polymer is 14.3 kg mol'¹, with a dispersity of 1.16 as determined by SEC-MALS. The specific refractive index increment value used for calculations is 0.0902 mL g'¹.

Alternatively, the polymer according to the invention may be synthesised via free radical polymerisation as disclosed below. 246.3 mg (15 mmol) of recrystallized azobisisobutyronitrile (AIBN) was weighed in a 250 mb round bottom Schlenk flask and equipped with a magnetic stirring bar. Then 58.1 mb of dimethyl sulfoxide (DMSO) were added and stirred under argon atmosphere until all AIBN crystals were dissolved. Next, 41.94 mb (400 mmol) of iPOx were added to the reaction mixture under argon atmosphere. The mixture was degassed with argon for 30 min. Afterwards, the reaction was heated to 65 °C in a heating block and stirred for 24 hours. The resulting reaction mixture was precipitated in an excess diethyl ether, centrifuged and the supernatant was discarded. The resulting solid was dried in the vacuum oven overnight to remove residual solvents. The obtained white powder was then redissolved in deionized water and dialyzed for 3 days for further purification. Lyophilization was performed with a Martin Christ freeze-dryer, model Alpha 2-4 LSC plus and yielded the final product as a white powder (yield: 25.2 g, 61%). The absolute number average molecular weight (Mn) of the analysed polymer was 36,000 Da with a dispersity of 1.48 as determined by SEC-MALS.

Post-modification reaction of PiPOx with the A-Boc-protected amino acids (NBAA) was performed using the following procedure, 1.8 mmol PiPOx (0.2 g) and 2.16 mmol of NBAA(s) were dissolved in 3.6 mL of dry DMF, flushed and sealed under argon. The feed molar ratio for homopolymers was PiPOx:NBAA = 1 :1.2 and for copolymers is PiPOx:NBAA 1 :NBAA 2 = 1 :0.6:0.6, and PiPOx molar concentration in the solution is 0.5 M. To reach full modification of PiPOx, the solutions were left to react at 100 °C for 72 h. Then, the solutions were cooled down to room temperature, diluted 2 mL of CHCL, and precipitated in diisopropyl ether. The resulting white powders were dried under vacuum overnight at 55 °C. The (co)polymers were obtained with high yields between 79% to 93%. The reaction route for homo and copolymers is given in scheme la and lb of Figure 1, respectively.

Synthesis of cationic charged PiPOx polymers with amino groups was performed using the following procedure, 1 mmol of modified PiPOx-NBAA or PiPOx-NBAAl_NBAA2 was dissolved in 5 mL of DCM, and TFA (20 equivalents) was added. The solution was left to react with vigorous stirring for 1 to 2 h at room temperature. The resulted precipitated polymer was further dissolved in MeOH and precipitated in diethyl ether. The isolated product was washed with 2 to 3 portions of diethyl ether and dried on the filter glass, then dissolved further in deionized water, filtered through a 0.2 pm polytetrafluorethylene filter, and freeze dried. The deprotected cationic polymers were obtained with yields between 81% to 97%. The general reaction route for the cationic charged (co)polymers is given in scheme 1c of Figure 1.

The solution for polarimetry measurement was prepared in a volumetric flask of 2 mL with 0.02 g of homopolymer and MeOH of spectrophotometric grade. The solution was left for 30 minutes to stand at room temperature, then carefully filled in the optical cell and measured at Z = 589 nm (Na line) with 2 seconds on the integration wheel. The measurements were carried out under identical conditions to be able to compare differences in measured specific rotation.

To determine the specific rotation of the homopolymers we used Biot’s equation:

[a]x = a-x €’¹-c’¹

Where [a],. is the specific rotation in units of degrees, a is the measured optical rotation in units of degrees for Na line, t is the length of the cell of 1 dm, and c is the sample concentration in units of g mL'¹. The values of the specific rotation are given as an average of five consecutive measurements.

Equal volumes of PiPOx-NBAA-TFA polymer solution in PBS-/- (phosphate buffered saline without calcium/magnesium) and siRNA (0.025 pg pL'¹) were mixed, vortexed for 10 seconds and incubated at room temperature for 30 min to allow complexation before Agarose gel electrophoresis was performed. JetPEI® was used as a control following manufacturer’s instructions (Polyplus, Illkirch, France). Briefly, siRNA and JetPEI reagent were first diluted in equivalent volumes of Rnase free water with 10% glucose, mixed and incubated at room temperature for 15 min. After incubation, 5 pL of Gel Loading Buffer (Invitrogen, Massachusetts, United States) was added to each sample (20 pL) prior to loading on a 1.2% agarose gel. The gel was run for 30 min at 100 V before imaging (Canon PowerShot A2300 IS 16.0 MP Digital Camera).

H1299-eGFP cells were seeded at a cell density of 7.500 cells/well in 96-well plates (Bioswisstec, Schaffhausen, Switzerland) and were allowed to settle overnight in order to perform quantification of eGFP gene silencing by flow cytometry. The following day, siRNA polyplexes were diluted 5x in Opti-MEM and applied on the cells for 4 hours. Polyplexes were prepared at a nitrogen-to-phosphate (N/P) ratio of 10. After 4 hours incubation, transfection medium was removed and cells were washed with PBS -/- and incubated for 44 hours in CCM at 37 °C. Expression of eGFP was detected via flow cytometry, using a CytoFLEX plate reader flow cytometer for 96-well plates (Beckman Coulter, Krefeld, Germany) and CytExpert software. Data analysis was performed using the FlowJo analysis software (Treestar, Costa Mesa, CA, USA) and the percentage of eGFP expression for each sample was calculated by normalizing the fluorescence signal of cells treated with siEGFP polyplexes to the fluorescence of cells treated with siCTRL polyplexes. For JetPRIME® transfection, cells were likewise incubated with the transfection reagent for 4 hours in CCM (see also figure 4).

To quantify the cellular internalization of the siRNA polyplexes by flow cytometry, Hl 299- eGFP cells were seeded in 96-well plates (Bioswisstec, Schaffhausen, Switzerland) at a density of 7.500 cells/well (lOOpL well'¹) and allowed to settle overnight. PiPOx-NBAA-TFA polymers were complexed with siCTRL:siCy5® (95:5 mol%) and diluted 5x in Opti-MEM (Gibco®-Life Technologies, Grand Island, NY, USA) prior to application on cells (4 hours, 37 °C, 5% CO2). Next, the cells were washed with dextran sulfate sodium salt (0.1 mg ml’¹ in PBS) to remove cell surface-bound fluorescence. Flow cytometry quantification of siCy5 fluorescence was carried out as described in this application.

Cell viability was analyzed using a CellTiter-Glo® assay (Promega). Hereto, the H1299-eGFP cells were seeded as previously described and allowed to settle overnight. The following day, cells were incubated for 4 hours in Opti-MEM with two selected PiPOx-NBAA-TFA polymers at mounting concentrations, followed by 44 hours incubation with CCM. Before initiating the assay, the CellTiter-Glo® buffer and the culture plates were placed at room temperature for 30 min. Next, the culture medium was replaced by 100 pL fresh medium, and an equivalent volume of assay buffer was added. To induce complete lysis, the plate was shaken for 2 min and left at room temperature for 10 min to allow signal stabilization. Subsequently, 100 pL from each well was transferred to an opaque 96-well plate (Greiner Bio-One, Kremsmunster, Austria) and the luminescence signal was measured with a GloMax® 96 Microplate Luminometer (Promega, Belgium). Data are presented as percentage of viable cells, calculated from the luminescence signal of each condition relatively to non-treated cells and by taking in account the background fluorescence of the medium.

Polyplexes were prepared by mixing equal volumes of siRNA solution (0.025 pg pL’¹) with polymers dissolved in PBS-/-. Following a 30 min complexation, the resulting polyplexes were diluted 5x in Hepes Buffer (20 mM, pH 7.4) to reach a final concentration corresponding to the highest concentration used for transfection experiments. The zeta potential and hydrodynamic diameter of the resulting PiPOx-NBAA-TFA siRNA polyplexes was measured by Dynamic Light Scattering (DLS) (Malvern, UK).

Aliphatic and aromatic NBAAs were used to modify PiPOx. To be able to control the copolymer composition by the NBAA feed ratio, kinetics studies were performed for selected NBAAs to calculate the reaction rate constants (kr) at 100 °C. The modification of PiPOx with valine (aliphatic NBAA), proline (cyclic aliphatic NBAA), and phenylalanine (aromatic NBAA) were monitored by 'H NMR spectroscopy and SEC chromatography. During the ring opening addition reaction of the NBAAs to the 2-oxazoline ring, a distinctive separation between the signals of the unreacted 2-oxazoline ring and the ester amide reaction product was observed. The signal of the protons of the formed ester amide structure (-NH-CH2-) is shifted downfield to 3.12 ppm compared to the corresponding protons of the unreacted 2-oxazoline ring (=N-CH2- ) at 3.45 ppm. By integrating these two signals, the chemical modification degree was determined. However, despite the variation in reactivity between Pro and Vai / phenylalanine (PhAla) observed in the first day, each reaction reached full conversion if heated for sufficient time. Thus, a 72-hour reaction time was used for all further experiments to reach full conversion. The calculated reaction rate constant value, k_r, for Pro (0.074 ± 0.004 x 10'³ mol L'¹ s'¹) was found to be 2.5 times slower compared to Vai and PhAla, with k_r values of 0.178 ± 0.003 x 10" ³ mol L'¹ s'¹ and 0.164 ± 0.007 x 10'³ mol L'¹ s'¹, respectively. Considering that the pK_a of the Boc-amino acids is in the same range, we can assume that the lower reactivity of Pro is due to steric effects induced by the strained proline ring. Despite the variations in k_r, the kinetic study indicates that the NBAAs have similar reactivity under these given conditions, except proline which is slower than the other amino acids. In addition, several polymers according to the invention were synthesized (see Table 1) using a similar synthesis route and were characterized similarly.

Table 1 : Physical properties for fully modified V-Boc protected (co)polymer series with the N- Boc amino acids. ^aThe yield was calculated gravimetrically based on the precipitated polymer; ^bThe compositions were estimated from H NMR spectra; ^cDetermined by SEC in DMAc, against PMMA standards; ^dSecond heating run with 10 °C min ¹ heating rate; ^eDecomposition temperature in helium flow at 10 °C min ¹ heating rate. ^fSpecific rotation calculated from polarimetry measurements in MeOH for Na line., n.d. stands for not determined.

It was found that well-defined homopolymers with an increased number average molar mass (Mn) and narrow dispersity were attained after post-modification reactions of PiPOx (see Table 1), indicating an increase in hydrodynamic volume due to the introduction of the large bulky side chains with no significant polymer chain coupling. The thermal properties of the polymer series were investigated via simultaneous TGA-DSC-MS analysis. The homopolymers exhibited both lower thermal stability and glass transition temperatures (T_g) compared to the starting polymer PiPOx (see Table 1). The decrease in T_g compared to starting polymer (PiPOx) can be attributed to the increased flexibility of the polymeric chain upon ring opening of the iPOx units. In addition to the A-Boc amino acid functionalized polymethacrylamide homopolymers, a series of A-Boc amino acid functionalized polymethacrylamide copolymers was prepared by one -pot modification reaction of PiPOx with equimolar amounts of two different NBAAs, as shown in Scheme lb of Figure 1, to provide a polymer with substantially 50:50 molar ratio of the amino acid derivatives. Parameters of the homopolymers according to the invention are summarized in Table 1.

The cationic polymethacrylamide homopolymer and copolymers according to the invention were obtained via deprotection of the A-Boc functionalized polymers as shown in Scheme 1c of Figure 1. The physical properties of said polymers are summarized in Table 2.

Table 2: Physical properties for cationic charged PiPOx (co)polymers series. ^aThe yield was calculated gravimetrically based on the precipitated polymer; ^bDetermined by SEC-MALS in MeOH; ^cCalculated theoretical value; ^dSecond heating run with 10 °C min^-1 heating rate; ^eDecomposition temperature in helium flow at 10 °C min^-1 heating rate. ^fSpecific rotation calculated from polarimetry measurements in MeOH (Na line), *exception for which Hg line was used. The specific rotation of the homopolymers was determined via polarimetry in methanol at room temperature. The values for the A -Boc-protected polymers are summarized in Table 1 , while the values for the cationic polymers are given in Table 2. After the post-modification with the N- Boc-L-amino acids, the resulting homopolymer series preserved their chiroptical properties along the polymer side chains. However, lower values were determined for the homopolymer series compared to the free /.-amino acids, which can be ascribed to polymer-polymer interactions in methanol, not being a theta solvent. The deprotection of the A-Boc groups may result in cationic (co)polymers also induced water-solubility as well as aqueous buffers, like phosphate buffered saline (PBS), as required for transfection.

The synthetized cationic PiPOx-NBAA-TFA polymethacrylamide (co)polymers have been tested for their ability to complex siRNA, using agarose gel electrophoresis using a commonly employed nitrogen-to-phosphate (N/P) ratio of 10. The least efficient complexation, leaving most of the siRNA migrating freely on the gel, was observed for the most hydrophobic polymers, i.e. those fully modified with phenylalanine and tryptophan. It is argued that large steric hindrance of the bulkier aromatic side chain could contribute to ineffective electrostatic interaction with the siRNA phosphate backbone or lower hydration of these most hydrophobic polymers might suppress the interactions. Surprisingly, applying copolymers in which Trp in combination with either Vai or Pro partly recovered the complexation efficiency, while copolymers comprising PhAla with Vai or Pro showed modest improvement of siRNA complexation. On the other hand, the homopolymer bearing Vai as a side chain was able to complex only half of the siRNA at a nitrogen to phosphate (N/P) ratio of 10. All the other polymers tested showed maximal siRNA complexation at the utilized experimental conditions. Polymers showing good complexation capacity were further tested for their ability to deliver the complexed siRNA into cells. Hereto, PiPOx-NBAA-TFA polyplexes with 50 nM of eGFP- targeting siRNA (siEGFP) and a N/P ratio of 10 were produced and evaluated to transfect H1299-eGFP cells. Surprisingly, PiPOx-Val Trp and PiPOx-Pro Trp demonstrated efficient gene silencing, leading to almost complete knockdown of the target eGFP gene (Fig. 2a and 2b). These data clearly demonstrate that siRNA complexation alone is not sufficient for efficient transfection. Indeed, polyplexes should be internalized by the target cell and overcome the endosomal barrier to allow cytosolic siRNA delivery, which is dependent on the type of polymer. Two copolymers (PiPOx-Val Trp and PiPOx-Pro Trp) were used to evaluate the cellular uptake of the resulting polyplexes (loaded with Cy5 -labeled siRNA) and to compare said polymers with the corresponding homopolymers, modified with only a single amino acid type. Little Cy5 fluorescence is detected in H1299-eGFP cells exposed to PiPOx-Trp (Fig. 2a). On the other hand, flow cytometry clearly indicates an efficient cellular internalization in > 90% of cells when transfected with, for example, both PiPOx-Val Trp and PiPOx-Pro Trp polyplexes, while uptake is almost absent for their homopolymer counterparts (Fig. 3a-c). When comparing both copolymers, the PiPOx-Pro Trp polyplexes are moderately more efficient in cellular uptake, showing a 25% higher MFI value compared to the PiPOx-Val Trp polyplexes (Fig. 3b).

In addition, compared to jetPEI the discovered copolymers are significantly more performant for siRNA transfection (Fig. 4) while the polymers show no cytotoxicity at the used concentrations for transfection that correspond to < 0.01 mg/mL (Fig. 5).

Similar methods were used to prepare PiPOx-Val Trp and PiPOx-Pro Trp copolymers with different chain length (degree of polymerization (DP); Fig. 6a and the corresponding polyplexes with siRNA for silencing of EGFP and an siRNA control. Transfection experiments revealed efficient silencing for all copolymers with DP 90 to 450 when cells were transfected with siRNA for EGFP while no silencing was observed for transfection with siRNA control or nontransfected controls (NTC) as shown in Fig. 6b. The present invention is by no means limited to the above described preferred embodiments and/or experiments thereof. The rights sought are defined by the following claims within the scope of which many modifications can be envisaged.

Claims

1. Polymer comprising m monomers according to formula (I), or a stereoisomer, a tautomer, a racemic, a salt, a hydrate, a N-oxide or solvate thereof, wherein: m is an integer in the range of 10 to 1000; n is an integer in the range of 0 to 50;

R¹ in combination with the adjacent carboxy group is for each monomer individually an amino acid derivative, wherein the polymer comprises at least two different amino acid derivatives; and

R² is for each monomer individually selected from hydrogen or methyl.

2. Polymer according to claim 1, wherein each amino acid derivative is independently selected from the group of tryptophan derivative, tyrosine derivative, arginine derivative, glycine derivative, alanine derivative, valine derivative, proline derivative, lysine derivative, phenylalanine derivative, and histidine derivative, preferably wherein each amino acid derivative is independently selected from the group of tryptophan derivative, tyrosine derivative, and arginine derivative.

3. Polymer according to any one of the preceding claims, wherein the at least two different amino acid derivatives are phenylalanine derivatives or tryptophan derivatives and valine derivatives or proline derivatives.

4. Polymer according to any one of the preceding claims, wherein one or more amino acid derivatives comprise an aromatic moiety.

5. Polymer according to any one of the preceding claims, wherein the amino acid derivatives are coupled via the carboxylic acid on the C-terminus.

6. Polymer according to any one of the preceding claims, wherein m is an integer in the range of 50 to 1000, preferably in the range of 100 to 500, more preferably in the range of 100 to 250.

7. Polymer according to any one of the preceding claims, wherein R² is methyl.

8. Polymer according to any one of the preceding claims, further comprising k monomers individually selected from the group of ethylene glycol, acrylate, and methacrylate, wherein k is an integer in the range of 1 to 1000, preferably wherein the k monomers are statistically or randomly distributed in the polymer or form blocks.

9. Polymer according to any one of the preceding claims, wherein one or more amino acid derivatives comprises a Boc group, trifluoroacetic acid counterion group, acetate counterion group.

10. Composition comprising a polymer according to any one of the claims 1 to 9, wherein the polymer is a delivery agent.

11. Composition according to claim 10, further comprising an active agent.

12. Composition according to claim 11, wherein the active agent comprises nucleic acids.

13. Composition according to claim 11 or 12, wherein the active agent is one or more selected from the group of RNA, siRNA, mRNA, circular RNA, self-amplifying mRNA, tRNA, rRNA, viral cRNA, miRNA, IncRNA, antisense oligonucleotides of DNA or antisense oligonucleotides of RNA, guide RNA, DNA, plasmid DNA, ribonucleoproteins, preferably the active agent may be one or more selected from the group of RNA, siRNA, mRNA, DNA, plasmid DNA, more preferably the active agent is siRNA.

14. Polymer according to any one of the claims 1 to 9, or composition according to any one of the claims 10 to 13, for use as a human or veterinary medicine.

15. Polymer according to any one of the claims 1 to 9, or composition according to any one of the claims 10 to 13, for use in the prevention and/or treatment of at least one disease or disorder selected from the group comprising oncological diseases, infectious diseases, genetic disorders.

16. Use of a polymer according to any one of claims 1 to 9, or a composition according to any one of the claims 10 to 13, as a delivery agent of an active agent, preferably as a delivery agent of siRNA; or as a delivery agent of plant protection agents.