SUCROSE ESTER BASED CATIONIC GENE VECTOR AND PREPARATION METHOD THEREOF

Abstract

The invention provides a class of sucrose ester based cationic lipids and preparation method thereof. The vector prepared using the cationic lipid can be used to deliver nucleic acid. The sucrose ester based cationic lipid is prepared by using a chemical synthesis method in the invention, wherein the synthesis method is simple, and has a relatively high product yield. A composition, including suspension, emulsion, micelle and liposome and the like, can be prepared by mixing the sucrose ester based cationic lipid compound of the invention with a co-lipid. Sucrose ester based cationic lipid complex can be prepared by using the said composition and nucleic acid, which has advantages such as simple preparation, low toxicity and high transfection efficiency, and is a novel and highly efficient gene vector.

Claims

1. A sucrose ester based cationic lipid compound wherein the sucrose ester based cationic lipid compound having a structure of a general formula I: ##STR00006## wherein, R is selected from C.sub.10-20 hydroxyl, bile acid group and cholesterol ester group; Y is selected from —NR.sup.aR.sup.b and —N.sup.+R.sup.aR.sup.bR.sup.cX.sup.−, wherein R.sup.a, R.sup.b and R.sup.c are the same or different, and are selected from hydrogen, C.sub.1-6 hydroxyl, C.sub.1-6 hydroxyalkyl, galactosyl, mannosyl and/or folic acid ester group; X.sup.− is selected from F.sup.−, Cl.sup.−, Br.sup.−, I.sup.−.

2. A method for preparing the sucrose ester based cationic lipid compound according to claim 1, wherein the method comprising the following steps: (1) Compound A is prepared by the reaction between sucralose and compound of formula i at a molar ratio of 1:1˜50: ##STR00007## the reaction temperature is 10 to 100° C., the reaction time is 10 to 50 h, the reaction solvent is a nitrogen-containing solvent, such as N, N-dimethylformamide, pyridine, acetonitrile, piperidine and triethylamine, etc.; and a weight-to-volume ratio of the reactant to the solvent is from 1:1 to 50; the compound of formula i is selected from C.sub.10-20 hydroxyl acid, bile acid or cholesterol derivative (cholesterol acid); (2) Compound B is prepared by the reaction between the compound A with the compound of formula ii at a molar ratio is from 1:1 to 8: ##STR00008## the reaction temperature is 10 to 120° C., the reaction time is 10 to 40 h, the reaction pressure is 1.0 to 5.0 atm, the solvent is N, N-dimethylformamide, methanol, ethanol, isopropanol or isobutanol, and a weight-to-volume ratio of the reactant to the solvent is from 1:1 to 50; R.sup.a, R.sup.b and R.sup.c of the compound of formula ii are the same or different, and are selected from hydrogen, C.sub.1-6 hydroxyl, C.sub.1-6 hydroxyalkyl, galactosyl, mannosyl and/or folic acid ester group, Y of the compound B is —NR.sup.aR.sup.b or —N.sup.+R.sup.aR.sup.bR.sup.cX; X.sup.− is F.sup.−, Cl.sup.−, Br.sup.−, I.sup.−.

3. The method for preparing the sucrose ester based cationic lipid compound according to claim 2, wherein when the synthesized product is a quaternary ammonium salt, equilibrium anions F.sup.−, Br.sup.− and I.sup.− can be obtained by replacing Cl.sup.− with an ion exchange column.

4. A cationic lipid composition, wherein the composition consisting of the above sucrose ester based cationic lipid compound and a co-lipid, a mass ratio of the said sucrose ester cationic lipid compound to the co-lipid is from 10:1 to 1:10.

5. The cationic lipid composition according to claim 4, wherein the co-lipid is one or a mixture of two or more selected from lecithin, phosphatidylethanolamine, glycolipid, dioleoylphosphatidyl chloride, palmitoyl oleoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, cholesterol, chitosan and sucrose ester.

6. The cationic lipid composition according to claim 4, wherein the composition is one or a mixture of two or more of suspension, emulsion, micelle or liposome.

7. A cationic lipid complex, wherein the cationic lipid complex is consisting of the above cationic lipid composition combined with nucleic acid, and a mass ratio of the cationic lipid composition to the nucleic acid is from 20:1 to 1:20.

8. The cationic lipid complex according to claim 7, wherein the nucleic acid delivered into a cell is one or a mixture of two or more of pDNA, microRNA and siRNA.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 is a structural formula I of a sucrose ester based cationic lipid compound of the invention.

[0040] FIG. 2 is the .sup.1H NMR spectrum of Example 1.

[0041] FIG. 3 is the .sup.13C NMR spectrum of Example 1.

[0042] FIG. 4 is the particle size diagram of the cationic liposome of Example 5.

[0043] FIG. 5 is the zeta potential diagram of the cationic liposome of Example 5.

[0044] FIG. 6 is the electrophoretogram of the cationic liposome-DNA complex of Example 7.

[0045] FIG. 7 is the graph showing the result of the green fluorescent protein detection after the Hep-2 cell is transfected by the cationic liposome-DNA complex in Example 8.

[0046] FIG. 8 is the graph showing the result of the luciferase assay after the Hep-2 cell is transfected by the cationic liposome-DNA complex in Example 8.

[0047] FIG. 9 is the Hep-2 cell viability diagram using the MTT assay in Example 9.

DETAILED DESCRIPTION OF THE INVENTION

[0048] The structure of the sucrose ester based cationic lipid compound synthesized by the above method in the invention is confirmed using nuclear magnetic resonance spectroscopy (.sup.1H NMR and .sup.13C NMR) or mass spectrometry.

Example 1 Preparation of Sucralose Laurate Quaternary Ammonium Salt (TSI12)

[0049] (1) Synthesis of Sucralose Laurate

[0050] In a 250 mL vessel equipped with a thermometer and a reflux tube, 3.9 g of sucralose, 3 g of anhydrous potassium carbonate, 5 mL of pyridine and N, N-dimethylformamide (DMF, 10 mL) were added followed by keeping the temperature of the oil bath at 75° C., after the full dissolution under magnetic stirring, 5 mL of lauric acid was dropwise added. After the reaction for 24 h, a pale yellow filtrate was obtained and then was subject to vacuum rotary evaporation to remove the solvent so as to obtain amber viscous oily liquid. After the column chromatographic purification, a white amorphous esterification product was obtained, of which the structure was characterized by the following: .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 5.44 (t, J=124.9, 2H), 6.37-4.56 (m, 6H), 6.37-4.28 (m, 10H), 6.37-3.89 (m, 20H), 6.37-3.59 (m, 26H), 6.37-2.37 (m, 28H), 2.32 (t, J=14.7, 4H), 2.07 (s, 2H), 1.99 (s, 2H), 1.77 (s, 2H), 1.74-1.58 (m, 6H), 1.42-1.15 (m, 33H), 1.52-0.58 (m, 39H), 1.43-0.58 (m, 39H), 1.11-0.53 (m, 6H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ: 174.44 (s), 107.88 (s), 94.90 (s), 82.14 (s), 80.40 (s), 74.63 (s), 74.02 (s), 73.37 (s), 71.64 (s), 65.88 (s), 64.52 (s), 44.29 (s), 42.93 (s), 33.92 (s), 31.73 (s), 29.07 (t, J=7.9), 25.42 (s), 23.16 (s), 14.00 (s). ESI-MS, m/z: Found [M+3H].sup.3+, 581.10, [M+2H−Cl].sup.+, 545.1, C.sub.24H.sub.41Cl.sub.3O.sub.9 calcd for [M]=578.1816, [M+3H]=581.1894, [M+2H−Cl]=545.2127.

[0051] (2) Synthesis of Sucralose Laurate Quaternary Ammonium Salt

[0052] In a 250 mL vessel equipped with a N.sub.2 protection, a thermometer, a reflux tube and a dropping funnel, 10 g of sucralose ester and 30 mL of N, N-dimethylformamide (DMF) were added, 30 mL of a trimethylamine solution was taken followed by keeping the temperature of the water bath at 100° C. The pressure is 1 atm. After the reaction for 24 h, the solvent was distilled off under reduced pressure. A reaction crude product was obtained which was separated and purified by column chromatography to obtain a product followed by dissolving in a solvent and crystallizing for several times to obtain a pale yellow powdery solid. The resulting solid was dried in an infrared rapid desiccator for 2 h to obtain sucralose laurate quaternary ammonium salt, of which the structure was characterized by the following: .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 5.40-5.53 (d, 1H), 4.54-4.56 (t, 1H), 4.43 (d, 1H), [4.35 (d, 1H), 4.23 (d, 1H)], 4.28-4.30 (d, 1H), 4.30-4.33 (d, 1H), 4.10 (s, 1H), 4.07 (s, 1H), 3.95-3.99 (m, 1H), [3.84 (d, 1H), 3.65 (d, 1H)], 3.61-3.78 (d, 2H), 3.22-3.30 (s, 9H), 2.36-2.41 (t, J=7.6 Hz, 2H), 1.60-1.66 (dd, J=14.6, 8.2 Hz, 2H), 1.28-1.30 (m, J=8.9 Hz, 16H), 0.88-0.89 (t, J=6.4 Hz, 3H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ: 172.97 (s), 103.57 (s), 92.22 (s), 75.12 (s), 74.94 (s), 74.44 (s), 71.14 (s), 68.03 (s), 67.43 (s), 66.91 (s), 63.53 (s), 63.06 (s), 52.94 (s), 42.93 (s), 32.95 (s), 31.05 (s), 28.19-28.72 (m), 23.99 (s), 21.71 (s), 12.43 (s). Q-TOF-MS, m/z: Found [M−Cl].sup.+, 602.2856, C.sub.27H.sub.50Cl.sub.3NO.sub.9 calcd for [M]=637.2551, [M−Cl]=602.2863.

Example 2 Preparation of Bile Acid-Sucrose Ester Based Cationic Lipid (TSB-2I)

[0053] (1) Preparation of Sucralose Bile Acid Ester

[0054] In a 250 mL vessel equipped with a thermometer and a reflux tube, 3.9 g of sucralose, 3 g of anhydrous potassium carbonate, 5 mL of acetonitrile and 20 mL of piperidine were added followed by keeping the temperature of the oil bath at 75° C., after the full dissolution under magnetic stirring, 10 mmol of bile acid was dropwise added. After the reaction was carried out for 24 h, a pale yellow filtrate was obtained which was subject to decompression rotary evaporation to remove the solvent so as to obtain amber viscous oily liquid. After the column chromatographic purification, a white amorphous mono-esterification product was obtained. .sup.1H NMR (400 MHz, CDCl3) δ: 4.86-4.29 (m, 4H), 4.86-3.92 (m, 8H), 3.89-3.68 (m, 3H), 3.68-3.41 (m, 2H), 3.37-3.18 (m, 2H), 2.38 (t, J=16.1, 2H), 2.38 (t, J=16.1, 2H), 3.08-1.78 (m, 5H), 3.08-0.98 (m, 29H), 3.08-0.91 (m, 35H), 3.08-0.44 (m, 39H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ: 174.51 (s), 107.88 (s), 94.90 (s), 82.14 (s), 80.40 (s), 74.63 (s), 74.09 (d), 73.56 (s), 73.37 (s), 71.64 (s), 70.69 (s), 67.02 (s), 65.88 (s), 64.52 (s), 50.15 (s), 47.48 (s), 45.82 (s), 44.29 (s), 42.93 (s), 41.84 (s), 40.62 (s), 40.36 (s), 38.98 (s), 35.64 (s), 34.24 (s), 31.84 (d, J=16.2), 30.78 (s), 29.76 (s), 29.33 (s), 28.14 (s), 24.87 (s), 18.78 (s), 17.69 (s), 11.94 (s). Q-TOF-MS, m/z: Found [M+Na].sup.+, 825.2860, C.sub.36H.sub.57Cl.sub.3O.sub.13 calcd for [M]=802.2865, [M+Na]=825.2865.

[0055] (2) Synthesis of Bile Acid—Sucrose Ester Based Cationic Lipid

[0056] In a 250 mL vessel equipped with a N.sub.2 protection, a thermometer, a reflux tube and a dropping funnel, 5 mmol of an isopropanol (20 mL) solution of sucralose bile acid ester and 0.7 g of anhydrous potassium carbonate were added followed by keeping the temperature of water bath at 60° C. Under the magnetic stirring, 10 mmol of an ethanol (10 mL) solution of diethanolamine was dropwise added for about 1 h. After the completion of dropwise addition, the temperature of water bath rose to 100° C. The reaction was carried out for 6 h. The reaction mixture was filtered to obtain a filtrate which was subject to decompression rotary evaporation to remove the solvent so as to obtain a mixture. Finally, high-purity bile acid-sucrose ester based cationic lipid was obtained by preparative chromatographic purification. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 5.57 (d, J=14.8, 2H), 4.77 (d, J=14.2, 2H), 4.56-4.35 (m, 7H), 4.30-4.05 (m, 6H), 3.81-3.60 (m, 4H), 3.59-3.37 (m, 10H), 3.37-3.18 (m, 4H), 2.92-2.40 (m, 12H), 2.35 (t, J=15.7, 4H), 2.15-1.82 (m, 10H), 1.82-1.28 (m, 43H), 1.24 (s, 6H), 1.18-1.03 (m, 2H), 0.96 (s, 12H), 0.88 (d, J=12.8, 6H), 0.75-0.44 (m, 2H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ: 174.51 (s), 107.88 (s), 94.90 (s), 80.40 (s), 78.39 (s), 78.09 (s), 74.09 (d), 73.56 (s), 73.37 (s), 71.64 (s), 70.69 (s), 67.02 (s), 65.88 (s), 64.52 (s), 59.18 (s), 58.37 (s), 57.48 (s), 50.15 (s), 47.48 (s), 45.82 (s), 44.29 (s), 41.84 (s), 40.62 (s), 40.36 (s), 38.98 (s), 35.64 (s), 34.24 (s), 31.84 (d), 30.78 (s), 29.76 (s), 29.33 (s), 28.14 (s), 24.87 (s), 18.78 (s), 17.69 (s), 11.94 (s). Q-TOF-MS, m/z: Found [M+Na].sup.+, 894.3891, C.sub.40H.sub.67Cl.sub.2NO.sub.15 calcd for [M]=871.3888, [M+Na]=994.3888.

Example 3 Preparation of Cholesterol-Sucrose Ester Based Cationic Lipid (TSD-2D)

[0057] (1) Preparation of Sucralose Cholesterol Ester

[0058] In a 250 mL vessel equipped with a thermometer and a reflux tube, 3.9 g of sucralose, 3 g of anhydrous potassium carbonate, 5 mL of trimethylamine and 20 mL of N, N-dimethylformamide (DMF) were added followed by keeping the temperature of the oil bath at 75° C. After the full dissolution under magnetic stirring, 10 mmol of cholesterol maleic acid was added. After the reaction was carried out for 24 h, a pale yellow filtrate was obtained, and was subject to decompression evaporation to remove the solvent so as to obtain amber viscous oily liquid. After column chromatographic purification, a pale yellow amorphous esterification product was obtained. .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 6.31 (s, 4H), 4.73-4.56 (m, 6H), 4.55-4.37 (m, 6H), 4.26 (ddd, J=23.6, 19.1, 12.2, 4H), 3.79-3.57 (m, 5H), 2.66-0.61 (m, 87H), 1.82-0.61 (m, 71H), 1.73-0.88 (m, 65H), 0.82 (s, 6H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ: 168.13 (s), 167.08 (s), 140.62 (s), 132.99 (s), 131.80 (s), 121.95 (s), 107.88 (s), 94.90 (s), 82.14 (s), 80.40 (s), 74.89 (s), 74.63 (s), 74.02 (s), 73.37 (s), 71.64 (s), 66.85 (s), 64.52 (s), 55.26 (s), 51.39 (s), 50.92 (s), 44.29 (s), 42.93 (s), 42.59 (s), 39.59 (s), 39.43 (s), 39.19 (s), 37.80 (s), 37.22 (s), 32.76 (d), 32.25 (s), 30.45 (s), 28.55-28.27 (m), 27.55 (s), 24.28 (s), 22.74 (s), 21.49 (s), 18.73 (s), 12.92 (s). Q-TOF-MS, m/z: Found [M+Na].sup.+, 971.3435, C.sub.42H.sub.63Cl.sub.3O.sub.11 calcd for [M]=848.3436, [M+Na]=971.3436.

[0059] (2) Synthesis of Cholesterol—Sucrose Ester Cationic Lipid

[0060] In a 250 mL vessel equipped with a N.sub.2 protection, a thermometer, a reflux tube and a dropping funnel, 5.2 mmol of an isobutanol (20 mL) solution of sucralose cholesterol ester and 1.0 g of anhydrous potassium carbonate were added. The temperature of the water bath was kept at 60° C. Under magnetic stirring, 16.5 mmol of a methanol solution (20 ml) of diethylamine was dropwise added for about 1 h. After the dropwise addition, the temperature of the water bath rose to 100° C. After increasing the pressure to 2 atm, the reaction was carried out for 6 h. The reaction mixture was filtered to obtain a filtrate which was subject to decompression evaporation to remove isobutanol to obtain a mixture. Finally, a high-purity cholesterol-sucrose ester based cationic lipid was obtained by preparative chromatographic purification.

[0061] .sup.1H NMR (400 MHz, CDCl.sub.3) δ: 6.31 (s, 4H), 5.01-4.24 (m, 10H), 5.01-3.73 (m, 24H), 2.85 (ddd, J=23.3, 12.5, 6.7, 6H), 2.39 (q, J=6.3, 4H), 2.24-2.14 (m, 4H), 2.00-1.83 (m, 8H), 2.68-0.75 (m, 107H), 2.46-0.75 (m, 105H), 1.72-1.45 (m, 20H), 1.44-1.35 (m, 6H), 1.34-0.88 (m, 53H), 0.82 (s, 6H). .sup.13C NMR (400 MHz, CDCl.sub.3) δ: 168.13 (s), 167.08 (s), 140.62 (s), 132.99 (s), 131.80 (s), 121.95 (s), 107.88 (s), 94.90 (s), 80.40 (s), 78.39 (s), 78.09 (s), 74.89 (s), 74.02 (s), 73.37 (s), 71.64 (s), 66.85 (s), 64.52 (s), 58.25 (s), 55.26 (s), 51.39 (s), 50.92 (s), 48.12 (s), 44.29 (s), 42.59 (s), 39.59 (s), 39.43 (s), 39.19 (s), 37.80 (s), 37.22 (s), 32.76 (d), 32.25 (s), 30.45 (s), 28.55-28.27 (m), 27.55 (s), 24.28 (s), 22.74 (s), 21.49 (s), 18.73 (s), 12.92 (s), 12.32 (s). Q-TOF-MS, m/z: Found [M+Na].sup.+, 908.4565, C.sub.46H.sub.73Cl.sub.2NO.sub.11 calcd for [M]=885.4561, [M+Na]=908.4561.

Example 4 Preparation of Cationic Liposomes

[0062] The sucrose ester based cationic lipids TSI12, TSB-2I, TSD-2D, TSI14 (sucralose myristate quaternary ammonium salt) and TSI16 (sucralose palmitate quaternary ammonium salt) obtained in the above Examples 1, 2 and 3 were irradiated under a UV light for 1 h. 1 mg of a cationic lipid and a certain amount of DOPE (the molar ratios of cationic lipid to DOPE were 2:1, 1:1 and 1:2) or cholesterol Chol, (the molar ratios of cationic lipid to Chol were 5:1, 4:1, 3:1, 2:1 and 1:1) were added to 1 mL of chloroform to be dissolved completely followed by blow-dry with the uniform nitrogen stream to form a thin film. The resulting film was dried in vacuum for 10 h, and the organic solvent was cleaned up. 1 mL of sterile deionized water was added. At a constant temperature of 55° C., the mixture was subject to ultrasound for 1-3 h until clear clarification so as to prepare cationic liposomes with a final concentration of 1.00 mM.

Example 5 Determination of Particle Size and Zeta Potential of Cationic Liposomes

[0063] Approximately 20 μL of the cationic liposomes prepared in Example 4 was taken and added to about 1 mL of water. The particle size and distribution of the cationic liposomes were measured using a Zeta-sizer-1000 laser particle size analyzer (Japan, HORIBA Scientific Company, the optical maser wavelength of 630 nm and the scattering angle of 90° to 173°), and the results were shown in FIG. 4. The zeta potential of the cationic liposomes was measured using a Zetaplus ζ-potential analyzer (HORIBA Scientific Company, Japan), and the results were shown in FIG. 5. In FIG. 4 and FIG. 5, Lipofectamine 2000 (Invitrogen Company, USA) and DOTAP (N-(2,3-dioleooyloxy-1-propyl) trimethylmethanesulfonate ammonia, Roche Company, Switzerland) are the commonly used cationic liposome in this field. The results in FIG. 4 show that the liposomes of the invention have a particle size in the range of 100 to 350 nm, and the particle size range falls within the effective transfection particle size range (<1 μm). The results in FIG. 5 show that the zeta potential of the liposomes of the invention is between 25 and 90 mV; the Zeta potential of the commercial reagent DOTAP is 29.8 mV and the Zeta potential of Lipofectamine 2000 is 46.4 mV; and they have an ability to bind to negatively charged ions (including nucleic acid and other molecules).

Example 6 Preparation of Liposome-DNA Complexes

[0064] Firstly, a certain amount of plasmid DNA (pGFP-N2 or pGL-3) was diluted in DMEM to prepare a 25 μL system. A certain volume of the cationic liposomes with a concentration of 1 mg/mL prepared in Example 4 was diluted in DMEM to prepare a 25 μL system, and then 25 μL of a liposome diluent was dropwise added to 25 μL of the plasmid DNA diluent so that the mass ratios (μg/μg) of liposome to DNA were 1:1, 2:1, 3:1, 4:1, 6:1, 8:1 and 10:1, respectively. The resulting mixture was mixed thoroughly with a vortex oscillator and was incubated at room temperature for 20 mins to form 50 μL of liposome-DNA complexes.

Example 7 Detection of the Binding Capacity of Cationic Liposomes to DNA

[0065] 20 μL of the complex solution prepared in Example 6 was uniformly mixed with 1.6 μL of loading buffer (Takara Company, Japan) followed by addition into 1.2% agarose gel. The mixture was electrophoresed at 90 V for 1 h. The DNA electrophoretogram was observed and photographed in a gel imaging system, as shown in FIG. 6. FIG. 6a shows the result of measurement of the DNA binding ability of an individual TSI14 liposome. FIGS. 6b, 6c and 6d show the results of measurement of the DNA binding ability of liposomes prepared by mixing TSI14 with DOPE at 2:1, 1:1 and 1:2, respectively.

[0066] FIGS. 6a, 6b, 6c and 6d: the first lane was a 2 kb DNA marker (λDNA/EcoR I+Hind III Markers, purchased from SABC); the second lane was naked DNA; and the 3th-10th lanes were liposome-DNA complexes in which the ratios of liposome to DNA were 1:1, 2:1, 3:1, 4:1, 6:1, 8:1 and 10:1, respectively. Without the addition of liposomes (naked DNA), a typical plasmid band appeared. After the addition of liposomes, the DNA band was significantly weakened. With the increase of the added amount of liposomes, the delay ability of DNA was obviously enhanced. It showed that the liposomes prepared by the invention had the capability of binding DNA.

Example 8 Determination of Transfection Efficiency

[0067] The liposome-DNA complex prepared in Example 6 was taken to carry out a condition experiment in a cell line Hep-2 (human laryngeal carcinoma epithelial cells) to investigate the transfection efficiency under different conditions and to optimize the use conditions of the reagent.

[0068] The cells were planted on a 96-well cell culture plate. An appropriate amount of cells is planted in each well, and the total volume of a cell culture fluid (comprising serum and antibiotics) was 100 μL. The cells were placed into an incubator with 5% of CO.sub.2 at a temperature of 37° C. for 24 h to achieve a cell density of 1 to 1.5×10.sup.4 on the day of transfection.

[0069] The growth medium was removed and was replaced with a medium with the equivalent amount (100 μL, without serum and antibiotics). 50 μL of the liposome-DNA complex sample of Example 6 was directly added into the plate wells, with 3 parallel wells per sample, followed by shaking the culture plate and gently mixing. The culture medium was cultivated in an incubator with 5% of CO.sub.2 at a temperature of 37° C. for 5 h, and was replaced with a medium containing serum and antibiotics. After incubation for 48 h, the gene expression analysis was carried out.

[0070] Gene Expression Analysis:

[0071] (1) Green fluorescent protein detection: The green fluorescent protein signal was observed by an inverted fluorescence microscope. Positive cells emitted bright green fluorescence, while negative cells failed. The results were shown in FIG. 7.

[0072] (2) Luciferase detection: The sample was firstly washed with PBS for 3 times, and then 100 μL of lysis buffer was added into each well. Lysate was collected, and was centrifuged at 12000 rpm and 4° C. for 5 min. 20 μL of supernatant was used for detecting the activity of luciferase. The relative light unit (RUL) value was detected on the Luminometer by a luciferase detection reagent. The luminescence time was measured to be 10 s. The results were shown in FIG. 8.

[0073] Experimental results were shown in FIGS. 7 and 8. The liposomes prepared according to the invention have the ability of delivering DNA. Wherein when the ratios of the liposome TSI14+C 3/1 to DNA were 6/1 and 8/1, and the ratio of the liposome TSI14+D 1/1 to DNA was 10/1, the transfection efficiency was higher than that of the commercial reagents Lipofectamine2000 and DOTAP.

Example 9 Determination of MTT Toxicity

[0074] Hep-2 (human laryngeal carcinoma epithelial cells) was planted on a 96-well cell culture plate, and 3 wells were parallel and averaged. 1.5×10.sup.4 Cells/100 μL of a culture solution were seeded in each well and were incubated to 90% confluency under the environment of 37° C. and 5% of CO.sub.2. The medium was removed followed by washing with D-Hanks once. 0.5 μg of the plasmid DNA (pGFP-N2) and the transfection reagent were diluted in 25 μL of DMEM, respectively, and then the liposome-DNA complex prepared in Example 6 was added to a 96-well cell culture plate. Similarly, the complex of Lipofectamine 2000 (Lipo) (its mass ratio to plasmid was 3:1) and DOTAP (its mass ratio to plasmid was 6:1) was added as a positive control into a 96-well cell culture plate. 100 μL of the medium free of cationic liposomes (without serum and antibiotics) was used as a negative control. The culture medium continued to be incubated in an incubator with 5% of carbon dioxide at 37° C. After 24 h, 20 μL, of 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL in PBS, pH 7.4) was added into each well and continued to be incubated for 5 h, followed by removing the medium. The resulting formazan crystals were dissolved with 150 μL of dimethylsulfoxide (DMSO) and a vigorous mixing rendered formazan dissolved. The reading was taken in a microplate reader, and the absorption wavelength was 570 nm. The microplate reader was adjusted to zero with a cell-free medium. The relative viability of the cationic liposomes relative to the control cells was calculated according to the following formula:

[A].sub.Sample/[A].sub.Control×100

[0075] [A].sub.Sample is a light absorption value of the test well, and [A].sub.Control is a light absorption value of the negative control well.

[0076] Experimental results were shown in FIG. 9. The cell viability of the sucrose ester based cationic liposomes prepared in the invention in Hep-2 cells was generally higher than that of the commercial reagents Lipofectamine 2000 and DOTAP.

Example 10 Preparation of Cationic Micelles

[0077] 1 mg of TSD-2D, and a certain amount of sucrose ester (the molar ratios of cationic lipid to sucrose ester were 2:1, 1:1 and 1:2), chitosan (the molar ratios of cationic lipid to chitosan were 2:1, 1:1 and 1:2) or palmitoyl oleoylphosphatidylethanolamine (the molar ratios of cationic lipid to palmitoyl oleoylphosphatidylethanolamine were 5:1, 4:1, 3:1, 2:1 and 1:1) were added and dissolved in DMF to prepare a solution at a concentration of 1 mg/mL. The deionized water was added dropwise under vigorous stirring until the solution became slightly cloudy to obtain a stable micellar solution. It was transferred to a dialysis bag and was placed in deionized water for dialysis during which the deionized water was replaced for several times. After 3 days, an aqueous solution with a certain concentration of 1 mg/mL was formulated.

Example 11 Preparation of Micelle-siRNA Complexes

[0078] Firstly, a certain amount of siRNA was diluted in DMEM to prepare a 25 μL system. A certain volume of the cationic micelles with a concentration of 1 mg/mL prepared in Example 10 were further diluted in DMEM to prepare a 25 μL system. Then 25 μL of the micelle diluent was added dropwise into 25 μL of the diluent of the plasmid DNA, so that the mass ratios (μg/μg) of the micelles to the siRNA were 1:1, 2:1, 3:1, 4:1, 6:1, 8:1 and 10:1, respectively. The mixture was fully mixed with a vortex oscillator and was incubated at room temperature for 20 min to form 50 μL of micelle-siRNA complexes.

Example 12 Detection of the Binding Ability of Cationic Micelles to siRNA

[0079] 20 μL of the micelle-siRNA complex solution prepared in Example 11 was mixed well with 1.6 μL of loading buffer (Takara Company, Japan). The mixture was added to 1.2% agarose gel, and was electrophoresed at 90 V for 1 h. The siRNA electrophoretogram was observed and photographed in a gel imaging system.