Composition for gene delivery comprising chitosan and liquid crystal formation material

Abstract

Disclosed herein is a composition for gene delivery which forms into liquid crystals in an aqueous fluid including chitosan and a liquid crystal formation material. The composition of the present invention including chitosan and a liquid crystal formation material improves the relatively low binding strength between chitosan and a gene, and also considerably increases the stability of a chitosan nanocomposite in blood serum, thereby performing highly efficient gene delivery and providing excellent stability, thus being useful as a gene therapeutic agent.

Claims

1. An aqueous injectable composition, comprising: (a) a liquid crystal formation material consisting of a sorbitan unsaturated fatty acid ester that has at least two hydroxyl groups in the polar head group; (b) a phospholipid; (c) a liquid crystal hardener; (d) chitosan or its salts; (e) a dispersing agent that is one or more selected from among a poloxamer, polyvinylalcohol, polyethylene glycol-glyceryl monooleate, hydrophobically modified starch, and a combination thereof; (f) a nucleic acid molecule comprising a gene or encoding a gene product for gene therapy; (g) protamine or pharmaceutically acceptable salts thereof, (h) sodium lauryl sulfate; and (i) water, wherein: the aqueous injectable composition is formulated to be administered via an intravenous injection, an intramuscular injection, a subcutaneous injection, an intrauterine injection, or a cerebrovascular injection for effecting gene therapy; the sorbitan unsaturated fatty acid ester is one or more selected from among sorbitan monooleate, sorbitan monolinoleate, sorbitan monopalmitoleate, sorbitan monomyristoleate, sorbitan sesquioleate, sorbitan sesquilinoleate, sorbitan sesquipalmitoleate, sorbitan sesquimyristoleate, sorbitan dioleate, sorbitan dilinoleate, sorbitan dipalmitoleate, sorbitan dimyristoleate, and a combination thereof; the phospholipid is at least one selected from among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, phosphatidylinositol, phosphatidic acid, sphingomyelin, and a combination thereof; the liquid crystal hardener is at least one selected from among tocopherol acetate, cholesterol, benzyl benzoate, ubiquinone, and a combination thereof; the salt of chitosan is at least one selected from among chitosan HCl, chitosan acetate, chitosan glutamate, and chitosan lactate; the nucleic acid molecule is at least one selected from among single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, plasmid DNA, single-stranded siRNA, double-stranded siRNA, an anti sense oligonucleotide, a ribozyme, a catalytic RNA, and a polymer of nucleotides; the sorbitan unsaturated fatty acid ester, the phospholipid, and the liquid crystal hardener are included in a weight ratio of 0.20-1:1-1.5:0.05-0.2; components (a)-(c) form a liquid crystal having a non-lamellar structure in an aqueous fluid; the liquid crystal consists of the liquid crystal formation material, the phospholipid and the liquid crystal hardener; the aqueous injectable composition comprises a nanocomposite having a size from about 10 to 500 nm; and the nanocomposite comprises the liquid crystal, the chitosan or its salts and the nucleic acid molecule.

2. The composition of claim 1, further comprising thiamine pyrophosphate or pharmaceutically acceptable salts thereof.

3. The composition of claim 1, further comprising at least one saccharide selected from among mannose, fructose, arabinose, trehalose, maltose, cellobiose, isomaltose, dextrin, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, dimethyl-β-cyclodextrin, trimethyl-β-cyclodextrin, and sulfobutylether β-cyclodextrin.

4. The composition of claim 1, further comprising a stabilizing agent, a buffer, a preservative, an analgesic agent, and a tonicity agent.

5. The composition of claim 1, wherein the chitosan is in the form of a coating.

6. A composition for gene delivery, comprising: a first solution capable of forming a liquid crystal having a non-lamellar structure in an aqueous fluid, comprising a liquid crystal formation material consisting of a sorbitan unsaturated fatty acid ester which has at least two hydroxyl groups in the polar head group, a phospholipid, and a liquid crystal hardener, wherein: the sorbitan unsaturated fatty acid ester is one or more selected from among sorbitan monooleate, sorbitan monolinoleate, sorbitan monopalmitoleate, sorbitan monomyristoleate, sorbitan sesquioleate, sorbitan sesquilinoleate, sorbitan sesquipalmitoleate, sorbitan sesquimyristoleate, sorbitan dioleate, sorbitan dilinoleate, sorbitan dipalmitoleate, sorbitan dimyristoleate, and a combination thereof; the phospholipid is at least one selected from among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, phosphatidylinositol, phosphatidic acid, sphingomyelin, and a combination thereof; the liquid crystal hardener is at least one selected from among tocopherol acetate, cholesterol, benzyl benzoate, ubiquinone, and a combination thereof; and the sorbitan unsaturated fatty acid ester, phospholipid, and the liquid crystal hardener are included in a weight ratio of 0.20-1:1-1.5:0.05-0.2; and a second solution comprising: chitosan or its salts; a dispersing agent selected from among one or more of a poloxamer, polyvinylalcohol, polyethylene glycol-glyceryl monooleate, hydrophobically modified starch, and a combination thereof; a nucleic acid molecule comprising a gene or encoding a gene product for gene therapy; protamine or pharmaceutically acceptable salts thereof; sodium lauryl sulfate; and water, wherein: the salt of chitosan is at least one selected from among chitosan HCl, chitosan acetate, chitosan glutamate, and chitosan lactate; the nucleic acid molecule is at least one selected from among single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, plasmid DNA, single-stranded siRNA, double-stranded siRNA, an antisense oligonucleotide, a ribozyme, a catalytic RNA, and a polymer of nucleotides; a nanocomposite is formed upon mixing the two solutions; the nanocomposite comprises a liquid crystal, the chitosan or its salts and the nucleic acid molecule; the liquid crystal consists of the liquid crystal formation material, the phospholipid and the liquid crystal hardener; and the composition is formulated to be administered via an intravenous injection, an intramuscular injection, a subcutaneous injection, an intrauterine injection, or a cerebrovascular injection for effecting gene therapy.

7. A lyophilized preparation of a composition for gene delivery, wherein the composition comprises: (a) a liquid crystal formation material consisting of a sorbitan unsaturated fatty acid ester which has at least two hydroxyl groups in the polar head group; (b) a phospholipid; (c) a liquid crystal hardener; (d) chitosan or its salts; (e) a dispersing agent, selected from among one or more of a poloxamer, polyvinylalcohol, polyethylene glycol-glyceryl monooleate, hydrophobically modified starch, and a combination thereof; (f) a nucleic acid molecule comprising a gene or encoding a gene product for gene therapy; (g) protamine or pharmaceutically acceptable salts thereof, and (h) sodium lauryl sulfate, wherein: the composition is formulated to be administered via an intravenous injection, an intramuscular injection, a subcutaneous injection, an intrauterine injection, or a cerebrovascular injection for effecting gene therapy; the sorbitan unsaturated fatty acid ester is one or more selected from among sorbitan monooleate, sorbitan monolinoleate, sorbitan monopalmitoleate, sorbitan monomyristoleate, sorbitan sesquioleate, sorbitan sesquilinoleate, sorbitan sesquipalmitoleate, sorbitan sesquimyristoleate, sorbitan dioleate, sorbitan dilinoleate, sorbitan dipalmitoleate, sorbitan dimyristoleate, and a combination thereof; the phospholipid is at least one selected from among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, phosphatidylinositol, phosphatidic acid, sphingomyelin, and a combination thereof; the liquid crystal hardener is at least one selected from among tocopherol acetate, cholesterol, benzyl benzoate, ubiquinone, and a combination thereof; the sorbitan unsaturated fatty acid ester, phospholipid, and the liquid crystal hardener are included in a weight ratio of 0.20-1:1-1.5:0.05-0.2; the salt of chitosan is at least one selected from among chitosan HCl, chitosan acetate, chitosan glutamate, and chitosan lactate; the nucleic acid molecule is at least one selected from among single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, plasmid DNA, single-stranded siRNA, double-stranded siRNA, an anti sense oligonucleotide, a ribozyme, a catalytic RNA, and a polymer of nucleotides; components (a)-(c) form a liquid crystal having a non-lamellar structure in an aqueous fluid; the liquid crystal consists of the liquid crystal formation material, the phospholipid, and the liquid crystal hardener; and the preparation is a solid lyophilized preparation.

8. The lyophilized preparation of claim 7, further comprising an excipient for lyophilization.

9. The lyophilized preparation of claim 8, wherein the excipient for lyophilization is a sugar alcohol or a saccharide.

10. The lyophilized preparation of claim 9, wherein the saccharide is at least one selected from among mannose, fructose, arabinose, trehalose, maltose, cellobiose, isomaltose, dextrin, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, dimethyl-β-cyclodextrin, trimethyl-β-cyclodextrin, and sulfobutylether β-cyclodextrin.

11. The composition of claim 1, wherein the nanocomposite is a gene therapeutic agent.

12. The composition of claim 1, wherein the nanocomposite has a size from about 200 to about 300 nm.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1A shows a picture of the structures of liquid crystals in an aqueous fluid prepared in Example 2, and FIG. 1B shows a picture of the structures of liquid crystals in an aqueous fluid prepared in Example 6;

(2) FIGS. 2A-2C show pictures of the structures of liquid crystals in an aqueous fluid prepared in Examples 2 (FIG. 2A), 6 (FIG. 2B), and 10 (FIG. 2C), respectively, via Cryo transmission electron microscopy (Cryo-TEM).

(3) FIG. 3 is a graph showing the particle size of liquid crystals in Examples 7, 10, 12, 13, 14, 17, 19 and Comparative Example 1;

(4) FIG. 4 is a graph showing the observation results of the inhibitory capability of the liquid crystals prepared in Examples 7, 10, 12, 13, 14, 17, 19 and Comparative Example 1 against target proteins;

(5) FIG. 5 is a graph showing the stability of particles in blood serum of the liquid crystals prepared in Examples 10, 17 18, 19, and Comparative Examples 2 and 3;

(6) FIG. 6 is a graph showing the size of particles of the liquid crystals prepared in Examples 23 and 24 after lyophilization; and

(7) FIG. 7 is a graph showing the observation results of the inhibitory capability of the liquid crystals prepared in Example 10 and Comparative Example 4 against tumor growth.

MODE FOR THE INVENTION

(8) Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to a specific disclosure type, but should be construed as including all modifications, equivalents, substitutes involved in the scope and the technological range of the present invention.

Examples 1-6

Preparation of Lipid Compositions Forming Liquid Crystal

(9) Phospholipid, sorbitan unsaturated fatty acid ester, a liquid crystal hardener, and ethanol were added in the amount as shown in Table 1 below, and the mixture was agitated at room temperature to obtain a uniform lipid solution.

(10) 0.15 mL of the lipid solution was added into 5 mL of 1.1% poloxamer 407 aqueous solution, and homogenized at 20-75° C. using an ultrasonicator or a homogenizer (PowerGenmode1123, Fisher) at a rate of 1,000-3,000 RPM for 5-10 minutes, and a composition for liquid crystals in a solution phase was obtained. Table 1 below shows the weight ratios for the final components of the compositions prepared thereof.

(11) TABLE-US-00001 TABLE 1 Unit (mg) Ex. 1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 Ex. 6 Lipoid 1.39 6.95 13.90  1.39 6.95 13.90  S-100.sup.1) Sorbitan 0.92 2.30 4.60 — — — monooleate Sorbitan — — — 0.92 2.30 4.60 sesquinoleate Polysorbate 0.05 — 0.50 — 0.25 0.50 80 Tocopherol 0.18 0.45 0.90 0.18 0.45 0.90 acetate Ethanol 0.36 0.45 0.60 0.90 0.36 0.45 Poloxamer 407 1.1  1.1  1.1  1.1  1.1  1.1  .sup.1)Lipoid S-100 consists of phosphotidylcholine, phospatidylethanolamine, phosphatidylinositol, and lysophosphatidylcholine, and includes phosphatidylcholine more than 94% as a major component.

Examples 7-13

Preparation of Composites with Gene, Chitosan, Protamine, Sodium Lauryl Sulfate, and Liquid Crystal Forming Lipid Composition

(12) Phospholipid, sorbitan unsaturated fatty acid ester, a liquid crystal hardener, and ethanol were added in the amount as shown in Table 2 below, and the mixture was agitated at room temperature to obtain a uniform lipid solution.

(13) 0.15 mL of the lipid solution was added into 5 mL of 1.1% poloxamer 407 aqueous solution, and homogenized at 20-75° C. using an ultrasonicator or a homogenizer (Power Gen Model 123, Fisher) at a rate of 1,000-3,000 RPM for 5-10 minutes, and obtained was a composition for liquid crystals in a solution phase.

(14) When gene delivery is to be performed by further adding sodium lauryl sulfate, survivin siRNA, which is to be delivered as an active material, was mixed with sodium lauryl sulfate in a weight ratio of 1:1 (w/w), and the mixture was prepared at the concentration of 20 mg/mL using water for injection. Chitosan HCl having a molecular weight of 150 kDa, protamine and poloxamer 407 were mixed at weight ratios shown in Table 2 below and prepared at a concentration of 8 mg/mL using water for injection, and then mixed with 20 mg/mL of the mixed solution including survivin siRNA and sodium lauryl sulfate, at a volume ratio of 1:2.5 (v/v) and agitated thereafter. The survivin siRNA mixed solution which does not include sodium lauryl sulfate was prepared in the same manner as above except for the step of mixing sodium lauryl sulfate.

(15) Finally, the thus prepared liquid crystal composition was added with survivin siRNA mixed solution according to the weight ratios shown in Table 2 below to obtain composites and the resulting composites were filtered using a 0.22 μm filter. The survivin siRNA used had a sense sequence of 5′-AAG GAG AUC AAC AUU UUC A(dTdT)-3′, and an antisense sequence of 5′-UGA AAA UGU UGA UCU CCU U(dTdT)-3′. Table 2 below shows the weight ratios of final components of the thus prepared compositions.

(16) TABLE-US-00002 TABLE 2 Unit (ug) Ex. 7 Ex. 8 Ex. 9 Ex. 10 Ex. 11 Ex. 12 Ex. 13 Lipoid 1.39 1.39 1.39 1.39 1.39 1.39 1.39 S-100.sup.1) Sorbitan 0.92 0.92 0.92 0.92 0.92 0.92 0.92 monooleate Polysorbate 0.05 0.05 0.05 0.05 0.05 0.05 0.05 80 Tocopherol 0.18 0.18 0.18 0.18 0.18 0.18 0.18 acetate Ethanol 0.36 0.36 0.36 0.36 0.36 0.36 0.36 Poloxamer 2.20 2.20 2.20 2.20 2.20 2.20 2.20 407 Survivin 1.00 1.00 1.00 1.00 1.00 1.00 1.00 siRNA Sodium — 1.00 — 1.00 1.00 1.00 1.00 lauryl sulfate Protamine — — 0.60 0.60 0.60 0.60 0.60 Chitosan 0.30 0.30 0.30 0.30 5.00 10.00 20.00 HCl

Examples 14-19

Preparation of Composites with Gene, Chitosan, Protamine, Thiamine Pyrophosphate and Liquid Crystal Forming Lipid Composition

(17) Phospholipid, sorbitan unsaturated fatty acid ester, a liquid crystal hardener, and ethanol were added in the amount as shown in Table 3 below, and the mixture was agitated at room temperature to obtain a uniform lipid solution.

(18) 0.15 mL of the lipid solution was added into 5 mL of 1.1% poloxamer 407 aqueous solution, and homogenized at 20-75° C. using an ultrasonicator or a homogenizer (PowerGenmode1123, Fisher) at a rate of 1,000-3,000 RPM, and a composition for liquid crystals in a solution phase was obtained.

(19) 20 mg of survivin siRNA, the gene to be delivered as an active material, 30 mg of chitosan HCl having a molecular weight of 150 kDa, 30 mg of thiamine pyrophosphate and 1 mg of protamine were dissolved in 1 mL of water for injection, respectively. The survivin siRNA mixed solution which does not include thiamine pyrophosphate was prepared in the same manner except for the step of dissolving thiamine pyrophosphate in 1 mL of water for injection.

(20) The thus prepared liquid crystal composition and each of the aqueous solutions prepared thereof were added according to the weight ratios shown in Table 3 to obtain a composite and the resulting composite was filtered using a 0.22 μm filter. Table 3 below shows the weight ratios of final components of the thus prepared compositions.

(21) TABLE-US-00003 TABLE 3 Unit (ug) Ex. 14 Ex. 15 Ex. 16 Ex. 17 Ex. 18 Ex. 19 Lipoid 1.39 1.39 1.39 1.39 0.35 0.35 S-100.sup.1) Sorbitan 0.92 0.92 0.92 0.92 0.23 0.23 monooleate Polysorbate 0.05 0.05 0.05 0.05 0.01 0.01 80 Tocopherol 0.18 0.18 0.18 0.18 0.05 0.05 acetate Ethanol 0.36 0.36 0.36 0.36 0.09 0.09 Poloxamer 407 0.28 0.28 0.28 0.28 0.28 0.50 Survivin 1.00 1.00 1.00 1.00 1.00 1.00 siRNA Thiamine — — 1.00 1.00 4.00 6.00 pyrophosphate Protamine — 0.60 — 0.60 0.60 0.60 Chitosan 0.30 0.30 0.30 0.30 0.30 0.30 HCl

Examples 20-24

Preparation of Lyophilizate of Composites with Liquid Crystal Forming Lipid Composition, Gene, Chitosan, Protamine and Sodium Lauryl Sulfate

(22) Phospholipid, sorbitan unsaturated fatty acid ester, a liquid crystal hardener, and ethanol were added in the amount as shown in Table 4 below, and the mixture was agitated at room temperature to obtain a uniform lipid solution.

(23) 0.15 mL of the lipid solution was added into 5 mL of 1.1% poloxamer 407 aqueous solution, and homogenized at 20-75° C. using an ultrasonicator or a homogenizer (PowerGenmode1123, Fisher) at a rate of 1,000-3,000 RPM, and a composition for liquid crystals in a solution phase was obtained.

(24) Survivin siRNA and sodium lauryl sulfate were mixed in a weight ratio of 1:1 (w/w), and the mixture was prepared at a concentration of 20 mg/mL using water for injection. Chitosan HCl with a molecular weight of 150 kDa, protamine and poloxamer 407 was mixed at a weight ratio of 1:2:4 (w/w) and prepared at a concentration of 8 mg/mL using water for injection, and then mixed with 20 mg/mL of the mixed solution including survivin siRNA and sodium lauryl sulfate, at a volume ratio of 1:2.5 (v/v) and agitated thereafter. The survivin siRNA mixed solution which does not include sodium lauryl sulfate was prepared in the same manner as above except for the step of mixing sodium lauryl sulfate.

(25) The thus prepared liquid crystal composition was added with the survivin siRNA mixed solution according to the weight ratios shown in Table 4 to obtain a composite and the resulting composite was filtered using a 0.22 μm filter. In order to lyophilize the thus prepared composite, an excipient prepared by using 30% solution in advance was mixed according to the weight ratios shown in Table 4, frozen at −70° C., and lyophilized to obtain a solid phase product.

(26) TABLE-US-00004 TABLE 4 Unit (ug) Ex. 20 Ex. 21 Ex. 22 Ex. 23 Ex. 24 Lipoid 1.39 1.39 1.39 1.39 1.39 S-100.sup.1) Sorbitan 0.92 0.92 0.92 0.92 0.92 monooleate Polysorbate 0.05 0.05 0.05 0.05 0.05 80 Tocopherol 0.18 0.18 0.18 0.18 0.18 acetate Ethanol 0.36 0.36 0.36 0.36 0.36 Poloxamer 2.20 2.20 2.20 2.20 2.20 407 Survivin 1.00 1.00 1.00 1.00 1.00 siRNA Sodium — — 1.00 1.00 1.00 lauryl sulfate Protamine — 0.60 — 0.60 0.60 Chitosan 0.30 0.30 0.30 0.30 0.30 Trehalose 23.00  23.00  23.00  23.00 Glucose 23.00

Comparative Example 1

Preparation of Composite with Liquid Crystal Forming Lipid Composition, Gene, Protamine, and Sodium Lauryl Sulfate

(27) In order to compare the producing capability of a composite for gene delivery according to its presence/absence of chitosan, composites for gene delivery without chitosan were manufactured and compared. In particular, phospholipid, sorbitan unsaturated fatty acid ester, a liquid crystal hardener, and ethanol were added in the amount as shown in Table 5 below, and the mixture was agitated at room temperature to obtain a uniform lipid solution.

(28) 0.15 mL of the lipid solution was added into 5 mL of 1.1% poloxamer 407 aqueous solution, and homogenized at 20-75° C. using an ultrasonicator or a homogenizer (PowerGenmode1123, Fisher) at a rate of 1,000-3,000 RPM, and a composition for liquid crystals in a solution phase was obtained.

(29) Survivin siRNA and sodium lauryl sulfate were mixed at a weight ratio of 1:1 (w/w), and the mixture was prepared at a concentration of 20 mg/mL using water for injection. Protamine and poloxamer 407 were mixed at a weight ratio of 2:4 (w/w) and prepared at a concentration of 8 mg/mL using water for injection, and then mixed with 20 mg/mL of the mixed solution including survivin siRNA and sodium lauryl sulfate, at a volume ratio of 1:2.5 (v/v) and agitated thereafter. The survivin siRNA mixed solution which does not include sodium lauryl sulfate was prepared in the same manner as above except for the step of mixing sodium lauryl sulfate.

(30) The thus prepared lipid solution was added with the survivin siRNA mixed solution according to the weight ratios shown in Table 5 to obtain a composite and the resulting composite was filtered using a 0.22 μm filter.

(31) TABLE-US-00005 TABLE 5 Unit (ug) Comp. Ex. 1 Lipoid S-100.sup.1) 1.39 Sorbitan monooleate 0.92 Polysorbate 80 0.05 Tocopherol 0.18 acetate Ethanol 0.36 Poloxamer 407 2.20 Survivin siRNA 1.00 Sodium lauryl sulfate 1.00 Protamine 0.60

Comparative Examples 2 and 3

Preparation of Composite with Genetic Drug Consisting of Gene, Chitosan, Thiamine Pyrophosphate and Protamine

(32) In order to confirm whether a composite for gene delivery can be formed in the absence of a material which forms liquid crystals, composites not including a material which forms liquid crystals were manufactured.

(33) First, 30 mg of chitosan HCl having a molecular weight of 150 kDa, 2 mg of thiamine pyrophosphate, and 0.5 mg of protamine were dissolved in 1 mL of water for injection, respectively, and the thus prepared aqueous solutions were mixed according to the weight ratios shown in Table 6 to obtain a composite.

(34) TABLE-US-00006 TABLE 6 Unit (ug) Comp. Ex. 2 Comp. Ex. 3 Survivin siRNA 1.00 1.00 Thiamine 17.10 17.10 pyrophosphate Protamine 0.60 Chitosan HCl 2.90 2.90

Comparative Example 4

Preparation of Composites Between a Gene, Water-Soluble Chitosan, Protamine, Sodium Lauryl Sulfate and a Liquid Crystal Forming Lipid Composition

(35) In order to confirm whether a gene included as an active material is effectively delivered for the intended purpose, a comparative group including fluorescein-labeled dsRNA oligomer (FL dsRNA, Invitrogen), which is a gene not capable of inhibiting tumor growth, was prepared instead of the survivin siRNA, and used as a control group to compare the effectiveness of gene delivery.

(36) First, phospholipid, sorbitan unsaturated fatty acid ester, a liquid crystal hardener, and ethanol were added in the amount as shown in Table 7 below, and the mixture was agitated at room temperature to obtain a uniform lipid solution.

(37) 0.15 mL of the lipid solution was added into 5 mL of 1.1% poloxamer 407 aqueous solution, and homogenized at 20-75° C. using an ultrasonicator or a homogenizer (PowerGenmode1123, Fisher) at a rate of 1,000-3,000 RPM, and a composition for liquid crystals in a solution phase was obtained.

(38) FL dsRNA and sodium lauryl sulfate were mixed at a weight ratio of 1:1 (w/w), and the mixture was prepared at a concentration of 20 mg/mL using water for injection. The nucleotide sequence used for FL dsRNA was 5′-UUG UUU UGG AGC ACG GAA A(dTdT)-3′. Chitosan HCl having a molecular weight of 150 kDa, protamine and poloxamer 407 were mixed at a weight ratio of 1:2:4 (w/w) and prepared at a concentration of 8 mg/mL using water for injection, and then mixed with 20 mg/ml of the mixed solution including survivin siRNA and sodium lauryl sulfate, at a volume ratio of 1:2.5 (v/v) and agitated thereafter.

(39) The thus prepared lipid solution was added with the FL dsRNA mixed solution according to the weight ratio shown in Table 7 to obtain a composite and the resulting composite was filtered using a 0.22 μm filter.

(40) TABLE-US-00007 TABLE 7 Unit (ug) Comp. Ex. 4 Lipoid S-100 1.39 Sorbitan monooleate 0.92 Polysorbate 80 0.05 Tocopherol acetate 0.18 Ethanol 0.36 Poloxamer 407 2.20 FL dsRNA 1.00 Sodium lauryl sulfate 1.00 Protamine 0.60 Chitosan HCl 0.30

Experimental Example 1

Confirmation of Structures of Liquid Crystals in an Aqueous Fluid Phase

(41) In order to confirm the structures of liquid crystals formed in an aqueous fluid phase by the lipid compositions prepared in Examples 1-6 using a polarization microscope (Motic, BA 300 Pol), the lipid compositions prepared in Examples 1-6 were very thinly applied on slide glasses, placed in Chalet including triple distilled water for 4 hours to form liquid crystals on slide glasses.

(42) The slide glasses were covered with cover glasses to prevent introduction of any air thereinto, and were observed under a polarization microscope (Motic, BA 300 Pol) at 200× magnification.

(43) The results are shown in FIG. 1A and FIG. 1B.

(44) As shown in FIGS. 1A and 1B, the structures of liquid crystals of the lipid compositions prepared in Examples 2 and 6, respectively, were confirmed to have the typical hexagonal structure which can provide excellent particle stability.

Experimental Example 2

Observation of Composites with Gene, Chitosan, Sodium Lauryl Sulfate, Protamine and Liquid Crystal Forming Lipid Composition Under Cryo Transmission Electron Microscope

(45) The lipid compositions prepared in Examples 2, 6 and 10 were observed under Cryo transmission electron microscopy (Cryo-TEM) (Tecnai 12 electron microscope, Philips, the Netherlands), and the formation of liquid crystal composite compositions for gene delivery was confirmed. The compositions were placed on top of grids in the form of thin meninges, and lyophilized at −170° C. The composites of the lyophilized compositions on top of the grids were observed according to the user's manual by the manufacturer.

(46) The results are shown in FIGS. 2A-2C.

(47) As shown in FIGS. 2A and 2B, it was confirmed that the lipid compositions prepared in Examples 2 and 6, respectively, formed the typical liquid crystals because they formed a spherically hexagonal type of a net in an aqueous fluid phase. Additionally, in Example 10, as shown in FIG. 2C, it was confirmed that the liquid crystal composite composition for gene delivery consisting of a gene, water-soluble chitosan, sodium lauryl sulfate, protamine, and a liquid crystal forming lipid composition formed a typical spherical type of a nanocomposite.

Experimental Example 3

Measurement of Particle Size Using Composites with a Gene, Chitosan, Sodium Lauryl Sulfate, Thiamine Pyrophosphate, Protamine, and a Liquid Crystal Forming Lipid Composition

(48) The particle size of the compositions prepared in Examples 7, 10, 12, 13, 14, 17, and 19, and Comparative Example 1 was measured using Electrophoretic Light Scattering Spectrophotometer (ELS) particle size and electric charge analyzer (ELS-Z, Otsuka, Japan). The particle size of the compositions was measured using particle size cell according to the user's manual by the manufacturer. The results are shown in FIG. 3.

(49) As shown in FIG. 3, it was confirmed that all the liquid crystal composite compositions for gene delivery which selectively do not include protamine in the composite compositions, which include a gene, water-soluble chitosan and a liquid crystal forming lipid composition (Examples 7 and 14), or those which further include sodium lauryl sulfate (Examples 10, 12, and 13) or thiamine pyrophosphate (Examples 17 and 19) were all shown to have a uniform particle size in the range of 200-300 nm. However, the composite compositions, which not include chitosan (Comparative Example 1) was shown to has ununiform particle size and the particle size became bigger. Accordingly, it was confirmed that chitosan is essential in forming a composite having a uniform particle diameter suitable for cell penetration.

Experimental Example 4

Observation of the Inhibitory Effect Against Target Protein Expression by Composites with a Gene, Chitosan, Sodium Lauryl Sulfate, Thiamine Pyrophosphate, Protamine, and a Liquid Crystal Forming Lipid Composition

(50) In order to evaluate the inhibitory effect against target protein expression by the groups treated with compositions prepared in Comparative Example 1, Examples 7, 10, 12, 13, 14, 17, and 19, and by groups treated with a gene alone, experiments were performed by a method described below using a survivin ELISA kit (R&D systems, catalog No. SVE00). A human prostate cancer cell line (PC-3, ATCC) was aliquoted into a 6-well plate so that each well has 1×105 cells, and incubated in a 10% fetal bovine serum(FBS)-containing medium (pH 7.2) at 37° C., 5% CO.sub.2 condition for 48 hours. Prior to administration of a genetic drug, the medium was replaced with a fresh 10% FBS-containing medium (pH7.2), and then stabilized for 2 hours. Then, compositions prepared in Comparative Example 1, Example 7, 10, 12, 13, 14, 17, and 19, and a gene alone were respectively added to the prostate cancer cell line and incubated at 37° C., 5% CO.sub.2 condition for 48 hours. The genetic drug administered was 2 μg/well. Upon completion of the incubation, the medium was removed and added with 500 μl of cell lysis buffer (cell signaling technology) to collect the prostate cancer cell line. Additionally, the collected material was centrifuged at 12,000 rpm for 20 minutes in order to separate cell residues from the supernatant. The inhibitory effect against target protein expression was compared by using 100 μl of the supernatant, and the amount of protein expression of survivin contained in the medium sample was measured using survivin ELISA kit according to the user's manual of the manufacturer.

(51) The results are shown in FIG. 4.

(52) As shown in FIG. 4, it was confirmed that the liquid crystal composite compositions for gene delivery which selectively do not include protamine in the composite compositions, which include a gene, water-soluble chitosan and a liquid crystal forming lipid composition (Examples 7 and 14), or those which further include sodium lauryl sulfate (Examples 10, 12, and 13) or thiamine pyrophosphate (Examples 17 and 19) were all shown to effectively inhibit the expression of target proteins via effective gene delivery into cells. In contrast, in a composite treated with a gene alone or in a composite where chitosan is absent (Comparative Example 1), the expression of target proteins was not inhibited at all. From the above, it was confirmed that cationic chitosan is essential for effective gene delivery although cationic protamine is present. That is, chitosan plays a crucial role in the composite according to the present invention, and the gene delivery function is completely lost in the absence of chitosan.

Experimental Example 5

Safety Evaluation in Blood Serum of Composites with a Gene, Chitosan, Sodium Lauryl Sulfate, Thiamine Pyrophosphate, Protamine, and a Liquid Crystal Forming Lipid Composition

(53) In order for chitosan to have in vivo biocompatibity as a gene delivery vector it is essential to secure its particle stability in blood serum but chitosan has the drawback that it causes precipitation in blood serum and is thus incapable of gene delivery. Accordingly, in order to confirm whether the liquid crystal composite compositions for gene delivery of the present invention can form stable particles in blood serum, the composites prepared in Examples of the present invention were compared with those in Comparative Examples 2 and 3, which do not include a liquid crystal formation material.

(54) More specifically, composites prepared in Comparative Examples 2 and 3 and Examples 10, 17, 18 and 19 were mixed so that blood serum can be 50%, and in 3 hours, the change in particle size was measured using Electrophoretic Light Scattering Spectrophotometer (ELS) particle size and electric charge analyzer (ELS-Z, Otsuka, Japan). The particle size of the compositions was respectively measured using particle size cell according to the user's manual by the ELS manufacturer.

(55) The results are shown in FIG. 5.

(56) As shown in FIG. 5, it was confirmed that the liquid crystal composite compositions for gene delivery which selectively further include sodium lauryl sulfate (Example 10) or thiamine pyrophosphate (Examples 17, 18, and 19) in the composite composition including a gene, water-soluble chitosan and a liquid crystal forming lipid composition maintained their particle size at the initial manufactured state to be in the range of 200-300 nm even in the blood serum. In contrast, in composites for gene delivery prepared excluding the lipid composition for forming liquid crystals (Comparative Examples 2 and 3), particles were 1,500 nm, 1,200 nm, respectively, were not capable of maintaining particle stability due to the severe agglutination with proteins present in the blood serum. Accordingly, it was confirmed that the present invention, by constituting a liquid crystal forming lipid composition along with chitosan, secures particle stability in the blood serum, and effectively performs delivery of a target gene while resolving the problems in the existing technology.

Experimental Example 6

Manufacture of Lyophilized Products Using Composites with a Gene, Chitosan, Sodium Lauryl Sulfate, Protamine, and a Liquid Crystal Forming Lipid Composition, and Measurement of Particle Size of the Same

(57) In order to confirm whether the liquid crystal composite composition of the present invention can be lyophilized for the purpose of easiness of storage and distribution, the compositions were lyophilized and then redispersed to confirm the particle size.

(58) More specifically, the compositions prepared in Examples 23 and 24 in which trehalose and glucose as saccharides were additionally included were lyophilized and then redispersed with purified water to prepare the gene at a concentration of 100 μg/mL, and the particle size was observed using an Electrophoretic Light Scattering Spectrophotometer (ELS) particle size and electric charge analyzer (ELS-Z, Otsuka, Japan). The particle size of the compositions was respectively measured using particle size cell according to the user's manual by the ELS manufacturer.

(59) The results are shown in FIG. 6.

(60) As shown in FIG. 6, it was confirmed that the compositions prepared in Examples 23 and 24 were able to maintain the initial particle size at about 200 nm even when they were rehydrated after lyophilization. Based on the above, particle stability at the level of before lyophilization can be maintained even when the compositions are lyophilized by further adding a saccharide and applied in vivo, thus confirming that the liquid crystal composite composition for gene delivery can be stably stored in the form of a lyophilized product during storage and distribution.

Experimental Example 7

Evaluation of In Vivo Tumor Growth Inhibitory Effect by Composites with a Gene, Chitosan, Sodium Lauryl Sulfate, Protamine, and a Liquid Crystal Forming Lipid Composition

(61) In order to confirm whether the gene being delivered by the liquid crystal composition for gene delivery of the present invention can be delivered inside a living body and effectively exhibit the intended purpose, the tumor inhibitory effect by the composition prepared in Example 10 which includes survivin siRNA as a gene, and the composition prepared in Comparative Example 4 which includes FL dsRNA, which is incapable of tumor inhibitory effect, as a gene. In order to evaluate the in vivo delivery capability of a genetic drug, an immunodeficient mouse model transplanted with a prostate cancer was used. In order to construct an immunodeficient mouse model transplanted with a prostate cancer, 2×10.sup.6 cells of prostate cancer cell line redispersed in 50 μl of RPMI culture medium was mixed with 50 μl of Matrigel (BD biosciences), and the mixture was subcutaneously injected into 5 week old immunodeficient mice. The size of tumor was grown to the size of 100 mm.sup.3, and then the compositions prepared in Example 10 and Comparative Example 4 to include the amount of gene at 200 μg/mL were respectively injected into the tail vein of mice. The amount of a single dose of the genetic drug was 40 μg and administered 6 times over a period of 2 weeks.

(62) The results are shown in FIG. 7.

(63) As shown in FIG. 7, when a composite composition for gene delivery consisting of a gene, water-soluble chitosan, sodium lauryl sulfate, protamine, and a liquid crystal forming lipid composition (Example 10) was intravenously injected into the immunodeficient mouse model transplanted with a prostate cancer, it was observed that in vivo tumor proliferation was considerably inhibited. In the group of mice intravenously injected with the composition prepared in Example 10 of the present invention, the tumor inhibitory rate was about 63% on the last day of the injection (Day 11) and about 70% on a week thereafter (Day 18), respectively, as compared with that of the untreated group. In contrast, in the mice belonging to the untreated group and in the group treated with a composite composition including a gene not capable of tumor suppression (Comparative Example 4), the tumor was shown to continue to grow without any noticeable inhibition. From the above, it was confirmed that the composite composition for gene delivery of the present invention can be effectively delivered via an in vivo intravenous injection, and also the gene delivered can effectively perform the intended effect.

(64) Conclusively, the compositions of the present invention were confirmed that they can exhibit a superior gene delivery effect via systemic administration, and also they resolved the bioincompatibility of the conventional chitosan as a gene delivery drug due to its relatively low binding strength with a gene and instability in blood serum.

INDUSTRIAL APPLICABILITY

(65) The compositions of the present invention including chitosan and a liquid crystal formation material for gene delivery have overcomed the low binding strength of chitosan with a gene, and also considerably improved the stability of chitosan nanocomposite in blood serum, enabling high efficiency gene delivery and superior stability, thus being useful as a gene therapeutic agent.