Amphoteric liposomes comprising imino lipids

Abstract

The invention concerns lipid assemblies, liposomes having an outer surface comprising a mixture of anionic and cationic moieties; wherein at least a portion of the cationic moieties are imino moieties that are essentially charged under physiological conditions, and their use for serum resistant transfection of cells.

Claims

1. Lipid assemblies, comprising: anionic and cationic amphiphiles, and a neutral lipid, wherein the cationic amphiphiles are selected from the group consisting of structures (11), (12), and (15): ##STR00023## wherein: R.sub.1 and R.sub.2 are each independently alkyl, alkenyl, or alkynyl, wherein the alkyl, alkenyl, or alkynyl has 8 to 24 carbon atoms and 0, 1, or 2 unsaturated bonds; A and B are each independently absent, —CH.sub.2—, —CH═, ═CH—, —O—, —NH—, —C(O)—O—, —O—C(O)—, —O—C(O)—NH—, or —NH—C(O)—O—; D is absent, —CH.sub.2—, —CH═, ═CH—, —O—, —NH—, —C(O)—O—, —O—C(O)—, —C(O)—NH—, —NH—C(O)—, —O—C(O)—NH—, —NH—C(O)—O—, m is an integer between 0 and 29; n is an integer between 0 and 29; and the imino group comprises a guanido moiety, wherein the imino group is substantially charged under physiological conditions; wherein the anionic amphiphiles are carboxyl or phosphate lipids, and wherein the charge ratio between the cationic and anionic amphiphiles is from 0.67 to 1.5.

2. The lipid assemblies of claim 1, wherein the charge ratio between the cationic and anionic amphiphiles is from 1 to 1.5.

3. The lipid assemblies of claim 1, wherein the charge ratio between the cationic and anionic amphiphiles is from 1 to 1.22.

4. The lipid assemblies of claim 1, wherein the lipid assemblies are pH-sensitive, have negative to neutral surface charge under physiological conditions, and mediate cell transfection in the presence of serum.

5. The lipid assemblies of claim 1, wherein the anionic amphiphiles are carboxyl lipids.

6. The lipid assemblies of claim 1, wherein the anionic amphiphiles are phosphate lipids.

7. The lipid assemblies of claim 1, wherein the imino group is: ##STR00024## wherein R.sub.11 and R.sub.12 are, independently of each other, H, —CH.sub.3 or —CH.sub.2CH.sub.3, n is 0 or 1, and L is an apolar region of the cationic amphiphile.

8. The lipid assemblies of claim 1, wherein the cationic amphiphiles are GUADACA.

9. The lipid assemblies of claim 1, wherein the anionic amphiphiles are selected from the group consisting of CHEMS, DMGS, DOGS, DOPA and POPA.

10. The lipid assemblies of claim 1, wherein the neutral lipid is selected from the group consisting of cholesterol, phosphatidylcholine, phosphatidylethanolamine, and mixtures thereof.

11. The lipid assemblies of claim 10, wherein the neutral lipid is cholesterol, and the molar fraction of cholesterol in the lipid assemblies is from 10 to 50 mol %.

12. The lipid assemblies of claim 1, further comprising PEG lipids.

13. The lipid assemblies of claim 1, wherein the charge ratio between the cationic and anionic amphiphiles is 1 or less and wherein no PEG lipids are present.

14. The lipid assemblies of claim 1, further comprising nucleic acid.

15. The lipid assemblies of claim 14, wherein the nucleic acid is an oligonucleotide.

16. The lipid assemblies of claim 14, wherein the nucleic acid is polynucleotide, DNA plasmid, linear DNA construct or mRNA.

17. The lipid assemblies of claim 1, wherein the charge ratio between the cationic and anionic amphiphiles is from 0.67 to 1.25.

18. The lipid assemblies of claim 1, wherein the guanido moiety is selected from the group consisting of: ##STR00025## ##STR00026## wherein L is an apolar region of the cationic amphiphile.

19. A method of transfecting cells, comprising applying the lipid assemblies of claim 1 to the cells in the presence of serum.

20. The method of claim 19, further comprising applying the lipid assemblies into bloodstream.

Description

FIGURE LEGENDS

(1) FIGS. 1-6 display the results of the screening experiment described in example 14. The nature of the cationic lipids is indicated in the smaller figures and other legends and axis are similar for all display items and are given in the separate smaller figure below. The double bars denote liposomes with 20% cholesterol (left bar) and 40% cholesterol (right bar), respectively.

(2) Bars represent the IC50 values for the respective liposome/siRNA combinations under the experimental conditions for each figure, that is, either in the presence of absence of mouse serum. These IC50 values denote the concentrations needed for a half-maximal inhibition of the cell growth and are given in nM. The maximum concentrations of the test items were 40 and 36 nM for the absence or presence of mouse serum, respectively.

(3) The order of the test items is as follows:

(4) FIG. 1 the anionic lipid is CHEMS−no addition of mouse serum

(5) FIG. 2 the anionic lipid is CHEMS+addition of mouse serum

(6) FIG. 3 the anionic lipid is DMGS−no addition of mouse serum

(7) FIG. 4 the anionic lipid is DMGS+addition of mouse serum

(8) FIG. 5 the anionic lipid is DOGS−no addition of mouse serum

(9) FIG. 6 the anionic lipid is DOGS+addition of mouse serum

EXAMPLES

(10) The teachings of this invention may be better understood with the consideration of the following examples. However, these examples should by no means limit the teachings of this invention.

Example 1—Liposome Production, Characterization and Encapsulation of siRNA

(11) Liposomes were prepared using methods as disclosed in WO071107304. More specifically, lipids were dissolved in isopropanol and liposomes were produced by adding siRNA solution in NaAc 20 mM, Sucrose 300 mM, pH 4.0 (pH adjusted with HAc) to the alcoholic lipid mix, resulting in a final alcohol concentration of 30%. The formed liposomal suspensions were shifted to pH 7.5 with twice the volume of Na.sub.2HPO.sub.4 136 mM, NaCl 100 mM (pH 9), resulting in a final lipid concentration of 3 mM and a final isopropanol concentration of 10%.

(12) Liposomes were characterized with respect to their particle size using dynamic light scattering (MALVERN 3000HSA).

(13) Active siRNA: 21mer blunt ended targeting mouse and human ILK-1 mRNA as in Haupenthal et al., int. J. Cancer (2007), 121:206-210.

(14) Control siRNA (SCR): 21 mer from the same source.

Example 2 General Cell Culture and Proliferation Assay

(15) HeLa cells were obtained from DSMZ (German Collection of Micro Organism and Cell Cultures) and maintained in DMFM (Gibco-Invitrogen) and supplemented with 10% FCS. The cells were plated at a density of 2.5×10.sup.4 cells/ml and cultivated in 100 μl medium at 37° C. under 5% CO.sub.2. After 16 h, the liposomes containing siRNA were diluted and 10 μl were added to the cells to yield final concentrations between 0.4 to 100 nM Plk1 or scrambled siRNA; 10 μl dilution buffer were also added to untreated cells and into wells without cells. Cell culture dishes were incubated for 72 h at 37° C. under 5% CO.sub.2

(16) Cell proliferation/viability was determined by using the CellTiter-Blue Cell Viability assay (Promega, US) according to the instructions of the supplier.

Example 3—Inhibition of Transfection by Sera

(17) Liposomes from DODAP:DMGS:Cholesterol (24:36:40 mol %) were loaded with active and control siRNA as above and 25 μl of the liposomes were incubated with 75 μl sera from different species (SIGMA-Aldrich) for 30 min. Following that, liposomes were added to the cells, incubation was continued for 72 h and cell viability was determined as above.

(18) When incubated without serum, administration of the active siRNA results in a strong inhibition of cell proliferation. As demonstrated in the Table 7 below, this process is inhibited in by the addition of sera.

(19) TABLE-US-00007 TABLE 7 Inhibition of cellular transfection by sera of different origin. siRNA type siRNA concentration Serum Cell viability (%) PLK1 50 nM no 7 PLK1 50 nM Human 98 PLK1 50 nM Hamster 80 PLK1 50 nM Rat 108 PLK1 50 nM Mouse 102 No No No 100

Example 4—Inhibition is Lipoprotein Dependent

(20) Liposomes as in Example 3 were incubated with human serum devoid of certain complement factors or lipoproteins (SIGMA-Aldrich) as above and analyzed for their ability to mediate the RNAi effect on HeLa cells.

(21) As demonstrated in Table 8, the efficacy of transfection can be restored by a depletion of lipoproteins. Removal of complement factors was ineffective.

(22) TABLE-US-00008 TABLE 8 Restoration of cellular transfection in sera being deficient of various factors. siRNA type siRNA concentration Serum Cell viability (%) PLK1 50 nM no 7 PLK1 50 nM Human, complete 98 PLK1 50 nM Human, no C3 91 complement factor PLK1 50 nM Human, no C9 98 complement factor PLK1 50 nM Human, lipoprotein 18 deficient No No No 100

Example 5—Serum Resistant Transfection Using a Guanido Lipid

(23) A series of liposomes was constructed from PONA:Anionic Lipid:Cholesterol (x:y:20 mol %) and loaded with active and control siRNA as in Example 1. Within that series, the ratio between the cationic component PONA and the anionic lipids CHEMS or DMGS was systematically varied between 0.33 and 2 as indicated in the table. Liposomes having a ratio of the cationic anionic lipid of 1 or greater were further supplied with 2 mol % DMPE-PEG2000 (Nippon Oils and Fats) to avoid aggregation of the particles. This modification is indicated by a “+” in the table. Control reactions with particles having C/A<1 did not reveal a change of transfection properties in the presence or absence of PEG lipids.

(24) HeLa cells were grown and maintained as in Example 2 and sera of human or mice origin (SIGMA-Aldrich) was added directly to the cells for 120 min. Following that, the liposomes were added to the cells in concentrations between 50 μM and 50 nM, incubation was continued for 72 h and cell viability was determined as above. The efficacy of transfection is expressed here as IC.sub.50, the concentration needed to inhibited cell proliferation by 50%, Low IC50 values therefore represent highly effective transfection.

(25) It becomes apparent from the results in the Table 9, that the addition of sera only marginally affects the transfection of siRNA mediated by the liposomes of the example. Some inhibition is still observed for liposomes from PONA:CHEMS comprising low amounts of the anionic lipid (ratios 0.33 and 0.5, particular strong inhibition with mouse serum).

(26) TABLE-US-00009 TABLE 9 Efficacy of transfection of liposomes comprising guanido moieties in the presence of sera. Ratio cationic/anionic lipid 0.33 0.50 0.67 0.82 1+ 1.22+ 1.5+ 2+ CHEMS No Serum  38.54  1.21 0.40 0.56 1.83 1.61 0.70 1.42 Human Serum 199.00  2.10 0.62 1.13 2.16 1.92 1.70 1.83 Mouse Serum 199.00 50.00 1.56 1.94 2.47 1.90 0.76 1.44 DMGS No Serum 0.23 0.54 0.01 0.01 Human Serum 1.50 2.39 2.88 2.21 Mouse Serum 0.67 0.69 1.41 1.81

Example 6—Criticality of the Guanido Head Group

(27) Series of liposomes having systematically varied ratios between the cationic and anionic lipid components were produced and loaded with siRNA as in Example 5. The cationic lipid components were PONA, PONamine and PONammonium, the anionic lipid was CHEMS and the cholesterol content was fixed to 20 mol %. Liposomes having a ratio of the cationic anionic lipid of 1 or greater were further supplied with 2 mol % DMPE-PEG2000 (Nippon Oils and Fats) to avoid aggregation of the particles. This modification is indicated by a “+” in the table.

(28) HeLa cells were grown and maintained as in Example 2 and sera of human or mice origin (SIGMA-Aldrich) was added directly to the cells for 120 min. Following that, the liposomes were added to the cells in concentrations between 50 pM and 50 nM, incubation was continued for 72 h and cell viability was determined as above. The efficacy of transfection is expressed here as IC.sub.50 as in Example 5.

(29) It becomes apparent from the data in Table 10, that only PONA, but neither PONamine and even less so PONammonium mediates the transfection of HeLa cells in the presence of serum. This is most striking in the case of mouse serum, which inhibits the transfection more aggressively. An excess of the cationic lipid components to some extent compensate the serum mediated loss of activity, but may be due to unspecific electrostatic adsorption of these liposomes to the cells.

(30) TABLE-US-00010 TABLE 10 Criticality of the guanido head group for the serum restant transfection of cells. C/A ratio 0.33 0.5 0.67 0.82 1+ 1.22+ 1.5+ 2+ PONA no serum 42.9 1.8 0.6 1.0 4.1 5.4 2.4 6.8 human serum 80.0 2.5 2.2 2.0 1.8 2.8 6.2 5.2 mouse serum 80.0 31.1 55.0 5.7 2.1 5.3 8.1 7.5 PONamine no serum 3.1 65.0 7.5 100.0 3.0 5.2 3.0 2.5 human serum 100.0 55.0 11.9 100.0 2.2 2.8 6.1 5.1 mouse serum 70.0 100.0 100.0 100.0 75.0 70.0 39.3 8.7 PONammonium no serum 80.0 100.0 90.0 90.0 65.0 9.5 9.5 5.2 human serum 95.0 90.0 90.0 80.0 90.0 11.8 12.4 15.7 mouse serum 85.0 100.0 100.0 100.0 100.0 90.0 75.0 55.0

Example 7—Optimization of the Liposome Composition

(31) Series of liposomes having systematically varied ratios between the cationic and anionic lipid components were produced and loaded with siRNA as in Example 5. The cationic lipid component was PUNA, the anionic lipids were CHEMS, DMGS or DOGS and the cholesterol content was varied between 0 and 40 mol %. Liposomes having a ratio of the cationic anionic lipid of 1 or greater but also some of the other liposomes were further supplied with 2 mol % DMPE-PEG2000 (Nippon Oils and Fats) to avoid aggregation of the particles. This modification is indicated by a “+” in the table.

(32) HeLa cells were grown and maintained as in Example 2 and liposomes were added to the cells in concentrations between 6 nM and 200 nM, incubation was continued for 72 h and cell viability was determined as above. The efficacy of transfection is expressed here as IC.sub.50 as in the examples above. In addition, the IC.sub.50 was determined for the liposomes carrying the inactive siRNA (SCR) and the ratio between IC.sub.50 (SCR) and IC.sub.50 (PLK1) was determined. A high value for this parameter indicates a very specific inhibition of the cellular viability by the PLK1 siRNA, low unspecific effects contributed by the carrier and low levels of cytotoxicity in general.

(33) TABLE-US-00011 TABLE 11 Optimization results for CHEMS. Lowest and highest detectable IC.sub.50 values are 6 and 200 nM, respectively. C/A 0.33 0.33+ 0.5 0.5+ 0.67 0.67+ 0.82 0.82+ 1+ 1.22+ 1.5+ 2+ PLK  0% Chol 44 77 6 6 6 6 6 6 6 6 6 6 20% Chol 54 79 6 6 6 6 6 6 6 6 6 6 40% Chol 67 94 6 6 6 6 6 6 6 6 6 6 SCR  0% Chol 90 86 113 152 23 200 16 21 15 16 14 11 20% Chol 73 90 109 128 200 200 26 23 21 11 16 10 40% Chol 94 117 198 200 200 200 6 6 30 14 27 12 SCR/  0% Chol 2.05 1.12 18.86 25.33 3.81 83.33 2.60 3.52 2.50 2.68 2.30 1.84 PLK 20% Chol 1.37 1.14 18.10 21.39 83.33 83.33 4.26 3.77 3.45 1.84 2.65 1.69 40% Chol 1.40 1.24 32.96 83.33 83.33 83.33 1.00 1.00 5.00 2.39 4.48 1.97

(34) TABLE-US-00012 TABLE 12 Optimization results for DMGS. Lowest and highest detectable IC.sub.50 values are 6 and 200 nM. respectively. C/A 0.33 0.5 0.67 0.8.2 1+ 1.22 1.5+ 2+ PLK  0% Chol 98 200 200 188 6 6 6 6 20% Chol 6 6 6 6 6 6 6 6 40% Chol 6 6 6 6 6 6 6 6 SCR  0% Chol 200 200 200 158 14 6 10 14 20% Chol 200 54 8 8 13 9 9 10 40% Chol 155 23 11 6 6 14 9 12 SCR/PLK  0% Chol 5.11 no effect no effect 0.84 2.26 1.00 1.66 2.36 20% Chol 83.33 9.01 1.27 1.26 2.20 1.55 1.45 1.69 40% Chol 25.85 3.90 1.83 1.00 1.00 2.27 1.54 1.97

(35) TABLE-US-00013 TABLE 13 Optimization results for DOGS. Lowest and highest detectable IC.sub.50 values are 6 nM, and 200 respectively. C/A 0.33 0.5 1 0.67 0.82 1.22+ 1.5+ 2+ PLK  0% Chol 200 200 200 200 6 6 6 6 20% Chol 22 200 200 200 6 6 6 6 40% Chol 6 170 200 200 6 6 6 6 SCR  0% Chol 200 200 200 200 14 10 16 10 20% Chol 200 200 200 200 21 10 12 8 40% Chol 15 197 200 200 12 7 9 9 SCR/PLK  0% Chol no effect no effect no effect no effect 2.40 1.59 2.65 1.63 20% Chol 22.42 no effect no effect no effect 3.45 1.65 2.07 1.29 40% Chol 2.48 1.16 no effect no effect 1.93 1.09 1.48 1.55

Example 6—Liposomes Comprising a Pyridinium Lipid

(36) SAINT-18 was used as the cationic lipid, its methylated pyridinium structure provides a charged imino moiety. CHEM, DMGS and DOGS were individually used as anionic lipids providing the carboxyl functional group. Series of liposomes having systematically varied ratios between the cationic and anionic lipid components were produced and loaded with siRNA as in Example 5. The lipid mixture was further supplied with 20 or 40 mol % cholesterol. Liposomes having a ratio of the cationic:anionic lipid of 1 or greater were further supplied with 2 mol % DMPE-PEG2000 (Nippon Oils and Fats) to avoid aggregation of the particles. This modification is indicated by a in the table.

(37) HeLa cells were grown and maintained as in Example 2 and liposomes were added to the cells in concentrations between 50 pM and 50 nM, incubation was continued for 72 h and cell viability was determined as above. The efficacy of transfection is expressed here as IC.sub.50 as in the examples above. In addition, the IC.sub.50 was determined for the liposomes carrying the inactive siRNA (SCR) and the ratio between IC.sub.50 (SCR) and IC.sub.50 (PLK1) was determined. A high value for this parameter indicates a very specific inhibition of the cellular viability by the PLK1 siRNA, low unspecific effects contributed by the carrier and low levels of cytotoxicity in general.

(38) TABLE-US-00014 TABLE 14 transfection results for liposomes from SAINT-18, CHEMS and cholesterol C/A ratio 0.33 0.5 0.67 0.82 1+ 1.22+ 1.5+ 2+ lipid anion CHEMS no serum PLK1 20% Chol 2.2 no eff. 1.7 33.9 17.8 7.2 4.1 2.7 40% Chol 7.8 no eff. 1.5 32.0 7.2 4.4 2.1 6.4 SCR 20% Chol no eff. no eff. 11.9 no off. 37.4 19.6 20.7 29.0 40% Chol no eff. no eff. no eff. no eff. no eff. 16.9 18.6 28.0 SCR/ 20% Chol >22.7 7.2 >1.5 2.1 2.7 5.0 10.6 PLK-1 40% Chol >26.4 32.5 >1.6 >7.0 3.9 8.7 4.4 lipid anion CHEMS plus mouse serum PLK1 20% Chol no eff. no eff. 14.4 no off. no off. no off. no eff. 31.7 40% Chol no eff. no eff. no eff. no eff. no eff. no eff. 35.5 23.1 SCR 20% Chol no eff. no eff. no eff. no eff. no eff. no eff. no eff. no eff. 40% Chol no eff. no eff. no eff. no eff. no eff. no eff. 41.5 38.1 SCR/ 20% Chol >3.5 >1.6 PLK-1 40% Chol 1.2 1.6

(39) TABLE-US-00015 TABLE 15 transfection results for liposomes from SAINT-18. DMGS and cholesterol C/A ratio 0.33 0.5 0.67 0.82 1+ 1.22+ 1.5+ 2+ lipid anion DMGS, no serum PLK1 20% Chol 0.8 2.3 1.7 43.6 24.3 7.5 5.2 3.8 40% Chol 1.6 2.3 1.8 2.2 11.4 8.9 3.8 5.8 SCR 20% Chol 7.7 8.2 5.3 36.0 28.1 27.6 10.5 10.3 40% Chol 4.7 no eff. 22.6 5.7 27.7 28.5 8.1 8.2 SCR/ 20% Chol 9.2 3.6 3.1 0.8 1.2 . 3.7 2.0 2.7 PLK-1 40% Chol 2.9 >22.1 12.6 2.5 2.4 3.2 2.1 1.4 lipid anion DMGS; plus mouse serum PLK1 20% Chol 4.0 8.0 2.7 no eff. 26.5 28.8 no eff. no eff. 40% Chol 2.0 2.2 1.6 1.6 no eff. 21.0 no eff. no eff. SCR 20% Chol 10.1 no eff. 23.4 no eff. 29.1 31.2 25.7 28.4 40% Chol 7.7 18.0 25.8 6.3 28.0 37.4 31.7 25.7 SCR/ 20% Chol 2.5 >6.2 8.6 1.1 1.1 PLK-1 40% Chol 3.9 8.0 16.5 3.9 1.8

(40) TABLE-US-00016 TABLE 16 transfection results for liposornes from SAINT-18, DOGS and cholesterol C/A ratio 0.33 0.5 0.67 0.82 1+ 1.22+ 1.5+ 2+ lipid anion DOGS, no serum PLK1 20% Chol 36.9 38.0 no eff. no eff. 9.2 8.1 7.0 6. 40% Chol 6.9 19.4 no eff. no eff. 22.7 8.7 6.6 8.5 SCR 20% Chol no eff. no eff. no eff. no eff. 27.5 20.5 10.2 25.9 40% Chol no eff. no eff. no eff. no eff. no eff. no eff. no eff. no eff. SCR/ 20% Chol >1.4 >1.3 3.0 2.5 1.5 4.3 PLK-1 40% Chol >7.3 >2.6 >2.2 >5.7 >7.6 >5.9 lipid anion DOGS, plus serum PLK-1 20% Chol 2.2 18.4 no eff. no eff. 27.5 30.5 26.3 28.1 40% Chol 2.7 7.7 no eff. no eff. 27.4 29.2 30.4 30.8 SCR 20% Chol no eff. 2.8 no eff. no eff. 32.6 34.4 30.9 33.2 40% Chol no eff. 8.2 no eff. no eff. 30.6 no eff. no eff. 42.8 SCR/ 20% Chol 1.3 >2.7 1.2 1.1 1.2 1.2 PLK-1 40% Chol >18.6 1.1 1.1 >1.7 >1.6 1.4

(41) As it becomes clear from the data in tables 14 to 16, a large number of amphoteric liposomes facilitate the transfection of cells even in the presence of mouse serum. Particularly useful are liposomes comprising SAINT-18 in combination with the diacylglycerols DMGS and DOGS, while the combination with CHEMS was only effective at C/A=0.67. As with the PONA combinations, the amphoteric constructs transfect the cells with high specificity, while the compositions having >1 do not provide a highly specific transfection as indicated by SCR/PLK1 being below 2.

(42) Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. The specification and examples should be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.

Example 9—Zeta Potential Measurements

9.1 Analysis of the Zeta Potential for Liposomes Formed from PONA:CHEMS:CHOL

(43) 100 μl of a lipidmix comprising x Mol % PONA, y mol % CHEMS and 20 Mol % cholesterol (20 mM total lipid concentration, solvent: isopropanol) was injected in 900 μl of a buffer comprising 10 mM acetic acid and 10 mM phosphoric acid pH4, X and Y, the molar percentages for PONA and CHEMS were adjusted to yield the C/A ratios in table 17. The suspension was immediately vortexed and 3 mL of a pH adjusting buffer was added. Buffers were selected from the group of 50 mM acetic acid and 50 mM phosphoric acid, adjusted to pH 4, 5, 6.5 or 7.5 using NaOH or 50 mM Na.sub.2HPC.sub.4/50 mM sodium acetate pH9.4. The mixing pH is was recorded and is given in the table 17 below together with the zeta potentials of the resulting lipid particles that were monitored using a Zetasizer HSA3000.

(44) TABLE-US-00017 TABLE 17 Zeta Potentials for lipid particles from PONA:CHEMS:CHOL C/A ratio Final pH 0.5 0.67 0.82 1.00 1.22 1.5 2.00 7.56 −54.40 −58.90 −58.20 −61.80 −21.80 22.60 #NV 7.20 −48.47 −46.00 −44.90 −50.00 −21.10 14.97 #NV 6.32 −44.33 −37.07 −31.37 0.64 23.43 9.60 #NV 4.84 19.67 18.00 22.15 31.80 32.77 32.57 28.37 3.93 35.53 41.73 43.75 46.63 46.20 43.40 43.23

(45) Clearly, the particles display amphoteric character even for mixtures having a C/A of 1.22, that is, greater than 1. Particles having a C/A of 0.67, 0.82 or 1 were also produced at pH7.4 and subsequently exposed to lower pH. There were no apparent changes to the zeta potentials shown in table 17.

9.2 Zeta Potential Measurements for Combinations Wherein DOPA is the Anionic Lipid

(46) Lipid particles were also prepared from binary mixtures of GUADACA and DOPA, an imino/phosphate combination of lipid head groups. The particles were prepared in the same fashion as described in 9.1 and the zeta potentials of table 18 were recorded for mixtures having different C/A ratios:

(47) TABLE-US-00018 TABLE 18 Zeta Potentials for lipid particles from GUADACA:DOPA C/A 0.65 0.75 0.98 1.16 1.4 final pH 4.5 21 13 38 46 51 5.32 −24 22 20 33 35 6.25 −8 −45 −30 2 24 7.02 −61 −67 −8 −56 −6 7.81 −67 −78 −76 −65 −21

(48) As with the particles obtained in 9.1, particles with amphoteric character are also obtained with C/A>1. Still, the drift in the isoelectric point follows the expectations.

9.3 Zeta Potential Measurements for DOTAP:CHEMS:CHOL

(49) For comparison, the same measurements were performed with lipid mixtures wherein PONA was substituted by DOTAP. The results are shown in table 19. In contrast to PONA: CHEMS, amphoteric particles from DOTAP:CHEMS are only found at C/A<1

(50) TABLE-US-00019 TABLE 19 Zeta potential for lipid particles from DOTAP:CHEMS:CHOL Ratio C/A 0.67 0.82 1 1.22 Final pH 7.56 −37.7 −21.63 4.9 13.25 7.20 −50.17 −24.1 #NV 12.55 6.32 #NV #NV 11.43 7.37 4.84 25.6 32.1 20.27 9.3 3.93 52.13 43.93 47.77 12.15

Example 10—Synthesis of CHOLGUA

(51) 25 g cholesterolchloroformiate and 50 equivalents (eq.) ethylendiamine were dissolved in dichloromethane and allowed to react for 6 h at 20° C. The aminoethylcarbamoyl-cholestererol was isolated using chromatography and crystallization. Yield was 28.7 g, purity 90%.

(52) CHOLGUA was synthesized from the aminaethylcarboarnoyl-cholesterol isolated before. 30 g of the substance were incubated with 1.5 eq. of 1H-pyrazole-1-carboxamidinium hydrochloride and 4 eq. N,N-diisopropylethylamin in dichloromethane/ethenol for 16 h at 20° C., after which the product was isolated by chromatography. Purity was 95%, Yield 16.5 g.

Example 11—Synthesis of DACA, PDACA and MPDACA

(53) 42.4 g of oleyl alcohol, 2.5 eq. of diisoproylazodicarboxylate, 2.5 eq, triphenylphosphine and 5 eq. LiI were reacted in tetrahydrofuran (THF) for 24 h at 20° C. Oleyliodid was isolated by chromatography with a purity of 90%, yield was 13.4 g.

(54) In a second step, 10 g oleic acid were mixed with 2.2 eq. of lithiumdiisopropylamide in THF for 0.5 h at 20° C., after which 1 eq. oleyliodide was added. The mixture was incubated for 2 h at 20° C. and DACA purified from the reaction mix using chromatography. Purity was 95%, Yield 14.96 g,

(55) For the synthesis of PDACA, 2 g of DACA, 1.2 eq. of 4-picolylamine, 1.4 eq, of O-benzotriazole-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate and 4 eq. of N-mathylmorpholine were mixed in THF for 24 h at 20° C. The reaction mixture was purified including chromatography. Purity of PDACA was 95%, yield was 1.72 g.

(56) For the synthesis of MPDACA, 2 g of PDACA was dissolved in THF together with 2 eq. of dimethylsulphate and the mixture was incubated for 16 h at 20° C., after which MPDACA was purified by chromatography. Purity of MPDACA: 95%, Yield:1.71 g

Example 12—Synthesis of GUADACA

(57) In a first step, 3.5 g DACA and 1.5 eq. of 1,1′-carbonyldiimidazol were dissolved in dichloromethane and incubated for 16 h at 20° C., after which 30 eq. ethylenediamine were added. The reaction mixture was incubated for 4 h at 20° C. after which aminoethyl-DACA was purified including chromatography. Purity was 90%, Yield 3.2 g.

(58) GUADACA was synthesized from aminoethyl-DACA and for that, 3.2 g of aminoethyl-DACA, 2.5 eq. 1H-pyrazole-1-carboxamidine hydrochloride and 12 eq. N,N-diisopropylethylamine were incubated for 3 h at 20° C., after which GUADACA was isolated. Purity: 95%, Yield: 2.24 g.

Example 13—Synthesis of BADACA

(59) BADACA was synthesized from DACA according to the following procedure: 4.1:5 g DACA, 1.2 eq, p-aminobenzamidine, 1.2 eq. N,N′-dicyclohexylcarbodiimid and 3 eq. of 4-Dimethylaminopyridine were mixed in dry dimethylformamide and incubated for 16 h at 70° C. BADACA was isolated from the reaction using chromatography. Purity: 95%, Yield: 1.62 g

Example 14 Serum Resistant Transfection of DACA or Cholesterol Based Cationic Lipids in Combination with Carboxyl Lipids

(60) Series of liposomes having systematically varied ratios between the cationic and anionic lipid components were produced and loaded with siRNA as in Example 5. The cationic lipid components were CHOLGUA, CHIM, DC-CHOL, TC-CHOL, GUADACA, MPDACA, BADACA end PDAGA. The anionic lipids were CHEMS, DMGS or DOGS and the cholesterol content was either 20 or 40 mol %, all lipid mixtures are identified in the data tables. Liposomes having a ratio of the cationic: anionic lipid of 1 or greater (C/A>=1) were further supplied with 1.5 mol % DMPE-PEG2000 (Nippon Oils and Fats).

(61) HeLa cells were grown and maintained as in Example 2 and mouse serum (SIGMA-Aldrich) was added directly to the cells for 120 min. Following that, the liposomes were added to the cells, incubation was continued for 72 h and cell viability was determined as above. The highest concentrations of liposomes were 40 nM and 36 nM for experiments in the absence or presence of mouse serum, respectively. The efficacy of transfection is expressed here as IC.sub.50 (in nM siRNA) as in Example 5. All results from this screening experiment are shown in FIGS. 1-6.

(62) Many of the transfecting mixtures resulted in very potent transfection of HeLa cells with siRNA, as indicated by the very low IC50 values. Combinations of lipids comprising imino lipids such as CHOLGUA, but more so MPDACA, GUADACA or PONA remain potent transfectants even in the presence of mouse serum.

Example 15—Serum Resistant Transfection of Several Cationic Lipids in Combination with Phosphate Lipid

(63) Series of liposomes having C/A ratios of either 0.75 or 1 were produced and loaded with siRNA as in Example 5. The cationic lipid components were CHOLGUA, CHIM, DC-CHOL, GUADACA, MPDACA, BADACA, PONA, DOTAP or DODAP. The anionic lipid was DOPA and the cholesterol content was 40 mol %, all lipid mixtures are identified in table 20. Liposomes were further supplied with 1.5 mol % DMPE-PEG2000 (Nippon Oils and Fats).

(64) HeLa cells were grown and maintained as in Example 2 and mouse serum (SIGMA-Aldrich) was added directly to the cells for 120-min. Following that, the liposomes were added to the cells, incubation was continued for 72 h and cell viability was determined as above. The efficacy of transfection is expressed here as IC.sub.50 (in nM of siRNA) as in Example 5.

(65) Many of the transfecting mixtures resulted in very potent transfection of HeLa cells with siRNA, as indicated by the very low IC50 values. Combinations of lipids comprising imino lipids such as CHOLGUA, but more so MPDACA, GUADACA or PONA remain potent transfectants even in the presence of mouse serum.

(66) TABLE-US-00020 TABLE 20 IC50 values (nM siRNA) for various liposomes in the presence and absence of mouse serum. Serum inhibition “not potent” refers to a lack of minimum potency in the presence of mouse serum. in these cases the inhibition factor cannot be defined. The highest concentration of siRNA in the test was 146 nM. −mouse serum +mouse serum IC50 IC50 IC50 IC50 serum C/A Cation PLK1 Scr. PLK1 Scr. inhibition 0.75 CholGUA 8 160 104 146 12 CHIM 26 160 146 146 not potent DC-Chol 28 160 146 146 not potent MPDACA 5 67 10 146 2 GUADACA 6 39 26 146 4 BADACA 159 160 146 146 not potent PONA 6 24 146 146 not potent DOTAP 21 152 146 146 not potent DODAP 160 160 146 146 not potent 1 CholGUA 9 141 125 146 14 CHIM 33 160 146 146 not potent DC-Chol 29 160 146 146 not potent MPDACA 12. 100 4 146 0.3 GUADACA 9 89 7 146 1 BADACA 38 160 146 146 not potent PONA 2 66 21 146 10 DOTAP 13 160 76 146 6 DODAP 94 160 146 146 not potent

Example 16—Serum Resistant Transfection is Poor in the Absence of Negatively Charged

(67) A series of liposornes was produced from cationic lipids and cholesterol as a neutral lipid. No anionic lipids were used in these preparations. The cationic lipid components were CHOLGUA, CHIM, DC-CHOL, ADACA, GUADACA, MPDACA, BADACA, PONA, DOTAP and DODAP and the liposomes were produced with the procedure described in example 5.

(68) The cholesterol content was 40 mol % and liposomes were further supplied with 1.5 mol % DMPE-PEG2000 (Nippon Oils and Fats) to avoid aggregate formation in the presence of siRNA.

(69) HeLa cells were grown and maintained as in Example 2 and mouse serum (SIGMA-Aldrich) was added directly to the cells for 120 min. Following that, the liposomes were added to the cells, incubation was continued for 72 h and cell viability was determined as above. The efficacy of transfection is expressed here as IC.sub.50 (in nM of siRNA) as in Example 5.

(70) The results obtained are shown in table 21 below. In all cases, the transfection efficacy is substantially lower than that of the mixtures further comprising an anionic lipid. With the exception of GUADACA or PONA, there was no activity detectable in the presence of mouse serum.

(71) TABLE-US-00021 TABLE 21 IC50 values (nM siRNA) for various liposomes in the presence and absence of mouse serum. Serum inhibition “not potent” refers to a lack of minimum potency in the presence of mouse serum, in these cases the inhibition factor cannot be defined. The highest concentration of siRNA in the test was 160 or 146 nM in the absence of presence of mouse serum, respectively. no mouse serum with mouse serum serum Cation IC50 PLK1 IC50 Scr. IC50 PLK1 IC50 Scr. inhibition CholGUA 93 160 146 146 not potent CHIM 160 160 146 146 not potent DC-Chol 101 109 146 146 not potent MPDACA 27 154 146 146 not potent GUADACA 22 69 95 146 4 BADACA 99 160 146 146 not potent PONA 30 100 70 99 2 DOTAP 160 160 146 146 not potent DODAP 160 160 146 146 not potent