Polymers for delivering activators, compositions containing said polymers, and their use

Abstract

This invention relates to a polymer for activator delivery. The polymer includes an amino acid derivative, such as a cationic polymer containing an amino acid derivative, and the polymer according to this invention may also be called PiPOx-XXX (where XXX refers to the amino acid derivative). Furthermore, this invention also relates to a composition containing the above polymer and its use.

Description

[Technical Field]

[0001] This invention relates to the field of smart biomaterials. More specifically, this invention relates to polymers containing amino acid derivatives, such as cationic polymers containing amino acid derivatives. Furthermore, this invention also relates to compositions containing such polymers and their uses. [Background technology]

[0002] Gene therapy holds great potential in treating genetic diseases such as cancer. For example, a major challenge in fully realizing the potential of cancer gene therapy is the lack of a safe and efficient system for delivering the nucleic acids used. Other examples where a safe and efficient delivery system is considered lacking include protein replacement therapy and nucleic acid-based vaccines. While viral vectors offer excellent transfection efficiency, their immunogenicity and the risk of insertional mutations raise safety concerns. In this context, non-viral vectors offer a safer alternative for gene delivery. Non-viral transfection of genetic material (e.g., nucleic acids such as DNA and RNA, or proteins such as ribonucleoprotein complexes) into cells, also known as gene delivery, has been simplified over many years by the development of synthetic vectors. Synthetic vectors are substances that electrostatically bind nucleic acids, condensing them into nanoparticles with a size of tens to hundreds of nanometers. This protects the nucleic acids from degradation and mediates their entry into cells. Cationic lipids and polymers can both be used to form complexes with nucleic acids, thereby creating lipoplexes (also called lipid nanoparticles (LNPs)) and polyplexes, respectively. Although LNP was rapidly brought to market as an mRNA vaccine for COVID-19, its manufacturing process is complex and costly.

[0003] The development of cationic polymers has played an important role in the delivery of genetic materials due to the great potential to adjust their structural and functional parameters. One of the most commonly used non-viral polymer vectors for nucleic acid transfection is linear polyethyleneimine (PEI). However, while high molecular weight PEI (with 500 repeating units) has shown high cytotoxicity in vitro and in vivo, low molecular weight PEI (with less than 250 repeating units) has been demonstrated to not only be less toxic in cell culture studies but also reduce transfection efficiency. Although alternative materials such as poly(β-amino ester), poly(lysine), cyclodextrin derivatives, dendrimers, poly(amidoamine), PEGylated polymers, methacrylate / methacrylamide polymers, poly(allylamine), and poly(2-oxazoline) (PiPOx) have been developed and improvements in effectiveness and reduction in toxicity have been obtained, satisfactory results have not been achieved. Therefore, it can be concluded that successful gene delivery is a complex multi-step process that requires the formation of nucleic acid complexes, the formation of nanoparticles, diffusion through biological barriers, cellular uptake, endosomal escape, and nucleic acid release. To date, efficiently handling all of these steps remains a major challenge for synthetic carriers, and thus the search for new polymers for efficient transfection remains the current goal.

[0004] Polymer-based transfection continues to attract interest due to the ease with which polyplexes are formed simply by mixing the polymer and nucleic acid, and the chemical diversity of polymers that allows for the obtainment of linear, branched, or dendritic polymer structures with multiple functions for optimizing transfection efficiency. It is known that the structural and functional parameters of polymers, such as length, dispersity, composition, bonding, sequence, and the ability to further self-organize into higher-order aggregates (e.g., micelles, polymersomes), determine binding efficiency, binding selectivity, cell permeability, and toxicity to transfected cells.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention aims to avoid or at least reduce the aforementioned problems and enable efficient and effective transfection / delivery.

[0006] The object of the present invention is to provide a (cationic) polymer for efficiently transfecting an activator, particularly a nucleic acid.

[0007] Surprisingly, it has been found that a (cationic) polymer containing an amino acid derivative provides efficient and effective transfection.

Means for Solving the Problems

[0008] In a first aspect, the present invention provides a polymer comprising m monomers of formula (I), or stereoisomers, tautomers, racemates, salts, hydrates, N-oxides, or solvates thereof:

Chemical formula

[0007] , , 2 ,

[0010] , ,

[0008] , , , , , 1 , , , 1 , , ,

[0009] , can be combined with adjacent carboxy groups to form an amino acid derivative for each monomer, and the polymer contains at least two different amino acid derivatives, R 2 can be individually selected from hydrogen or methyl for each monomer).

[0009] R 1 It should be noted that R is individually selected for each monomer. Therefore, different amino acid derivatives can be present in the polymer according to the present invention.

[0010] In a more preferred embodiment of the present invention, each amino acid derivative can be independently selected from the group consisting of tryptophan derivatives, tyrosine derivatives, arginine derivatives, glycine derivatives, alanine derivatives, valine derivatives, proline derivatives, lysine derivatives, phenylalanine derivatives, histidine derivatives, isoleucine derivatives, leucine derivatives, methionine derivatives, threonine derivatives, cysteine ​​derivatives, glutamine derivatives, aspartic acid derivatives, asparagine derivatives, glutamic acid derivatives, serine derivatives, selenocysteine ​​derivatives, and pyrrolicin derivatives.

[0011] In a more preferred embodiment of the present invention, each amino acid derivative can be independently selected from the subgroups of aromatic amino acid derivatives, carboxyl amino acid derivatives, charged amino acid derivatives, hydrophobic amino acid derivatives, hydrophilic amino acid derivatives, and polar uncharged amino acid derivatives. It should be noted that amino acid derivatives may exist in multiple subgroups. Preferably, charged amino acid derivatives have a positive charge and more preferably include a positively charged group and / or a protonable group.

[0012] Examples of aromatic amino acid derivatives include histidine derivatives, phenylalanine derivatives, tyrosine derivatives, and tryptophan derivatives. Examples of charged amino acid derivatives include arginine derivatives, glycine derivatives, histidine derivatives, lysine derivatives, proline derivatives, and selenocysteine ​​derivatives. Examples of hydrophobic amino acid derivatives include alanine derivatives, valine derivatives, isoleucine derivatives, leucine derivatives, methionine derivatives, phenylalanine derivatives, tyrosine derivatives, and tryptophan derivatives. Examples of hydrophilic amino acid derivatives include arginine derivatives, lysine derivatives, asparagine derivatives, histidine derivatives, and proline derivatives. Examples of polar uncharged amino acid derivatives include serine derivatives, threonine derivatives, asparagine derivatives, and glutamine derivatives.

[0013] In a more preferred embodiment of the present invention, each amino acid derivative can be independently selected from the group consisting of tryptophan derivatives, tyrosine derivatives, arginine derivatives, glycine derivatives, alanine derivatives, valine derivatives, proline derivatives, lysine derivatives, phenylalanine derivatives, and histidine derivatives. Preferably, each amino acid derivative can be independently selected from the group consisting of tryptophan derivatives, tyrosine derivatives, and arginine derivatives.

[0014] In a more preferred embodiment of the present invention, at least two different amino acid derivatives include tryptophan, and more preferably, at least two different amino acid derivatives are a phenylalanine derivative or a tryptophan derivative and a valine derivative or a proline derivative. In other words, the two different amino acid derivatives are a phenylalanine derivative and a valine derivative, or a phenylalanine derivative and a proline derivative, or a tryptophan derivative and a valine derivative, or a tryptophan derivative and a proline derivative. It should be noted that the polymer according to the present invention preferably includes a tryptophan derivative and a valine derivative, or a tryptophan derivative and a proline derivative.

[0015] In a more preferred embodiment of the present invention, one or more amino acid derivatives include an aromatic moiety.

[0016] In a more preferred embodiment of the present invention, the amino acid derivative is bonded via a C-terminal carboxylic acid.

[0017] In a more preferred embodiment of the present invention, the amino acid derivative includes a hydrophobic amino acid derivative. Preferably, at least 20 mol% of the amino acid derivative present in the polymer is hydrophobic, more preferably at least 25 mol% of the amino acid derivative present in the polymer is hydrophobic, even more preferably at least 30 mol% of the amino acid derivative present in the polymer is hydrophobic, even more preferably at least 35 mol% of the amino acid derivative present in the polymer is hydrophobic, and most preferably at least 40 mol% of the amino acid derivative present in the polymer is hydrophobic.

[0018] In a more preferred embodiment of the present invention, up to 100 mol% of the amino acid derivative present in the polymer is hydrophobic, preferably up to 95 mol% of the amino acid derivative present in the polymer is hydrophobic, more preferably up to 90 mol% of the amino acid derivative present in the polymer is hydrophobic, and most preferably up to 85 mol% of the amino acid derivative present in the polymer is hydrophobic.

[0019] In a more preferred embodiment of the present invention, 20 mol% to 100 mol% of the amino acid derivative present in the polymer may be hydrophobic, preferably 20 mol% to 95 mol% of the amino acid derivative present in the polymer may be hydrophobic, more preferably 25 mol% to 95 mol% of the amino acid derivative present in the polymer may be hydrophobic, even more preferably 30 mol% to 95 mol% of the amino acid derivative present in the polymer may be hydrophobic, even more preferably 30 mol% to 90 mol% of the amino acid derivative present in the polymer may be hydrophobic, even more preferably 35 mol% to 90 mol% of the amino acid derivative present in the polymer may be hydrophobic, and most preferably 40 mol% to 85 mol% of the amino acid derivative present in the polymer may be hydrophobic.

[0020] In a more preferred embodiment of the present invention, m can be an integer in the range of 50 to 1000, preferably 100 to 500, and more preferably 100 to 250.

[0021] In a more preferred embodiment of the present invention, n may be an integer in the range of 1 to 40, more preferably an integer in the range of 1 to 30, even more preferably an integer in the range of 1 to 20, even more preferably an integer in the range of 1 to 10, and most preferably an integer in the range of 1 to 5.

[0022] In a more preferred embodiment of the present invention, n is 1.

[0023] In a more preferred embodiment of the present invention, R 2 It can be represented as methyl.

[0024] In a more preferred embodiment of the present invention, the polymer further comprises k monomers individually selected from the group consisting of ethylene glycol, acrylate, and methacrylate, where 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.

[0025] In a more preferred embodiment of the present invention, one or more amino acid derivatives include a Boc group, a trifluoroacetate counterionic group, and an acetate counterionic group.

[0026] In a second aspect, the present invention provides a composition containing a polymer according to the present invention, wherein the polymer is a delivery agent.

[0027] In a more preferred embodiment of the present invention, the composition further includes an activator.

[0028] In a more preferred embodiment of the present invention, the activator comprises nucleic acid.

[0029] In a more preferred embodiment of the present invention, the activator can be one or more selected from the group consisting of RNA, siRNA, mRNA, self-amplified mRNA, circular RNA, tRNA, rRNA, viral cRNA, miRNA, lncRNA, antisense oligonucleotide of DNA, antisense oligonucleotide of RNA, guide RNA, DNA, plasmid DNA, and ribonucleoprotein. Preferably, the activator may be one or more selected from the group consisting of RNA, siRNA, mRNA, DNA, and plasmid DNA, and more preferably, the activator is siRNA.

[0030] In a third aspect, the present invention provides polymers or compositions according to the present invention for use as pharmaceuticals for humans or animals, or for use in protecting crops.

[0031] In a fourth aspect, the present invention provides the use of a polymer or composition according to the present invention as a delivery agent for activators, preferably as a delivery agent for siRNA, or as a delivery agent for plant protection agents such as siRNA molecules used in plant protection applications.

[0032] In a more preferred embodiment of the present invention, the activator is a pharmaceutical product.

[0033] In a more preferred embodiment of the present invention, the polymer or composition of the present invention is intended for use in the prevention and / or treatment of at least one disease or disorder selected from the group including neoplastic diseases, infectious diseases, and genetic disorders.

[0034] The present invention also provides the use of the polymer or composition of the present invention in the prevention and / or treatment of at least one disease or disorder selected from the group including neoplastic diseases, infectious diseases, genetic disorders, or crop diseases.

[0035] In a fifth aspect, the present invention provides a method for preventing and / or treating at least one disease or disorder selected from the group including neoplastic diseases, infectious diseases, and genetic disorders, the method comprising administering a therapeutically effective amount of a compound or composition according to the present invention to a subject in need thereof.

[0036] With regard to the drawings, it is emphasized that the details shown are illustrative and intended only to illustrate various embodiments of the present invention. The drawings are presented to provide the most useful and simplest explanation of the principles and conceptual aspects of the present invention. In this regard, structural details of the present invention are not intended to be shown more specifically than necessary for a basic understanding of the invention. By examining this specification in conjunction with the drawings, it will become clear to those skilled in the art how some embodiments of the present invention can be actually implemented. [Brief explanation of the drawing]

[0037] [Figure 1] This figure shows the reaction pathways for (a) synthesis of fully modified PiPOx homopolymer using N-(tert-butoxycarbonyl)amino acid (NBAA), (b) synthesis of fully modified PiPOx copolymer using NBAA, and (c) preparation of the final cationic polymethacrylamide (co)polymer. [Figure 2] This figure shows flow cytometry quantification of the silencing efficiency in H1299-eGFP cells of polymers shown to form complexes with siRNA. Transfection was performed at an siRNA concentration of 50 nM for each sample, and silencing is expressed as the mean ± standard deviation over three technical replicates (a); a representative histogram of eGFP silencing induced by PiPOx-Pro_Trp complexed with eGFP-targeted siRNA (sieGFP) compared with complex formation using non-targeted siRNA (siCTRL) (b). [Figure 3]This figure shows the quantification of the percentage of H1299-eGFP cells in which Cy5-labeled siRNA (siCy5) could be detected (a); the mean fluorescence intensity (MFI) of the Cy5-positive cell population (b); and a representative dot plot of cells transfected with the indicated polyplex loaded with Cy5-labeled siRNA (c). Data are expressed as mean ± standard deviation over three technical replications. [Figure 4] This figure shows the eGFP silencing efficiency of PiPOx-AA copolymer compared to commercially available JetPRIME® transfection reagents. Data are expressed as mean ± standard deviation. [Figure 5] This figure shows the cell viability of H1299-eGFP cells treated for 4 hours at the indicated PiPOx polymer concentrations. The concentrations used in the cell transfection experiments corresponded to less than 0.01 mg / ml, and cell viability of over 90% was maintained with both polymers. [Figure 6-1] This figure shows the quantitative analysis of the silencing efficiency of PiPOx-Val_Trp copolymers and PiPOx-Pro_Trp copolymers of different chain lengths in H1299-eGFP cells by flow cytometry: a) details of the polymers investigated; b) mean fluorescence intensity of transfection for each sample at a 50 nM siRNA concentration. Silencing is expressed as mean ± standard deviation over three technical replicates. siCTRL samples were transfected with non-eGFP silencing siRNA, and siEGFP samples were transfected with EGFP silencing siRNA. NTC is the non-transfected control. [Figure 6-2] Same as above [Figure 6-3] Same as above [Modes for carrying out the invention]

[0038] The present invention will be described further below. Different aspects of the present invention will be defined in more detail in the following text. Each of the aspects defined in this manner may be combined with any other aspect or more of the invention unless explicitly stated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or more of the features indicated as preferred or advantageous.

[0039] When used herein, the terms "about" or "approximately" refer to measurable values ​​such as parameters, quantities, and time intervals, and mean that they include variations of ±10%, preferably ±5%, more preferably ±1%, and even more preferably ±0.1% or less from a specified value, provided that such variations are appropriate for carrying out the disclosed invention. Naturally, the values ​​to which the modifiers "about" or "approximately" apply are themselves specifically and preferably disclosed.

[0040] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural subjects unless the context clearly indicates otherwise.

[0041] When describing the compounds of the present invention, unless otherwise specified in the context, the terms used shall be interpreted according to the following definitions.

[0042] As used in the present invention, the term “substituted” is always intended to mean that one or more hydrogen atoms on the atom specified by “substituted” in this expression are replaced by a group selected from the specified group, provided that the substitution does not exceed the normal valence of the atom and that the substitution results in a chemically stable compound, i.e., a compound robust enough to be isolated from the reaction mixture in a useful purity and formulated into a therapeutic agent.

[0043] As described herein, some of the compounds of the present invention may contain one or more chiral central asymmetric carbon atoms that can result in various optical forms (e.g., enantiomers or diastereoisomers). The present invention encompasses all such optical forms and mixtures thereof of all possible stereochemistrys.

[0044] More generally, it will be apparent to those skilled in the art that the compounds of the present invention may exist in the form of various isomers and / or tautomers, including, but not limited to, geometric isomers, conformational isomers, E / Z isomers, stereochemical isomers (i.e., enantiomers and diastereoisomers), and isomers corresponding to the presence of the same substituent at different positions on the ring present in the compounds of the present invention. All such conceivable isomers, tautomers, and mixtures thereof are within the scope of the present invention.

[0045] As already detailed herein, the present invention provides polymers comprising m monomers of formula (I), or their stereoisomers, tautomers, racemates, metabolites, prodrugs or predrugs, salts, hydrates, N-oxides, or solvates: [ka] (In the formula, m is an integer in the range of 10 to 1000. n is an integer in the range of 0 to 50. R 1 It combines with an adjacent carboxyl group, and each monomer is individually an amino acid derivative, and the polymer contains at least two different amino acid derivatives. R 2 (For each monomer, hydrogen or methyl is selected individually.)

[0046] Note that m monomers in a bonded state form a polymer, and the weight of the polymer is (partially) defined by the amount of monomers bonded to each other.

[0047] Also, it should be noted that the carboxyl group of formula I may sometimes be regarded as an ester. Therefore, R combined with an adjacent carboxy group 1 may be regarded as R combined with an adjacent ester 1 being an amino acid derivative individually for each monomer.

[0048] Also, it should be noted that R combined with an adjacent carboxy group 1 forms an amino acid derivative. Throughout this application, an amino acid derivative refers to a compound or chemical group structurally derived from a parent compound through one or more steps. For example, a polymer containing an amino acid derivative can be obtained by reacting an (protected, e.g., Boc-protected) amino acid or its salt with a starting polymer.

[0049] In other words, despite this specific definition of an amino acid or its derivative, derivatives of these amino acids with similar structural and physicochemical properties result in functional analogs with similar biological activities and thus clearly still form part of the gist of the present invention. Therefore, an amino acid derivative refers to a part of a molecule, such as a monomer unit, derived from an amino acid covalently bonded to another part of the molecule.

[0050] Also, it should be noted that R 1 is selected individually for each monomer. Therefore, different amino acid derivatives are present in the polymer according to the present invention.

[0051] Furthermore, it should be noted that throughout this application, the polymers according to the present invention may also be referred to as PiPOx-XXX, where XXX refers to an amino acid derivative or may be replaced with a letter code for the amino acid derivative, such as a three-letter code. In addition, a polymer containing two different amino acid derivatives may be referred to as PiPOx-XXX_XXX. For example, PiPOx-Pro refers to PiPOx containing a proline derivative. However, the same polymer can be obtained by polymerization (radical polymerization or anionic polymerization) of the corresponding (meth)acrylamide monomer containing (protected) amino acid side chains.

[0052] Note that the following three-letter codes apply to amino acid derivatives.

[0053] [Table A] Tryptophan Valine Isoleucine Tyrosine Proline Leucine Arginine Lysine Methionine glycine Phenylalanine Threonine Alanine Histidine Selenocysteine glutamine Serine Asparagine Pyrrolicin

[0054] An advantage of the polymer according to the present invention is that the polymer can serve as a non-viral vector for transfection. As a result, it can form stable complexes with other molecules, such as activators containing nucleotides and / or nucleic acids. Furthermore, the polymer according to the present invention exhibits efficient and effective transfection capabilities for over 90% of cells, and IC12. 50It was found to exhibit potent gene silencing, with the value decreasing to 5.5 nM. In addition, the polymer according to the present invention provides high gene silencing without reducing cell viability, compared to commercially available polycationic transfection reagents such as JetPRIME (trademark) (Figure 4).

[0055] In addition, the toxicity of the polymer according to the present invention is lower than that of conventional polymers and / or delivery agents when used in humans, animals, or plants (Figure 5).

[0056] A further advantage of the polymer according to the present invention is that it exhibits excellent thermal stability and retains optical activity. As a result, the polymer according to the present invention can be applied to the development of chiral materials for applications in asymmetric catalytic reactions, selective release of enantiomers, and chiral resolution.

[0057] It has been found that amino acid derivatives provide efficient and effective transfection of molecular structures, including nucleic acids.

[0058] In a more preferred embodiment, each amino acid derivative can be independently selected from the group consisting of tryptophan derivatives, tyrosine derivatives, arginine derivatives, glycine derivatives, alanine derivatives, valine derivatives, proline derivatives, lysine derivatives, phenylalanine derivatives, histidine derivatives, isoleucine derivatives, leucine derivatives, methionine derivatives, threonine derivatives, cysteine ​​derivatives, glutamine derivatives, aspartic acid derivatives, asparagine derivatives, glutamic acid derivatives, serine derivatives, selenocysteine ​​derivatives, and pyrrolicine derivatives. Preferably, each amino acid derivative can be independently selected from the group consisting of tryptophan derivatives, tyrosine derivatives, arginine derivatives, glycine derivatives, alanine derivatives, valine derivatives, proline derivatives, lysine derivatives, phenylalanine derivatives, and histidine derivatives. More preferably, each amino acid derivative can be independently selected from the group consisting of tryptophan derivatives, tyrosine derivatives, and arginine derivatives, and even more preferably, the amino acid derivative is a tryptophan derivative.

[0059] In a more preferred embodiment, one or more amino acid derivatives can be hydrophobic amino acids, preferably one or more hydrophobic amino acids independently selected from the group consisting of alanine derivatives, valine derivatives, leucine derivatives, isoleucine derivatives, proline derivatives, and phenylalanine derivatives.

[0060] In a more preferred embodiment, the amino acid derivative is a combination of a valine derivative and a tryptophan derivative, or a combination of a proline derivative and a tryptophan derivative.

[0061] The advantage of amino acid derivatives such as tryptophan derivatives, tyrosine derivatives, lysine derivatives, histidine derivatives, and arginine derivatives, preferably tryptophan derivatives, tyrosine derivatives, and arginine derivatives, is that they can efficiently disperse positive charges. As a result, molecules containing nucleotides can be efficiently and effectively delivered to the desired tissue.

[0062] A further advantage of the above amino acids and / or all of the aforementioned amino acids is that polymer-based transfection is realized. Furthermore, the above amino acid derivatives do not affect the chemical diversity of the polymer. Therefore, the chemical diversity of the polymer according to the present invention is realized, for example, the use of linear, branched, hyperbranched, and / or dendritic polymer structures having multiple functions to optimize transfection efficiency.

[0063] The above amino acids were found to influence the structural and functional parameters of the polymer according to the present invention, and these parameters could be tuned to desired properties. For example, length, dispersion, composition, binding, sequence, and the ability to further self-assemble into higher-order aggregates (e.g., micelles, polymerosomes) were found to determine binding efficiency, binding selectivity, cell permeability, and toxicity to transfected cells.

[0064] In a more preferred embodiment, the polymer may include monomers according to formula II, or their stereoisomers, tautomers, racemates, salts, hydrates, N-oxides, or solvates: [ka] (In the formula, R 3 (For each monomer, hydrogen or methyl is selected individually.)

[0065] Preferably, R 3 It is methyl.

[0066] It should be noted that monomers produced by formula II can serve as starting materials for forming monomers produced by formula I.

[0067] It has been found that monomers according to formula II do not affect the properties of the polymer according to the present invention when they are present in amounts of less than 15% of all monomers (monomer units) in the polymer according to the present invention, preferably less than 10% of all monomers (monomer units) in the polymer according to the present invention, and more preferably less than 5% of all monomers (monomer units) in the polymer according to the present invention.

[0068] In a more preferred embodiment of the present invention, one or more amino acid derivatives include one or more of the following in the side chain of the monomer: an aromatic moiety and / or a positive charge, a carboxyl group, and one or more primary amino groups. Preferably, the one or more amino acid derivatives that include a positive charge in the side chain include a positive charge and / or a protonable group.

[0069] While not bound by theory, it should be noted that aromatic units can interact with the bases of nucleic acids. The amine moiety can provide a positive charge, which, through interaction with negatively charged nucleic acid units, can result in solubility, such as solubility in water.

[0070] Preferably, the polymer according to the present invention is a cationic polymer and / or comprises one or more amino acid derivatives containing an aromatic group and / or one or more primary amino groups.

[0071] The advantage of the cationic polymer according to the present invention is the potential of the polymer to modulate structural and functional parameters.

[0072] Cationic polymers containing amino acid moieties with exposed primary amino groups in their side chains were found to offer efficient transfection, exhibit low cytotoxicity, and strong binding ability to molecules such as nucleic acids. These effects were further enhanced with polymers containing tryptophan derivatives. The above polymers exhibited low water solubility and induced aggregation.

[0073] Furthermore, it was found that incorporating tryptophan derivatives (indole units) into the polymer sequence improved polyplex formation and increased cellular uptake and transfection efficiency. In addition, tryptophan derivatives enable membrane interactions of cell-permeable peptides, which determines increased uptake into cells. Therefore, partially incorporating amino acid derivatives containing aromatic moieties as side chain groups into the polymer facilitates payload uptake and endosomal escape, thereby enabling more effective delivery of nucleotide-containing molecules such as genes.

[0074] In a more preferred embodiment at present, the amino acid derivative is bonded via a C-terminal carboxylic acid.

[0075] An advantage of amino acid derivatives linked via a C-terminal carboxylic acid is that the polymer according to the present invention becomes positively charged in water.

[0076] In a more preferred embodiment, the polymer according to the present invention is soluble in an aqueous solvent. Preferably, the solvent contains at least 25% by volume of water, more preferably at least 30% by volume of water, even more preferably at least 35% by volume of water, even more preferably at least 40% by volume of water, even more preferably at least 45% by volume of water, and most preferably at least 50% by volume of water.

[0077] In a more preferred embodiment, the solvent contains up to 100% by volume of water, preferably up to 90% by volume of water, even more preferably up to 80% by volume of water, and most preferably up to 70% by volume of water.

[0078] In a more preferred embodiment of the present invention, the amino acid derivative includes a hydrophobic amino acid derivative. Preferably, at least 20 mol% of the amino acid derivative present in the polymer is hydrophobic, more preferably at least 25 mol% of the amino acid derivative present in the polymer is hydrophobic, even more preferably at least 30 mol% of the amino acid derivative present in the polymer is hydrophobic, even more preferably at least 35 mol% of the amino acid derivative present in the polymer is hydrophobic, and most preferably at least 40 mol% of the amino acid derivative present in the polymer is hydrophobic.

[0079] In a more preferred embodiment of the present invention, up to 100 mol% of the amino acid derivative present in the polymer is hydrophobic, preferably up to 95 mol% of the amino acid derivative present in the polymer is hydrophobic, more preferably up to 90 mol% of the amino acid derivative present in the polymer is hydrophobic, and most preferably up to 85 mol% of the amino acid derivative present in the polymer is hydrophobic.

[0080] By providing a polymer containing one or more hydrophobic amino acids, efficient and effective transfection becomes possible, enabling efficient and effective delivery of molecules containing nucleotides. The amount of hydrophobic amino acid derivative is standard 1 It is determined using 1H-NMR.

[0081] In a more preferred embodiment of the present invention, m can be an integer in the range of 50 to 1000, preferably in the range of 100 to 500, and more preferably in the range of 100 to 250.

[0082] When the value of m is an integer in the range of 50 to 1000, preferably 50 to 500, more preferably 60 to 400, more preferably 75 to 350, and more preferably 100 to 250, efficient and effective transfection becomes possible.

[0083] In a more preferred embodiment of the present invention, R 2 can be methyl. Preferably, R 2 The fact that it is methyl is combined with the fact that the polymer according to the present invention is cationic.

[0084] R 2 The advantage of the group being methyl is that this group improves the stability of the polymer according to the present invention.

[0085] In a more preferred embodiment of the present invention, the polymer further comprises k monomers individually selected from the group consisting of ethylene glycol, acrylates, and methacrylates, where 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.

[0086] The advantage of including k monomers in the polymer according to the present invention is that the properties of the polymer as a delivery agent can be further tailored to the user's needs. Therefore, a wide range of activators, including proteins, can be delivered using the polymer according to the present invention.

[0087] In a more preferred embodiment of the present invention, one or more amino acid derivatives include a Boc group, a trifluoroacetate counterionic group, and an acetate counterionic group.

[0088] It should be noted that one or more amino acid derivatives containing a Boc group, a trifluoroacetate counterion group, or an acetate counterion group may not be deprotected, or may be introduced to increase transfection or reduce toxicity.

[0089] As has already been described in detail herein, the present invention provides a composition containing a polymer according to the present invention, wherein the polymer is a delivery agent.

[0090] The composition provides the same effects and advantages as those described for the polymer according to the present invention.

[0091] The advantages of the composition according to the present invention are that the polymer has lower toxicity compared to conventional delivery agents and is an efficient and effective delivery agent.

[0092] In a preferred embodiment of the present invention, the composition further comprises an activator. Preferably, the activator comprises a nucleic acid.

[0093] In a preferred embodiment, the present invention provides a composition containing one or more polymers of the present invention that form a complex with nucleic acids, wherein the N / P ratio is particularly 1 to 40, preferably 1 to 30, more preferably 1 to 25, and most preferably 1 to 20.

[0094] It has been found that the activator comprising the polymer and nucleic acid according to the present invention provides an efficient composition for transfection.

[0095] In a more preferred embodiment of the present invention, the activator may be one or more selected from the group consisting of RNA, siRNA, mRNA, circular RNA, self-amplified mRNA, tRNA, rRNA, miRNA, lncRNA, antisense oligonucleotide (DNA or RNA), guide RNA, viral cRNA, DNA, plasmid DNA, and ribonucleoprotein. Preferably, the activator may be one or more selected from the group consisting of RNA, siRNA, mRNA, DNA, plasmid DNA, and protein, and more preferably, the activator is siRNA.

[0096] The polymer according to the present invention has been found to provide an efficient and effective composition comprising one or more activators selected from the group consisting of RNA, siRNA, mRNA, tRNA, rRNA, viral cRNA, DNA, plasmid DNA, and protein. In particular, the polymer according to the present invention provides an effective and efficient composition comprising siRNA.

[0097] As already described in detail herein, the present invention provides polymers or compositions according to the present invention for use as pharmaceuticals for humans or animals.

[0098] Use as a pharmaceutical product for human or animal use provides the same effects and benefits as those described for the polymers and compositions according to the present invention.

[0099] In preferred embodiments of the present invention, the use of the polymer or composition is in the prevention and / or treatment of at least one disease or disorder selected from the group including neoplastic diseases, infectious diseases, and genetic disorders.

[0100] As already detailed herein, the present invention provides for the use of polymers or compositions according to the present invention as delivery agents for activators, preferably as delivery agents for siRNA, or as delivery agents for plant protectants. Preferably, the activator is a pharmaceutical.

[0101] The use of activators, for example, as drug delivery agents, preferably as siRNA delivery agents, or as plant protection agent delivery agents, provides the same effects and advantages as described for the polymers, compositions, and their use as pharmaceuticals for humans or animals, or for their use in the protection of crops according to the present invention.

[0102] Furthermore, the activator can be used as a preventative measure.

[0103] As already described in detail herein, the present invention provides a method for the prevention and / or treatment of at least one disease or disorder selected from the group including neoplastic diseases, infectious diseases and genetic disorders, comprising administering a therapeutically effective amount of a compound or composition according to the present invention to a subject in need thereof.

[0104] The preventive and / or therapeutic methods according to the present invention provide the same effects and advantages as those described for the use of polymers, compositions, or other pharmacopoeias in humans or animals, or for the protection of crops, and for activators, such as drug delivery agents, preferably for the delivery of siRNA according to the present invention.

[0105] The compounds of the present invention can be prepared according to the reaction schemes presented in the following examples, but these are merely examples of the present invention, and those skilled in the art will understand that the compounds of the present invention can also be prepared by any of several standard synthesis processes commonly used by those skilled in organic chemistry.

[0106] In preferred embodiments, polymers according to formula I can be prepared by radical polymerization or anionic polymerization of the corresponding acrylamide monomer or methacrylamide monomer having an amino acid side chain.

[0107] The present invention will be described here using the following synthetic and biological examples, but these examples do not limit the scope of the present invention in any way. [Examples]

[0108] High-purity grade N-Boc-amino acids (NBAA) (N-Boc-glycine, N-Boc-L-alanine, N-Boc-L-valine, N-Boc-L-lysine, N-Boc-L-proline, N-Boc-L-phenylalanine, N-Boc-L-tryptophan, N-Boc-L-histidine), solvents (N,N'-dimethylformamide (DMF), methanol, dichloromethane (DCM), diethyl ether), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich and used as is unless otherwise specified. 2-Isopropenyl-2-oxazoline (Sigma-Aldrich, 98%, iPOx) was distilled under reduced pressure from CaH2 before use. Tetrahydrofuran (Sigma-Aldrich, THF) was freshly distilled from Na / benzophenone under an Ar gas stream before use. 2.5 mol L in hexane -1 The N-butyllithium solution (Sigma-Aldrich, n-BuLi) was used as received.

[0109] High-sensitivity green fluorescent protein (siEGFP)-targeting 21-nucleotide double-stranded siRNA and negative control siRNA (siCTRL) were purchased from Eurogentec (Slan, Belgium). The siCTRL sequence does not show homology to known eukaryotic genes. siEGFP sequence: sense strand = 5'-CAAGCUGACCCUGAAGUUCtt-3'; antisense strand = 5'-GAACUUCAGGGUCAGCUUGtt-3'. siCTRL sequence: sense strand = 5'-UGCGCUACGAUCGACGAUGtt-3'; antisense strand = 5'-CAUCGUCGAUCGUAGCGCAtt-3'. Uppercase letters represent ribonucleotides, and lowercase letters represent 2'-deoxyribonucleotides. The fluorescently labeled siRNA used in cell uptake experiments consisted of a siCTRL sequence (siCy5) with the 5' end of the sense strand modified with the Cy5® dye. Labeling and quality control were performed by Eurogentec (Slan, Belgium). 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 from absorbance measurements at 260 nm (1 OD260 = 40 μg / mL) using a NanoDrop 2000c UV-Vis spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA).

[0110] Human non-small cell lung cancer cell line (H1299-eGFP) that stably expresses eGFP is tested in 10% fetal bovine serum (FBS, Hyclone®, GE Healthcare, Belgium, Maheren), 2 mM L-glutamine, and 100 U mL -1Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (i.e., "complete cell culture medium" or CCM) supplemented with penicillin / streptomycin. The cell lines were maintained in a humidified atmosphere at 37°C with 5% CO2, and the culture medium was changed every other day. Once the confluence level reached 80%–90%, the cells were divided using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA). Cells were periodically examined to confirm that they were mycoplasma-negative.

[0111] 1 ¹H NMR spectra were recorded at 25°C using a Bruker instrument operating at 300 MHz. Chemical shifts (δ) are relative to CDCl3 (δ 7.26 ppm) or D2O (δ 4.79 ppm). Infrared spectra were measured using a Bruker Vertex 70 spectrometer equipped with a Harrick MVP2 diamond ATR instrument, with wavenumber (cm²) -1 The results were reported in [publication name]. Optical rotation was measured using a Perkin Elmer 241 polarimeter with a 20 mg / 2 mL MeOH solution.

[0112] Size exclusion chromatography (SEC) of Boc-protected polymers was performed using an Agilent 1260-HPLC system. This system included a 1260 refractive index detector (RID), a 1260 online degasser, a 1260 isocratic pump, a 1260 automated liquid sampler, and a 50°C thermostat column compartment (with two PSS Novema Max linear M columns (8 mm × 300 mm) and a guard column in series). The mobile phase used was DMAc containing 50 mM LiCl at a flow rate of 0.5 mL / min. Spectra were analyzed using Agilent Chemstation software with the GPC add-on. Molar mass and dispersion values ​​were calculated relative to PMMA standards.

[0113] Size exclusion chromatography (SEC) of cationic polymers was performed using an Agilent 1260 Infinity II HPLC system. This system included a 1260 refractive index detector (RID), a 1260 online degasser, a 1260 isocratic pump, a 1260 automated liquid sampler, and a 30°C thermostat column compartment (with one Phenomenex PolySep-P3000 (7.8 mm × 300 mm) column and a guard column in series). The mobile phase used was a mixture of methanol (MeOH) and sodium acetate buffer (0.1 M, pH 6.5) (80:20 vol%), at a rate of 0.5 mL min. -1 The flow rate was [value missing]. The spectrum was analyzed using OpenLab CDS software with the GPC add-on.

[0114] Light scattering (LS) measurements were performed using a Wyatt Technology Wyatt Heleos II multi-angle light scattering detector (MALS). The detector was connected online to an Agilent 1260 Infinity II HPLC system (MeOH-sodium acetate-SEC) and used to determine the absolute molar mass of the polymer sample being analyzed. Measurements were performed at ambient temperature; that is, the LS detector does not have a temperature control unit. Refractive index (RI) increment (dn / dc) values ​​were determined by online size exclusion chromatography (SEC) with an RI detector. This was performed on 1 mg mL of the polymer reported herein. -1 ~10 mg mL -1 The increment of RI in the concentration series was measured. The LS results were analyzed using Astra 7 software (Wyatt Technology).

[0115] Thermal analysis and glass transition temperature (T g Simultaneous TGA-DSC and MS coupling were performed on a NETZSCH STA 449C Jupiter system connected to an Aeolos II MS detector. For all samples, a helium stream (25 mL min) was used. -1) below, 10℃ min -1 Decomposition was performed in scanning mode from ambient temperature to 750°C at the specified heating rate. Differential scanning calorimetry (DSC) was performed under a nitrogen atmosphere at 10°C min. 1 (10℃ min- 1 The heating / cooling rate was set using a Setaram DSC 131 instrument.

[0116] PiPOx, for example, PiPOx with an average molecular weight of 14300 Da, was prepared by living anionic polymerization as disclosed below. In a typical preparation, the anionic polymerization of iPOx and all operations were carried out under an Ar gas stream in clean / dry glassware. 1.6 mol L of 5 mL (47.7 mmol) of iPOx in 25 mL of THF. -1 The solution was cooled to -40°C. Then, 146.8 μL of n-BuLi (0.367 mmol) in hexane was added to the reaction flask. Polymerization was maintained at this temperature for 10 minutes, after which the reaction mixture was allowed to return to room temperature naturally. Polymerization was stopped by adding 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 as a white fine powder in 96% yield. The absolute number-average molecular weight (Mn) of the analyzed polymer, determined by SEC-MALS, was 14.3 kg mol. -1 The dispersion was 1.16. The specific refractive index increment used in the calculation was 0.0902 mL g. -1 That is the case.

[0117] Alternatively, the polymer according to the present invention can also be synthesized by free radical polymerization as disclosed below.

[0118] 246.3 mg (15 mmol) of recrystallized azobisisobutyronitrile (AIBN) was weighed into a 250 mL round-bottom Schlenk flask, and a magnetic stirring bar was added. 58.1 mL of dimethyl sulfoxide (DMSO) was then added, and the mixture was stirred under an argon atmosphere until all AIBN crystals were dissolved. Next, 41.94 mL (400 mmol) of iPOx was added to the reaction mixture under an argon atmosphere. The mixture was degassed using argon for 30 minutes. The reaction mixture was then heated to 65°C in a heating block and stirred for 24 hours. The resulting reaction mixture was precipitated in excess diethyl ether, centrifuged, and the supernatant was discarded. The resulting solid was dried overnight in a vacuum oven to remove residual solvent. The resulting white powder was then dissolved again in deionized water and dialyzed for 3 days for further purification. Freeze-drying was performed using a Martin Christ Alpha 2-4 LSC plus freeze-dryer, yielding the final product as a white powder (yield: 25.2 g, 61%). The absolute number-average molecular weight (Mn) of the analyzed polymer, determined by SEC-MALS, was 36,000 Da, and the degree of dispersion was 1.48.

[0119] The post-modification reaction of PiPOx with N-Boc protected amino acids (NBAA) was carried out using the following procedure. 1.8 mmol of PiPOx (0.2 g) and 2.16 mmol of NBAA (or more) were dissolved in 3.6 mL of dry DMF, flushed under argon, and sealed. The molar supply ratio for the homopolymer was PiPOx:NBAA = 1:1.2, and the molar supply ratio for the copolymer was PiPOx:NBAA 1:NBAA 2 = 1:0.6:0.6, with a PiPOx molar concentration of 0.5 M in the solution. To completely modify PiPOx, the solution was reacted at 100°C for 72 hours. The solution was then cooled to room temperature, diluted with 2 mL of CHCl3, and precipitated in diisopropyl ether. The resulting white powder was vacuum-dried overnight at 55°C. The (co)polymer was obtained in high yields of 79% to 93%. The reaction pathways for the homopolymer and copolymer are shown in Scheme 1a and Scheme 1b in Figure 1, respectively.

[0120] The synthesis of cationic amino group-containing PiPOx polymers was carried out using the following procedure. 1 mmol of modified PiPOx-NBAA or PiPOx-NBAA1_NBAA2 was dissolved in 5 mL of DCM, and TFA (20 equivalents) was added. The solution was reacted at room temperature for 1 to 2 hours with vigorous stirring. The resulting precipitated polymer was further dissolved in MeOH and precipitated in diethyl ether. The separated product was washed 2 to 3 times with diethyl ether, dried on a glass filter, then dissolved in deionized water, filtered through a 0.2 μm polytetrafluoroethylene filter, and lyophilized. Deprotected cationic polymers were obtained in yields of 81% to 97%. The schematic reaction pathway for cationic (co)polymers is shown in Scheme 1c in Figure 1.

[0121] The solution for optical rotation measurement was prepared in a 2 mL volumetric flask using 0.02 g of homopolymer and spectroscopic-grade MeOH. After allowing the solution to stand at room temperature for 30 minutes, it was carefully packed into an optical cell and measured for 2 seconds using an integration wheel at λ=589 nm (Na line). The measurements were performed under identical conditions to allow for comparison of the difference in measured specific rotation.

[0122] Biot's formula was used to determine the specific rotation of the homopolymer: [α] λ =α×l -1 ×c -1 (In the formula, [α] λ is the specific rotation (in degrees), α is the optical rotation (in degrees) measured for the Na line, l is the cell length (1 dm), and c is the concentration of the sample (g mL). -1 The unit is ( ). The specific rotation value is shown as the average of 5 consecutive measurements.

[0123] PiPOx-NBAA-TFA polymer solution and siRNA (0.025 μg μL) in PBS- / - (calcium / magnesium-free phosphate-buffered saline) -1Equal volumes of siRNA and JetPEI were mixed, vortexed for 10 seconds, incubated at room temperature for 30 minutes to form a complex, and then agarose gel electrophoresis was performed. JetPEI® was used as a control according to the manufacturer's instructions (Polyplus, France, Il Kirsch). Briefly, the siRNA and JetPEI reagents were first diluted with equal volumes of RNase-free water containing 10% glucose, mixed, and incubated at room temperature for 15 minutes. After incubation, 5 μL of gel loading buffer (Invitrogen, Massachusetts, USA) was added to each sample (20 μL), and then loaded onto a 1.2% agarose gel. The gels were electrophoresed at 100 V for 30 minutes before imaging (Canon PowerShot A2300 IS 16.0 MP digital camera).

[0124] H1299-eGFP cells were seeded at a cell density of 7500 cells / well in 96-well plates (Bioswisstec, Schaffhausen, Switzerland) and left to stand overnight for quantification of eGFP gene silencing by flow cytometry. The following day, an siRNA polyplex was diluted 5-fold with Opti-MEM and applied to the cells for 4 hours. The polyplex was prepared with a nitrogen-to-phosphate (N / P) ratio of 10. After 4 hours of incubation, the transfection medium was removed, the cells were washed with PBS - / -, and incubated in a CCM at 37°C for 44 hours. eGFP expression was detected by 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 FlowJo analysis software (Treestar, Costa Mesa, California, USA). The eGFP expression rate for each sample was calculated by normalizing the fluorescence signal of cells treated with siEGFP polyplexes with the fluorescence signal of cells treated with siCTRL polyplexes. For JetPRIME® transfection, cells were incubated with the transfection reagent in CCM for 4 hours (see also Figure 4).

[0125] To quantify the intracellular uptake of siRNA polyplexes by flow cytometry, 7500 H1299-eGFP cells were placed in a 96-well plate (Bioswisstec, Schaffhausen, Switzerland) (100 μL per well). -1 Seeds were seeded at a density of ) and left to stand overnight. A complex was formed with siCTRL:siCy5(trademark) (95:5 mol%) on PiPOx-NBAA-TFA polymer, diluted 5-fold in Opti-MEM (Gibco(trademark)-Life Technologies, Grand Island, New York, USA), and applied to the cells (4 hours, 37°C, 5% CO2). Next, to remove the fluorescence bound to the cell surface, dextran sulfate sodium salt (0.1 mg ml in PBS) was used. -1 The cells were washed with ). Flow cytometry quantification of siCy5 fluorescence was performed as described in this application.

[0126] Cell viability was analyzed using the CellTiter-Glo® assay (Promega). Here, H1299-eGFP cells were seeded as described above and left to stand overnight. The following day, the cells were incubated in Opti-MEM with two selected mount concentrations of PiPOx-NBAA-TFA polymer for 4 hours, followed by incubation with CCM for 44 hours. Before starting the assay, the CellTiter-Glo® buffer and culture plate were left at room temperature for 30 minutes. Next, the culture medium was replaced with 100 μL of fresh medium and an equal volume of assay buffer was added. To induce complete lysis, the plate was shaken for 2 minutes and left at room temperature for 10 minutes to stabilize the signal. Subsequently, 100 μL from each well was transferred to an opaque 96-well plate (Greiner Bio-One, Kremsmünster, Australia), and the luminescence signal was measured using a GloMax® 96 microplate luminometer (Promega, Belgium). The data is expressed as the percentage of viable cells calculated from the luminescence signals of untreated cells under each condition, taking into account the background fluorescence of the culture medium.

[0127] Polyplex is prepared by dissolving the polymer in PBS- / - and an equal volume of siRNA solution (0.025 μg μL). -1 The polyplex was prepared by mixing the following: After allowing the complex to form for 30 minutes, the resulting polyplex was diluted fivefold with Hepes buffer (20 mM, pH 7.4) to reach the final concentration corresponding to the maximum concentration used in transfection experiments. The zeta potential and hydrodynamic diameter of the resulting PiPOx-NBAA-TFA siRNA polyplex were measured by dynamic light scattering (DLS) (Malvern, UK).

[0128] Aliphatic and aromatic NBAAs were used to modify PiPOx. To control the copolymer composition by adjusting the NBAA supply ratio, kinetic studies were performed on selected NBAAs to calculate the reaction rate constant (kr) at 100°C. Modification of PiPOx with valine (aliphatic NBAA), proline (cyclic aliphatic NBAA), and phenylalanine (aromatic NBAA) was performed as follows: 1 The reaction was monitored by 1H NMR spectroscopy and SEC chromatography. During the ring-opening addition reaction of NBAA to the 2-oxazoline ring, the signals of the unreacted 2-oxazoline ring and the ester amide reaction product were clearly separated and observed. The proton signal of the formed ester amide structure (-NH-CH2-) shifted to 3.12 ppm at a lower field compared to the 3.45 ppm proton corresponding to the unreacted 2-oxazoline ring (=N-CH2-). The degree of chemical modification was determined by integrating these two signals. However, despite the difference in reactivity between Pro and Val / phenylalanine (PhAla) observed on the first day, each reaction reached complete transformation with sufficient heating time. Therefore, a reaction time of 72 hours was used in all subsequent experiments to achieve complete transformation. The calculated reaction rate constant k for Pro r (0.074±0.004×10 -3 mol L -1 s -1 ) are Val and PhAla (0.178 ± 0.003 × 10⁻¹⁰, respectively). -3 mol L -1s -1 and 0.164±0.007×10 -3 mol L -1 s -1 k r It was found to be 2.5 times slower compared to the value. Boc-amino acid pK a Considering that they are in the same range, the low reactivity of Pro can be inferred to be due to steric effects induced by the strained proline ring. From kinetic studies, k r Despite these differences, it is suggested that NBAAs exhibit similar reactivity under these given conditions, except that proline reacts more slowly than the other amino acids. Furthermore, several polymers according to the present invention were synthesized using similar synthetic routes (see Table 1) and similarly characterized.

[0129] [Table 1] Table 1: Physical properties of a series of N-Boc protective (co)polymers fully modified with N-Boc amino acids. polymer c degree of dispersion a The yield is calculated by gravimetric analysis based on the precipitated polymer; b The composition is 1 Estimated from 1H NMR spectrum; c Determined against the PMMA standard by SEC in DMAc; d 10℃min -1 The second heating operation at the heating rate; e 10℃min -1 Decomposition temperature in a helium stream at the heating rate; f This is the specific rotation calculated from the optical rotation measurement of the Na line in MeOH, and indicates that nd could not be determined.

[0130] Following the post-modification reaction of PiPOx, it was confirmed that homopolymers with increased number-average molar mass (Mn), narrow dispersion, and clearly defined structure were obtained (see Table 1). This suggests that the introduction of large, bulky side chains increases the hydrodynamic volume, resulting in minimal polymer chain coupling. The thermal properties of the series of polymers were investigated by simultaneous TGA-DSC-MS analysis. The homopolymers showed improved thermal stability and glass transition temperature (T) compared to the starting polymer, PiPOx. g Both of the following were shown to be low (see Table 1). T compared to the starting polymer (PiPOx) g The decrease may be due to increased flexibility of the polymer chain resulting from ring-opening of the iPOx units.

[0131] In addition to polymethacrylamide homopolymers functionalized with N-Boc amino acids, a series of N-Boc amino acid-functionalized polymethacrylamide copolymers were prepared by a one-pot modification reaction with PiPOx and two equimolar amounts of two different NBAA, as shown in Scheme 1b of Figure 1, thereby obtaining polymers with a substantially 50:50 molar ratio of amino acid derivatives. The parameters of the homopolymers according to the present invention are summarized in Table 1.

[0132] The cationic polymethacrylamide homopolymers and copolymers according to the present invention were obtained by deprotecting N-Boc-functionalized polymers as shown in Scheme 1c of Figure 1. The physical properties of these polymers are summarized in Table 2.

[0133] [Table 2] Table 2: Physical properties of a series of cationically charged PiPOx(co)polymers. polymer cord a The yield is calculated by gravimetric analysis based on the precipitated polymer; b Determined in MeOH by SEC-MALS; c Calculated theoretical value; d 10℃min -1The second heating operation at the heating rate; e 10℃min -1 Decomposition temperature in a helium stream at the heating rate; f Specific rotation (Na line) calculated from optical rotation measurements in MeOH; * An exception using Hg wire.

[0134] The specific rotation of the homopolymers was determined by optical rotation measurement in methanol at room temperature. The values ​​for the N-Boc protected polymers are summarized in Table 1, and the values ​​for the cationic polymers are shown in Table 2. After post-modification with N-Boc-L-amino acids, the resulting series of homopolymers retained chiral optical properties along the polymer side chains. However, lower values ​​were measured for the series of homopolymers compared to free L-amino acids. This may be due to polymer-polymer interactions in methanol, which is not a theta solvent. Deprotection of the N-Boc group yields cationic (co)polymers, which induce solubility not only in water but also in aqueous buffers such as phosphate-buffered saline (PBS), which is necessary for transfection.

[0135] The ability of synthesized cationic PiPOx-NBAA-TFA polymethacrylamide (co)polymers to complex with siRNA was tested by agarose gel electrophoresis using a commonly used nitrogen-to-phosphate ratio (N / P) of 10. The most hydrophobic polymers, i.e., those fully modified with phenylalanine and tryptophan, exhibited the lowest efficiency of complex formation, with most of the siRNA remaining freely migrated on the gel. This may be due to the significant steric hindrance of the bulky aromatic side chains in neutralizing electrostatic interactions with the siRNA phosphate backbone, or the low hydration of these most hydrophobic polymers suppressing the interaction. Surprisingly, the application of copolymers combining Trp with Val or Pro restored some degree of complex formation efficiency, while copolymers containing PhAla along with Val or Pro showed only slight improvement in siRNA complex formation. On the other hand, homopolymers with Val in the side chains could only complex with half of the siRNA at a nitrogen-to-phosphate ratio (N / P) of 10. All other polymers tested showed the greatest complex formation with siRNA under the experimental conditions used.

[0136] For polymers that showed excellent complex-forming ability, further testing was conducted regarding their ability to deliver the complexed siRNA to cells. For this purpose, PiPOx-NBAA-TFA polyplexes containing 50 nM eGFP-targeted siRNA (siEGFP) with an N / P ratio of 10 were fabricated, and transfection into H1299-eGFP cells was evaluated. Surprisingly, PiPOx-Val_Trp and PiPOx-Pro_Trp exhibited efficient gene silencing, almost completely knocking down the target eGFP gene (Figures 2a and 2b). These data clearly demonstrate that complex formation with siRNA alone is insufficient for efficient transfection. In fact, the polyplex needs to be taken up by the target cell and be able to deliver the siRNA across the endosomal barrier into the cytoplasm, which depends on the type of polymer.

[0137] The intracellular uptake of polyplexes (loaded with Cy5-labeled siRNA) obtained using two copolymers (PiPOx-Val_Trp and PiPOx-Pro_Trp) was evaluated and compared with the corresponding homopolymer modified with only a single amino acid type. In H1299-eGFP cells exposed to PiPOx-Trp, Cy5 fluorescence was hardly detected (Figure 2a). On the other hand, flow cytometry clearly showed efficient intracellular uptake in over 90% of cells when transfected with both PiPOx-Val_Trp and PiPOx-Pro_Trp polyplexes, but hardly any uptake was observed with the corresponding homopolymer polyplexes (Figures 3a-3c). Comparing the two copolymers, the PiPOx-Pro_Trp polyplex showed slightly higher intracellular uptake efficiency and a 25% higher MFI value compared to the PiPOx-Val_Trp polyplex (Figure 3b).

[0138] Furthermore, the discovered copolymer showed significantly superior performance in siRNA transfection compared to jetPEI (Figure 4), while exhibiting no cytotoxicity at transfection concentrations equivalent to 0.01 mg / mL or less (Figure 5).

[0139] Using a similar method, PiPOx-Val Trp and PiPOx-Pro Trp copolymers with different chain lengths (degrees of polymerization (DP)) (Figure 6a), as well as corresponding polyplexes using siRNA and siRNA controls for EGFP silencing, were prepared. Transfection experiments revealed that, as shown in Figure 6b, cells were efficiently silenced by all copolymers having DPs from 90 to 450 when transfected with siRNA for EGFP, while no silencing was observed with transfection using siRNA controls or with an untransfected control (NTC). The present invention is by no means limited to the preferred embodiments and / or experiments thereof described above. The rights sought are defined by the appended claims, within which many modifications can be envisioned. [Explanation of Symbols]

[0140] Drawing translation Figure 1 glycine alanine valine proline Lysine phenylalanine tryptophane Figure 2 eGFP silencing % EGFP expression % EGFP expression rate Figure 3 % Cy5 Positive cells % Cy5 Positive cells Figure 4 % EGFP expression % EGFP expression rate siRNA concentration (nM) siRNA concentration (nM) Figure 5 Viability % Survival rate % PiPOx concentration mg / ml PiPOx concentration (mg / ml) Figure 6(a) Polymer Polymer Code Composition Mn (theoretic) Mn (theoretical value) Figure 6(b) polymer polymer

Claims

1. Polymers comprising m monomers of formula (I), or their stereoisomers, tautomers, racemates, salts, hydrates, N-oxides, or solvates: 【Chemistry 1】 (In the formula, m is an integer in the range of 10 to 1000. n is an integer in the range of 0 to 50. R 1 It is combined with an adjacent carboxyl group to form an amino acid derivative for each monomer, and the polymer contains at least two different amino acid derivatives. R 2 (Each monomer is individually selected from the group consisting of hydrogen or methyl.)

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

3. The polymer according to claim 1 or 2, wherein the at least two different amino acid derivatives are a phenylalanine derivative or a tryptophan derivative and a valine derivative or a proline derivative.

4. The polymer according to any one of claims 1 to 3, wherein one or more amino acid derivatives include an aromatic moiety.

5. The polymer according to any one of claims 1 to 4, wherein the amino acid derivative is bonded via a C-terminal carboxylic acid.

6. The polymer according to any one of claims 1 to 5, wherein m is an integer in the range of 50 to 1000, preferably in the range of 100 to 500, and more preferably in the range of 100 to 250.

7. R 2 The polymer according to any one of claims 1 to 6, wherein is methyl.

8. The polymer according to any one of claims 1 to 7, further comprising k monomers individually selected from the group consisting of ethylene glycol, acrylates, and methacrylates, wherein k is an integer in the range of 1 to 1000, and preferably the k monomers are statistically or randomly distributed in the polymer or form blocks.

9. The polymer according to any one of claims 1 to 8, wherein one or more amino acid derivatives include a Boc group, a trifluoroacetate counterionic group, and an acetate counterionic group.

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

11. The composition according to claim 10, further comprising an activator.

12. The composition according to claim 11, wherein the activator comprises nucleic acid.

13. The composition according to claim 11 or 12, wherein the activator is one or more selected from the group consisting of RNA, siRNA, mRNA, circular RNA, self-amplified mRNA, tRNA, rRNA, viral cRNA, miRNA, lncRNA, antisense oligonucleotide of DNA or antisense oligonucleotide of RNA, guide RNA, DNA, plasmid DNA, and ribonucleoprotein, preferably the activator is one or more selected from the group consisting of RNA, siRNA, mRNA, DNA, and plasmid DNA, and more preferably the activator is siRNA.

14. A polymer according to any one of claims 1 to 9, or a composition according to any one of claims 10 to 13, for use as a pharmaceutical product for humans or animals.

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

16. Use of the polymer according to any one of claims 1 to 9 or the composition according to any one of claims 10 to 13 as a delivery agent for activators, preferably as a delivery agent for siRNA, or as a delivery agent for plant protective agents.

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