Aza-naphthalene functionalized polymers for gene and drug delivery

Abstract

The present invention relates to nanoparticles comprising an aza-naphthalene functionalized polymer and a biologically active component wherein the polymer comprises a polyamine segment based on a bis (alpha, beta-unsaturated carbonyl) monomer, an aza-naphthalene-containing amine monomer comprising an aza-naphthalene moiety, and an amine monomer. The invention also relates to polymer nanoparticles for use in medical therapy, such as in vivo delivery of bioactive components.

Description

[0001] The present invention is in the field of polymer nanoparticles for delivering biologically active components (e.g. polynucleotides, oligonucleotides, peptides and / or proteins) to cells. In particular, the present invention relates to polyamine-containing polymers of these nanoparticles.

[0002] Polymer nanoparticles, and in particular polycationic nanoparticles, are used in the medical field to deliver biologically active components (e.g., polynucleotides, oligonucleotides, peptides and / or proteins) to cells. See, for example, Pudlarz and Szemraj (OpenLife Sci. 2018: 13: 285-298). These polymer nanoparticles include polymers, such as polyamine-containing polymers that can be positively charged under physiological conditions, in which case the resulting nanoparticles can be referred to as polycationic nanoparticles or simply cationic nanoparticles.

[0003] The process of transporting, delivering and activating a payload by nanoparticles is referred to as transfection. In the case where the payload comprises nucleic acid, transfection can be used to indicate the process of intentionally introducing nucleic acid into a eukaryotic cell. The success of a medical method using nanoparticles depends particularly on transfection efficiency. In this regard, see, for example, Kim and Eberwine, Anal. Bioanal. Chem. 2010 Agu: 397 (8): 3173-8. Transfection efficiency depends on a variety of factors, including particle and payload stability, endocytosis, intracellular transport, positioning, and payload unpacking.

[0004] A variety of specific polyamine-containing polymers are known from WO 2012 / 165953 and references cited therein as well as US 2012 / 0149630 and WO 2021 / 069920. These polymers are known for their ability to deliver siRNA, DNA, etc.

[0005] Other types of polymers for DNA delivery are known from Piotrowski-Daspit et al. Advanced Drug Delivery Reviews 156 (2020) 119-132 and Van Bruggen et al., PNAS, 117 (2020) (52), 32919-32928.

[0006] Quinoline-functionalized polymers are known, for example, from Aderibigbe et al. (J. Inorg. Organomet. Polym. (2012), 22: 429-438).

[0007] Polyamine-containing polymers, such as those described in detail in the aforementioned WO 2012 / 165953, are known for their good transfection efficiency. However, it remains desirable to further improve the transfection efficiency of such polymers.

[0008] The present inventors have unexpectedly discovered that the transfection efficiency of polymer nanoparticles based on polyamine-containing polymers can be improved by functionalizing a portion of these polyamine-containing polymers with one or more azaphthalene moieties.

[0009] Thus, in one aspect, the present invention relates to nanoparticles comprising an azine-functionalized polymer, wherein the polymer comprises a polyamine segment of formula (I),

[0010] -[XB]n-[XQ]m—

[0011] (I)

[0012] wherein X is based on a bis(α,β-unsaturated carbonyl) monomer, B is based on an amine monomer, Q is based on an aza-naphthyridine-containing amine monomer comprising an aza-naphthyridine moiety, and wherein m is 1 or greater, and n is 1 or greater.

[0013] It was found that polymers with an increased content of azanaphthalene moieties (i.e. with an increased number m) resulted in a concomitant increase in transfection efficiency in cells. However, it was also found that this effect leveled off. Considering that it was also found that polymers based on amine monomers that do not contain azanaphthalene moieties (i.e. polymers where n is at least 1) in addition to azanaphthalene amine monomers can be more easily synthesized in aqueous solvents than polymers based entirely on azanaphthalene amine monomers (i.e. polymers where n is 0), this is preferred. Polymers where n is 0 typically produce poorly water-soluble polymers in the slightly acidic to neutral pH range. Therefore, it is considered impractical to synthesize these polymers. Polymers where n is 0 can be obtained in anhydrous solvent systems (e.g. dimethyl sulfoxide). However, this generally provides polymers that may not be easily purified using commonly used aqueous purification methods.

[0014] Incorporation of an amine monomer (i.e. the monomer on which B is based) to obtain a polymer according to the invention, i.e. n is at least 1, results in an improved synthesis. Typically, the water-soluble polymer is provided in a wide range of nitrogen-containing naphthyridine amine monomer (Q) to amine monomer (B) ratios. Thus, the polymer can be synthesized in a preferred aqueous solvent and purified using commonly used purification methods.

[0015] The aza-naphthalene moiety herein refers to a naphthalene moiety (molecular formula C 10 H 7-x , where x is the number of optional substituents), wherein at least one carbon is replaced by a nitrogen atom - for example, the formula is C9NH 6-x Monoazine, molecular formula C8N2H 5-xwherein x is the number of optional substituents. The naphthyridine moiety may be substituted or unsubstituted. Preferably, it is substituted with an alkoxy or halide substituent.

[0016] Particularly suitable and preferred monoazanaphthalenes for use in the present invention include benzopyridines, which include quinoline (also known as 1-azanaphthalene or 2,3-benzopyridine) and isoquinoline (also known as 2-azanaphthalene or 3,4-benzopyridine). The quinoline moiety gives particularly good results and is most preferred. In the case where the azanaphthalene moiety is a quinoline moiety, which is preferred, the polymers of the present invention may be referred to as quinoline functionalized polymers.

[0017] Particularly suitable naphthidines for use in the present invention include 1,2-naphthyridine (also known as cinnoline), 1,3-naphthyridine (also known as quinazoline), 1,4-naphthyridine (also known as quinoxaline), 2,3-naphthyridine (also known as phthalazine), 1,8-naphthyridine, 1,7-naphthyridine, 2,7-naphthyridine, 1,6-naphthyridine, 1,5-naphthyridine and 2,6-naphthyridine.

[0018] The term "segment" as used herein is understood to be a polymer structure of any length. The polyamine segment according to formula (I) is based on a di(α,β-unsaturated carbonyl) monomer (X) and an amine monomer (Q and B), the combination of which produces repeating units. Thus, in each repeating unit, there may be a Q amine (i.e. [XQ]) or a B amine (i.e. [XB]), which may be randomly located in the polyamine segment. The amine monomer B does not contain a naphthalene-containing portion and differs from the naphthalene-containing amine monomer Q at least in this respect. Therefore, the polyamine segment can be regarded as a copolymer, in particular a random copolymer.

[0019] The designations n and m represent the number of repeating units in each polyamine. In a polyamine, Q is always present so that m is equal to 1 or greater, and B is always present: n is equal to 1 or more. The ratio of n to m can be determined by proton nuclear magnetic resonance spectroscopy ( 1 H-NMR), where the integral of the aromatic signal of Q can be compared with the total polymer CH2 integral in a limited spectral window of 1.0 to 2.0 ppm. From this ratio, the m / n ratio can be derived. In some typical embodiments, the sum of n and m is from 2 to 250, preferably from 5 to 100. The typical weight-average molecular weight of the polyamine is from 1 to 100 kD, preferably from 5 to 40 kD. The weight-average molecular weight can be determined by a method based on gel permeation chromatography (GPC). In particular, the weight-average molecular weight can be determined using three G1316B thermostated (35°C) columns connected in series. (NOVEMA MAX medium combination, having a molar mass range of 100 to 1 000 000 Da; PSS NOVEMAMax 5μm, 8×300mm+2×NOVEMAMax 5 μm column 8×300 mm, PSS Polymer Standards Service GmbH) with 100 mM NaCl + 0.3% formic acid as eluent, flow rate 1 mL / min and equipped with a refractive index detector. Molecular weight can be determined relative to dextran standards.

[0020] X and B groups

[0021] The polyamine may be based on a combination of Q and two or more different amine monomers B (e.g., three different types of amine monomers B). In addition, Q may represent a mixture of different nitrogen-containing naphthyridinyl amine monomers. The same applies to the X component of the structure derived from a di(α,β-unsaturated carbonyl) monomer. Thus, X, B, and Q in formula (I) may represent a mixture of di(α,β-unsaturated carbonyl) monomers, general amine monomers, and nitrogen-containing naphthyridinyl amine monomers, respectively. It will be appreciated that B and Q are typically based on primary and secondary amines, since such amines are typically sufficiently reactive in polymerization reactions leading to polymers (i.e., aza-Michael addition type reactions, see below).

[0022] The polyamine segments according to formula (I), and in particular X, Q and B thereof, are defined by the monomers on which they are based, as is common in the art for the sake of clarity and conciseness. It will be appreciated that in the preparation of the polyamines, the X monomers react with the Q and B monomers, respectively, and thus their chemical structures change. For example, during the preparation of the polyamines, the monomers on which Q and B are based are typically reacted with the di(α,β-unsaturated carbonyl) monomer X in a (aza-) Michael addition type reaction, after which the double bond in the α,β-unsaturated carbonyl moiety becomes a single bond.

[0023] Monomer X is preferably according to formula (I x ) di(α,β-unsaturated carbonyl) monomers,

[0024]

[0025] in

[0026] A is selected from C(R 1 )2、O、N(R 1 ), S and their combinations,

[0027] R 1 is selected from H, halide, and C1-C1 optionally substituted with one or more halide 22 Hydrocarbyl (eg, alkyl, alkenyl, and polyalkenyl), and combinations thereof; and

[0028] R 2is selected from C1-C1 optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms 40 Straight chain, cyclic and branched alkylene groups, and combinations thereof.

[0029] Preferably, A in the polyamine segment comprises N(R1), in which case the polymer segment can be referred to as poly(aminoamide). In other words, the polyamine segment is preferably a poly(aminoamide) (PAA) segment.

[0030] Hydrocarbyl and hydrocarbylene groups as used in the present invention include monovalent and divalent groups formed by removing one or two hydrogen atoms from hydrocarbons (e.g., ethyl, phenyl and ethylene, phenylene), respectively. When intervening with one or more heteroatoms, these groups may be heteroaliphatic and / or heteroaromatic, as well as straight chain, branched and / or cyclic.

[0031] Monomer B is based on the formula (I B1 ) of the primary amine,

[0032]

[0033] Or based on formula (I B2 ) of diamine,

[0034]

[0035] in

[0036] R 3 independently selected from H, optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms C1-C 40 Straight chain, cyclic and branched hydrocarbon groups, and combinations thereof; and R 4 is selected from C1-C1 optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms 40 Straight chain, cyclic and branched alkylene groups, and combinations thereof.

[0037] Preferably, R 3 Having an overall neutral or negative charge at slightly acidic to neutral pH. Wherein slightly acidic to neutral pH means herein a pH of about 6 to about 7.

[0038] In some specific embodiments of the present invention, X is degradable under physiological conditions. Therefore, X preferably comprises a moiety in which one or more bonds are easily broken (e.g., due to hydrolysis, reduction, oxidation, etc.) under physiological conditions. Some examples of such moieties include esters, thioesters, and disulfide moieties. In this regard, disulfide moieties are particularly preferred. Therefore, X preferably comprises a disulfide moiety, more preferably a compound of formula (I x ) 2contains a disulfide moiety, even more preferably R 2 selected from C1-C 10 Hydrocarbylene groups, preferably straight chain alkylene groups, and combinations thereof.

[0039] In some specific embodiments of the present invention, B comprises a hydroxyl moiety, preferably a primary hydroxyl moiety. B1 ) and / or (I B2 ) 3 At least one or more of the formula (I B1 ) and / or (I B2 ) 3 At least one or more of C1-C 10 Hydroxyl, most preferably straight chain alkyl C1-C 10 Hydroxides, and combinations thereof.

[0040] Particularly suitable monomers on which B can be based have biological activity and / or impart water solubility. Particularly suitable monomers include 4-aminobutanol (ABOL) and 5-aminopentanol (APOL) and other hydroxyl-containing monomers. Preferably, when incorporated into a polymer, the monomer is neutral (i.e., uncharged) under physiological conditions (i.e., at 20° C. at pH 7). When incorporated into a polymer, the incorporation of these neutral monomers such as ABOL and APOL leads to better transfection efficiency compared to monomers that are positively charged under physiological conditions (see, e.g., Lin et al. Bioconjugate Chem. 2007, 18, 1, 138-145). Some examples of such monomers that are positively charged include amine monomers containing additional amine groups (e.g., aliphatic tertiary amines), such as dimethylaminopropylamine and N-(3-aminopropyl)-morpholine. Therefore, when incorporated into a polymer, monomers that are positively charged under physiological conditions are less preferred for B.

[0041] Q and the aza-naphthalene moiety

[0042] Q in the polyamine comprises an azanaphthyridine moiety. The azanaphthyridine moiety may be optionally substituted as further elucidated herein.

[0043] In a preferred embodiment, Q is based on Q1 ) of a primary amine,

[0044]

[0045] And / or based on formula (I Q2 ) of diamine,

[0046]

[0047] in

[0048] Z 1 represents a group comprising an azanaphthalene moiety;

[0049] Z 2 represents a group comprising an aza-naphthalene moiety, or is selected from H, optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms C1-C 40 Straight chain, cyclic and branched hydrocarbon groups, and combinations thereof; and

[0050] R 4 As described above for formula (I B2 ) is limited.

[0051] In formula (I Q2 ), there is at least one aza-naphthalene moiety. In certain embodiments, according to formula (I Q2 )'s Q in Z 1 It contains only the naphthalene azide part and in Z 2 In other embodiments, according to formula (I Q2 )'s Q in Z 1 and Z 2 Both contain azanaphthene moieties. The polyamines according to the invention may also contain a mixture of various Q groups, each group having a different structure and / or containing a different amount of azanaphthene moieties.

[0052] In some specific embodiments, Z 1 and optional Z 2 independently represents a part according to any of the following structures,

[0053]

[0054] Where R 5 is selected from C1-C1 optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms 40 , preferably C1-C 10 , more preferably C1-C6 straight chain, cyclic and branched alkylene groups, and combinations thereof; and wherein the azanaphthalene moiety is optionally substituted by one or more substituents, the substituents are preferably selected from halides, alkyls and alcoholates, which are optionally substituted by one or more heteroatoms.

[0055] The naphthalene azamethine moiety can be combined with the formula (I Q1 ) and (I Q2 ) is connected to a primary or secondary amine group. Preferably, the moiety is connected via a spacer R 5 Connected to the backbone of the polyamine segment.

[0056] More preferably, formula (I Q1) and (I Q2 ) in any of the 1 and optionally Z 2 Independently express the formula (I Z1 )、(I Z2 ) or (I Z3 ) of the quinoline portion, preferably of formula (I Z3 ) of the quinoline portion.

[0057]

[0058] Where R 5 is selected from C1-C1 optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms 40 , preferably C1-C 10 , more preferably C1-C6 straight chain, cyclic and branched alkylene groups, and combinations thereof, wherein preferred examples are in I Z3 Given in.

[0059] The azanaphthalene moiety may optionally be substituted with one or more substituents, preferably selected from halides, alkyls and alcoholates, both of which may optionally be substituted again with one or more heteroatoms. In a particularly preferred embodiment, the quinoline moiety comprises a substituent at the 7-position, and formula (I Q1 ) and (I Q2 ) in any of the 1 and optional Z 2 Independently express the formula (I Z4 )part

[0060]

[0061] Where R 6 is selected from alkyl, alcoholate, halide, and mixtures thereof, which are optionally substituted with one or more heteroatoms, preferably halide, more preferably wherein R 6 For Cl.

[0062] The inventors found that the spacer group R 5 Can have a relatively simple structure, such as a straight chain unsubstituted alkylene group, such as butylene (C4H8), hexylene (C6H 12 ) or octyl (C8H 16 ), while retaining the beneficial effect of improving transfection. Thus, although a spacer comprising a quinuclidine ring (such as that found in the natural product quinine) can be used, it is not required.

[0063] General polymer formula and core

[0064] Wherein X, B and Q are based on the formula (I X )、(I B1) or (I B2) , and (I Q1 ) or (I Q2 In some embodiments of the monomer of the present invention, the polyamine can be represented by any one of formulas (IIa) to (IId) and combinations thereof, wherein n, m, p, A, R 1 , R 2 , R 3 , R 4 , Z 1 and Z 2 As for (I), (I X )、(I B1 )、(I B2 )、(I Q1 ), and (I Q2 ) is limited.

[0065]

[0066] In some specific embodiments of the present invention, the polyamine is linked to a core, preferably a polymer core (T). Thus, in some specific embodiments of the present invention, the polyamine segments and preferably polymers according to the present invention have a structure according to formula (III), wherein n and m may be as defined for formula (I), and q represents the number of poly(aminoamide) segments per core (T), i.e. the number of arms. In a typical embodiment, q is from 1 to 64.

[0067]

[0068] In a particularly preferred embodiment, the polyamine segments and preferably polymers according to the present invention have a structure according to any one of formulas (IVa), (IVb), (IVc), (IVd) and combinations thereof, wherein n, m, A, R 1 , R 2 , R 3 , R 4 , Z 1 and Z 2 For formula (I), (I X )、(I B1 )、(I B2 )、(I Q1 ), and (I Q2 ) is limited.

[0069]

[0070] In formulae (III) and (Iva) to (IVd), T represents a preferred weight average molecular weight M. w is a nucleus of about 60 to about 25,000; q is 1 to 64; and R 6is selected from H, optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms C1-C 40 Straight chain, cyclic and branched hydrocarbon groups, and combinations thereof.

[0071] The core T is usually based on a structure having one or more primary and / or secondary amines. These amines are represented in formulae (IVa) to (IVd) by the group NR 6 Representation. Particularly suitable cores include oligoamine cores or polyamine cores, such as 1,2-ethylenediamine, tris(2-aminoethyl)amine, and polymer cores with primary and / or secondary amines, such as polyethyleneimine (PEI), poly(aminoamide) polymers or dendritic polymers (PAMAM), polypropyleneimine (PPI), poly(esteramine) polymers (PEAN) and poly(etheramine) polymers (PEAC) and other polymers described in WO 2012 / 165953 (referred to as POL therein). PEI is particularly preferred as a core, and in this regard, PEI 800 with an average of 6 primary amines is most preferred, because this gives particularly good results. It is understood that both ends of the poly(aminoamide) polymer can react with POL, which can result in two or more cores being incorporated into the same macromolecule. However, in certain embodiments, reaction conditions can be selected to limit crosslinking.

[0072] The present inventors have found that the relative amount of the nitrogen-containing naphthyridine amine monomer Q in the polyamine and / or polymer relative to the amine monomer B affects the transfection efficiency and toxicity. In addition, it was found that the higher the relative amount of Q becomes, the more the solubility decreases concomitantly during the synthesis of the polymer, and also causes the polymer to have lower solubility in aqueous systems.

[0073] The relative amount of Q relative to B can be expressed as a ratio of m to n. Due to the presence of the amine monomer B, n is at least 1, and thus the ratio can be 1:1, i.e. 1. However, preferably the polyamine segment and / or polymer comprises more B than Q. In some specific embodiments, the ratio of m to n is 1:20 or greater (i.e. 0.05 or greater, e.g. 0.05 to 20). Preferably, the ratio of m to n is 1:10 to 10:1, more preferably 1:5 to 2:1, and most preferably 1:4 to 1:1, e.g. about 1:3, as particularly good results are obtained within this range.

[0074] The polymers according to the formula (I x ) and (I B1 ) and (I B2Some suitable embodiments of monomers of ) are described in WO 2012 / 165953, the contents of which are incorporated herein in their entirety. Therefore, preferably, the following applies to the polyamine segments and / or polymers according to the present invention and / or the monomers on which they are based:

[0075] -R 2 Independently selected from the following and combinations thereof:

[0076] a) C1-C 40 Alkylene, wherein the alkylene may be linear or branched and is optionally substituted and / or is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S, and / or wherein the alkylene is interrupted by one or more -SS- groups;

[0077] b) C3-C 40 Cycloalkylene, wherein the cycloalkylene is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one or more heteroatoms in the ring, wherein the heteroatoms are independently selected from O, N and S, and / or wherein the cycloalkylene is interrupted by one or more -SS- groups outside the ring;

[0078] c) C6-C 40 arylene, wherein the arylene group is optionally substituted;

[0079] d) C6-C 40 heteroarylene, wherein the heteroarylene comprises one or more heteroatoms independently selected from O, N and S and / or wherein the heteroarylene is optionally substituted;

[0080] e) C7-C 40 Alkylarylene, wherein the alkylarylene is optionally substituted and / or wherein the alkyl part of the alkylarylene is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S, and / or wherein the alkyl part of the alkylarylene is interrupted by one or more -SS- groups;

[0081] f)C7-C 40 alkylheteroarylene, wherein the alkylheteroarylene comprises one or more heteroatoms independently selected from O, N and S, and / or wherein the alkylheteroarylene is optionally substituted, and / or wherein the alkyl part of the alkylheteroarylene is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms independently selected from O, N and S, and / or wherein the alkyl part of the alkylheteroarylene is interrupted by one or more -SS- groups; and

[0082] g) Two of them C7-C40 (Hetero)arylene and / or C7-C 40 Alkyl(hetero)arylene groups linked to each other via -SS- groups, wherein the alkyl part of the alkyl(hetero)arylene group is linear or branched and is optionally (partially) unsaturated and / or optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

[0083] -R 3 Independently selected from the following and combinations thereof, preferably wherein R 3 Has an overall neutral or negative charge at slightly acidic to neutral pH:

[0084] a) H;

[0085] b) C1-C 10 Alkyl, wherein the alkyl group may be linear or branched and is optionally substituted and / or optionally (partially) unsaturated and / or optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

[0086] c) C3-C 12 Cycloalkyl, wherein the cycloalkyl is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one or more heteroatoms in the ring, wherein the heteroatoms are independently selected from O, N and S;

[0087] d) C6-C 12 Aryl, wherein the aryl group is optionally substituted;

[0088] e) C6-C 12 heteroaryl, wherein the heteroaryl group comprises one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S, and wherein the heteroaryl group is optionally substituted;

[0089] f)C7-C 14 Alkylaryl, wherein the alkylaryl group is optionally substituted and / or wherein the alkyl part of the alkylarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S; and

[0090] g) C7-C 14 Alkylheteroaryl, wherein the alkylheteroaryl comprises one or more heteroatoms independently selected from O, N and S and / or wherein the alkylheteroarylene is optionally substituted, and / or wherein the alkyl part of the alkylheteroarylene is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

[0091] -R 4 Independently selected from the following and combinations thereof:

[0092] a) C1-C 12 Alkylene, wherein the alkylene may be linear or branched and is optionally substituted and / or optionally (partially) unsaturated and / or optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

[0093] b) C3-C 12 Cycloalkylene, wherein the cycloalkylene is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one or more heteroatoms in the ring, wherein the heteroatoms are independently selected from O, N and S;

[0094] c) C6-C 12 arylene, wherein the arylene group is optionally substituted;

[0095] d) C6-C 12 heteroarylene, wherein the heteroarylene group comprises one or more heteroatoms independently selected from O, N and S and / or wherein the heteroarylene group is optionally substituted;

[0096] e) C7-C 12 Alkylarylene, wherein the alkylarylene is optionally substituted and / or wherein the alkyl part of the alkylarylene is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S; and

[0097] f)C7-C 12 Alkylheteroarylene, wherein the alkylheteroarylene comprises one or more heteroatoms independently selected from O, N and S, and / or wherein the alkylheteroarylene is optionally substituted, and / or wherein the alkyl part of the alkylheteroarylene is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

[0098] -R 6 Independently selected from the following and combinations thereof:

[0099] a) H;

[0100] b) C1-C 10 Alkyl, wherein the alkyl group may be linear or branched and is optionally substituted and / or optionally (partially) unsaturated and / or optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S;

[0101] c) C3-C 12Cycloalkyl, wherein the cycloalkyl is optionally substituted and / or is optionally (partially) unsaturated and / or optionally comprises one or more heteroatoms in the ring, wherein the heteroatoms are independently selected from O, N and S;

[0102] d) C6-C 12 Aryl, wherein the aryl group is optionally substituted;

[0103] e) C6-C 12 heteroaryl, wherein the heteroaryl group comprises one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S, and wherein the heteroaryl group is optionally substituted;

[0104] f)C7-C 14 Alkylaryl, wherein the alkylaryl group is optionally substituted and / or wherein the alkyl part of the alkylarylene group is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S; and

[0105] g) C7-C 14 Alkylheteroaryl, wherein the alkylheteroaryl contains one or more heteroatoms independently selected from O, N and S and / or wherein the alkylheteroarylene is optionally substituted, and / or wherein the alkyl part of the alkylheteroarylene is linear or branched and is optionally (partially) unsaturated and / or is optionally interrupted by one or more heteroatoms, wherein the heteroatoms are independently selected from O, N and S.

[0106] Nanoparticles and medical uses

[0107] The present invention relates to nanoparticles comprising an aza-naphthalene functionalized polymer comprising a polyamine segment as described herein.

[0108] Under certain conditions, the aza-naphthalene functionalized polymers can self-assemble into nanoparticles. In addition to the aza-naphthalene functionalized polymers, these nanoparticles also contain bioactive components that can be used for medical purposes. Typically, the bioactive components are embedded in the aza-naphthalene functionalized polymers. The bioactive components can be any substance or combination of substances that can induce biological or physical responses in the human body. Some examples of bioactive components include nucleotides, polynucleotides, oligonucleotides, peptides, proteins, and combinations thereof. For example, these include all types of RNA and DNA and their derivatives and analogs, i.e., plasmid DNA, dbDNA, hpDNA, c3DNA, microcircles, phosphorodiamidate morpholino oligomers (phosphorodiamidate morpholino oligomers, PMO), siRNA, mRNA, miRNA, endless RNA, circular RNA, single-stranded and / or double-stranded RNA, including the designed guide RNA (gRNA or sgRNA) used in gene editing technology. The sources of these substances are irrelevant, so any artificially constructed or chemically modified nucleotides, oligonucleotides, polynucleotides, oligopeptides, polypeptides and / or proteins should also be understood to be included in the term biologically active payload. Therefore, one or more gene constructs are also considered to bioactive payloads. In addition, polynucleotides, oligonucleotides, peptides, nucleotides and / or proteins can be based on naturally occurring building blocks, also can be based on non-naturally occurring building blocks, for example non-natural nucleosides or non-natural amino acids. Although nanoparticles of the present invention are particularly suitable for polymeric and oligomeric bioactive components, therefore they are preferred, bioactive components can also be non-polymeric and non-oligomeric, and can comprise any active pharmaceutical ingredient.

[0109] Nanoparticles according to the present invention may also comprise non-functionalized polymers. Wherein non-functionalized polymers in this article mean polymers that are not functionalized with a naphthalene azide moiety. The structure of non-functionalized polymers may be similar to a naphthalene azide functionalized polymers, the main difference being that there is no naphthalene azide moiety. Suitable polymers in this regard are described in WO 2012 / 165953.

[0110] For example, in some preferred embodiments, the non-functionalized polymer comprises a non-functionalized polyamine segment according to formula (V), more preferably according to formula (VIa), (VIb), or a combination thereof.

[0111]

[0112] Furthermore, similar to the nitrogen-containing naphthalene polymers, the non-functionalized polymers used in the nanoparticles according to the present invention may comprise a core (T) and thus may have a structure according to formula (VII), more preferably a structure according to formula (IIXa), (IIXb) or a combination thereof.

[0113]

[0114]

[0115] In formulas (V), (VIa), (VIb), (VII), (IIXa) and (IIXb), the variables X, B, n, A, R1, R2, R3, R4, R6, T and q may be independently as defined herein for the aza-naphthalene functionalized polymer. Wherein independently means that the substituents and groups represented by the variables may be the same or different for the aza-naphthalene functionalized polymer and the non-functionalized polymer present in the nanoparticle. In some preferred embodiments, both the aza-naphthalene functionalized polymer and the non-functionalized polymer present in the nanoparticle have the same substituents and groups represented by X, B, A, R1, R2, R3, R4, R6, T and q, and preferably wherein n of the non-functionalized polymer is also equal to n+m of the aza-naphthalene functionalized polymer.

[0116] The non-functionalized polymer is optionally present in the nanoparticles, and in a typical embodiment, the nanoparticles are based only on aza-naphthalene functionalized polymers, not on non-functionalized polymers. This is particularly preferred for the good homogeneity of the nanoparticles and their properties. If non-functionalized polymers are present, the ratio of non-functionalized polymers to aza-naphthalene functionalized polymers in the nanoparticles can be appropriately varied. Factors that may affect the optimal ratio include the ratio of Q and B in the aza-naphthalene functionalized polymer, the (structural) similarity between the non-functionalized polymer and the aza-naphthalene functionalized polymer, and the biologically active ingredients. Preferably, the ratio of non-functionalized polymers to aza-naphthalene functionalized polymers in the nanoparticles is such that the molecular ratio of Q to B (calculating the sum of B units in the non-functionalized polymer and the aza-naphthalene functionalized polymer) is 1:20, or has a higher Q content, preferably 1:10 to 10:1, more preferably 1:5 to 2:1, most preferably 1:4 to 1:1, for example about 1:3.

[0117] Compared to nanoparticles based only on non-functionalized polymers, aza-naphthalene functionalized polymers and nanoparticles show improved transfection efficiency. The nanoparticles can be used for in vitro or in vivo transfection with nucleic acids in eukaryotic cells to express proteins, which is beneficial for medical purposes. Therefore, another aspect of the present invention relates to methods (medical methods) using aza-naphthalene functionalized polymers and nanoparticles in medical treatments. In other words, the present invention also relates to aza-naphthalene functionalized polymers and nanoparticles for use as drugs.

[0118] The term medical treatment herein includes in vivo transfection with nucleic acids in eukaryotic cells to express proteins, and includes curative treatment, preventive treatment, prophylactic treatment, diagnostic treatment, and the like.

[0119] In particular, the azine-functionalized polymers and / or nanoparticles can be used as vaccines, e.g., prophylactic vaccines and / or as therapeutic vaccines. Thus, vaccines comprising nanoparticles can be used to express therapeutic proteins. The most preferred method of administration for vaccines is generally injection, e.g., intradermal, subcutaneous, intramuscular injection using parenteral administration, or alternatively microneedle administration or needle-free injection methods. Other methods are also included, e.g., oral administration or intranasal administration. Preferably, the polymer-coated nanoparticles are used as vaccines by injection, e.g., by intramuscular injection.

[0120] Unless the context clearly indicates otherwise, nouns as used herein without quantifier modification mean one / kind or more / kind. The term "and / or" includes any and all combinations of one or more of the relevant listed items. It should be understood that the terms "include" and / or "comprising" indicate the presence of the stated features, but do not exclude the presence or addition of one or more other features.

[0121] For clarity and concise description, features are described herein as part of the same or separate embodiments, however it is to be understood that the scope of the invention may include embodiments having a combination of all or some of the described features.

[0122] The invention is illustrated by the following non-limiting examples.

[0123] Example 1 - Synthesis of N1-(7-chloroquinoline-4-yl)-hexane-1,6-diamine (Q6)

[0124] While stirring, 1,6-diaminohexane (20.6g, 0.18mol) and 4,7-dichloroquinoline (10.3g, 0.052mol) are heated to approximately 150 DEG C, so that solid is melted, and gained mixture is stirred for 3 hours under nitrogen.In reaction mixture, 50mL methanol is added, and 50mL1M NaOH solution is slowly added while stirring subsequently.Visible precipitate is formed.Precipitate is filtered out, and filtrate is further purified by crystallization or silica gel column chromatography to provide N1-(7-chloroquinoline-4-yl)-hexane-1,6-diamines (Q6).

[0125] Example 2 - Synthesis of p(CBA-ABOL-Q) / PEI

[0126] 4-Amino-1-butanol (ABOL, 0.60 g, 6.75 mmol), Q6 (0.60 g, 2.16 mmol) and cystamine bis(acrylamide) (CBA, 2.60, 10.0 mmol) were dissolved in methanol (10 mL) under N2 atmosphere at 50° C. MilliQ (2 mL) and CaCl2 (0.44 g, 4.0 mmol) were added, and the mixture was stirred at 50° C. for 48 hours.

[0127] Branched PEI 800 PEI solution in MilliQ water was prepared by weighing 100 mg of PEI in a test tube, adding 1 mL of MilliQ water and mixing to dissolve. Then, 0.680 mL of the prepared PEI solution (100 mg / mL) was added to the reaction mixture and stirred at 50°C for 48 hours.

[0128] The reaction mixture was then acidified to pH 4 using 6 M HCl and the solvent was evaporated to obtain the crude quinoline functionalized polymer, which was further purified by dialysis.

[0129] Example 3 - In vitro transfection efficiency of nanoparticles loaded with mRNA-GFP as the amount of Q6 in the polymer used increases

[0130] Polymer p(CBA-ABOL-Q) / PEI was synthesized similarly to Example 2, using 5 mmol CBA and the amounts of ABOL and Q as described in Table 1 as starting materials for polymerization, and 220 mg of CaCl as catalyst. After 48 hours, 340 μL of PEI stock solution (100 mg / mL) was added and stirred for 72 hours.

[0131] Table 1 - Amounts of ABOL and Q6 in different tested polymers

[0132]

[0133] 120 ng / μL of mRNA encoding eGFP (Trilink, catalog number L-7201-1000) was made into a solution in 10 mM histidine pH 6.5. The polymers from Table 1 were dissolved in 10 mM histidine pH 6.5 at a concentration of 3 mg / mL. The 120 ng / μL mRNA solution was added 1:1 (v / v) to the cationic polymer. The solution was incubated at room temperature for at least 15 minutes before use for transfection.

[0134] COS7 cells were seeded in 48-well cell culture plates (1.6×10 4 24 hours after seeding the cells, the medium was replaced with DMEM with 10% FBS and 2% 1M HEPES pH 7.2 and 15 μg polymer / mL of the polymers listed in Table 1 with mRNA. After 24 hours, the cells were analyzed for eGFP expression using FACS analysis. Figure 1As shown in Figure 2, the percentage of eGFP-positive cells increased with increasing amounts of Q6 in the polymer. The viability of the cells was determined using Alamar blue reagent. The viability of the cells decreased with increasing amounts of Q6 in the polymer used. The percentage of eGFP-positive cells (vs. Figure 1 The percentages are the same as shown) and cell viability is as Figure 2 shown.

[0135] Example 4 - Number of transfected cells with nanoparticles loaded with mRNA-EGFP in the presence and absence of Q6

[0136] Polymers p(CBA-ABOL-Q6)PEI and p(CBA-ABOL)PEI were synthesized and tested in transfection as described in Examples 2 and 3. Polymer p(CBA-ABOL-Q6)PEI containing 25% Q6 was compared to polymer without Q6. C2C was transfected with 50 μg polymer / mL medium over a 24-hour period. 12 The number of eGFP-positive cells was quantified by FACS analysis. Figure 3 Given in.

[0137] Example 5 - Transfection efficiency of polymers with Q6 and N1-(7-chloroquinolin-4-yl)-butane-1,4-diamine (Q4)

[0138] Q4 was prepared following a similar procedure as described in Example 1, substituting 1,4-diaminobutane for 1,6-diaminohexane. The polymer comprising Q4 was synthesized following the same procedure as described in Example 2. The in vitro transfection efficiency of mRNA-eGFP loaded nanoparticles based on 25% Q6 and 25% Q4 was determined and compared. COS7 cells were transfected with 30 μg polymer / mL culture medium during 4 hours, after which the cell culture medium was changed. The number of eGFP positive cells was quantified by FACS analysis after 24 hours. The results are shown in Figure 2. Figure 4 Given in.

[0139] Example 6 - RNA encapsulation stability of nanoparticles with and without Q6

[0140] As described in Examples 2 and 3, polymers p(CBA-ABOL-Q6)PEI and p(CBA-ABOL)PEI were synthesized and used in nanoparticle formulations with RNA. The polymer p(CBA-ABOL-Q6)PEI containing 25% Q6 was compared to a polymer incorporating only ABOL monomer without Q6. The formed nanoparticles were used in a release assay that used heparin competition to displace RNA nucleic acids from the nanoparticles. Increasing the heparin concentration resulted in increased competition for RNA binding to the polymer, resulting in the release of RNA from the nanoparticles. The released RNA was then quantified using RiboGreen reagent. Higher concentrations of heparin were required to release RNA from nanoparticles containing Q6, indicating a stronger interaction between the polymer containing Q6 and RNA and improved nanoparticle stability. The results were Figure 5 Given in.

Claims

1. A nanoparticle comprising an aza-naphthalene functionalized polymer and a biologically active component selected from the group consisting of nucleotides, polynucleotides, oligonucleotides, peptides, proteins, and combinations thereof, wherein the polymer comprises a polyamine segment of formula (I), -[X-B] n -[X-Q] m - (I) wherein X is based on a bis(α,β-unsaturated carbonyl) monomer, B is based on an amine monomer, Q is based on an aza-naphthylidene-containing amine monomer comprising an aza-naphthylidene moiety, m is at least 1, and n is at least 1, preferably n+m is 2 to 250.

2. The nanoparticle according to claim 1, wherein the naphthyridine moiety is selected from benzopyridine and diazine, preferably benzopyridine, more preferably quinoline, the benzopyridine including 1-naphthyridine (quinoline) and 2-naphthyridine (isoquinoline), the diazine including 1,2-naphthyridine (cinnoline), 1,3-naphthyridine (quinazoline), 1,4-naphthyridine (quinoxaline), 2,3-naphthyridine (phthalazine), 1,8-naphthyridine, 1,7-naphthyridine, 2,7-naphthyridine, 1,6-naphthyridine, 1,5-naphthyridine and 2,6-naphthyridine.

3. The nanoparticle according to any one of the preceding claims, wherein X is based on x ) di(α,β-unsaturated carbonyl) monomers, in A is selected from C(R 1 )2、O、N(R 1 ), S and their combinations, R 1 is selected from H, halide, and C1-C1 optionally substituted with one or more halide 22 Hydrocarbyl groups such as alkyl, alkenyl and polyalkenyl groups, and combinations thereof; and R 2 is selected from C1-C1 optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms 40 Straight chain, cyclic and branched alkylene groups, and combinations thereof.

4. The nanoparticle according to any one of claims 1 to 2, wherein X comprises a disulfide moiety, preferably wherein X is as defined in claim 3, and wherein R 2 comprises a disulfide moiety, more preferably wherein R 2 selected from C1-C 10 Hydrocarbylene groups, preferably straight chain alkylene groups, and combinations thereof.

5. The nanoparticle according to any one of the preceding claims, wherein B is based on a B1 ) of the primary amine, Or based on formula (I B2 ) of diamine, in R 3 independently selected from H, optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms C1-C 40 Straight chain, cyclic and branched hydrocarbon groups, and combinations thereof; and R 4 is selected from C1-C1 optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms 40 Straight chain, cyclic and branched alkylene groups, and combinations thereof.

6. The nanoparticle according to any one of claims 1 to 4, wherein B comprises a hydroxyl moiety, preferably wherein B is as defined in claim 5, and wherein R 3 contains a hydroxyl moiety, more preferably wherein R 3 Selected from C1-C 10 Hydrocarbyl hydroxide, preferably a linear alkyl C1-C 10 Hydroxides, and combinations thereof.

7. The nanoparticle according to any one of the preceding claims, wherein Q is based on the formula (I Q1 ) of the primary amine, And / or based on formula (I Q2 ) of diamine, in Z 1 represents a group comprising said azanaphthene moiety; Z 2 represents a group comprising the aza-naphthalene moiety, or is selected from H, optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms C1-C 40 Straight chain, cyclic and branched hydrocarbon groups, and combinations thereof; R 4 As defined in claim 5.

8. Nanoparticles according to the preceding claims, wherein Z 1 and optional Z 2 independently represents a part according to any of the following structures, Where R 5 is selected from C1-C1 optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms 40 , preferably C1-C 10 , more preferably C1-C6 straight chain, cyclic and branched alkylene groups, and combinations thereof; and Wherein the azanaphthene moiety is optionally substituted by one or more substituents, preferably selected from halides, alkyls and alcoholates, which are optionally substituted by one or more heteroatoms.

9. The nanoparticle according to any one of claims 7 to 8, wherein Z 1 and optional Z 2 Independently express the formula (I Z2 ) part, preferably formula (I Z3 )part, Where R 5 is selected from C1-C1 optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms 40 , preferably C1-C 10 , more preferably C1-C6 straight chain, cyclic and branched alkylene groups, and combinations thereof; and Wherein the azanaphthene moiety is optionally substituted by one or more substituents, preferably selected from halides, alkyls and alcoholates, which are optionally substituted by one or more heteroatoms.

10. The nanoparticle according to any one of claims 7 to 9, wherein Z 1 and optional Z 2 Independently express the formula (I Z4 )part Where R 5 As defined in claim 9, preferably wherein R 5 Represents C1-C 10 Alkylene, more preferably C1-C6 alkylene, most preferably C1-C4 alkylene, and combinations thereof; and Where R 6 is selected from alkyl, alcoholate, halide, and mixtures thereof, which are optionally substituted with one or more heteroatoms, preferably halide, more preferably wherein R 6 For Cl.

11. The nanoparticle according to any one of the preceding claims, wherein the polyamine segment has a structure according to any one of formulas (IIa), (IIb), (IIc) and (IId) and combinations thereof, Where n, m, A, R 1 , R 2 , R 3 , R 4 , Z 1 and Z 2 Each independently as defined in any one of the preceding claims.

12. The nanoparticle according to any one of the preceding claims, wherein the polyamine segment, preferably the polymer, has a structure according to formula (III), preferably according to any one of formula (IVa), (IVb), (IVc), (IVd) and combinations thereof, {[X-B] n -[X-Q] m } q -T (III) Where n, m, A, R 1 , R 2 , R 3 , R 4 , Z 1 and Z 2 each individually as defined in any one of the preceding claims; Where T represents the weight average molecular weight M w From about 60 to about 25,000 nuclei; q is 1 to 64; and R 6 is selected from H, optionally substituted by one or more heteroatoms and / or interrupted by one or more heteroatoms C1-C 40 Straight chain, cyclic and branched hydrocarbon groups, and combinations thereof.

13. Nanoparticles according to any one of the preceding claims, wherein the ratio of m to n is 1 :20 or more, preferably 1 :10 to 10:1 , more preferably 1 :5 to 2:1 , most preferably 1 :4 to 1 :1 , such as about 1 :

3.

14. The nanoparticle according to any one of the preceding claims, wherein the biologically active component comprises RNA, DNA or a derivative thereof.

15. Nanoparticle according to any one of the preceding claims for use in a medical method, preferably as a vaccine.

16. Use of the nanoparticles according to any one of the preceding claims 1 to 14 for in vitro transfection of eukaryotic cells with nucleic acids.

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