CATIONIC POLYMERS WITH D-FRUCTOSE SUBSTITUENTS

Abstract

The invention relates to new cationic polymers conjugated with D-fructose, as a result of which they can selectively interact with specific structure elements on cell surfaces. The problem was that of creating novel, biocompatible, easy-to-produce, D-fructose-conjugated cationic polymers that have a higher selectivity with respect to certain cell types. To solve this problem, the invention proposes cationic polymers with covalently bonded D-fructose of general formula (I) with the following components: a) cationic polymer: macromolecular compounds of n repeat units with one or more positive charges; b) linker: a unit that links the cationic polymer with D-fructose or derivatives of D-fructose by means of any alkyl or aryl group, any alkenyl or alkinyl group, an ether, thioether or amine, an ester, amide or other carboxylic acid derivative, a heterocycle (e.g. triazole or m maleimide), a disulphide, an imine or an imide; c) D-fructose: one or more D-fructoses or D-fructose derivatives in an open-chain, furanoid or pyranoid structure, not glycosidically linked via one of the five possible carbon atoms (1, 3, 4, 5, 6).

Description

The invention relates to new cationic polymers conjugated with D-fructose which can selectively interact with specific structure elements on cell surfaces.

Known cationic polymers, such as poly(ethyleneimine) (PEI) or poly-L-lysine (PLL), have significant disadvantages (H. Lv et al. (2006): “Toxicity of cationic lipids and cationic polymers in gene delivery” Journal of Controlled Release 114: 100-109):

• • general and non-specific cytotoxicity to cells,
  • triggering aggregation and hemolysis of blood cells.

Cationic polymers have the ability to complex negatively charged, genetic material, such as siRNA (small interfering ribonucleic acid) or pDNA (plasmid desoxyribonucleic acid), due to their high density of positive charges. The resulting adducts between cationic polymers and genetic material are called polyplexes, which can help to transport genetic material (e.g. siRNA) into cells.

Cationic polymers have already been used successfully as an additive in sugar surfactants for improving the sensory properties and the foam sensation in cosmetic applications (patent application: Use of cationic biopolymers to improve the sensory properties of sugar surfactant-containing preparations, DE19605355A1, Joerg Kahre, Rolf Wachter).

Furthermore, cationic polymers can be functionalized with small protein sequences (e.g.: RGD peptides) and thus the selectivity can be influenced (C L Waite et al. (2009): “PAMAM-RGD Conjugates Enhance siRNA Delivery Through a Multicellular Spheroid Model of Malignant Glioma” Bioconjugate Chemistry: 20: 1908-1916). The exact mechanism is unclear and the presence of integrin (transport membrane protein in animal cells) is necessary.

A covalent linkage of cationic polymers with sugars has also become known.

Poly(propyleneimine) polymers could be functionalized with D-mannose and their properties studied in successful use against HIV. This approach is used exclusively for the targeted activation of immune cells, so-called macrophages, with lectin receptors. (T. Dutta et al (2007): “Targeting potential and anti-HIV activity of lamivudine loaded mannosylated poly(propyleneimine) dendrimer” Biochimica et Biophysica Acta (BBA)—General Subjects: 1770, 681-686).

The synthesis of D-galactose-conjugated poly(ethylene glycol) poly(ethyleneimine) copolymers for the transfection of genetic material into hepatocytes has been described. This approach is only suitable for liver cells which have ASGP receptors (patent application: Cellular targeting poly(ethylene glycol)-grafted polymeric gene carrier, WO2003008555A2, Kazyyoshi Sagara; publication: K. Sagara et al. (2002). “A new synthesis of galactose-poly(ethylene glycol) polyethyleneimines for gene delivery to hepatocytes” Journal of Controlled Release 79 (1-3): 271-281).

Lactose and α-cyclodextrin were coupled to a cationic, star-shaped poly(amidoamine) (PAMAM) dendrimer for the treatment of familial, amyloidotic polyneuropathy. The approach aims only at the transthyretin gene expression in hepatocytes (Y. Hayashi et al (2012): “Potential Use of Lactosylated Dendrimer (G3)/α-Cyclodextrin Conjugates as Hepatocyte-Specific siRNA Carriers for the Treatment of Familial Amyloidotic Polyneuropathy” Molecular Pharmaceutics: 9, 1645-1653).

Cationic liposomes were modified with D-fucose and examined for their influence on adenovirus-induced immune responses. This approach is exclusively for targeted delivery of the specific transcription factor NF-κB to spleen and liver macrophages. (Huang, H. et al. (2009): “Suppressive effects of sugar-modified cationic liposomes/NF-κB decoy complexes on adenovirus vector-induced innate immune responses” Journal of Controlled Release: 133, 139-145).

Acrylate- or methacrylate-based cationic polymers comprising glycosidically bound saccharides have been described. The approach describes only the chemical composition of such polymers and in no way mentions a possible biological application. With the glycosidically bound sugar residues described there, an interaction with sugar transporters in cell membranes cannot be achieved (patent application: Novel Glycopolymers, Uses Thereof, and Monomers Useful for Preparation Thereof, US20080281064A1, Stephanie Chiron, Marie-Pierre LaBeau, Etienne Fleury, David Viet, Sylvain Cottaz, Hugues Driguez, Sami Halila).

Nucleic acids and their polyplexes with cationic polymers have been described. In this approach, sugar molecules were present in solution in polyplex formation, but are not covalently bound to the cationic polymer. So they do not fulfill any direct targeting functions. (Patent application: Nucleic acid-cationic polymer compositions and methods of making and using the same, WO2016178233A1, Abraham Hochberg, Jennifer Gallula).

The aim of the invention is to provide novel, biocompatible, easily producible, D-fructose-conjugated cationic polymers having an increased selectivity with respect to certain cell types.

The term selectivity relates in one aspect to the interaction of novel D-fructose-conjugated, cationic polymers with certain structural elements on the cell surface and, in another aspect, to the cytotoxic effect on certain cell types. A non-limiting example of particular interest is herein the selective, cytotoxic effect on GLUT5-overexpressing cell types, such as, for example, a majority of breast cancer cell types.

According to the invention, cationic polymers having D-fructose substituents are found to solve this problem which contain general formula (I) as basic structure.

The cationic polymer herein is a macromolecular compound of n repeating units (preferably n=10 to 1000) having one or more positive charges.

Non-limiting examples of preferred cationic polymers can include poly-L-lysine (PLL), polyethylenimine (PEI) or dextrans such as diethylaminoethyl-dextran (DEAE-D) or dextran-spermine (D-SPM) or polymethacrylates such as poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) and poly(dimethylaminoethyl methacrylate) (PDAMA).

Linkers are herein one or more atoms or functional groups that connect the cationic polymer to the D-fructose unit. Suitable for this purpose are, for example, any alkyl or aryl residue, any alkenyl or alkynyl residue, an ether or thioether, an amine, an ester-, amide- or another carboxylic acid derivative, a heterocycle (e.g. triazole or maleimide), a disulfide, an imine or an imide.

Under D-fructose and its derivatives are understood all chemical molecules which are based on D-fructose and maintain the stereochemistry at positions 3, 4 and 5 in open or closed form.

Chemical modifications, in particular the introduction of functional groups, such as thiol, azide, carboxylic acids and their derivatives and/or amino groups, to one or more positions of the sugar (but not at the glycosidic C2 atom of the D-fructose), while maintaining the stereochemistry of positions 3, 4 and 5 are herein also understood as D-fructose derivatives.

D-fructose has a keto functionality in open-chain form which completely changes the chemical properties. It is, like in other sugars, introduced into the cells via specific transport proteins (GLUTs) and metabolized. The transporter responsible for D-fructose is the GLUT5 transporter (A. Godoy et al. (2006): Differential subcellular distribution of glucose transporters GLUT1-6 and GLUT9 in human cancer: Ultrastructural localization of GLUT1 and GLUT5 in breast tumor tissues.” Journal of Cellular Physiology 207 (3): 614-627).

Surprisingly, the D-fructose-substituted, cationic polymer P3 has shown to be advantageous:

In comparison to unmodified, cationic polymers (e.g. L-PEI), P3 contains:

• • an increased water solubility;
  • cytotoxicity to breast cancer cells, such as MDA-MB-231;
  • no cytotoxicity to non-cancer cells, such as HUVEC or L929;
  • greatly reduced hemolytic activity with respect to blood cells;
  • no triggering of the aggregation of blood components;
  • and the ability to form polyplexes with negatively charged biomolecules, such as pDNA or siRNA.

The invention is illustrated in the following by the synthesis of D-fructose-conjugated cationic polymers (based on linear poly(ethyleneimine) (L-PEI, (I)) and branched poly(ethyleneimine) (B-PEI, (II)).

(I) Synthesis of D-Fructose-Conjugated (Unbranched) L-PEI

1. Synthesis of the SH-Functionalized D-Fructose Derivative in a Four-Step Synthesis

Schematic representation of the four-step synthesis of 1-O-(2-mercapto-ethyl)-2,3:4,5-di-O-isopropylidene-β-D-fructopyranoside: a) Benzyl 2-bromoethyl ether, NaH, THF, rt; b) H₂/Pd (C), CH₃OH, rt; c) mesyl chloride, Et₃N, 4-DMAP, CH₂Cl₂, 0° C.; d) 1. Thiourea, butanone, 95° C., 2. K₂S₂O₅, CH₂Cl₂/H₂O, 50° C.

The D-fructose derivative 5 was fully characterized and all individual steps could be performed in high yields. The introduction of the thiol serves to attach the sugar to the polymer via a photocatalyzed thiol-ene click reaction.

2. Synthesis of the Block Copolymer Followed by Thiol-Ene Click Between D-Fructose and Polymer Precursor and Deprotection of the Sugar Unit

Schematic representation of the synthesis of P (EI-stat-ButEnOx-stat-FruButOx): a) 6 M HCl, 100° C., reflux; b) pyridine, 4-DMAP, 80° C.; c) D-fructose derivative (5), methanol, 2,2-dimethoxy-2-phenylacetophenone, 25° C., UV=365 nm; d) THF/H₂O, 2M HCl, 40° C.

The copolymers and corresponding intermediates have been extensively characterized. As precursor used was a copolymer containing ethyleneimine (EI) and with double bonds functionalized EI. In the last step, the sugar derivative 5 was attached via a photocatalyzed thiol-ene click reaction. Acid deprotection resulted in the water-soluble polymer P3.

(II) Synthesis of D-Fructose Conjugated, Branched Poly(Ethyleneimine) (B-PEI)

1. Synthesis of Epoxy-Functionalized D-Fructose

Proceeding from commercially available, isopropylidene-protected D-fructose, Williamson etherification with epichlorohydrin can be used to produce the epoxy-functionalized D-fructose.

2. Coupling of Epoxy-Functionalized D-Fructose with (Branched) B-PEI

Schematic representation of the general ring-opening reaction between epoxides and primary amines.

Schematic representation of a possible repeating unit of branched poly (ethyleneimine) (B-PEI).

By stirring at room temperature in methanol for 3 days, B-PEI can be functionalized by a ring-opening reaction with the previously synthesized D-fructose derivative. D-fructose-conjugated B-PEIs were prepared with 14%, 23%, 28%, 39% and 76% functionalized primary amino groups.

3. Cleavage of the Protecting Groups on the Fructose Residues

Acidic cleavage of the isopropylidene protecting groups in the presence of water was carried out after heating the cationic polymers with bound D-fructose derivatives at 40° C. for several days using 2M HCl. Dialysis (cellulose ester, MWCO: 500-1000 Da) against water resulted in D-fructose-functionalized B-PEIs.

The polymer P3 was subjected to intensive, biological evaluation.

a) Cytotoxicity and Hemocompatibility

FIG. 1 shows cell type dependent cytotoxicity studies by alamarBlue assay of polymers P1, P2 and P3. Untreated cells were used as a reference for 100% vitality. The cells were treated for 24 h with the indicated polymer concentrations.

Surprisingly, the D-fructose-conjugated polymer P3 showed increased toxicity to the breast cancer cell line MDA-MB-231, while non-cancer cells (HUVEC and L929) showed no significant reduction in cell vitality. Polymers P2 and P1 showed no selectivity (FIG. 1).

FIG. 2A shows the erythrocyte aggregation assay of the polymers at indicated concentrations. B-PEI was used as a positive control, PBS as a negative control. FIG. 2B shows the hemolysis assay of the erythrocytes after incubation with the polymers at the indicated concentrations. Triton X-100 was used as a positive control (100% hemolysis) and PBS as a negative control (1.99%). A value less than 2% hemolysis is classified as non-hemolytic, 2 to 5% as slightly hemolytic, and >5% as hemolytic. The values represent the mean of three measurements (±standard deviation).

The polymer P3 causes no aggregation of erythrocytes and shows no hemolysis in contrast to P1 and P2 (FIG. 2).

b) Formation Rate and Stability of Polyplex Formation

The ability to complex genetic material is of major interest with respect to the cationic polymer used. To check this, various ratios (N/P ratios) of the sum of all the nitrogen atoms (N) of the cationic polymer and of the phosphorus atoms (P) of the genetic material were tested. FIGS. 3A and 3B show the polyplex formation and stability with pDNA of polymers P1, P2 and P3. FIG. 3A particularly shows the binding affinity at indicated N/P ratios (ethidium bromide quenching assay) and FIG. 3B shows the dissociation assay of the polyplexes at an N/P ratio of 20 using heparin (0 to 60 UmL⁻¹). The values reflect the mean of three measurements again ±SD (n=3).

The D-fructose conjugated polymer P3 shows stable polyplex formation at an N/P ratio >15 and further shows rapid release of the genetic material in the presence of heparin (FIG. 3).

c) Size of Polyplexes

	Z-average		Numeric average	Zeta potential
Polymer	[d/nm]	PDI	[d/nm]	[mV]
P1	217 ± 8	0.47	71 ± 13	24.0 ± 0.4
P2	264 ± 11	0.35	109 ± 33	24.3 ± 1.1
P3	165 ± 1	0.26	83 ± 29	17.6 ± 0.4

The table shows the size and zeta potential of the polyplexes of P1 to P3 at N/P 20 in HBG buffer (measured by dynamic and electrophoretic light scattering).

d) Cell Uptake

To support the results of the cell toxicity studies, the polymers were marked with different dyes (Cy-5 and rhodamine-SCN), incubated with the mentioned cell lines, and the results were evaluated by flow cytometry (FACS) and confocal laser scanning microscopy (CLSM).

FIG. 4 shows the cell uptake studies. Polyplexes of polymers P1 to P3 were incubated with pDNA marked with YOYO-1, and L929-, HUVEC- and MDA-MB-231 cells. It is shown the relative, mean fluorescence intensity (MFI) of all living cells compared to the pDNA control without polymer (dots). The values reflect the mean of three measurements ±SD (n=3).

P1 and P2 show herein a nonspecific uptake into all cell lines (5-60%) at all N/P ratios. P3, however, shows a significantly increased uptake into the breast cancer cell line MDA-MB-231 for N/P=50 (60%) in comparison to P1 and P2 (20-30%). Furthermore, P3 shows a clearly decreased uptake into the non-breast cancer cell line L929 (20%) and the human primary cell line HUVEC (5%) for N/P=50. The clear difference in uptake behavior in MDA-MB-231 between the immediate precursor P2 and the D-fructose-conjugated P3 underlines a successful targeting function of the sugar molecule. The columns in FIG. 4 reflect the percentage of cells that have fluorescence by pDNA uptake.

These results were also observed by confocal laser scanning microscopy of the cells when incubated with the dye-marked polymers. For N/P 50, the fluorescence intensity of P3 in L929 was low and high in MDA-MB-231 cells, whereas polymers P1 and P2 showed a reverse trend. The results of the uptake studies in living cells are consistent with the results of the cytotoxicity assays and thus show a cell type specificity of the D-fructose-conjugated polymer P3.

Claims

1. Cationic polymers with covalently bonded D-fructose of general formula (I) with the components: a) cationic polymer: macromolecular compounds of n repeating units having one or more positive charges;b) linker: a unit linking the cationic polymer to D-fructose or to derivatives of D-fructose by any alkyl or aryl radical, any alkenyl or alkynyl radical, an ether, thioether or amine, an ester, Amide or another carboxylic acid derivative, a heterocycle (e.g. triazole or maleimide), a disulfide, an imine or an imide;c) D-fructose: one or more D-fructose or D-fructose derivatives in open-chain, furanoid or pyranoid structure, non-glycosidically linked via one of the five possible carbon atoms (1, 3, 4, 5, 6).

2. Cationic polymers with covalently bonded D-fructose according to claim 1, characterized in that they have functional groups which have positive charges under appropriate conditions.

3. Cationic polymers with covalently bonded D-fructose according to at least one of claims 1 to 2, characterized in that they contain functional groups which can carry positive charges at different positions once or several times in the polymer.

4. Cationic polymers with covalently bonded D-fructose according to at least one of claims 1 to 3, characterized in that they can be described both as a homopolymer or as a copolymer (random and/or block and/or gradient).

5. Cationic polymers with covalently bonded D-fructose according to at least one of claims 1 to 4, characterized in that they are linear or branched, wherein the latter form includes, for example, stars (dendrimers), brushes, combs, etc.

6. Cationic polymers with covalently bonded D-fructose according to at least one of claims 1 to 5, characterized in that one or more D-fructose residues are bonded to individual, a plurality of or all repeating units of the cationic polymer via linkers according to claim 1.

7. Cationic polymers with covalently bonded D-fructose according to at least one of claims 1 to 6, characterized in that the D-fructose can contain next to free OH groups further substituents on the carbon atoms 1, 2, 3, 4, 5 and/or 6.

8. Cationic polymers with covalently bonded D-fructose according to at least one of claims 1 to 7, characterized in that a biologically active material from the group of nucleic acids is bonded electrostatically and/or covalently.

9. Cationic polymers with covalently bonded D-fructose according to claim 8, characterized in that the bonded nucleic acid is from the group of DNA, RNA, a ribosome and/or a DNA-RNA hybrid and is double-stranded and/or single-stranded.

10. Use of the cationic polymer with covalently bonded D-fructose according to any one of claims 1 to 9 for the transport and delivery of a biologically active material into a living cell.

11. The use of the cationic polymers with covalently bonded D-fructose according to any one of claims 1 to 9 for the selective killing of certain cell types.