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, the shell polymer including poly(lactic-co-glycolic acid (PLGA); a complexing polymer, the complexing polymer including a polymethacrylate; and a genetic material, wherein 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, characterized in that 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, and the genetic material includes a siRNA.

3. The nanostructured active ingredient carrier system of claim 1, wherein the genetic material includes siRNA, mRNA, ncRNA, saRNA, short hairpin-RNA, micro-RNA, or plasmid-DNA.

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

5. The nanostructured active ingredient carrier system of claim 1, wherein the at least one hydrophobic shell polymer has a layered structure in the carrier system.

6. 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.

7. The nanostructured active ingredient carrier system according to claim 1, wherein the at least one hydrophobic shell polymer is a biodegradable polymer.

8. The nanostructured active ingredient carrier system of claim 1, wherein the complexing polymer comprises is a copolymer.

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

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

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

12. The nanostructured active ingredient carrier system of claim 3, wherein the genetic material includes siRNA.

13. 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.

14. The nanostructured active ingredient carrier system according to claim 1, wherein the shell polymer is a biocompatible polymer.

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

16. A method of transporting nucleic acids into a cell, the method comprising transfecting the cell with the nucleic acids using a nanostructured active ingredient carrier system, wherein the nanostructure active ingredient carrier system comprises at least one hydrophobic shell polymer, the shell polymer including poly(lactic-co-glycolic acid (PLGA); at least one complexing polymer; and the nucleic acids, wherein the complexing polymer comprises primary amino groups, secondary amino groups, or a combination of primary and secondary amino groups.

17. The method of claim 16, wherein the nucleic acids include siRNA.

Description

(1) The invention will be explained in more detail below with reference to the figures and the exemplary embodiments. In the figures:

(2) 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,

(3) FIG. 2: shows the elution diagram of the size exclusion chromatography of the Boc-protected polymers taken by means of refractive index detection,

(4) FIG. 3: shows the reaction scheme of the complexing polymer preparation according to exemplary embodiment 1,

(5) FIG. 4: shows the reaction scheme of the cleavage reaction of the Boc group according to exemplary embodiment 2,

(6) FIG. 5: shows the 2-D HSQC-NMR of the cleavage reaction of the Boc group according to exemplary embodiment 2,

(7) FIG. 6: shows the elution diagram of the size exclusion chromatography of the complexing polymers recorded by means of refractive index detection,

(8) 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,

(9) FIG. 8: shows the titration curves of the complexing polymers 2 to 7 in a diagram vs. the total volume of the titrant used,

(10) 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,

(11) FIG. 10. shows the reaction scheme of the NHS ester coupling reaction,

(12) 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,

(13) 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,

(14) 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.

(15) FIG. 12A: shows the relative viability of L929 fibroblast cells, 24 h after the addition of interaction complexes,

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

(17) FIG. 13: shows the time-dependent uptake of the interaction complexes P1 to P6 in HEK cells,

(18) FIG. 14: shows the time-dependent uptake of P6 and siRNA encapsulated nanoparticles in HEK cells compared to PEI nanoparticles, and

(19) 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).

EXEMPLARY EMBODIMENT 1

Synthesis of Complexing Polymers

(20) 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.

(21) 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.

(22) The reaction solution was then heated with stirring in an oil bath, preheated to 70° C., for 38 h.

(23) The copolymer was precipitated twice from tetrahydrofuran in n-hexane and then dried under reduced pressure.

(24) The conversion was determined using the .sup.1H-NMR spectrum against the internal standard.

(25) 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.

(26) 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.

(27) 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

(28) 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.

(29) 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.

(30) The synthetic scheme is shown in FIG. 10.

EXEMPLARY EMBODIMENT 3

Production of Nanoparticles

(31) 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).

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

(33) A final concentration of 0.3% PVA in ultrapure water is used.

(34) 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.

(35) Subsequently, the particles are incubated for up to 48 h at room temperature to allow the organic solvent to evaporate.

(36) 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

(37) Nanoparticles of PLGA, polymethacrylates and siRNA are reproducibly produced with constant parameters.

(38) 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.

(39) 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

(40) 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.

(41) This is followed by the addition of the corresponding complexes or nanoparticles in various concentrations.

(42) After 24 h, the medium is exchanged with fresh medium, treated with the reagent AlamarBlue.

(43) 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

(44) 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).

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

(46) 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.

(47) 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.

(48) The results are shown in FIG. 13 and FIG. 14.

(49) 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

(50) 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.

(51) 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).

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

(53) Thereafter, the supernatant is removed and the cells are incubated in fresh growth medium for a further 24 h to 72 h.

(54) 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.

(55) 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.

(56) 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.