Complexes containing shielding moieties

Abstract

The present invention relates to novel polymer conjugates comprising at least one anionic poly(methionine sulfoxide)-based copolymer, useful as non-viral vectors for the delivery of active ingredients, including nucleic acids, to cells.

Description

[Technical field]

[0001] This application claims the benefit of European Patent Application No. 21383069.8, filed November 25, 2021.

[0002] The present disclosure relates to novel polymer conjugates that contain shielding moieties that are anionic copolymers that contain methionine sulfoxide-based blocks. These polymer conjugates can be used as non-viral vectors for the delivery of active ingredients, such as nucleic acids, to cells. [Background technology]

[0003] The use of cationic polymers as non-viral synthetic carriers for the delivery of active ingredients, more particularly nucleic acids, to target cells has attracted considerable attention. Thus, polyion complexes (PICs), specifically formed by electrostatic interactions between nucleic acids functioning as polyanions and cationic polymers (polycations), have been given the name polyplexes and have been widely disclosed in the prior art. However, applicability and transition from bench to commercialization remain challenges.

[0004] One of the main reasons for the limited development of polycation-based non-viral vectors is that cationic polymers are required to exhibit different functions at different stages of the delivery process. For example, the potential energy change due to proton transfer of nanoparticles may cause endosomal buffering and membrane destabilization, so polymer carriers may need to have high amine density and suitable pKa to overcome the endosomal membrane barrier. On the contrary, the positive charge of nanoparticles may cause aggregation in the bloodstream and nonspecific interactions with negatively charged serum components, which may cause thrombosis in capillaries. This has the risk of interfering with the construction of plasma membranes and inducing high cytotoxicity and excessive immune responses. In addition, these positively charged nanoparticles may cause severe serum inhibition and are quickly cleared from the blood, which prevents their in vivo application.

[0005] A well-known attempt to solve these problems is the introduction of a neutral charge shield by coating the surface of nanoparticles with polyethylene glycol (PEG), which is widely used in drug delivery and nanotechnology due to its stealth and biocompatibility properties. Thanks to these properties, particulate delivery systems containing PEG can avoid aggregation, evade the immune system, and consequently have a long circulation time in the body.

[0006] It is also known that a polycation having a polyethylene glycol (PEG) moiety can condense a nucleic acid to form a core moiety through the interaction between the nucleic acid and the polycation moiety in the block copolymer, and a polyplex having a structure in which the hydrophilic and biocompatible PEG moiety in the block copolymer forms a shell surrounding the core moiety can be obtained. Thus, PICs can stably encapsulate nucleic acids and avoid foreign body recognition mechanisms present in vivo.

[0007] However, the presence of covalently attached PEG can significantly reduce transfection efficiency in the case of polyplexes, as the neutral surface of the nanoparticles can reduce the cellular uptake efficiency, or can cause activity loss in the case of proteins due to hindered spacing of the active sites. PEG is also known to produce anti-PEG antibodies, which can cause immunogenicity, allergic reactions, and anaphylactic shock.

[0008] Polyion complex polypeptide hydrogels have been disclosed that include at least two diblock, triblock, or pentablock copolypeptides and water. Thus, for example, WO2019067676 discloses polyion complex polypeptide hydrogels in which each copolypeptide has an ionic segment with opposite charges to each other, and both copolypeptides are methionine sulfoxide (M o In particular, WO 2019067676 discloses hydrogels comprising (M o A) n K x , (M o A) n E x and water-based PIC hydrogels are described.

[0009] Hydrogel systems have potential applications as bioinks for tissue regeneration and injectable depots / carriers for small molecule or protein controlled release applications. Gels are colloidal materials in which a small amount of solid, microphase separated component network (gelator) can immobilize the bulk flow of a larger amount of liquid-like phase. This microphase separation is induced by nucleation. As a result, the material becomes "solid-like" in its hydrodynamic behavior; that is, the storage (elastic) modulus (G') is greater than the loss (viscous) modulus (G'') over a range of shear frequencies within the linear viscoelastic region.

[0010] Therefore, from the state of the art, there remains a need to develop other polycation-based non-viral vectors that overcome the shortcomings of the prior art. Summary of the Invention

[0011] The inventors have found that several anionic copolymers containing methionine sulfoxide-based blocks are useful as shields for positively charged protein or polycation-based non-viral vectors to deliver proteins or active ingredients (including nucleic acids) to cells due to low cytotoxicity, high efficiency, potentially suitable plasma half-life times, potentially high permeability and retention, high solubility in aqueous solutions, high stability with limited or even completely suppressed aggregation problems in the bloodstream, and potentially different cell and tissue tropism.

[0012] The object of the present invention is to provide a novel polycation-based non-viral vector, i.e. a polymer conjugate comprising a cationic polymer, at least one pharma- ceutical, veterinary or cosmetically active ingredient and a shielding component which is an anionic copolymer containing a methionine sulfoxide-based block.

[0013] Thus, according to one aspect of the present invention, a) positively charged nanoparticles comprising a cationic polymer covalently or electrostatically bound to at least one pharma- ceutical, veterinary or cosmetically active ingredient; b) Below: i. a copolymer comprising substructure I; ii. a copolymer comprising substructure II, and iii. Copolymers containing partial structure III At least one anionic copolymer selected from Including, Here, substructures I, II and III are -A m -B n -(substructure I), -(B n -A m ) p -B n -(Substructure II), -(A m -B n ) p -Am -(partial structure III), each example of A is an amino acid residue independently selected from methionine sulfoxide, ethionine sulfoxide, S-alkyl-cysteine ​​sulfoxide, S-alkylcysteine ​​sulfone, S-alkylhomocysteine, S-alkylhomocysteine ​​sulfoxide, glycosylated cysteine, serine, homoserine, homomethionine sulfoxide, sarcosine, glycine, and alanine; At least 50 mol % of the A amino acid residues are methionine sulfoxide; each instance of B is independently selected from glutamic acid, aspartic acid, and salts thereof; m is an integer from 10 to 600; n is an integer from 5 to 200; p is an integer from 1 to 2; A polymer composite is provided.

[0014] It is also an object of the present invention to provide novel protein-based conjugates that include a cationically charged protein and a shielding moiety that is an anionic copolymer that includes a methionine sulfoxide-based block.

[0015] Thus, according to one aspect of the present invention, a) a cationically charged protein; and b) Below: i. a copolymer comprising substructure I; ii. a copolymer comprising substructure II, and iii. Copolymers containing partial structure III At least one anionic copolymer selected from Including, wherein substructures I, II and III are as defined herein. Protein-based conjugates are provided.

[0016] According to another aspect of the present invention, As a shield for positively charged proteins or positively charged nanoparticles comprising a cationic polymer covalently or electrostatically bound to at least one pharma- ceutical, veterinary or cosmetically active ingredient for the delivery of the protein or pharma-ceutical, veterinary or cosmetically active ingredient to a biological target; i. a copolymer comprising substructure I; ii. a copolymer comprising substructure II, and iii. Copolymers containing partial structure III Use of at least one anionic copolymer selected from Provided herein is the use, wherein substructures I, II and III are as defined herein.

[0017] A further aspect of the present invention relates to a pharmaceutical, veterinary or cosmetic composition comprising at least one polymer conjugate or at least one protein-based conjugate as defined herein, together with one or more suitable acceptable excipients.

[0018] In a further aspect of the invention, a) providing a polymer conjugate, protein-based conjugate or composition as described herein; b) contacting the biological target with the polymer conjugate, the protein-based conjugate, or the composition;

[0023] A method for delivering at least one pharma- ceutical, veterinary, or cosmetically active ingredient to a biological target is provided, comprising:

[0019] This embodiment may also be referred to as a method for delivering at least one pharma- ceutical, veterinary, or cosmetically active ingredient to a target cell, comprising the steps of administering to an animal, including a human, a solution containing the polymer conjugate, the protein-based conjugate, or the composition, such that the polymer conjugate, the protein-based conjugate, or the composition as defined herein may be introduced into the target cell, translocating the polymer conjugate, the protein-based conjugate, or the composition from an endosome to the cytoplasm, dissociating the polymer conjugate, the protein-based conjugate, or the composition within the cell, and releasing the active ingredient into the cytoplasm.

[0020] In a further aspect of the invention, a) providing a cationic polymer in a first liquid; b) providing at least one pharma- ceutical, veterinary or cosmetically active ingredient in a second liquid; c) contacting the cationic polymer in the first liquid with at least one pharma- ceutical , veterinary or cosmetically active ingredient in a second liquid to form positively charged nanoparticles; d) Below: i. a copolymer comprising substructure I; ii. a copolymer comprising substructure II, and iii. Copolymers containing partial structure III providing an anionic copolymer selected from e) contacting the positively charged nanoparticles with an anionic copolymer to form a polymer complex; Including, wherein substructures I, II and III are as defined herein. A method for the preparation of a polymer composite is provided.

[0021] or, a) providing a cationic polymer in a first liquid; b) providing at least one pharma- tically, veterinarily or cosmetically active ingredient in a second liquid and i. a copolymer comprising substructure I; ii. a copolymer comprising substructure II, and iii. Copolymers containing partial structure III and mixing with an anionic copolymer selected from c) contacting the cationic polymer in a first liquid with at least one pharma- ceutical, veterinary or cosmetically active ingredient and the anionic copolymer in a second liquid to form shielded nanoparticles; Including, wherein substructures I, II and III are as defined herein. A method for the preparation of a polymer composite is provided.

[0022] In one aspect of the invention there is provided a polymer conjugate, protein-based conjugate or composition as defined herein for use as a medicament.

[0023] In one aspect of the invention, there is provided a polymer conjugate, protein-based conjugate or composition as defined herein for (i) use as a transfection reagent for transfecting at least one active agent into a cell, (ii) use in in vivo or ex vivo therapy encoding recombinant proteins, peptides or antibodies, (iii) use in the production of peptides, proteins, antibodies or recombinant viruses, (iv) use as a therapeutic or prophylactic vaccine against viral infections or a therapeutic vaccine against cancer, and (v) use in genome engineering for cell reprogramming, cell differentiation, or gene editing.

[0024] In another aspect of the invention, there is provided a protein-based complex or a composition containing same for use in protein-based therapy.

[0025] In a further aspect of the invention, there is provided a device for delivering at least one pharmaceutical, veterinary or cosmetically active ingredient into a cell, comprising the polymer conjugate or a composition containing same.

[0026] Non-limiting examples of the present disclosure are described below with reference to the accompanying drawings. [Brief description of the drawings]

[0027] [Figure 1] Shielded complexes: A) ternary complexes formed by genetic material, polycations and shielding block anionic copolymers; B) schematics of the formation of an assembly of proteins and shielding block polyanionic copolymers.

[0028] [Diagram 2]The results of polyplexes PXN1_8_V1_1 pDNA and PXN1_8_V1_0.5 pDNA (top) and PXN1_15_V1_1 pDNA and PXN1_15_V1_0.5 pDNA (bottom) with shielding polymer V1 analyzed by agarose gel electrophoresis technique are shown. This technique is a qualitative method to show the ability of polyplexes to form complexes with genetic material (DNA). It also shows the ability to release genetic material at low and high concentrations in the presence of low and high concentrations of polyanionic competitor (heparin). In the lane labeled M, free pDNA was added. As shown in the figure, the free genetic material glows under a UV transilluminator. In lanes 1 and 4 (top) and 4 and 7 (bottom), polyplexes were added and it can be seen that when polycations are present and polyplexes are formed, the genetic material is encapsulated and no signal can be observed. In lanes 2 and 5 (top) and 5 and 8 (bottom), polyplexes were added in the presence of low concentrations of heparin competitor and show no release under those conditions. In lanes 3 and 6 (top) and 6 and 9 (bottom), polyplexes were added with high concentrations of anionic heparin competitor and release of genetic material is observed. This behavior is ideal because polyplexes must be stable at low concentrations of competitor molecule outside the cell but unstable enough to release cargo when an intracellular stimulus is applied. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] All terms used herein in this application are to be understood in their ordinary meanings known in the art unless otherwise specified. Other more specific definitions for certain terms used in this application are set forth below, and are intended to be applied uniformly throughout this specification and claims, unless an explicitly stated definition gives a broader definition.

[0030] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more." Unless otherwise specified, definite articles used herein, such as "the," also include plural nouns.

[0031] The term "substituted" means that one or more hydrogen atoms on the specified atom or group are replaced with one selected from the indicated group, provided that the normal valence of the specified atom under the existing circumstances is not exceeded. Combinations of substituents and / or variables are permitted. The term "optionally substituted" means that the number of substituents can be equal to or different from zero. Unless otherwise specified, an optionally substituted group can be substituted with as many optional substituents as can be accommodated by replacing hydrogen atoms with non-hydrogen substituents on any available carbon or nitrogen atom. Groups in the compounds of the present invention can be substituted with one, two, three, four or five identical or different substituents, particularly one, two or three substituents.

[0032] The phrase "natural amino acid" as used herein refers to any of the 20 amino acids that occur naturally in proteins. Such natural amino acids include the non-polar or hydrophobic amino acids glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Cysteine ​​is non-polar or hydrophobic but is sometimes classified as polar. Natural amino acids also include polar or hydrophilic amino acids such as tyrosine, serine, threonine, aspartic acid (also known as aspartate when charged), glutamic acid (also known as glutamate when charged), asparagine, and glutamine. Certain polar or hydrophilic amino acids have side chains that are charged depending on the pH of the environment. Such charged amino acids include lysine, arginine, and histidine. One of skill in the art will recognize that the protection of the polar or hydrophilic amino acid side chains can make an amino acid non-polar. For example, suitably protected tyrosine hydroxyl groups can be rendered non-polar and hydrophobic by protecting the hydroxyl group.

[0033] The phrase "unnatural amino acid" as used herein refers to an amino acid that is not included in the list of 20 naturally occurring amino acids in proteins described above. Such amino acids include any D-isomer of the 19 naturally occurring amino acids, with glycine being achiral. Unnatural amino acids also include homoserine and ornithine. Other unnatural amino acid side chains are well known to those skilled in the art, including unnatural aliphatic side chains. Other unnatural amino acids include modified amino acids, including those that are N-alkylated, cyclized, phosphorylated, acetylated, amidated, azidylated, and labeled.

[0034] As used herein, the term "repeat unit" or "block" refers to a repeating monomer unit. A repeat unit or block may be composed of a single monomer, or may be composed of one or more monomers occurring in a "mixed block."

[0035] Those of ordinary skill in the art will recognize that monomeric repeat units are defined by brackets ([ ]) shown around the repeating monomeric unit. The number (or letter representing a numerical range) to the right of the brackets represents the number of monomeric units present in the polymer chain.

[0036] According to some embodiments, optionally in combination with any of the embodiments provided above or below, at least one anionic copolymer comprising substructure I, II or III electrostatically interacts with positively charged proteins acting as a shielding layer on or between the proteins.

[0037] Considering the nomenclature used herein for the shielding polymers according to the present invention, it should be noted that the numerical values ​​mentioned in brackets refer to the degree of polymerization (DP) of each monomer unit as a statistical number. The DP of a particular monomer unit is calculated by dividing the molecular weight of the polymer by the molecular weight of the monomer unit. The DP values ​​are subject to reasonable uncertainty due to the ring-opening polymerization mechanism, which may be considered in the context of the present invention to be within ±20%, preferably ±15%, more preferably ±10%, even more preferably ±5%, and especially preferably ±2%.

[0038] The at least one anionic copolymer is selected from copolymers containing substructure I, copolymers containing substructure II, and copolymers containing substructure III, where substructures I, II, and III are shown above.

[0039] In the copolymers of substructures I, II and III, m is an integer between 10 and 600. In one embodiment, optionally in combination with any of the embodiments provided above or below, m is an integer between 10 and 400, more particularly between 15 and 300, even more particularly between 20 and 200, even more particularly between 25 and 180. Other specific ranges are 40 to 380, 50 to 350, 75 to 300, 90 to 250 and 100 to 200. The integer m corresponds to the degree of polymerization of the A amino acid residues, which can be measured by SEC or 1H-NMR spectroscopy, as shown in the examples.

[0040] In the copolymers of substructures I, II and III, n is an integer from 5 to 200. In one embodiment, optionally in combination with any of the embodiments provided above or below, n is an integer from 8 to 150, more particularly from 10 to 120, even more particularly from 10 to 110, even more particularly from 10 to 100. Other specific ranges are 5 to 190, 6 to 180, 7 to 170, 12 to 120 and 15 to 80. The integer n corresponds to the degree of polymerization of the B amino acid residues, which can be measured by SEC or 1H-NMR spectroscopy, as shown in the examples.

[0041] In certain embodiments, optionally in combination with any of the embodiments provided above or below, the m:n ratio in the copolymer of moieties I, II and III defined herein ranges from 1:1 to 120:1, preferably from 2:1 to 60:1, more preferably from 4:1 to 20:1, and even more preferably from 5:1 to 10:1.

[0042] In the copolymers of substructures I, II and III, each instance of A is an amino acid residue independently selected from methionine sulfoxide, ethionine sulfoxide, S-alkyl-cysteine ​​sulfoxide, S-alkylcysteine ​​sulfone, S-alkylhomocysteine, S-alkylhomocysteine ​​sulfoxide, glycosylated cysteine, serine, homoserine, homomethionine sulfoxide, sarcosine, glycine and alanine, wherein at least 50 mol % of the A amino acid residues are methionine sulfoxide.

[0043] In one embodiment, optionally in combination with any of the embodiments provided above or below, for each instance of A, 100 mol % of the A amino acid residues are methionine sulfoxide.

[0044] In one embodiment, optionally in combination with any of the embodiments provided above or below, for each instance of A, at least 50 mol % of the A amino acid residues are methionine sulfoxide, more particularly between 60 mol % and 98 mol %, even more particularly between 75 mol % and 95 mol %, and even more particularly between 80 mol % and 90 mol % of the A amino acid residues are methionine sulfoxide.

[0045] In one embodiment, optionally in combination with any of the embodiments provided above or below, for each instance of A, 50 mol% to 98 mol% of the A amino acid residues are methionine sulfoxide and the remaining amino acid residues are independently selected from sarcosine, glycine and alanine, more particularly 60 mol% to 98 mol% of the A amino acid residues are methionine sulfoxide and the remaining A amino acid residues are independently selected from sarcosine, glycine and alanine, and even more particularly 75 mol% to 95 mol% of the A amino acid residues are methionine sulfoxide and the remaining A amino acid residues are independently selected from sarcosine, glycine and alanine.

[0046] In one embodiment, optionally in combination with any of the embodiments provided above or below, each instance of B is glutamic acid.

[0047] In one embodiment, optionally in combination with any of the embodiments provided above or below, the at least one anionic copolymer is (1) Poly((L-methionine sulfoxide)138-stat-(L-alanine)19-block-poly(L-glutamic acid)65, (M O )138A19E65, (2) Poly((L-methionine sulfoxide) 167-stat-(L-alanine) 25-block-poly(L-glutamic acid) 82, (M O )167A25E82, (3) Poly((L-methionine sulfoxide) 154-stat-(L-alanine) 25-block-poly(L-glutamic acid) 84, (M O )154A25E84, (4) Poly((L-methionine sulfoxide) 139-stat-(L-alanine) 20-block-poly(L-glutamic acid) 98, (M O )139A20E98, (5) Poly((L-methionine sulfoxide) 167-stat-(L-alanine) 25-block-poly(L-glutamic acid) 80, (M O )167A25E80, (6) Poly(L-methionine sulfoxide 0.88-stat-L-alanine 0.12) 155-block-poly(L-glutamic acid) 30, (M O A) 155E30, (7) Poly(L-methionine sulfoxide 0.88-stat-L-alanine 0.12) 155-block-poly(L-glutamic acid) 60, (M O A) 155E60, (8) Poly(L-methionine sulfoxide 0.88-stat-L-alanine 0.12) 155-block-poly(L-glutamic acid) 65, (M O A) 155E65, (9) Poly(L-methionine sulfoxide 0.88-stat-L-alanine 0.12) 155-block-poly(L-glutamic acid) 90, (M O A) 155E90, (10) Poly(L-methionine sulfoxide 0.88-stat-L-alanine 0.12) 155-block-poly(L-glutamic acid) 120, (M O A) 155E120, (11) Poly(L-methionine sulfoxide 0.88-stat-L-alanine 0.12) 100-block-poly(L-glutamic acid) 30, (M O A) 100E30, (12) Poly(L-methionine sulfoxide) 155-block-poly(L-glutamic acid) 60, (M O )155E60, (13) Poly(L-methionine sulfoxide) 60-block-poly(L-glutamic acid) 10, (M O )60E10, (14) Poly(L-methionine sulfoxide) 60-block-poly(L-glutamic acid) 20, (M O )60E20, (15) Poly(L-methionine sulfoxide) 60-block-poly(L-glutamic acid) 30, (M O )60E30, (16) Poly(L-methionine sulfoxide) 60-block-poly(L-glutamic acid) 40, (M O )60E40, (17) Poly(L-methionine sulfoxide)60-block-poly(L-glutamic acid)60, (M O )60E60, (18) Poly(L-methionine sulfoxide)60-block-(racemic)poly(L-glutamic acid)60, (M O )60(racemic-E)60, (19) Poly(L-methionine sulfoxide 0.88-stat-L-alanine 0.12) 50-block-poly(L-glutamic acid) 30-block-poly(L-methionine sulfoxide 0.88-stat-L-alanine 0.12) 50, (M O A) 50E30(M O A) 50, (20) Poly(L-methionine sulfoxide 0.88-stat-L-alanine 0.12) 50-block-poly(L-glutamic acid) 30-block-poly(L-methionine sulfoxide 0.88-stat-L-alanine 0.12) 100-block-poly(L-glutamic acid) 30-block-poly(L-methionine sulfoxide 0.88-stat-L-alanine 0.12) 50, (M O A) 50E30(M O A) 100E30(M O A) 50, (21) Poly(L-glutamic acid) 30-block-poly(L-methionine sulfoxide 0.88-stat-L-alanine 0.12) 100-block-poly(L-glutamic acid) 30, E30 (MOA) 100E30 The structure comprises a backbone selected from:

[0048] Anionic copolymers containing substructures I, II and III are polymer structures that contain methionine sulfoxide-based moieties as repeating units. These polymers are characterized by molecular weight (which may be average molecular weight (MW) or number average molecular weight (Mn)), degree of polymerization and polydispersity index. Molecular weight may be measured by methods well known in the art, such as size exclusion chromatography (SEC) (also called gel permeation chromatography (GPC)), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) or 1H-NMR spectroscopy. Some of these methods are shown in more detail in the following examples.

[0049] The term "polydispersity index" (PDI) is used as a measure of the broadness of the molecular weight distribution. The higher the PDI, the broader the molecular weight. The PDI of a polymer is calculated as the ratio of the weight average (MW) / number average (Mn) molecular weight.

[0050] According to another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the anionic copolymer comprising substructures I, II and III has an average molecular weight (Mw) measured by SEC as disclosed in the examples of 2000 to 50000 Da, more particularly 5000 to 40000 Da, even more particularly 10000 to 35000 Da or 20000 to 30000 Da.

[0051] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the polydispersity index of the anionic copolymer comprising substructures I, II and III as measured by SEC as disclosed in the examples is between 1.01 and 2.00, or between 1.01 and 1.8, or between 1.01 and 1.50, or between 1.01 and 1.30, or between 1.01 and 1.25.

[0052] The polymer complexes of the present invention may be nanoparticles, in which the active ingredient, preferably a nucleic acid, interacts with a cationic polymer forming a positively charged nanoparticle to form a core portion, and an anionic copolymer forms a shell portion around the core portion (see FIG. 1A).

[0053] Positively charged nanoparticles can be easily prepared in any given buffer solution, for example by mixing an active ingredient with a cationic polymer. The conditions for preparation, such as aqueous medium, pH, temperature and ionic strength, can be appropriately adjusted by those skilled in the art.

[0054] As used herein, the term "nanoparticle" refers to a particle having at least two dimensions in the nanoscale, and in particular all three dimensions in the nanoscale. In particular, a nanoparticle is substantially rod-shaped with a substantially circular cross-section, such as a nanowire or nanotube, and "nanoparticle" refers to a particle having at least two dimensions in the nanoscale, the two dimensions being the cross-section of the nanoparticle.

[0055] The term "size" as used herein refers to a characteristic physical dimension. For example, for a substantially spherical nanoparticle, the size of the nanoparticle corresponds to the diameter of the nanoparticle. For a substantially rod-shaped nanoparticle with a substantially circular cross section, such as a nanowire or nanotube, the size of the nanoparticle corresponds to the diameter of the cross section of the nanoparticle. For a substantially box-shaped nanoparticle, such as a nanocube, nanobox or nanocage, the size of the nanoparticle corresponds to the maximum side length. When referring to a collection of nanoparticles as having a particular size, it is understood that the collection of nanoparticles may have a size distribution of approximately the specified size. Thus, the size of a collection of nanoparticles as used herein can refer to the size distribution mode, such as the peak size of the size distribution.

[0056] Anionic copolymers comprising partial structures I, II and III may exist as geometric isomers (i.e., cis-trans isomers), optical isomers, or stereoisomers, such as diastereomers, as well as tautomers. Thus, it should be understood that these definitions include each and every individual isomer, including cis-trans isomers, stereoisomers and tautomers, as well as racemic mixtures thereof and pharma-ceutically acceptable salts thereof. Thus, the definitions of cationic polymers and anionic copolymers comprising partial structures I, II and III are intended to encompass all R and S isomers of the chemical structures in any ratio, including, for example, enrichment of one of the possible isomers and a corresponding smaller ratio of the other isomer (i.e., enantiomeric excess or diastereomeric excess). In the particular case of amino acids, they may be in the L or D form.

[0057] The anionic copolymers containing substructures I, II and III may be provided in any form suitable for the intended administration, including, in particular, pharma- ceutically acceptable salts thereof.

[0058] Pharmaceutically acceptable salts refer to both salts of cationic polymers or anionic copolymers containing substructures I, II and III, which are deemed acceptable for clinical, veterinary and / or cosmetic use. Exemplary pharmaceutically acceptable salts include those salts prepared by reaction of cationic polymers or anionic copolymers containing substructures I, II and III with inorganic or organic acids or organic or inorganic bases. Such salts are known as acid addition salts and base addition salts, respectively. It will be recognized that the particular counterion or counterions forming part of any salt are not critical properties, so long as the salt as a whole is pharmaceutically acceptable and the counterion does not impart undesirable qualities to the salt as a whole. These salts can be prepared by methods known to those skilled in the art.

[0059] Examples of pharma- ceutically acceptable addition salts include those with inorganic acids, such as hydrochloric, hydrobromic, sulfuric, nitric, hydroiodic, metaphosphoric or phosphoric acid, as well as organic acids, such as succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, trifluoroacetic, malic, lactic, formic, propionic, glycolic, gluconic, camphorsulfuric, isethionic, mucic, gentisic, isonicotinic, saccharic, glucuronic, furoic, glutamic, ascorbic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic) acid, ethanesulfonic ... Examples of such salts include acid addition salts formed with carboxylic acids, such as carboxylic acid, pantothenic acid, stearic acid, sulfanilic acid, alginic acid, galacturonic acid, and arylsulfonic acids, e.g., benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, methanesulfonic acid, or naphthalenesulfonic acid, as well as base addition salts formed with organic bases, such as alkali metals and alkaline earth metals, N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), lysine, and procaine, as well as internally formed salts.

[0060] According to certain embodiments, optionally in combination with any of the embodiments provided above or below, the polymer conjugate is obtained when mixed in an aqueous medium at a pH in the range of 4 to 9, preferably 4.5 to 8.5, more preferably 5 to 7.5, and particularly preferably 6.5 to 7.4. The pH can be easily adjusted using a buffer as the solvent.

[0061] According to certain embodiments, optionally in combination with any of the embodiments provided above or below, the ionic strength of the solution to be mixed may be appropriately adjusted within a range that does not destroy the structure of the nanoparticles or inhibit the encapsulation of the substance to be encapsulated in the nanoparticles, which is preferably within the range of 0 to 1000 mM, preferably 0 to 300 mM, more preferably 0 to 150 mM, and particularly preferably 0 to 50 mM.

[0062] The polymer conjugates may have a particle hydrodynamic diameter in the range of 10 nm to 2000 nm, preferably 20 nm to 800 nm, more preferably 25 nm to 350 nm, 30 nm to 300 nm, and 30 nm to 200 nm, as measured by dynamic light scattering.

[0063] The protein-based complexes may have particle hydrodynamic diameters in the range of 2 nm to 2000 nm, preferably 5 nm to 1000 nm, more preferably 10 nm to 800 nm, 15 nm to 700 nm and 20 nm to 600 nm as measured by dynamic light scattering.

[0064] In the context of the present disclosure, the term "polyplex" refers to a polymer complex formed by electrostatic interaction between a cationic polymer as described herein and at least one polyanionic genetic material (preferably a nucleic acid). In the context of the present disclosure, the term "stabilized polyplex" or "shielded polyplex" refers to a polymer complex formed by electrostatic interaction between a cationic polymer as described herein, an anionic shielding polymer, and at least one polyanionic genetic material (preferably a nucleic acid).

[0065] According to a more specific embodiment, optionally in combination with any of the embodiments provided above or below, the N / P ratio in the polyplex of the present disclosure, defined as [total number of cationic groups in the block copolymer (N)] / [total number of phosphate groups in the nucleic acid (P)], ranges from 1 to 100, preferably from 2 to 50, more preferably from 2 to 30. The N / P ratio refers to the ratio between the molar concentration of protonatable amino groups derived from the side chains of the cationic polymer (N) and the molar concentration of phosphate groups derived from the nucleic acid (P) in the mixed solution.

[0066] Examples of cationic polymers include poly-L-lysine (PLL), poly-L-ornithine (PLO), poly-L-histidine, polyamidoamines, polyarginine, poly[2-{(2-aminoethyl)amino-ethyl-aspartamide] (pAsp(DET)), poly(dimethylaminoethyl methacrylate) (pDMAEMA), polyethyleneimine (PEI), chitosan, poly(β-amino esters), cationic or cationically ionized lipids or lipid-like substances, as well as block copolymers of polyethylene glycol and polyarginine, block copolymers of polyethylene glycol and polylysine. Examples of suitable cationic polymers include block, random or graft polycationic combinations based on polyamino acids, such as block copolymers of polyethylene glycol and poly[2-{(2-aminoethyl)amino}-ethyl-aspartamide] (PEG-pAsp(DET)), and any other suitable cationic polymers, as long as the cationic polymer is capable of forming a complex with at least one anionic active ingredient, and this complex is then capable of forming a polymer complex with at least one shielding anionic copolymer comprising the moiety of formula I, II or III as defined in the present invention.

[0067] Using appropriate surface functionalities, the compounds of the present disclosure may be further modified with cell targeting groups and / or penetration enhancers that can actively target cells and aid in cell entry, thereby resulting in conjugates with improved cell-specific delivery. Optionally, the compounds of the present disclosure may be further modified with labeling or imaging agents to facilitate visualization and / or detection.

[0068] Thus, in one embodiment, optionally in combination with any of the embodiments provided above or below, at least one cellular targeting agent, at least one labeling or imaging agent, or at least one cellular targeting agent plus at least one labeling or imaging agent are covalently attached to the polypeptide backbone of the cationic polymer or anionic copolymer via an amino acid side residue, i.e., C- or N-terminal group, via an amide, ester, anhydride bond, or via a linker containing one or more functional groups, including but not limited to, alkyne, azide, reactive disulfide, maleimide, hydrazide, hydrazone, Schiff base, acetal, aldehyde, carbamate, and reactive ester.

[0069] The term "cell targeting agent" refers to any biological or chemical structure that exhibits affinity for molecules present in the human or animal body that can direct functionalized nanoparticles by directing them toward a target site for therapeutic treatment, for example because they selectively bind to a receptor expressed or overexpressed in a specific cell type. Thus, the term includes antibodies against a specific antigen, ligands for a specific receptor or antigen, such as folic acid for its receptor, or sugars, such as galactose for its liver receptor. The targeting agent may be attached to the functionalized end group of the anionic polymer via the A and / or A' moieties, or may be attached to the cationic polymer.

[0070] Cell targeting groups are well known in the art. Examples of targeting agents include, but are not limited to, monoclonal and polyclonal antibodies (e.g., IgG, IgA, IgM, IgD, IgE antibodies), fragment antibodies, nanobodies, sugars (e.g., mannose, mannose-6-phosphate, galactose, galactosamine, mannosamine), proteins (e.g., transferrin), oligopeptides (e.g., cyclic and acyclic RGD-containing oligopeptides), oligonucleotides (e.g., aptamers), vitamins (e.g., folic acid), Her-2 binding peptides, TLR agonists, β-D-glucose, Asn-Gly-Arg peptides, angiopep-2, aptamers (A-9, A10, anti-gp120, T TA1, sgc8, anti-MUC-1, AS1411), primaquine, zidovudine, superoxide dismutase, prednisolone, platinum, cisplatin, sulfamethoxazole, amoxicillin, etoposide, mesalazine, doxorubicin, paclitaxel, 5-aminosalicylic acid, denosumab, docetaxel, calcitonin, proanthocyanidins, methotrexate, camptothecin, galactose, glycyrrhetinic acid, lactose, hyaluronic acid, octreotide, lactobionic acid, β-galactosyl moiety, arabinogalactan, chitosan, azo-based polyphosphazenes, azo groups and 4-amino-benzyl-carbamate, succinate, 4,4'-Dihydroxyazobenzene-3-carboxylic acid, cyclic RGD pentapeptide, aspartic acid octapeptide, alendronate, transferrin, bisphosphonate alendronate, monosialoganglioside GM1, glutathione, E-selectin thioaptamer, poloxamer-407, urokinase-type plasminogen activator receptor (uPAR) antagonist, CXCR4 chemokine receptor antagonist, GRP78 peptide antagonist, RGD peptide, RGD cyclic peptide, luteinizing hormone releasing hormone (LHRH) antagonist peptide, aminopeptidase targeting peptide, brain homing peptide, kidney homing peptide, heart homing peptide, gut homing peptide, integrin homing peptide, angiogenesis tumor endothelium homing peptides, ovary homing peptides, uterus homing peptides, sperm homing peptides, microglia homing peptides, synovium homing peptides, urothelium homing peptides, prostate homing peptides, lung homing peptides (e.g., RCPLSHSLICY), laminin receptor binding peptides (e.g., YIGSR), skin homing peptides, retina homing peptides, pancreas homing peptides, liver homing peptides, lymph node homing peptides, adrenal homing peptides, thyroid homing peptides, bladder homing peptides, breast homing peptides, neuroblastoma homing peptides, lymphoma homing peptides, muscle homing peptides, wound vasculature homing peptides, adipose tissue homing peptides, virus binding peptides or fusion peptides.

[0071] The term "labeling or imaging" as used herein refers to a molecule that facilitates the visualization and / or detection of the targeting molecule disclosed herein. Thus, the expression "labeling agent or imaging agent" in the context of this disclosure refers to any substance that is used as a label or enhances specific structure in any imaging technology. Thus, imaging agents include optical imaging agents, magnetic resonance imaging agents, radioisotopes and contrast agents. Imaging agents or labeling agents are well known in the art. Specific examples of imaging agents or labeling agents are gases such as sterile air, oxygen, argon, nitrogen, fluorine, perfluorocarbons, carbon dioxide, nitrogen dioxide, xenon and helium, as well as commercially available agents used in positron emission tomography (PET), computed tomography (CAT), single photon emission computed tomography, X-ray, fluoroscopy and magnetic resonance imaging (MRI). Examples of materials suitable for use as contrast agents in MRI include currently available gadolinium chelates such as diethylenetriaminepentaacetic acid (DTPA) and gadopentetate dimeglumine, as well as iron, magnesium, manganese, copper and chromium. Examples of materials useful for CAT and X-ray include iodine-based materials for intravenous administration, such as ionic monomers typified by diatrizoate and iothalamate, non-ionic monomers such as iopamidol, iohexol and ioversol, non-ionic dimers such as iotrol and iodixanol, and ionic dimers, such as ioxaglic acid. Other useful materials include insoluble salts such as barium and zinc acetate for oral use. In some molecules, the imaging agent is a dye. In some molecules, the imaging agent is a fluorescent moiety. In some molecules, the fluorescent moiety is selected from fluorescent proteins, fluorescent peptides, fluorescent dyes, fluorescent materials or combinations thereof.Examples of fluorescent dyes include, but are not limited to, xanthenes (e.g., rhodamine, rhodol, fluorescein and their derivatives), bimanes, coumarins and their derivatives (e.g., umbelliferone and aminomethylcoumarin), aromatic amines (e.g., dansyl, squaric acid dyes), benzofurans, fluorescent cyanines, indocarbocyanines, carbazoles, dicyanomethylenepyrans, polymethines, oxabenzanthrans, xanthenes, pyryliums, carbostyryls, perylenes, acridones, quinacridones, rubrenes, anthracenes, coronenes, phenanthrenes, pyrenes, butadienes, stilbenes, porphyrins, phthalocyanines, lanthanide metal chelate complexes, rare earth metal chelate complexes and derivatives of such dyes. Examples of fluorescein dyes include, but are not limited to, 5-carboxyfluorescein, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate and 6-carboxyfluorescein. Examples of rhodamine dyes include, but are not limited to, tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxyrhodol derivatives, tetramethyl, tetraethylrhodamine, diphenyldimethyl, diphenyldiethylrhodamine, dinaphthylrhodamine, rhodamine 101 sulfonyl chloride (sold under the trade name TEXAS RED®). Examples of cyanine dyes include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, IRDYE680, Alexa Fluor 750, IRDye800CW, ICG. Examples of fluorescent peptides include GFP (green fluorescent protein) or derivatives of GFP (e.g., EBFP, EBFP2, Azurite, mKalama1, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, YPet). The fluorescent label is detected by any suitable method.For example, fluorescent labels may be detected by exciting the fluorochrome with light of an appropriate wavelength and detecting the resulting fluorescence, for example, by microscopy, visual inspection, or by using electronic detectors such as photographic film, charge-coupled device (CCD), photomultiplier tubes, etc. In some molecules, imaging agents are labeled with positron-emitting isotopes (e.g., 18F) for positron emission tomography (PET), gamma-emitting isotopes (e.g., 99mTc) for single-photon emission computed tomography (SPECT), or paramagnetic molecules or nanoparticles (e.g., Gd3+ chelates or coated magnetite nanoparticles) for magnetic resonance imaging (MRI). In some molecules, imaging agents are labeled with gadolinium chelates, iron oxide particles, superparamagnetic iron oxide particles, ultrasmall paramagnetic particles, manganese chelates, or gallium-containing agents. Examples of gadolinium chelates include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA). In some molecules, the imaging agent is a near-infrared fluorophore for near-IR imaging, luciferase (firefly, bacteria or coelenterate) or other luminescent molecules for bioluminescence imaging or perfluorocarbon-filled vesicles for ultrasound. In some molecules, the imaging agent is a nuclear probe. In some molecules, the imaging agent is a SPECT or PET radionuclide probe. In some molecules, the radionuclide probe is selected from technetium chelates, copper chelates, radioactive fluoride, radioactive iodine, indium chelates. Examples of Tc chelates include, but are not limited to, HYNIC, DTPA and DOTA. In some molecules, the imaging agents contain a radioactive moiety, such as a radioisotope, such as 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 64Cu, Lu radioisotopes.

[0072] In one embodiment, optionally in combination with any of the embodiments provided above or below, at least one active agent is covalently attached to the polypeptide backbone of the cationic or anionic polymer via an amino acid side residue, i.e., C- or N-terminal group, via an amide, ester, anhydride bond, or via a linker containing one or more functional groups, including but not limited to, alkyne, azide, reactive disulfide, maleimide, hydrazide, hydrazone, Schiff base, acetal, aldehyde, carbamate, and reactive ester. In another embodiment, the covalent bond is a bioresponsive bond.

[0073] In another preferred embodiment, optionally in combination with any of the embodiments provided above or below, at least one active agent is attached to the polypeptide backbone of the cationic or anionic polymer by electrostatic interactions.

[0074] According to certain embodiments, optionally in combination with any of the embodiments provided above or below, the at least one active agent is selected from the group consisting of low molecular weight drugs, peptides, antibodies, hormones, enzymes, nucleic acids, proteins, and combinations thereof.

[0075] According to certain embodiments, optionally in combination with any of the embodiments provided above or below, the active ingredient is a nucleic acid, and thus the positively charged nanoparticles are herein named polyplexes. In certain embodiments, the positively charged nanoparticles comprise a combination of two or more nucleic acids.

[0076] The term "nucleic acid" as used herein refers to DNA or RNA. In certain embodiments, optionally in combination with any of the embodiments provided above or below, the nucleic acid is a DNA / RNA hybrid, small interfering RNA (siRNA), microRNA (miRNA), single guide RNA (sgRNA), donor DNA, self-amplifying / replicating RNA, circular RNA (oRNA), plasmid DNA (pDNA), closed linear DNA (lcDNA), small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA (aRNA), messenger RNA (mRNA), CRISPR guide RNA, antisense nucleic acid, decoy nucleic acid, aptamer and ribozyme, to name a few, including both their nucleotide sequence and any structural embodiment, such as double-stranded, single-stranded, helical, hairpin, and may include modified or unmodified bases.

[0077] When separate nucleic acids are provided, they may be all DNA molecules or all RNA molecules, or may be a mixture of DNA and RNA molecules or molecules that contain an association of DNA and RNA strands.

[0078] The nucleic acid may be a poly or oligonucleotide, such as an oligo or poly double-stranded RNA, an oligo or poly double-stranded DNA, an oligo or poly single-stranded RNA, an oligo or poly single-stranded DNA, etc. Each of the nucleotides contained in the nucleic acid may be a naturally occurring nucleotide or a chemically modified non-naturally occurring nucleotide.

[0079] The length of the nucleic acid is not particularly limited, and the nucleic acid may have a short chain in the range of 10 to 200 bases, preferably 20 to 180 bases, preferably 25 to 100 bases, preferably 30 to 50 bases, or the nucleic acid may have a relatively long chain of 200 to 20,000 bases, more preferably 250 to about 15,000 bases.

[0080] According to certain embodiments, optionally in combination with any of the embodiments provided above or below, the nucleic acid is a closed linear DNA (lcDNA), i.e. a molecule in which the double-stranded region is flanked and protected by two single-stranded loops, thereby generating a dumbbell-shaped molecule.

[0081] In a more particular embodiment, optionally in combination with any of the embodiments provided above or below, the lcDNA consists of a stem region comprising a double-stranded DNA sequence of interest covalently closed at both ends by hairpin loops, and the lcDNA comprises at least two modified nucleotides.

[0082] The term "closed linear DNA" or "lcDNA" as used herein refers to a single-stranded, covalently closed DNA molecule that forms a "dumbbell" or "dogbone" shaped structure under conditions that allow nucleotide hybridization. Thus, lcDNA is formed by a single-stranded DNA molecule, but the formation of a "dumbbell" structure by hybridization of two complementary sequences within the same molecule generates a structure consisting of a double-stranded intermediate segment flanked by two single-stranded loops. Those skilled in the art know how to generate lcDNA from open or closed double-stranded DNA using routine molecular biology techniques. For example, those skilled in the art know that lcDNA can be generated by attaching a hairpin DNA adapter to both ends of an open double-stranded DNA, for example by the action of a ligase. A "hairpin DNA adapter" refers to a single-stranded DNA that forms a stem-loop structure by hybridization of two complementary sequences, where the stem region formed is closed at one end by a single-stranded loop and open at the other end.

[0083] A "modified nucleotide" is any nucleotide (e.g., adenosine, guanosine, cytidine, uracil, and thymidine) that has been chemically modified by modification of the base, sugar, or phosphate group, or that has incorporated a non-natural moiety into its structure. Thus, modified nucleotides can be naturally or non-naturally occurring, depending on the modification.

[0084] According to one embodiment, optionally in combination with any of the embodiments provided above or below, the - / + ratio of the positively charged nanoparticle or the positively charged protein and at least one anionic shielding copolymer comprising substructure I, II or III according to the invention, defined as [total number of negative charges (-) caused by polyanionic blocks in the shielding polymer] / [total negative charges (-) caused by cationic groups in the block copolymer], is in the range of 0 to 2, preferably 0.1 to 2, more preferably 0.3 to 2, more preferably 0.5 to 1.75, more preferably 0.75 to 1.5. The - / + ratio refers to the ratio of the molar concentration of negative charges (-) caused by anionic blocks in the mixed solution to the molar concentration of positive charges (+) caused by protonatable amino groups from the side chains of the cationic polymer or the positively charged protein.

[0085] In one embodiment, optionally in combination with any of the embodiments provided above or below, the polymer conjugate may contain at least an active agent in an amount ranging from 1 to 70% w / w, based on the weight ratio of active agent to the polymer conjugate. In a preferred embodiment, the range is 1 to 60% w / w. In an even more preferred embodiment, the polymer conjugate contains an active agent in an amount ranging from 2 to 50% w / w. Other preferred ranges are 2 to 40% w / w, 3 to 35% w / w, and 3 to 30% w / w.

[0086] In a more specific embodiment, optionally in combination with any of the embodiments provided above or below, the protein-based complex may contain an amount of protein in the range of 5-99% w / w based on the mass ratio of active agent:protein-based complex. In a preferred embodiment, this range is 15-98% w / w. In an even more preferred embodiment, the protein-based complex contains an amount of protein in the range of 30-95% w / w. Other preferred ranges are 40-92% w / w, 50-90% w / w and 60-90% w / w.

[0087] The polymer or protein-based conjugates of the present disclosure constitute useful tools for therapeutic or diagnostic indications, where at least one anionic copolymer comprising substructure I, II or III acts as a protective shield for the positively charged nanoparticles carrying the active ingredient or the positively charged protein, thereby improving certain properties such as improved circulation time, safety or toxicological profile or release profile under physiological conditions, as well as, in the case of polyplexes, improved transfection efficiency into the desired cells.

[0088] The pharmaceutical, diagnostic or therapeutic compositions of the present disclosure may be prepared in solid form or as an aqueous suspension in a pharma- ceutically acceptable diluent. These preparations may be administered by any suitable route of administration, and for this reason, the preparations are formulated in a suitable pharmaceutical form for the selected route of administration. In more specific embodiments, optionally in combination with any of the embodiments provided above or below, administration is by oral, topical, rectal or parenteral routes (including subcutaneous, intraperitoneal, intradermal, intramuscular, intravenous routes, etc.). The pharmaceutical, diagnostic or therapeutic compositions may be applied to various kinds of animals, including humans. Various conditions, such as dosage, number of administrations and duration of administration, may be determined according to the type of animal and its condition, as necessary.

[0089] The pharmaceutical, diagnostic or therapeutic composition may be prepared according to a general method by selecting and using, as necessary, agents commonly used in drug manufacturing, such as excipients, fillers, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, disinfectants, flavoring agents, soothing agents, stabilizers and isotonicity agents.As the form of such pharmaceutical composition, intravenous injection (including drip infusion) is generally adopted.For example, the pharmaceutical composition of the present invention is provided in the form of a single-dose ampoule or a multi-dose container.

[0090] When the method refers to diagnosis, this embodiment can also be devised as a method for diagnosing a disease in an isolated sample of a subject, the method comprising administering to said subject an effective amount of either the polymer conjugate or a pharmaceutical composition having one or more imaging agents as defined above to the isolated sample of the subject. The detection of these imaging agents may be performed by well-known techniques, such as imaging diagnostic techniques. Examples of imaging diagnostic techniques suitable for the present disclosure include, but are not limited to, ultrasound imaging, magnetic resonance imaging (MRI), fluoroscopy, X-ray, positron emission tomography (PET), single photon emission computed tomography (SPECT), fluorescence microscopy and in vivo fluorescence.

[0091] Thus, the present disclosure also refers to the use of the polymer conjugates or pharmaceutical compositions of the present disclosure as bioimaging tools, particularly for tracking the internalization and delivery of active or imaging agents.

[0092] "Bioimaging tools" according to the present invention are to be understood as reagents used in imaging techniques used in biology to track cells or certain compartments of a particular tissue. Examples of bioimaging tools include chemiluminescent compounds, fluorescent and phosphorescent compounds, X-ray or alpha, beta or gamma emitting compounds, etc.

[0093] A further aspect of the present disclosure relates to the use of the polymer complexes defined herein as non-viral vectors useful for transfection of host eukaryotic cells in culture, in vivo or ex vivo, monocytic parasites and bacteria, including gene editing using CRISPR / Cas9 methodology, and commonly used for biomedical applications such as vaccines or gene therapy.

[0094] A further aspect of the present disclosure relates to the use of a protein-based conjugate as defined herein as a carrier commonly used for protein-based treatments such as vaccines and protein replacement therapy.

[0095] In certain embodiments, optionally in combination with any of the embodiments provided above or below, the present invention refers to the use of the polymer complexes defined herein as transfection reagents for delivering active agents (preferably nucleic acids, regardless of size and structure, i.e. circular and linear nucleic acids) to target cells in vivo, in vitro or ex vivo. In certain embodiments, optionally in combination with any of the embodiments provided above or below, the active agent is selected from the group consisting of low molecular weight drugs, peptides, proteins, antibodies, nucleic acids, aptamers and combinations thereof.

[0096] The transfection reagent is also useful for co-transfecting two or more active agents (e.g., two or more nucleic acids) at the same time. Transfection compositions (such as kits) as well as methods of using the transfection reagent to deliver nucleic acids to target cells are also within the scope of the present invention. Further embodiments will become apparent upon review of this disclosure.

[0097] The present invention also relates to methods for the transfer of active agents in vitro, ex vivo, and in vivo that involve the use of the polymer conjugates disclosed herein.

[0098] The present invention also provides the composition for use as a pharmaceutical composition to induce a modulatory effect on the expression of one or more target proteins involved or associated with a genetic or complex genetic disease, immune disease, cancer, viral infection or tumor in various tissues / organs.

[0099] The present invention also relates to the in vitro or ex vivo use of the composition according to the present invention in the production of biological products, in particular biological products coding recombinant protein, peptide or antibody, or in the production of recombinant viruses such as adeno-associated virus (AAV), lentivirus (LV), adenovirus, oncolytic virus or baculovirus or viral or virus-like particles, said composition comprising polymer complex as defined herein and comprising at least one nucleic acid molecule for transfection.The term "biological product" as used herein refers to protein or nucleic acid or combination thereof, living entities such as cells or viruses, cell compartments, organoids and tissues.

[0100] The present invention also relates to the in vitro or ex vivo use of the polymer complexes according to the invention for genome engineering, cell reprogramming, cell differentiation or gene editing.

[0101] Compositions for transfecting cells include a polymer conjugate as defined herein and an acceptable excipient, buffer, cell culture medium or transfection medium.

[0102] The present invention also relates to a composition as defined herein for use as a therapeutic or prophylactic vaccine against viral infection or a therapeutic vaccine against cancer.Generally, in this embodiment, the vaccine is delivered by direct administration, such as systemic, intramuscular, intradermal, intraperitoneal, intratumoral, oral, topical or subcutaneous administration, in which the composition is mixed with a pharma- ceutically acceptable vehicle.In other words, the vaccine may be directly injected into the body, particularly into a human individual, to induce cellular and / or humoral responses.

[0103] Cellular targeting is achieved by different mechanisms and depends on the nature and characteristics of the transfection reagent, the method or protocol, the composition or formulation and the route of administration.

[0104] In a more particular embodiment, optionally in combination with any of the embodiments provided above or below, the present invention refers to a polymer conjugate for use in the prevention and / or treatment of different diseases, such as, inter alia, neurodegenerative diseases, neurological diseases, cancer, infectious diseases, ageing-related diseases, neuroinflammation, demyelinating diseases, multiple sclerosis, ischemic diseases, immune deficiencies, inflammatory diseases, rare diseases, depending on the active agent it carries.

[0105] The compounds described in this disclosure, their pharma- ceutically acceptable salts and solvates, and pharmaceutical compositions containing them, may be used with other additional drugs to provide a combination therapy, which may be part of the same pharmaceutical composition or, alternatively, may be provided in the form of a separate composition for simultaneous or non-simultaneous administration with the pharmaceutical composition containing the compound having formula (I), its pharma- ceutically acceptable salts, stereoisomers, or solvates.

[0106] In one embodiment, the present invention provides a nucleic acid delivery kit comprising the polymer complex of the present invention. This kit may be preferably used in gene therapy for various types of target cells, such as cancer cells. In the kit of the present invention, the storage state of the block copolymer is not particularly limited. Taking into account their stability (preservative properties), usefulness, etc., the block copolymer may be stored in the form of a solution, powder, etc.

[0107] The kit of the present invention may contain other components as well as the above-mentioned polymer complex.Examples of such other components include various kinds of buffers, various kinds of nucleic acids (plasmid DNA, antisense oligo DNA, siRNA, etc.) that are introduced into cells, buffers used for dissolution, various kinds of proteins and instructions (manuals for use).

[0108] All terms used herein in this application are to be understood in their ordinary meanings known in the art unless otherwise specified. Other more specific definitions for certain terms used in this application are set forth below, which are intended to be applied uniformly throughout this specification and claims, unless a definition expressly set forth otherwise gives a broader definition.

[0109] The term "disorder" as used herein is intended to be generally synonymous with, and is used interchangeably with, the terms "disease," "syndrome," and "condition (as in medical condition)," in that all reflect an abnormal condition of the human or animal body or one of its parts that impairs normal functioning, and is typically made clear by distinguishing between signs and symptoms.

[0110] As used herein, the terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", "physiologically acceptable carrier" or "physiologically acceptable excipient" refer to a pharma- ceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each component must be "pharmaceutically acceptable" in the sense of being compatible with the other components of a pharmaceutical formulation, suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0111] The terms "cosmetically acceptable carrier" or "dermatologically acceptable carrier", as used interchangeably herein, refer to an excipient or carrier that is suitable for use in contact with human skin without causing excessive toxicity, incompatibility, instability, or allergic reaction.

[0112] The term "therapeutically acceptable" refers to compounds that are suitable for use in contact with the tissues of a patient without excessive toxicity, irritation, allergic response, or immunogenicity, commensurate with a reasonable benefit / risk ratio, and that are effective for their intended use.

[0113] As used herein, the terms "treat," "treating," and "treatment" refer to ameliorating the symptoms associated with a disease or disorder, including preventing or delaying the onset of a symptom of a disease or disorder and / or reducing the severity or frequency of a symptom of a disease or disorder.

[0114] The term "protecting group" as used herein refers to a group of atoms that, when attached to a reactive group in a molecular shield, reduces or prevents reactivity.Protective groups for carboxyl and amino groups are well known in the art.Suitable amine protecting groups known in the art can be used without limitation, examples of which include acyl-based groups, carbamate-based groups, imide-based groups and sulfonamide-based groups. Among them, methyloxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, t-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (FMOC), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl group (Troc), benzoyl (Bz), benzyl (Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trimethylsilylethyloxycarbonyl (Teoc), benzhydryl, triphenylmethyl (trityl), (4-methoxyphenyl)diphenylmethyl (MMT), dimethoxytrityl (DMT) and diphenylphosphino group are preferred. The introduction of the protecting group may be carried out under basic conditions by adding a protecting agent corresponding to each protecting group to the reaction solution after the reduction reaction.

[0115] In the present invention, the "subject" may be a mammal, including a human. The subject may be a healthy subject or a subject suffering from some disease. In the present invention, "treatment" refers to the cure, prevention or induction of remission of a disease or disorder, or the reduction of the rate of progression of a disease or disorder. The treatment may be achieved by administering a therapeutically effective amount of a pharmaceutical composition.

[0116] Throughout the specification and claims, the word "comprises" and variations of that word are not intended to exclude other technical features, additives, ingredients, or steps. Furthermore, the word "comprises" encompasses the case of "consisting of". Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon consideration of the specification or may be learned by practice of the present invention. The following examples and drawings are given by way of illustration and are not intended to limit the invention. Reference signs associated with the drawings and placed in parentheses in the claims are merely intended to enhance the comprehension of the claims and should not be construed as limiting the scope of the claims. Furthermore, the present invention encompasses all possible combinations of the specific preferred embodiments described herein. EXAMPLES

[0117] Only a few examples have been disclosed herein, and other alternatives, modifications, uses and / or equivalents thereof are possible. Moreover, all possible combinations of the examples described are also encompassed. Thus, the scope of the disclosure should not be limited by the specific examples, but should be determined only by a fair interpretation of the following claims.

[0118] General procedure for the preparation of anionic copolymers Anionic shielding copolymers were prepared according to the general preparation method described below. The block copolymer was obtained by ROP (ring-opening polymerization). In the first step, methionine N-carboxyanhydride (NCA) and alanine-NCA were polymerized to obtain the copolymer, then the second block (glutamic acid (OtBu)NCA) was added. The oxidation of methionine is then carried out by using tert-butyl hydroperoxide (TBHP). The last step consists of deprotection of glutamic acid (OtBu) under acidic conditions.

[0119] Example 1: Synthesis of the shielding block copolymer (PMet(O)-co-PAla)-b-PGluOtBu 1.1. General procedure for polymerization of (PMet-co-PAla)-b-PGluOtBu [ka] The block copolymer was obtained by ROP (ring-opening polymerization). Methionine N-carboxyanhydride (NCA) and alanine-NCA were added to a Schlenk flask equipped with a stir bar and a stopper. After three cycles of vacuum / N2, the mixture was dissolved in anhydrous THF. The initiator (i-propylamine) was then diluted in THF (2 mL) and added to the reaction mixture, which was stirred at room temperature for three days. Once NCA consumption was confirmed by IR, the corresponding second blocked amino acid-NCA (glutamic acid (OtBu)NCA) was added to the reaction mixture dissolved in anhydrous THF. The mixture was stirred at room temperature for two days. Complete conversion of the monomer was detected by IR. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was lyophilized and the block copolymer was isolated as a white solid. Yield: 80-98%.

[0120] 1.2.Oxidation of methionine [ka] The oxidation reaction was carried out by suspending the block copolymer in 16 equivalents of 80% TBHP (tert-butyl hydroperoxide) and 0.2 equivalents of CSA (camphorsulfonic acid) for each Met unit in MiliQ water. The oxidation was quenched with Na2S2O3 (0.1 M) and the product was purified by TFF (tangential flow filtration) and lyophilized.

[0121] 1.3. General procedure for the Glu(OtBu) deprotection step [ka] The PMet(O)-co-PAla-b-PGluOtBu block copolymer was dissolved in trifluoroacetic acid at 0° C. (100 mg / mL) and the mixture was stirred at 5° C. for 1 h. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried in vacuum. The block copolymer was isolated as a white solid. Yield: 70-95%. [V1~V5] 1 H NMR(D2O): δ1.50(d,CH3Ala),1.90-2.49(m,2CH2Glu+CH2Met(O)),2.85-3.20(m,CH2Met(O)),4.25-4.65(m,CHMet(O)+CHAla+CHGlu). [Table 1] a Determined by NMR. b Determined by SEC-MALS. c Determined by SEC column calibration. V1=iPr-P[Met(O)138-co-Ala19]-b-PGlu(ONa)65 V2=iPr-P[Met(O)167-co-Ala25]-b-PGlu(ONa)82 V3=iPr-P[Met(O)154-co-Ala25]-b-PGlu(ONa)84 V4=iPr-P[Met(O)139-co-Ala20]-b-PGlu(ONa)98 V5=iPr-P[Met(O)167-co-Ala25]-b-PGlu(ONa)80 The DP numbers given here are subject to reasonable uncertainties within ±20%.

[0122] Example 2: Synthesis and description of polycationic carriers To demonstrate the stabilizing ability and improved transfection characteristics conferred to polyplexes by the shielding polymer, the universal standard jetPEI® (Polyplus-transfection, Illkirch, France) (see Polyplus:101-10N) was used. JetPEI® is a powerful reagent that ensures robust, efficient and reproducible DNA transfection into mammalian cells with low toxicity. jetPEI® is mainly composed of linear polyethyleneimine manufactured by Polyplus-transfection. jetPEI® is provided as a 7.5 mM sterile, non-pyrogenic aqueous solution (expressed as the concentration of nitrogen residual). In addition, other commercially available cationic polymers were tested, namely nbu-poly-L-ornithine (PLO) hydrobromide (supplied by Polypeptide Therapeutic Solutions, catalog batch number: CM-CC1004-01-42B), chitosan (CHI) (purchased from Kytozyme (15-20 kDa)), linear poly-L-lysine (nbu-PLL) (supplied by Polypeptide Therapeutic Solutions, batch number: CM-CC1030-02-08A) and star-shaped poly-L-lysine (St-PLL) (supplied by Polypeptide Therapeutic Solutions, batch number: PI03-01-166C). To prove the stabilization of other polycations of very different structure and composition, different polycationic NVVs based on star-shaped polyamino acids were also synthesized and their polyplexes were also stabilized and assayed. The following examples describe the preparation of the above polycationic compounds used to complex and deliver genetic material.

[0123] Example 2.1: Preparation of Compound N1 [ka] Generally speaking, to synthesize the compound of formula (N1) according to the present disclosure, a three-arm star initiator was first obtained in two or three steps. Then, such an initiator was used to polymerize y-benzyl-L-aspartate NCA and L-phenylalanine NCA to obtain a protected star random copolymer benzyl (St-PAsp(Bz)-co-PPhe). The benzyl group was removed by aminolysis reaction to obtain the corresponding Star-PAsp-oligoamine-co-PPhe.

[0124] Scheme 1 shows a specific example of the polymerization and aminolysis steps. [ka]

[0125] 2.1.A. Synthesis of 3-arm star initiators Synthetic routes to 3-arm star initiators are described below.

[0126] 2.1.A.1. Trifluoroacetate salt of N,N,N-tris(2-((2-aminoethyl)disulfanyl)ethyl)benzene-1,3,5-tricarboxamide (St-SS-initiator) (5) Following the general procedure disclosed in Scheme 2, the trifluoroacetate salt of N,N,N-tris(2-((2-aminoethyl)disulfanyl)ethyl)benzene-1,3,5-tricarboxamide (St-SS-initiator) (5) was synthesized. [ka]

[0127] The synthesis of the trimer amine initiator began with a coupling reaction followed by amine deprotection.

[0128] Step (a): Synthesis of ((((benzenetricarbonyltris-(azanediyl))tris(ethane-2,1-diyl))tris(disulfanediyl))tris(ethane-2,1-diyl))-tri-tert-butyltricarbamate [ka] N-(tert-Butyloxycarbonyl)cystamine (7.99, 27 mmol, 3.3 equiv.) was weighed into a flame-dried two-necked round-bottom flask and dissolved in 56 mL of anhydrous THF. Freshly distilled DIPEA (4.75 mL, 27 mmol, 3.3 equiv.) was added and stirred at room temperature for 15 min. 1,3,5-Benzenetricarbonyl trichloride (2.25 g, 8.3 mmol, 1 equiv.) was weighed into a flame-dried two-necked round-bottom flask and dissolved in 28 mL of anhydrous THF. The trichloride solution was added slowly via syringe to the N-(tert-Butyloxycarbonyl)cystamine mixture. The progress of the reaction was monitored by thin layer chromatography (TLC). After 4 h, the solvent was evaporated under vacuum and the residue was dissolved in ethyl acetate. The organic layer was washed successively with Milli-Q water, 1 M hydrochloric acid and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate and concentrated in vacuo to give tri-tert-butyl ((((benzenetricarbonyltris-(azanediyl))tris(ethane-2,1-diyl))tris-(disulfanediyl))tris(ethane-2,1-diyl))-tricarbamate as a white foam (7.5 g, η = 98%). 1 H NMR(CDCl3):δ=1.39(brs,27H,-C(CH3)3),2.84(t,J=6.26Hz,6H,CH2),2.96(t,J=6.84Hz,6 H.CH2),3.46(m,6H,CH2),3.79(m,6H,CH2),5.18(brs,3H,-NHBoc),7.39(brs,3H,arylCH).

[0129] Step (b): Synthesis of the trifluoroacetate salt of N,N,N-tris(2-((2-aminoethyl)disulfanyl)ethyl)benzene-1,3,5-tricarboxamide (St-SS-initiator) (5) [ka] 7.5 g (8.19 mmol) of initiator (5) was dissolved in anhydrous dichloromethane (180 mL) and 90 mL of TFA was added. The reaction was stirred under nitrogen for 60 min and monitored by TLC for completion. The solvent was evaporated under vacuum. The TFA salt of initiator (5) (7 g, 7.31 mmol) was obtained in quantitative yield and dried under vacuum. 1 H NMR (D2O): δ=2.86(m,12H),3.25(t,J=6.49Hz,8H),3.60(t,J=6.85Hz,8H),8.02(brs,3H,arylCH).

[0130] 2.1.B. Synthesis of Star-Shaped PAsp(Bz)(6) Copolymers Containing Hydrophobic Segments [ka] To synthesize copolymers bearing hydrophobic residues, polymerization was carried out via a ring-opening polymerization mechanism using the trifluoroacetate salt of N,N,N-tris(2-((2-aminoethyl)disulfanyl)ethyl)-benzene-1,3,5-tricarboxamide as an initiator.

[0131] General procedure for the synthesis of St-SS-PAsp(Bz)(45)-co-PPhe(5)(N1) β-Benzyl-L-aspartate-N-carboxyanhydride (3.5 g, 14.15 mmol) and L-phenylalanine-N-carboxyanhydride (1.57 mmol) were added to a Schlenk tube equipped with a stir bar and stopper, purged with three vacuum / N2 cycles, and dissolved in a mixture of anhydrous chloroform (100 mL) and DMF (6 mL). The star-shaped initiator was then dissolved in DMF (4 mL) and added to the reaction mixture. The mixture was stirred at 50° C. for 16 h. Upon completion, the reaction mixture became clear and complete conversion of the monomer could be detected by IR. The reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) and dried under vacuum. The copolymer was isolated as a white solid. Yield: 70-80%. 1H NMR (TFA): δ = 2.99 (s, 2H, CH), 3.94 (brs, 1H, CH), 4.93 (s, 1H, CH), 5.15 (m, 2H, benzyl CH), 7.20 (s, 5H, aryl CH), 8.42 (s, aryl CH).

[0132] The ratio of repeat units introduced was adjusted by varying the mixture ratio of the corresponding monomer units that could be reacted. In this precursor, the hydrophobic residues were 1 H-NMR is consistent with polyaspartic acid protecting groups. This system will be analyzed following aminolysis (Example 5.1.C below).

[0133] 2.1.C. Synthesis of amphiphilic polyaspartamide derivative St-SS-PAspDET-co-PR18 (10) Polyamino acids were prepared by the simultaneous aminolysis reaction of PBLA and DET, as shown in the synthetic route below. [ka]

[0134] As an example, we describe herein a synthetic method in which R18 represents a phenylalanine group. Copolymer of St-S-SPAsp(Bz)45-co-PPhe (5) (500 mg copolymer, 470 mg PBLA, DP: 45) was dissolved in NMP (10 mL) and cooled to 4°C. The resulting copolymer solution was added dropwise to a mixture of DET (12 mL, 50 equivalents per unit of PAsp(Bz)) and the solution was stirred for 4 hours at 4°C under nitrogen atmosphere. After this time, the reaction mixture was added dropwise into cold HCl (6 M) for neutralization (pH 3.5). The polymer product was purified by centrifuge-assisted ultrafiltration. After filtration, the remaining aqueous polymer solution was freeze-dried to obtain the final product. Yield: 70-80%. 1H NMR (DO) [R18 = Phe side chain]: δ = 2.91 (brs, 2H, CH2), 3.84-3.18 (m, 2H, CH2), 7.34 (brs, 5H, aryl CH of Phe), 8.33 (s, aryl CH).

[0135] Table 2 refers to the amphiphilic copolymer St-SS-PAspDET-co-R18 according to formula (N1). [Table 2] a Determined by NMR. b Determined by SEC. Mn and DP refer to number average molar mass and degree of polymerization, respectively. D stands for polydispersity determined by SEC-MALS software analysis.

[0136] Example 2.2: Preparation of Compound N3 Similar to the synthesis of St-SS-PAspDET-co-R1 (N1), the same experimental procedure is used to generate St-SS-PAspDET-co-PAsp imidazole amine (N3). [ka] Following the experimental procedure described in the previous section, the synthesis of St-SS-poly(β-benzyl-L-aspartate) (star-shaped PAsp(Bz)) (7) was carried out. Yield: 70-90%. 1 H NMR (TFA): δ = 2.92 (m, 2H, CH2), 4.85 (s, 1H, CH), 5.05 (m, 2H, benzyl CH2), 7.13 (s, 5H, aryl CH), 8.38 (s, aryl CH). [Table 3] a Determined by NMR. b Determined by SEC. Mn and DP refer to number average molar mass and degree of polymerization, respectively. D stands for polydispersity determined by SEC-MALS software analysis.

[0137] The aminolysis reaction of poly(β-benzyl-L-aspartate) (7) to generate St-PAspDET-co-PAsp imidazole amine (N3) was carried out following the same experimental procedure as for N1, but in this case using two amines, namely diethylenetriamine and 1-(3-aminopropyl)imidazole. 1 H NMR [St-PAspDET / imidazole amine] (DO): δ = 2.14 (brs, 2H, CH2), 2.87 (brs, 2H, CH2), 3.22 (m, 2H, CH2), 4.30 (brs, 2H, CH2), 7.52 (s, imidazole CH), 7.57 (s, imidazole CH), 8.34 (s, aryl CH), 8.78 (s, imidazole CH). [Table 4] a Determined by NMR. b Determined by SEC. Mn and DP refer to number average molar mass and degree of polymerization, respectively. D stands for polydispersity determined by SEC-MALS software analysis.

[0138] Example 3: Polyplex Formulation Polyplex formulations are called "PX Nn_比1_遮蔽用ポリマー_比2 nuc " and here we will name it "N n" corresponds to the nomenclature of the polycationic compounds provided herein (i.e., N1 is Star-PAsp-oligoamine-co-PPhe cationic polymer described in Example 2.1 above, N2 is the standard jetPEI®, N3 is St-SS-PAspDET-co-PAsp imidazole amine cationic polymer described in Example 2.2 above), N4 is star-shaped poly-L-lysine (St-PLL Curapath catalog, product number: 1075), N5 is linear poly-L-lysine (nbu-PLL, Curapath catalog, product number: 1019), N6 is nbu-poly-L-ornithine (PLO Curapath catalog, product number: 1018), N7 is chitosan (CHI, taken from Kitozyme catalog, chitosan 15-20 kDa) was used to form the polyplex), "ratio 1" refers to the N / P ratio of cationic polymer:genetic material, "shielding polymer" refers to the polyanionic shielding diblock copolymer (Vn), "ratio 2" refers to the + / - ratio of cationic polymer:shielding (anionic) polymer, and "nuc" refers to the type of nucleic acid, i.e., pDNA, mRNA or lcDNA.

[0139] In the following examples, pDNA containing 6233 bp of expressed luciferase (purchased from PlasmidFactory, see PF461 (pCMV-luc)), commercially available mRNA (luc) (purchased from Trilink Biotechnologies), commercially available pDNA (GFP) (obtained from PlasmidFactory LLC) and lcDNA (SEQ ID NO: 1) (obtained according to standard molecular biology methods such as those disclosed in Heinrich, M. et al., "Linear closed mini DNA generated by the prokaryotic cleaving-joining enzyme TelN is functional in mammalian cells," J Mol Med, 2002, 80th edition, pp. 648-654) were used.

[0140] The sequence of the lcDNA according to SEQ ID NO:1 in this example is the sequence in Table 5. [Table 5] JPEG2024542574000017.jpg244159JPEG2024542574000018.jpg242159JPEG2024542574000019.jpg226159

[0141] 3.1. Polyplex Formulation Procedure 1 To investigate the stability, size, toxicity and transfection ability, shielded polyplex formulations were prepared in situ (mixing in a pipette) as follows.

[0142] The desired amount of pDNA, mRNA or lcDNA and the calculated amount of cationic polymer with the indicated charge ratio (+ / -) or amine:phosphate ratio (N / P) were diluted in PBS (pH 7.4) in a separate tube. Only protonatable nitrogens, not amide nitrogens, were considered in the calculation of + / - and N / P ratios. Prior to polyplex formation, the corresponding amount of shielding polymer was added to the nucleic acid tube and mixed. For the formation of shielded polyplexes, the cationic polymer solution and the genetic material + shielding polymer solution were mixed by rapidly pipetting up and down (10 times) and incubated at room temperature for 20 min. The formed polyplexes were then characterized by DLS to determine their size.

[0143] As a specific example, shielded polyplex PX loaded with 20 μg of pDNA N1_8_V1_1 pDNA (Final polyplex volume of 200 μl) is shown. Other polyplex formulations were performed in a similar manner. The amount of shielding anionic polymer according to the present invention is calculated as follows: once the amount of amine required for the NP8 ratio is established, divide it by 2, half of which is used for polymer-DNA interaction, and the other half is compared to the required shielding anionic polymer NP ratio (NP1).

[0144] First, a 10 mg / ml stock solution of the shielding polymer and a 4 mg / ml stock solution of the polycationic polymer were prepared. The experimental procedure was as follows. 1.80 μl of PBS is added into the Eppendorf tube. Then 20 μl of pDNA from a 1 mg / ml stock is diluted and 15.8 μl of shielding polymer from a 10 mg / ml stock is added (final volume of 115.8 μl). 2. Add 18.2 μl of the polycationic polymer solution (stock 4 mg / ml) to the pDNA-shielding polymer solution and complete with PBS to a final volume of 200 μl (66 μl PBS). 3. Incubate at room temperature for 20 minutes. 4. The polyplex is ready for use.

[0145] The samples formulated in this first method were prepared in a similar manner for their in-vitro testing. After 24 hours of incubation, toxicity and transfection efficiency were evaluated. The ratios investigated for each polymer were N / P 8, 15 or 30. As a positive control for transfection, jetPEI® (Polyplus-transfection, Illkirch, France) (see Polyplus:101-10N) was used at a nitrogen:phosphorus ratio (NP5). Cell transfections were performed with jetPEI® according to the manufacturer's instructions. jetPEI® is mainly composed of linear polyethyleneimine produced by Polyplus-transfection. jetPEI® is provided as a 7.5 mM sterile and non-pyrogenic aqueous solution (expressed as the concentration of the nitrogen residual).

[0146] Example 4: Size and stability of shielded polyplexes Polyplex stability is a major aspect in developing efficient therapeutics. The assurance of mid- to long-term stability of the pharmaceutical formulation will be tracked by a panel of assays to mimic the physiological conditions encountered by the drug along the route of administration required to be stable during circulation to the target site of action. It is well known that polyplexes exhibiting positive surface charge undergo salt-induced aggregation when administered systemically, which can lead to inaccurate cell biology evaluations and serious toxicity issues. Initial stability studies are currently under development during this project and are aimed at monitoring polyplex particle properties (size).

[0147] Size measurements of stabilized polyplexes formed with different N / P ratios of pDNA, mRNA or lcDNA, different polycations and shielding polymers were performed using a Malvern Zetasizer NanoZS instrument equipped with a 532 nm laser at a fixed scattering angle of 173. 20 μl samples were measured using quartz glass high-performance cuvettes (Hellma Analytics). Size distribution was determined by measuring n>3 (diameter: nm). For stability measurements, polyplexes were kept in a refrigerator (2-8 °C) during the experiment and the stability of polyplexes was measured at different times.

[0148] 4.1. Stabilization of polyplexes formed by N1 and V1 and pDNA as cargo The stability and formation of N1 polyplexes at different times according to polyplex formulation procedure 1 (as reported above) was investigated using different NP ratios (8 and 15) and shielding polymer V1 with different - / + charge ratios in PBS (pH 7.4). Different amounts of genetic material were also used for this experiment (shown in Table 3). The final polyplex solution (200 μl) was left to stabilize for 20 min before measuring the size by DLS (Malvern Panalytical, Spain). The polyplexes were kept in a refrigerator during the experiment and the stability of the polyplexes was measured at different times.

[0149] As shown in Table 6, the presence of the shielding polymer provided improved stability to the polyplexes for up to at least several days in solution, maintaining a consistent size over extended periods of time and avoiding aggregation. [Table 6] D(n) represents the hydrodynamic diameter measured by DLS, and N / A represents the inability to measure due to the presence of aggregation.

[0150] As can be observed in this table, the size of the polyplexes is determined by the mass of genetic material and the ratio of shielding polymers present in the final formulation. Those polyplexes formulated without a shielding polymer (i.e., PX N1_8_V1_0 pDNA ) formed large aggregates that could not be measured by DLS techniques.

[0151] 4.2. Stabilization of polyplexes formed by N2 and V1 and pDNA as cargo As shown in Table 7, the presence of the shielding polymer provided improved stability to the polyplexes formulated by the procedure set forth in Example 4.1 in solution for up to at least several days, maintaining a consistent size for extended periods of time and avoiding aggregation. [Table 7] D(n) represents the hydrodynamic diameter measured by DLS, and N / A represents the inability to measure due to the presence of aggregation.

[0152] 4.3. Stabilization of Polyplexes Formed by N1 or N3 and V1 and mRNA as Cargo Polyplex stability in PBS (pH 7.4) with NP=15 with mRNA was investigated at different times using shielding polymer V1 + / -=0.5, 1, 2. The final polyplex solution was left to stabilize for 20 min before measuring the size by DLS (Malvern Panalytical, Spain). The polyplexes were kept in a refrigerator during the experiment and the stability of the polyplexes was measured at different times.

[0153] These results demonstrate that N1 and N3 complexed polymers at NP ratio of 20 are stabilized by V1, whatever the + / - ratio used for 24 hours polyplex stabilization, using mRNA as genetic material. Notably, the stability period extends by at least 5 days with increasing + / - ratio. This phenomenon is related to the fact that increasing the concentration of shielding improves the stabilization properties. As shown in Table 8, the presence of shielding polymer enhances the stabilization of polyplexes. [Table 8] D(n) represents the hydrodynamic diameter measured by DLS, and N / A represents the inability to measure due to the presence of aggregation.

[0154] 4.4. Stabilization of polyplexes formed by N1 or N3 and V1 and lcDNA as cargo Polyplex stability in PBS (pH 7.4) with lcDNA using NP=20 was investigated at different times using shielding polymer V1 + / -=0.5, 1, 2. The final polyplex solution was left to stabilize for 20 min before size measurement by Stunner (Unchained Labs, Belgium). Polyplexes were kept in a refrigerator during the experiment and polyplex stability was measured at different times.

[0155] Note that the use of different types of genetic material yields similar results to those previously described in Table 9, with V1+ / -1 and 2 yielding stable polyplexes for at least 7 days. As shown in Table 9, the presence of the shielding polymer enhances polyplex stabilization when lcDNA is used. [Table 9] Z-ave represents the hydrodynamic diameter measured by Stunner, N / A represents the inability to measure due to the presence of aggregation.

[0156] 4.5. Stabilization of polyplexes formed by N4, N5, N6 or N7 and V1 and pDNA as cargo Polyplex stability in PBS (pH 7.4) with NP=15 with pDNA and different complexing polymers was investigated with shielding polymer V1 at different times + / -=0.5, 1, 2. The final polyplex solution was left to stabilize for 20 min before measuring the size by DLS (Malvern Panalytical, Spain). The polyplexes were kept in a refrigerator during the experiment and the stability of the polyplexes was measured at different times.

[0157] As shown in Table 10, V1 can also be used to stabilize polyplexes made with different chemistries. These systems were stable for at least 1 day when PBS was used for their formulation. Furthermore, some of these polymers, N6 nbu-poly-L-ornithine (PLO) hydrobromide and N7 CHI, gave stable polyplexes for 5 days when they were prepared in acetate buffer. The use of acetate buffer is required to promote their complete protonation of their residues or their limited solubility at neutral pH. Linear N5 poly-L-lysine and N4 star poly-L-lysine were also tested. [Table 10] D(n) represents the hydrodynamic diameter measured by DLS, N / A represents not measurable due to the presence of aggregation, and the Ac superscript indicates formulation in acetate buffer (pH=5).

[0158] Example 5: Complex formation / disassembly experiments Furthermore, electrophoretic gels were used as a first screening method to evaluate the efficiency of complex formation and the possible presence of free pDNA in polyplexes. To perform the electrophoresis, an E-gel Power Snap electrophoresis device and an E-Gel Power snap camera (Invitrogen) were used. 1.2% agarose gels prepared with SYBR safe DNA marker (E-Gel® 1.2% with SYBR safe, Invitrogen) were used. The complex formation efficiency of polyplexes (20 μl) at different NPs and different - / + shielding polymer ratios was evaluated, as was the degradation of polyplexes in the presence of low (0.075 IU / ml) and high (200 IU / ml) heparin concentrations (PanReacAppliChem, Spain). For low concentrations, 0.1 μl of 15 IU / ml heparin solution was added to 20 μl of already formed polyplexes, and for high concentrations, 0.8 μl of 5000 IU / ml heparin solution was added to 20 μl of polyplexes. Once the gel (20 μl / well) is loaded, the instrument protocol is selected according to the type of gel used (in our case it was a protocol of about 40 minutes, but the time can be modified according to the sample). [Table 11]

[0159] In all cases, no free pDNA is observed for the different NPs or at low concentrations of heparin, but at high concentrations of heparin, a free pDNA signal is observed due to the competition between heparin and pDNA for binding to the polymer, indicating the ability of the polymers to release their cargo (representative images of the gel can be observed in Figure 1). [Table 12]

[0160] PX N2_5_V1_1 pDNA In all cases, pDNA is degraded. PEI_5_V1_0.5 pDNA In the present study, free pDNA is observed at high concentrations of heparin (representative images of the gel can be seen in Figure 2).

[0161] Example 6A: Cell Culture HeLa cells were cultured in DMEM high glucose with Glutamax (Gibco-Thermo Fisher #61965-059) supplemented with 10% fetal bovine serum (Hyclone #SV30160.03HI, provided by GE Healthcare Europe GmbH). Transfections were performed on 96-well plates with 10000 cells / well in a final volume of 100 μl and cells were incubated for 24 h at 37°C and 5% CO2. After 24 h the medium was removed and refreshed with 90 μl complete medium. Transfection mixtures were prepared with PBS and, in the case of the positive control (JetPEI), 10 μl of each formulation was added to the cells after 20 min stabilization according to the manufacturer's guidelines (#101-10N, Polyplus Transfection). After 24 h the cells were harvested and processed.

[0162] Example 6B: ATP Evaluation for Cytotoxicity Assessment After 24 hours of incubation, the medium was aspirated and 50 μl / well of ATPLite reagent (ATPLite (PerkinElmer) #6016731) was added. Plates were incubated for 10 minutes at room temperature and in the dark. Luminescence was read spectrophotometrically using a VictorNivo (PerkinElmer) and data was expressed as cell viability with untreated control cells taken as 100%.

[0163] Example 6C: Luciferase Assay After 24 hours of incubation, 100 μl of BrightGlo Reagent (Promega #E2620) was added to each well according to the manufacturer's instructions. Luciferase activity was measured using VictorNivo (PerkinElmer) after 5 minutes of incubation at room temperature. Data was expressed as luminescence versus transfection percentage versus transfection positivity.

[0164] Example 6D: Biological activity of polyplexes formed by N1 and V1 in HeLa cells The transfection efficiency and cell viability of polyplexes formed by N1 and V1 in HeLa cells are reported in the following table. Transfection data are expressed as a percentage (%) of the positive control. jetPEI® is a 100% positive control after 24 hours of treatment, and when comparing cell viability with untreated (NT) cells, the ATP content reading for NT cells is equal to 100%. [Table 13]

[0165] As can be extracted from the above data, the presence of the shielding polymer not only enhances cell viability in HeLa cells, but also increases the transfection efficiency by up to 3-fold while imparting little toxicity to the polymer complex.

[0166] Prior art documents International Publication No. 2019067676 J Mol Med, 2002, 80th edition, pp.648-654

Claims

1. a) positively charged nanoparticles comprising a cationic polymer, a pharmaceutically acceptable salt thereof, or any stereoisomer or mixture of stereoisomers, any of said cationic polymer compounds or pharmaceutically acceptable salts thereof, wherein said cationic polymer is covalently or electrostatically bound to at least one pharmaceutical, veterinary or cosmetically active ingredient; b) Below: i. a copolymer comprising substructure I; ii. a copolymer comprising substructure II, and iii. Copolymers containing the partial structure III and at least one anionic copolymer selected from Including, The partial structures I, II and III are -A ｍ -B ｎ -(Substructure I), -(B ｎ -A ｍ ) ｐ -B ｎ -(Substructure II), -(A ｍ -B ｎ ) ｐ -A ｍ -(partial structure III), each instance of A is an amino acid residue independently selected from methionine sulfoxide, ethionine sulfoxide, S-alkyl-cysteine ​​sulfoxide, S-alkylcysteine ​​sulfone, S-alkylhomocysteine, S-alkylhomocysteine ​​sulfoxide, glycosylated cysteine, serine, homoserine, homomethionine sulfoxide, sarcosine, glycine, and alanine; at least 50 mol % of the A amino acid residues are methionine sulfoxide; each instance of B is independently selected from glutamic acid, aspartic acid, and salts thereof; m is an integer from 20 to 600; n is an integer from 5 to 200, and p is an integer from 1 to 2; Polymer composites.

2. 2. The polymer conjugate of claim 1, wherein 60 mol % to 98 mol % of the A amino acid residues are methionine sulfoxide, and the remainder of the A amino acid residues are selected from sarcosine, glycine, and alanine.

3. 2. The polymer conjugate of claim 1, wherein 100 mol % of the A amino acid residues are methionine sulfoxide.

4. m is an integer from 25 to 300; n is an integer from 8 to 150; The polymer composite of claim 1 .

5. m is an integer from 25 to 180; n is an integer from 10 to 100; The polymer composite of claim 1 .

6. 10. The polymer conjugate of claim 1, wherein at least one cell targeting agent, at least one labeling or imaging agent, or at least one cell targeting agent plus at least one labeling or imaging agent is covalently attached to the polypeptide backbone of the cationic polymer or the anionic copolymer via an amino acid side residue, i.e., C- or N-terminal group, via an amide, ester, or anhydride bond or linker.

7. 10. The polymer conjugate of claim 1, wherein the positively charged nanoparticles form a core portion and the anionic copolymer forms a shell portion.

8. 10. The polymer conjugate of claim 1, wherein the at least one pharmaceutical, veterinary, or cosmetically active ingredient is selected from the group consisting of low molecular weight drugs, peptides, antibodies, nucleic acids, aptamers, and combinations thereof.

9. 2. The polymer conjugate of claim 1, wherein the nucleic acid is selected from the group consisting of DNA / RNA hybrids, small interfering RNA (siRNA), microRNA (miRNA), sgRNA, donor DNA, self-amplifying / replicating RNA, circular RNA (oRNA), plasmid DNA (pDNA), closed linear DNA (lcDNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and antisense RNA (aRNA), messenger RNA (mRNA), CRISPR guide RNA, antisense nucleic acid, decoy nucleic acid, aptamer, and ribozyme.

10. As a shield for positively charged nanoparticles comprising a cationic polymer covalently or electrostatically bound to at least one pharmaceutical, veterinary or cosmetically active ingredient for delivery of the pharmaceutical, veterinary or cosmetically active ingredient to a biological target; i. a copolymer comprising substructure I; ii. a copolymer comprising substructure II, and iii. Copolymers containing the partial structure III Use of at least one anionic copolymer selected from 10. The use according to claim 1, wherein the substructures I, II and III are as defined in claim 1.

11. 10. A pharmaceutical, veterinary or cosmetic composition comprising at least one polymer conjugate according to claim 1 together with one or more suitable acceptable excipients.

12. 1. A method for delivering at least one pharmaceutical, veterinary or cosmetic active ingredient to a biological target, comprising: a) providing a polymer composite according to claim 1; b) contacting the biological target with the polymer conjugate or the composition; A method comprising:

13. a) providing a cationic polymer in a first liquid; b) providing at least one pharmaceutically, veterinarily or cosmetically active ingredient in a second liquid and iv. a copolymer comprising substructure I; v. a copolymer comprising substructure II, and vi. Copolymers containing substructure III and mixing with an anionic copolymer selected from c) contacting the cationic polymer in the first liquid with the at least one pharmaceutically, veterinarily, or cosmetically active ingredient and the anionic copolymer in the second liquid to form shielded nanoparticles; Including, wherein said substructures I, II and III are as defined herein. Methods for the preparation of polymer composites.

14. 10. The polymer conjugate of claim 1 for use in (i) as a transfection reagent for transfecting at least one active agent into cells, (ii) in in vivo or ex vivo therapy encoding a peptide or antibody, (iii) in the production of peptides, antibodies or recombinant viruses, (iv) as a therapeutic or prophylactic vaccine against viral infection or a therapeutic vaccine against cancer, and (v) in genome engineering, cell reprogramming, cell differentiation or gene editing.

15. 10. A device for delivering at least one pharmaceutical, veterinary or cosmetic active ingredient into a cell, comprising the polymer conjugate of claim 1.

16. A method for delivering at least one pharmaceutical, veterinary or cosmetic active ingredient to a target cell, comprising: Administering a solution containing the polymer conjugate of claim 1 to an animal, including a human, thereby introducing the polymer conjugate or composition into the target cells; Translocating the polymer conjugate or composition from the endosome to the cytoplasm; dissociating the polymer conjugate or composition within the cell; and releasing the active ingredient into the cytoplasm; A method comprising:

17. A method for diagnosing a disease in a sample isolated from a subject, comprising:

10. A method comprising: administering to the subject an effective amount of the polymer conjugate of claim 1, wherein the polymer conjugate comprises one or more imaging agents in an isolated sample of the subject.

18. A method for treating or preventing a disease selected from a neurodegenerative disease, a neurological disease, cancer, an infectious disease, an aging-related disease, neuroinflammation, a demyelinating disease, multiple sclerosis, an ischemic disease, an immunodeficiency, an inflammatory disease, and a rare disease, comprising: administering to a subject in need thereof a therapeutically effective amount of the polymer conjugate of claim 1. A method comprising:

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