NANOPARTICLES CONTAINING COMPLEXES OF NUCLEIC ACIDS AND CATIONIC COPOLYMERS, PROCESS FOR PREPARING THEM AND THEIR USE FOR GENE TRANSFER IN CELLS

Abstract

##STR00001##

The invention relates to nanoparticles containing complexes constituted by nucleic acids and cationic copolymers containing the recurring structural units of formulae (Ia) and (Ib) wherein R.sup.1 and R.sup.6 independently represent hydrogen, alkyl or —COOR.sup.9, R.sup.2 and R.sup.7 independently represent hydrogen or alkyl, R.sup.3 is selected from the group consisting of —O—R.sup.10—, —COO—R.sup.10, —CONH—R.sup.10- or —R.sup.10—, R.sup.4 represents hydrogen, alkyl, cycloalkyl, aryl, aralkyl or alkylaryl, R.sup.5 represents hydrogen, alkyl, cycloalkyl, aryl, aralkyl, alkylaryl or —(alkylene-NH—).sub.malkyl, or R.sup.4 and R.sup.5 together with the nitrogen atom they have in common form a heterocyclic ring, R.sup.8 is selected from the group consisting of —O—R.sup.11, —COO—R.sup.11, —CONH—R.sup.11 or —R.sup.11, R.sup.9 and R.sup.11 independently represent hydrogen or a monovalent organic residue, R.sup.10 represents a bivalent organic residue, and m is an integer from 1 to 5, with the proviso that the nanoparticles have a diameter (z-average) of less than or equal to 900 nm as determined by dynamic light scattering and that the molar ratio of nitrogen atoms in the copolymer to the phosphate groups in the nucleic acid ranges between 1 and 200. The nanoparticles according to the invention allow the transfer of nucleic acids into cells with great efficiency.

Claims

1. Nanoparticles comprising complexes formed from nucleic acids and cationic copolymers containing the recurring structural units of the formulae (Ia) and (Ib) ##STR00004## wherein R.sup.1 and R.sup.6 are, independent of each other, hydrogen, alkyl or —COOR.sup.9, R.sup.2 and R.sup.7 are, independent of each other, hydrogen or alkyl, R.sup.3 is selected from the group consisting of 613 O—R.sup.10—, —COO—R.sup.10—, —CONH—R.sup.10— or —R.sup.10—, R.sup.4 is hydrogen, alkyl, cycloalkyl, aryl, aralkyl or alkylaryl, R.sup.5 is hydrogen, alkyl, cycloalkyl, aryl, aralkyl, alkylaryl or —(alkylene-NH—).sub.m-alkyl, or R.sup.4 and R.sup.5 form a heterocyclic ring together with the nitrogen atom they have in common, R.sup.8 is selected from the group consisting of —O—R.sup.11, —COO—R.sup.11, —CONH—R.sup.11 or —R.sup.11, R.sup.9 and R.sup.11 are, independent of each other, hydrogen or a monovalent organic radical, R.sup.10 represents a bivalent organic radical, and m is an integer from 1 to 5, with the requirement that the nanoparticles have a diameter (z-average) of less than or equal to 900 nm, determined by dynamic light scattering, and that the molar ratio of nitrogen atoms in the copolymer to phosphate groups in the nucleic acid is between 1 and 200.

2.-7. (canceled)

8. The nanoparticles according to claim 1, characterized in that the copolymers comprise a recurring structural unit of the formula (Ia) and two different recurring structural units of the formula (Ib), in which R.sup.1 and R.sup.6 are hydrogen, R.sup.2 and R.sup.7, independent of each other, are hydrogen or methyl, in particular methyl, R.sup.3 is —COO—R.sup.10—, R.sup.10is ethylene, R.sup.4 and R.sup.5, independent of each other, are C.sub.1-C.sub.6 alkyl, in particular methyl, and R.sup.8 is —COO—R.sup.11, where, in one recurring structural unit of the formula (Ib), R.sup.11 is C.sub.1-C.sub.3 alkyl, in particular methyl, and, in another recurring structural unit of the formula (Ib), R.sup.11 is C.sub.4-C.sub.6 alkyl, in particular n-butyl.

9. The nanoparticles according to claim 1, characterized in that, in addition to the recurring structural units of the formulae (Ia) and (Ib), the copolymers also contain further recurring structural units of the formula (Ic) ##STR00005## wherein R.sup.12, R.sup.13 and R.sup.14, independent of each other, are hydrogen or alkyl, preferably hydrogen or C.sub.1-C.sub.6 alkyl, particularly preferably hydrogen or methyl, and BG represents a bivalent organic bridging group with ether, ester, amide, sulfide, phosphate or disulfide groups.

10. The nanoparticles according to claim 1, characterized in that the molar proportion of the recurring structural units of the formula (Ia) is between 10 and 75%, preferably between 15 and 65% and very particularly preferably between 20 and 55%, based on the total cationic copolymer, and in that the molar proportion of the recurring structural units of the formula (Ib) is between 90 and 25%, preferably between 85 and 45% and very particularly preferably between 80 and 45%, based on the total cationic copolymer.

11. The nanoparticles according to claim 1, characterized in that their particle diameters (z-average) range between 40 and 250 nm, determined by light scattering.

12. The nanoparticles according to claim 1, characterized in that their polydispersity index of particle size distribution, measured with the Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, United Kingdom) using cumulant analysis of the correlation function (ISO13321, ISO22412), ranges between 0.05 and 0.4, preferably between 0.1 and 0.4 and particularly preferably between 0.1 and 0.3.

13. The nanoparticles according to claim 1, characterized in that the polydispersity index of molar mass distribution of the cationic copolymers used ranges between 1.0 and 3.0, preferably between 1.01 and 2.6.

14. The nanoparticles according to claim 1, characterized in that they have a transfection efficiency for pDNA of 15 to 50% (viable fluorescent cells), in particular of 20 to 45%, after 1 hour incubation time of cells with the nanoparticles and 23 hours subsequent incubation of the cells in growth medium without nanoparticles.

15. The nanoparticles according to claim 1, characterized in that the molar ratio of nitrogen atoms in the copolymer to phosphate groups in the nucleic acid is between 1 and 100, preferably between 2.5 and 100 and very particularly preferably between 5 and 50.

16. The nanoparticles according to claim 15, characterized in that they have diameters between 40 and 250 nm, determined by DLS, and a polydispersity index of particle diameters between 0.1 and 0.3.

17. The nanoparticles according to claim 16, characterized in that their molar ratio of nitrogen atoms in the copolymer to phosphate groups in the nucleic acid is between 10 and 30.

18. The nanoparticles according to claim 1, characterized in that they are present dispersed in water, and in that their proportion by weight in the dispersion is between 0.01 and 20%, preferably between 0.05 and 5%.

19. A method for the production of nanoparticles comprising the following measures: i) production of an aqueous solution of a cationic copolymer containing the recurring structural units of the formulae (Ia) and (Ib) according to claim 1 having a pH between 3 and 6.5, ii) production of an aqueous solution of a nucleic acid, iii) mixing of the two solutions produced in steps i) and ii) in a selected quantity ratio of nucleic acid and copolymer to give a desired molar N/P ratio of nitrogen atoms in the copolymer to phosphate groups in the nucleic acid between 1 and 200, and iv) agitation of the resulting mixture.

20. The method according to claim 19, characterized in that it comprises, as step v), an incubation of the obtained mixture.

21. The method according to claim 19, characterized in that the aqueous solution of the cationic copolymer for step i) contains a buffer, in particular an acetate buffer, citrate buffer, lactate buffer, phosphate buffer, phosphate-citrate buffer or mixtures of the buffers.

22. The method according to claim 19, characterized in that the aqueous solution of the nucleic acid for step ii) has a pH from 6.5 to 8.5, in particular from 6.8 to 7.5.

23. The method according to claim 22, characterized in that the aqueous solution of the nucleic acid for step ii) contains a buffer, in particular an HBG, HEPES, BIS-TRIS propane or TRIS buffer.

24. A method for gene delivery into cells comprising the following steps: A) bringing cells into contact with an aqueous suspension comprising the nanoparticles according to claim 1, and B) subsequent incubation.

25. The method according to claim 24, comprising the following steps: C) provision of a cell culture in a bioreactor or incubator, D) addition of an aqueous suspension comprising the nanoparticles according to at least one of claims 1 to 18, E) distribution of the aqueous suspension in the cell culture, and F) subsequent incubation.

26. The method according to claim 24, characterized in that the cells used are selected from the group consisting of single cells, tissues or cell cultures.

27. (canceled)

Description

EXAMPLE 1A

Synthesis of (nBMA-st-MMA-st-DMAEMA) copolymer (PBMD) by RAFT polymerization (st=statistically distributed)

[0173] CPAETC (130.7 mg, 4.96×10.sup.−4 mol), nBMA (3.5265 g, 2.48×10.sup.−2 mol), MMA (2.5218 g, 2.52×10.sup.−2 mol), DMAEMA (7.8165 g, 4.97×10.sup.−2 mol), 1,4-dioxane (6.2113 g), a 1.0% by weight ACVA solution in 1,4-dioxane (1.436 g, 14.36 mg ACVA, 5.12×10.sup.−5 mol) and 1,3,5-trioxane (external NMR standard, 23.7 mg) were introduced into a 20 ml microwave vial equipped with a magnetic stirrer. The solution was deoxygenated by bubbling with argon for 10 minutes. The vial was sealed, placed in a 70° C. oil bath and stirred for 21 hours, with samples taken at predetermined times for .sup.1H—NMR and DMAc-SEC analysis. The polymer was precipitated three times from THF into cold hexane and dried under reduced pressure to give a yellow solid. DMAc-SEC: M.sub.n,SEC=25.1 kg mol.sup.−1, Ð=1.13.

EXAMPLE 1B

[0174] Synthesis of DMAEMA homopolymer by RAFT polymerization

[0175] CPAETC (50.0 mg, 1.9×10-4 mol), DMAEMA (4.54 g, 2.88×10-2 mol), 1,4-dioxane (2.5 g), 1% by weight ACVA in 1,4-dioxane (426 mg, 1.5×10-5 mol) and 1,3,5-trioxane

[0176] (external NMR standard, 21 mg) were introduced into a 20 mL microwave vial with magnetic stirrer. The vial was sealed and the solution was deoxygenated by bubbling with argon for approx. 10 min. The vial was placed in an oil bath set at 70° C. and stirred for 7 hours, with samples taken at set times for .sup.1H—NMR and CHCl3-SEC analysis. The polymer was precipitated three times from THF into cold hexane and dried under reduced pressure to give a yellow solid. CHCl.sub.3-SEC: M.sub.n,SEC=14.2 kg mol-1, Ð=1.19.

[0177] FIG. 1A shows the structure of the copolymer Eudragit® E100.

[0178] FIG. 1B shows the structure of the copolymer represented by RAFT polymerization according to Example 1A.

[0179] FIG. 2 shows the molecular weight distribution of the copolymer Eudragit® E100 and the copolymer represented by RAFT polymerization according to Example 1A, determined by size exclusion chromatography (SEC).

EXAMPLE V1

Formation of nanoparticles and complexation with pDNA (not according to the invention)

[0180] For nanoparticle formation, the copolymers produced in Example 1 were dissolved in 2.5 mL acetone (2 mg mL.sup.−1). The polymer solution was manually dropped into 5 mL ultrapure water, the resulting nanoparticle suspension was stirred overnight at 800 rpm to remove the organic solvent, and stored at 4° C. until use. To achieve the respective N/P ratio, pDNA was either added in different concentrations during the formation process during the acetone phase and subsequent nanoparticle formation or pDNA was added in different concentrations to the final particle suspension.

EXAMPLE 2

Formation of nanoparticulate copolymer-nucleic acid complexes (according to the invention)

[0181] Stock solutions of the copolymers produced according to Example 1 were produced by dissolving in 0.2 M acetate buffer (pH 5.8). pDNA, siRNA or mRNA were dissolved in ultrapure water. To produce nucleic acid-polymer complexes, different dilutions of the copolymer as well as the nucleic acid were prepared in HBG buffer (20 mM HEPES, 5% glucose (w/v), pH 7.4) or in 20 mM HEPES buffer to achieve the respective N/P ratios (molar ratio of nitrogen atoms in the copolymer to the phosphate groups in the nucleic acid). After mixing the copolymer solution and the nucleic acid solution, the mixtures were immediately vortexed for 10 seconds. Before use, the resulting copolymer-nucleic acid complexes were incubated at room temperature for at least 15 minutes.

[0182] FIG. 3 schematically shows the formation of nucleic acid-copolymer complexes in nanoparticles from the copolymer according to Example 1A (DMAEMA:BMA:MMA=2:1:1).

[0183] The use of the cationic and hydrophobic copolymer results in binding, stabilization and protection of the genetic material, formation of stable nanoparticles, stability against competing polyanions and causes endosomal release after uptake by the cell.

EXAMPLE 3

General rule for the characterization of nanoparticles and of nanoparticulate copolymer-pDNA complexes

EXAMPLE 3A

[0184] Diameter (z-average) and zeta potential of nanoparticles and pDNA-copolymer complexes were determined by dynamic or electrophoretic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, Worcestershire, United Kingdom). For size determination, a refractive index of 1.33 was assumed for ultrapure water and 1.59 for the copolymer. The zeta potential of the nanoparticles produced by precipitation in the presence of organic solvents was determined on the same samples.

Example 3B

[0185] Gel retardation examination

[0186] The pDNA binding ability at different N/P ratios was determined by agarose gel electrophoresis. Samples were produced as described for the pDNA-polymer complexes in Examples V1 and 2, ran at 80 V for 1.5 hours on a 1% agarose gel stained with ethidium bromide (EtBr, 0.1 μg mL.sup.−1) and imaged using a gel imager (Red™ Imaging System, Alpha Innotech, Kasendorf, Germany).

[0187] Example 3C

[0188] Ethidium bromide binding assay (EBA) and heparin release assay (HRA)

[0189] pDNA complexation and stability of the nanoparticulate copolymer-pDNA complexes were investigated by using an ethidium bromide binding assay and a heparin release assay.

[0190] For this purpose, pDNA at a concentration of 15 μg mL.sup.−1 was incubated with ethidium bromide for 10 minutes. The polymer stock solutions were diluted in a black 96-well plate (Nunc, Thermo Fisher) to adjust N/P ratios from 1 to 50. Then pDNA was added and the nanoparticulate copolymer-pDNA complexes were incubated at 37° C. for 15 minutes. Ethidium bromide fluorescence intensity was measured at λ.sub.Ex=525 nm/λ.sub.EM=605 nm. pDNA without copolymer was defined as 100% free DNA. The release of complexed DNA was investigated by gradual addition of heparin and measurement of the resulting changes in ethidium bromide fluorescence intensity. The influence of pH on pDNA binding and pDNA release was investigated by performing the experiment at different pH values in the respective buffers (acetate buffer pH 5 and 5.8 and HBG buffer pH 6.5; 7 and 7.4).

Example 3D

[0191] Transfection of HEK293T cells with EGFP pDNA

[0192] The HEK293T cell line was cultured in Dulbecco's modified Eagle's medium (DMEM, 1 g L.sup.−1 glucose, 10% (v/v) FBS, 100 g mL.sup.−1 penicillin/streptomycin) at 37° C. in a humidified 5% CO.sub.2 atmosphere. For transfection experiments, 0.2*10.sup.6 cells per mL were seeded into a 24-well plate in 500 μL DMEM supplemented with 10 mM HEPES and left to recover for 24 hours. 1 hour before treatment, the treatment medium was replaced with 450 μL fresh DMEM (10 mM HEPES). Nanoparticulate copolymer-pDNA complexes were freshly prepared as described in Example 2 using egfp pDNA encoding for EGFP or pkmyc pDNA not encoding for a fluorescent protein as negative controls. The cells were treated with 50 μL of a dispersion of nanoparticulate copolymer-pDNA complexes of the indicated N/P ratio and pDNA concentration or with HBG buffer as a control (ctrl) and incubated for 1 or 4 hours. The supernatant was then removed, cultured by ctrl and incubated. The supernatant was then removed, replaced with fresh DMEM (10 mM HEPES) and the cells were further incubated for up to 24 hours. After incubation, cells were separated by trypsin-EDTA, resuspended in HBSS (2% FBS (v/v), 20 mM HEPES) and fluorescence was measured using a flow cytometer (Cytoflex S, Beckmann coulter, Calif., U.S.A.). EGFP expression of viable cells was analyzed by excitation at 488 nm and measurement of emission at 610 nm (bandpass filter 610/20). Fluorescent cells were identified by gating to the negative control.

Example 3E

[0193] Knock-down of GFP in HEK-GFP cells using anti-EGFP siRNA

[0194] The HEK-GFP cell line was cultured in Dulbecco's modified Eagle's medium (DMEM, 1 g L.sup.−1 glucose, 10% (v/v) FBS, 100 g mL.sup.−1 penicillin/streptomycin) at 37° C. in a humidified 5% CO.sub.2 atmosphere. For transfection experiments, 0.1*10.sup.6 cells per mL were seeded into a 24-well plate in 500 μL DMEM supplemented with 10 mM HEPES and left to recover for 24 hours. 1 hour before treatment, the treatment medium was replaced with 450 μL fresh DMEM (10 mM HEPES). Copolymer-siRNA complexes were freshly prepared as described in Example 2. The cells were treated with 50 μL of the freshly prepared complexes. Complexes with siRNA not directed against GFP and HBG buffer were used as negative controls. 72 hours after treatment with copolymer-siRNA complexes, the supernatant was removed and the cells were separated with trypsin-EDTA. After resuspension in HBSS (2% FBS (v/v), 20 mM HEPES), the cells were analyzed by flow cytometry at an excitation wavelength of 488 nm at 525 nm (bandpass filter 525/40).

Example 3F

[0195] Transfection of HEK293T cells with GFP mRNA

[0196] The HEK293T cell line was cultured in Dulbecco's modified Eagle's medium (DMEM, 1 g L.sup.−1 glucose, 10% (v/v) FBS, 100 g mL.sup.−1 penicillin/streptomycin) at 37° C. in a humidified 5% CO.sub.2 atmosphere. For transfection experiments, 0.2*10.sup.6 cells per mL were seeded into a 24-well plate in 500 μL DMEM supplemented with 10 mM HEPES and left to recover for 24 hours. 1 hour before treatment, the treatment medium was replaced with 450 μL fresh DMEM (10 mM HEPES). Copolymer-mRNA complexes were freshly prepared as described in Example 2. 50 μL of the copolymer-mRNA complexes were added to the cells and incubated for 4 hours. The supernatant was then removed and the cells were incubated for a further 2 or 20 hours. After incubation, the supernatant was removed, the cells were detached using trypsin-EDTA and resuspended in HBSS (2% FBS (v/v), 20 mM HEPES). Subsequently, the fluorescence of the cells was analyzed by flow cytometry. For this purpose, an excitation wavelength of 488 nm was used and the emission was measured at 525 nm (bandpass filter 525/40).

Example 3G

[0197] PrestoBlue® test to determine viability

[0198] For cytotoxicity assays, HEK293T cells were seeded into 24-well plates at a density of 0.2 10.sup.6 cells per mL (HEK293T) in 500 μL DMEM (10 mM HEPES) and incubated for 24 hours to enable recovery. 50 μL nanoparticulate copolymer-pDNA complexes were added as described in Example 2 to test a concentration range of 0.25-1.5 g mL.sup.−1 pDNA.

[0199] The cells were incubated with the nanoparticulate copolymer-pDNA complex for 1 or 4 hours. The supernatant was then removed and replaced with 500 μL fresh DMEM (10 M HEPES). 24 hours after treatment, the supernatant was removed and this was replaced with a 10% (v/v) solution of PrestoBlue® (Invitrogen, Calif., U.S.A.) diluted with DMEM medium. The cells were incubated for 45 min and the supernatant was transferred to a 96-well plate (100 μL per well) to determine fluorescence intensity (λ.sub.Ex 560 nm, λ.sub.EM 590 nm). Cells treated with the HBG buffer were used as control and viability was calculated relative to the buffer control after subtracting the blank (PrestoBlue® without cells).

[0200] Investigations of Nanoparticulate Polymer Particles and Nanoparticulate DNA-Polymer Complexes

[0201] The binding of genetic material is a crucial step in the gene delivery process, as the genetic material must overcome extracellular and intracellular barriers to reach their target cells. Encapsulation and complexation by cationic polymers, for example, enable protection from nucleases in the bloodstream and the crossing of cell membranes (see in this regard H. Yin, R. L. Kanasty, A. A. Eltoukhy, A. J. Vegas, J. R. Dorkin, D. G. Anderson, Nat Rev Genet 2014, 15, 541-555).

EXAMPLE 4

Experiments on the pDNA-binding ability of polymers and the release of pDNA from the complexes formed

[0202] FIG. 4 describes results of experiments on the DNA-binding ability of polymers and the release of pDNA from the complexes formed.

[0203] FIG. 4A shows results of an ethidium bromide binding assay (EBA) on DNA-polymer complexes in HBG buffer at pH 7.4. DNA-IPEI complexes, DNA-PDMAEMA complexes and DNA-PBMD complexes were examined.

[0204] The binding of the genetic material is crucial for its application in gene delivery. Therefore, the pDNA-binding ability of the polymer was investigated by agarose gel electrophoresis at different N/P ratios. FIG. 4B shows results of agarose gel electrophoresis tests with DNA-PBMD complexes at different N/P ratios.

[0205] FIGS. 4C-4E show results of heparin release assays (HRA) for pH-dependent DNA binding and DNA release in the pH range from 5 to 7.4. DNA-IPEI complexes, DNA-PDMAEMA complexes and DNA-PBMD complexes were examined.

[0206] The test shown in FIG. 4B confirmed the complete binding of pDNA at N/P ratios above 1. The DNA-polymer interaction can be further characterized by the reversible intercalation of ethidium bromide into the DNA. Strong fluorescence can be observed when this is intercalated into the pDNA. However, this is reduced by the interaction between polymer and genetic material (EBA). The test was carried out at pH 7.4 and N/P ratios from 1 to 50 in order to investigate the effect of an increasing polymer amount on complex formation. In addition to the PBMD polymer, IPEI and a PDMAEMA homopolymer were investigated (FIG. 4A). The latter is composed of the same number of cationic groups as the PBMD polymer in order to evaluate the influence of hydrophobic side chains in the PBMD polymer. It was observed that the relative fluorescence intensity (RFI) decreased with increasing N/P ratios, reaching a plateau at an N/P ratio of 10 (FIG. 4A). IPEI showed the lowest plateau values, followed by PDMAEMA (13% compared to 42%) while PBMD only decreased the RFI to 72%. Since the electrophoresis test showed complete DNA binding above N/P 1, these differences could be due to differences in DNA condensation and thus differently packed complexes, which may lead to different levels of EtBr displacement. Therefore, it can be assumed that the PBMD polymer forms a less dense DNA-copolymer complex compared to IPEI and PDMAEMA.

[0207] To investigate the stability of the complexes in the relevant physiological pH range, the dissociation of the complexes was examined at pH values from 5 (endosomal) to 7.4 (blood). Preformed DNA-copolymer complexes with an N/P ratio of 20 were incubated with increasing amounts of heparin as the competing polyanion, and the fluorescence intensity was measured. Dissociation, and thus the release of pDNA from the complex, caused re-intercalation of EtBr. For all polymers, it was observed that ambient pH has an influence on complex formation prior to the addition of heparin. For IPEI (upper FIG. 4C), a notable displacement of EtBr and subsequent release of pDNA was observed at relatively low heparin concentrations (20 U mL.sup.−1). pDNA complexes formed at lower pH values required slightly less heparin addition to release the pDNA, but at high heparin concentrations (100 U/ml), pDNA was fully released at all pH values. In general, pDNA release from PDMAEMA complexes required higher amounts of heparin (middle FIG. 4D) and was more strongly influenced by ambient pH in comparison to IPEI. At low pH values (5 and 5.8), 100 U/ml heparin resulted in full release of pDNA, whereas at pH values of 6.5 and above, only 66-79% was released. The PBMD copolymer (lower FIG. 4E) showed an even more pronounced pH-dependent behavior. RFI values before heparin addition range from 27 to 60% and are lower at pH values of 5 to 5.8. The release of the entirety of pDNA was only observed at pH 5 and decreased gradually with increasing pH. At neutral pH, a slight decrease in RFI after heparin addition indicates even stronger complexation of pDNA in the presence of competing polyanions. In general, IPEI was hardly affected by pH, while PDMAEMA and PBMD copolymer showed a weak and the strongest influence of pH, respectively, on the initial RFI values and the subsequent pDNA release.

[0208] These results suggest an influence of the pK.sub.a value and the hydrophobicity of the copolymer on pDNA binding, arrangement in the complex and subsequent pDNA release. For IPEI, a pK.sub.a value of 8.5 is reported in the literature, while PDMAEMA has a pK.sub.a of about 7.5. For PBMD, only an apparent pK.sub.a of 6.8 could be determined, as the polymer precipitated in this pH range during titration.

[0209] When calculating the percentage of charges from these pK.sub.a values, a charge level of 100 to 90% was calculated for IPEI over the entire pH range tested, while PDMAEMA showed a decrease from 100% at pH 5 to 56% at pH 7.4. When calculating the charge level for PBMD with the apparent pK.sub.a value, the effect is even more pronounced (98% at pH 5 and 20% at pH 7.4). As the charge level and thus the number of cationic groups in the polymer decreases, the ratio of hydrophilic to hydrophobic groups changes, leading to additional hydrophobic interactions and to different arrangements in the complex and thus to altered release behavior of the DNA. This effect is most clearly illustrated in the PBMD copolymer, where the additional hydrophobic side chains of the nBMA and MMA units promote complex stability, especially at neutral pH values, through strong hydrophobic interactions (see in this regard E. J. Adolph, C. E. Nelson, T. A. Werfel, R. Guo, J. M. Davidson, S. A. Guelcher, C. L. Duvall, J. Mater Chem B 2014, 2, 8154-8164). Thus, the PBMD copolymer shows high potential as a gene delivery vector, as high stability and prevention of dissociation at neutral pH in the blood stream is advantageous for systemic application of complexes.

Example 5

Experiments on the complexation of pDNA

[0210] FIGS. 5 and 6 describe results of experiments on the complexation of pDNA in different polymers and the formation of nanoparticles.

[0211] FIG. 5A schematically shows the formation of nanoparticles by nanoprecipitation with solvent evaporation technique.

[0212] The left side of FIG. 5B shows an electron micrograph (scanning electron microscopy, SEM) of nanoparticles of Eudragit® E100 precipitated from acetone-water mixtures.

[0213] The right side of FIG. 5B shows an electron micrograph (scanning electron microscopy, SEM) of nanoparticles of PBMD copolymer precipitated from acetone-water mixtures.

[0214] FIG. 6 schematically shows the formation of nanoparticulate DNA complexes of PBMD with pDNA depending on the formulation method used. The left side of FIG. 6 shows the nanoprecipitation with solvent evaporation techniques combined with different techniques of pDNA addition (in-process and post-process addition). The right side of FIG. 6 shows the water-based solvent-free pH-dependent formulation for complexation of pDNA. The obtained nanoparticles were characterized by dynamic light scattering.

[0215] The upper FIG. 7A shows the diameter (z-average) of nanoparticles of PBMD copolymer and pDNA at different N/P ratios produced by precipitation from acetone or aqueous solution. The middle FIG. 7B shows the PDI values of the diameters of nanoparticles of PBMD copolymer and pDNA at different N/P ratios produced by precipitation from acetone or from aqueous solution. The lower FIG. 7C shows the surface charge (zeta potential) of nanoparticles of PBMD copolymer and pDNA produced by means of the solvent evaporation technique from acetone.

[0216] The left two FIGS. 7D show electron micrographs (scanning electron microscopy, SEM) of nanoparticles of PBMD copolymer produced at two different N/P ratios by nanoprecipitation from acetone in the presence of pDNA. The right FIG. 7D shows an electron micrograph (cryogenic transmission electron microscopy, cryo-TEM) of nanoparticles of PBMD copolymer produced by nanoprecipitation from aqueous solution.

[0217] Since the PBMD copolymer showed high potential for pDNA binding and high complex stability at the pH of blood, the copolymer was used to develop a stable formulation for encapsulating the pDNA. Three different formulation approaches were investigated. The commonly used nanoprecipitation with addition of DNA [0218] a) for the purpose of final nanoparticle suspension or [0219] b) during the formulation process was compared with [0220] c) the complexation of the DNA by the polymer dissolved in the acidic buffer as a pH-dependent water-based nanoprecipitation.

[0221] Nanoprecipitation is a widely used method for the formulation of polymeric nanoparticles that allows easy adjustment of nanoparticle size and complexation of a variety of components. Nanoprecipitation of the PBMD copolymer without the addition of pDNA results in particle diameters of about 125 nm (N/P ratio 0) with a positive surface charge (+55 mV) (see FIG. 7C). It was expected that the positive surface charge of the nanoparticles should facilitate the binding and complexation of pDNA to the particle surface. Addition of pDNA to the pre-formulated positively charged nanoparticles (NP+pDNA) generally resulted in increased size and polydispersity of the nanoparticles (FIGS. 7A and 7B). Only at the lowest and highest N/P ratios investigated (N/P 5 and N/P 100) were nanoparticles found with sizes below 200 nm and with acceptable PDI values (PDI<0.250).The surface charge of the particles decreased with increasing DNA amount and shifted to negative values at N/P 10 (FIG. 7C).These results indicate that pre-formulated nanoparticles (NP+pDNA) have limited binding ability for pDNA and do not completely condense the pDNA during complexation. Pre-incubation of the DNA with the polymer dissolved in acetone and subsequent nanoprecipitation in water (NP(pDNA)) leads to a similar N/P ratio-dependent trend of nanoparticle size and homogeneity. Interestingly, the zeta potential remains in the positive range compared to the NP+pDNA formulation. In general, particles obtained by adding the pDNA in the procedure showed a slightly smaller z-average value than those obtained by adding the pDNA afterwards. This could be due to improved interaction of the pDNA with the dissolved polymer chains during the nanoprecipitation process. Overall, this formulation approach did not significantly improve DNA encapsulation, and the amount of complexed pDNA that resulted in stable nanoparticles in the size range below 200 nm with low polydispersity was limited to relatively high N/P ratios (N/P 100).

[0222] The copolymer Eudragit® E100 was also used for nanoparticle formation and pDNA complexation, in addition to the polymers PDMAEMA, IPEI and PBMD copolymer described above.

[0223] Formulation methods based on polymers and organic solvents for nanoprecipitation and related formulation methods are known in principle (see R. Jain, P. Dandekar, B. Loretz, M. Koch, C.-M. Lehr, MedChemComm 2015, 6, 691-701; N. Kanthamneni, B. Yung, R.J. Lee, Anticancer research 2016, 36, 81-85; M. Gargouri, A. Sapin, S. Bouali, P. Becuwe, J. Merlin, P. Maincent, Technology in cancer research & treatment 2009, 8, 433-443).

[0224] According to the invention, a new organic solvent-free, water-based formulation method is provided, which was inspired by the joint complexation of DNA with water-soluble polymers. The Eudragit® E100 copolymer used here and the PBMD copolymer produced by RAFT are soluble under acidic conditions due to the protonation of the DMAEMA groups.

[0225] To implement the method according to the invention, sodium acetate buffer (pH 5.8) was used to dissolve the copolymers before mixing with pDNA. This formulation approach resulted in nanoparticles in the range of 100 nm with decreasing diameter at higher N/P ratios and with PDI values around 0.25 for all N/P ratios tested (see FIGS. 7A and 7B). Overall, much lower N/P ratios were obtained with this formulation method, indicating better complexation and condensation of the pDNA by the PBMD copolymer. This could be due to a higher charge fraction of the polymer when dissolved in the acetate buffer, and thus better complexation and condensation of the pDNA. Low N/P ratios are preferred for gene delivery. At lower N/P ratios, lower amounts of copolymer can be used, resulting in more efficient gene delivery and lower toxic potential due to reduced copolymer concentrations. Since the complex formulation meets these criteria, this formulation was tested for in vitro transfection compared to the NP+pDNA formulation. The NP(pDNA) formulation was not further investigated as the low N/P ratios tested in the in vitro experiments (10 and 20) could not be produced due to failure and strong aggregation during the formulation process.

Example 6

[0226] Experiments on pDNA transfection efficiency in HEK293T cells

[0227] FIGS. 8, 9 and 10 describe results of experiments on the transfection efficiency of pDNA-copolymer complexes in cells.

[0228] FIG. 8A shows comparisons of formulation methods and their transfection efficiency measured by flow cytometry.

[0229] FIG. 8B describes results obtained by fluorescence microscopy.

[0230] FIG. 8C describes the results of the optimization of the N/P ratio and the pDNA concentration in complexes with PBMD copolymer.

[0231] FIG. 9 describes results of transfection experiments with complexes of pDNA with IPEI.

[0232] FIG. 10 describes results of transfection experiments with complexes of pDNA with PBMD copolymer as well as with various commercially available polymers such as Eudragit® 100.

[0233] The transfection efficiency of the pH-dependent formulation prepared by pDNA addition after nanoparticle generation was investigated by transferring EGFP-encoding pDNA into HEK293T cells and measuring the EGFP expression after 24 hours. The transfection efficiency of the formulation was determined by the addition of pDNA after nanoparticle generation. Two different N/P ratios were tested in the non-toxic region (FIG. 8A) of the copolymer. Complexes were used which had been generated by precipitation of the nanoparticles from acetone followed by pDNA addition (right part of FIG. 8A and C) and which had been generated by precipitation of the nanoparticles from acidic aqueous solution in the presence of pDNA (left part of FIG. 8A). Two different media containing different levels of FCS were also used. One medium contained 10% FCS and another medium was serum-reduced (Opti-MEM, 2% FCS). The left two columns in each part of FIG. 8A describe the results in the serum-containing medium; the right two columns in each part of FIG. 8A describe the results in serum-free medium.

[0234] FIG. 8B shows fluorescence microscopy examinations of the transfected cells after 1+23 incubation time (without sample) or after 4+20 hours incubation time (sample). Complexes were used which had been generated by precipitation of the nanoparticles from acetone followed by addition of pDNA (bottom row of FIG. 8B) and which had been generated by precipitation of the nanoparticles from acidic aqueous solution in the presence of pDNA (middle row of FIG. 8B). N/P ratios of 20 were used in each case.

[0235] In addition, the top row of FIG. 8B presents results showing cells that received HBG buffer instead of nanoparticles or complexes, serving as an internal control within the experiment (ctrl).

[0236] In general, both formulations, those produced by precipitation from acetone and those produced by precipitation from acidic aqueous solution, showed transfection in HEK293T cells with higher efficiency under serum-reduced conditions and at higher N/P ratios. The pH-dependent formulation showed higher and more consistent transfection efficiencies compared to the formulation produced by precipitation from acetone. This could be due to aggregates formed by the formulation of pDNA addition after the procedure with particle diameters>4 500 nm and with higher polydispersity in the DLS measurements (FIGS. 7A and 7B). Therefore, the sedimentation of the different species in the formulation differs depending on their size, leading to variations in cellular uptake and transfection efficiency.

[0237] The pH-dependent formulation leads to nanoparticles with less variation in sizes without the formation of aggregates. This could lead to more controlled uptake into cells and more consistent transfection rates.

[0238] Since the pH-dependent formulation yields particles with preferred N/P ratio, size and homogeneity, this formulation was further investigated and optimized for transfection efficiency. The conditions during transfection with PBMD-copolymer complexes were optimized for high transfection efficiency while maintaining high cell viability. Furthermore, the minimum amount of copolymer and pDNA was to be identified. Different pDNA concentrations were investigated, keeping the N/P ratio constant at 20, as this was found to be optimal (see left half of FIG. 8C). Subsequently, the ideal pDNA concentration (0.5 mg mL.sup.−1) was further optimized by varying the N/P ratio (see right half of FIG. 8C).

[0239] The results from FIG. 8C were compared with complexes formed with p-DNA and IPEI as the gold standard (see FIG. 9). Overall, complexes formed with PBMD-copolymer showed high transfection efficiencies at lower pDNA concentrations and shorter incubation times compared to complexes formed with pDNA and IPEI.

[0240] Incubation times of 4 hours resulted in higher transfection efficiencies for complexes of pDNA and PBMD copolymer compared to complexes of p-DNA and IPEI over the range of concentrations and N/P ratios investigated, but also resulted in reduced cell viability at higher polymer concentration (=larger N/P ratio) (see FIGS. 8C and 9). When the pDNA concentration was increased (8.59% at 0.25 μg mL.sup.−1 to 64.3% at 1.5 μg mL.sup.−1, 4 hours incubation time), an increase in transfection efficiency was observed (see left part of FIG. 8C). Increasing the N/P ratio at constant pDNA concentration resulted in higher efficiencies (see right part of FIG. 8C), but led to increased cell detachment and cell death. Therefore, 0.5 μg mL.sup.−1 at an N/P ratio of 20 was chosen as the ideal conditions for effective pDNA transfection with the PBMD-copolymer complex.

[0241] Compared to the gold standard IPEI, the pDNA concentration could be reduced to 0.5 μg mL.sup.−1, which still resulted in 22.5% fluorescent cells after 1 hour incubation, and which was not possible with IPEI in this short incubation time and in this investigated pDNA concentration range (0.5 to 5 μg mL.sup.−1) (see left part of FIG. 8C with FIG. 9). To achieve higher transfection efficiencies, either the pDNA concentration must be increased or incubation times of up to 24 hours are required (see FIG. 9). PDMAEMA showed almost no transfection even with very high pDNA concentrations of 5 μg mL.sup.−1 and incubation times of 24 hours. These results support the hypothesis of the influence of hydrophobic side chains on the in vitro performance of cationic polymers as gene delivery vectors.

[0242] FIG. 10 shows the transfection efficiency of pDNA complexes with PBMD copolymer, with PDMAEMA homopolymer and with the commercial polymers Eudragit® E100, Viromer red and IPEI.

[0243] The formulations for PBMD and EUDRAGIT® E100 were produced by precipitation from acidic aqueous solution in the presence of pDNA. Expression of eGFP in HEK293T cells after transfection was measured by flow cytometry. HEK293T cells were transfected at an N/P ratio of 20 and a pDNA concentration of 0.5 μg mL.sup.−1.

[0244] The pDNA-PBDM complex formulation technique was further applied to the commercial polymer Eudragit® E100 and investigated under the optimized transfection conditions. In addition, the transfection efficiency was compared with the commercial transfection agent Viromer Red. Overall, the pH-dependent formulation was applicable to the commercial polymer EUDRAGIT® E100, which showed comparable transfection efficiencies after 1 hour incubation with the pDNA-PBMD-copolymer complex, but showed a slightly lower efficiency after 4 hours incubation. This could be due to the slightly higher DMAEMA content in the PBMD copolymer. A higher cationic content would lead to higher DNA binding and increased membrane activity.

[0245] Both copolymers are clearly superior in performance to the commercial transfection agents Viromer Red and IPEI.

[0246] FIG. 11 describes results of experiments on the transfection efficiency of siRNA complexes in cells.

[0247] To determine knock-down efficiency, anti-GFP siRNA was introduced into HEK-GFP cells showing stable expression of GFP by copolymer-siRNA complexes produced by precipitation from aqueous acidic solution. Knock-down efficiency after 72 hours incubation was compared with the gold standard Lipofectamine. Cells treated only with HBG buffer served as control. Both Lipofectamine and copolymer-siRNA complexes showed a reduction in the number of fluorescent cells (GFP positive cells) compared to the control (100%). Copolymer-siRNA complexes showed a reduction of GFP positive cells to 42.9% at an N/P ratio of 10 and a further reduction to 25.2% at an N/P ratio of 5. Lipofectamine resulted in a reduction to 8.6%. Thus, the PBMD copolymer also shows a high potential for the introduction of siRNA into cells, which can still be improved by further optimization of the transfection conditions such as incubation time, N/P ratio and siRNA quantity.

[0248] FIG. 12 describes results of experiments on the transfection efficiency of mRNA complexes in cells.

[0249] The precipitation of complexes of genetic material and copolymer was also applied to the production of copolymer-mRNA complexes. The transfection efficiency of these complexes was determined by introducing GFP mRNA into HEK293T cells. Different N/P ratios (5, 10, 20) and mRNA concentrations (0.25 to 0.75 μg mL.sup.−1) were tested. The cells were incubated for 4 hours with the complexes. 6 and 24 h after the start of treatment, GFP expression was determined by fluorescence measurement in flow cytometry. The transfection efficiency of the copolymer-mRNA complexes was compared with the commercially available Viromer Red, which was developed for the transfection of mRNA and shows high efficiencies in the literature. Cells incubated only with HBG buffer or mRNA without copolymer served as negative controls. Both Viromer Red and copolymer-mRNA complexes led to mRNA concentration-dependent GFP expression in HEK293T cells. A higher mRNA concentration entailed higher expression levels, while an increase in the N/P ratio in the copolymer-mRNA complexes also entailed an increase. Viromer Red showed the highest transfection efficiency at an mRNA concentration of 0.75 μg mL.sup.−1and an incubation time of 24 hours (55.8%). The highest transfection efficiency was achieved after 24 hours incubation with copolymer-mRNA complexes with an N/P ratio of 20 and an mRNA concentration of 0.75 μg mL.sup.−1 (73.1%). In addition to introducing pDNA and siRNA into cells, the PBMD copolymer thus also shows a high potential for introducing mRNA into cells.

[0250] FIGS. 13A-13C describe results for the synthesis and characterization of different PBMD copolymers with varying molecular weights.

[0251] They show the synthesis route for the production of the PBMD copolymers (described in Example 1A, FIG. 13A) and the molecular weight distribution and polydispersity of the copolymers prepared by RAFT polymerization analogous to Example 1A, as determined by size exclusion chromatography (SEC) (FIGS. 13B and 13C).

[0252] FIG. 14 shows the transfection efficiency of pDNA complexes with PBMD copolymers of different molecular weight in HEK293T cells.

[0253] The transfection efficiency of the PBMD polymer library was investigated in HEK293T cells under the optimized conditions of PBMD182 copolymer. egfp-pDNA was complexed by the copolymers dissolved in acetate buffer and incubated with the cells for either 1 or 4 hours. Overall, all copolymer-pDNA complexes within the PBMD library showed transfection efficiency in HEK293T cells. The results show a clear influence of molar mass and molar content of DMAEMA on transfection efficiency. Copolymer-pDNA complexes with a molecular weight of >15 kDa and a molar DMAEMA content of 50% show transfection efficiencies of 18% and higher depending on the incubation time. The highest transfection efficiencies were achieved with the PBMD182 copolymer (the copolymer prepared according to Example 1A) after 4 hours incubation with the copolymer-pDNA complexes (34.6%). The PBMD185(40%) copolymer with a molar content of DMAEMA of 40% and with a molar mass >15 kDa showed comparatively slightly lower transfection efficiencies (13% after 1 hour incubation, 21.7% after 4 hours incubation) compared to polymers with 50% molar DMAEMA content. The formation of copolymer-pDNA complexes can thus also be transferred to polymers of different molar mass.