Carboxylated polyamine derivatives as transfection reagents

Abstract

The invention provides polyamine derivatives, a use of the polyamine derivatives for the transfection of polyanions into cells, and a method of transfecting cells with a polyanion, comprising mixing said polyanion with a polyamine derivative e.g. in a buffer and treating said cells with the mixture obtained in the previous step.

Claims

1. A polyalkylenimine derivative for the transfection of polyanions into cells comprising: a plurality of carboxylated substituents comprising a carboxyl group bonded via a hydrophobic linker to amino groups of a polyalkylenimine, wherein each of said carboxylated substituents comprises from 6 to 40 carbon atoms, preferably from 6 to 20 carbon atoms, and more preferably from 8 to 16 carbon atoms; and a plurality of hydrophobic substituents bonded to amino groups of said polyalkylenimine, wherein each of said hydrophobic substituents comprises at least 2 carbon atoms.

2. The polyalkylenimine derivative of claim 1, wherein any one or more of said hydrophobic linkers comprises from 1 to 3 heteroatoms selected from O, N, and S.

3. The polyalkylenimine derivative of claim 1, wherein any one or more of said hydrophobic substituents comprises from 1 to 3 heteroatoms selected from O, N, and S provided said hydrophobic substituent has at least 6 carbon atoms.

4. The polyalkylenimine derivative of claim 1 further being complexed to a nucleic acid or a protein.

5. The polyalkylenimine derivative of claim 1 wherein each of said hydrophobic substituents comprises from 6 to 40 carbon atoms.

6. The polyalkylenimine derivative according to claim 1, wherein said carboxylated substituents are carboxyalkyl substituents and said hydrophobic substituents are alkyl substituents.

7. The polyalkylenimine derivative according to claim 6, wherein the number of carbon atoms of said carboxyalkyl substituent together with the number of carbon atoms of said alkyl substituent is between 10 and 30.

8. The polyalkylenimine derivative according to claim 7, wherein the number of carbon atoms of said carboxyalkyl substituent together with the number of carbon atoms of said alkyl substituent is between 15 and 25.

9. The polyalkylenimine derivative according to claim 6, wherein a uniform carboxyalkyl substituent and no mixture of different carboxyalkyl substituents is used and wherein a uniform alkyl substituent and no mixture of alkyl substituents is used.

10. The poylalkylenimine derivative according to claim 9, wherein the number of carbon atoms of said uniform carboxyalkyl substituent together with the number of carbon atoms of said uniform alkyl substituent is between 10 and 30, preferably between 15 and 25.

11. The poylalkylenimine derivative according to claim 10, wherein the number of carbon atoms of said uniform carboxyalkyl substituent together with the number of carbon atoms of said uniform alkyl substituent is between 15 and 25.

12. A polyvinylamine derivative for the transfection of polyanions into cells, comprising: a plurality of carboxylated substituents comprising a carboxyl group bonded via a hydrophobic linker to amino groups of a polyvinylamine, wherein each of said carboxylated substituents comprises from 6 to 40 carbon atoms; and a plurality of hydrophobic substituents bonded to amino groups of said polyvinylamine, wherein each of said hydrophobic substituents comprises at least 2 carbon atoms, preferably from 6 to 40 carbon atoms.

13. The polyvinylamine derivative of claim 12 wherein each of said carboxylated substituents comprises from 6 to 20 carbon atoms.

14. The polyvinylamine derivative of claim 13 wherein each of said carboxylated substituents comprises from 8 to 16 carbon atoms.

15. The polyvinylamine derivative of claim 12 wherein each of said hydrophobic substituents comprises from 6 to 40 carbon atoms.

16. The polyvinylamine derivative of claim 12, wherein any one or more of said hydrophobic linkers comprises from 1 to 3 heteroatoms selected from O, N, and S.

17. The polyvinylamine derivative of claim 12, wherein any one or more of said hydrophobic substituents comprises from 1 to 3 heteroatoms selected from O, N, and S provided said hydrophobic substituent has at least 6 carbon atoms.

18. A polyallylamine derivative for the transfection of polyanions into cells, comprising: a plurality of carboxylated substituents comprising a carboxyl group bonded via a hydrophobic linker to amino groups of a polyallylamine, wherein each of said carboxylated substituents comprises from 6 to 40 carbon atoms; and a plurality of hydrophobic substituents bonded to amino groups of said polyallylamine, wherein each of said hydrophobic substituents comprises at least 2 carbon atoms.

19. The polyallylamine derivative of claim 18 wherein each of said carboxylated substituents comprises from 6 to 20 carbon atoms.

20. The polyallylamine derivative of claim 19 wherein each of said carboxylated substituents comprises from 8 to 16 carbon atoms.

21. The polyallylamine derivative of claim 18 wherein each of said hydrophobic substituents comprises from 6 to 40 carbon atoms.

22. The polyallylamine derivative of claim 18, wherein any one or more of said hydrophobic linkers comprises from 1 to 3 heteroatoms selected from O, N, and S.

23. The polyallylamine derivative of claim 18, wherein any one or more of said hydrophobic substituents comprises from 1 to 3 heteroatoms selected from O, N, and S provided said hydrophobic substituent has at least 6 carbon atoms.

Description

DESCRIPTION OF THE FIGURES

(1) FIGS. 1-4: The figures show various designs for transfectants arrayed in microplates. The numbers in the schematic microplates denote an amount of transfectant in nmol (based on nitrogen atoms) deposited in each well. The figures take 96 well plates as an example and place the transfectants in certain vertical rows. It is understood that similar designs can easily be derived from this general structure, e.g. where the patterns are organized horizontally, with more or less empty wells, with partial use of the plate and the like.

(2) FIG. 1 Amount of transfectant in nmol placed into certain wells of a 96 well plate, even distribution.

(3) FIG. 2 Amount of transfectant in nmol placed into certain wells of a 96 well plate, to simplify optimization of the N/P ratio.

(4) FIG. 3 Amount of transfectant in nmol placed into certain wells of a microplate, including empty wells for serial dilutions of the transfection complexes.

(5) FIG. 4 Amount of transfectant in nmol placed into certain wells of a microplate, facilitating a simple optimization of the N/P ratio and serial dilution of the transfection complexes.

EXAMPLES

(6) The invention is now further illustrated with the following examples without being limited to these.

Example 1

Modification of Polyamines

(7) Linear PEI (free base) having an average molecular mass of 2 kDa or 25 kDa was from Polysciences, catalog items 24313 and 23966, respectively. Branched PEI (free base) of 10 kDa size was from Aldrich, catalog item 408727. The polymers were dissolved in absolute ethanol at a concentration of 250 mono-mM; that is the concentration of nitrogens was 250 mM. Propylbromide, hexylbromide, nonylbromide and dodecylbromide (all from SIGMA) were dissolved in absolute ethanol at 250 mM and 3-bromopropanoic acid methyl ester, 6-bromohexanoic acid methyl ester, 8-bromooctanoic acid ethylester, 11-bromoundecanoic acid methyl ester or 16-bromohexadecanoic acid methyl ester were dissolved in absolute ethanol at 500 mM. K.sub.2CO.sub.3 was used as a 6 M solution in water.

(8) For the modification of 100 mol polyamine (based on monomer), the following solutions were pipetted per reaction:

(9) 400 l polyamine solution

(10) X l of the -bromo carboxylic acid ester

(11) Y l of the 1-bromoalkane

(12) wthanol up to 650 l

(13) 33 l K.sub.2CO.sub.3

(14) All mixtures were sealed and incubated for 7 days at 60 C. in an oven. Then, 250 l of 2N NaOH were added, the mixtures were sealed again and incubated for another 2 days at 60 C. For initial tests, small aliquots of the reaction mixture were taken and diluted 1:100 in 75% ethanol/water. 42 l of each diluted sample were transferred into a 96 well plate and dried.

Example 2

Complexation with siRNA

(15) Modified polyamines were hydrated for 20 min in 50 l of buffer pH4 (10 mM HAc, 10 mM NaH.sub.2PO.sub.4, pH4 with NaOH). 50 l of a siRNA (2 M in buffer pH4) were added and after 2-3 min the polymer-siRNA solution was brought to pH7 using 10 l of 160 mM NaOH.

Example 3

Cultivation of Cells

(16) HeLa cells were cultivated in 100 l of RPMI1640 medium (PAA Lab GmbH) supplemented with 10% FCS (Sigma-Aldrich), 1 Pen/Strep (PAA lab GmbH) solution (according to the instructions of the manufacturer) and seeded at a density of 4000 cells/well in a 96 well-plate. The density of live cells was measured using the Countess Cell counter (Invitrogen). Cells were cultivated in a humidified incubator at 37 C. and 5% CO2. 24 h after plating cells were supplied with fresh complete medium and transfected the same day.

Example 4

Transfection of Cells

(17) HeLa cells were transfected with the polyamine-siRNA-complexes by transferring 10 l material as obtained under example 2 per well. This addition results in a concentration of 100 nM PLK-1 siRNA in the cell culture medium. Cells were cultivated for three days at 37 C. and 5% CO.sub.2 in a humidified incubator without a change of the cultivation medium.

Example 5

Type of siRNA

(18) The siRNA used here is targeting the PLK-1 gene, the product of which is an essential component in the cell cycle. Downregulation of the target protein results in mitotic arrest and apoptosis. The transfection of cells is therefore monitored using a cell viability assay. The siRNA targeting PLK-1 and an unrelated control siRNA used herein were published by Haupenthal et al. (2007) Int J Cancer, 121, 206-210.

Example 6

Viability Assay

(19) Cells were tested for viability after three days by using the Cell Titer Blue assay (CTB, Promega). The medium was discarded and cells were supplied with fresh complete medium supplemented with the CTB reagent (5:1, vol/vol). 90-100 min after incubation at 37 C. and 5% CO2 in a humidified incubator the samples were transferred to a black fluorescence plate and color change of the samples was measured by a fluorescence reader at Ex560 nm/Em590 nm. The signal was normalized to 100% using untreated cells grown on the same culture plate and 0% with wells in which no cells were seeded.

Example 7

Transfection with Modified Polyamines

(20) The following tables show the % of live cells relative to an untreated culture as a metric for the PLK-1 downregulation following cell transfection. In the experiment, carboxylated, alkylated and co-modified polyamines were tested in parallel.

(21) TABLE-US-00004 TABLE 4 Transfection using carboxylated polyethylenimines Eq. carboxyl.sup.1 0 0.1 0.2 0.3 0.4 0.6 0.8 PEI2k C3 103 105 109 108 105 106 106 C6 103 110 111 111 112 108 C8 98 99 102 99 103 103 C11 98 96 75 98 108 114 C16 23 93 99 102 103 104 PEI25k C3 102 94 99 101 101 102 102 C6 96 99 104 107 105 105 C8 98 100 105 102 105 105 C11 94 56 49 60 106 113 C16 27 40 98 103 105 105 bPEI C3 79 92 94 91 97 104 102 C6 93 94 91 92 109 107 C8 85 90 92 93 103 101 C11 44 43 30 67 102 105 C16 56 106 104 106 107 107 .sup.1equivalents -bromo-carboxylic acid methyl ester added per nitrogen of the PEI polymer

(22) TABLE-US-00005 TABLE 5 Transfection using alkylated polyethylenimines Eq. alkyl.sup.2 0 0.05 0.1 0.15 0.2 0.3 0.4 0.5 0.7 PEI2k C3 110 107 108 112 112 111 105 105 111 C6 100 93 78 83 84 86 91 106 C9 101 107 109 99 72 74 67 107 C12 114 108 92 66 73 112 68 112 PEI25k C3 110 104 106 109 109 108 102 103 108 C6 100 84 61 39 36 37 70 86 C9 95 93 91 88 55 64 78 87 C12 93 89 77 62 87 90 102 109 bPEI C3 88 94 94 87 93 88 92 91 85 C6 70 55 53 57 57 55 71 102 C8 45 43 55 55 78 79 99 104 C11 78 80 93 104 105 108 107 107 .sup.2equivalents -bromoalkanes added per nitrogen of the PEI polymer

(23) TABLE-US-00006 TABLE 6 Transfection using co-modified polyethylenimines eq carboxyl + alkane.sup.3 0 0.063 0.13 0.19 0.25 0.38 0.51 0.63 0.89 PEI2k C11.sup.- C3 94 93 99 97 98 102 100 106 106 C11.sup.- C6 92 44 26 25 50 60 40 88 C11.sup.- C9 95 72 31 20 19 40 26 85 C11.sup.- C12 98 73 24 18 16 12 35 104 PEI25k C11.sup.- C3 96 90 90 90 90 82 64 87 100 C11.sup.- C6 99 36 23 21 26 31 31 54 C11.sup.- C9 84 30 24 23 21 26 29 52 C11.sup.- C12 72 42 22 24 20 23 22 27 bPEI C11.sup.- C3 74 83 65 34 36 31 28 23 97 C11.sup.- C6 48 39 27 29 25 #NV 52 97 C11.sup.- C9 18 16 20 #NV #NV 47 78 106 C11.sup.- C12 34 26 29 28 22 26 50 104 .sup.3combined equivalents -bromo-undecanoic acid methyl ester and 1-bromoalkane added per nitrogen of the PEI polymer, ratio of -bromocarboxylic acid to bromoalkane 1.25:1

(24) The polyethylenimines used here are essentially inactive transfectants under the assay conditions, only the branched PEI displays weak activity. Certain of the single modifications with either alkyl or carboxyl moieties yield improved activity and reductions of the cell viability below 50% are highlighted in the tables. The combined modifications with both carboxyalkyl and alkyl moieties display much greater activity on cells and the improvement is apparent for a wide range of DOS.

Example 8

Transfection Efficiency on Different Cell Types

(25) Transfectant:

(26) 5 mmol of branched PEI was modified with 11-bromoundecanoic acid methyl ester and nonylbromid as described in example 1, all other reagents were brought to scale. The degree of substitution was about 40%, the ratio between the modifiers about 1. Modified Pei was purified from the reaction mixture using size exclusion chromatography in 10 mM NaOH, 70% ethanol.

(27) Complexation:

(28) Purified transfectant was provided in a solution containing 30% ethanol and 10 mM NaOH. Polymers were diluted in buffer D (10 mM citrate, adjusted to pH5.0 with NaOH) or buffer F (10 mM phosphate, adjusted to pH6.5 with NaOH) to a concentration of 4 mM. To obtain a NP-ratio of 10, 150 l of the diluted polymers were complexed with 150 l of 10 M siRNA solution (Plk1 or scrambled control) in buffer D or F. Serial dilutions of these complexes were made in the respective buffers in a 96 well-plate to obtain concentrations from 500 nM to 2 nM on cells.

(29) Transfection:

(30) Cells were transfected with the polymer-siRNA-complexes by transferring a volume of 10 l/well from the complexation plate to the cell plate (column 1-10). Cells were cultivated for three days at 37 C. and 5% CO2 in a humidified incubator without a change of the cultivation medium.

(31) Viability Assay:

(32) Cells were tested for viability after three days by using the Cell Titer Blue assay (CTB, Promega). The medium was discarded and cells were supplied with fresh complete medium supplemented with the CTB reagent (120 l, 5:1, vol/vol). For NIH3T3, Jurkat and THP-1 cells the CTB-medium reagent was added directly to the cell medium, without discarding. 90-100 min (240 min for NIH3T3, Jurkat and THP-1 cells) after incubation at 37 C. and 5% CO2 in a humidified incubator the samples were transferred to a black fluorescence plate and color change of the samples was measured by a fluorescence reader at Ex560 nm/Em590 nm.

(33) TABLE-US-00007 TABLE 7 List of cell types Name Type Species Growth Source HeLa Cervix-Carcinom Human adherent DSMZ HepG2 Hepato-Carcinom Human adherent CLS Neuro 2a Neuroblastom Mouse adherent CLS

(34) The performance of the modified polyamine was measured as EC50, the concentration of siRNA needed for an inhibition of cell growth of 50%. In parallel, the same test was carried out using an unrelated siRNA and the EC50 value was also calculated. The signal-to-noise ratio is then defined as the ratio between the EC50 (control) and EC50 (PLK-1).

(35) TABLE-US-00008 TABLE 8 Transfection and signal-to-noise ratio for a modified polyamine Cell type EC50 (PLK-1, nM) EC50 (CTR, nM) Signal/Noise HeLa 3 27 9 HepG2 54 145 3 Neuro2A 38 142 4 CHO 15 71 5

(36) The data demonstrate a very efficient transfection of the siRNA into different cell types, including cell growing in suspension. The transfection reaction is very well tolerated by the cells with little or almost no signs of toxicity, indicated by signal-to-noise ratios of 3 or higher.

Example 9

Transfection Efficiency and Signal-to-Noise Ratio of Commercial Reagents

(37) In the same experiment as described in example 8, several commercial transfectants were tested in parallel, the results of such test are listed in table 9.

(38) Commercial Transfectants:

(39) Interferin (Polyplus, France), Ribocellin (BioCellChallenge, Germany), TranslT-TKO (Mirus, US), siPort NeoFX (Ambion/Life technologies Corp.), Dharmafect (Dharmacon/Thermo Fisher) and Lipofectamine RNAiMAX (Invitrogen/Life Technologies Corp.) were handled and complexed to Plk1 and scr siRNA according to the manufacturer's instructions. Serial dilutions were made in Optimem I (Gibco/Life Technologies) to obtain siRNA concentrations from 100 nM to 2 nM on cells.

(40) TABLE-US-00009 TABLE 9 Transfection and signal-to-noise ratio for commercial reagents Reagent siPort NeoFX Dharmafect RNAiMAX EC50 (nM) EC50 (nM) EC50 (nM) Cell type PLK SCR S/N PLK SCR S/N PLK SCR S/N HeLa 42 >100 3 13 24 2 57 300 5 HepG2 500 211 0 94 95 1 153 326 2 Neuro 500 >500 n.a. 159 144 1 360 0 0 CHO >500 >500 n.a. 13 27 2 43 204 5

(41) Although transfection could be achieved in many cases in particular with the reagents Dharmafect and RNAiMAX, the signal-to-noise ratios are substantially lower throughout the test.

Example 10

Analytical Characterization

(42) Transfectant:

(43) 5 mmol of branched PEI was modified with 11-bromoundecanoic acid methyl ester and nonylbromid as described in example 1, all other reagents were brought to scale. Alkylating reagents were added to target a degree of substitution was about 40%, the ratio between the modifiers about 1. Modified Pei was purified from the reaction mixture using size exclusion chromatography in 10 mM NaOH, 70% ethanol.

(44) Analysis:

(45) The estimated concentration of PEI was about 60 mM (based on monomer) at this point. 1 mL of the solution was mixed with 0.5 mL of 100 mM methoxyacetic acid and 25 l of 2N NaOH. The mixture was dried under vacuum, dissolved in 700 l CD3OD and analyzed by 1H NMR. Signals: methoxyacetic acid 4.0 ppm; bPEI backbone 2.4-2.7 ppm, terminal CH3 of the alkyl moiety 0.8 ppm and terminal CH2-COOH of the carboxyalkyl 2.1 ppm.

(46) TABLE-US-00010 TABLE 10 Analysis of the 1H NMR spectrum methoxy-acetic Signal Example 10 acid Methoxyacetic acid integral 2 mol 100 PEI backbone integral 4.8 mol 120 CH3 integral 0.7 mol 23.33 % of PEI 19% CH2COOH integral 0.4 mol 20 % of PEI 17% C/A 0.86 Total DOS 36%

Example 11

More Polyamine Derivatives

(47) The following polyamines were subjected to the modification reaction outlined in example 1:

(48) TABLE-US-00011 TABLE 11 Polyamines Architec- Molecular Polymer ture weight Source PEI (free base) Linear 2 kDa Polysciences, Cat 24313 PEI (free base) Linear 25 kDa Polysciences, Cat 23966 PEI (free base) Linear 100 kDa Polysciences, Cat 25414 PEI (free base) Linear 250 kDa Polysciences, Cat 24314 Polyethylenimine Branched 600 Da Polysciences, Cat 02371 (free base) Polyethylenimine Branched 1.2 kDa Polysciences, Cat 06088 (free base) Polyethylenimine Branched 1.8 kDa Polysciences, Cat 06089 (free base) PEI (free base) Branched 10 kDa Aldrich, Cat 408727 PEI (30% solution Branched 70 kDa Polysciences, Cat 06090 in water) PEI (50% solution Branched 750 kDa Polysciences, Cat 25448 in water) Poly-(N- Linear 500 kDa Polysciences, Cat 24038 methylvinylamine), free base Poly(allylamine), Linear 15 kDa Polysciences, Cat 24826 15% solution in water

(49) The polymers were dissolved in absolute ethanol at a concentration of 250 mono-mM; that is the concentration of nitrogen was 250 mM.

(50) The carboxyl compounds for the modification of polyamines were chosen from the following table 12 and were dissolved in absolute ethanol at a concentration of 250 mM:

(51) TABLE-US-00012 TABLE 12 Carboxyl compounds for the modification of polyamines. Carboxyl Compound Symbol Type Source 6-bromohexanoic acid methyl C6 Alkyl Acros Organices ester 392690250 8-bromooctanoic acid C8 Alkyl Nanjieng ethylester Pharmatechs HA- 04H79 11-bromoundecanoic acid C11 Alkyl Aldrich 447463 methyl ester 16-bromohexadecanoic acid C16 Alkyl Aldrich 684511 methyl ester Methyl 4-(2-Bromoethyl) CR1 Aryl Chempur 043959 benzoate Methyl 4-(Bromomethyl) CR2 Aryl ABCR AB246432 phenylacetate Methyl 4-(2-Bromomethyl) CR3 Aryl ABCR AB109915 benzoate Methyl 3-(4-Bromomethyl) CR4 Aryl Combi-Blocks OR- cinnamate 2146 5 g

(52) The hydrophobic compounds for the modification of the polyamines were chosen from the following table 13 and were also dissolved in absolute ethanol at a concentration of 250 mM:

(53) TABLE-US-00013 TABLE 13 Hydrophobic compounds for the modification of polyamines. Hydrophobic Compound Symbol Type Source Propylbromide A3 n-alkyl SIGMA B78106 Hexylbromide A6 n-alkyl Aldrich B68240 Nonylbromide A9 n-alkyl Aldrich B74607 Dodecylbromide A12 n-alkyl Aldrich B65551 Benzylbromide BB Aryl SIGMA B17905 2-bromoethylbenzene 2-BEB Alkylaryl SIGMA B65780 4-isopropylbenzylbromide 4-IPBB Alkylaryl Aldrich 563285 1-bromo-2-cyclohexyl- B2CHE Cyclic Alkyl Aldrich 467952 ethane 3-(2-bromomethyl)-indole 3-2-BEI Heterocyclic Aldrich 376523 Aryl 1-(2-bromomethyl)- 1-2-BEN Aryl Aldrich 559318 naphtaline
K.sub.2CO.sub.3 was used as a 6 M solution in water.

(54) For the modification of 100 mol polyamine (based on monomer), the following solutions were pipetted per reaction:

(55) 400 l polyamine solution

(56) X l of the -bromo carboxylic acid ester

(57) Y l of the brominated hydrocarbon compound

(58) Absolute ethanol up to 650 l

(59) 33 l K.sub.2CO.sub.3

(60) The combined amounts of X+Y were chosen for a degree of substitution of the amino groups of the polyamine between 0 and 100% and X and Y were further selected to complement each other and give various C/A ratios between 0 (only hydrophobic substituents) and the exclusive use of the carboxyl substituent, denoted as C/A=99 in the tables below.

(61) All mixtures were sealed and incubated for 7 days at 60 C. in an oven. Then, 250 l of 2N NaOH were added, the mixtures were sealed again and incubated for another 2 days at 60 C. For initial tests, small aliquots of the reaction mixture were taken and diluted 1:100 in 75% ethanol/water. 42 l of each diluted sample were transferred into a 96 well plate and dried.

Example 12

Transfection of siRNA Using Modified Polyamines

(62) The modified polyamines from example 11 were complexed with siRNA targeting PLK-1 and tested for their transfection properties as described in the examples 2 to 6 with the exception of using Buffer F (10 mM NaH2PO4, 225 mM sucrose, pH7.2 (adjusted with NaOH)) for siRNA complexation and transfection.

(63) The results of the transfection reactions are described in the table 14 for modified polyamines where the hydrophobic moiety is an aliphatic or cyclic alkyle and in table 15 where said hydrophobic group is an aryle or alkylaryle.

(64) TABLE-US-00014 TABLE 14 transfection of siRNA using certain polyamines wherein the lipophilic group is an alkyle. Lower numbers indicate successful transfection of the cells and are highlighted for values <=50. Mol Hydro % cell viability weight Carbox. phobic at certain C/A ratios Polymer in KDa moiety moiety DOS 99 6 2.5 1.3 0.75 0.4 0.16 0 lin PEI 2 C16 A9 0.2 89 66 42 64 84 95 98 96 2 C16 B2CHE 0.2 104 43 43 98 103 106 109 107 25 C16 A6 0.2 63 30 19 79 103 100 102 101 25 C16 A9 0.2 93 59 36 61 84 104 105 103 25 C16 B2CHE 0.2 92 36 24 71 96 89 104 107 100 C16 A9 0.2 84 34 46 76 97 92 96 101 250 C16 A9 0.2 90 43 42 91 94 95 95 96 bPEI 0.6 C16 A12 0.5 75 72 70 62 62 66 51 79 1.2 C16 A6 0.35 82 74 38 23 36 73 87 88 1.2 C16 A9 0.35 83 89 47 24 23 63 50 80 1.2 C16 B2CHE 0.35 109 107 79 42 28 91 90 96 1.8 C16 A9 0.35 104 101 95 94 66 41 73 91 10 C16 A9 0.2 102 102 88 39 43 81 90 88 10 C11 A9 0.35 91 88 78 58 32 54 95 103 10 C11 A12 0.35 103 93 70 26 27 77 109 109 10 C11 B2CHE 0.5 86 88 85 88 49 53 21 91 10 C16 B2CHE 0.35 105 104 109 107 38 18 19 33 70 C16 A9 0.35 99 88 81 58 49 29 29 91 70 C16 B2CHE 0.35 92 99 100 94 27 22 27 75 750 C11 A12 0.35 100 58 24 46 50 97 90 94

(65) The data of this table 14 demonstrate that many of the modified polyamines are capable of transfecting cells with siRNA. The hydrophobic group was selected from aliphatic or cyclic alkyl moieties and both resulted in the formation of active carriers regardless of their structural differences.

(66) The active carriers were formed from polyamines having widely different molecular weights, and different architecture (linear or branched). It is also apparent from the data that the carboxyl and hydrophobic components act synergistically, while single modifications contribute little, if any, to the carrier properties.

(67) TABLE-US-00015 TABLE 15 transfection of siRNA using certain polyamines wherein the hydrophobic group is an aryle, alkylaryl or heteroaryle. mol Hydro % cell viability weight Carbox phobic at certain C/A ratios Polymer in kDa moiety moiety DOS 99 6 2.5 1.3 0.75 0.4 0.16 0 lin PEI 2 C16 BB 0.2 104 54 71 103 108 99 106 110 2 C16 2-BEB 0.2 96 54 46 93 104 108 109 110 2 C16 4-IPBB 0.2 99 37 82 102 104 103 102 110 2 C16 1-2-BEN 0.2 99 58 52 97 93 92 100 104 2 C16 3-2 BEI 0.2 101 67 74 103 102 93 101 106 25 C16 BB 0.2 103 88 76 104 109 104 96 108 25 C16 2-BEB 0.2 68 26 31 84 96 97 100 112 25 C16 4-IPBB 0.2 89 30 52 78 98 99 108 113 25 C16 1-2-BEN 0.2 93 46 41 68 86 84 92 99 25 C16 3-2 BEI 0.2 95 55 48 97 99 86 92 100 bPEI 1.2 C16 BB 0.35 108 110 96 49 93 93 97 107 1.2 C16 2-BEB 0.35 106 102 92 46 45 80 99 106 1.2 C16 4-IPBB 0.35 107 104 76 44 45 34 31 80 1.8 C16 BB 0.35 105 104 100 50 30 72 78 104 1.8 C16 2-BEB 0.35 105 98 95 84 43 77 85 109 1.8 C16 4-IPBB 0.35 103 94 94 52 36 25 28 67 10 C16 BB 0.2 87 104 72 28 20 33 65 101 10 C16 2-BEB 0.35 107 108 109 106 39 19 41 108 10 C11 4-IPBB 0.2 94 86 85 73 42 21 25 45 10 C16 4-IPBB 0.2 105 105 66 23 19 18 16 21 10 C16 1-2-BEN 0.2 85 82 86 86 88 31 22 99 10 C16 3-2 BEI 0.35 91 94 99 94 84 41 94 96 70 C16 BB 0.35 98 89 99 88 21 45 98 94 70 C16 2-BEB 0.35 105 98 101 88 27 38 94 104 70 C16 4-IPBB 0.35 101 99 99 86 44 30 19 76

(68) The data of table 15 demonstrate that many of the modified polyamines are capable of transfecting cells with siRNA. The hydrophobic group was selected from aryle or alkylaryl moieties and all resulted in the formation of active carriers regardless of their structural differences.

(69) The active carriers were formed from polyamines having widely different molecular weights, and different architecture (linear or branched). It is also apparent from the data that the carboxyl and hydrophobic components act synergistically, while single modifications contribute little, if any, to the carrier properties.

Example 13

Transfection of Plasmids

(70) Cell culture: HeLa cells were cultivated in 100 l of RPMI1640 medium (PAA Lab GmbH) supplemented with 10% FCS (Sigma-Aldrich), 1 Pen/Strep (PAA lab GmbH) solution (according to the manufacturer instructions) and seeded at 8000 cells/well of a 96 well-plate. Cells were cultivated in a humidified incubator at 37 C. and 5% CO2. 24 h after plating cells were supplied with fresh complete medium and transfected the same day.

(71) Complexation: Polyamines were provided in 96 well plates, each well containing 3 mM polymer in 70% ethanol, Plasmid-DNA (pCMV-Luc) was provided as 0.011 g/l stock solution in Buffer F (10 mM NaH2PO4, 225 mM sucrose, pH7.2 (adjusted with NaOH)). 10 l of polyamines were complexed with to 90 l of Plasmid-DNA by mixing. 10-15 minutes after complexation, 10 l of the mixture was used for transfection of HeLa cells resulting in 100 ng of Plasmid-DNA/well.

(72) Determination of luciferase expression: Cells were prepared for quantitation of luciferase 24 h after transfection. Therefore, culture plates were equilibrated to room temperature for ca. 10 min. After discarding the medium cells were washed once with PBS. Cells were lysed using 100 l of 1 Beetle Lysis Juice (PJK GmbH, Germany) and prepared for luminescence measurement 5 min later. Expression of luciferase was quantified using a TECAN luminescence plate reader.

Example 14

Results for the Transfection of Plasmids

(73) The modified polyamines from example 11 were complexed with plasmid and tested for their transfection properties as described in example 13.

(74) The results of the transfection reactions are described in the table 16 for modified polyamines where the lipophilic moiety is an aliphatic or cyclic alkyle; in table 17 where said lipophilic group is an aryle or alkylaryle and in table 18 where the carboxyl moiety comprises an aryl or alkylaryl.

(75) TABLE-US-00016 TABLE 16 luminescence measured upon transfection of HeLa cells with a plasmid using certain polyamines wherein the hydrophobic group is an aliphatic alkyle. Signals lower than 100 light units were omitted for clarity, signals over 5000 light units are underlined. mol Hydro luminescense signal weight Carbox phobic at certain C/A ratios Polymer in kDa moiety moiety DOS 99 6 2 1.3 0.75 0.4 0.16 0 lin PEI 2 C16 A6 0.25 8.9E+02 4.8E+03 8.6E+03 3.6E+02 2 C16 B2CHE 0.25 1.1E+03 1.2E+03 2.3E+02 2.5E+03 2.7E+03 25 C16 A6 0.25 1.3E+03 1.0E+04 1.6E+04 5.9E+03 1.1E+03 7.6E+02 25 C6 A9 0.25 3.7E+03 4.4E+04 4.0E+04 3.5E+04 2.6E+04 25 C16 B2CHE 0.25 1.4E+03 5.1E+03 3.3E+03 1.0E+04 4.1E+03 4.0E+04 100 C16 A3 0.25 3.9E+03 8.8E+02 6.3E+04 5.8E+04 1.1E+06 1.3E+06 2.2E+06 4.8E+03 100 C16 A3 0.35 1.2E+02 3.7E+02 4.8E+03 4.3E+03 2.0E+05 1.8E+06 1.6E+05 1.5E+03 100 C11 A3 0.25 7.3E+02 2.1E+03 4.0E+03 3.1E+03 1.1E+05 1.0E+05 3.8E+04 4.5E+03 250 C16 A3 0.25 3.1E+03 4.2E+03 7.9E+03 5.4E+05 9.4E+05 8.4E+03 250 C16 A3 0.35 2.3E+02 4.1E+04 1.5E+05 3.0E+05 4.1E+03 250 C16 A3 0.5 1.6E+03 9.7E+04 1.2E+05 1.5E+03 250 C16 A6 0.25 2.4E+03 4.4E+04 6.4E+04 1.5E+04 6.1E+03 1.1E+04 5.4E+02 250 C11 A3 0.25 4.6E+02 7.0E+03 3.1E+02 3.0E+04 1.2E+05 5.6E+04 5.0E+04 bPEI 0.6 C16 A12 0.35 2.5E+02 2.2E+04 9.0E+04 4.8E+02 0.6 C11 A12 0.5 3.9E+02 5.0E+02 3.4E+03 7.4E+03 1.4E+04 7.1E+04 5.1E+03 1.0E+04 0.6 C16 B2CHE 0.35 7.9E+02 4.0E+03 8.7E+03 1.6E+03 1.2 C11 A9 0.35 1.2E+03 5.3E+03 7.3E+02 3.2E+04 7.4E+04 3.2E+04 6.8E+02 1.2 C11 A12 0.35 1.8E+03 2.0E+04 4.4E+04 4.9E+05 1.3E+05 3.2E+04 6.5E+02 1.2 C11 A12 0.5 1.7E+03 2.2E+04 5.9E+04 8.4E+04 1.5E+05 1.3E+05 2.8E+03 1.2 C16 A6 0.35 1.5E+03 3.4E+04 2.1E+04 1.2E+04 3.3E+03 3.2E+02 1.2 C16 A9 0.35 2.4E+03 4.1E+03 3.0E+04 3.4E+04 3.8E+04 2.3E+02 1.2 C16 A12 0.35 4.5E+02 1.6E+02 3.1E+03 4.5E+04 2.6E+04 1.0E+02 1.2 C16 B2CHE 0.35 3.8E+03 3.1E+04 1.5E+05 1.3E+05 4.9E+04 1.8E+04 3.0E+03 1.8 C11 A12 0.35 6.3E+02 5.6E+04 4.2E+04 2.6E+04 2.9E+05 2.5E+05 1.4E+04 1.0E+02 1.8 C16 A6 0.35 1.2E+02 7.6E+03 1.0E+05 9.8E+04 4.6E+04 7.1E+02 1.8 C16 A9 0.35 2.3E+02 2.2E+03 7.7E+04 5.9E+04 4.1E+04 1.9E+04 1.8 C16 B2CHE 0.35 1.8E+03 8.0E+04 1.1E+05 7.5E+04 4.7E+04 1.5E+03 1.8 C16 A12 0.25 7.9E+02 1.0E+03 3.8E+03 1.9E+04 8.5E+04 1.1E+04 1.3E+03 10 C6 A6 0.35 2.1E+02 6.7E+03 7.5E+04 2.2E+04 2.4E+03 6.6E+02 9.3E+02 10 C8 A6 0.25 6.8E+03 1.1E+04 1.4E+04 1.2E+04 2.2E+03 5.3E+02 10 C11 A9 0.35 9.4E+02 4.3E+03 2.6E+04 1.1E+04 4.8E+03 4.3E+03 1.2E+02 10 C16 B2CHE 0.35 2.8E+02 8.2E+03 2.6E+04 1.4E+04 1.1E+03 70 C6 A6 0.35 2.1E+04 8.9E+04 2.0E+03 9.3E+02 4.3E+02 70 C8 A6 0.25 6.7E+02 4.3E+03 5.9E+04 2.6E+04 1.3E+04 2.2E+04 9.4E+02 1.7E+02 70 C11 A9 0.35 7.0E+02 6.5E+03 1.8E+04 1.6E+04 1.4E+03 7.2E+03 4.5E+02 70 C11 A12 0.35 8.9E+02 4.2E+04 4.3E+04 1.2E+04 5.9E+03 3.5E+03 2.4E+02 70 C16 B2CHE 0.35 5.0E+02 3.4E+03 4.4E+04 3.8E+04 1.7E+04 1.9E+03 750 C11 A12 0.35 9.1E+02 1.4E+04 7.6E+03 5.1E+03 poly-N- 500 C11 A3 0.35 7.1E+02 2.2E+02 3.3E+03 methyl 500 C11 A6 0.35 3.9E+02 1.1E+04 vinyl- amine Poly- 15 C8 A6 0.35 1.0E+03 3.6E+03 allyl- 15 C11 A6 0.25 1.7E+02 1.6E+03 8.5E+02 1.3E+03 4.3E+02 amine 15 C11 B2CHE 0.25 1.0E+03 1.0E+03 1.7E+02 9.5E+03 2.1E+02

(76) The data of this table 16 demonstrate that many of the carboxylated, hydrophobized polyamines are capable of transfecting a plasmid into cells. The hydrophilic group was selected from aliphatic or cyclic alkyl moieties and both resulted in the formation of active carriers regardless of their structural differences.

(77) The active carriers were formed from polyamines having widely different molecular weights, different architecture (linear or branched) and different chemistry (polyethylenimines, polyallymine, polyvinylamine). It is also apparent from the data that the carboxyl and hydrophobic components act synergistically, while single modifications contribute little, if any, to the carrier properties.

(78) TABLE-US-00017 TABLE 17 transfection of a plasmid using certain polyamines wherein the hydrophobic group is an aryl or alkylaryl. Signals lower than 100 light units were omitted for clarity, signals over 5000 light units are underlined. mol Hydro luminescense signal weight Carbox phobic at certain ratios Polymer in kDa moiety moiety DOS 99 6 2.5 1.3 0.75 0.4 0.16 0 lin PEI 2 C16 BB 0.25 1.4E+02 6.7E+03 1.5E+04 1.1E+03 2 C16 2-BEB 0.25 5.0E+02 4.2E+03 1.9E+03 2.1E+03 1.5E+03 2 C16 4-IPBB 0.25 5.9E+02 1.0E+04 6.4E+02 3.0E+02 1.2E+03 1.2E+03 2 C16 1-2-BEN 0.25 3.2E+03 1.3E+04 3.5E+03 2.3E+03 5.5E+02 25 C16 BB 0.25 1.4E+03 2.9E+04 3.2E+04 25 C16 2-BEB 0.25 2.9E+02 1.3E+04 2.9E+04 2.8E+04 2.0E+04 1.9E+03 25 C16 4-IPBB 0.25 1.4E+03 1.8E+04 2.2E+03 1.7E+03 9.4E+02 1.6E+03 25 C16 1-2-BEN 0.25 1.7E+02 1.5E+04 7.0E+04 4.3E+04 1.8E+04 1.9E+03 1.9E+02 bPEI 10 C16 BB 0.35 4.0E+02 2.5E+04 2.3E+03 10 C16 2-BEB 0.35 6.3E+02 5.7E+03 10 C16 4-IPBB 0.35 9.0E+02 3.6E+03 10 C16 1-2-BEN 0.35 5.4E+02 5.2E+03 3.3E+03 70 C16 BB 0.25 6.0E+03 6.3E+02 2.7E+04 8.4E+04 3.1E+04 1.1E+04 1.7E+02 70 C16 2-BEB 0.25 1.3E+03 1.5E+04 1.9E+04 1.9E+04 4.5E+03 4.9E+03 2.3E+03 70 C16 4-IPBB 0.25 2.9E+03 4.4E+04 4.9E+04 3.5E+04 8.7E+03 3.6E+03 6.7E+03 0.6 C16 BB 0.35 2.8E+02 1.8E+04 3.8E+04 9.0E+03 0.6 C16 2-BEB 0.35 2.4E+02 1.3E+04 1.8E+04 5.2E+02 0.6 C16 4-IPBB 0.35 2.5E+03 3.3E+04 1.7E+04 2.7E+03 1.7E+03 1.2 C16 BB 0.35 5.3E+02 4.6E+03 3.6E+04 3.1E+05 2.9E+05 1.2E+04 4.6E+03 3.3E+02 1.2 C16 2-BEB 0.35 2.7E+02 2.8E+04 1.1E+05 2.1E+05 3.0E+04 3.1E+02 1.2 C16 4-IPBB 0.25 2.1E+02 1.3E+04 7.2E+04 4.8E+04 3.2E+04 8.1E+03 2.7E+03 5.1E+02 1.2 C11 4-IPBB 0.5 7.2E+04 6.7E+04 5.0E+04 3.6E+04 5.0E+04 9.3E+03 5.4E+02 1.8 C16 BB 0.35 5.8E+02 8.4E+03 8.1E+04 6.2E+05 4.0E+04 1.5E+03 5.2E+02 1.8 C16 2-BEB 0.35 1.3E+02 6.2E+03 1.1E+05 1.8E+05 1.6E+04 2.1E+02 6.9E+02 1.8 C16 4-IPBB 0.35 3.1E+03 3.8E+02 9.9E+03 1.1E+05 9.6E+04 1.8E+04 1.6E+04 Poly- 15.0 C11 BB 0.25 9.2E+02 3.5E+04 1.4E+04 3.6E+03 allyl- 15.0 C11 2-BEB 0.25 1.6E+02 1.6E+03 1.7E+03 8.1E+02 4.8E+02 8.7E+02 amine

(79) The data of this table 17 demonstrate that many of the carboxylated, hydrophobized polyamines are capable of transfecting cells with a plasmid. The hydrophobic group was selected from aryle or alkylaryl moieties and all resulted in the formation of active carriers regardless of their structural differences.

(80) The active carriers were formed from polyamines having widely different molecular weights, different architecture (linear or branched) and different chemistry (polyethylenimines, polyallymine). It is also apparent from the data that the carboxyl and hydrophilic components act synergistically, while single modifications contribute little, if any, to the carrier properties.

(81) TABLE-US-00018 TABLE 18 transfection of a plasmid using certain polyamines wherein the carboxyl group comprises an aryl or alkylaryl. Signals lower than 100 light units were omitted for clarity, signals over 5000 light units are underlined. mol Hydro luminescense signal weight Carbox phobic at certain ratios Polymer in KDa moiety moiety DOS 99 6 2.5 1.3 0.75 0.4 0.16 0 lin PEI 2 CR1 A9 0.25 2.3E+04 1.5E+04 7.4E+03 9.1E+03 2.0E+03 3.0E+02 25 CR1 A3 0.25 1.0E+03 6.1E+02 2.0E+03 7.1E+02 1.4E+03 1.3E+03 25 CR1 A9 0.25 4.8E+02 9.5E+03 8.7E+04 5.2E+04 2.1E+04 8.9E+03 1.1E+03 25 CR1 A12 0.25 2.6E+04 1.4E+04 2.4E+03 3.3E+02 4.8E+02 3.8E+02 bPEI 10 CR1 A3 0.25 8.4E+02 3.4E+03 3.4E+03 2.1E+03 9.1E+02 2.4E+02 10 CR1 A6 0.25 1.7E+02 1.3E+03 5.7E+03 2.5E+03 1.8E+04 2.6E+04 8.2E+03 3.0E+03

(82) The data of this table 18 demonstrate that many of the carboxylated, hydrophobized polyamines are capable of transfecting cells with a plasmid. The carboxyl group was selected from carboxylic acids comprising aryle or alkylaryl moieties and all resulted in the formation of active carriers regardless of their structural differences.

(83) The active carriers were formed from polymers having widely different molecular weights or different architecture. It is also apparent from the data that the carboxyl and hydrophobic components act synergistically, while single modifications contribute little, if any, to the carrier properties.

Example 15

Synthesis of Various Carboxylated, Hydrophobized Oligospermines and Homologues Thereof

(84) Starting from commercially available 1,4-diamino butane (1) the central intermediate 21 was obtained in a three step synthesis.

(85) ##STR00002##

(86) Compound 2 is easily accessible by addition of acrylonitrile to 1,4-diamino butane (1) in step a. Following this, two boc-protection groups were introduced into the molecule, leading to compound 3. The nitrile 3 was then reduced with hydrogen in the presence of excess butylamine to give 21.

(87) ##STR00003##

(88) Unfortunately, the NCH.sub.2CC moiety is not stable and susceptible to cleavage. Therefore, the triple bond of compounds 41, 42 or the higher oligomers were reduced followed by removal of the Boc-groups.

(89) Analogue reactions were carried out using 1,5-diaminopentane or 1,6-diaminohexane as the starting material 1, leading to a series of oligospermine homologues or polyalkylenimines wherein the variable x denoting the spacing between the nitrogen groups oscillates between 2 and 3, 2 and 4, or 2 and 5.

(90) In addition to the formation of the oligospermine homologues, the size of the hydrophobic substituents was systematically varied between butyl (as illustrated in this example), ethyl, hexyl and decyl, yielding a matrix of different polyalkylenimines having variations in their alkylen groups, their hydrophobic substituents and their degree of oligomerization.

(91) The oligospermines and their homologues (altogether the polyalkylenimines of this example) were desalted using Sephadex G25 and the individual oligomers were purified using ion exchange chromatography on CM-Sepharose Fast Flow and SP-Sepharose Fast Flow. The oligospermines elute from the ion exchange column according to their degree of polymerization and are denoted with roman numerals I to XII, whereby higher numbers stand for longer oligomers. The degree of polymerization was determined with mass spectroscopy and ranged from trimers through to about eicosamers.

(92) Fractions containing the separated oligomers were extracted from the buffer using dichlormethan under basic conditions, dried under vacuum and dissolved in absolute ethanol at a concentration of 250 mM nitrogen.

(93) In a subsequent step, samples of 50 mol of each oligomer were derivatized with 0, 10, 15, 20 or 25 mol of the -bromocarboxylic acids C3, C6, C8, C11 and C16 using the general protocol of example 11.

Example 16

Transfection of Cells Using Carboxyl-Hydrocarbon-Oligospermines

(94) The transfection testing of the various carboxylated and hydrophobized oligospermines and their respective homologues was performed using the siRNA targeting PLK-1, the HeLa cells and the cell culture conditions and assays described in the examples 2 to 7.

(95) The following table 19 is a list of the results obtained using the carboxyl-hydrocarbon-oligospermines as transfectants for siRNA.

(96) TABLE-US-00019 TABLE 19 Inhibition of the cell viability of HeLa cells upon transfection of a cytotoxic siRNA using carboxylated, hydrophobized oligospermines. Numerals x denote the oscillating lengths of the alkylen units according to formula (1), the roman numerals for the oligomers denote the degree of polymerization of the oligomers. The numbers in the table show the remaining cell viability, low numbers indicate an efficient transfection. % substitution % substitution with C11 with C16 x alkyl oligomer 0 20 30 40 50 0 20 30 40 50 2; 4 butyl III 105 87 81 79 70 100 31 39 34 37 IV 91 56 63 32 37 99 25 36 26 29 V 77 81 83 81 15 79 80 76 47 41 VI 100 101 98 100 99 98 101 102 101 105 2; 5 butyl II 77 70 79 83 13 82 82 71 49 37 2; 3 hexyl I 51 61 72 35 2 57 29 27 56 67 II 85 95 92 35 31 90 63 47 79 52 III 82 88 104 37 23 107 40 40 64 55 IV 86 44 84 41 27 64 33 29 62 97 V 100 79 91 34 35 101 75 43 54 88 VI + VII 80 82 81 84 87 85 85 86 88 87 VIII + IX 93 92 87 48 5 102 93 69 92 23 X 54 37 0 0 2 73 24 1 2 2 XI 19 0 0 1 4 102 68 94 85 83 XII 62 70 63 28 11 104 71 104 92 37 2; 4 hexyl II 102 108 97 84 106 102 103 105 57 60 III 87 104 89 39 43 90 97 65 81 56 IV 100 105 90 81 43 95 82 79 66 52 VI 98 94 93 86 86 96 0 2 93 89 VII 100 93 93 88 88 70 58 23 74 90 VIII 30 59 0 3 34 17 39 23 7 61 XI 104 100 104 106 104 107 107 110 112 109 X 84 85 73 31 13 62 46 16 53 53 IX 101 97 84 88 88 30 26 12 80 96 2; 5 hexyl III 104 77 49 2 31 36 7 73 60 92 IV 110 105 89 98 108 103 89 68 43 70 V 92 83 67 24 39 87 64 59 42 35 VI 97 93 97 99 96 99 99 96 100 99 2; 3 decyl II + III 102 86 78 94 93 104 105 105 113 104 VI 107 108 95 88 79 109 108 109 109 109 VII 109 99 69 80 92 105 108 113 108 119 VIII 109 2 6 92 13 107 105 107 111 106 IX 109 111 108 108 107 104 104 104 103 106

(97) The data of this table 19 demonstrate that many of the carboxylated, hydrophobized oligospermines are capable of transfecting cells. The active carriers were formed from oligospermines having widely different molecular weights and are of different molecular architecture as the spacing between the nitrogen atoms was varied. It is also apparent from the data that the carboxyl and hydrophobic components act synergistically, as the alkylated oligospermins display little, if any activity in the cell transfection while the introduction of the carboxyl moieties lead to active carrier structures.

Example 17

Neutral and Anionic Particles from Modified Polyamines and Nucleic Acid

(98) Buffers containing 280 mM sucrose, 10 mM sodium dihydrogenphosphate and 3 mM sodium hydroxide (pH6.5) or 7 mM sodium hydroxide (pH7.2) was prepared. Stock solutions of the siRNA from example 5 were prepared in the buffers to obtain 10 fold the concentrations listed in table 20.

(99) The modified polyamine of example 8 was provided as a solution having a concentration of 56 mM modified polyamine (as mono-mM) in 70% ethanol, 10 mM sodium hydroxide; 50 l of said solution of the modified polyamine were rapidly mixed with 4.1 ml of either buffer. A colloid forms and the particle size and the zeta potential of the dispersed phase were determined using MALVERN Zetasizer 3000 HSA.

(100) 900 l of the various siRNA solutions were rapidly mixed with the dispersions of the modified polyamine in buffer so to obtain the N/P ratios listed in table 20 and size and zeta potential were recorded again.

(101) TABLE-US-00020 TABLE 20 Size and zeta potential of complexes between a modified polyamine and siRNA having various ratios of N/P. pH 6.5 pH 7.2 Zeta Zeta polyamine siRNA Size potential Size potential [M] [M] N/P [nm] [mV] [nm] [mV] 560 6.2 2 496 29.5 503 32.5 560 3.1 4 537 30.2 519 29.2 560 2.1 6 858 7.5 546 34.9 560 1.4 9 1268 2.8 1292 1.5 560 1.04 12 857 2.1 615 0.1 560 0.83 15 656 2.1 606 0.4 560 0.62 20 521 0.3 625 1.5 560 0.41 30 516 2.3 596 1.3 560 0 99 532 2.1 538 1.8

(102) General observations: the modified polyamines do from particles having a size of about 500 nm and an almost neutral zeta potential. Addition of small amounts of siRNA resulting in N/P ratios of 12 or higher do not substantially alter the size or surface charge of the particles. However, addition of larger amounts of siRNA led to the formation of anionic particles of about the same size. Intermediate amounts of siRNA lead to intermediate zeta potential and the formation of aggregates. The systems behavior can be understood as the interaction of two polyelectrolytes of opposite charge, where aggregation occurs under the conditions of charge neutralization as described in Endert et al. (2004) Nanocapsules from liposomal templates, pp. 238-248 in Carrier Based Drug Delivery, Oxford University Press. Specific observations: At pH6.5 the charge reversal towards the fully anionic state requires a N/P of 4, whereas the same process is completed at N/P6 at a pH of 7.2. This observation is in line with the stronger ionization of the PEI backbone amines at lower values of pH.

Example 18

Lyophilization of Transfectants

(103) The modified polyamine from Example 8 was provided as solution in 70% ethanol, 10 mM sodium hydroxide having a concentration of 56 mM nitrogen. Various amounts of the clear solution of the transfectant were rapidly injected into a homogenous phase comprising 280 mM sucrose and 20 mM sodium phosphate adjusted with NaOH to pH 6.5 so that the final concentration of transfectant was 0.15 mM or 0.03 mM. The mean particles sizes and polydispersities were 389 nm (PI of 0.09) and 332 nm (PI of 0.11) for the samples having 0.15 mM or 0.03 mM nitrogen, respectively.

(104) Aliqouts of 100 l each were placed in 96 well plates. The materials were frozen for 4 hours at 18 C. and transferred into a freeze-dryer. Lyophilization occurred at a constant pressure of 0.12 mbar for 20 hours. After removal from the lyophilization chamber, the plates were quickly sealed using a heat sealer.

(105) One the next day, seals were broken and the samples were rehydrated with water. The lyophilized product dissolved quickly and homogeneously. Particle size and polydispersity were determined as above.

(106) Within 15 minutes after starting rehydration, the resulting colloidal transfectants had a mean particle size of 423 nm (PI 0.13) when 15 nmol of transfectant were placed in each well. The resulting colloidal transfectants had a mean particle size of 356 nm (PI 0.12) when 3 nmol of transfectant were placed in each well.

(107) The content of European patent application No. 12 006 963.8, filed on Oct. 8, 2012, the priority of which is claimed, is herewith incorporated by reference including all claims and entire description.