JP6358661B2 - Surfactant-like compound - Google Patents

Description

  The present invention relates to a surfactant-like compound, and more specifically, has high biocompatibility and stability in blood, low toxicity due to excess cation, and is easily transported to a target site with a small particle size. The present invention relates to a surfactant-like compound capable of forming a nucleic acid complex with high expression efficiency.

In recent years, gene therapy using nucleic acids such as plasmid DNA and antisense DNA has attracted attention. An important element of gene therapy is a drug delivery system (drug delivery system (DDS)) that efficiently sends nucleic acids to a target site.
In conventional nucleic acid drug delivery systems, a complex of a nucleic acid and a nucleic acid carrier has been used for nucleic acid delivery.
The complex has low toxicity in vivo, and is required to protect the nucleic acid from the nucleolytic enzyme and efficiently transport it to the target site. For the formation of the complex, many cationic groups are present in the molecule. Techniques such as formation of a polyion complex of a nucleic acid carrier having a nucleic acid and a nucleic acid complex have been used.
For example, Patent Document 1 discloses that a hydrophilic polymer chain block is covalently bonded to one end of the main chain of the polyamino acid chain block as a copolymer that provides a stable association with siRNA under physiological conditions. And a copolymer in which a hydrophobic group is bonded via a covalent bond to a side chain of 10% to 70% of amino acid repeating units in the polyamino acid chain block. Yes.
Patent Document 2 includes a polyion complex formed from a nucleic acid and a cationized pullulan derivative as a preparation for increasing the efficiency of targeting and uptake of the nucleic acid to the liver in vivo. A preparation for liver delivery characterized by a cationization rate of 10-27% has been proposed.
Patent Document 3 discloses a polyion complex in the form of a non-polymeric micelle in which double-stranded ribonucleic acid and a block copolymer having a large number of nitrogen-containing groups are electrostatically bonded, and a dynamic light scattering measurement method. A polyion complex has been proposed having an average particle size of less than 100 nm when measured by.
Patent Document 4 discloses a cationic polymer containing a plurality of nitrogen-containing cationic groups as a polyion complex that sufficiently retains a photosensitizing substance in serum and excellent in structural stability, and a nucleic acid polyplex that is a constituent component thereof. There have been proposed polyion complexes containing.

International Publication No. 2009/113645

JP 2006-028061 A

JP 2010-233499 A

Special table 2007-99661 gazette

However, in the proposals according to Patent Documents 1 to 4, there is a problem that there is toxicity to the living body, and because the particle size of the ion complex is large, the transfer efficiency from the blood vessel to the target site is poor, and the expression efficiency at the target site is poor. There was a problem.
For this reason, there is a demand for the development of a compound that can form a nucleic acid complex that has low toxicity to a living body, is easily transported to a target site with a small particle size, and has high expression efficiency at the target site.

  Accordingly, an object of the present invention is to provide a surfactant-like compound that can form a nucleic acid complex that has low toxicity to a living body, is easily transported to a target site with a small particle size, and has high expression efficiency at the target site. There is to do.

As a result of diligent studies to solve the above-mentioned problems, the present inventors have controlled the number of cationic groups and nitrogen-containing groups in the structure of a compound that forms a complex with a nucleic acid, thereby combining the compound with the nucleic acid. As a result of finding out and further examining the physical and biological properties of the body, the present invention has been completed.
That is, the present invention provides the following inventions.
1. A surfactant-like compound comprising a hydrophilic group, a hydrophobic group, and 1 to 5 nitrogen-containing groups located therebetween.
2. 2. The surfactant-like compound according to 1, wherein the hydrophilic group is a nonionic hydrophilic group.
3. The surfactant-like compound according to 1 or 2, wherein the hydrophilic group is an ethylene glycol group represented by the following formula (1).
R— (CH ₂ —CH ₂ —O) _n − (1)
(In the formula, n represents an integer of 40 to 120. R represents a hydrocarbon group having 1 to 18 carbon atoms.)
4). The surfactant-like compound according to any one of 1 to 3, wherein the hydrophobic group is a linear or branched alkyl group having 1 to 18 carbon atoms or a cholesterol group.
5). The hydrophobic group has a non-hydrophobic substituent in a part thereof,
The surfactant-like compound according to any one of 1 to 4.
6). 6. The surfactant-like compound according to 5, wherein the substituent is a hydrogen-bonding functional group-containing substituent.
7). 7. The surfactant-like compound according to 6, wherein the substituent is located at the end of the hydrophobic group.
8). 8. The surfactant-like compound according to 7, wherein the hydrophobic group is a 1-amido-alkyl group, 1-hydroxy-alkyl group, or 1-thymine-alkyl group represented by the following formula 1.
In formula, n is an integer of 1-20.


9. The surfactant-like compound according to any one of 1 to 8, wherein the nitrogen-containing group is an amide group or an imidazolium group.
10. The surfactant-like compound according to any one of 1 to 9, which has a cationic group between the hydrophobic group and the nitrogen-containing group.
11. 11. The surfactant-like compound according to 10, wherein the cationic group is an imidazolium group.
12 The surfactant-like compound according to any one of 1 to 11, which is used for forming a nucleic acid complex.

  The surfactant-like compound of the present invention is a nucleic acid having high biocompatibility and stability in blood, low toxicity to the living body, small particle size, easy transport to the target site, and high expression efficiency at the target site. A surfactant-like compound capable of forming a complex and can be preferably used for forming a nucleic acid complex.

1 is a drawing-substituting photograph showing the results of agarose gel electrophoresis of a complex of a surfactant-like compound and DNA obtained in Example 1. FIG. FIG. 2 is a drawing-substituting photograph showing the result of agarose gel electrophoresis of the complex of the surfactant-like compound and DNA obtained in Example 2. FIG. 3 is a drawing-substituting photograph showing the result of agarose gel electrophoresis of the complex of the surfactant-like compound and DNA obtained in Example 3. FIG. 4 is a drawing-substituting photograph showing the results of agarose gel electrophoresis of the complex of the surfactant-like compound and DNA obtained in Example 4. FIG. 5 is a drawing-substituting photograph showing the result of agarose gel electrophoresis of the complex of the surfactant-like compound and DNA obtained in Example 5. FIG. 6 is a chart of the CD spectrum of the complex of the surfactant-like compound and DNA obtained in Example 1. FIG. 7 is a graph showing the results of measuring the in vivo gene expression of the complex of the surfactant-like compound obtained in Example 3 and DNA at the time of mouse administration by luciferase assay. FIG. 8 is a diagram showing the results of measurement of gene expression in vivo by luciferase assay during administration of a complex of the surfactant-like compound and DNA obtained in Examples 1 and 3 to mice. FIG. 9 is a chart showing ¹ H-NMR measurement results of the surfactant-like compound obtained in Example 6. FIG. 10 is a drawing-substituting photograph showing the result of agarose gel electrophoresis of the complex of the surfactant-like compound and DNA obtained in Example 6. FIG. 11 is a drawing-substituting photograph showing the results of agarose gel electrophoresis in the stability evaluation of the complex of the surfactant-like compound and DNA obtained in Example 6. FIG. 12 is a graph showing the results of measurement of gene expression in vivo by luciferase assay during administration of the complex of the surfactant-like compound obtained in Example 6 and DNA to mice.

Hereinafter, the present invention will be described in more detail.
The surfactant-like compound of the present invention is characterized by comprising a hydrophilic group, a hydrophobic group, and a specific number of nitrogen-containing groups located therebetween.

<Hydrophilic group>
As the hydrophilic group, a nonionic hydrophilic group is particularly preferable.
Examples of the nonionic hydrophilic group include an ethylene glycol group, a dextran group, and a polyacrylamide group, and an ethylene glycol group represented by the following formula (1) is preferable.
R— (CH ₂ —CH ₂ —O) _n − (1)
(In the formula, n represents an integer of 40 to 120. R represents a linear or branched alkyl group having 1 to 18 carbon atoms.)
This provides biocompatibility, enhances the solubility of the complex in water when it forms a complex with nucleic acid, and is non-specific to serum proteins and cell surfaces under physiological conditions such as blood. Suppresses natural interactions and increases stability in blood.
Specifically, the following ethylene glycol groups are preferred.
_{CH 3 - (CH 2 -CH 2} -O) n -,
_{C 2 H 5 - (CH 2} -CH 2 -O) n -,
_{C 3 H 7 - (CH 2} -CH 2 -O) n -,
_{C 4 H 9 - (CH 2} -CH 2 -O) n -,
_{C 5 H 11 - (CH 2} -CH 2 -O) n -,
In these exemplary groups, n indicating the degree of polymerization is in the range of 40 to 120.

<Hydrophobic group>
Examples of the hydrophobic group include a hydrophobic hydrocarbon group, a linear or branched alkyl group, an aryl group, a cholesterol group, a porphyrin group, a deprotonated imidazole group, and the like. A chain or branched alkyl group is preferred.
Specific examples include the following hydrophobic groups.
Linear or branched alkyl group: methyl group, ethyl group, butyl group, propyl group, pentyl group, hexyl group, octyl group, nonyl group, decyl group, lauryl group, stearyl group, isopropyl group, tert-butyl group aryl Group: phenyl group, benzyl group, naphthyl group, alkylphenyl group, pyrenyl group cholesterol group: cholesterol group shown in the following formula: porphyrin group: porphyrin group shown in the following formula: deprotonated imidazole group: imidazole shown in the following formula By this, hydrophobic interaction with DNA can be performed, and a stable complex with nucleic acid can be formed.

The hydrophobic group may have a non-hydrophobic substituent in a part thereof.
Preferred examples of the non-hydrophobic substituent include hydrogen-bonding functional group-containing substituents. Among them, amide groups, hydroxyl groups, nucleobases (thymine, cytosine, adenine, guanine, uracil), modified nucleobases ( A hydrogen-containing functional group-containing substituent such as 5-methylcytosine and 5-hydroxymethylcytosine) is preferable.
When the hydrophobic group has a non-hydrophobic substituent in a part thereof, the position of the substituent is preferably located at the end of the hydrophobic group.
As such a hydrophobic group having a non-hydrophobic substituent in part, any hydrophobic group in which the non-hydrophobic substituent is introduced into the above-described alkyl group or the like can be used without particular limitation. The 1-amido-alkyl group, 1-hydroxy-alkyl group, 1-thymine-alkyl group and the like shown in the following chemical formula can be particularly preferably mentioned.
Thereby, a more stable complex can be formed with the nucleic acid.
In the formula, n is an integer of 1 to 20, and preferably an integer of 3 to 20.


Specifically, the following hydrophobic groups can be mentioned.

<Nitrogen-containing group>
The nitrogen-containing group is a group containing one or more nitrogen atoms in the molecule, and specific examples include an amide group, an imide group, a urea group, an imidazolium group, and the like. Preferably mentioned.
Thereby, hydrogen bonds can be formed between the DNA grooves, and a stable complex with DNA can be formed.
Moreover, the said specific number of the said nitrogen-containing group is 1-5 pieces, and it is preferable that it is one piece. By setting it within this range, it is possible to form a nucleic acid complex having a small particle diameter with low toxicity to a living body.
Specific examples of the amide group include the following substituents.

<Cationic group>
The surfactant-like compound of the present invention preferably has a cationic group between the hydrophobic group and the nitrogen-containing group.
Examples of the cationic group include an imidazolium group, a quaternary amino group, a guanidinium group, and the following substituents, and an imidazolium group is preferable.
Thereby, the nucleic acid phosphate group and the electrostatic interaction can suppress the cross-linking between the nucleic acids, and a stable complex can be formed. Moreover, since it is a monocation, a complex free from toxicity due to cation excess can be formed.
Specific examples of the cationic group include the following groups (imidazolium groups) and the like.

Specific examples of the surfactant-like compound of the present invention include the following compounds.
In addition, the surfactant-like compound of the present invention means a compound having a hydrophilic group and a hydrophobic group at both ends of the molecule in the same manner as the surfactant, and does not necessarily have a function as a surfactant. Absent.

In the following formula, n is as described above.

<Manufacturing method>
A method for producing the surfactant-like compound of the present invention will be described.
In the case where the surfactant-like compound of the present invention does not have the cationic group, the hydrophilic group-containing compound is aminated, and the hydrophobic group-containing carboxylic acid anhydride, carboxylic acid halide, carboxylic acid, and the like. It can be produced by reacting an acid azide or an active ester to form an amide bond.
When the cationic group is included, an activated ester is synthesized by reacting the carboxylic acid compound of the cationic group with N-hydroxysuccinimide (NHS), and the activated ester and the end of the hydrophilic group are synthesized. It is produced by synthesizing an amide-bonded compound by reacting with an aminated compound, and by bonding the hydrophobic group by reacting the compound with a halide having the hydrophobic group. be able to. In addition, a commercial item can also be used as the said halide.

The surfactant-like compound of the present invention can be preferably used for nucleic acid complex formation.
Here, the “nucleic acid” that can form a complex with the surfactant-like compound of the present invention is DNA or RNA having an effect of treating or preventing various diseases, for example, DNA or RNA encoding a protein, antisense. Examples thereof include DNA and RNA such as DNA and RNA, and so-called artificial nucleic acids, which may be partially modified, or chimeras thereof.
When used for forming a nucleic acid complex, a complex of the surfactant-like compound of the present invention and a nucleic acid such as DNA or RNA is prepared by mixing the surfactant-like compound of the present invention with DNA or RNA. can do.
The complex of the surfactant-like compound of the present invention and the nucleic acid is a stable complex having a small particle size. As a result, the efficiency of transporting the target nucleic acid to the target site and the gene expression efficiency in the body are high, which is suitable as a drug delivery system.
The compounding ratio of the surfactant-like compound of the present invention and the nucleic acid in forming the complex is the ratio of the number of molecules of the surfactant-like compound to the number of nucleic acid phosphate groups. = 0.1 to 64: 1 is preferable, and 0.1 to 2: 1 is more preferable.
The chain length of the nucleic acid when forming the complex is preferably 4000 to 6000 bp for double-stranded DNA and 20 to 30 base for single-stranded DNA or RNA.
The complex can be formed in the presence of various solvents, and examples of the solvent that can be used in this case include arbitrary buffer solutions (PBS, HEPES, etc.). The formation of the complex can be performed using a known technique without any particular limitation.
In addition, the complex formed using the surfactant-like compound of the present invention has no toxicity due to cation excess, and has a biocompatible hydrophilic group such as a polyethylene glycol group, thereby providing biocompatibility and It is highly stable in blood and is preferable for drug delivery.
Examples of uses for drug delivery include uses such as gene therapy using nucleic acids, RNA interference, and mRNA delivery, and these uses can be dealt with by administering the complex.
The complex is administered by subcutaneous, intradermal, intravenous, arterial or intramuscular injection, transmucosal (oral, nose, lung, eye, rectum, uterus, etc.) administration, intracerebral administration, intratumoral administration, etc. be able to. In that case, additives, such as a diluent and a gel, can also be used as needed.
The dose and period of the complex are not particularly limited, and can be appropriately selected according to various conditions such as the type of nucleic acid as an active ingredient, the body weight and age of the administration target.
In administration, it is also possible to give delivery efficiency and target tissue selectivity by combination with physical energy. The physical energy at this time means a magnetic field, light, ultrasonic waves, electricity, and pressure.

  EXAMPLES Hereinafter, although an Example and a comparative example are shown and this invention is demonstrated more concretely, this invention is not restrict | limited to these at all.

[Example 1] Synthesis of BuIm-PEG (synthesis of imidazole NHS activated ester)
126 mg of 1-imidazole acetic acid and 115 mg of N-hydroxysuccinimide (NHS) were dissolved in 10 mL of N, N-dimethylformamide (DMF) to obtain a solution. To this solution, 3 mL of a DCC solution in which 206 mg of N, N′-dicyclohexylcarbodiimide (DCC) was dissolved in 3 mL of DMF was added, mixed, reacted at 50 ° C. for 16 hours, and NHS esterified to obtain an activated ester.
An explanatory diagram of the reaction is shown below.

(Synthesis of Im-PEG by coupling reaction of NHS ester and PEG-NH ₂ )
To the reaction solution after the NHS esterification reaction, 400 mg of PEG-NH ₂ (MW2000, manufactured by NOF Corporation) shown below was directly added and mixed, and reacted at 50 ° C. for 24 hours. In order to complete the reaction, 89.2 mg of the NHS-activated ester was directly added to the solution and mixed, and the reaction was repeated at 50 ° C. for 24 hours to obtain Im-PEG (an amide-bonded compound).
An explanatory diagram of the reaction is shown below.

(Synthesis of BuIm-PEG by partial alkylation of Im-PEG) After the reaction after the above coupling reaction, 100 mg of isolated Im-PEG was dissolved in 7 mL of DMF, and 1-bromobutane (halide having a hydrophobic group) was obtained. 7 μL (68.5 mg) was added and mixed, followed by reaction for 24 hours at 50 ° C. This addition of 1-bromobutane was repeated twice, and the product after the reaction was purified by reprecipitation and dialysis. As a surfactant-like compound, 59.6 mg (55% yield) of BuIm-PEG was obtained.
An explanatory diagram of the reaction is shown below.

Example 2 Synthesis of OcIm-PEG Same as Example 1 except that 1-bromobutane used for partial alkylation of Im-PEG of Example 1 was changed to 86.4 μL (96.6 mg) of 1-bromooctane. As a result, 70.0 mg (yield 63%) of OcIm-PEG as the surfactant-like compound of the present invention was obtained.
An explanatory diagram of the reaction is shown below.

[Example 3] Synthesis of Chol-PEG 120 mg of PEG-NH ₂ (MW2000, manufactured by NOF Corporation, a compound in which the end of a hydrophilic group was aminated) and cholesteryl chloroformate (a cholesterol group was introduced into a hydrophobic group) Monocarboxylic acid halide (53.9 mg) was dissolved in chloroform (10 mL), and triethylamine (TEA) 8.36 μL (6.07 mg) was added to the solution, followed by reaction at 40 ° C. for 48 hours. The product after the reaction was purified by filtration and dialysis to obtain 143 mg (99% yield) of Chol-PEG represented by the following formula as a surfactant-like compound of the present invention.
An explanatory diagram of the reaction is shown below.

[Example 4] Synthesis of Oc-PEG 120 mg of PEG-NH ₂ (MW2000, manufactured by NOF Corporation) and 39.4 μL (35.8 mg) of nonanoic acid anhydride were dissolved in 10 mL of DMF, and triethylamine (TEA) was dissolved in the solution. 8.36 μL (6.07 mg) was added and reacted at 50 ° C. for 48 hours. The product after the reaction was purified by dialysis to obtain 42 mg (yield 35%) of Oc-PEG as the surfactant-like compound of the present invention.
An explanatory diagram of the reaction is shown below.

[Example 5] Synthesis of St-PEG The surface activity of the present invention was the same as in Example 4 except that nonanoic anhydride of Example 4 was changed to 100 mg of stearic anhydride and the reaction temperature was changed to 40 ° C. 66.8 mg (yield 59%) of St-PEG as an agent-like compound was obtained.
An explanatory diagram of the reaction is shown below.

[Example 6] Synthesis of HeA-Im-PEG (may be abbreviated as "APe-Im-PEG") 1-Bromobutane used for partial alkylation of Im-PEG of Example 1 is 6-bromohexanamide The surfactant-like surfactant of the present invention having a hexaneamide group (HeA) (synonymous with “1-amido-pentyl group (APe)”) as a hydrophobic group in the same manner as in Example 1 except that the amount was changed to 54.3 mg. 50 mg (yield 80%) of HeA-Im-PEG (APe-Im-PEG) as a compound was obtained.

An explanatory diagram of the reaction is shown below.

In addition, the obtained HeA-Im-PEG was confirmed for synthesis by measuring a ¹ H-NMR spectrum using an NMR apparatus (apparatus name: AV500, manufactured by Bruker).
The result is shown in FIG.

[Test Example 1] Evaluation of BuIm-PEG / DNA complex (preparation of surfactant-like compound / DNA complex)
0.3 μg of pGL3 (Promega), a commercially available plasmid as DNA, and BuIm-PEG as the surfactant-like compound of the present invention obtained in Example 1 were combined with BuIm for the number of phosphate groups of DNA. -Phosphate buffer solution (composition 50 mM NaH ₂ PO ₄ +50 mM Na ₂ HPO ₄ ) so that the number of PEG molecules is 0.1, 0.5, 1, _{2, 4,} 8, 16, 32 and 64 The final solution volume was 15 μL. After dissolution, the mixture was incubated at 37 ° C. for 24 hours to prepare a BuIm-PEG / DNA complex as a surfactant-like compound / DNA complex.

(Anarose gel electrophoresis analysis of surfactant-like compound and DNA complex)
The BuIm-PEG • DNA complex was analyzed by agarose gel electrophoresis to confirm the formation of the DNA complex.
The resulting BuIm-PEG / DNA complex solution was mixed by pipetting, and the entire amount was loaded on an agarose gel containing 1 μg / mL ethidium bromide, and electrophoresis was performed under the following conditions.
conditions:
Agarose gel: 1% by weight of agarose, buffer phosphate buffer (composition 50 mM NaH ₂ PO ₄ +50 mM Na ₂ HPO ₄ )
Voltage: 50mV
Electrophoresis time: 30 minutes The agarose gel after the completion of electrophoresis was developed with a UV transilluminator, and an image was obtained with a gel imaging device (device name: GelDoc2000, manufactured by BIO-RAD).
In addition, as a comparative sample, a sample containing only DNA containing no surfactant-like compound was also tested.
The result is shown in FIG.
In the figure, PEG / P means the ratio of the number of DNA phosphate groups to the number of PEG molecules in the surfactant-like compound / DNA complex. Represents the number of molecules. Moreover, DNA in the figure means a sample containing only DNA that does not contain a surfactant-like compound.

(Results and discussion)
From the results shown in FIG. 1, a band shift toward the cathode is observed as indicated by an arrow when PEG / P is 0.1 or more, the amount of the shifted band increases in 8 and 16, and the band in 32 and 64 Disappearance was observed.
The band shift means that BuIm-PEG and DNA form a complex to increase the molecular weight, and the DNA charge is partially canceled and the migration distance is shifted in a shorter direction.
The disappearance of the band means that BuIm-PEG and DNA form a complex and the DNA is in an aggregated state, so that ethidium bromide cannot be intercalated and DNA is not stained.
From these results, it can be seen that BuIm-PEG and DNA form a complex, and the DNA is aggregated. In particular, in 32 and 64, the stability of the BuIm-PEG • DNA complex is high.

[Test Example 2] Evaluation of OcIm-PEG / DNA complex (analysis of surfactant-like compound / DNA complex by agarose gel electrophoresis)
An OcIm-PEG / DNA complex was prepared and analyzed by agarose gel electrophoresis in the same manner as in Test Example 1 except that the OcIm-PEG obtained in Example 2 was used as the surfactant-like compound.
The result is shown in FIG.

(Results and discussion)
From the results shown in FIG. 2, a band shift toward the cathode was observed as indicated by an arrow when PEG / P was 0.1 or more, and the amount of the band shifted when it was 8 or more increased.
From this, it can be seen that OcIm-PEG and DNA form a complex.

[Test Example 3] Evaluation of Chol-PEG / DNA Complex Using the Chol-PEG obtained in Example 3 as a surfactant-like compound, Chol-PEG was converted into a PEG molecule with respect to the number of phosphate groups in DNA. Solubilize so that the final volume is 15 μL with phosphate buffer (composition 50 mM NaH ₂ PO ₄ +50 mM Na ₂ HPO ₄ ) so that the number is 1, 4, 8, 16, 32, 64 and 96. Except for the above, in the same manner as in Test Example 1, a Chol-PEG • DNA complex was prepared and analyzed by agarose gel electrophoresis.
The result is shown in FIG.

(Results and discussion)
From the results shown in FIG. 3, as indicated by an arrow when PEG / P is 1 or more, a band shift toward the cathode is observed despite the absence of DNA charge cancellation. The amount of shifted bands increased, and disappearance of bands was seen at 64 and 96.
From this, it can be seen that Chol-PEG and DNA form a complex, and the DNA is aggregated. In particular, in 64 and 96, the stability of the Chol-PEG • DNA complex is high.

[Test Example 4] Evaluation of Oc-PEG / DNA complex Oc-PEG / DNA complex as in Test Example 3 except that the Oc-PEG obtained in Example 4 was used as the surfactant-like compound. Body was prepared and analyzed by agarose gel electrophoresis.
The result is shown in FIG.

(Results and discussion)
From the results shown in FIG. 4, when PEG / P is 1 or more, a band shift toward the cathode is observed as indicated by an arrow, and when it is 64 or more, the amount of the shifted band increases.
From this, it can be seen that Oc-PEG and DNA form a complex.

[Test Example 5] Evaluation of St-PEG / DNA complex A St-PEG / DNA complex was prepared in the same manner as in Test Example 3 except that St-PEG obtained in Example 5 was used as the surfactant-like compound. Body was prepared and analyzed by agarose gel electrophoresis.
The result is shown in FIG.

(Results and discussion)
From the results shown in FIG. 5, a band shift toward the cathode was observed as indicated by an arrow when PEG / P was 1 or more, and the amount of the shifted band increased when 16 or more.
From this, it can be seen that St-PEG and DNA form a complex.

[Test Example 6] Particle size measurement of surfactant-like compound / DNA complex: 0.7 μg of commercially available plasmid pDNA3 as DNA and the surfactant-like compound of the present invention obtained in Examples 1 to 5 as DNA Prepared with 100 μL of phosphate buffer (composition 50 mM NaH ₂ PO ₄ +50 mM Na ₂ HPO ₄ +130 mM NaCl) so that the number of PEG molecules is 64 with respect to the number of phosphate groups of 1 and 24 at 37 ° C. After incubation for a time, a surfactant-like compound / DNA complex was prepared.
The particle size of the obtained surfactant-like compound / DNA complex was measured by a dynamic light scattering method.
Measurement of the particle diameter by the dynamic light scattering method was performed by measuring the scattered light intensity under the following conditions and calculating the particle diameter from the scattered light intensity.
conditions:
Device: Device name ELSZ2 (Otsuka Electronics Co., Ltd.)
In addition, as a comparative sample, a sample containing only a DNA containing no surfactant-like compound and a sample prepared using PEG-NH ₂ instead of the surfactant-like compound were also tested.
The results are shown in Table 1.
In the table, PEG / P means the ratio of the number of DNA phosphate groups to the number of PEG molecules in the surfactant-like compound / DNA complex. Represents the number of molecules of N. D indicates that the particle size could not be measured.

(Results and discussion)
The particle size of the complex of the surfactant-like compound of the present invention and DNA obtained in Examples 1 to 5 was as small as 13.0 ± 1.1 to 137.5 ± 28.7 nm. .
This result shows that the complex of the surfactant-like compound of the present invention and DNA has a size that can be transferred from a blood vessel to a cell at the time of biological administration and has a particle size suitable for intravenous injection. Yes.
Moreover, the particle diameters of the complex of the surfactant-like compound of Example 1 and 2 having a cationic group and DNA were 13.0 ± 1.1 nm and 87.3 ± 1.1 nm, respectively.
This result shows that the surfactant-like compound of the present invention having a cationic group can form a DNA complex having a small particle size, thereby increasing the transfer efficiency from blood vessels to cells at the time of biological administration. .

[Test Example 7] Circular dichroism spectrum measurement of surfactant-like compound / DNA complex The surfactant-like compound (BuIm-PEG) of the present invention obtained in Example 1 was converted to DNA (pGL3) phosphate. Prepare a surfactant-like compound / DNA complex by preparing 1000 μL of pure water so that the number of PEG molecules is 64 per group of 1 and incubating at 37 ° C. for 24 hours. The circular dichroism spectrum (CD spectrum measurement) was measured.
Condition: DNA amount: 12 μg
Apparatus: J-820 (manufactured by JASCO Corporation)
In addition, as a comparative sample, a sample containing only DNA containing no surfactant-like compound was also tested.
The results are shown in FIG. 6 (chart diagram) and Table 2 (wavelength (λ _max ) indicating maximum molar ellipticity and molar ellipticity at 300 nm ([θ] ₃₀₀ )).

(Results and discussion)
From the results of [θ] _{300 in} Table 2 and the part indicated by 1 in FIG. 6, it can be seen that a negative cotton effect appears in DNA in the surfactant-like compound / DNA complex. _It can be seen that a blue shift can be seen from the results of _max and the part indicated by 2 in FIG.
From these results, it can be seen that the DNA in the surfactant-like compound / DNA complex undergoes a conformational change due to dehydration.

[Test Example 8] Gene transfer efficiency evaluation test 1
An evaluation test of gene transfer efficiency of a surfactant-like compound / DNA complex prepared using the surfactant-like compound (Chol-PEG) of the present invention obtained in Example 3 was performed.
(Preparation of surfactant-like compound / DNA complex)
PGL3 (Promega) 3.5 μg obtained by ligating a modified luciferase gene downstream of the SV40 promoter with a commercially available plasmid as DNA, and Chol-PEG as the surfactant-like compound of the present invention obtained in Example 3 Phosphate buffered saline (PBS, composition 137 mM NaCl + 2.P) so that the number of cholesterol groups is 0.1, 0.5, 1.0 and 2.0 with respect to the number of phosphate groups in DNA. 7 mM KCl + 10 mM Na ₂ HPO ₄ +1.76 mM KH ₂ PO ₄ ) so that the final volume was 35 μL. After dissolution, the mixture was incubated at 37 ° C. for 24 hours to prepare a Chol-PEG / DNA complex as a surfactant-like compound / DNA complex.
(Administration to mice)
The prepared Chol-PEG / DNA complex was sterilized by filtration, and 35 μL was injected intradermally into the intradermal region of the back skin of a mouse (line ICR, 5 weeks old, body weight 20-25 g).
In addition, as a comparative sample, a sample injected with PBS alone and a sample containing only DNA not containing a surfactant-like compound were also tested.
(Tissue extraction)
Two days after the administration, the mice were anesthetized according to the ethical guidelines for animal experiments, dissected, and the skin was removed.
The removed skin was stored at −80 ° C. until luciferase assay.
(Protein extraction-Luciferase assay)
Protein extraction from the skin was performed with a homogenate.
The luciferase assay was performed by quantifying luminescence using a luciferase assay system (manufactured by Promega) with a luminometer (device name: LB96V, manufactured by Bertrud). The amount of luminescence was standardized by the amount of protein in the sample.
In addition, the protein amount of the sample was measured by BCA method using BCA Protein Assay Kit (Pierce).
The result is shown in FIG.
In the figure, Chol / P means the ratio of the number of DNA phosphate groups to cholesterol groups in the surfactant-like compound / DNA complex, and cholesterol when the number of DNA phosphate groups is 1. Represents the number of molecules of the group, PBS represents a sample administered with PBS alone, and DNA represents a sample of DNA alone containing no surfactant-like compound.
(Results and discussion)
FIG. 7 shows that administration of the Chol-PEG / DNA complex prepared using the surfactant-like compound (Chol-PEG) of the present invention is luciferase in vivo when PEG / P is 1 and DNA alone is administered. The luminescence by was high.
From this result, administration of a chol-PEG / DNA complex prepared using the surfactant-like compound (Chol-PEG) of the present invention is more effective in gene expression in vivo than administration of DNA with clinical application examples. Is high.

[Test Example 9] Evaluation test 2 of gene transfer efficiency
An evaluation test of gene transfer efficiency of a surfactant-like compound / DNA complex prepared using the surfactant-like compounds (BuIm-PEG and Chol-PEG) of the present invention obtained in Examples 1 and 3 was performed. .
(Preparation of surfactant-like compound / DNA complex)
The BuIm-PEG as the surfactant-like compound of the present invention obtained in Example 1 was replaced with a positive charge (cation group charge) of BuIm-PEG with respect to the number of negative charges (phosphate group charges) of DNA (pGL3). ) The mixture was mixed so that the number was 0.5, and Chol-PEG as the surfactant-like compound of the present invention obtained in Example 3 was changed to the number 1 of the phosphate group of DNA (pGL3). However, BuIm-PEG and Chol-PEG / DNA complex as a surfactant-like compound / DNA complex were the same as in Test Example 8 except that mixing was performed so that the number of cholesterol group molecules was 1.0. The body was prepared.
(Administration to mice)
The prepared BuIm-PEG • DNA complex and Chol-PEG • DNA complex were administered to mice in the same manner as in Test Example 8. Administration to mice was performed on 2 mice per sample.
In addition, as a comparative sample, a test using only a DNA containing no surfactant-like compound was also conducted.
(Tissue extraction)
The skin was extracted and stored in the same manner as in Test Example 8.
(Protein extraction-Luciferase assay)
The amount of luminescence was measured in the same manner as in Test Example 8.
The result is shown in FIG.
(Results and discussion)
FIG. 8 shows that administration of a BuIm-PEG / DNA complex prepared using the surfactant-like compound (BuIm-PEG) of the present invention produces about 7 times as much light emission by luciferase in vivo as that of administration of DNA alone. In the administration of the Chol-PEG / DNA complex prepared using the surfactant-like compound (Chol-PEG) of the present invention, the luminescence by luciferase in the living body is higher than that in the administration of DNA alone. It was.
From these results, administration of BuIm-PEG • DNA complex and Chol-PEG • DNA complex prepared using the surfactant-like compounds of the present invention (BuIm-PEG and Chol-PEG) has clinical application examples. It can be seen that gene expression in vivo is higher than administration of DNA alone, and that gene expression in vivo is particularly high in administration of BuIm-PEG • DNA complex.

[Test Example 9] Evaluation of HeA-Im-PEG / DNA complex 1
(Anarose gel electrophoresis analysis of surfactant-like compound and DNA complex)
As the surfactant-like compound, the HeA-Im-PEG obtained in Example 6 was used, and the HeA-Im-PEG had a number of PEG molecules (PEG / P) of 0.5 with respect to the number of phosphate groups of DNA. , 1, 2, 4, 8, 16, 32, 64 so that the final solution amount was 15 μL with a phosphate buffer (composition 50 mM NaH ₂ PO ₄ +50 mM Na ₂ HPO ₄ ). Except for the above, a HeA-Im-PEG • DNA complex solution was prepared in the same manner as in Test Example 1, and analyzed by agarose gel electrophoresis.
The result is shown in FIG.
In addition, when HeA-Im-PEG is mixed in an amount of 0.5, 1, 2, 4, 8, 16, 32, 64 of the number of PEG molecules with respect to the number of phosphate groups of DNA, The number of positive charges (charge of cationic groups) (charge ratio) of HeA-Im-PEG with respect to the number 1 of acid groups is also the same value as the number of molecules of PEG with respect to the phosphate groups.
(Results and discussion)
From the results shown in FIG. 10, when PEG / P is 0.5 or more, a band shift toward the cathode is observed as indicated by an arrow, when 4 is a band smeared toward the anode, and when 8 or more, Naked DNA is observed. The disappearance of the band was observed. The smear band suggests the formation of a net negatively charged microparticle DNA complex. Furthermore, the band shifted toward the anode from the Naked DNA band seen in 8 suggests the formation of a net negatively charged ultrafine particle size DNA complex. It is considered a complex.
From these results, it can be seen that HeA-Im-PEG forms a complex with DNA when PEG / P is 0.5 or more. In particular, when PEG / P is 8 or more, the HeA-Im-PEG • DNA complex is found to have high stability.
In HeA-Im-PEG, the band shift toward the cathode at a low charge ratio of PEG / P of 0.5 or higher when compared with the results of the surfactant-like compounds of Examples 1 to 5. (Complex formation with DNA) was observed.
Further, the disappearance of the band indicating that the DNA was in an aggregated state and the stability of the HeA-Im-PEG • DNA complex was high was also observed at a low charge ratio of PEG / P of 16 or more.
From these results, it can be seen that HeA-Im-PEG has higher ability to form a complex with DNA and to aggregate DNA than the surfactant-like compounds of Examples 1 to 5.

[Test Example 10] Evaluation 2 of HeA-Im-PEG / DNA complex
(Stability evaluation of surfactant-like compound / DNA complex against polyanion)
The stability of the surfactant-like compound / DNA complex was evaluated. The stability was evaluated using the stability of the surfactant-like compound / DNA complex with the polyanion against the substitution reaction as an index.
(Preparation of surfactant-like compound and DNA complex)
HeA-Im-PEG as the surfactant-like compound of the present invention obtained in Example 6 was used in an amount of 1 or 8 in terms of the number of PEG molecules (PEG / P) relative to the number of phosphate groups in DNA ( A HeA-Im-PEG / DNA complex solution was prepared in the same manner as in Test Example 1 except that the mixture was mixed so that the charge ratio was 1 and the charge ratio was 8), respectively.
(Polyanion treatment)
To the adjusted HeA-Im-PEG / DNA complex solution, a dextran sulfate (model number: D4911, manufactured by SIGMA-ALDRICH) solution as a polyanion was mixed and added to the HeA-Im-PEG / DNA complex, 37 ° C., The reaction was incubated for 60 minutes. For samples with PEG / P of 1, the dextran sulfate solution was mixed and added so that the final concentration was 1, 2, 3 mM, and for samples with PEG / P of 8, the final concentration of dextran sulfate was 1, The mixture was added to 2, 3, and 8 mM.
(Agarose gel electrophoresis analysis)
After the HeA-Im-PEG • DNA complex and dextran sulfate were reacted, the mixture was analyzed by agarose electrophoresis in the same manner as in Test Example 1.
The result is shown in FIG.

(Results and discussion)
From the results shown in FIG. 11, in the HeA-Im-PEG • DNA complex with PEG / P = 1, the main band indicating the formation of the HeA-Im-PEG • DNA complex is present at a dextran sulfate concentration of 2 mM or less. In addition, a minor band derived from the supercoil was observed under the main band (on the anode side) at a concentration of 3 mM. This result indicates that the HeA-Im-PEG • DNA complex is stable to dextran sulfate as high as 2 mM, and DNA is released to 3 mM dextran sulfate.
On the other hand, in the HeA-Im-PEG • DNA complex having PEG / P of 8, no band change was observed when the concentration of dextran sulfate was 1 to 8 mM. This result shows that the HeA-Im-PEG • DNA complex is stable against dextran sulfate at a high concentration of 8 mM and forms a very stable complex. Further, at this time, as described in [Test Example 10], since the band shifted from the Naked DNA band in the anode direction is maintained, the stability of the DNA complex having an ultrafine particle diameter charged net negative is improved. It was suggested.
Further, as can be seen from these results, the complex of HeA-Im-PEG and DNA as the surfactant-like compound of the present invention can be obtained by changing the PEG / P ratio (charge ratio). It is possible to control the characteristics. Thereby, drug delivery characteristics can be controlled. For example, when the purpose of drug delivery is to introduce a gene into cultured cells in a short period of time, the stability of the complex is controlled so as not to be too high. In the case of drug delivery to low cells or tissues, it can be controlled to increase the stability of the complex, such as changing the PEG / P ratio (charge ratio). Can be done easily. From the above, it can be seen that the surfactant-like compound of the present invention can be suitably used for nucleic acid complex formation and drug delivery.

[Test Example 11] Evaluation test 3 of gene transfer efficiency
An evaluation test of the gene transfer efficiency of the surfactant-like compound / DNA complex prepared using the surfactant-like compound (HeA-Im-PEG) of the present invention obtained in Example 6 was performed.
(Preparation of surfactant-like compound / DNA complex)
HeA-Im-PEG as the surfactant-like compound of the present invention obtained in Example 6 has a number of PEG molecules (PEG / P) of 0.5, 0.6 with respect to the number of phosphate groups of DNA. , 0.7, 0.8, 0.9, 1.0, 1.2, 1.5 HeA-Im-PEG / DNA complex as in Test Example 8 A body solution was prepared.
(Administration to mice)
The prepared HeA-Im-PEG / DNA complex solution was administered to mice in the same manner as in Test Example 8, except that it was changed to intra-tibial muscle administration (35 μL). Administration to mice was performed on 2 mice per sample.
In addition, as a comparative sample, a sample containing only a DNA that does not contain a surfactant-like compound, a sample containing only a phosphate buffer, and a straight-chain that is a cationic water-soluble polymer instead of the surfactant-like compound of the present invention Tests of samples prepared using a commercially available in vivo nucleic acid introduction reagent (trade name: jetPEI, manufactured by Polyplus transfection) based on polyethyleneimine (hereinafter sometimes referred to as a group using commercially available products). ) Also went. Sample adjustment by jetPEI was performed according to the attached manual.
(Tissue extraction, protein extraction to luciferase assay)
Tissue extraction after administration and the steps of protein extraction to luciferase assay were performed in the same manner as in Test Example 8 except that tissue extraction was performed 6 days after administration.
The result is shown in FIG.

(Results and discussion)
From FIG. 12, the administration group of the HeA-Im-PEG • DNA complex prepared using the surfactant-like compound (HeA-Im-PEG • DNA) of the present invention is the comparative experimental group (PBS only). The amount of luminescence due to luciferase in the living body was higher than that of the group administered with DNA and the group administered with DNA alone). In particular, the amount of luminescence is high when PEG / P is 0.6 to 1.0, and in particular, in the group where PEG / P is 0.8, more than 10 times the group administered with DNA alone, and more than the group using commercially available products An extremely high light emission amount of 1000 times or more was exhibited.
From the above results, it can be seen that the surfactant-like compound of the present invention can be suitably used for nucleic acid complex formation and drug delivery.

The surfactant-like compound of the present invention is useful for nucleic acid complex formation and can be preferably used for drug delivery of nucleic acid therapeutics.
The complex of the surfactant-like compound of the present invention and DNA is a stable complex having a small particle size. Thereby, the transfer efficiency and gene expression efficiency of the target DNA in the body to the target site are high.
In addition, since the surfactant-like compound of the present invention contains a biocompatible substituent such as a polyethylene glycol group, it is highly stable in blood and has no toxicity due to excessive cation. Since it has these functions, it is useful for drug delivery.

Claims (2)

A surfactant-like compound which is any compound represented by the following chemical formula.

(In the formula, n represents an integer of 40 to 120.)
Surfactant-like compound of claim 1 wherein the nucleic acid complex formation.