Azanaphthalene-functionalized polymers for gene and drug delivery

Abstract

The present invention is directed to nanoparticles comprising an azanaphthalene-functionalized polymer and a biologically active component, where the polymer comprises a polyamine segment based on a bis(α,β-unsaturated carbonyl) monomer, an azanaphthalene-containing amine monomer that includes an azanaphthalene moiety, and an amine monomer. The present invention is further directed to polymeric nanoparticles for use in medical procedures, such as in vivo delivery of biologically active agents.

Description

[Technical field]

[0001] Inventions in the field of polymeric nanoparticles for the delivery of biologically active components, such as polynucleotides, oligonucleotides, peptides and / or proteins, to cells. In particular, the present invention is directed to polyamine-containing polymers for these nanoparticles. [Background technology]

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

[0003] The process of transport, delivery and activation of a payload by a nanoparticle is called transfection. Where the payload includes a nucleic acid, transfection can be used to refer to the process of deliberately introducing a nucleic acid into a eukaryotic cell. The success of nanoparticle-based medical procedures depends, inter alia, on transfection efficiency. In this regard, see especially Kim and Eberwine, Anal. Bioanal. Chem. 2010 Agu:397(8):3173-8. Transfection efficiency depends on a variety of factors, including particle and payload stabilization, endocytosis, intracellular trafficking, localization, and payload unpackaging.

[0004] Various specific polyamine-containing polymers are known from WO 2012 / 165953 and the 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. 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.

[0005] Quinoline-functionalized polymers are known, inter alia, from Aderibigbe et al. (J. Inorg. Organomet. Polym. (2012), 22:429-438). Polyamine-containing polymers, such as those specifically described in the aforementioned WO 2012 / 165953, are known for their good transfection efficiency. However, there remains a need to further improve the transfection efficiency of such polymers. The present inventors have surprisingly found that the transfection efficiency of polymeric nanoparticles based on polyamine-containing polymers can be improved by functionalizing some of these polyamine-containing polymers with one or more azanaphthalene moieties. Summary of the Invention

[0006] Thus, in one aspect, the present invention is directed to a nanoparticle comprising an azanaphthalene-functionalized polymer, wherein the polymer comprises a polyamine segment of formula (I): [ka] wherein X is based on a bis(α,β-unsaturated carbonyl) monomer, B is based on an amine monomer, Q is based on an azanaphthalene-containing amine monomer that includes an azanaphthalene moiety, and wherein m is 1 or greater, and n is 1 or greater.

[0007] Polymers with increasing content of azanaphthalene moieties (ie, polymers with increasing numbers of m) were found to concomitantly increase the efficiency of transfection into cells. However, this effect was also found to plateau. It has also been found that polymers based on azanaphthalene-containing amine monomers as well as amine monomers that do not contain azanaphthalene moieties (i.e., polymers where n is at least 1) are easier to synthesize in aqueous solvents and are preferred over polymers based solely on azanaphthalene-containing amine monomers (i.e., polymers where n is 0). Polymers where n is 0 generally result in polymers that are poorly soluble in water in the slightly acidic to neutral pH range. Therefore, the synthesis of these polymers is considered impractical. Polymers where n is 0 can be obtained in anhydrous solvent systems such as dimethylsulfoxide. However, this typically provides a polymer that cannot be readily purified by commonly used aqueous purification methods.

[0008] In accordance with the present invention, the incorporation of amine monomers (ie, monomers on which B is based), ie, n is at least 1, to obtain polymers has been improved in synthesis. Generally, the water-soluble polymers are provided in a wide range of ratios of the azanaphthalene-containing amine monomer (Q) and the amine monomer (B). Thus, the polymers may be synthesized in a preferred aqueous solvent and purified using commonly available purification methods.

[0009] In this specification, an azanaphthalene moiety is a naphthalene moiety in which at least one carbon is replaced by a nitrogen atom (molecular formula C 10 H7-x where X is the number of optional substituents, for example, 6-x Monoazanaphthalene, with the molecular formula C8N2H 5-x where X is the number of optional substituents. The azanaphthalene moiety can be substituted or unsubstituted. Preferably, it is substituted with an alkoxy or halide substituent.

[0010] Preferred monoazanaphthalenes particularly suitable for the present invention include the benzopyridines, including quinoline (also called 1-benzadine or 2,3-benzopyridine) and isoquinoline (also called 2-benzadine or 3,4-benzopyridine). The quinoline moiety gives particularly good results and is most preferred. When the azanaphthalene moiety is a quinoline moiety, which is preferred, the polymer of the invention can be referred to as a quinoline-functionalized polymer.

[0011] Diazanaphthalenes particularly suitable for the present invention include 1,2-diazanaphthalene (also known as cinnoline), 1,3-diazanaphthalene (also known as quinazoline), 1,4-diazanaphthalene (also known as quinoxaline), 2,3-diazanaphthalene (also known as phthalazine), 1,8-diazanaphthalene, 1,7-diazanaphthalene, 2,7-diazanaphthalene, 1,6-diazanaphthalene, 1,5-diazanaphthalene, and 2,6-diazanaphthalene.

[0012] As used herein, the term "segment" is understood as a polymeric structure of any length. The polyamine segment according to formula (I) is based on bis(α,β-unsaturated carbonyl) monomers (X) and amine monomers (Q and B), the combination of which provides the repeat unit. Thus, in each repeat unit there is either a Q amine (ie, [XQ]) or a B amine (ie, [XB]), which may be randomly positioned within the polyamine segment. Amine monomer B does not contain an azanaphthalene moiety and differs from azanaphthalene-containing amine monomer Q in at least this aspect. Thus, the polyamine segments of the present invention can be considered to be copolymers, particularly random copolymers.

[0013] The labels n and m represent the number of each repeat unit in the polyamine. In polyamines, Q is always present, therefore m is 1 or greater, and B is always present, therefore n is 1 or greater. The ratio of n to m can be determined by proton nuclear magnetic resonance spectroscopy ( 1 The Q aromatic signal can be determined by H-NMR (H NMR), where the integral of the Q aromatic signal can be compared to the whole polymer CH integral in a defined spectral window between 1.0 and 2.0 ppm. From this ratio, the m / n ratio can be derived. In a typical embodiment, the sum of n and m is in the range of 2-250, preferably 5-100. Typical weight average molecular weights of the polyamines range 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 may be determined using three G1316B thermostatted (35° C.) columns connected in series. (Combination of NOVEMA MAX Medium, molar mass range: 100-1 000 000 Da; PSS NOVEMA Max 30Å, 5μm, 8X300mm + 2XNOVEMA Max 1000Å, 5μm columns, 8X300mm, PSS Polymer Standards Service GmbH), 100mM NaCl + 0.3% formic acid as eluent, at a flow rate of 1mL / min and equipped with a refractive index detector. Molecular weight can be determined relative to dextran standards.

[0014] X and B groups The polyamine may be based on Q in combination with two or more different amine monomers B, for example three different types of amine monomers B. Additionally, Q may represent a mixture of different azanaphthalene-containing amine monomers. The same applies to the X moiety which is derived from the structure of a bis(α,β-unsaturated carbonyl) monomer. Thus, X, B and Q in formula (I) may each represent a mixture of bis(α,β-unsaturated carbonyl) monomers, general amine monomers and azanaphthalene-containing amine monomers. It will be appreciated that B and Q are typically based on primary and secondary amines; because such amines are generally sufficiently reactive in polymerization reactions (i.e., aza-Michael addition type reactions, see below) to result in polymers.

[0015] 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 brevity. It will be appreciated that in preparing the polyamine, the X monomers react with the Q and B monomers, respectively, resulting in a change in their chemical structure. For example, during the preparation of polyamines, the monomers on which Q and B are based typically undergo an (aza)Michael addition type reaction with a bis(α,β-unsaturated carbonyl) monomer X, followed by conversion of the double bond of the α,β-unsaturated carbonyl moiety to a single bond.

[0016] The monomer X preferably has the formula (I x ) is a bis(α,β-unsaturated carbonyl) monomer represented by the formula [ka] During the ceremony, A is C(R 1 )2, O, N(R 1 ), S, and combinations thereof; R 1 is H, halide, and C1-C such as alkyl, alkenyl, and polyalkenyl. 22 hydrocarbyl, which are optionally substituted with one or more halides, and combinations thereof; and R 2 is C1~C 40 linear, cyclic and branched hydrocarbylenes, which are optionally substituted and / or interrupted with one or more heteroatoms, and combinations thereof.

[0017] Preferably, A in the polyamine segment comprises N(R1), in which case the polymer segment may be referred to as a poly(aminoamide). In other words, the polyamine segment is preferably a poly(aminoamide) (PAA) segment. Hydrocarbyl and hydrocarbylene as used herein include monovalent and divalent groups formed by removing one or two hydrogen atoms from a hydrocarbon, such as ethyl, phenyl and ethylene, phenylene, respectively. When interrupted by one or more heteroatoms, these groups can be heteroaliphatic and / or heteroaromatic, as well as linear, branched and / or cyclic.

[0018] The monomer B is represented by the formula (I B1 Based on a primary amine represented by [ka] or formula (I B2 Based on the diamine represented by [ka] During the ceremony, R3 are independently H, C1 to C 40 linear, cyclic and branched hydrocarbyls, which are optionally substituted and / or interrupted with one or more heteroatoms, and combinations thereof; and R 4 is C1~C 40 linear, cyclic and branched hydrocarbylenes, which are optionally substituted and / or interrupted with one or more heteroatoms, and combinations thereof.

[0019] Preferably, R 3 has an overall neutral or negative charge at slightly acidic to neutral pH. As used herein, slightly acidic to neutral pH means a pH between about 6 and about 7. In a specific embodiment of the invention, X is degradable under physiological conditions. Thus, X preferably contains a moiety in which one or more bonds are readily cleaved (eg, by hydrolysis, reduction, oxidation, etc.) under physiological conditions. Examples of such moieties include ester, thioester, disulfide moieties, and the like. Disulfide moieties are particularly preferred in this regard. Thus, X preferably comprises a disulfide moiety, more preferably a group represented by the formula (I x ) in R 2 contains a disulfide moiety, and even more preferably R 2 is interrupted by at least one disulfide moiety, C1-C 10 It is selected from the group consisting of hydrocarbylene, preferably linear alkylene, and combinations thereof.

[0020] In a particular embodiment of the invention, B contains a hydroxyl moiety, preferably a primary hydroxyl moiety. Therefore, the formula (I B1 ) and / or (I B2 ) at least one R 3contains a hydroxyl moiety, and more preferably has the formula (I B1 ) and / or (I B2 ) at least one R 3 is C1~C 10 Hydroxyhydrocarbyl, most preferably linear alkyl C1-C 10 hydroxides, and combinations thereof.

[0021] Particularly suitable monomers on which B may be based have biological activity and / or impart water solubility. Particularly suitable monomers include 4-aminobutanol (ABOL), 5-aminopentanol (APOL), and other hydroxyl-containing monomers. Preferably, the monomers are neutral (ie, uncharged) under physiological conditions (ie, pH 7 at 20° C.) when incorporated into the polymer. Incorporation of these neutral monomers, such as ABOL and APOL, into polymers resulted in improved transfection efficiency compared to monomers that are positively charged under physiological conditions (see for example Lin et al. Bioconjugate Chem. 2007, 18, 1, 138-145). Examples of such positively charged monomers include amine monomers that contain an additional amine (eg, an aliphatic tertiary amine), such as dimethylaminopropylamine and N-(3-aminopropyl)-morpholine. Thus, monomers that become positively charged under physiological conditions when incorporated into the polymer are less favorable for B.

[0022] Q and the azanaphthalene moiety The polyamine, Q, contains an azanaphthalene moiety. The azanaphthalene moiety may be optionally substituted, as further described herein.

[0023] In a preferred embodiment, Q is a group represented by the formula (I Q1 Based on a primary amine represented by [ka] and / or formula (I Q2 Based on the diamine represented by [ka] During the ceremony, Z 1 represents a group containing an azanaphthalene moiety; Z 2 represents a group containing an azanaphthalene moiety, or H, C1-C 40 linear, cyclic and branched hydrocarbyls, which are optionally substituted and / or interrupted with one or more heteroatoms, and combinations thereof; and R 4 is represented by the formula (I B2 ) can be as defined above.

[0024] Formula (I Q2 ) has at least one azanaphthalene moiety. In one embodiment, the compound of formula (I Q2 ) by Q is Z 1 Only Z contains the azanaphthalene moiety. 2 Does not include. In another embodiment, the compound of formula (I Q2 ) Q is the azanaphthalene moiety Z 1 and Z 2 Includes both. Polyamines according to the invention can also contain a mixture of different Q groups, each group having a different structure and / or containing different amounts of azanaphthalene moieties.

[0025] In a specific embodiment, Z 1 and optionally Z 2 independently represent a moiety according to any of the following structures: [ka] In the formula, R 5 is C1~C 40 , preferably C1 to C 10 , more preferably selected from the group consisting of C1-C6 linear, cyclic and branched hydrocarbylenes, which are optionally substituted and / or interrupted with one or more heteroatoms, and combinations thereof; and wherein the azanaphthalene moiety is optionally substituted with one or more substituents, preferably selected from the group consisting of halides, alkyls and alkoxides, and optionally substituted with one or more heteroatoms.

[0026] The azanaphthalene moiety has the formula (I Q1 ) and (I Q2 ) in a variety of ways. Preferably, this portion is a spacer R 5 is attached to the backbone of the polyamine segment via

[0027] More preferably, the compound of formula (I Q1 ) and (I Q2 ) in either 1 and optionally Z 2 are independently represented by the formula (I Z1 ), (I Z2 ) or (I Z3 ), preferably a quinoline moiety of the formula (I Z3 ) represents the quinoline moiety of [ka] In the formula, R 5 is C1~C 40 , preferably C1 to C 10 , more preferably C1-C6 linear, cyclic and branched hydrocarbylenes, which are optionally substituted and / or interrupted by one or more heteroatoms, and combinations thereof, preferred examples of which are I Z3 As shown in.

[0028] The azanaphthalene moiety may be optionally substituted with one or more substituents, preferably one or more substituents selected from the group consisting of halides, alkyls and alkoxides, both of which may be optionally substituted again with one or more heteroatoms. In a particularly preferred embodiment, the quinoline moiety contains a substituent at the 7-position and has the formula (I Q1 ) and (I Q2 ) in either 1 and optionally Z 2 are independently represented by the formula (I Z4 ) part, [ka] In the formula, R 6 is selected from the group consisting of halides, alkyls, alkoxides and mixtures thereof, optionally substituted with one or more heteroatoms, preferably halides, more preferably wherein R 6 is Cl.

[0029] The present inventors have determined that the spacer R 5 However, it is possible to use dimethyl ether such as butylene (C4H8), hexylene (C6H 12 ), or octylene (C8H 16 It has been found that the structure may be relatively simple, such as a straight-chain unsubstituted alkylene such as . Thus, although it is possible to use a spacer containing quinuclidine, such as that present in the natural product quinine, this is not essential.

[0030] General polymer formula and core X, B, and Q are of the formula (I X ), (I B1 ) or (I B2 ), and (I Q1 ) or (I Q2 In an embodiment based on a monomer according to formula (IIa)-(IId), the polyamine can be represented by any one of formulas (IIa)-(IId) and combinations thereof, wherein n, m, p, A, R 1 , R2 , R 3 , R 4 , Z 1 and Z 2 (I), (I X ), (I B1 ), (I B2 ), (I Q1 ) and (I Q2 ) can be as defined for [ka]

[0031] In a particular embodiment of the invention, the polyamine is linked to a core, preferably a polymer core (T). Thus, in a particular embodiment of the invention, the polyamine segment, preferably the polymer, according to the invention has a structure according to formula (III), where n and m are as defined in formula (I) and q represents the number of poly(aminoamide) segments, i.e., the number of arms, per core (T). In typical embodiments, q ranges from 1-64. [ka]

[0032] In a particularly preferred embodiment, the polyamine segment, preferably a polymer, according to the present invention has a structure according to any of formulas (IVa), (IVb), (IVc), (IVd), and combinations thereof, where n, m, A, R 1 , R 2 , R 3 , R 4 , Z 1 and Z 2 is represented by the formula (I), X ), (I B1 ), (I B2 ), (I Q1 ) and (I Q2 ) may be as defined for [ka]

[0033] Furthermore, in formulas (III) and (IVa) to (IVd), T is preferably a weight average molecular weight M w represents a core having a value of about 60 to about 25,000; q ranges from 1 to 64; and R 6 H, C1~C 40 linear, cyclic and branched hydrocarbyls, which are optionally substituted and / or interrupted with one or more heteroatoms, and combinations thereof.

[0034] Core T is generally based on structures having one or more primary and / or secondary amines. These amines are 6 The compound is represented by formulae (IVa) to (IVd) having a group. Particularly suitable cores include oligo- or polyamine cores, such as 1,2-ethylenediamine, tris-(2-aminoethyl)amine, and polymer cores having primary and / or secondary amines, such as polyethyleneimine (PEI), poly(aminoamide) polymers or dendrimers (PAMAM), polypropyleneimine (PPI), poly(esteramine) polymers (PEAN) and poly(etheramine) polymers (PEAC), and other polymers described in WO 2012 / 165953 (referred to therein as POL). PEI is particularly preferred as the core, with PEI800, having an average of 6 primary amines, being most preferred in this regard and giving particularly good results. It can be appreciated that both ends of the poly(aminoamide) polymer can potentially be reacted with a POL, such that two or more cores can be incorporated into the same macromolecule. In certain embodiments, however, reaction conditions can be selected to limit crosslinking.

[0035] The present inventors have found that the relative amounts of azanaphthalene-containing amine monomer Q and amine monomer B in the polyamine and / or polymer affect transfection efficiency and toxicity. Furthermore, it was found that a higher relative amount of Q resulted in a concomitant decrease in solubility during the synthesis of the polymer, similarly resulting in a polymer with low solubility in aqueous systems.

[0036] The relative amounts of Q and B can be expressed as the ratio of m to n. Because amine monomer B is present, n is at least 1, and therefore the ratio is 1:1, or 1. Preferably, however, the polyamine segment and / or polymer contains more B than Q. In a specific embodiment, the ratio of m to n is in the range of 1:20 or more (ie, 0.05 or more, for example, 0.05 to 20). Preferably, the ratio of m to n is in the range of 1:10 to 10:1, more preferably in the range of 1:5 to 2:1, and most preferably in the range of 1:4 to 1:1, e.g., about 1:3, within which particularly good results have been obtained.

[0037] The formula (I) for the polymer according to the present invention x ) and (I B1 ) and (I B2 Suitable embodiments of monomers according to the formula (I) are described in WO 2012 / 165953, which is incorporated herein in its entirety. Preferably, therefore, the following applies to the polyamine segments and / or polymers according to the invention and / or their monomers based thereon:

[0038] -R 2 is independently selected from the group consisting of: a) C1~C 40alkylene, where the alkylene group may be linear or branched, optionally substituted and / or optionally (partially) unsaturated and / or optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where the alkylene group is interrupted by one or more -SS- groups; b) C3~C 40 cycloalkylene, where the cycloalkylene group is optionally substituted and / or optionally (partially) unsaturated and / or optionally contains one or more heteroatoms in the ring, where the heteroatoms are independently selected from O, N and S, and / or where the cycloalkylene group is interrupted on the outside of the ring by one or more -SS- groups;

[0039] c) C6~C 40 arylene, where the arylene group is optionally substituted; d) C6~C 40 heteroarylene, where said heteroarylene group contains one or more heteroatoms independently selected from O, N and S, and / or where said heteroarylene group is optionally substituted; e) C7~C 40 alkylarylene, where the alkylarylene group is optionally substituted, and / or where the alkyl portion of the alkylarylene group is linear or branched, optionally (partially) unsaturated, and / or optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S, and / or where the alkyl portion of the alkylarylene group is interrupted by one or more -SS- groups;

[0040] f) C7~C 40alkylheteroarylene, where the alkylheteroarylene group contains one or more heteroatoms independently selected from O, N, and S, and / or where the alkylheteroarylene group is optionally substituted, and / or where the alkyl moiety of the alkylheteroarylene group is linear or branched, optionally (partially) unsaturated, and / or optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N, and S, and / or where the alkyl moiety of the alkylheteroarylene group is interrupted by one or more -SS- groups; and

[0041] g) Two C7-C 40 (Hetero)arylene groups and / or C7-C 40 alkyl(hetero)arylene groups linked together by -SS- groups, wherein the alkyl moieties of said alkyl(hetero)arylene groups are linear or branched, optionally (partially) unsaturated, and / or optionally interrupted by one or more heteroatoms, wherein said heteroatoms are independently selected from O, N and S; and combinations thereof;

[0042] -R 3 are independently selected from the group consisting of: a) H; b) C1~C 10 alkyl, where the alkyl group may be linear or branched, optionally substituted and / or optionally (partially) unsaturated and / or optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S; c) C3~C 12 cycloalkyl, where the cycloalkyl group is optionally substituted and / or optionally (partially) unsaturated and / or optionally contains one or more heteroatoms in the ring, where the heteroatoms are independently selected from O, N and S;

[0043] d) C6~C 12aryl, where the aryl group is optionally substituted; e) C6~C 12 heteroaryl, where said heteroaryl group contains one or more heteroatoms, where said heteroatoms are independently selected from O, N and S, and where said heteroaryl group is optionally substituted; f) C7~C 14 alkylaryl, where the alkylaryl group is optionally substituted, and / or where the alkyl portion of the alkylarylene group is linear or branched, optionally (partially) unsaturated, and / or optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S; and

[0044] g) C7~C 14 alkylheteroaryl, where said alkylheteroaryl group contains one or more heteroatoms independently selected from O, N and S, and / or where said alkylheteroarylene group is optionally substituted, and / or the alkyl portion of said alkylheteroarylene group is linear or branched, optionally (partially) unsaturated, and / or optionally interrupted by one or more heteroatoms, where said heteroatoms are independently selected from O, N and S; and combinations thereof, preferably where R 3 has an overall neutral or negative charge at slightly acidic to neutral pH;

[0045] -R 4 are independently selected from the group consisting of: a) C1~C 12 alkylene, where the alkylene group may be linear or branched, optionally substituted and / or optionally (partially) unsaturated and / or optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S; b) C3~C 12cycloalkylene, where the cycloalkylene group is optionally substituted and / or optionally (partially) unsaturated and / or optionally contains one or more heteroatoms in the ring, where the heteroatoms are independently selected from O, N and S;

[0046] c) C6~C 12 arylene, where the arylene group is optionally substituted; d) C6~C 12 heteroarylene, where said heteroarylene group contains one or more heteroatoms independently selected from O, N and S, and / or where said heteroarylene group is optionally substituted; e) C7~C 12 alkylarylene, where the alkylarylene group is optionally substituted and / or the alkyl portion of the alkylarylene group is linear or branched, optionally (partially) unsaturated and / or optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S; and

[0047] f) C7~C 12 alkylheteroarylene, where said alkylheteroarylene group contains one or more heteroatoms independently selected from O, N and S, and / or where said alkylheteroarylene group is optionally substituted, and / or where the alkyl moiety of said alkylheteroarylene group is linear or branched, optionally (partially) unsaturated, and / or optionally interrupted by one or more heteroatoms, where said heteroatoms are independently selected from O, N and S; and combinations thereof;

[0048] -R 6 are independently selected from the group consisting of: a) H; b) C1~C 10alkyl, where the alkyl group may be linear or branched, optionally substituted and / or optionally (partially) unsaturated and / or optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S; c) C3~C 12 cycloalkyl, where the cycloalkyl group is optionally substituted and / or optionally (partially) unsaturated and / or optionally contains one or more heteroatoms in the ring, where the heteroatoms are independently selected from O, N and S;

[0049] d) C6~C 12 aryl, where the aryl group is optionally substituted; e) C6~C 12 heteroaryl, where said heteroaryl group contains one or more heteroatoms, where said heteroatoms are independently selected from O, N and S, and where said heteroaryl group is optionally substituted; f) C7~C 14 alkylaryl, where the alkylaryl group is optionally substituted, and / or where the alkyl portion of the alkylarylene group is linear or branched, optionally (partially) unsaturated, and / or optionally interrupted by one or more heteroatoms, where the heteroatoms are independently selected from O, N and S; and

[0050] g) C7~C 14 alkylheteroaryl, where said alkylheteroaryl group contains one or more heteroatoms independently selected from O, N and S, and / or where said alkylheteroarylene group is optionally substituted, and / or the alkyl portion of said alkylheteroarylene group is linear or branched, optionally (partially) unsaturated, and / or optionally interrupted by one or more heteroatoms, where said heteroatoms are independently selected from O, N and S; and combinations thereof.

[0051] Nanoparticles and Medical Applications The present invention is directed to nanoparticles comprising azanaphthalene-functionalized polymers containing polyamine segments as described herein.

[0052] Under certain conditions, azanaphthalene-functionalized polymers can self-assemble into nanoparticles. These nanoparticles further contain biologically active components in addition to the azanaphthalene-functionalized polymer that can be used for medical purposes. Generally, the biologically active moiety is embedded within the azanaphthalene-functionalized polymer. A biologically active ingredient can be any substance or combination of substances capable of eliciting a biological or physical response in the human body. Examples of biologically active moieties include nucleotides, polynucleotides, oligonucleotides, peptides, proteins, and combinations thereof. These include, for example, all kinds of RNA and DNA as well as their derivatives and analogues, especially plasmid DNA, dbDNA, hpDNA, c3DNA, minicircles, phosphorodiamidate morpholino oligomers (PMOs), siRNA, mRNA, miRNA, endless RNA, circular RNA, single-stranded and / or double-stranded RNA such as designed guide RNAs (gRNAs or sgRNAs) used in gene editing techniques. The origin of these substances is irrelevant, and therefore, artificially constructed or chemically modified nucleotides, oligonucleotides, polynucleotides, oligopeptides, polypeptides, and / or proteins are also understood to be encompassed by the term biologically active payload. Thus, one or more gene constructs are also considered to be biologically active payloads. Moreover, the polynucleotides, oligonucleotides, peptides, nucleotides and / or proteins may be based on naturally occurring building blocks, but also on non-naturally occurring building blocks, such as non-natural nucleosides or non-natural amino acids. Although the nanoparticles of the invention are particularly suitable, and therefore preferred, for polymeric and oligomeric biologically active ingredients, the biologically active ingredients may be non-polymeric and non-oligomeric and may include any active pharmaceutical ingredient.

[0053] The nanoparticles according to the present invention may further comprise a non-functionalized polymer. By non-functionalized polymer is meant herein a polymer that is not functionalized with an azanaphthalene moiety. The structure of the unfunctionalized polymer may be similar to the azanaphthalene-functionalized polymer, with the main difference being the absence of the azanaphthalene moiety. Suitable polymers in this regard are described in WO 2012 / 165953.

[0054] For example, in a preferred embodiment, the non-functionalized polymer comprises a non-functionalized polyamine segment according to formula (V), more preferably formula (VIa), (VIb), or a combination thereof. [ka]

[0055] Moreover, similar to the azanaphthalene-containing polymers, the non-functionalized polymers used in the nanoparticles according to the present invention can comprise a core (T) and thus have a structure according to formula (VII), more preferably formula (IIXa), (IIXb), or a combination thereof. [ka]

[0056] In formulas (V), (VIa), (VIb), (VII), (IIXa) and (IIXb), the variables X, B, n, A, R1, R2, R3, R4, R6, T and q may independently be as defined herein for an azanaphthalene-functionalized polymer. "Independently" means that the substituents and groups represented by the variables can be the same or different for the azanaphthalene-functionalized and non-functionalized polymers present in the nanoparticles. In a preferred embodiment, both the azanaphthalene-functionalized polymer and the non-functionalized polymer present in the nanoparticles have the same substituents and groups represented by X, B, A, R1, R2, R3, R4, R6, T and q, preferably where n of the non-functionalized polymer is equal to n+m of the azanaphthalene-functionalized polymer.

[0057] Non-functionalized polymers are optionally present in the nanoparticles, and in typical embodiments, the nanoparticles are based solely on azanaphthalene-functionalized polymers and not on non-functionalized polymers. This is particularly preferred due to the good homogeneity of the nanoparticles and their properties. When non-functionalized polymer is present, the ratio of non-functionalized polymer to azanaphthalene-functionalized polymer in the nanoparticles can be varied accordingly. Factors that may influence the optimal ratio include the ratio of Q to B in the azanaphthalene-functionalized polymer, the (structural) similarity between the unfunctionalized polymer, the azanaphthalene-functionalized polymer, and the biologically active moiety. Preferably, the ratio of non-functionalized polymer to azanaphthalene-functionalized polymer in the nanoparticles is such that the molecular ratio of Q to B (counting the sum of the B units in the non-functionalized polymer and the azanaphthalene-functionalized polymer) is in the range of 1:20 or higher Q content, preferably in the range of 1:10 to 10:1, more preferably in the range of 1:5 to 2:1, most preferably in the range of 1:4 to 1:1, for example about 1:3.

[0058] Azanaphthalene-functionalized polymers and nanoparticles show improved transfection efficiency compared to nanoparticles based only on non-functionalized polymers. The nanoparticles can be suitably used to transfect nucleic acids in eukaryotic cells in vitro or in vivo to express proteins that are advantageous for medical purposes. Thus, a further aspect of the present invention is directed to methods of using the azanaphthalene-functionalized polymers and nanoparticles in medical procedures (medical methods). In other words, the present invention is further directed to azanaphthalene-functionalized polymers and nanoparticles for use as pharmaceuticals.

[0059] As used herein, medical treatment includes the in vivo transfection of a nucleic acid into a eukaryotic cell to express a protein, and includes therapeutic treatment, preventive treatment, prophylactic treatment, diagnostic treatment, and the like. In particular, the azanaphthalene-functionalized polymers and / or nanoparticles can be used as vaccines, such as prophylactic vaccines, and / or for use as therapeutic vaccines. Thus, vaccines comprising nanoparticles can be used to express therapeutic proteins. The most preferred method of administration of the vaccine is typically by injection, for example parenterally, intradermally, subcutaneously, intramuscularly, or alternatively, using microneedle or needle-free injection techniques. Other methods such as oral or intranasal administration are also included. Preferably, the polymer-coated nanoparticles are used as a vaccine, for example by injection, such as intramuscular injection.

[0060] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The term "and / or" includes any and all combinations of one or more of the associated listed items. It will be understood that the terms "comprise" and / or "comprising" specify the presence of stated features but do not exclude the presence or addition of one or more other features.

[0061] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, but it will be understood that the scope of the invention may include embodiments having all or a partial combination of the described features. The present invention can be further illustrated by the following non-limiting examples. [Brief description of the drawings]

[0062] FIG. 1 is a graph showing the % GFP positive cells versus the amount of Q6 in the polymer. FIG. 2 is a graph showing % GFP positive cells and cell viability versus the amount of Q6 in the polymer. FIG. 3 is a graph showing the % GFP positive cells in p(CBA-ABOL-Q6)PEI and p(CBA-ABOL)PEI. FIG. 4 is a graph showing the % GFP positive cells in Q6 and Q4. FIG. 5 is a graph showing % RNA release versus heparin concentration. EXAMPLES

[0063] Example 1. Synthesis of N1-(7-chloroquinolin-4-yl)-hexane-1,6-diamine (Q6) 1,6-Diaminohexane (20.6 g, 0.18 mol) and 4,7-dichloroquinoline (10.3 g, 0.052 mol) were heated with stirring to approximately 150° C. to melt the solids, and the resulting mixture was stirred under nitrogen for 3 hours. To the reaction mixture was added 50 mL of methanol followed by slow addition of 50 mL of 1 M NaOH solution with stirring. A visible precipitate formed. The precipitate was filtered off, and the filtrate was further purified by crystallization or silica column chromatography to give N1-(7-chloroquinolin-4-yl)-hexane-1,6-diamine (Q6).

[0064] Example 2. Synthesis of p(CBA-ABOL-Q) / PEI 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) at 50 °C under a N atmosphere. MilliQ (2 mL) and CaCl2 (0.44 g, 4.0 mmol) were added and the mixture was stirred at 50 °C for 48 h. A solution of branched PEI 800 PEI in MilliQ water was prepared by weighing 100 mg of PEI into 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 h. The reaction mixture was then acidified to pH 4 using 6 M HCl and the solvent was evaporated to give the crude quinoline-functionalized polymer, which was further purified by dialysis.

[0065] Example 3. In vitro transfection efficiency of mRNA-GFP loaded nanoparticles with increasing amounts of Q6 in the polymer used The synthesis of the polymer p(CBA-ABOL-Q) / PEI was carried out similarly to Example 2, using 5 mmol of CBA and the amounts of ABOL and Q listed in Table 1 as feedstocks for polymerization, and 220 mg of CaCl2 as catalyst. 340 μL of PEI stock (100 mg / mL) was added after 48 hours and stirred for 72 hours. [Table 1]

[0066] A 120 ng / μL solution of mRNA encoding eGFP (Trilink, cat# L-7201-1000) was made in 10 mM histidine, pH 6.5. The polymers in Table 1 were dissolved in 10 mM histidine, pH 6.5 at a concentration of 3 mg / mL. A 120 ng / μL mRNA solution was added to the cationic polymer at a 1:1 (v / v) ratio. The solution was incubated at room temperature for at least 15 minutes before being used for transfection.

[0067] Culture COS7 cells in DMEM medium containing 10% FBS in a 48-well cell culture plate (1.6X10 cells per well). 4 Cells) were seeded. 24 hours after seeding the cells, the culture medium was replaced with DMEM containing 10% FBS and 2% 1 M HEPES, pH 7.2, and the polymers (15 μg polymer / mL) and mRNA listed in Table 1. After 24 hours, cells were analyzed for eGFP expression using FACS analysis. As the amount of Q6 in the polymer increased, the percentage of eGFP-positive cells increased as shown in Figure 1. Cell viability was measured with Alamar Blue reagent. As the amount of Q6 in the polymer used increased, the cell viability decreased. The percentage of eGFP positive cells (same percentages as shown in FIG. 1) and cell viability are shown in FIG.

[0068] Example 4. Number of cells transfected with mRNA-EGFP-loaded nanoparticles with or without Q6 The polymers p(CBA-ABOL-Q6)PEI and p(CBA-ABOL)PEI were synthesized and tested in transfection as described in Examples 2 and 3. The polymer p(CBA-ABOL-Q6)PEI containing 25% Q6 was compared to the polymer without Q6. C2C 12 Cells were transfected with 50ug polymer / mL culture medium for 24 hours and the number of eGFP positive cells was quantified by FACS analysis. The results are shown in Figure 3.

[0069] Example 5. Transfection efficiency of polymers containing Q6 and N1-(7-chloroquinolin-4-yl)-butane-1,4-diamine (Q4) Q4 was prepared following a similar procedure as described in Example 1, using 1,4-diaminobutane instead of 1,6-diaminohexane. The Q4-containing polymer is 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 is determined and compared. COS7 cells were transfected with 30ug polymer / mL culture medium for 4 hours, after which the cell culture medium was replaced. The number of eGFP-positive cells was quantified 24 hours later by FACS analysis. The results are shown in Figure 4.

[0070] Example 6. RNA encapsulation stability in nanoparticles with or without Q6 The polymers p(CBA-ABOL-Q6)PEI and p(CBA-ABOL)PEI were synthesized and used in the formulation of nanoparticles containing RNA as described in Examples 2 and 3. The polymer p(CBA-ABOL-Q6)PEI containing 25% Q6 was compared to a polymer incorporating only non-Q6 containing ABOL monomers. The nanoparticles formed were used in a release assay to displace the RNA nucleic acid from the nanoparticles using heparin competition. Increasing heparin concentration increases competition between RNA and polymer binding, thereby releasing RNA from the nanoparticles. The released RNA was then quantified using Ribogreen reagent. Higher concentrations of heparin were required to release RNA from the Q6-containing nanoparticles, indicating stronger interactions between the Q6-containing polymer and RNA and increased stability of the nanoparticles. The results are shown in Figure 5.

Claims

1. 1. A nanoparticle comprising an azanaphthalene-functionalized polymer and a biologically active moiety selected from the group consisting of nucleotides, polynucleotides, oligonucleotides, peptides, proteins, and combinations thereof, wherein the polymer comprises a polyamine segment of formula (I): 【Chemistry 1】 wherein X is based on a bis(α,β-unsaturated carbonyl) monomer, B is based on an amine monomer, Q is based on an azanaphthalene-containing amine monomer that includes an azanaphthalene moiety, m is at least 1, and n is at least 1, preferably n+m is in the range of 2 to 250.

2. 2. The nanoparticle of claim 1, wherein the azanaphthalene moiety is selected from the group consisting of benzopyridines, including 1-benzazine (quinoline) and 2-benzazine (isoquinoline), and diazanaphthalenes, including 1,2-diazanaphthalene (cinnoline), 1,3-diazanaphthalene (quinazoline), 1,4-diazanaphthalene (quinoxaline), 2,3-diazanaphthalene (phthalazine), 1,8-diazanaphthalene, 1,7-diazanaphthalene, 2,7-diazanaphthalene, 1,6-diazanaphthalene, 1,5-diazanaphthalene, and 2,6-diazanaphthalene, preferably benzopyridine, more preferably quinoline.

3. X is a group represented by the formula (I x Based on a bis(α,β-unsaturated carbonyl) monomer represented by 【Chemistry 2】 During the ceremony, A is C(R 1 ) 2 , O, N(R 1 ), S, and combinations thereof; R 1 is H, halide, and C such as alkyl, alkenyl, and polyalkenyl. 1 ~C 22 hydrocarbyl, which are optionally substituted with one or more halides, and combinations thereof; and R 2 is C 1 ~C 40 and combinations thereof, Nanoparticles according to any one of claims 1 to 2.

4. X comprises a disulfide moiety, preferably X is as defined in claim 3, and R 2 contains a disulfide moiety, more preferably R 2 interrupted by at least one disulfide moiety, C 1 ~C 10 The nanoparticles according to any one of claims 1 to 2, which are selected from the group consisting of hydrocarbylenes, preferably linear alkylenes, and combinations thereof.

5. B is a compound represented by the formula (I B1 ) based on a primary amine represented by 【Chemistry 3】 or formula (I B2 Based on the diamine represented by 【Chemistry 4】 During the ceremony, R 3 are independent of each other, H, C 1 ~C 40 linear, cyclic and branched hydrocarbyls, which are optionally substituted and / or interrupted with one or more heteroatoms, and combinations thereof; and R 4 is C 1 ~C 40 and combinations thereof, Nanoparticles according to any one of claims 1 to 4.

6. Nanoparticles according to any one of claims 1 to 4, wherein B comprises a hydroxyl moiety, preferably B is as defined in claim 5, and R 3 contains a hydroxy moiety, more preferably R 3 But, C 1 ~C 10 Hydrocarbyl hydroxide, preferably linear alkyl C 1 ~C 10 The nanoparticles according to any one of claims 1 to 4, selected from the group consisting of: hydroxides, hydrates, and combinations thereof.

7. Q is a compound represented by the formula (I Q1 Based on a primary amine represented by 【Chemistry 5】 and / or formula (I Q2 Based on the diamine represented by 【Chemistry 6】 During the ceremony, Z 1 represents a group containing an azanaphthalene moiety; Z 2 represents a group containing an azanaphthalene moiety, or H, C 1 ~C 40 linear, cyclic and branched hydrocarbyls, which are optionally substituted and / or interrupted with one or more heteroatoms, and combinations thereof; R 4 is as defined in claim 5, Nanoparticles according to any one of claims 1 to 6.

8. Z 1 and optionally Z 2 independently represent a moiety according to any of the following structures: 【Chemistry 7】 In the formula, R 5 is C 1 ~C 40 , preferably C 1 ~C 10 , more preferably C 1 ~C 6 linear, cyclic and branched hydrocarbylenes, which are optionally substituted and / or interrupted with one or more heteroatoms, and combinations thereof; and The azanaphthalene moiety is optionally substituted with one or more substituents, preferably one or more substituents selected from the group consisting of halide, alkyl and alkoxide, and is optionally substituted with one or more heteroatoms.

8. The nanoparticles according to claim 7.

9. Z 1 and optionally Z 2 is independently represented by the formula (I Z2 ), preferably the moiety of formula (I Z3 ) part, 【Chemistry 8】 In the formula, R 5 is C 1 ~C 40 , preferably C 1 ~C 10 , more preferably C 1 ~C 6 linear, cyclic and branched hydrocarbylenes, which are optionally substituted and / or interrupted with one or more heteroatoms, and combinations thereof; and The azanaphthalene moiety is optionally substituted with one or more substituents, preferably one or more substituents selected from the group consisting of halide, alkyl and alkoxide, and is optionally substituted with one or more heteroatoms. Nanoparticles according to any one of claims 7 to 8.

10. Z 1 and optionally Z 2 is independently represented by the formula (I Z4 ) part 【Chemistry 9】 In the formula, R 5 is as defined in claim 9, preferably R 5 is C 1 ~C 10 Alkylene, more preferably C 1 ~C 6 Alkylene, most preferably C 1 ~C 4 alkylene, and combinations thereof; and R 6 is selected from the group consisting of halides, alkoxides, alkyls and mixtures thereof, optionally substituted with one or more heteroatoms, preferably a halide, more preferably R 6 is Cl, Nanoparticles according to any one of claims 7 to 9.

11. the polyamine segment has a structure according to any of formulas (IIa), (IIb), (IIc), and (IId), and combinations thereof; 【Chemistry 10】 In the formula, n, m, A, R 1 , R 2 , R 3 , R 4 , Z 1 and Z 2 are each independently as defined in any preceding claim, Nanoparticles according to any one of claims 1 to 10.

12. The polyamine segment, preferably the polymer, has a structure represented by formula (III): 【Chemistry 11】 Preferably, the compound has a structure represented by any one of formulas (IVa), (IVb), (IVc), and (IVd), and a combination thereof. 【Chemistry 12】 In the formula, n, m, A, R 1 , R 2 , R 3 , R 4 , Z 1 and Z 2 are each independently as defined in any preceding claim; T is the weight average molecular weight M w represents a core having a Mn of about 60 to about 25,000; q ranges from 1 to 64; R 6 , H, C 1 ~C 40 and combinations thereof, wherein the linear, cyclic and branched hydrocarbyls are optionally substituted and / or interrupted by one or more heteroatoms. Nanoparticles according to any one of claims 1 to 11.

13. 13. The nanoparticles according to any one of claims 1 to 12, wherein the ratio of m to n is in the range of 1:20 or more, preferably in the range of 1:10 to 10:1, more preferably in the range of 1:5 to 2:1, most preferably in the range of 1:4 to 1:1, such as about 1:

3.

14. The nanoparticle of any one of claims 1 to 13, wherein the biologically active component comprises RNA, DNA, or derivatives thereof.

15. Nanoparticles according to any one of claims 1 to 14 for use in a medical method, preferably as a vaccine in a medical method.

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

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