NANOSTRUCTURED ACTIVE INGREDIENT CARRIER SYSTEM

Abstract

The invention relates to a nanostructured active ingredient carrier system, in particular for reducing cytotoxic properties owing to the use of sheath polymer and the transport resulting therefrom, for interactions with cell membranes during the transport of hydrophilic constituents and, in connection therewith, the generation of an early endosomal release of the interaction complex from the carrier system. The problem addressed by the present invention is that of specifying a nanostructured active ingredient carrier system which avoids the disadvantages of the prior art and in particular permits a reduction in cytotoxic properties owing to the use of a sheath polymer and the transport resulting therefrom. This problem is solved in that a nanostructured active ingredient carrier system is provided in the form of a particle consisting of a carrier sheath, wherein the carrier sheath comprises at least one or more hydrophobic sheath polymers, one or more charged complexing polymers and one or more hydrophilic active ingredients, wherein the complexing polymer interacts with the active ingredient.

Claims

1. A nanostructured active ingredient carrier system comprising at least one hydrophobic shell polymer, at least one complexing polymer, and at least one hydrophilic active ingredient, wherein the complexing polymer interacts with the active ingredient, and the complexing polymer comprises primary amino groups, secondary amino groups, or a combination of primary and secondary amino groups.

2. The nanostructured active ingredient carrier system according to claim 1, wherein an interaction between the complexing polymer and the active ingredient is caused by one or more non-covalent interactions in the form of electrostatic bonds, ionic bonds, hydrogen bonds, or van der Waals forces.

3. The nanostructured active ingredient carrier system according to claim 1, wherein the hydrophilic active ingredient includes a genetic material.

4. The nanostructured active ingredient carrier system according to claim 3, wherein the genetic material includes siRNA, mRNA, ncRNA, saRNA, short hairpin-RNA, micro-RNA, or plasmid-DNA, or a combination thereof.

5. The nanostructured active ingredient carrier system according to claim 4, wherein the genetic material includes siRNA.

6. The nanostructured active ingredient carrier system according to claim 1, wherein the hydrophilic active agent includes a peptide or a protein.

7. The nanostructured active ingredient carrier system according to claim 6, wherein the hydrophilic active ingredient includes antibodies, interferons, or cytokines.

8. The nanostructured active ingredient carrier system according to claim 1, wherein the complexing polymer includes at least one of a polypeptide, a poly(methacrylate), a polystyrene, a polyamide, a polyacrylamide, a polyurethane, a polyacrylonitrile, a polyethylene glycol, a polyethylene oxide, a polyoxazoline, or any copolymer thereof.

9. The nanostructured active ingredient carrier system according to claim 8, wherein the complexing polymer includes poly-N,N-dimethyl-(2-aminoethyl)-methacrylate (PDMAEMA), poly-(2-aminoethyl)-methacrylate (PAEMA), poly-N-methyl-(2-amioethyl)-methacrylate (PMAEMA), or a copolymer thereof, or any combination thereof.

10. The nanostructured active ingredient carrier system according to claim 1, characterized in that the hydrophobic shell polymer includes at least one of a polyester, a poly(meth)acrylate, a polystyrene, a polyamide, a polyurethane, a polyacrylonitrile, a polytetrafluoroethylene, a silicone, a polyethylene glycol, a polyethylene oxide, a polyoxazoline, or any combination or copolymer thereof.

11. The nanostructured active ingredient carrier system according to claim 1, characterized in that the hydrophobic shell polymer is a biocompatible polymer.

12. The nanostructured active ingredient carrier system according to claim 1, further comprising sucrose, trehalose, or glucose.

13. The nanostructured active ingredient carrier system of claim 1, wherein the nanostructured active ingredient carrier system is free of polyethyleneimine.

14. The nanostructured active ingredient carrier system of claim 1, wherein the active ingredient carrier system is a nanoparticle having a size not greater than 1 micron.

15. The nanostructured active ingredient carrier system of claim 1, wherein the complexing polymer comprises at least two types of amino groups, the two types of amino groups being selected from primary amino groups, secondary amino groups and tertiary amino groups.

16. The nanostructured active ingredient carrier system of claim 1, wherein the complexing polymer comprises primary amino groups and secondary amino groups.

17. The nanostructured active ingredient carrier system of claim 1, wherein the complexing polymer comprises primary amino groups, secondary amino groups, and tertiary amino groups.

18. A nanostructured active ingredient carrier system comprising a shell polymer, the shell polymer including poly(lactic-co-glycolic acid (PLGA); a complexing polymer, the complexing polymer including a polymethacrylate; and a hydrophilic active ingredient.

19. The nanostructured active ingredient carrier system according to claim 18, wherein the hydrophilic active agent includes a genetic material, a peptide or a protein.

20. A nanostructured active ingredient carrier system comprising at least one hydrophobic shell polymer, at least one complexing polymer, and at least one hydrophilic active ingredient, wherein the complexing polymer interacts with the active ingredient, a transfection efficiency is at least 20%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The present disclosure may be better understood, and its numerous features and advantages made apparent to those skilled in the art by referencing the accompanying drawings.

[0040] FIG. 1 includes a table showing the molecular weights and polydispersities of Boc-protected polymers according to embodiments.

[0041] FIG. 2 includes a graph showing the elution diagram of the size exclusion chromatography of the Boc-protected polymers taken by means of refractive index detection according to embodiments.

[0042] FIG. 3 includes the reaction scheme of the complexing polymer preparation according to one embodiment.

[0043] FIG. 4 includes the reaction scheme of the cleavage reaction of the Boc group according to one embodiment.

[0044] FIG. 5 includes a graph illustrating the 2-D HSQC-NMR of the cleavage reaction of the Boc group according to one embodiment.

[0045] FIG. 6 includes a graph showing the elution diagram of the size exclusion chromatography of the complexing polymers recorded by means of refractive index detection.

[0046] FIG. 7 includes a table showing the molecular weights and polydispersities of the Boc-protected polymers determined by a) AF4 by means of MALS detection, b) and c) by SEC (0.1% TFA, 0.1M NaCl dextran calibration/c DmAc, 0.21% LiCl, PMMA calibration), and d) the degree of polymerization determined by AF4 and .sup.1H NMR spectroscopy according to embodiments.

[0047] FIG. 8 includes a graph showing the titration curves of the complexing polymers 2 to 7 in a diagram vs. the total volume of the titrant used, according to embodiments.

[0048] FIG. 9 includes a graph showing the superimposition of the .sup.1H-NMR spectra A) copolymer of the complexing polymers with Boc group, B) complexing polymer after elimination reaction with hydrochloric acid, C) complexing polymer after the cleavage reaction with trifluoroacetic acid, according to embodiments.

[0049] FIG. 10 includes a graph showing the reaction scheme of the NHS ester coupling reaction according to one embodiment.

[0050] FIG. 11A includes a graph showing the size of the particles produced directly after the preparation and removal of the solvent and after freeze-drying and resuspension in ultrapure water according to embodiments.

[0051] FIG. 11B includes a graph showing the polydispersity of the particles produced directly after preparation and removal of the solvent and after freeze-drying and resuspension in ultrapure water according to embodiments.

[0052] FIG. 11C includes a graph showing the zeta potential of the particles produced directly after the preparation and removal of the solvent and after freeze-drying and resuspension in ultrapure water, according to embodiments.

[0053] FIG. 12A includes a graph showing the relative viability of L929 fibroblast cells, 24 h after the addition of interaction complexes, according to embodiments.

[0054] FIG. 12B includes a graph showing the relative viability of L929 fibroblast cells, 24 h after the addition of nanoparticles (B) in the corresponding concentrations, according to embodiments.

[0055] FIG. 13 includes a graph showing the time-dependent uptake of the interaction complexes P1 to P6 in HEK cells according to embodiments.

[0056] FIG. 14 includes a graph showing the time-dependent uptake of P6 and siRNA encapsulated nanoparticles in HEK cells compared to PEI nanoparticles according to embodiments.

[0057] FIG. 15 includes a graph showing the transfection efficiency of the interaction polyplexes with GFP-coded pDNA in HEK cells under the influence of serum-reduced medium (OptiMEM) and serum-containing standard growth medium (RPMI) according to embodiments.

DETAILED DESCRIPTION

[0058] In the present disclosure, the terms below are to be understood as follows:

[0059] Nanoparticles are structures that are smaller than 1 μm and can be composed of multiple molecules. They are generally characterized by a higher surface to volume ratio and thus offer a higher chemical reactivity. These nanoparticles can consist of polymers.

[0060] Polymers are characterized by the repetition of certain units (monomers), but can also consist of several different repeat units. The monomers are covalently bonded together by the chemical reaction (polymerization) and form from the linking polymerizable unit. the so-called polymer backbone. The unconnected groups form the side chains where functional groups can be located. If these polymers have hydrophobic properties to some extent, they can form nanoscale structures (e.g., nanoparticles, micelles, vesicles) in an aqueous environment. “Shell polymer” is understood to mean one or more different polymers present in layers or as a blend wherein a random mixture of two or more polymers results in the formation of a hydrophobic nanostructured active ingredient carrier system, with an interaction complex is included and/or incorporated therein.

[0061] “Interaction complex” is understood to mean a complex formed by electrostatic interaction of one or more hydrophilic active ingredients and one or more complexing polymers.

[0062] “Active ingredient” describes at least one pharmaceutically active ingredient selected from the group, consisting of low molecular weight substances, inhibitors, inducers, or contrast agents, and in particular also of higher molecular weight substances, for example, in the form of nucleic acid, wherein the hydrophilic agents contain potentially therapeutically useful nucleic acids (e.g., short interferin RNA, short hairpin RNA, microRNA, plasmid DNA) and proteins (e.g., antibodies, interferons, cytokines).

[0063] The term “pharmaceutical active ingredient” is understood to mean any inorganic or organic molecule, substance or compound that has a pharmacological effect. The term “pharmaceutical active ingredient” is used synonymously herein with the term “medicine” and “medicament.”

[0064] The pharmaceutical active ingredient can be those which have little or no bioavailability without inclusion in a nanoparticle or a liposome or have little or no stability in vivo.

[0065] The essence of the invention consists in providing a nanostructured active ingredient carrier system in the form of a shell polymer-enclosed, highly reactive nucleic acid-polymer complex, inter alia, for gene delivery.

[0066] This nano-structured active ingredient delivery system in the form of a particle consists of a carrier shell, wherein the carrier shell comprises at least one or more hydrophobic shell polymers, one or more charged complexing polymers, and one or more hydrophilic agents, wherein the complexing polymer interacts with the active ingredient.

[0067] The at least one shell polymer is selected from the group consisting of polyesters, poly(meth)acrylates, polystyrene derivatives, polyamides, polyurethanes, polyacrylonitriles, polytetrafluoroethylenes, silicones, polyethylene glycols, polyethylene oxides, and polyoxazolines and the copolymers thereof in various compositions, e.g., random, gradient, alternating, block, graft, or star copolymers.

[0068] Preferably, the at least one shell polymer is a biocompatible polymer.

[0069] The at least one shell polymer is particularly preferably a hydrophobic, biodegradable polymer, particularly preferably selected from the group consisting of PLGA, PLA, PCL, PGA, PEG, or PDX.

[0070] The “complexing polymer” represents one or more hydrophilic polymers which, through electrostatic interaction, are able to produce an interaction complex with one or more hydrophilic active ingredients.

[0071] More preferably, the complexing polymer consists of linear, water-soluble, cationic polymers with a proportion of 0 to 70% secondary amine functionalities in the polymer backbone. This at least one complexing polymer is selected from the group, consisting of polypeptides, poly(meth)acrylates, polystyrene derivatives, polyamides, polyurethanes, polyacrylonitriles, polyethylene glycols, polyethylene oxides, and polyoxazolines and their copolymers in various compositions, e.g., statistical, gradient, alternating, block, and graft copolymers. The invention expressly excludes the use of polyethyleneimine.

[0072] After incorporation of the nanostructured active ingredient carrier system (=nanoparticles) into the target tissue, there is the release of the interaction complex and an optionally included pharmaceutically active ingredient.

[0073] The release of a nanoparticle as part of the nanostructured active ingredient carrier system takes place as follows: [0074] 1. Acidification of the endosome, destabilization of the nano-structured carrier system, degradation of the shell polymer of the carrier shell by pH-dependent or enzymatic cleavage; [0075] 2. Release of the active interaction complex from the carrier shell, thereby allowing the complexing polymer to penetrate the endosome, the vesicular membrane; [0076] 3. Release of the active ingredient from the endosome. In this case, the active ingredient can already be unbound or else bound to complexing polymer; final release of the active ingredient follows [0077] 4. Polymer components are supplied to various metabolic pathways

[0078] The advantage of this nanostructured active ingredient carrier system is that it allows a reduction in cytotoxic properties, the interactions with cell membranes during transport of hydrophilic components, and thus the generation of early endosomal release of the interaction complex from the carrier system, resulting in optimal active ingredient release.

[0079] Another object of the present invention relates to a pharmaceutical composition comprising such a nanostructured active ingredient carrier system as well as suitable auxiliaries and additives.

[0080] The term “auxiliaries and additives” according to the invention means any pharmacologically acceptable and therapeutically meaningful substance which is not a pharmaceutical active ingredient but can be formulated together with the pharmaceutical active ingredient in the pharmaceutical composition to influence, in particular to improve, qualitative properties of the pharmaceutical composition.

[0081] Preferably, the auxiliaries and/or additives do not develop any significant or at least no undesirable pharmacological effect with regard to the intended treatment.

[0082] Suitable auxiliaries and additives are, for example, pharmaceutically acceptable inorganic or organic acids, bases, salts, and/or buffer substances. Examples of inorganic acids are hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid, with hydrochloric acid and sulfuric acid in particular being preferred.

[0083] Examples of suitable organic acids are malic acid, tartaric acid, maleic acid, succinic acid, acetic acid, formic acid, and propionic acid, and particularly preferably ascorbic acid, fumaric acid, and citric acid. Examples of pharmaceutically acceptable bases are alkali hydroxides, alkali metal carbonates, and alkali ions, preferably sodium. Mixtures of these substances can be used in particular for adjusting and buffering the pH value.

[0084] Preferred buffer substances are furthermore PBS, HEPES, TRIS, MOPS, and other physiologically acceptable buffer substances.

[0085] Further suitable auxiliaries and additives are solvents or diluents, stabilizers, suspension agents, preservatives, fillers, cryoprotectants, emulsion mediators and/or binders, and other conventional auxiliaries and additives known in the art. The choice of auxiliaries and the amounts thereof to be used will depend on the pharmaceutical active ingredient and the mode of administration.

[0086] A nanostructured active ingredient carrier system, which forms the pharmaceutical composition with the suitable auxiliaries and additives, can be achieved, for example, by the double emulsion method known per se.

[0087] The nanostructured active ingredient carrier system and the pharmaceutical composition, which comprises such a nanostructured active ingredient carrier system and suitable auxiliaries and additives, represent a hitherto unique, combinable therapeutic systems to transport a variety of substances, especially pharmaceutical active ingredients (e.g., nucleic acids, but also hydrophilic molecules) into a cell, wherein the combination with the interaction complex leads to an early endosomal release. The safe transport into the cell is realized by the shell protein and the interaction complex. The result is a fast endosomal release, which is ensured by the interaction of the interaction complex with the endosomal membrane. In this way, it is possible to efficiently and rapidly introduce and release one or more active ingredients into cells.

[0088] The invention will be explained in more detail below with reference to the figures and the exemplary embodiments. In the figures:

[0089] FIG. 1: shows the molecular weights and polydispersities of the Boc-protected polymers determined by a) SEC (DmAc, 0.21% LiCl, PMMA calibration), and b) the degree of polymerization determined by means of SEC and .sup.1H-NMR spectroscopy,

[0090] FIG. 2: shows the elution diagram of the size exclusion chromatography of the Boc-protected polymers taken by means of refractive index detection,

[0091] FIG. 3: shows the reaction scheme of the complexing polymer preparation according to exemplary embodiment 1,

[0092] FIG. 4: shows the reaction scheme of the cleavage reaction of the Boc group according to exemplary embodiment 2,

[0093] FIG. 5: shows the 2-D HSQC-NMR of the cleavage reaction of the Boc group according to exemplary embodiment 2,

[0094] FIG. 6: shows the elution diagram of the size exclusion chromatography of the complexing polymers recorded by means of refractive index detection,

[0095] FIG. 7: shows the molecular weights and polydispersities of the Boc-protected polymers determined by a) AF4 by means of MALS detection, b) and c) by SEC (0.1% TFA, 0.1M NaCl dextran calibration/c DmAc, 0.21% LiCl, PMMA calibration), and d) the degree of polymerization determined by AF4 and .sup.1H NMR spectroscopy. In addition, the pKA value of the polymers was determined by titration,

[0096] FIG. 8: shows the titration curves of the complexing polymers 2 to 7 in a diagram vs. the total volume of the titrant used,

[0097] FIG. 9: shows the superimposition of the .sup.1H-NMR spectra A) copolymer of the complexing polymers with Boc group, B) complexing polymer after elimination reaction with hydrochloric acid, C) complexing polymer after the cleavage reaction with trifluoroacetic acid,

[0098] FIG. 10: shows the reaction scheme of the NHS ester coupling reaction,

[0099] FIG. 11A: shows the size of the particles produced directly after the preparation and removal of the solvent and after freeze-drying and resuspension in ultrapure water,

[0100] FIG. 11B: shows the polydispersity of the particles produced directly after preparation and removal of the solvent and after freeze-drying and resuspension in ultrapure water,

[0101] FIG. 11C: shows the zeta potential of the particles produced directly after the preparation and removal of the solvent and after freeze-drying and resuspension in ultrapure water.

[0102] FIG. 12A: shows the relative viability of L929 fibroblast cells, 24 h after the addition of interaction complexes,

[0103] FIG. 12B: shows the relative viability of L929 fibroblast cells, 24 h after the addition of nanoparticles (B) in the corresponding concentrations,

[0104] FIG. 13: shows the time-dependent uptake of the interaction complexes P1 to P6 in HEK cells,

[0105] FIG. 14: shows the time-dependent uptake of P6 and siRNA encapsulated nanoparticles in HEK cells compared to PEI nanoparticles, and

[0106] FIG. 15: shows the transfection efficiency of the interaction polyplexes with GFP-coded pDNA in HEK cells under the influence of serum-reduced medium (OptiMEM) and serum-containing standard growth medium (RPMI).

EXAMPLES

Exemplary Embodiment 1

Synthesis of Complexing Polymers

[0107] Homo- and copolymers of N-tert-butyloxycarbonyl-(2-aminoethyl) methacrylates (hereinafter BocAEMA), N-methyl-N-tert-butyloxycarbonyl-(2-aminoethyl) methacrylates (hereinafter BocMAEMA) and N,N-dimethyl-(2-aminoethyl) methacrylates (hereinafter DMAEMA) were prepared by reversible addition-fragmentation chain transfer polymerization.

[0108] In a typical reaction, 0.73 g of BocAEMA (3.18×10.sup.−3 mol), 0.77 g of BocMAEMA (3.18×10.sup.−3 mol), 0.98 mg of azobis(isobutyronitrile) initiator (5.96×10.sup.−5 mol), 5.68 mg of 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (20.33×10.sup.−5 mol), and 5.03 mL of dimethylformamide were added together with anisole as internal standard (0.34 mL) in a 25 mL microwave reaction vessel, and this was degassed for 30 min using an argon stream.

[0109] The reaction solution was then heated with stirring in an oil bath, preheated to 70° C., for 38 h.

[0110] The copolymer was precipitated twice from tetrahydrofuran in n-hexane and then dried under reduced pressure.

[0111] The conversion was determined using the .sup.1H-NMR spectrum against the internal standard.

[0112] The analytical data are shown in FIG. 1, the size exclusion chromatography is shown in FIG. 2, and the synthetic scheme is shown in FIG. 4.

[0113] The tert-butyloxycarbonyl (Boc) protected polymers were deprotected in 1M methanolic hydrochloric acid over 16 h, the solvent removed under reduced pressure, dissolved in deionized water, and lyophilized for 24 h.

[0114] The synthetic scheme is shown in FIG. 3, the 2-D NMR is shown in FIG. 5, the size exclusion chromatography is shown in FIG. 6, the analytical data is shown in FIG. 7, the titration curves with the pK.sub.A values are shown in FIG. 8, and the .sup.1H-NMR comparison of deprotection methods is shown in FIG. 9.

Exemplary Embodiment 2

Functionalization of Complexing Polymers and Shell Polymers

[0115] The linkage of the polymers to fluorescent dyes by means of N-hydroxysuccinimide (hereinafter, NHS) activated coupling of carboxylic acids with primary amines was used for the visualization of the shell and complexing polymers.

[0116] For this purpose, in a typical reaction, the NHS-ester derivative of cyanine 5® (0.5 mg, 8.1×10.sup.−4 mol) (together with the copolymer PMAEMA-co-AEMA 37.0 mg, 6.8×10.sup.−4 mol) and triethylamine (0.3 mL) were stirred in methanol (9.7 mL) for 24 h at room temperature under the exclusion of light. The solvent was then removed under reduced pressure, the solid residue dissolved in water and dialyzed against distilled water for 7 days in a regenerated cellulose dialysis membrane tube (Carl Roth, exclusion limit 3500 g.Math.mol.sup.−1), and then freeze-dried for 24 h.

[0117] The synthetic scheme is shown in FIG. 10.

Exemplary Embodiment 3

Production of Nanoparticles

[0118] The particles used are prepared, for example, by means of double emulsion. High-frequency ultrasound is used, which favors the formation of nanoscale particles with the aid of the surface-active substance polyvinyl alcohol (PVA).

[0119] For this purpose, the hydrophobic shell polymers are dissolved in ethyl acetate, a water-immiscible solvent (10-20 mg.Math.mL.sup.−1).

[0120] A final concentration of 0.3% PVA in ultrapure water is used.

[0121] The interaction complex is previously formed from the complexing polymer and the genetic material, here the siRNA, in ultrapure water. Subsequently, a first emulsion of interaction complex and shell polymer is formed, which is subsequently transferred to water and, after reapplication of high-frequency ultrasound, a second emulsion is formed.

[0122] Subsequently, the particles are incubated for up to 48 h at room temperature to allow the organic solvent to evaporate.

[0123] The particles formed are washed by centrifugation and resuspension in ultrapure water, treated with 5% sucrose, and then frozen (−80° C.) to be lyophilized.

Exemplary Embodiment 4

Characterization of the Nanoparticles

[0124] Nanoparticles of PLGA, polymethacrylates and siRNA are reproducibly produced with constant parameters.

[0125] The results are shown in FIGS. 11A to 11C, where A=size, B=polydispersity, C=zeta potential surface charge. For the size and size distribution measurement, dynamic light scattering is used; the charge is determined by ζ-potential.

[0126] FIG. 11A shows that the size of the particles remains constant regardless of the preparation or the shell or complexing polymer used.

Exemplary Embodiment 5

Toxicity of Interaction Complex and Nanoparticles

[0127] The cytotoxicity studies is carried out according to the ISO10993-5 protocol with L929 mouse fibroblast cells. The cells are seeded in DMEM growth medium (Dulbecco's modified Eagle's medium) at a cell concentration of 10.sup.4 cells per well in a 96-well plate, and were incubated for 24 h at 37° C. and 5% CO.sub.2.

[0128] This is followed by the addition of the corresponding complexes or nanoparticles in various concentrations.

[0129] After 24 h, the medium is exchanged with fresh medium, treated with the reagent AlamarBlue.

[0130] After a further incubation period of 4 h at 37° C., the fluorescence measurement of the individual wells was carried out using a microplate reader (Tecan) at an excitation wavelength of 570 nm and an emission wavelength of 610 nm. Untreated cells served as a negative control, with their measurements corresponding to a viability of 100%. The results are shown in FIGS. 12A and 12B. It can be clearly seen that encapsulation led to an increase in biocompatibility.

Exemplary Embodiment 6

Cellular Uptake of Interaction Complex and Nanoparticles

[0131] To study the cellular uptake of complexes and nanoparticles, human embryonic kidney cells (HEK) are seeded into 24-well plates in RPMI 1640 growth medium (containing 10% fetal calf serum and 1% antibiotic).

[0132] After 24 h, the medium is exchanged for serum-reduced medium (OptiMEM) and incubated for a further hour.

[0133] Polyplexes with YOYO-labeled pDNA or nanoparticles with Nile Red and complexing polymer is added to the HEK cells and incubated for up to 4 h at 37° C., 5% CO.sub.2.

[0134] Cellular uptake is assessed by flow cytometry. A total of 10,000 cells is measured, and all living cells (FSC/SSC scattering) with a positive signal (FL1) are counted.

[0135] The results are shown in FIG. 13 and FIG. 14.

[0136] The investigation of the cellular particle uptake was performed via confocal laser scanning microscopy. For this purpose, HEK cells are seeded in microscopy vessels with a glass bottom and the microscopic uptake by the cells takes place 1 to 4 hours after addition of the samples. Furthermore, the cell nuclei are stained with Hoechst 33342, lysosomes with LysoTracker Red DND-99 or LysoTracker Green DND-26, and cell membranes with CellMask Orange plasma membrane stain.

Exemplary Embodiment 7

Transfection and Knockdown

[0137] For the transfection studies, HEK cells or a stable GFP-CHO cell line are seeded in 24-well plates with a cell concentration of 10.sup.15 cells/mL.

[0138] One hour before the addition of the samples, the medium is exchanged either with serum-reduced medium (OptiMEM) or serum-containing growth medium (RPMI 1640).

[0139] Polyplexes or nanoparticles are added to the cells (50 μL per well) and incubated for 4 h at 37° C., 5% CO.sub.2.

[0140] Thereafter, the supernatant is removed and the cells are incubated in fresh growth medium for a further 24 h to 72 h.

[0141] The analysis of the transfection efficiency is carried out by flow cytometry. To this end, the cells are trypsinized and stained with propidium iodide for a live/dead determination.

[0142] To determine the transfection efficiency, 10,000 cells are measured and all living cells with positive GFP signal (E.sub.x 488 nm; E.sub.m 525 nm) are counted. The transfection results are shown in FIG. 15.

[0143] All features described in the description, the exemplary embodiments, and the following claims can be essential to the invention both individually and in any combination with one another.