JP6872782B2 - Cationic glucan nanospheres, complexes, nucleic acid-introducing agents and cancer therapeutics - Google Patents

Description

The present invention relates to cationic glucan nanospheres, complexes, nucleic acid-introducing agents and cancer therapeutic agents.

Currently, low-molecular-weight drugs obtained by chemical synthesis are widely used as pharmaceuticals, but the development of new drugs is required due to problems such as low targetivity and side effects. Proteins and synthetic peptides are expected as new medicines, but they also have problems such as molecular stability and synthesis cost. Therefore, attention is being paid to nucleic acid drugs called plasmid DNA and siRNA (small interfering RNA). Nucleic acids have the advantages of being relatively inexpensive to synthesize and specifically recognizing base sequences. However, as with other drugs, stability in vivo is an issue, and the development of carriers that transport nucleic acids to target locations is required.

The amphipathic polymer forms particles by associating its hydrophobic groups in water by hydrophobic interaction and self-assembling. In recent years, nano-sized polymer gels have been attracting attention from various fields such as medical treatment and biotechnology (Patent Document 1).

In addition, amino sugar-containing glucans in which glucosamine is introduced into a plurality of non-reducing ends are synthesized with respect to cyclic branched glucans (GD) synthesized from starch by an enzymatic reaction, and complex formation with nucleic acids is disclosed. (Patent Document 2).

Japanese Patent Application Laid-Open No. 2008-056638 WO2012 / 060110

An object of the present invention is to provide a technique for efficiently introducing nucleic acid into a cell.

The present invention provides the following cationic glucan nanospheres, complexes, nucleic acid-introducing agents and cancer therapeutic agents.
Item 1. A glucandendrimer (GD) having a spherical structure is modified with a polyvalent basic compound, and the polyvalent basic compound is selected from the group consisting of spermidine, spermine, diethylaminoethylamine (DEAE), polybasic amino acids and polyethyleneimine. Cationic glucan nanospheres, which is at least one of the following species.
Item 2. Item 2. The cationic glucan nanosphere according to Item 1, wherein the multivalent basic compound is spermine or spermidine.
Item 3. Item 2. The cationic glucan nanosphere according to Item 1 or 2, further modified with a hydrophobic group.
Item 4. Item 3. The cationic glucan nanosphere according to any one of Items 1 to 3, wherein the hydrophobic group is a cholesteryl group or an alkylcarbonyl group having 6 to 18 carbon atoms.
Item 5. Item 4. A complex of a cationic glucan nanosphere and a nucleic acid according to any one of Items 1 to 4.
Item 6. Item 5. The complex according to Item 5, wherein the nucleic acid is DNA, RNA, plasmid, siRNA, miRNA or antisense nucleic acid.
Item 7. Item 8. A nucleic acid introducing agent comprising the cationic glucan nanosphere according to any one of Items 1 to 4.
Item 8. A cancer therapeutic agent containing the complex according to Item 5 or 6 as an active ingredient.

The cationic glucan nanospheres of the present invention in which a cationic group (a polyvalent basic group introduced into GD by modifying with a polyvalent basic compound) and, if necessary, a hydrophobic group are further introduced into the glucan dendrimer (GD). Can form a complex with nucleic acids (DNA, RNA) and is excellent as an agent for introducing nucleic acids into cells.

Interaction between cationic GD and nucleic acid Structural formula of DEAE-substituted GD Structural formula of spermine substitution GD Electrophoresis of the complex of DEAE-substituted GD and siRNA Electrophoresis of the complex of spermine-substituted GD and siRNA Complex size of DEAE derivative and nucleic acid Complex size of spermine derivative and nucleic acid Relative amount of VEGF mRNA after addition of siRNA / DEAE-substituted GD complex Relative amount of VEGF mRNA after addition of siRNA / spermine-substituted GD complex Average fluorescence intensity of siRNA taken up by cells Average fluorescence intensity when endocytosis inhibitor is added PGL3 compounding with GD15-spe and CHP-spe PGL3 compounding with CHGD-spe and C12GD-spe Complex size of pGL3 and GD15-spe Luminescence of luciferase after delivery of pGL3 Changes in mouse body weight after administration of siRNA / cationic GD complex Tumor size changes after administration of siRNA / cationic GD complex Photograph of cancer-bearing mice on day 20 of administration of siRNA / cationic GD complex Tumor weight removed 20 days after administration of siRNA / cationic GD complex

The glucan dendrimer (GD) having a spherical structure used in the present invention can be synthesized from a partial decomposition product of starch by three kinds of enzymes (branching enzyme, sucrose phosphorylase, α-glucan phosphorylase). The obtained GD is α-1,4: α-1,6-glucan. GD has extremely high branching property, forms monodisperse nanoparticles having a spherical structure with a high sugar chain density, and is highly soluble in water. GD can form particles having different molecular weights and sizes by controlling the conditions of the enzymatic reaction.

The diameter of the cationic glucan nanospheres of the present invention is preferably about 1 to 200 nm, more preferably about 2 to 100 nm, still more preferably about 3 to 80 nm, and particularly preferably about 5 to 60 nm.

The cationic glucan nanospheres of the present invention may be modified with a hydrophobic organic group. Examples of the hydrophobic organic group include a group represented by R, COR or CONHR (R is an alkyl group having a linear or branched carbon number of 8 to 20 and an alkenyl having a linear or branched carbon number of 8 to 20). Group or cholesteryl group). Modification with a hydrophobic organic group (R, COR or CONHR) causes the OH group and the hydrophobic organic group of the glucose residue constituting GD to form an ether bond (OR), an ester bond (OCOR) and a urethane bond (OCONHR). It is done by doing.

Examples of the alkyl group having a linear or branched carbon number of 8 to 20 include octyl, 2-ethylhexyl, nonyl, decyl, undecyl, dodecyl, tridecylic, tetradecyl, hexadecyl, octadecyl, icosyl, and isostearyl.

Examples of the alkenyl group having a linear or branched carbon number of 8 to 20 include octenyl, decenyl, undecenyl, dodecenyl, tetradecenyl, palmitooleyl, and oleyl.

GD modified with a hydrophobic organic group includes GD and halogen compounds (R-Cl, R-Br, RI, etc.), acid halides (Cl-COR, Br-COR, etc.), and isocyanate compounds (R-NCO). Or, a reaction product of a diisocyanate compound and ROH) is reacted in a suitable aprotic solvent in the presence of a base, if necessary, at a temperature of about 0 ° C. to the boiling point of the solvent for about 1 to 24 hours. Can be done. Examples of the aprotic solvent include tetrahydrofuran, pyridine, acetonitrile, acetone, diethyl ether, dimethylformamide, dimethyl sulfoxide, chloroform, methylene chloride, dioxane, N-methylpyrrolidone, etc., which may be used alone or in combination of two or more. Can be used. Examples of the base include alkali metal carbonates such as sodium carbonate and potassium carbonate, alkali metal hydrogen carbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate, triethylamine and diisopropylethylamine. Examples of the diisocyanate compound include aliphatic diisocyanates such as hexamethylene diisocyanate, alicyclic diisocyanates such as isophorone diisocyanate, and aromatic diisocyanates such as xylylene diisocyanate.

GD is composed of glucose. The hydrophobic organic group preferably contains 0.01 to 50, more preferably 0.1 to 10, and even more preferably 0.2 to 5, particularly 0.3 to 3 per 100 units of glucose. GD having a hydrophobic group is preferable because it increases the efficiency of introducing nucleic acid into cells.

Furthermore, the GD and the GD modified with a hydrophobic group are modified with a polyvalent basic group to give the cationic glucan nanospheres of the present invention.

The polyvalent basic compound may form a cationic group of trivalent or higher under acidic conditions, and is a polybasic amino acid such as spermidine, spermine, diethylaminoethylamine (DEAE), polyethyleneimine (PEI), polylysine, and polyarginine. (The basic amino acid is lysine or arginine, and the number of basic amino acids is 3 or more, for example, 3 to 20, preferably 3 to 15, more preferably 4 to 10) and the like. Spermidine, spermine, polylysine and polyarginine are preferred, spermidine and spermine are more preferred, and spermine is most preferred. The polyvalent basic group preferably contains 1 to 100, more preferably 3 to 80, still more preferably 5 to 60, particularly 6 to 40, per 100 units of glucose. When there are many polyvalent basic groups, a complex can be formed with a large number of nucleic acids (for example, siRNA) or a nucleic acid having a large molecular weight (for example, a plasmid), and the efficiency of nucleic acid introduction is improved. Too many basic groups tend to increase cytotoxicity.

The interaction between the cationic glucan nanospheres of the present invention and nucleic acid is schematically shown in FIG.

The complex of the present invention is a complex of a cationic glucan nanosphere and a nucleic acid. Examples of nucleic acids include DNA and RNA. Specific examples of the nucleic acid include antisense nucleic acids, plasmids, gene constructs, siRNA, miRNA, shRNA, dsRNA and the like. Preferred nucleic acids are plasmids, siRNAs, with siRNA being more preferred. The ratio of the number of phosphate residues (P) to the number of polyvalent basic groups (C) in the nucleic acid is P / C = about 0.5 to 5, preferably about 0.6 to 3, and more preferably 0. It is about 7 to 2, more preferably about 0.8 to 1.5. The P / C is most preferably around 1. The ratio of the number of cationic glucan nanospheres to the number of nucleic acids in the complex is about 1:10 to 10: 1, preferably about 1: 5 to 5: 1. The ratio (+/-) of the total total number of positive charges (+) to the total number of negative charges (-) in the complex is about +/- = 0.5 to 30, preferably about 0.8 to 20, and more. It is preferably about 1 to 15, more preferably about 1.2 to 10, and even more preferably about 1.5 to 8.

Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited to the examples.

In the examples, the following reagents and abbreviations were used.
-Spe: spermin-CH: cholesteryl group or cholesterol-CHGD (GD modified with cholesteryl group: cholesteryl group is a urethane bond with two OH groups of GD and OH group of cholesterol using hexanediisocyanate (-OCONH (CH). ₂ ) ₆ NHCOO-) bonded.)
-CHP (Pullulan modified with cholesteryl group: The cholesteryl group is a combination of the OH group of pullulan and the OH group of cholesterol with two urethane bonds (-OCONH (CH ₂ ) ₆ NHCOO-) using hexane diisocyanate. is there.)
-C12GD (GD modified with a dodecyl group; the dodecyl group is bonded to the OH group of GD by a urethane bond using dodecyl isocyanate.)

Manufacturing example 1
1. Synthesis of DEAE-substituted GD
1-1. Reagent ・ GD15 (Molecular weight Mw 220,000 size 15nm)
・ CHP1.2 (cholesteryl group: 1.2 pieces / 100 monosaccharides)
・ CHGD15 (cholesteryl group: 1.9 pieces / 100 monosaccharides)
・ C12GD15 (dodecyl group: 3.2 pieces / 100 monosaccharides)
・ 1,1'-carbonyl diimidazole (CDI) (SIGMA)
・ N, N-diethylethylenediamine (DEAE) (Aldrich)
・ DMSO (super dehydration) (WAKO)
In the following, CHGD15 may be abbreviated as "CHGD", C12GD15 may be abbreviated as "C12GD", CHGD15-DEAE may be abbreviated as "CHGD-DEAE", and C12GD15-DEAE may be abbreviated as "C12GD-DEAE". Sometimes abbreviated, CHGD15-spe may be abbreviated as "CHGD-spe", and C12GD15-spe may be abbreviated as "C12GD-spe".

1-2. Method
GD15, CHP, CHGD15, and C12GD15 were _{dissolved in dehydrated DMSO under N 2} at the weight ratios shown in Table 1 and CDI was added in a proportion of 32/100 monosaccharides. After reacting at room temperature for 3 hours, DEAE was added in an amount 10 times that of CDI, and the reaction was carried out for another 24 hours. After the reaction, it was dialyzed against pure water (MWCO: 3,500), purified, and freeze-dried to obtain a synthetic product. For the compound, ^{the substitution rate of DEAE was calculated by 1} H NMR (10 mg / mL in D ₂ O).

1-3. Results and Discussion By the above method, 1043 mg of GD15-DEAE, 1203 mg of CHP-DEAE, 183 mg of CHGD-DEAE, and 217 mg of C12GD-DEAE could be recovered. The DEAE substitution rate of each derivative is shown in Table 2, and the structural formula of DEAE-substituted GD is shown in FIG.

2. Synthesis of spermine-substituted GD
2-1. Reagent ・ GD15 (Molecular weight Mw 220,000 size 15nm)
・ CHP1.2 (cholesteryl group: 1.2 pieces / 100 monosaccharides)
・ CHGD15 (cholesteryl group: 1.9 pieces / 100 monosaccharides)
・ C12GD15 (dodecyl group: 3.2 pieces / 100 monosaccharides)
・ 1,1'-carbonyl diimidazole (CDI) (SIGMA)
・ Spermine (Aldrich)
・ DMSO (super dehydration) (WAKO)

2-2. Method
GD15, CHP, CHGD15 and C12GD15 were _{dissolved in dehydrated DMSO under N 2} at the weight ratios shown in Table 3 and CDI was added with 32/100 monosaccharides. After reacting CHP for 3 hours and GD for 5 hours at room temperature, spermine was added 10 times as much as CDI and reacted for another 24 hours. After the reaction, it was dialyzed against pure water (MWCO: 3,500), purified, and freeze-dried to obtain a synthetic product. For the compound, ^{the substitution rate of spermine was calculated by 1} H NMR (10 mg / mL in D ₂ O).

2-3. Results and discussion By the above method, 1043 mg of GD15-spe, 1203 mg of CHP-spe, 183 mg of CHGD-spe, and 217 mg of C12GD-spe were obtained. The spermine substitution rate of each derivative is shown in Table 4, and the structural formula of the spermine substitution GD is shown in FIG.

Test Example 1
1. Evaluation of physical properties of the compound The particle size (dynamic light scattering method DLS) and ζ potential of the synthesized DEAE-substituted GD and spermine-substituted GD were measured.
1-1. Measurement sample and reagent ・ GD15-DEAE
・ CHP-DEAE
・ CHGD-DEAE
・ C12GD-DEAE
・ GD15-spe
・ CHP-spe
・ CHGD-spe
・ C12GD-spe
・ PBS (gibco)

1-2. Measurement method
The DEAE derivative and spermine derivative were dissolved in PBS at 2 mg / mL each, and ultrasonically, centrifuged (20,000 G), and filtered (0.22 μm, PVDF), and the particle size and ζ potential were measured.

1-3. Results and discussion
The sizes and ζ potentials of DEAE-substituted GD and spermine-substituted GD are shown in Tables 5 and 6. It was found that the spermine-substituted GD having three amino groups had a stronger positive charge, and all of them were relatively monodisperse particles.

Test Example 2
・ SiRNA complexing with cationic GD
Complexity with siRNA was performed using DEAE-substituted GD and spermine-substituted GD, and the hybridization was confirmed by electrophoresis.

1-1. Reagent ・ GD15-DEAE
・ CHP-DEAE
・ CHGD-DEAE
・ C12GD-DEAE
・ GD15-spe (spe: 25/100 monosaccharides)
・ CHP-spe (cholesteryl group: 1.2 pieces / 100 monosaccharides, spe: 23 pieces / 100 monosaccharides)
・ CHGD-spe (cholesteryl group: 1.9 pieces / 100 monosaccharides, spe: 33 pieces / 100 monosaccharides)
・ C12GD-spe (dodecyl group: 3.2 pieces / 100 monosaccharides, spe: 22 pieces / 100 monosaccharides)
・ SiRNA
・ PBS (gibco)
・ SYBR Green I Nucleic Acid Gel Stain (TAKARA)

1-2. Method
GD15-DEAE, CHP-DEAE, CHGD-DEAE, and C12GD-DEAE were dissolved in PBS at 20 mg / mL and treated with ultrasonic waves, centrifugation (20,000 G), and filters (0.22 μm, PVDF). The DEAE1 molecule was C and one base of siRNA was P, and the C / P ratio was adjusted to 0.5 to 32 for 30 min at room temperature (siRNA = 0.3 μg). A loading buffer was added to the complex, applied to a 2% agarose gel, and electrophoresed at 100 V for 15 min. The gel was stained with SYBR Green for 10 min and measured by LAS.

1-3. Results and Discussion The results of gel electrophoresis are shown in FIGS. 4 and 5. Free siRNA was observed when the C / P ratio was 1 or less, but when the C / P ratio was high, complexation of DEAE-substituted GD and spermine-substituted GD with siRNA was observed.

2. Nucleic acid complex sizing
2-1. Measurement method
Four types of DEAE-substituted GD and spermine-substituted GD were used as 20 mg / mL solutions. As a negative control, siRNA and nanogel (CHP-DEAE, CHP-spe) were mixed and DLS measurement was performed (nanogel final concentration = 1 mg / mL).

2-2. Results and discussion
The complex sizes when the C / P ratio is changed are shown in FIGS. 6 and 7. When cationic nanogel (CHP-DEAE) and anionic siRNA were mixed, aggregates were formed near neutrality, and the size of the CHP-DEAE complex increased. On the other hand, since there was almost no change in complex size between DEAE-substituted GD and spermine-substituted GD, it is considered that both the complex with siRNA formed stable fine particles.

3. SiRNA delivery by cationic GD
3-1. Evaluation of RNA interference by siRNA delivery In an intracellular siRNA delivery experiment using cationic GD, the knockdown efficiency of mRNA targeted by siRNA was evaluated.

3-1-1. Reagent ・ GD15-DEAE (DEAE: 13 pieces / 100 monosaccharides)
・ CHP-DEAE (cholesteryl group: 1.2 pieces / 100 monosaccharides, DEAE: 29 pieces / 100 monosaccharides)
・ CHGD-DEAE (cholesteryl group: 1.9 pieces / 100 monosaccharides, DEAE: 8 pieces / 100 monosaccharides)
・ C12GD-DEAE (dodecyl group: 3.2 pieces / 100 monosaccharides, DEAE: 11 pieces / 100 monosaccharides)
・ GD15-spe (spe: 25/100 monosaccharides)
・ CHP-spe (cholesteryl group: 1.2 pieces / 100 monosaccharides, spe: 23 pieces / 100 monosaccharides)
・ CHGD-spe (cholesteryl group: 1.9 pieces / 100 monosaccharides, spe: 33 pieces / 100 monosaccharides)
・ C12GD-spe (dodecyl group: 3.2 pieces / 100 monosaccharides, spe: 22 pieces / 100 monosaccharides)
・ ReverTraAce (Toyobo)
・ VEGF siRNA (siRNA (Invitrogen) that targets mRNA encoding VEGF)
-Alexa488-siRNA (fluorescently labeled siRNA)
・ Universal Probe Library # 12 (Roche)
・ 18S rRNA Probe (FAM-atccattggagggcaagtctggtgc-BHQ)
・ VEGFA Fw (gcagcttgagttaaacgaacg)
・ VEGFA Rv (ggttcccgaaaccctgag)
・ 18S rRNA Fw (atgagtccactttaa atcctttaacga)
・ 18S rRNA Rv (ctttaatatacgctattggagctggaa)
・ Light Cycler 480 Probes Master (Roche)
・ AllStars Neg. SiRNA AF488 (QIAGEN)
・ Maxwell RSC simply RNA Cells

3-1-2. Method
A 20 mg / mL solution of 4 DEAE derivatives and 4 spermine derivatives was used. Mouse VEGF siRNA was dissolved in RNase free water and complexed with C / P = 4, 8, 16 for DEAE derivatives and C / P = 2, 4, 8 for spermine derivatives (siRNA = 50 pmol). Complex was performed at room temperature for 30 minutes, mixed with 990 μL of RPMI medium, and the medium was replaced. Transfection was performed at 37 ° C. for 24 hours.

After transfection, the medium was removed and total RNA was extracted using the Maxwell RSC simply RNA Cells kit. RNA concentration was measured with NanoDrop and diluted with water to 100 ng / μL. This RNA aqueous solution was mixed with the reverse transcription reagent kit ReverTraAce, and PCR was performed at 37 ° C. for 15 min, 95 ° C. for 1 min, and 4 ° C. to synthesize cDNA. Of the mouse RNAs, the knockdown VEGF mRNA and the housekeeping gene 18S rRNA sequences were used, added to a 96-well plate, and real-time-PCR was performed by the TaqMan probe method. Knockdown efficiency was evaluated relative to the amount of 18S rRNA produced by VEGF RNA.

3-1-3. Results and discussion
The knockdown efficiency of the DEAE derivative is shown in FIG. 8, and the knockdown efficiency of the spermine derivative is shown in FIG. Knockdown was hardly observed with DEAE derivatives due to low intracellular delivery efficiency. On the other hand, all spermine derivatives showed remarkable knockdown, and in particular, spermine derivatives (CHGD-spe, C12GD-spe) into which a hydrophobic group was introduced showed RNA interference efficiently, and high knockdown efficiency with respect to VEGF mRNA was observed. Was done.

3.2. Cell uptake behavior of siRNA complex (flow cytometry)
Using fluorescently labeled siRNA (Alexa488-siRNA), the amount of uptake into cells by DEAE derivative and spermine derivative was evaluated by a flow cytometer.

3-2-1. Method
A 20 mg / mL solution of 4 DEAE derivatives and 4 spermine derivatives was used. Alexa488-siRNA was dissolved in RNase free water and complexed with nanogel at C / P = 4 (siRNA = 50 pmol). The complex was added to the cells under the same conditions as above and measured by flow cytometry at t = 24 h.

3-2-2. Results and Discussion The average fluorescence intensity of fluorescently labeled siRNA taken up into cells by spermine derivatives and DEAE derivatives is shown in FIG. All spermine derivatives (GD-spe, CHGD-spe, C12GD-spe) efficiently introduced siRNA into cells. It was found that the uptake efficiency of the DEAE derivative was low.

From the results of the knockdown efficiency of the spermine derivative and the DEAE derivative (Fig. 9) and the result of the uptake efficiency (Fig. 10), it was found that there is a good correlation between the intracellular introduction amount of siRNA and the knockdown efficiency.

3.3. Specific endocytosis pathway of siRNA / GD complex Inhibition experiments were conducted to investigate the endocytosis pathway in cell introduction.

3-3-1. Reagent ・ C12GD-spe (dodecyl group: 3.2 pcs / 100 monosaccharide, spe: 22 pcs / 100 monosaccharide)
・ AllStars Neg. SiRNA AF488 (QIAGEN)
・ Renca cell
・ RPMI1640 (Gibco)
・ Chlorquine (WAKO)
・ Methyl-β-cyclodextrin (WAKO)
・ Cytochalasin D (WAKO)
・ Chlorpromazine (WAKO)
・ EIPA (sigma)
・ PBS (Gibco)
・ Stain buffer

3-3-2. Experiment
C12GD-spe was lysed in PBS and complexed with fluorescently labeled siRNA (Alexa488-siRNA) at C / P = 4 (siRNA = 25 pmol, total 50 μL). Renca cells were seeded on a glass-based dish at 4 × 10 ⁴ _{cells / well and pre-cultured at 37 ° C. under 5% CO 2} for about 20 h. As endocytosis inhibitors, chlorquine, methyl-β-cyclodextrin, cytochalasin D, chlorpromazine and EIPA were dissolved in PBS. Add to pre-cultured cells so that [chlorquine] = 50 μM, [methyl-β-cyclodextrin] = 5 mM, [cytochalasin D] = 5 μM, [chlorpromazine] = 50 μM, [EIPA] = 50 μM. And incubated for 30 minutes. After washing twice with 1 mL of RPMI, 950 μL of RPMI and 50 μL of siRNA complex were mixed, added to cells, complexed for 4 hours, and measured by flow cytometry.

3-3-3. Results and discussion Figure 11 shows the decrease in fluorescence intensity of cells to which an inhibitor was added to the positive control. Inhibition of uptake by methyl-β-CD occurred in the siRNA complex as well as the raw material of GD, suggesting that the endocytosis pathway of intracellular uptake is caveolae endocytosis. This is considered to be due to the small particle size even when combined with siRNA.

Test example 3
1. plasmid DNA compositing with cationic GD A plasmid DNA (pGL3) compositing experiment was performed using spermine-substituted GD.

1-1. Confirmation of compounding by electrophoresis
1-1-1. Spermine Substitution GD and Reagents ・ GD15 Molecular Weight Mw 220,000 Size 15nm
・ GD15-spe (spe: 25/100 monosaccharides)
・ CHP-spe (cholesteryl group: 1.2 pieces / 100 monosaccharides, spe: 23 pieces / 100 monosaccharides)
・ CHGD-spe (cholesteryl group: 1.9 pieces / 100 monosaccharides, spe: 33 pieces / 100 monosaccharides)
・ C12GD-spe (dodecyl group: 3.2 pieces / 100 monosaccharides, spe: 22 pieces / 100 monosaccharides)
・ PBS (gibco)
・ PGL3-Control Vector (Promega)

1-2. Complex detection method
GD15-spe, CHP-spe, CHGD-spe, and C12GD-spe were dissolved in PBS and treated with ultrasonic waves, centrifugation (20,000 G), and filter (0.22 μm, PVDF). With 1 molecule of spermine as C and 1 base of plasmid (pGL3) as P, the plasmid (pGL3 = 0.2 μg) and spermine-substituted GD are mixed for 30 min at room temperature so that the mixing ratio (C / P ratio) is 0.125 to 8. did. A loading buffer was added to the complex, applied to a 2% agarose gel, and electrophoresed at 100 V for 30 min. The gel was stained with SYBR Gold for 40 min and measured by LAS.

1-3. Results and Discussion The images of electrophoresis are shown in FIGS. 12 and 13. It was found that all spermine-substituted GDs formed a complex with C / P = 1 or higher.

2. Cationic GD and plasmid DNA complex size
2-1. Measurement method
GD15-spe was dissolved in water and complexed with plasmid (pGL3) at C / P = 0.5-32 (GD-spe = 1 mg / mL). After standing at room temperature for 30 minutes, the particle size was measured by dynamic light scattering DLS.

2-2. Results and discussion
The DLS result of the complex of pGL3 and GD15-spe is shown in FIG. It was found that aggregates were formed in the complex near C / P = 1 as in the conventional cationic CHP. This is thought to be because the plasmid DNA is larger than the siRNA, so it has aggregated through the GD-spe particles. At C / P = 4 and above, a stable complex with a relatively small size was formed.

3. plasmid DNA delivery by cationic GD
Gene transfection experiments were performed using spermine-substituted GD. Using a plasmid containing the luciferase gene, the expression of the luciferase protein was used as its enzymatic activity, and the luciferase luminescence amount was measured and evaluated.

3-1. Spermine-substituted GD and reagents ・ GD15-spe (spe: 25/100 monosaccharides)
・ CHP-spe (cholesteryl group: 1.2 pieces / 100 monosaccharides, spe: 23 pieces / 100 monosaccharides)
・ CHGD-spe (cholesteryl group: 1.9 pieces / 100 monosaccharides, spe: 33 pieces / 100 monosaccharides)
・ C12GD-spe (dodecyl group: 3.2 pieces / 100 monosaccharides, spe: 22 pieces / 100 monosaccharides)
・ PGL3 (E. coli synthesis)
・ Lipofectamine 2000
・ COS7 cell
・ DMEM (10% FBS, Gibco)
・ PBS (Gibco)
・ Glo-lysis
・ Bright-glo
・ BCA Protein assay kit

3-2. Measurement method
COS7 cells were seeded on a 12-well plate at 4 × 10 ⁴ _{cells / well and pre-cultured at 37 ° C. under 5% CO 2} for about 20 h. The spermine derivative was dissolved in PBS and complexed with pGL3 at C / P = 4.8 (pGL3 = 1 μg, total 50 μL). After removing the medium of the precultured cells, 950 μL of OptiMEM and 50 μL of the plasmid complex were added and incubated for 2 hours. After washing twice with RPMI, 1 mL of RPMI was added to the cells and cultured for 22 hours. The medium was removed, cells were lysed with 200 μL of Glo-lysis, protein concentration was measured with BCA assay, and luciferase expression level was measured with Bright-Glo.

3-3. Results and discussion Figure 15 shows the amount of luciferase luminescence after introduction of the plasmid into cells by spermine-substituted GD. Higher expression of luciferase than the PEI system was observed in GD15-spe and C12GD-spe, demonstrating that the plasmid was efficiently delivered to cells by complex formation of spermine-substituted GD and plasmid.

Test Example 4
1. Treatment experiment of cancer-bearing mice based on siRNA delivery by cationic GD
A therapeutic experiment was conducted on cancer-bearing mice using cationic GD as the delivery carrier of siRNA. For cationic GD, select C12GD-spe, which is a spermine-substituted GD having a dodecyl group, and GD15-spe, which does not have a dodecyl group, and siRNA is siRNA (siVEGF) that targets mRNA encoding VEGF and as a negative control. I chose siNega. A therapeutic experiment was performed by administering to cancer-bearing mice four types of siRNA / cationic GD complexes consisting of a combination of two types of spermine-substituted GD and two types of siRNA.

1-1. Preparation of cancer-bearing mice
1-1-1. Reagents / BALB / C: 19 animals (female, 7-9 weeks old)
・ Renca cells (mouse kidney cancer cells)
RPMI 1640 (10% FBS, 100 μg / mL streptomycin, 100 U / mL penicillin)

1-1-2. Experiment for producing cancer-bearing mice
The backs of 19 BALB / C mice were shaved and 1.0 × 10 ⁶ cells of Renca cells were subcutaneously transplanted. Tumors were grown to an average of 50 mm ^{3 while observing tumor formation.} The tumor size was measured using a caliper with a major axis of A (mm) and a minor axis of B (mm) ^{using the formula (A 2} x B) / 2.

1-2. Topical administration of VEGF mRNA-targeted siRNA (siVEGF) by cationic GD
1-2-1. Reagents / Cancer-bearing mice: 19 / VEGF mRNA target siRNA (siVEGF, macrogen)
・ Negative control siRNA (siNega, invitrogen)
・ C12GD-spe
・ GD15-spe

1-2-2. SiRNA administration experiment
C12GD-spe and GD15-spe are dissolved in water and mixed with siVEGF and siNega at C / P = 8, respectively, and siRNA / cationic GD complexes (siVEGF / C12GD-spe, siNega / C12GD-spe, siVEGF / GD15-spe, siNega / GD15-spe,). 20 μg (50 μL) of siRNA per mouse was topically administered to the tumor 5 times at t = 0, 4, 8, 12, 16 days. The administration groups (A to F) are shown in Table 7. Body weight and tumor size were measured at t = 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 days. Twenty days later, he was euthanized, and blood was collected, a tumor was removed, and a splenectomy was performed. The resected tumor was weighed and cryopreserved.

1-2-3. Results and Discussion Fig. 16 shows the change in body weight when siRNA / cationic GD complex was administered to cancer-bearing mice, Fig. 17 shows the change in tumor size, and Fig. 18 shows the photograph of cancer-bearing mice. The weight of the tumor removed 20 days after administration is shown in FIG.

From FIG. 16, administration of siRNA and cationic GD complex did not reduce body weight and no toxicity was observed.

From FIGS. 17 and 18, tumor size in the group administered with siRNA (siVEGF) targeting VEGF-encoding mRNA and two cationic GD complexes (siVEGF / C12GD-spe, siVEGF / GD15-spe) The increase was suppressed, and an antitumor effect was observed. Tumor size was not suppressed and increased over time in the group administered with siRNA (siNega) as a negative control and the cationic GD complex.

In addition, from FIG. 19, the group to which siVEGF and the cationic GD complex (siVEGF / C12GD-spe, siVEGF / GD15-spe) were administered and the cationic GD complex as a negative control (siNega / C12GD-spe, siNega / GD) From the comparison of the excised tumor weights of the group to which -spe) was administered, an antitumor effect was observed with the siVEGF and the cationic GD complex.

Spermine-substituted cationic GD (C12GD-spe, GD15-spe) exhibits antitumor activity by forming a complex with siRNA (siVEGF) that targets mRNA encoding VEGF and delivering siVEGF to tumor cells. It became clear.

Claims (8)

Multivalent with a basic compound will be modified glucan dendrimer (GD) having a spherical structure, wherein the polybasic compound is spermine, or Ri spermidine der, the polybasic compound via an amide bond glucan dendrimers Cationic glucan nanospheres bound to. The cationic glucan nanosphere according to claim 1, wherein the multivalent basic compound is spermine. The cationic glucan nanosphere according to claim 1 or 2, further modified with a hydrophobic group. The cationic glucan nanosphere according to claim 3 , wherein the hydrophobic group is a cholesteryl group or an alkylcarbonyl group having 6 to 18 carbon atoms. A complex of a cationic glucan nanosphere according to any one of claims 1 to 4 and a nucleic acid. The complex according to claim 5, wherein the nucleic acid is DNA, RNA, plasmid, siRNA, miRNA or antisense nucleic acid. A nucleic acid-introducing agent comprising the cationic glucan nanosphere according to any one of claims 1 to 4. A cancer therapeutic agent containing the complex according to claim 5 or 6 as an active ingredient.

Images