JP5721130B2 - Asymmetric nanotube-forming asymmetric double-headed lipid molecule, asymmetric nanotube formed by the lipid molecule, and drug encapsulated product using the asymmetric nanotube - Google Patents

Description

  The present invention relates to an asymmetric double-headed lipid molecule capable of forming a hollow fiber-like asymmetric nanotube structure suitable for drug encapsulation, and a method for producing the same, and further includes a hollow fiber-like organic nanotube formed by self-assembly of the lipid molecule, and The present invention relates to use for drug encapsulation using the nanotube.

It is known that organic molecules such as certain lipid molecules form a hollow fiber structure (referred to as organic nanotube) by self-assembly in a solvent (Non-Patent Documents 1 and 2). These form a tube structure having an outer diameter of about 10 nm to 1000 nm and a length of about 100 nm to several mm, and have a minute hollow space inside, so that various materials (proteins, drugs, DNA, etc.) are encapsulated. be able to. These organic nanotubes are formed by self-assembly due to weak interactions such as hydrogen bonds and van der Waals forces, so that they have a specific temperature or pH ranging from room temperature to 100 ° C depending on the structure of the constituent molecules. It is possible to reversibly decompose the tube structure depending on the above conditions. Since this temperature and pH include the range of environmental temperature and pH in the living body, it means that the temperature and pH can be adjusted to decompose it in the living body. Moreover, even if there is no decomposition | disassembly of a tube, it has the characteristic that the material encapsulated inside is discharge | released gradually from the opening part of both ends of a tube (nonpatent literatures 3 and 4).
By using these characteristics, it is expected to be applied to drug delivery capsules and drug sustained release capsules, fixed matrices such as proteins, and storage capsules (Non-Patent Documents 5 and 6). However, since most of the conventional organic nanotubes have inner and outer surfaces coated with the same hydrophilic portion, it has been difficult to selectively and efficiently encapsulate materials such as drugs and proteins inside the organic nanotube. (Non-Patent Documents 7, 8, and 9). Specifically, when encapsulating various materials, the water inside the organic nanotubes is removed by lyophilization under reduced pressure, and then an aqueous solution in which the materials are dispersed and dissolved is added to rehydrate (so-called capillary phenomenon). A complicated operation is required (Non-Patent Documents 7 and 8).

The present inventors have conventionally proposed various organic nanotubes (hereinafter referred to as asymmetric nanotubes) that are characterized by having inner and outer surfaces coated with different functional groups as organic nanotubes capable of easily and efficiently encapsulating drugs. (Non-Patent Documents 10, 11, 12, 13). These asymmetric nanotubes are both asymmetric two-heads in which two hydrophilic parts differing at both ends of the hydrophobic part alkylene chain, specifically, 1-glucopyranosylamine at one end and a carboxyl group or amino group at the other end, respectively. Type lipid molecules (hereinafter referred to as “asymmetric double-headed lipids”) (Patent Document 1). These asymmetric nanotubes can encapsulate a material having a charge opposite to that of the inner surface of the nanotube, usually by simply mixing at room temperature. When a carboxyl group-based lipid molecule with a carboxyl group at one end is used, the inner surface of the tube is selectively coated with a carboxyl group, making it negatively charged, and on the other hand, using an amine-based lipid molecule having an amino group. The former is preferable as a drug delivery capsule or drug carrier that encloses and transports a positively charged drug, and the latter is preferable to a negatively charged drug carrier for transporting a drug, as well as into a cell. Use as a nucleic acid carrier such as gene transport is also expected.
The present inventors have so far reported studies on selective encapsulation of proteins, fluorescent molecules, oligo DNA, etc., in vitro release behavior, etc. using these asymmetric nanotubes (Non-Patent Documents). 10, 11, 12, 13). Such various asymmetric nanotubes have been provided, and encapsulation by these asymmetric nanotubes has enabled simple, selective and efficient encapsulation of unstable materials such as biopolymers and drugs.
However, the practical application of these asymmetric nanotubes as encapsulating materials has problems to be solved, such as improved stability in the hydrated state of asymmetric nanotubes and improved encapsulation performance, and it is difficult to produce asymmetric nanotubes. In some cases, the synthesis process of the asymmetric double-headed lipid molecule that is the raw material of the tube may be complicated, and purification may be difficult.
That is, as a practical drug-encapsulating asymmetric nanotube, it is essential to develop an asymmetric nanotube that satisfies all of the following points.
1. The synthesis and purification process of the raw asymmetric bihead lipid molecule is simple.
2. The production of asymmetric nanotubes can be carried out selectively at room temperature or at least under mild conditions.
3. The asymmetric nanotube has long-term storage stability in a hydrated state.
4). The inclusion target compound can be efficiently encapsulated at a high concentration and can be easily released.
So far, even though some of these problems have been partially solved, reports of asymmetric nanotubes that have solved all these problems have used carboxylic acid lipid molecules or amine lipid molecules. Even if it was not seen.

On the other hand, conventionally, liposome-based drug capsules have been mainly used as drug capsules. However, since a pH gradient is used for encapsulation and subsequent purification is required, preparation is complicated and requires a long time. Therefore, it has been desired to develop an encapsulating agent that can easily and efficiently encapsulate a drug, stably hold the drug, and can be rapidly and slowly released in target cells such as cancer cells. In particular, anthracycline anticancer agents such as doxorubicin, idarubicin, epirubicin, daunorubicin, and pirarubicin are highly toxic, particularly cardiotoxic, and therefore have excellent encapsulating agents that can be delivered directly to cancer cells and can be sustainedly released. It was sought after.
As described above, the asymmetric nanotube formed of carboxylic acid lipid molecules has an outer surface covered with a sugar hydrophilic portion, and the inner surface is composed of a carboxyl group and has a negative charge, so an amino group such as doxorubicin is not present. It is expected as an effective capsule for the delivery of anticancer agents. Further, such nanotubes can be expected to release functional drugs due to ionic / non-ionic changes in the carboxyl group on the inner surface or changes in the shape of the tube depending on the external environment such as pH.
However, a lipid molecule in which the 1-glucopyranosylamino group, which is a carboxylic acid-based asymmetric bihead lipid molecule reported by the present inventors, is bonded to one end of a long-chain dicarboxylic acid via an amide bond is formed. Since asymmetric nanotubes (Non-patent Document 10) simultaneously produce microtubes and tape-like aggregates during the manufacturing process of asymmetric nanotubes, a nanotube separation / purification process such as centrifugation is required, and the resulting nanotubes are aqueous. Dispersibility in a solvent is poor, and there are disadvantages that long-term stability is poor due to easy crystallization due to long-term storage even at room temperature or refrigeration. Asymmetric nanotubes (Patent Document 3) using the same lipid molecule and intercalating a low molecular weight organic compound in the production process also have the same shortcoming of poor long-term stability.
In addition, by introducing an oligoglycine residue into a lipid molecule having a carboxyl group as a hydrophilic part, the water dispersibility of the resulting asymmetric nanotube can be increased, and long-term storage in an aqueous solvent has also been improved (non-patented) Reference 14) describes that for the production of asymmetric nanotubes from the same lipid, lipid molecules are dispersed in milli-Q water (ultra pure water) and heated to around 100 ° C., or an equal amount of sodium hydroxide is added to the lipid. It requires not only a complicated and advanced technique that requires a step of neutralizing at room temperature by heating to 60 ° C or higher, and then synthesizing the lipid molecules of the nanotube raw material with seven or more steps. Therefore, it is extremely difficult to synthesize in large quantities, and it is not practical.
As one of the attempts to synthesize a simple asymmetric bihead lipid molecule, a nanotube using 2-glucosamine as a glucopyranosylamine residue at the other end of a carboxylic acid asymmetric bihead lipid molecule containing no glycine residue (Non-Patent Document 15). The synthesis of the lipid molecule itself was very simple and could be synthesized by one-step synthesis and recrystallization from commercially available compounds such as 2-glucosamine and long-chain dicarboxylic acid. As a result of detailed analysis of the nanotubes, the functional groups covering the inner and outer surfaces are the same and asymmetric nanotubes cannot be formed (unpublished data).
Also, when a diacetylene residue that is a polymerizable functional group is introduced into the hydrophobic part of a carboxylic acid lipid molecule having a 1-glucopyranosyl group as a hydrophilic part, a nanotube is formed (Patent Document 2). According to detailed structural analysis, asymmetric nanotubes were not formed (unpublished data).

  From the above technical background, it has been strongly expected to provide an asymmetric organic nanotube whose inner surface is coated with a carboxyl group and a novel asymmetric double-headed lipid molecule therefor, which can solve all the above problems. That is, it is an asymmetric bihead lipid molecule that can be easily synthesized from readily available raw materials, and can produce asymmetric nanotubes selectively and in high yield under mild conditions, and the resulting asymmetric nanotubes have long-term storage stability. At the same time, there has been a strong demand to provide an asymmetric bihead lipid molecule that can efficiently encapsulate a cationic drug such as doxorubicin at a high concentration and can produce an encapsulated product that can be easily released by changing pH conditions.

Japanese Patent No. 4174702 JP 2005-239632 A JP2008-264897

Toshimi Shimizu, Mitsutoshi Masuda, Hiroyuki Minamikawa, Chemical Review, 2004, 105, 1401-1443. “Organic / Inorganic / Metal Nanotubes Latest Technology and Application Development of Non-Carbon Nanotubes”, Toshimi Shimizu, Takeshi Kijima, Frontier Publishing, Tokyo, 2008. Mitsutoshi Masuda, Naohiro Kameda, Network Polymer, 2010, 31, 191-200. Naohiro Kameta, Hiroyuki Minamikawa, Mitsutoshi Masuda, Go Mizuno, Toshimi Shimizu, Soft Matter, 2008, 4, 1681-1687. Chia-Chun Chen, Yao-Chung Liu, Chia-Hsuan Wu, Chun-Chia Yeh, Ming-Tsan Su, Yi-Chun Wu, Advanced Materials, 2005, 17, 404-407. Mohammand Reza Abidian, Dong-Hwan Kim, David C Martin, Advanced Materials, 2006, 18, 405-409. Bo Yang, Shoko Kamiya, Yoshiki Shimizu, Naoto Koshizaki, Toshimi Shimizu, Chemistry of Materials, 2004, 16, 2826-2831. Hiroharu Yui, Yoshiki Shimizu, Shoko Kamiya, Ichiro Yamashita, Mitsutoshi Masuda, Kohzo Ito, Toshimi Shimizu, Chemistry Letters, 2005, 34, 232-233. "Applied technology of new materials for cyclic and cylindrical supramolecules", Toshikazu Takada, CMC Publishing, Tokyo, 2006, P138-149. Mitsutoshi Masuda, Toshimi Shimizu, Langmuir, 2004, 20, 5969-5977. Naohiro Kameta, Mitsutoshi Masuda, Hiroyuki Minamikawa, Nikolay V. Gotev, Jeong A. Rim, Jong H. Jung, Toshimi Shimizu, Advanced Materials, 2005, 17, 2732-2736. Naohiro Kameta, Go Mizuno, Mitsutosi Masuda, Hiroyuki Minamikawa, Masaki Kogiso, Toshimi Shimizu, Chemistry Letters, 2007, 36, 896-897. Naohiro Kameta, Kaname Yoshida, Mitsutoshi Masuda, Toshimi Shimizu, Chemistry of Materials, 2009,21,5892-5898. Soo Jing Lee, Naohiro Kameta, Hiroyuki Minamikawa, Mitsutoshi Masuda, Toshimi Shimizu, 58th Polymer Symposium Proceedings, Presentation No. 1 Pe047, “Function of Metal Cation-Doped Nanotube by using Unsymmetrical Bolaamphiphiles”. Mitsutoshi Masuda, Hyakuyo Wada, Toshimi Shimizu, 59th Annual Polymer Conference, Publication No. 2Pd002, “Organic Nanotube Formation of Glucosamine-based Double-headed Lipids Containing Anomer Mixtures”. Naohiro Kameta, Mitsutoshi Masuda, Hiroyuki Minamikawa, Toshimi Shimizu, Langmuir, 2007, 23,4634-4641. George Fotakis and John A. Timbrell, Toxocology Letters, 2006, 160, 171-177.

  The present invention is intended to provide an asymmetric double-headed lipid molecule that can be easily synthesized from readily available raw materials and can selectively produce asymmetric nanotubes under mild conditions in a high yield. As an asymmetric nanotube obtained by self-assembly of type lipid molecules, it is stable even in long-term storage at high purity and relatively high concentration, and selectively and efficiently encapsulates drugs with amino groups such as doxorubicin at high concentration An object of the present invention is to provide an asymmetric nanotube capable of producing an encapsulated product that can be easily released by changing pH conditions. Then, an object of the present invention is to provide an encapsulated drug composition having a high stimulus-responsive sustained release function in which a cationic drug such as doxorubicin is selectively encapsulated at a high concentration by the asymmetric nanotube.

As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention have synthesized the lipid molecule itself, which was previously developed by the inventors, but cannot form asymmetric organic nanotubes. It was thought to produce a derivative of an asymmetric bihead lipid molecule (Non-patent Document 15) comprising a long-chain dicarboxylic acid bonded to the amino group of 2-glucosamine. Specifically, oligoglycine is further linked to the terminal carboxyl group of the asymmetric bihead lipid molecule by a dehydration condensation reaction to synthesize a lipid molecule (compound-1) represented by the following general formula (1). As a result, the purification process was not necessary and it was easy to produce under mild conditions, and the obtained lipid molecules had good water dispersibility and formed only asymmetric nanotubes with high selectivity. It was found that even in long-term dispersion storage over 6 months at room temperature or refrigeration at a relatively high concentration of about 5 mg / ml in MilliQ water, the dispersion state can be maintained while maintaining the tube form stably. . It is surprising that the production of nanotubes from the lipids obtained in the present invention could be achieved completely at room temperature not only by heating / cooling from milli-Q water but also by neutralization. That is, the present lipid could be quickly dissolved in an equimolar equivalent amount of sodium hydroxide aqueous solution at room temperature, and this solution could be neutralized with hydrochloric acid or the like at room temperature to produce asymmetric nanotubes. Furthermore, the synthesis of lipid molecules can be completed in only three stages, even when counted from commercially available raw materials, and lipids with sufficient purity can be obtained by filtration of solids generated during the progress of the reaction and washing of the obtained solids in all purification steps. It is also a very surprising feature. These excellent characteristics as asymmetric nanotubes have not been achieved in any conventional carboxylic acid-based asymmetric nanotubes, but are also the first in all asymmetric organic nanotubes including amine-based asymmetric nanotubes.
And it confirmed that the asymmetric nanotube obtained by this functioned also as a drug capsule which encapsulates doxorubicin efficiently, and completed this invention.

That is, the present invention relates to an asymmetric bihead lipid molecule (also referred to as Compound-1) represented by the following general formula (1) or a salt thereof.


(In the formula, n represents an integer of 8 to 16, m represents an integer of 1 to 4, and the 1-position hydroxyl group of 2-glucosamine represented by a wavy line represents either α or β anomer, or an equilibrium mixture thereof. .)
And a synthesis method thereof, and an asymmetric nanotube having high purity and long-term stability, produced by self-assembly characterized by neutralization from the sodium salt of the same lipid at room temperature, The present invention provides an encapsulated product of an asymmetric nanotube obtained from the lipid molecule and a drug having an amino group or a cationic drug.

Specifically, the present invention is as follows.
[1] An asymmetric bihead lipid molecule represented by the following general formula (1) or a salt thereof;
(In the formula, n is an integer of 8 to 16, and m is an integer of 1 to 4. In addition, the 1-position hydroxyl group of 2-glucosamine represented by a wavy line in the formula is either α or β anomer, or Represents the equilibrium mixture).
[2] α, ω-long-chain dicarboxylic acid HOOC- (CH ₂ ) _n —COOH represented by the following general formula (2) General formula (2)
(In the formula, n represents an integer of 8 to 16.)
Is condensed with 2-glucosamine in aqueous methanol, and the resulting solid is filtered and washed, whereby intermediate-3 represented by the following general formula (3)
(In the formula, n represents an integer of 8 to 16. In addition, the 1-position hydroxyl group of 2-glucosamine represented by a wavy line in the formula represents either α or β anomer, or an equilibrium mixture thereof.)
Manufacture and
Intermediate-3 and an oligoglycine ester represented by the following general formula (4) in which the C-terminus of oligoglycine is protected with an ester or the like

(In the formula, m represents an integer of 1 to 4. R is a residue of a compound having an alcoholic hydroxyl group, for example, a methyl group, an ethyl group, a tertiary butyl group, or a benzyl group.)
Intermediate 5 represented by the following general formula (5)
(In the formula, n represents an integer of 8 to 16, and m represents an integer of 1 to 4. R represents a residue of a compound having an alcoholic hydroxyl group, for example, a methyl group, an ethyl group, or a tertiary butyl group. (In addition, the 1-position hydroxyl group of 2-glucosamine represented by a wavy line in the formula represents either α, β anomer, or an equilibrium mixture thereof.)
And is deprotected by alkaline hydrolysis to obtain an asymmetric bihead lipid molecule represented by the following general formula (1) or a salt thereof
(In the formula, n is an integer of 8 to 16, and m is an integer of 1 to 4. In addition, the 1-position hydroxyl group of 2-glucosamine represented by a wavy line in the formula is either α or β anomer, or Represents the equilibrium mixture.)
How to manufacture.
[3] A structure formed from the asymmetric bihead lipid molecule or the salt thereof according to [1], wherein the inner surface is coated with a carboxyl group and the outer surface is coated with a 2-glucosamide group. Asymmetric nanotubes.
[4] The asymmetric double-headed lipid molecule according to [1] is dispersed in water and cooled after heating and dissolved, or dispersed and dissolved in water or an alkaline solution at room temperature, and then neutralized with an acid. A method for producing an asymmetric nanotube.
[5] The method for producing an asymmetric nanotube according to [4], further comprising forming a short tube by performing freeze-drying treatment or ultrasonic treatment.
[6] A cationic drug-asymmetric nanotube complex, wherein the asymmetric nanotube of [3] includes a cationic drug.
[7] A cationic drug composition comprising the cationic drug-asymmetric nanotube complex according to [6] as an active ingredient.
[8] The cationic drug composition according to [7], wherein the cationic drug is an anthracycline anticancer agent having an amino group.

According to the present invention, an asymmetric nanotube whose inner surface is coated with a carboxyl group and whose outer surface is coated with a sugar residue is produced selectively and in high yield under mild conditions such as pH change at room temperature, which has been difficult in the past. be able to. In addition, since it has high dispersibility, it does not cause crystallization even at high concentrations in an aqueous solvent, and is very stable in long-term storage, and even in a relatively high concentration aqueous dispersion such as 5 mg / ml, it is stable for more than 2 months. It can be saved. The asymmetric double-headed lipid molecule constituting the asymmetric nanotube is a novel compound, and the raw materials 2-glucosamine, long-chain dicarboxylic acid and oligoglycine are inexpensive and can be used under mild conditions of only 3 steps. It can be produced by a synthesis reaction, and it is not necessary to use column chromatography for purification, and high-purity lipids can be obtained in all reprecipitation steps.
The synthesis and purification of this asymmetric nanotube-forming lipid molecule is easy, and asymmetric nanotubes can also be easily produced under mild conditions, because the mass synthesis of capsules as drug carriers and the final cost reduction of the drug composition This is also important.
The long-term storage stability and high dispersibility at high concentrations of the asymmetric nanotubes of the present invention enable the total amount of liquid administered intravenously to reduce the burden on patients and the safety of drugs in anticancer drug treatment, respectively. It is essential to reduce as much as possible. The asymmetric nanotube has an outer diameter of only 15 nm, and its length can be adjusted from 100 nm to 2 μm by physical disturbance such as sonication, so that it can be delivered to cancer tissue by EPR (Enhanced Permiability and Retation) effect. Can be increased. Thus, the characteristics such as stability and good dispersibility of the asymmetric nanotube of the present invention are one of excellent characteristics as a drug carrier.
In addition, since the asymmetric nanotube of the present invention is covered with a carboxyl group on the inside and is negatively charged at a pH near neutrality, just mixing a drug having an amino group such as doxorubicin or a cationic drug, It has an excellent property that it can encapsulate drugs. Conventional liposome-based drug capsules that are complicated to prepare cannot be mixed at an arbitrary ratio at the time of treatment, and only those encapsulated drugs in advance have been marketed. In the present invention, however, Encapsulation is possible by quickly adjusting the nanotubes at the medical site, or by mixing the nanotube dispersion and the drug at an arbitrary ratio during treatment. As described above, since lipid synthesis of raw materials and preparation of nanotubes are extremely easy, they can be provided as inexpensive drug capsules.
The asymmetric nanotube of the present invention can be used in a condition-selective and efficient manner in response to an external stimulus such as pH change, by changing the ionization state of the carboxyl group covering the inner surface of the tube or changing the shape of the tube structure itself. Can be released. For this reason, in addition to the selective delivery of the nanotube to the cancer tissue by the EPR effect, it is possible to efficiently release the drug in a low pH environment unique to the cancer tissue. Furthermore, in the case of the nanotube complex taken up into the cancer cell, it becomes possible to promptly release in a low pH environment in the endosome.
As a result, it can be used as a functional drug sustained-release carrier in a drug delivery system for medical use, and can also be expected to be used as a carrier for emulsifiers, dispersants, stabilizers and the like in the cosmetics and food fields.

¹ H-NMR of Intermediate-3 (n = 10) (DMSO-d ₆ and 1 drop D ₂ O, room temperature) ¹ H-NMR of Compound-1 (n = 10, m = 2) (DMSO-d ₆ and 1 drop D ₂ O, room temperature) ¹ H-NMR of Compound-1 (n = 10, m = 3) (DMSO-d ₆ and 1 drop D ₂ O, room temperature) ¹ H-NMR of Compound-1 (n = 10, m = 3) (DMSO-d ₆ and 1 drop D ₂ O, room temperature) ¹ H-NMR of Intermediate-3 (n = 12) (DMSO-d ₆ and 1 drop D ₂ O, room temperature) ¹ H-NMR (DMSO-d ₆ , room temperature) of Compound-1 (n = 12, m = 2) ¹ H-NMR (DMSO-d ₆ , room temperature) of compound-1 (n = 12, m = 3) ¹ H-NMR of compound-1 (n = 12, m = 4) (DMSO-d ₆ and 1 drop D ₂ O, room temperature) ¹ H-NMR of Intermediate-3 (n = 14) (DMSO-d ₆ and 1 drop D ₂ O, room temperature) ¹ H-NMR (DMSO-d ₆ , room temperature) of compound-1 (n = 12, m = 3) ¹ H-NMR of compound-1 (n = 14, m = 4) (DMSO-d ₆ and 1 drop D ₂ O, room temperature) An electron microscope observation image (transmission image) of an asymmetric nanotube obtained by self-assembly of compound-1 (in the case of n = 10, m = 2) by (A) heating and cooling and (B) pH adjusting method. An electron microscope observation image (transmission image) of an asymmetric nanotube obtained by (A) heating and cooling of compound-1 (in the case of n = 10, m = 3) and (B) self-assembly by a pH adjusting method. An electron microscope observation image (transmission image) of an asymmetric nanotube obtained by self-assembly of compound-1 (in the case of n = 10, m = 4) by (A) heating and cooling and (B) pH adjustment method. An electron microscope observation image (transmission image) of an asymmetric nanotube obtained by (A) heating and cooling and (B) self-assembly by pH adjustment method of Compound-1 (in the case of n = 12, m = 2). The inset in the left figure is a hydrogel composed of nanotubes prepared by heating and cooling. An electron microscope observation image (transmission image) of an asymmetric nanotube obtained by self-assembly of Compound-1 (in the case of n = 12, m = 3) by (A) heating and cooling and (B) pH adjusting method. An electron microscope observation image (transmission image) of an asymmetric nanotube obtained by self-assembly of compound-1 (in the case of n = 12, m = 4) by (A) heating and cooling and (B) pH adjusting method. The electron microscope observation image (transmission image) of the asymmetric nanotube obtained by the self-assembly of the compound-1 (in the case of n = 14, m = 3) by the pH adjustment method. An electron microscope observation image (transmission image) after ultrasonic treatment of an asymmetric nanotube obtained from Compound-1 (n = 12, m = 4) obtained by heating and cooling. The inset in the left figure shows the title nanotube dispersion. Electron microscope observation image (transmission image) after ultrasonication, lyophilization, and rehydration of an asymmetric nanotube obtained from Compound-1 (n = 12, m = 4) obtained by heating and cooling. The inset (left) shows the title dispersion. The inset (right) is an enlarged image of the electron microscope observation H image. (A) An electron microscope observation image of an asymmetric nanotube encapsulating doxorubicin (when n = 12, m = 4 in compound-1) and (B) an encapsulation rate of doxorubicin by an asymmetric nanotube. Doxorubicin release experiment from asymmetric nanotubes (compound-1 with n = 12, m = 4. Encapsulation was performed with ONT / DOX = 7/1). The parentheses in the figure represent the pH of the buffer used. Anti-cancer properties of doxorubicin nanotubes to Hela cells (MTS assay).

1. About Asymmetrical Double-headed Lipid Molecules of the Present Invention (1-1) Characteristics of Asymmetrical Double-headed Lipid Molecules of the Present Invention: Compound of Formula (1) (Compound-1)
The asymmetric nanotube-forming lipid molecule of the present invention is a novel compound not described in any literature, and its shape is such that a 2-glucosamine residue is linked to one end of a long-chain carboxylic acid via an amide group and an oligoglycine is connected to the other end. It is an asymmetric bihead lipid molecule condensed at the N-terminus.
The asymmetric bihead lipid molecule of the present invention is very easy to synthesize, and can produce an asymmetric nanotube structure having an outer diameter of around 15 nm and an inner diameter of about 8-9 nm selectively and with high purity by changing the pH at room temperature. Therefore, the chain length of the alkylene chain of the lipid molecule of the present invention needs to be in the range of 8 to 16, and an integer of 10 to 14 is preferable from the viewpoint of good dispersibility during lipid synthesis and ease of purification. More preferably, it is an even number. The number of oligoglycine residues may be in the range of 1 to 4, but 2 to 4 is desirable from the viewpoint of ease of purification during nanotube production and the purity of the resulting tube. The asymmetric bihead lipid molecule of the present invention may be in the form of a water-soluble pharmaceutically acceptable salt. For example, sodium salt and ammonium salt are preferable.
As the 2-glucosamine used as the hydrophilic part, either D-form or L-form may be used, but D-form is preferred because of its availability. Since the D-form is used in the embodiment of the present invention, the case where the D-form is used will be described below, but the present invention is not limited to this.
That is, the asymmetric bihead lipid molecule of the present invention is represented by the following general formula (1).
(In the formula, n is an integer of 8 to 16, and m is an integer of 1 to 4. In addition, the 1-position hydroxyl group of 2-glucosamine represented by a wavy line in the formula is either α or β anomer, or Represents the equilibrium mixture.)

(1-2) Method for Producing Asymmetric Double-headed Lipid Molecules of the Present Invention The asymmetric double-headed lipid molecule represented by the general formula (1) or a salt thereof can be easily produced by, for example, the following method.
Α, ω-long-chain dicarboxylic acid HOOC- (CH ₂ ) _n —COOH represented by the following general formula (2)
(In the formula, n represents an integer of 8 to 16.)
Is condensed with 2-glucosamine in aqueous methanol, and the resulting solid is filtered and washed, whereby intermediate-3 represented by the following general formula (3)
(In the formula, n represents an integer of 8 to 16. The wavy line of the hydroxyl group in the formula represents the 1-position hydroxyl group of 2-glucosamine represented by the wavy line in the formula, either α or β anomer, or an equilibrium mixture thereof. Represents.)
Can be manufactured.
Next, an oligoglycine ester represented by the following general formula (4) in which the intermediate 3 of the general formula (3) and the C-terminal of oligoglycine are protected with an ester or the like

(In the formula, m represents an integer of 1 to 4. R is a residue of a compound having an alcoholic hydroxyl group, for example, a methyl group, an ethyl group, a tertiary butyl group, or a benzyl group.)
Intermediate 5 represented by the following general formula (5)
(In the formula, n represents an integer of 8 to 16, and m represents an integer of 1 to 4. R represents a residue of a compound having an alcoholic hydroxyl group, for example, a methyl group, an ethyl group, a benzyl group, (In addition, the 1-position hydroxyl group of 2-glucosamine represented by a wavy line in the formula represents either α or β anomer, or an equilibrium mixture thereof.)
Got.
Next, this is deprotected by alkaline hydrolysis, and an asymmetric bihead lipid molecule (also referred to as Compound-1) represented by the following general formula (1) or a salt thereof:
(In the formula, n is an integer of 8 to 16, and m is an integer of 1 to 4. In addition, the 1-position hydroxyl group of 2-glucosamine represented by a wavy line in the formula is either α or β anomer, or Represents the equilibrium mixture.)
Can be synthesized.

(1-3) Condensation Step for Obtaining Intermediate-3 (General Formula (3)) Intermediate-3 (general formula (3)) of the present invention is typically described in Non-Patent Document 15. Since it contains a lipid molecule, it can be produced according to the method described in Non-Patent Document 15 or by a similar method.
(A) About α, ω-long-chain dicarboxylic acid represented by the following general formula (2) of the raw material HOOC- (CH ₂ ) _n —COOH General formula (2)
(In the formula, n represents an integer of 8 to 16.)
As the raw material long-chain dicarboxylic acid, those having 8 to 16 carbon atoms in the central hydrophobic alkylene chain can be used. Specifically, dicarboxylic acid sebacic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, and octadecanedioic acid can be used. These dicarboxylic acids can be used in the form of salts or monoesters, but in the case of monoesters, hydrolysis is required after condensation of glucosamine.
In addition, when these acid anhydrides or acid chlorides of these dicarboxylic acids are used, a condensation reaction occurs without a condensing agent. In this case, however, hydrolysis of the active substance is required by adding water after the condensation.

(A) Condensing agent As the condensing agent used in the condensation reaction to obtain Intermediate-3 (general formula (3)), various condensing agents used for peptide synthesis can be used. Specifically, “4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride” (DMT-MM), 1-ethyl-3- (3 -Dimethylaminopropyl) carbodiimide hydrochloride, dicyclohexylcarbodiimide, diisopropylcarbodiimide and the like can be used. Even if such a condensing agent is not used, an acid anhydride or acid chloride of the corresponding carboxylic acid can also be used. However, DMT-MM, which gives a very high yield of condensate even in reactions in water or methanol, is desirable. In the same reaction, methanol, ethanol, isopropanol, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, etc. can be used. In order to isolate and purify intermediate-3 by precipitation, methanol, ethanol containing dicarboxylic acid, 2-glucosamine, 5 to 40% by volume of water having moderate solubility in intermediate-3, Isopropanol is preferred.

(C) About 2-glucosamine The amine component of the present invention is 2-glucosamine, and the hydroxyl group at the 1-position is an α anomer or β anomer, or an equilibrium mixture thereof, or a salt such as hydrochloride. There may be.
For example, when 2-glucosamine hydrochloride is used, it is dissolved in water and then converted into an amine with sodium methoxide or the like and used in the reaction. Alternatively, if the disodium salt of α, ω-long chain dicarboxylic acid or the like is used as a raw material in advance, neutralization of 2-glucosamine hydrochloride is not necessary.
2-glucosamine may be either D-form or L-form, but D-form is preferred because it is easily available. Therefore, D-form is used in the embodiment of the present invention. Therefore, D-form is shown for 2-glucosamine displayed as an amine component such as the general formula (1).

(D) Condensation reaction The condensation reaction of the long-chain dicarboxylic acid of general formula (2) and 2-glucosamine is carried out in an aqueous solvent, preferably in a water-containing methanol solution, with or without the addition of the condensing agent. Let it be done.
The reaction temperature at that time is selected in the range of 0 to 50 degrees, and the reaction time is usually in the range of 10 minutes to 30 hours.
In the above condensation reaction, only Intermediate-3 precipitates with high purity during the reaction, so there is no need for purification, but if necessary, reprecipitation by heating / cooling using methanol or water-containing methanol again. Further, it can be of high purity.
In such a reaction, 2-glucosamine may react at both ends of the dicarboxylic acid of the general formula (2), but the ¹ H-NMR spectrum of the obtained reaction product (in heavy dimethyl sulfoxide, at room temperature) From the integration ratio of the methylene signal adjacent to the 2.2 to 2.3 ppm carboxyl group and the methylene signal adjacent to the 2.1 ppm sugar amide, it can be confirmed that Intermediate-3 is obtained very selectively. ing.
Thus, according to the present invention, the desired intermediate-3 is obtained in a yield of about 25 to 65%.

(E) About Intermediate-3 Intermediate-3 of the present invention is a lipid molecule represented by the following general formula (3),
(In the formula, n represents an integer of 8 to 16. The wavy line of the hydroxyl group in the formula represents the 1-position hydroxyl group of 2-glucosamine represented by the wavy line in the formula, either α or β anomer, or an equilibrium mixture thereof. Represents.)
Non-patent document 15 describes typical n = 10,12 lipid molecules.
Intermediate-3 itself is also a carboxylic acid-based asymmetric biceps lipid molecule similar to the asymmetric biceps lipid molecule of the present invention, but only a mixture of nanotubes and fragments thereof can be obtained by self-assembly. As described in [0004] above, the asymmetric nanotubes could not be formed.
Intermediate-3 of the present invention has characteristic absorptions derived from sugar hydroxyl groups at 3700-3300 cm ⁻¹ in the infrared absorption spectrum, characteristic absorptions derived from oligomethylene chains at 2917-2922 cm ⁻¹ , and 2848-2852 cm ⁻¹ , 1675 ～1600Cm ^-1 in absorption derived from carboxyl group, absorption derived from amide carbonyl group 1640～1620Cm ^-1, indicating the absorption derived from the sugar skeleton 1100~1000cm ^-1.
Furthermore, in ¹ H-NMR (DMSO-d ₆ (deuterated dimethyl sulfoxide) and 1 drop D ₂ O, room temperature), δ value was 7.7 (β anomeric NH), 7.5 (α anomeric NH) , 4.9 (the 1st proton of the α anomer), 4.4 (the 1st proton of the β anomer), 3.2-3.7 (methine of the pyranose ring of the sugar, methylene, positions 2 to 6), 3.04-3.13 (methylene on the sugar pyranose ring, 6th position), 2.28 (methylene next to the carboxyl group of the α-form), 2.2 (methylene next to the carboxyl group of the beta-form), Since signals of 1 (methylene adjacent to the amide), 1.5 (methylene next to the amide) and 1.2 (other methylenes) can be observed, it can be identified as intermediate-3.

(1-4) Steps for obtaining the asymmetric bihead lipid molecule (Compound-1) of the present invention from Intermediate-3 via Intermediate-5 (a) Step for obtaining Intermediate-5 Intermediate-3 Is subjected to dehydration condensation with an oligoglycine ester of the following general formula (4) to obtain intermediate-5.
(In the formula, m represents an integer of 1 to 4. R is a residue of a compound having an alcoholic hydroxyl group, for example, a methyl group, an ethyl group, a tertiary butyl group, or a benzyl group.)
As the oligoglycine ester of the general formula (4), esters such as methyl ester, ethyl ester, tertiary butyl ester, and benzyl ester of glycine polymer such as glycine or glycylglycine are used. Ethyl ester or methyl ester is preferred because of the ease of alkaline hydrolysis.

Also in the reaction for dehydrating condensation of Intermediate-3 and the oligoglycine ester of the general formula (4), the same condensing agent as in the above (1-3) (a) can be used. Specifically, DMT-MM, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, dicyclohexylcarbodiimide, diisopropylcarbodiimide and the like can be used, but DMT-MM giving a high yield of condensate Alternatively, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is desirable. In the same reaction, dimethylformamide (DMF), dimethylacetamide, N-methylpyrrolidone, methanol, ethanol, isopropanol, dimethyl sulfoxide, and the like can be used, but DMF is preferred from the viewpoint of solubility of raw materials.
Similarly, in the condensation reaction, the reaction temperature is selected in the range of 0 to 50 degrees, and the reaction time is usually in the range of 10 minutes to 30 hours.
In order to wash the solid obtained by distilling off DMF after the condensation reaction to obtain Intermediate-5 and remove impurities as a dissolved component, methanol, ethanol, isopropanol, etc. are used. In order to suppress solubility, it is desirable to use methanol.

(A) Intermediate-5 As shown in the following general formula (5), intermediate-5 has a protective carboxyl group (methyl group, ethyl group, tertiary butyl) as shown in the following general formula (5). Group or benzyl group).
(In the formula, n represents an integer of 8 to 16, and m represents an integer of 1 to 4. R represents a residue of a compound having an alcoholic hydroxyl group, for example, a methyl group, an ethyl group, or a tertiary butyl group. (In addition, the 1-position hydroxyl group of 2-glucosamine represented by a wavy line in the formula represents either α, β anomer, or an equilibrium mixture thereof.)
In the above condensation reaction system, only the intermediate-5 has low solubility and is selectively precipitated, so that it is not necessary to purify it except for washing the precipitated solid after filtration. Higher purity can be achieved by reprecipitation by heating and cooling using methanol or water-containing methanol.

(C) Hydrolysis Step for Obtaining Target Compound-1 The asymmetric bihead lipid molecule (Compound-1) of the present invention can be easily obtained by deprotecting Intermediate-5 by hydrolysis. In the hydrolysis at that time, a methanol / water mixed solvent is preferable from the viewpoint of solubility and reactivity. The reaction temperature is selected in the range of 20 to 80 degrees, and the reaction time is usually in the range of 30 minutes to 6 hours. In addition, when R of intermediate-5 is a benzyl group, debenzyl ester in catalytic hydrogenation reduction using palladium on carbon as a catalyst in DMF or methanol, methanol / water, or the like may be used. When R is a tertiary butyl group, deesterification by acid treatment in DMF is also possible.
In addition, since the hydrolysis reaction proceeds quantitatively and almost no side reaction is observed, the target compound-1 can be obtained with high purity by neutralization by addition of an acid after the reaction. Purification by heating and cooling may be performed with methanol, ethanol, DMF, or the like.

(D) Identification of Asymmetric Double-headed Lipid Molecule (Compound-1) of the Present Invention The measured value of elemental analysis of such a compound agrees with the calculated value within an error range, and it is 3700-3300 cm ⁻¹ in the infrared absorption spectrum. characteristic absorption derived from sugar hydroxyl, 2917-2922Cm ^-1, and 2848-2852Cm ^-1 to the characteristic absorption derived from oligo methylene chain, absorption derived from carboxyl group 1675-1600Cm ^-1, glycine 1650～1620Cm ^-1 Absorption derived from an amide carbonyl group linked to a residue or glucosamine, and absorption derived from a sugar skeleton at 1100 to 1000 cm ⁻¹ are shown. Furthermore, in ¹ H-NMR (DMSO-d ₆ and 1 drop D ₂ O or DMSO-d ₆ only, room temperature), the δ value is around 8.2 (glycine NH), 7.6 (glucosamine NH), Around 4.9 (proton at the 1st position of the α anomer), around 4.4 (the 1st proton at the β anomer), around 3.2-3.7 (the pyranose ring of the sugar, positions 2 to 6, methylene of glycine) ), 3.0-3.1 (sugar pyranose ring, 6-position methylene), 2.2-2.1 (methylene next to the amide carbonyl group), 1.5 (two adjacent to the amide) Methylene), signal around 1.2 (other methylene) can be observed. From this, the said compound can be identified as the target compound-1.

2. About Asymmetric Nanotubes of the Present Invention (2-1) Asymmetric Nanotube Production Method of the Present Invention (A) Heat Dissolution / Cooling Method One of the methods for producing asymmetric nanotubes using the asymmetric double-headed lipid molecule of the present invention is Heat dissolution / cooling method for self-assembly of typical self-assembling lipid molecules can be applied.
The asymmetric bihead lipid molecule (0.5 mg to 5 mg) of the present invention is dispersed in 1 ml of pure water (such as Milli-Q water), heated and dissolved at 80 to 100 ° C., and then cooled (every 0.1 to 10 ° C. To obtain a hydrogel composed of asymmetric nanotubes. From the electron microscope observation at this time, an intertwined structure of nanotubes having a length of 10 μm or more has been confirmed.
Since the asymmetric nanotube of the present invention has high structural stability, it can withstand physical disturbance such as freeze-drying treatment or ultrasonic treatment. By these physical disturbance treatments, it is possible to convert into asymmetrical nanotubes with good yield and short axial ratio and with good dispersibility.
The form, size, etc. of the obtained asymmetric nanotube can be confirmed by dropping the dispersion on a grid for an electron microscope and staining the inside with a stain that is opaque to electron beams. In this way, an asymmetric nanotube having an outer diameter of about 15 nm, an inner diameter of about 8-9 nm, and a length of 100 nm-2 μm could be obtained.

(I) pH adjustment method The asymmetric bihead lipid molecule of the present invention is dispersed and dissolved in an equimolar to 2 equivalent aqueous sodium hydroxide solution at room temperature, and then an acid solution such as hydrochloric acid is equimolar to 2 etc. at room temperature. Asymmetric nanotubes can be obtained in the same manner by adding about an amount and adjusting the pH to about 5-6. As said hydroxide aqueous solution, hydroxide aqueous solutions, such as lithium, potassium, rubidium, cesium, can also be used besides sodium hydroxide. In addition to hydrochloric acid, phosphoric acid, formic acid, acetic acid, citric acid, ascorbic acid, sulfuric acid and the like can be used as the acid solution.
In addition, when using the salt (for example, sodium salt) etc. of an asymmetric bihead type lipid molecule as a raw material, although the said sodium hydroxide aqueous solution may be used, many acids are required for neutralization. For this reason, more preferably, the same salt is dispersed and dissolved in pure water (such as Milli-Q water) at room temperature, and then an arithmetic liquid such as hydrochloric acid is added at about room temperature to about 1.5 equivalents at room temperature as described above. It may be prepared similarly by adjusting the pH to about 5-6.
Even when this pH adjustment method is applied, it can be converted into an asymmetric nanotube with a short axial ratio by ultrasonic treatment, freeze-drying, etc., and the sizes are almost the same. Particularly in the latter case, the lipid concentration can be increased to about 10 mg / ml in a well-dispersed state depending on the amount of water added during rehydration. Further, it is possible to increase the concentration beyond that (for example, 50 mg / ml), but it changes into a gel.

(2-2) Stability of Asymmetric Nanotubes of the Present Invention The dispersion of asymmetric nanotubes thus obtained does not give a precipitate for about 6 months or more even when stored at room temperature or in a refrigerator at any of the above concentrations. It can be stored as a good dispersion. Furthermore, if the dispersion is freeze-dried, it can be stored at room temperature for one year or more, and a similar dispersion can be obtained by adding Milli-Q water or the like to rehydrate.

(2-3) Structure of the asymmetric nanotube of the present invention Confirmation of the tube-like structure can be made by visualizing the space in the tube by observation with an electron microscope after negative staining (Non-Patent Documents 1 and 10). That is, when a staining agent that does not transmit electron beams is introduced into the inner space of the tube by capillary action and a transmission image is obtained by observation with an electron microscope, the inner space of the tube in which the staining agent is distributed gives a dark image, and the wall of the tube Because it is made of organic material that allows easy transmission of lines, it gives a thin contrast. This can be confirmed.
In addition, the confirmation of the asymmetric nanotube structure (that is, the structure in which the carboxyl group is arranged on the inner surface of the tube or the 2-glucosamide group is arranged on the outer surface) is confirmed by the period d of the nanotube film thickness obtained from the powder X-ray diffraction pattern of the nanotube And the molecular length L estimated from the molecular model. That is, when the asymmetric double-headed lipid forms an asymmetric nanotube structure, it is known that the membrane period d and the molecular length L of the nanotube are substantially the same or d slightly smaller (Non-patent Documents 9 and 10). ). Therefore, as a result of comparing and examining d and L as described below, it was found that the above relationship was satisfied in any case (Table 1).
From the above, it was found that the organic nanotube of the present invention is an asymmetric nanotube.

<Table 1> Relationship between film period d obtained from XRD of asymmetric nanotube and molecular length L obtained from molecular model

  Although not described in Table 1, it was confirmed that when n = 14, 16 of Compound-1 of the present invention, the same tube form and size as in the case of n = 10, 12 were exhibited. In general, it has been clarified from previous studies that the longer the chain length, the easier it is to form an asymmetric nanotube structure (Non-patent Document 17). A series of lipid groups differing only in the molecular chain length can be obtained by self-assembly. It is also known that when the resulting nanotube structures have substantially the same form and size (inner diameter, outer diameter, wall thickness), the molecular arrangement is the same (Non-patent Document 10). Therefore, it is suggested that an asymmetric nanotube structure similar to the case of n = 10, 12 is formed also in the case of n = 14, 16.

3. Encapsulation in asymmetric nanotubes of the present invention (3-1) Regarding target substances that can be encapsulated Since the inside of the asymmetric nanotubes of the present invention is coated with carboxyl groups, the drug preferably included therein is carboxyl A compound having a cationic functional group such as an amino group capable of ion bonding with a group is preferable, and forms a stable cationic drug-asymmetric nanotube complex. In addition, since the inside of the tube is filled with a negative charge, the entire substance is a positively charged cationic substance, even if it is not a compound having a functional group capable of ionic bonding. In addition, if the substance is about 0.1 to 5 nm in size, it can be included, and it can be said that the asymmetric nanotube including the cationic substance also forms a cationic substance-asymmetric nanotube complex. .
As a specific cationic drug forming the cationic drug-asymmetric nanotube complex, anthracycline anticancer drugs such as doxorubicin, idarubicin, epirubicin, daunorubicin, pirarubicin, and other drugs having an amino group are particularly preferably used.
Also preferred are cationic drugs such as cisplatin, nedaplatin, carboplatin, oxaliplatin.
Moreover, it is applicable not only to such drugs having a pharmacological action as pharmaceuticals but also to various additives such as cationic emulsifiers, dispersants, stabilizers, humectants for cosmetics and foods.

(3-2) Encapsulation method into asymmetric nanotubes A cationic substance to be encapsulated dissolved or suspended in pure water (Milli Q water) to a concentration of 0.1 mg / mL to 50 mg / Equivalent amounts of the asymmetric nanotube dispersion of the invention (concentration: 0.1 mg / mL to 20 mg / mL) are mixed and left at room temperature for about 10 minutes to 3 hours. Thereby, since the encapsulation rate of about 30 to 95% can be achieved, the total amount of the composition for containing an effective amount can be kept low, for example, when used as a composition for an anticancer agent.
Moreover, the substance which was not encapsulated can be efficiently removed by filtering the mixed solution through a 200 nm porous membrane. As a result, only the tube encapsulating the cationic substance can be isolated as a solid, and the concentration can be adjusted by redispersing it in milli-Q water at an arbitrary concentration. The concentration can also be adjusted by dispersing a solid obtained by freeze-drying a tube encapsulating a cationic substance in milliQ water at an arbitrary concentration.
The encapsulation rate of the drug is quantified by encapsulating by the above method, filtering the nanotubes, and redispersing the nanotubes in MilliQ water to measure the UV absorption specific to the drug. be able to.
For example, in the case of doxorubicin, 200 μL of doxorubicin solution (dissolved in 2.0 mg, 1.0 mg, 0.714 mg, 0.625 mg, 0.555 mg, and 0.50 mg per mL of milliQ water) and a nanotube dispersion (200 μL, 5 mg / mL) and mixed and left at room temperature for 30 minutes. From this, 200 μL was taken, filtered through a 200 nm porous membrane, and further washed with milliQ water (400 μL). The solid (nanotube) separated by filtration was redispersed in 200 μL of milliQ water together with the filter paper. The encapsulation rate was determined by comparing the absorption of the solution before filtration and the solution after filtration by UV absorption (480 nm) of doxorubicin.
In the case of doxorubicin, the drug encapsulation rate of the present invention can be encapsulated at an extremely high concentration of about 62 to 95%.

(3-3) Release of Drugs from Nanotubes The drug encapsulated in the present invention changes the ionization state of the carboxyl group covering the inner surface of the tube or the tube structure itself in response to external stimuli such as pH change. The drug can be released selectively and efficiently by changing the shape.
Specifically, by adding a buffer solution to the dispersion of Milli-Q water and adjusting the pH value to about 4 to 6, the carboxyl group on the inner surface is protonated, so that the release can be accelerated. Moreover, it is possible to release slowly by adjusting the pH to about 7-8.
In addition, as an asymmetric nanotube of the present invention, a nanotube whose size is reduced to about 50 nm to 500 nm by ultrasonic treatment or the like is used, so that it can be applied to target cancer cells and tissues by an EPR (Enhanced Permiability and Retation) effect. It is possible to increase the amount delivered.
The amount of drug released from the nanotube is determined by sealing the tube dispersion in the inner aqueous phase of a dialysis membrane tube (fractionated molecular weight of about 1200 to 20000), and the drug released through the membrane and released into the outer aqueous phase is subjected to spectroscopy such as UV. It is possible to measure quantitatively by measuring by analysis.
For example, in the case of an asymmetric nanotube encapsulating doxorubicin (nanotube / Dox = 8/1) in a PBS buffer (pH 7.4), this is sealed in a dialysis membrane (molecular weight cut off 14000) and similarly in a PBS buffer. The amount of released doxorubicin in the outer aqueous phase was monitored by UV absorption while stirring in the outer aqueous phase of (pH 7.4).
In that case, a sustained release effect of about 10 to 12% per hour in PBS buffer (pH 7.4) and about 50% in 4 hours could be confirmed.
On the other hand, in the weakly acidic state (pH 5.5), rather than 33% per hour, 82% or more is released in the 4th hour, and almost all is released in 8 hours. It was confirmed that the release of the anticancer drug can be accelerated by the protonation of the carboxylate generated from the stimulation. The point that the release of the anticancer drug is accelerated under this low pH condition is considered to be extremely effective when the nanotube is used as a drug capsule. This is because cancer tissues and cancer cells generally have the property of being weakly acidic (low pH) compared to normal cells.

4). Encapsulated drug composition (4-1) Drug composition Since the asymmetric nanotube of the present invention has excellent properties as a drug carrier as described above, the asymmetric nanotube containing an effective amount of a therapeutic drug The complex can be used as an active ingredient to form a pharmaceutical composition together with a pharmacologically acceptable carrier. The pharmaceutical composition includes a pharmaceutical composition for preventing, treating or treating cancer such as an anticancer agent. In addition, since it is effective when it is desired to gradually release a drug in an aqueous environment, the drug composition includes a cationic drug composition formulated for cosmetics and foods.
Typical drugs as the drug in the capsule of the present invention are the above-mentioned anthracycline anticancer drugs such as doxorubicin, idarubicin, epirubicin, daunorubicin, pirarubicin and other drugs having an amino group. In addition, cationic drugs such as cisplatin, nedaplatin, carboplatin, and oxaliplatin can be used in the same manner.
Here, pharmaceutically acceptable carriers include diluents or excipients such as water or physiologically acceptable buffers.
The pharmaceutical composition of the present invention can be administered orally, such as a tablet, but can also be administered parenterally, such as intravenously or directly into the affected area, or an external preparation.
The dosage at the time of administering the pharmaceutical composition of the present invention to a patient can be appropriately determined according to the type of drug used, the age, weight and medical condition of the patient to be medically treated, the route of administration, etc. The effective amount of the drug per administration is 1 μg to 1 g, preferably 100 μg to 10 mg.

(4-2) Function of drug composition The drug-encapsulated product of the asymmetric nanotube of the present invention has a sustained release action that can be gradually released in the vicinity of neutrality, and an accelerated drug release effect under acidic conditions. Two effects can be expected. That is, in the case of cancer cells, two effects can be expected: a selective drug release effect in the vicinity of a slightly acidic cancer tissue, and a high anticancer activity expression by rapid drug release inside the cancer cell.
In the present invention, the term “cell” typically refers to mammalian cells including humans. In vitro cultured cells may be used, but in the case of drug treatment, tissues in organs, cells in organs, immune cells such as blood cells and lymphocyte cells in body fluids such as blood, and various cancers Cells are targeted.

(4-3) Measurement of anticancer activity As an example, the anticancer activity of an anticancer agent composition using an asymmetric nanotube encapsulating doxorubicin of the present invention was confirmed as follows.
For anticancer activity, asymmetric nanotubes encapsulating doxorubicin are added to Hela cells dispersed in the culture solution, and after 24 hours of incubation, the number of cells or their alternatives (intracellular protein content, mitochondrial activity, etc.) are measured. However, as a control experiment, it can be performed by comparing with a solution containing only doxorubicin. As a measurement method at that time, typically, a method of measuring the mitochondrial activity of a cell (MTT assay, Non-Patent Document 18) can be used.
In this example, asymmetric nanotubes encapsulating doxorubicin were added to the cells and cultured, followed by quantification according to the mitochondrial activity of the cells by MTS assay. Both the MTT assay and the MTS assay quantify the number of active cells by measuring the concentration of a dye called formazan produced by an enzyme in mitochondria in the cells. In the MTT assay, insoluble formazan is generated after the drug reaction, and therefore, the solution needs to be processed before the absorbance is measured. In contrast, the MTS assay does not produce insoluble formazan and does not require post-treatment, so that the operation is simpler and quicker.

EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited at all by these examples.
Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and various techniques used to implement the present invention include those that clearly indicate the source. Except for this, it can be easily and reliably carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, kit instruction manuals, catalogs, and the like.
In addition, the description content in the technical literature, the patent gazette, and the patent application specification cited in this specification shall be referred to as the description content of the present invention.

(Example 1) Synthesis of an asymmetric nanotube-forming asymmetric double-headed lipid molecule (Formula (1) Compound-1, n = 10, m = 2) Among the asymmetric double-headed lipid molecules represented by the general formula (1), Lipid molecules (compound-1 (n = 10, m = 2)) in the case of n = 10 and m = 2 were synthesized according to a synthesis method via intermediate-5 shown in the following reaction formula (1).
(In the reaction formula, “DMT-MM” is an abbreviation of “4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride” “MeONa” is an abbreviation for “sodium methoxide”.

(1-1) Synthesis of Intermediate-3 (n = 10) Dodecanedioic acid (11.9 g, 46 mmol) was dissolved in methanol (400 ml). To this solution was added D (+)-glucosamine hydrochloride (5 g, 23 mmol) and sodium methoxide (1.25 g, 23 mmol) dissolved in water (ml). DMT-MM (6.4 g, 23 mmol) dissolved in methanol (20 ml) was added and stirred at room temperature. While monitoring the reaction by TLC, DMT-MM (9.6 g, 35 mmol) and sodium methoxide (2.5 g, 46 mmol) were added and reacted for 30 hours. After completion of the reaction, the reaction solution was filtered, and the residue was washed with water and methanol. Reprecipitation was performed with ethanol and water 1/1 to obtain a product (5.79 g). Yield 25%.
FIG. 1 shows a chart of ¹ H-NMR (DMSO-d ₆ and 1 drop D ₂ O) after purification.
¹ H-NMR (DMSO-d ₆ and 1 drop D ₂ O) 7.68 (dd, J = 8.4 Hz, NH), 7.52 (d, J = 8.1 Hz, NH), 4.92 ( d, J = 3.4 Hz, 0.7H, H-1α), 4.43 (d, J = 8.1 Hz, 0.3H, H-1β), 3.24-3.69 (m, 5H) , 3.04-3.13 (m, 1H), 2.28 (t, J = 7.4Hz, 0.3H, -CH 2 -COOHα), 2.18 (t, J = 7.4Hz, 1 .7H, —CH ₂ COOHβ), 2.09 (t, J = 7.5 Hz, —CH ₂ —CONH, 2H), 1.47 (d, J = 6.8 Hz, —CH ₂ CH ₂ CO, 4H) ), 1.24 (s, 12H, -CH 2).

(1-2) Synthesis of Compound-1 (n = 10, m = 2)
Intermediate-3 (n = 10) (0.5 g, 1.2 mmol) obtained in (1-1) above was dissolved in DMF by heating, cooled to room temperature, and then glycylglycine hydrochloride methyl ester (0. 22 g, 1.23 mmol), DMT-MM (0.44 g, 1.35 mmol) and triethylamine (1.25 mmol) were added. After stirring at room temperature for 2 hours, DMF was distilled off. Methanol was added to the obtained solid, and an insoluble white solid was filtered off to obtain Intermediate-5 (n = 10, m = 2). This was dispersed in a heated methanol / water solvent and hydrolyzed by adding an aqueous sodium hydroxide solution (1.5 ml, 1 mol / L) at 60 ° C. After the reaction, the solvent was distilled off, methanol was added to disperse the solid, hydrochloric acid was added to adjust the pH to 4, and filtration was performed to obtain the target compound-1 as a white solid (0 .35 g, 58%: from Intermediate-3 (n = 10)). A chart of ¹ H-NMR (DMSO-d ₆ and 1 drop D ₂ O) after purification is shown in FIG.

Elemental analysis _{(as C 22 H 39 N 3 O 10} H 2 O)
C H N
Calculated value (%) 50.47 7.89 8.03
Actual value (%) 50.59 7.75 7.9

Example 2 Synthesis of Asymmetric Nanotube Forming Asymmetric Double-head Lipid Molecule (Formula (1) Compound-1, n = 10, m = 3) Intermediate-3 obtained in Example (1-1) ( n = 10) (0.5 g, 1.2 mmol), glycylglycylglycine hydrochloride methyl ester (0.29 g, 1.23 mmol), DMT-MM (0.44 g, 1.35 mmol), triethylamine (1.25 mmol) ) Was added. After stirring at room temperature for 2 hours, DMF was distilled off. Methanol was added to the obtained solid, and an insoluble white solid was filtered off to obtain Intermediate-5 (n = 10, m = 3). This was dispersed in a heated methanol / water solvent and hydrolyzed by adding an aqueous sodium hydroxide solution (1.5 ml, 1 mol / L) at 60 ° C. After the reaction, the solvent was distilled off, methanol was added to disperse the solid, hydrochloric acid was added to adjust the pH to 4, and filtration was performed to obtain the target compound-1 as a white solid (0 .43 g, 63%: from Intermediate-3 (n = 10)). FIG. 3 shows a chart of ¹ H-NMR (DMSO-d ₆ and 1 drop D ₂ O) after purification.

Elemental analysis _{(as C 24 H 42 N 4 O 11} H 2 O)
C H N
Calculated value (%) 51.05 7.85 9.92
Actual value (%) 50.93 7.95 9.88

Example 3 Synthesis of Asymmetric Nanotube Forming Asymmetric Double-head Lipid Molecule (Formula (1) Compound-1, n = 10, m = 4) Intermediate-3 obtained in Example (1-1) ( n = 10) (0.5 g, 1.2 mmol), glycylglycylglycylglycine hydrochloride methyl ester (0.65 g, 1.23 mmol), DMT-MM (0.44 g, 1.35 mmol), triethylamine (1 .25 mmol) was added. After stirring at room temperature for 2 hours, DMF was distilled off. Methanol was added to the obtained solid, and an insoluble white solid was filtered off to obtain Intermediate-5 (n = 10, m = 4). This was dispersed in a heated methanol / water solvent and hydrolyzed by adding an aqueous sodium hydroxide solution (1.5 ml, 1 mol / L) at 60 ° C. After the reaction, the solvent was distilled off, methanol was added to disperse the solid, hydrochloric acid was added to adjust the pH to 4, and filtration was performed to obtain the target compound-1 as a white solid (0 .48 g, 65%: from Intermediate-3 (n = 10)). FIG. 4 shows a chart of ¹ H-NMR (DMSO-d ₆ and 1 drop D ₂ O) after purification.

Elemental analysis _{(as C 26 H 45 N 5 O 12} H 2 O)
C H N
Calculated value (%) 50.3948.97 7.43 10.98
Actual value (%) 48.75 7.11 11.15

Example 4 Synthesis of Asymmetric Nanotube Forming Asymmetric Double-head Lipid Molecule (Formula (1) Compound-1, n = 12, m = 2) (4-1) Synthesis of Intermediate-3 (n = 12) Tetradecane Diacid (4.8 g, 18 mmol) was dissolved in methanol (200 ml). Thereafter, an aqueous sodium hydroxide solution (0.4 mol / L) was added in 100 ml of water. To this solution was added D (+)-glucosamine hydrochloride (2 g, 9.2 mmol). DMT-MM (3.1 g, 9.5 mmol) was added thereto, and the mixture was stirred at room temperature for 30 hours. After completion of the reaction, the reaction solution was filtered, and the residue was washed with water and methanol. Since the pH was not adjusted to 4, reprecipitation was performed with ethanol and water 1/1 to obtain a product (2.2 g). Yield 64%. FIG. 5 shows a chart of ¹ H-NMR (DMSO-d ₆ and 1 drop D ₂ O) after purification.

¹ H-NMR (DMSO-d ₆ and 1 drop D ₂ O) 7.68 (dd, J = 8.4 Hz, NH), 7.52 (d, J = 8.1 Hz, NH), 4.92 ( d, J = 3.4 Hz, 0.7H, H-1α), 4.43 (d, J = 8.1 Hz, 0.3H, H-1β), 3.24-3.69 (m, 5H) , 3.04-3.13 (m, 1H), 2.28 (t, J = 7.4Hz, 0.3H, -CH 2 -COOHα), 2.18 (t, J = 7.4Hz, 1 .7H, —CH ₂ COOHβ), 2.09 (t, J = 7.5 Hz, —CH ₂ —CONH, 2H), 1.47 (d, J = 6.8 Hz, —CH ₂ CH ₂ CO, 4H) ), 1.24 (s, 16H, -CH 2).

(4-2) Synthesis of Compound-1 (n = 12, m = 2) Intermediate-3 (n = 12) (0.4 g, 0.95 mmol) obtained in the above (4-1) After heating and dissolving in DMF and cooling to room temperature, glycylglycine hydrochloride methyl ester (200 mg, 1.0 mmol), DMT-MM (340 mg, 1.1 mmol), and triethylamine (1.0 mmol) were added. After stirring at room temperature for 2 hours, DMF was distilled off. Methanol was added to the obtained solid, and an insoluble white solid was filtered off to obtain Intermediate-5 (n = 12, m = 2).
This was dispersed in a heated methanol / water solvent, and hydrolyzed by adding an aqueous sodium hydroxide solution (1 mol / L, 1.5 ml) at 60 ° C. After the reaction, the solvent was distilled off, methanol was added to disperse the solid, hydrochloric acid was added to adjust the pH to 4, and the target compound-1 (n = 12, m = 2) was filtered. ) Was obtained as a white solid (355 mg, 70%). FIG. 6 shows a chart of ¹ H-NMR (DMSO-d ₆ and 1 drop D ₂ O) after purification.

Elemental analysis _{(as C 24 H 43 N 3 O 10} H 2 O)
C H N
Calculated value (%) 52.26 8.22 7.64
Actual value (%) 52.29 8.18 7.48

Example 5 Synthesis of Asymmetric Nanotube Forming Asymmetric Double-head Lipid Molecule (Formula (1) Compound-1, (n = 12, m = 3)) Intermediate obtained in Example (4-1) 3 (n = 12) (0.2 g, 0.48 mmol) was dissolved in DMF by heating, cooled to room temperature, and then glycylglycylglycine hydrochloride (114 mg, 0.52 mmol) and DMT-MM (170 mg, 170 mg, 0.52 mmol) and triethylamine (0.52 mmol) were added. After stirring at room temperature for 2 hours, DMF was distilled off. Methanol was added to the obtained solid, and an insoluble white solid was filtered off to obtain Intermediate-5 (n = 12, m = 3).
This was dispersed in a heated methanol / water solvent and hydrolyzed by adding an aqueous sodium hydroxide solution (1 mol / L, 0.75 ml) at 60 ° C. After the reaction, the solvent was distilled off, methanol was added to disperse the solid, hydrochloric acid was added to adjust the pH to 4, and the target compound-1 (n = 12, m = 3) was filtered. ) Was obtained as a white solid (210 mg, 68%). FIG. 7 shows a chart of ¹ H-NMR (DMSO-d ₆ ) after purification.

Elemental analysis _{(as C 26 H 46 N 4 O 11} H 2 O)
C H N
Calculated value (%) 51.30 7.95 9.20
Actual value (%) 51.15 7.99 9.05

Example 6 Synthesis of Asymmetric Nanotube Forming Asymmetric Double-head Lipid Molecule (Formula (1) Compound-1, (n = 12, m = 4)) Intermediate Obtained in Example (4-1) 3 (n = 12) (0.4 g, 0.95 mmol) was dissolved in DMF by heating, cooled to room temperature, and then glycylglycine hydrochloride ethyl ester (200 mg, 1.0 mmol) and DMT-MM (340 mg, 1 0.0 mmol), triethylamine (1.0 mmol) was added. After stirring at room temperature for 2 hours, DMF was distilled off. Methanol was added to the obtained solid, and an insoluble white solid was filtered off to obtain Intermediate-5 (n = 12, m = 4).
This was dispersed in a heated methanol / water solvent, and hydrolyzed by adding an aqueous sodium hydroxide solution (1.5 mol / L, 1 ml) at 60 ° C. After the reaction, the solvent was distilled off, methanol was added to disperse the solid, hydrochloric acid was added to adjust the pH to 4, and the target compound-1 (n = 12, m = 4) was filtered. ) As a white solid (357 mg, 58%). FIG. 8 shows a chart of ¹ H-NMR (DMSO-d ₆ and 1 drop D ₂ O) after purification.

Elemental analysis _{(as C 28 H 49 N 5 O 12} H 2 O)
C H N
Calculated Value (%) 49.18 7.81 10.24
Actual value (%) 49.09 7.63 10.37

Example 7 Synthesis of Asymmetric Nanotube Forming Asymmetric Double-head Lipid Molecule (Formula (1) Compound-1 (n = 14, m = 3) (7-1) Synthesis of Intermediate-3 (n = 14) Hexadecane Diacid (1.3 g, 4.6 mmol) was dissolved in methanol (90 ml) and to this solution was added D (+)-glucosamine hydrochloride (0.5 g, 2.3 mmol) and sodium dissolved in water (2 ml). Methoxide (0.125 g, 2.3 mmol) was added DMT-MM (0.641 g, 2.3 mmol) dissolved in methanol (1 ml) was added and stirred at room temperature, while the reaction was monitored by TLC, DMT-MM (0.96 g, 3.5 mmol) and sodium methoxide (0.25 g, 4.6 mmol) were added and reacted for 29 hours After completion of the reaction, the reaction solution was filtered and the residue was mixed with water and methanol. Washed. Ethanol, reprecipitated with water 1/1, product (0.34 g) was obtained. Yield 33%. ¹ after purification H-NMR _{(DMSO-d 6} and 1 drop of _D 2 O) The chart is shown in FIG.

¹ H-NMR (DMSO-d ₆ and 1 drop D ₂ O) 7.70 (dd, J = 8.5 Hz, NH), 7.53 (d, J = 8.0 Hz, NH), 4.92 ( d, J = 3.3 Hz, 0.7H, H-1α), 4.43 (d, J = 8.2 Hz, 0.3H, H-1β), 3.24-3.69 (m, 5H) , 3.03-3.14 (m, 1H), 2.28 (t, J = 7.3 Hz, 0.3 H, —CH ₂ —COOHα), 2.18 (t, J = 7.3 Hz, 1 .7H, —CH ₂ COOHβ), 2.10 (t, J = 7.6 Hz, —CH ₂ —CONH, 2H), 1.48 (d, J = 6.8 Hz, —CH ₂ CH ₂ CO, 4H) ), 1.24 (s, 20H, -CH 2).

(7-2) Synthesis of asymmetric bihead lipid molecule (Compound-1 of formula (1) -1 (n = 14, m = 3)) Intermediate-3 (n = 14) obtained in (7-1) above (400 mg, 0.9 mmol) was dissolved in DMF by heating and cooled to room temperature, and then glycylglycylglycine hydrochloride (250 mg, 1.0 mmol), DMT-MM (340 mg, 1.1 mmol), triethylamine ( 1.0 mmol) was added. After stirring at room temperature for 2 hours, DMF was distilled off. Methanol was added to the obtained solid, and an insoluble white solid was filtered off to obtain Intermediate-5 (n = 12, m = 3).
This was dispersed in a heated methanol / water solvent and hydrolyzed by adding an aqueous sodium hydroxide solution (1 mol / L, 1.5 ml) at 80 ° C. After the reaction, the solvent was distilled off, methanol was added to disperse the solid, hydrochloric acid was added to adjust the pH to 4, and the target compound-1 (n = 14, m = 3) was filtered. ) As a white solid (460 mg, 78%). A chart of ¹ H-NMR (DMSO-d ₆ ) after purification is shown in FIG.

Elemental analysis value (as C ₂₈ H ₅₀ N ₄ O ₁₁ H ₂ O)
C H N
Calculated value (%) 52.82 8.23 8.80
Actual value (%) 52.99 8.09 8.62

Example 8 Synthesis of Asymmetric Nanotube Forming Asymmetric Double-headed Lipid Molecule (Compound-1 of Formula (1) -1 (n = 14, m = 4)) Intermediate obtained in Example (7-1) 3 (n = 14) (0.1 g, 0.22 mmol) was dissolved in DMF by heating, cooled to room temperature, and then glycylglycylglycylglycine hydrochloride ethyl ester (61 mg, 0.23 mmol) and DMT-MM (68 mg, 0.24 mmol) was added. After stirring at room temperature for 19 hours, DMF was distilled off. Methanol was added to the obtained solid, and an insoluble white solid was filtered off to obtain Intermediate-5 (n = 14, m = 4).
The obtained intermediate-5 (n = 14, m = 4) (50 mg, 0.07 mmol) was dispersed in a heated methanol / water solvent, and an aqueous sodium hydroxide solution (0.1 mol / L, 2.7 ml) was added. Hydrolysis was performed by adding at 65 ° C. After adjusting the pH to 4 by adding hydrochloric acid, the solvent was distilled off. Methanol was added and the insoluble white solid was filtered off to obtain the target compound-1 (n = 14, m = 4) as a white solid (25 mg, 41%). FIG. 11 shows a chart of ¹ H-NMR (DMSO-d ₆ and 1 drop D ₂ O) after purification.
¹ H-NMR (DMSO-d ₆ and 1 drop D ₂ O, room temperature) of compound-1 (n = 14, m = 4) obtained in this example 4.91 (d, J = 3.3 Hz, 0.7H, H-1α), 4.42 (d, J = 8.2 Hz, 0.3H, H-1β), 3.34-3.76 (m, 13H), 3.04-3.14. (M, 1H), 2.28 (t, J = 7.3 Hz, 0.3 H, —CH ₂ —COOHα), 2.07-2.14 (m, 3.7 H, —CH ₂ COOHβ, —CH _{2 -CONH), 1.47 (d,} J = 6.8Hz, -CH 2 CH 2 CO, 4H), 1.23 (s, 20H, -CH 2).

Elemental analysis _{(as C 30 H 53 N 5 O 12} H 2 O)
C H N
Calculated value (%) 51.94 7.99 10.09
Actual value (%) 51.88 8.12 10.01

Example 9 Formation of Asymmetric Nanotubes by Self-Assembly of Asymmetric Bihead Lipid Molecule (Compound-1) First, among the asymmetric bihead lipid molecules of the present invention produced in Example 1, Compound-1 (n = 10, m = 2), the following two methods: (1) Dissolution by heating, followed by cooling, and (2) Adjusting pH from sodium salt at room temperature. Asymmetric nanotubes were formed.
(9-1) Method by Heating / Cooling Method Compound-1 (n = 10, m = 2) (5 mg) was dispersed in water (Milli Q water, 1 ml) and dissolved by heating (about 100 ° C.). The solution was cooled to room temperature to obtain a transparent gel-like material containing nanotubes. After negative staining with phosphotungstic acid, a nanotube structure having an outer diameter of about 15 nm, an inner diameter of about 8 nm, and a length of 5 μm or more was confirmed by scanning electron microscope observation (transmission mode) (FIG. 12A).

(9-2) Method by pH adjustment method Compound-1 (n = 10, m = 2) (5 mg) was dispersed in water (Milli-Q water, 1 ml). At room temperature, an aqueous solution of sodium hydroxide (1 mol / L, 0.01 mL) was added to the solution to dissolve, and further an aqueous hydrochloric acid solution (1 mol / L, 0.01 mL) was added to adjust the pH to 5. A dispersion containing was obtained. After negative staining with phosphotungstic acid, a nanotube structure having an outer diameter of about 15 nm, an inner diameter of about 8 nm, and a length of 1 μm or more was confirmed by observation with a scanning electron microscope (transmission mode) (FIG. 12B).

(9-3) Confirmation of Asymmetric Nanotube Formability in Other Asymmetric Double-headed Lipid Molecules Furthermore, the above-mentioned (9-1) and (9- It was confirmed that a nanotube structure of the same size was formed by self-assembly by the same method as in 2).
FIG. 4 shows electron microscope observation images (transmission images) of asymmetric nanotubes obtained by heating and cooling of compound-1 produced in Example 1 (in the case of n = 10, m = 2) and self-assembly by a pH adjustment method, respectively. 12A and B.
FIG. 4 shows electron microscope observation images (transmission images) of asymmetric nanotubes obtained by heating and cooling of compound-1 produced in Example 2 (in the case of n = 10, m = 3) and self-assembly by a pH adjustment method, respectively. Shown in 13A and B.
FIG. 4 shows electron microscope observation images (transmission images) of asymmetric nanotubes obtained by heating and cooling of compound-1 produced in Example 3 (in the case of n = 10, m = 4) and self-assembly by a pH adjustment method, respectively. Shown in 14A and B.
Electron microscope observation images (transmission images) of the asymmetric nanotubes obtained by heating and cooling of the compound-1 produced in Example 4 (in the case of n12, m = 2) and self-assembly by the pH adjustment method are shown in FIG. 15A and FIG. Shown in B. Also, the inset shows that the tube is intertwined to give a well-dispersed transparent gel, indicating that the tube is highly dispersible.
FIG. 4 shows electron microscope observation images (transmission images) of asymmetric nanotubes obtained by heating and cooling of compound-1 produced in Example 5 (in the case of n = 12, m = 3) and self-assembly by a pH adjustment method, respectively. Shown in 16A and B.
Electron microscope observation images (transmission images) of the asymmetric nanotubes obtained by self-assembly of the compound-1 produced in Example 6 (in the case of n = 12, m = 4) by the pH adjustment method are shown in FIGS. 17A and 17B, respectively. Shown in
FIG. 18 shows an electron microscope observation image (transmission image) of the asymmetric nanotube obtained by self-assembly of the compound-1 produced in Example 7 (in the case of n = 14, m = 3) by the pH adjustment method.

(Example 10) Shortening of asymmetric nanotube and freeze-drying (10-1) Shortening of asymmetric nanotube In Example (9-3), Compound-1 (n = 12, m = 4) was heated and cooled. The asymmetric nanotube formed by applying the method was subjected to ultrasonic treatment to obtain a shortened nanotube. That is, the solution obtained by treating the nanotube solution (5 mg / mL) with a probe-type sonicator at 4 ° C. for 1 minute was a transparent dispersion. When this solution was observed with a scanning electron microscope, it was found that short nanotubes having a length of 100 to 500 nm were obtained. Furthermore, when the time was 2 minutes, the length was shortened to 50 to 200 nm. Further, it was confirmed that the nanotube structure was stable even when the dispersion obtained by the same treatment was stored at 4 degrees for 60 days or more. FIG. 19 shows an electron microscope observation image after ultrasonic treatment of an asymmetric nanotube obtained from Compound-1 (n = 12, m = 4) obtained by heating and cooling. The inset shows the nanotube dispersion prepared by heating and cooling.

(10-2) Freeze-drying of asymmetric nanotube Here, in order to show that the asymmetric nanotube of the present invention can be stored for a long time by freeze-drying, ultrasonic treatment of the asymmetric nanotube obtained in (10-1) above The product was freeze-dried and then rehydrated to obtain an asymmetric nanotube dispersion.
Specifically, the sonicated product of the asymmetric nanotube obtained in (10-1) above was freeze-dried and kept at room temperature for 30 days, and then rehydrated by adding milli-Q water to a high concentration. A dispersion (5 mg / mL) was obtained (FIG. 21). This dispersion was confirmed to be stable at 4 degrees for 60 days or more. FIG. 20 shows an electron microscope observation image after ultrasonication, lyophilization, and rehydration of an asymmetric nanotube obtained from Compound-1 (n = 12, m = 4) obtained by heating and cooling.

Example 11 Drug Encapsulation Experiment Using the asymmetric nanotube produced in Example 10 above, doxorubicin was encapsulated by the following experiment.
200 μL of doxorubicin solution (dissolved in 2,1,0.714, 0.625, 0.555, 0.5 mg / mL of milli-Q water) and a dispersion of the asymmetric nanotube (200 μL, 5 mg / mL) Mix and leave at room temperature for 30 minutes. Take 200 μL, filter through a 200 nm porous membrane, and wash with milliQ water (400 μL). The solid was redispersed with 200 μL of milli-Q water together with the filter paper. The encapsulation rate was determined by comparing the absorption of the solution before filtration and the solution after filtration by UV absorption (480 nm) of doxorubicin. FIG. 21 shows an electron microscope observation image of the asymmetric nanotube after encapsulation of doxorubicin and its encapsulation rate.
As a result, as shown in FIG. 21 (B), when ONT / DOX = 10/1 to 7/1, 90% or more is encapsulated. It was done. Further, as is clear from the image of FIG. 21A, the shape of the nanotubes is not changed even after doxorubicin is encapsulated. And the aggregation in a solution was not recognized but it also confirmed that the high dispersibility is maintained.

(Example 12) Drug Release Experiment Next, the release experiment of doxorubicin from the asymmetric nanotube in which doxorubicin was encapsulated in the above (Example 11) was performed by dialysis in PBS buffer (pH 7.4 and pH 5.5). Went by. As a result, as shown in FIG. 22, in the vicinity of neutrality (pH 7.4), about 62% of the total encapsulated amount was finally released after 10 hours, and the released amount was 70% of the whole even after 32 hours. However, in the weakly acidic state (pH 5.5), 82% or more was released in the 4th hour, and almost the entire amount was released in the 8th hour, covering the inner surface of the asymmetric nanotube. This indicates that the protonation of the carboxylate generated from the carboxyl group can be stimulated to accelerate the release of the anticancer agent. The point that the release of an anticancer drug is accelerated under this low pH condition is that, in general, cancer tissue and cells have the property of being acidic (low pH) compared to normal cells. It can be expected that the effectiveness of using nanotubes as drug capsules is considered extremely high.

(Example 13) Functional as an anticancer agent composition Hela cells were used to encapsulate doxorubicin-encapsulated asymmetric nanotubes (in the case of compound-1 where n = 12, m = 4. Encapsulation was performed with ONT / DOX = 7/1) In contrast, cytotoxicity (anticancer activity) to Hela cells was examined by MTS assay. As a result, it was found that the activity was higher than that of free doxorubicin as shown in FIG. This is because asymmetric nanotubes encapsulating doxorubicin are taken up by endosomes in cancer cells and exposed to a low pH environment peculiar to endosomes, so that the carboxylate on the inner surface of the tube is protonated and doxorubicin is released rapidly. Conceivable.
Thus, the cationic anticancer agent encapsulation product such as doxorubicin by the asymmetric nanotube of the present invention has a selective drug release effect in the vicinity of a slightly acidic cancer tissue as described in Example 12, and this Example Two effects of high anticancer activity due to rapid drug release in cancer cells can be expected. That is, it was shown that the cationic anticancer agent encapsulated product of the asymmetric nanotube of the present invention has an excellent function as an anticancer agent composition.

Claims (8)

An asymmetric bihead lipid molecule represented by the following general formula (1) or a salt thereof;
(In the formula, n is an integer of 8 to 16, and m is an integer of 1 to 4. In addition, the 1-position hydroxyl group of 2-glucosamine represented by a wavy line in the formula is either α or β anomer, or Represents the equilibrium mixture). Α, ω-long-chain dicarboxylic acid HOOC- (CH ₂ ) _n —COOH represented by the following general formula (2)
(In the formula, n represents an integer of 8 to 16.)
Is condensed with 2-glucosamine in aqueous methanol, and the resulting solid is filtered and washed, whereby intermediate-3 represented by the following general formula (3)
(In the formula, n represents an integer of 8 to 16. In addition, the 1-position hydroxyl group of 2-glucosamine represented by a wavy line in the formula represents either α or β anomer, or an equilibrium mixture thereof.)
Manufacture and
Intermediate-3 and an oligoglycine ester represented by the following general formula (4) in which the C-terminus of oligoglycine is protected with an ester.
(In the formula, m represents an integer of 1 to 4. R is a methyl group, an ethyl group, a tertiary butyl group, or a benzyl group.)
Intermediate 5 represented by the following general formula (5)
(In the formula, n represents an integer of 8 to 16, and m represents an integer of 1 to 4. R represents a methyl group, an ethyl group, a tertiary butyl group, or a benzyl group. (The 1-position hydroxyl group of 2-glucosamine shown represents either α or β anomer, or an equilibrium mixture thereof.)
And is deprotected by alkaline hydrolysis to obtain an asymmetric bihead lipid molecule represented by the following general formula (1) or a salt thereof
(In the formula, n is an integer of 8 to 16, and m is an integer of 1 to 4. In addition, the 1-position hydroxyl group of 2-glucosamine represented by a wavy line in the formula is either α or β anomer, or Represents the equilibrium mixture.)
How to manufacture.   An asymmetric nanotube formed from the asymmetric bihead lipid molecule according to claim 1 or a salt thereof, wherein the inner surface is coated with a carboxyl group and the outer surface is coated with a 2-glucosamide group.   An asymmetric nanotube characterized in that the asymmetric bihead lipid molecule according to claim 1 is dispersed in water, heated and then cooled, or dispersed and dissolved in water or an alkaline solution at room temperature, and then neutralized with an acid. Production method.   5. The method for producing an asymmetric nanotube according to claim 4, wherein the tube is formed into a short tube shape by performing freeze-drying treatment or ultrasonic treatment.   A cationic drug-asymmetric nanotube complex, wherein the asymmetric nanotube of claim 3 includes a cationic drug.   A cationic drug composition comprising the cationic drug-asymmetric nanotube complex according to claim 6 as an active ingredient.   The cationic drug composition according to claim 7, wherein the cationic drug is an anthracycline anticancer drug having an amino group.