JPWO2017154767A1 - Organic nitroxide radical compound, production method thereof, nanoemulsion particle and use thereof - Google Patents

Abstract

To provide a nanoemulsion particle that can be used as a metal-free contrast agent such as MRI, and an organic nitroxide radical compound capable of easily preparing the nanoemulsion particle. The organic nitroxide radical compound represented by the formula (1) (wherein R1 represents a linear alkyl group having 10 to 26 carbon atoms), and the organic nitroxide radical compound and the nonionic surfactant are 0. Nanoemulsion particles composed of a molar ratio of 8: 1 to 1.2: 1 and encapsulating nitroxide radicals.

Description

  The present invention relates to an organic nitroxide radical compound, a production method thereof, nanoemulsion particles, and use thereof.

  Currently, gadolinium preparations such as Gd-DTPA are used as intravascularly administered contrast agents used in magnetic resonance imaging (MRI). However, since gadolinium preparations are known to have side effects, it is desirable to use metal-free contrast agents that do not contain metals.

Since organic nitroxide radicals have paramagnetism, their application as metal-free contrast agents such as MRI has recently been studied. For example, in Non-Patent Documents 1 and 2, an amphiphilic block copolymer polymer compound in which an organic nitroxide radical called TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl) is covalently bonded is synthesized. It was disclosed that core / shell nanoparticles were prepared in water using
Patent Document 1 discloses a polymer having a nitroxide radical in the side chain and a polymer micelle comprising the polymer. However, in any case, the synthesis and purification of the polymer compound is complicated, the particle size distribution is wide, and the nitroxide radical is easily reduced by vitamin C or the like in the blood, so that the function of the radical cannot be exhibited. is there.

International Publication No. 2009/133647

X. Zhuang et al., Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 48, 5404-5410 (2010) T. Yoshitomi et al., Biomacromolecules, 10, 596-601 (2009)

  Under such circumstances, the main problem to be solved by the present invention is to provide nanoemulsion particles that can be used as a metal-free contrast agent such as MRI, and organic nitroxide radical compounds that can easily prepare the nanoemulsion particles. It is to be.

  As a result of intensive studies to develop nanoemulsion particles that can be used as a metal-free contrast agent, the present inventors have determined that a specific ratio of an organic nitroxide radical compound having a specific structure and a nonionic surfactant in an aqueous solvent. It was found that the nanoemulsion particles encapsulating nitroxide radicals can be produced by mixing with, thereby solving the above problems. The present invention has been completed based on such findings.

That is, this invention relates to the organic nitroxide radical compound, manufacturing method, nanoemulsion particle, etc. which are shown to the following items 1-7.
Item 1. Formula (1):

(In the formula, R ¹ represents a linear alkyl group having 10 to 26 carbon atoms.)
An organic nitroxide radical compound represented by:
Item 2. A method for producing an organic nitroxide radical compound according to item 1, wherein the formula (2):

(In the formula, R ¹ represents a linear alkyl group having 10 to 26 carbon atoms.)
The manufacturing method which oxidizes the compound represented by these in presence of a bivalent copper compound.
Item 3. A nanoemulsion particle in which the organic nitroxide radical compound according to Item 1 and the nonionic surfactant are included in a molar ratio of 0.8: 1 to 1.2: 1 and the nitroxide radical is included.
Item 4. Item 4. The nanoemulsion particle according to Item 3, wherein the nonionic surfactant is a nonionic surfactant having a linear alkyl group having 10 to 26 carbon atoms.
Item 5. The nonionic surfactant is represented by the formula (3):

(In the formula, R ² represents a linear alkyl group having 10 to 26 carbon atoms. N is an integer of 2 or more.)
Item 5. The nanoemulsion particle according to Item 3 or 4, which is a polyoxyethylene alkyl ether represented by:
Item 6. The contrast agent for MRI containing the nano emulsion particle of any one of said items 3-5.
Item 7. 6. A drug delivery system carrier comprising the nanoemulsion particles according to any one of items 3 to 5.

  The organic nitroxide radical compound of the present invention is a novel compound not described in any literature, which was first synthesized by the present inventors. By mixing the organic nitroxide radical compound with a nonionic surfactant at a specific ratio in an aqueous solvent, nanoemulsion particles encapsulating the nitroxide radical can be easily produced.

This nanoemulsion particle is an amphiphilic particle with the outside of the molecule being hydrophilic and the inside of the molecule being hydrophobic, and has magnetism due to the inclusion of nitroxide radicals inside the molecule. It can be suitably used as a contrast agent.
Moreover, the nanoemulsion particles of the present invention are extremely stable in an aqueous solvent and are not easily reduced by vitamin C or the like. Furthermore, since a hydrophobic substance such as an anticancer agent can be encapsulated inside the molecule, it can also be used as a drug delivery system carrier.

It is a schematic diagram of the nested structure which a compound (1) and a nonionic surfactant form. The result of EPR and DLS measured 1 day after the nanoemulsion particles obtained in Example 1 is shown. The result of EPR and DLS measured 3 days after the nanoemulsion particles obtained in Example 1 is shown. The result of EPR and DLS measured 5 days after the nanoemulsion particles obtained in Example 1 is shown. The result of EPR and DLS measured 7 days after the nanoemulsion particles obtained in Example 1 is shown. The result of EPR and DLS measured 1 day after the nanoemulsion particles obtained in Comparative Example 1 is shown. The result of EPR and DLS measured 3 days after the nanoemulsion particles obtained in Comparative Example 1 is shown. The result of EPR and DLS measured 5 days after the nanoemulsion particles obtained in Comparative Example 1 is shown. The result of EPR and DLS measured 7 days after the nanoemulsion particles obtained in Comparative Example 1 is shown. The result of EPR and DLS measured 1 day after the nanoemulsion particles obtained in Comparative Example 2 is shown. The result of EPR and DLS measured 3 days after the nanoemulsion particles obtained in Comparative Example 2 is shown. The result of EPR and DLS measured 5 days after the nanoemulsion particles obtained in Comparative Example 2 is shown. The result of EPR and DLS measured 7 days after the nanoemulsion particles obtained in Comparative Example 2 is shown. The result of EPR and DLS before dilution of the nanoemulsion particles obtained in Example 1 and after 10-fold dilution is shown. The EPR and DLS results measured one day after nanoemulsion particles prepared in a phosphate buffered aqueous solution are shown. The EPR and DLS results measured 3 days after the nanoemulsion particles prepared in phosphate buffered water are shown. The EPR and DLS results measured after 4 days for nanoemulsion particles prepared in an aqueous phosphate buffer solution are shown. The EPR and DLS results measured after 7 days of nanoemulsion particles prepared in phosphate buffered water are shown. It is a graph which shows the stability with respect to ascorbic acid of the nanoemulsion particle | grains prepared in the phosphate buffer aqueous solution. It is an absorption spectrum of only phosphate buffer aqueous solution (Only (-)-PBS). It is an absorption spectrum of a supernatant solution (Pyrene / (−)-PBS) after adding pyrene to an aqueous phosphate buffer solution. The absorption spectrum of the micelle (Pyrene / 5 / (-)-PBS) containing only pyrene and the result of DLS are shown. The absorption spectrum of the nanoemulsion particle (Pyrene / (±) -10c / 5 / (−)-PBS) encapsulating Compound 1a and pyrene and the result of DLS are shown. It is an enlarged view of the absorption spectrum of the range of 350-500 nm of FIG. It is a fluorescence spectrum of only phosphate buffer aqueous solution (Only (-)-PBS). It is a fluorescence spectrum of a supernatant solution (Pyrene / (−)-PBS) after adding pyrene to an aqueous phosphate buffer solution. It is a fluorescence spectrum of a micelle (Pyrene / 5 / (-)-PBS) containing only pyrene. It is a fluorescence spectrum of nanoemulsion particles (Pyrene / (±) -10c / 5 / (−)-PBS) encapsulating Compound 1a and pyrene. It is an enlarged view of the fluorescence spectrum of the range of 425-500 nm of FIG. (-)-It is an evaluation of resistance to ascorbic acid of magnetic nanoemulsion of surfactant 1 encapsulating (±) -2 (n = 18) in PBS. Surfactant 1 magnetic nanoemulsion, (1) (±) -2 (n = 18) encapsulating (±) -2 (n = 18) encapsulated in a magnetic field of 4.7 T 18) T ₁ weighted image magnetic nanoemulsion composed of and the determination of longitudinal relaxation rate constant of water. Magnetic nanoemulsion of surfactant 1 encapsulating (±) -2 (n = 18), HeLa of magnetic nanoemulsion composed of surfactant 1 encapsulating 7 and (±) -2 (n = 18) Toxicity assessment for cells. (-)-Is the results of (a) EPR and (b) DLS measured 2 hours and 3 hours after preparation of magnetic nanoemulsion particles of surfactant 1 encapsulating (±) -3 in PBS, respectively. . The concentrations of Surfactant 1 and (±) -3 are 10 mM each. (A) EPR and (b) measured 3 hours and 3 and a half hours after preparation of surfactant 1 magnetic nanoemulsion particles encapsulating (±) -2 (n = 4) in PBS It is a result of DLS. The concentrations of surfactant 1 and (±) -2 (n = 4) are 10 mM and 5 m, respectively. From the preparation of surfactant 1 magnetic nanoemulsion particles encapsulating (±) -2 (n = 8) in (-)-PBS (a) 1 hour and a half later and (b) EPR measured after 2 days It is a result. The concentrations of Surfactant 1 and (±) -2 (n = 8) are 10 mM and 5 mM, respectively. (-)-Results of DLS measured after preparation of magnetic nanoemulsion particles of surfactant 1 encapsulating (±) -2 (n = 8) in PBS (a) 1 hour later and (b) 2 days later It is. The concentrations of Surfactant 1 and (±) -2 (n = 8) are 10 mM and 5 mM, respectively. (-)-EPR results measured from (a) 2 hours and (b) 2 days after preparation of magnetic nanoemulsion particles of surfactant 1 encapsulating (±) -2 (n = 14) in PBS It is. The concentrations of surfactant 1 and (±) -2 (n = 14) are each 10 mM. (-)-DLS results measured immediately after (a) preparation and (b) two days after preparation of surfactant 1 magnetic nanoemulsion particles encapsulating (±) -2 (n = 14) in PBS is there. The concentrations of surfactant 1 and (±) -2 (n = 14) are each 10 mM. (A) 4 hours, (b) 2 days and (c) 6 days after preparation of magnetic nanoemulsion particles of surfactant 1 encapsulating (±) -2 (n = 16) in PBS It is a result of EPR measured later. The concentrations of surfactant 1 and (±) -2 (n = 16) are 10 mM each. (A) 2 hours, (b) 2 days and (c) 6 days from the preparation of magnetic nanoemulsion particles of surfactant 1 encapsulating (±) -2 (n = 16) in PBS It is the result of DLS measured later. The concentrations of surfactant 1 and (±) -2 (n = 16) are 10 mM each. (A) 2 hours after preparation of surfactant 1 magnetic nanoemulsion particles encapsulating (±) -2 (n = 18) in PBS (b) 2 days later, (c) 7 days later And (d) EPR results measured after 14 days. The concentrations of Surfactant 1 and (±) -2 (n = 18) are 10 mM each. (A) 1 hour, (b) 2 days, (c) 7 days after preparation of magnetic nanoemulsion particles of surfactant 1 encapsulating (±) -2 (n = 18) in PBS And (d) DLS results measured after 14 days. The concentrations of Surfactant 1 and (±) -2 (n = 18) are 10 mM each. (A) 2 hours, (b) 2 days, and (c) 6 days after preparation of magnetic nanoemulsion particles of surfactant 1 encapsulating (±) -2 (n = 20) in PBS It is a result of EPR measured later. The concentrations of Surfactant 1 and (±) -2 (n = 20) are 10 mM each. (A) 1 hour after preparation of surfactant 1 magnetic nanoemulsion particles encapsulating (±) -2 (n = 20) in PBS (b) 2 days later and (c) 6 days It is the result of DLS measured later. The concentrations of Surfactant 1 and (±) -2 (n = 20) are 10 mM each.

  Hereinafter, the organic nitroxide radical compound of the present invention, its production method, nanoemulsion particles and its use will be described in detail.

Organic Nitroxide Radical Compound The organic nitroxide radical compound of the present invention has the formula (1):

(In the formula, R ¹ represents a linear alkyl group having 10 to 26 carbon atoms.)
(Hereinafter, also simply referred to as “compound (1)”). The organic nitroxide radical compound has an amphipathic property in which R ¹ is a hydrophobic part and the other part is a hydrophilic part.

R ¹ is a linear alkyl group having 10 to 26 carbon atoms, and examples thereof include n-decyl, lauryl, myristyl, cetyl, stearyl, and icosyl groups.
R ¹ is preferably a linear alkyl group having 16 to 22 carbon atoms, more preferably a linear alkyl group having 18 to 22 carbon atoms, and particularly preferably a linear alkyl group having 18 to 20 carbon atoms.

  The organic nitroxide radical compound of the present invention can be either racemic or optically active.

Method for Producing Organic Nitroxide Radical Compound Compound (1) is, for example, formula (2):

(In the formula, R ¹ is the same as above.)
(Hereinafter also simply referred to as “compound (2)”) to generate radicals by oxidation.

  The oxidation can be performed, for example, in a suitable solvent using a divalent copper compound as a catalyst in the presence of a base and using an oxidizing agent.

  As the solvent, a compound in which the compound (2) is easily dissolved or dispersed and does not adversely affect the oxidation reaction can be used. Examples of such a solvent include alcohols such as methanol and ethanol, tetrahydrofuran, and the like. The usage-amount of a solvent is about 40-60 mass parts normally with respect to 1 mass part of compounds (2), and about 35-55 mass parts is preferable.

  Examples of the base include aqueous ammonia and sodium hydroxide. The usage-amount of a solvent is about 3-5 mass parts normally with respect to 1 mass part of compounds (2), and about 2-4 mass parts is preferable.

  Examples of the catalyst include divalent copper compounds such as copper (II) acetate and copper (II) nitrate. The usage-amount of a catalyst can be selected suitably. For example, about 2 to 5 mol, preferably about 3 to 4 mol can be used per 1 mol of compound (2).

  Examples of the oxidizing agent include oxygen, ozone, air, silver oxide, and hydrogen peroxide. The amount of the oxidizing agent to be used is generally about 2 to 5 mol, preferably about 3 to 4 mol, per 1 mol of compound (2).

  The reaction temperature and reaction time can be appropriately adjusted depending on the amount of the compound (2) used, the oxidizing agent, and the like. For example, the reaction temperature is usually about 10 to 40 ° C, preferably about 20 to 30 ° C. The reaction time is usually about 1 to 5 minutes, preferably about 1 to 2 minutes.

As the compound (2), a commercially available product can be used. Alternatively, the compound (2) can be produced by applying a known production method. For example, it can be produced by a Grignard reaction between nitrone and a Grignard reagent in which the 1- and 4-positions of the benzene ring are substituted with a BrMg group and an OR ¹ group, respectively.
The production of the Grignard reagent and the Grignard reaction can be carried out according to the conditions normally used in the Grignard reaction. Detailed reaction conditions and the like are described in the examples.

Nanoemulsion particles and use thereof The nanoemulsion particles of the present invention comprise the organic nitroxide radical compound and the nonionic surfactant in a molar ratio of 0.8: 1 to 1.2: 1, and contain nitroxide radicals. It is what has been.

The nanoemulsion particles of the present invention are composed of the organic nitroxide radical compound and a nonionic surfactant, and the organic nitroxide radical compound is 0.8 to 1.2 with respect to 1 mol of the nonionic surfactant. Mole is contained, and nitroxide radicals are included in the particles.
When the molar ratio of the organic nitroxide radical compound to the nonionic surfactant is 0.8: 1 to 1.2: 1, the organic nitroxide radical compound can exist as emulsion particles stable in an aqueous solvent. In order to obtain more stable emulsion particles, the molar ratio of the organic nitroxide radical compound to the nonionic surfactant is preferably 1: 1.

  The organic nitroxide radical compound is as described above.

Nonionic surfactants are amphiphilic compounds having a hydrophobic portion and a hydrophilic portion.
Examples of the nonionic surfactant include ester type surfactants such as glyceryl laurate, glyceryl monostearate, sorbitan fatty acid ester, and sucrose fatty acid ester; ethers such as polyoxyalkylene alkyl ether and polyoxyalkylene alkylphenyl ether Type surfactants; ester ether type surfactants such as polyoxyethylene sorbitan fatty acid ester and sorbitan fatty acid ester polyalkylene glycol; alkanolamide type surfactants such as lauric acid diethanolamide and stearic acid diethanolamide; decyl glycoside, lauryl glycoside And higher alcohols such as cetanol and stearyl alcohol.

  Among these nonionic surfactants, those having a linear alkyl group having 10 to 26 carbon atoms are preferred. More preferably, the following formula (3):

(In the formula, R ² represents a linear alkyl group having 10 to 26 carbon atoms. N is an integer of 2 or more.)
It is polyoxyethylene alkyl ether represented by these. In the nonionic surfactant of the formula (3), R ² is a hydrophobic part, and the other part is a hydrophilic part.

R ² is a linear alkyl group having 10 to 26 carbon atoms, and examples thereof include n-decyl, lauryl, myristyl, cetyl, stearyl, icosyl and the like.
The number of carbon atoms in R ² is preferably close to the number of carbon atoms in R ^1, and are preferably the same as the number of carbon atoms in R ^1. R ² is preferably a linear alkyl group having 16 to 22 carbon atoms, more preferably a linear alkyl group having 18 to 22 carbon atoms, and particularly preferably a linear alkyl group having 18 to 20 carbon atoms.
Examples of the polyoxyethylene alkyl ether having a linear alkyl group having 10 to 26 carbon atoms include polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene tridecyl ether and the like.

  n is an integer greater than or equal to 2, Preferably it is an integer of 10-20.

  The nonionic surfactant is preferably biocompatible.

  A commercially available product can be used as the nonionic surfactant. Examples include Brij (registered trademark) 30, Brij (registered trademark) 35, Brij (registered trademark) 56, Brij (registered trademark) 58, Brij (registered trademark) 78, Brij (registered trademark) 98, and the like.

  The nanoemulsion particles of the present invention are obtained by mixing the organic nitroxide radical compound (compound (1)) and the nonionic surfactant (compound (2)) in an aqueous solvent, and then heating the mixture. be able to.

Examples of the aqueous solvent used for producing the nanoemulsion particles of the present invention include water, a hydrophilic organic solvent, or a mixed solvent of water and a hydrophilic organic solvent.
Examples of the hydrophilic organic solvent include alcohol solvents such as methanol, ethanol, isopropyl alcohol, and tert-butanol; ketone solvents such as acetone, methyl ethyl ketone, and methyl isobutyl ketone; ester solvents such as methyl acetate, ethyl acetate, and butyl acetate; Aprotic polar solvents such as N, N-dimethylformamide, dimethyl sulfoxide, sulfolane, N-methylpyrrolidone; glycol ethers such as diethylene glycol monomethyl ether and propylene glycol monomethyl ether acetate; glycol solvents such as ethylene glycol and propylene glycol; Ether solvents such as diethyl ether, tetrahydrofuran, dioxane and the like can be used.
As the aqueous solvent, water is preferable.

Concentration of the organic nitroxide radical compound in the solvent is, 1 × preferably ^{10 -3 ~1 × 10 -2 mol /} L, 1 × 10 -2 mol / L is more preferable. By setting the concentration within the above range, nanoemulsion particles having good stability can be obtained.

The molar concentration of the nonionic surfactant in the solvent is preferably 1 × 10 ⁻³ to 1 × 10 ⁻¹ mol / L, and more preferably 1 × 10 ⁻² mol / L. By setting the concentration within the above range, nanoemulsion particles having good stability can be obtained.

The mixing ratio of the organic nitroxide radical compound and the nonionic surfactant is preferably within the range of a molar ratio of organic nitroxide radical compound: nonionic surfactant = 0.8: 1 to 1.2: 1. A molar ratio of 1: 1 is more preferred. By mixing the organic nitroxide radical compound and the nonionic surfactant in the above ratio, nanoemulsion particles having good stability can be obtained.
From this, it is surmised that the nanoemulsion particles of the present invention are formed by a nested structure of the organic nitroxide radical compound and the nonionic surfactant as shown in FIG.
Furthermore, when the carbon chain (R ² ) of the nonionic surfactant and the carbon chain (R ¹ ) of the organic nitroxide radical compound are substantially equal in length, a nested structure as shown in FIG. 1 is formed. In doing so, the carbon chains are closely combined and the interaction between the molecules increases, so that it is conceivable that more stable nanoemulsion particles can be obtained.

The organic nitroxide radical compound (compound (1)) and the nonionic surfactant (compound (2)) are mixed in an aqueous solvent. The mixing of the compound (1) and the compound (2) is preferably performed with stirring. More preferably, the compound (1) and the compound (2) are stirred under normal pressure for several seconds to several hours.
Stirring and mixing can be performed, for example, using a magnetic stirrer or the like, or by irradiation with ultrasonic waves or microwaves. For example, when water is used as an aqueous solvent, the organic nitroxide radical compound is added dropwise to the stirring aqueous solution of the nonionic surfactant and stirred, and then both are mixed by ultrasonic irradiation. It is preferable to do.
In the case where the organic nitroxide radical compound is hardly soluble in water, the organic nitroxide radical compound is dissolved in advance in a hydrophilic organic solvent in which the organic nitroxide radical compound is soluble, and the resulting solution is dissolved in an aqueous solution of the nonionic surfactant. It is preferable to drop it into As a temperature heated after mixing, about 60-90 degreeC is preferable, and about 85-90 degreeC is more preferable. The heating time is preferably about 15 to 40 minutes, more preferably about 30 minutes.

  The formation of nanoemulsion particles can be confirmed by changing the color of the solution from cloudy to transparent, but is not limited thereto.

The nanoemulsion particles of the present invention are amphiphilic particles in which the outside of the particles is hydrophilic and the inside of the particles is hydrophobic, and nitroxide radicals are encapsulated inside the molecule. Therefore, the nanoemulsion particles of the present invention have a stable radical structure and are not easily reduced by vitamin C or the like.
The nanoemulsion particles of the present invention have paramagnetism due to a nitroxide radical, and can be used as a functional organic material that can be controlled by a magnetic field. In addition, since the radical structure can be efficiently organized, it can be applied to substance transport by magnetic field control, and can be used for drug drug delivery systems (DDS) containing stable radicals.

  Specifically, the nanoemulsion particles of the present invention can be used as a contrast agent for MRI because magnetic particles can be traced in real time to the deep part of the body by MRI.

Moreover, since the inside of a molecule | numerator is hydrophobic in the nanoemulsion particle | grains of this invention, hydrophobic substances, such as an anticancer agent, can be included in a molecule | numerator inside. Furthermore, since the particle diameter is about 70 nm and the particles are stable, it is possible to induce the anticancer agent to the tumor tissue by applying an external magnetic field to the nanoemulsion particles encapsulating the anticancer agent. It becomes. Therefore, the nanoemulsion particles of the present invention can be used as a DDS carrier.
According to this method, it is possible to efficiently deliver an anticancer drug to cancer cells by installing a magnet in the vicinity of the cancer cells, particularly directly under the skin of skin cancer, breast cancer, etc. It is expected to show its power in targeting cancer cells. In addition, if a fluorescent agent is encapsulated in the nanoemulsion particles of the present invention, the position of cancer cells can be specified.

  Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples. In addition, this invention is not limited to a following example.

  Analysis and evaluation in the examples were carried out by the following methods. In addition, unless otherwise indicated, the reagent used in the Example purchased the commercial item, and used it as it was, without refine | purifying.

Analysis and evaluation in the examples were carried out by the following methods.
(1) Electron paramagnetic resonance (EPR)
EPR was measured using a pulse ESR device (JES-PX1050 type, manufactured by JEOL Ltd.). Specifically, the sample was enclosed in an EPR capillary tube and both ends were closed with putty. Next, this capillary was inserted into an EPR tube, and this was installed in the cavity. The EPR measurement was performed by irradiating the sample with microwaves at room temperature and under a magnetic field application condition of 0.33T.
(2) Dynamic light scattering measurement (DLS)
DLS was measured for hydrodynamic diameter by a dynamic light scattering (DLS) method using a dynamic light scattering analyzer (manufactured by Microtrack Bell, UPA-UT151).
(3) Absorption spectrum The absorption spectrum was measured using an ultraviolet-visible near-infrared spectrophotometer (manufactured by Shimadzu Corporation, UV-3600 type).
(4) Fluorescence spectrum The fluorescence spectrum was measured using BioTek Instruments, Inc., Cytation 5 Cell Imaging Multi-Mode Reader.

Production Example 1 (Synthesis of nitrone)
3-Buten-2-one (22.0 g, 0.314 mol) distilled at 110 ° C. under normal pressure was dissolved in 2-nitropropane (139 g, 1.56 mol). Tetramethylguanidine (10.8 g, 0.094 mol) was slowly added to the resulting solution at 0 ° C., and the mixture was stirred overnight at room temperature, and then 2N hydrochloric acid was added and extracted with diethyl ether.
The organic layer was washed with a saturated aqueous solution of sodium bicarbonate and a saturated aqueous solution of sodium chloride, dried over anhydrous magnesium sulfate, and then the solvent was removed. Furthermore, unreacted 2-nitropropane was removed by distillation under reduced pressure to obtain 5-methyl-5-nitrohexane-2-one (14.4 g, 0.090 mol, yield 29%).

Next, water (139 mL) and ammonium chloride (17.301 g, 0.323 mol) were added to 5-methyl-5-nitrohexane-2-one (20.1 g, 0.125 mol). Zinc powder (77.9 g, 0.236 mol) was slowly added to the resulting mixture at 0 ° C. and allowed to react for 4 hours. The solution was filtered with suction and the solid was washed with methanol.
The filtrate was extracted with chloroform (50 mL × 3) and dried over anhydrous magnesium sulfate. After removing chloroform, it was dried by benzene azeotropy. After vacuum drying for 3 hours, nitrone ((±) -11) was obtained (4.83 g, 0.038 mol, yield 30%).

Example 1
(1) Synthesis of organic nitroxide radical compound (Synthesis example of 18 carbon atoms and results of elemental analysis)
An organic nitroxide radical compound (compound 1a) in which R ¹ is a linear alkyl group having 14 carbon atoms in the formula (1) was synthesized as follows.

  Magnesium (0.506 g, 20.0 mmol) was placed in a two-necked flask and dried under reduced pressure, and the flask was filled with argon. To this was added tetrahydrofuran (20 mL) and 1-bromo-4-tetradecyloxybenzene (7.10 g, 20.0 mmol). Furthermore, a small amount of 1,2-dibromoethane was added to start the reaction, and the mixture was stirred at room temperature for 4 hours to prepare a Grignard reagent.

  On the other hand, the nitrone (1.27 g, 10.0 mmol) synthesized in Production Example 1 was placed in a two-necked flask and azeotroped with a few drops of benzene. The inside of the two-necked flask was evacuated and then filled with argon. Tetrahydrofuran (20 mL) was added thereto, cooled to −78 ° C., and a Grignard reagent was added dropwise. The mixture was slowly warmed and then stirred at room temperature overnight. The reaction mixture was poured into a saturated aqueous ammonium chloride solution (50 mL) and extracted with dichloromethane (50 mL). After drying over anhydrous magnesium sulfate, the solvent was distilled off.

The residue was dissolved in methanol (30 mL), 25% concentrated aqueous ammonia (2 mL) and copper acetate (0.805 g, 4.00 mmol) were added, and oxygen was bubbled for 1 minute. After the solution turned dark blue, the reaction solution was concentrated under reduced pressure, and methanol was distilled off. To the residue was added saturated aqueous sodium hydrogen carbonate solution (50 mL), and the mixture was extracted with dichloromethane (50 mL). The obtained organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off.
Finally, the residue was purified by silica gel column chromatography (n-hexane / diethyl ether = 90/10 (volume ratio)) to obtain Compound 1a (0.478 g, 1.15 mmol, yield 11.5%).

Elemental Analysis Calculated (%): C, 77.83; H, 11.13; N, 3.36.Found (%): C, 77.61; H, 11.01; N, 3.34.
EPR (THF, 25 ° C): g = 2.0067, a _N = 1.38 mT

(2) Preparation of nanoemulsion particles As a surfactant, Brij (registered trademark) 58 (polyoxyethylene cetyl ether, n = 20) (0.114 g) manufactured by Sigma-Aldrich was put in a vial, and a necessary amount of water was added. In addition, ultrasonic irradiation was performed for 2 minutes to prepare 10 mL of a 1 × 10 ⁻² M surfactant aqueous solution.

  Compound 1a (8.52 mg, 20.4 μmol) was dissolved in a small amount of diethyl ether. The obtained diethyl ether solution was dripped little by little into the surfactant aqueous solution (2.0 mL) vigorously stirred, and was stirred for 30 minutes. Thereafter, ultrasonic irradiation was performed for 3 minutes, and then nanoemulsion particles were prepared by heating at 90 ° C. for 30 minutes.

Comparative Example 1
In the same manner as in Example 1, except that 1-bromo-4-butoxybenzene (4.58 g, 20.0 mmol) was used instead of 1-bromo-4-tetradecyloxybenzene, R in formula (1) ^An organic nitroxide radical compound in which ¹ is a linear alkyl group having 4 carbon atoms was synthesized (0.396 g, 1.43 mmol, 14.3% yield).

Elemental Analysis Calculated (%): C, 73.87; H, 9.48; N, 5.07.Found (%): C, 73.63; H, 9.66; N, 5.02.
EPR (THF, 25 ° C): g = 2.0067, a _N = 1.39 mT

To the surfactant aqueous solution (1 × 10 ⁻² M) prepared in Example 1 (2), the organic nitroxide radical compound (2.76 mg, 10 μmol) obtained above was added dropwise with a microsyringe. When this was irradiated with ultrasonic waves for 5 seconds, a macroemulsion (particle diameter of about 1 mm) was formed and settled to the bottom of the vial. This macroemulsion responded to permanent magnets (up to 500 mT) and moved freely in water.
This is based on the organic nitroxide compound in the macroemulsion having magnetism. From this, it can be easily predicted that the nanoemulsion particles prepared in Example 1 having the same organic nitroxide compound also have magnetism.

  Thereafter, the emulsion was again irradiated with ultrasonic waves (20 seconds), and then heated at 90 ° C. for 30 minutes to prepare nanoemulsion particles.

Comparative Example 2
In the formula (1), as in Example 1, except that 1-bromo-4-octoxybenzene (5.71 g, 20.0 mmol) was used instead of 1-bromo-4-tetradecyloxybenzene. An organic nitroxide radical compound in which R ¹ is a linear alkyl group having 8 carbon atoms was synthesized (0.686 g, 2.06 mmol, yield 20.6%).

Elemental Analysis Calculated (%): C, 75.86; H, 10.31; N, 4.21.Found (%): C, 75.99; H, 10.35; N, 4.19.
EPR (THF, 25 ° C): g = 2.0066, a _N = 1.39 mT

  Using the organic nitroxide radical compound obtained above, nanoemulsion particles were prepared in the same manner as in Example 1 (2).

Test Example 1 (Stability evaluation of nanoemulsion particles)
For the nanoemulsion particles prepared in Example 1, Comparative Example 1 and Comparative Example 2, EPR and DLS were measured after 1 day, 3 days, 5 days, and 7 days. The results of the nanoemulsion particles of Example 1 are shown in FIGS. 2 to 5, the results of the nanoemulsion particles of Comparative Example 1 are shown in FIGS. 6 to 9, and the results of the nanoemulsion particles of Comparative Example 3 are shown in FIGS.

  From FIG. 2 to FIG. 5, the nanoemulsion particles of Example 1 rapidly increased in particle size from the first day to the second day, and the particle size became constant (about 70 nm) after the second day. It was found to exist stably over 7 days. Since no peak (three lines) derived from the compound 1a was observed in the EPR spectrum, it was found that almost all of the dropped compound 1a was included in the nanoemulsion particles.

  6-9, the particle size was constant (about 9 nm) over 7 days and no significant change was observed in the linearity of the EPR spectrum, indicating that nanoemulsion particles exist stably over 7 days. I understood. However, since three EPR spectrum peaks were seen, it was found that the compound 1a leaked from the nanoemulsion particles.

  As shown in FIGS. 10 to 13, the particle size was constant (approximately 100 nm) over 7 days, and the EPR spectrum linearity did not change significantly, indicating that nanoemulsion particles exist stably over 7 days. I understood. However, since two EPR spectrum peaks were seen, it was found that the compound 1a leaked from the nanoemulsion particles.

  From the above results, it was found that the nanoemulsion particles of Example 1 exist stably in water.

Test Example 2 (Stability evaluation of nanoemulsion particles (10-fold dilution))
After preparing nanoemulsion particles in Example 1, the concentration of the aqueous solution was diluted 10 times, and EPR and DLS were measured. The result is shown in FIG.

  From FIG. 14, it was found that the nanoemulsion particles prepared in Example 1 exist stably even after 10-fold dilution because there is no significant change in the linearity of the EPR spectrum before and after dilution and the particle diameter does not change. .

Example 2
Nanoemulsions were prepared in phosphate buffer.

Put a surfactant Brij (R) 58 (0.114 g) in a vial, the required amount of phosphate buffer solution ((-) - PBS) was added, subjected to ultrasonic irradiation for 2 minutes, 1 × 10 ^- 10 mL of a phosphate buffered aqueous solution of ² M surfactant was prepared.

  Compound 1a (8.52 mg, 20.4 μmol) was dissolved in a small amount of ether. The obtained ether solution was added dropwise little by little into a phosphate buffer aqueous solution (2.0 mL) of a surfactant that was vigorously stirred and allowed to stir for 30 minutes. Then, the nanoemulsion solution (2.0 mL) was prepared by ultrasonically irradiating for 3 minutes, and heating at 90 degreeC for 1 hour after that.

Test Example 3 (Stability evaluation of nanoemulsion particles in phosphate buffer)
For the purpose of evaluating practicality, the stability (time-dependent change) of the nanoemulsion particles of the surfactant encapsulating Compound 1a in a phosphate buffer aqueous solution was evaluated by EPR spectroscopy and DLS measurement. The results are shown in FIGS.

  From FIG. 15-18, unlike pure water, the emulsion particles were united on the 7th day. This is considered to have changed to another structure other than the emulsion (associated micelle, lamellar structure, etc.). Therefore, it is considered that nanoemulsion particles cannot be stabilized as much as pure water in a phosphate buffered aqueous solution.

Test Example 4 (Stability evaluation of nanoemulsion particles in phosphate buffer against ascorbic acid)
In order to evaluate the stability to ascorbic acid, ascorbic acid (3.59 mg, 0.02 mmol) was added to the phosphate buffer aqueous solution (2.0 mL) of nanoemulsion particles prepared in the same manner as in Example 2, and EPR The spectrum was measured. The area change (double integration value) of the EPR spectrum in the range of 314.5 mT to 339.5 mT (3145 to 3395 gauss) immediately after the start of measurement was taken as 100%, and the time change of the spectrum area value was measured. The results are shown in FIG.

  From FIG. 19, it took about 150 minutes until the concentration of Compound 1a became constant. Therefore, since the nanoemulsion particles are gradually reduced with respect to the reducing agent, it is considered to be advantageous for application to a contrast agent for DDS, MRI and the like.

Example 3
Surfactant nanoemulsion particles encapsulating pyrene (pseudo-drug) and compound 1a in a phosphate buffer were prepared.

  Compound 1a (8.52 mg, 20.4 μmol) and pyrene (0.20 mg, 0.99 μmol) were dissolved in a small amount of ether. The obtained ether solution was added dropwise little by little into a phosphate buffer aqueous solution (2.0 mL) of a surfactant that was vigorously stirred and allowed to stir for 30 minutes. Then, the nanoemulsion solution (2.0 mL) was prepared by ultrasonically irradiating for 3 minutes, and heating at 90 degreeC for 1 hour after that.

Test Example 5 (Measurement of absorption spectrum and fluorescence spectrum of nanoemulsion particles encapsulating pyrene in phosphate buffer)
In order to confirm that pyrene was encapsulated inside the emulsion, the absorption spectrum of the nanoemulsion particles (Pyrene / (±) -10c / 5 / (−)-PBS) prepared in Example 3 was measured.
For comparison, phosphate buffer solution only (Only (-)-PBS), supernatant solution after adding pyrene to phosphate buffer solution (Pyrene / (-)-PBS), micelle containing pyrene (Pyrene / 5 / The absorption spectrum of (-)-PBS) was measured.
In addition, DLS was measured for nanoemulsion particles (Pyrene / (±) -10c / 5 / (-)-PBS) and micelles containing pyrene (Pyrene / 5 / (-)-PBS). The results are shown in FIGS.
Moreover, about nanoemulsion particle | grains (Pyrene / (±) -10c / 5 / (-)-PBS), the enlarged view of the absorption spectrum in the range of 350-500 nm is shown in FIG.

  From FIG. 23, the absorption spectrum derived from pyrene was confirmed in the nanoemulsion particles (Pyrene / (±) -10c / 5 / (−)-PBS) of the surfactant in which pyrene was encapsulated together with the compound 1a. Pyrene is usually very insoluble in water and precipitates at the bottom of the vial, but in the emulsion solution prepared in Example 3, no precipitate was confirmed at the bottom of the vial. Therefore, it is considered that the added pyrene is encapsulated in the magnetic emulsion.

From the DLS measurement, the particle size of the nanoemulsion particles (Pyrene / (±) -10c / 5 / (−)-PBS) encapsulating Compound 1a and pyrene was about 15 nm (FIG. 23).
Since the particle size of micelle (Pyrene / 5 / (−)-PBS) containing only pyrene without compound 1a is about 10 nm (FIG. 22), the amount of pyrene contained in one emulsion particle is very small. Conceivable.

Furthermore, the fluorescence spectrum of the nanoemulsion particles (Pyrene / (±) -10c / 5 / (−)-PBS) of surfactant in which pyrene was encapsulated together with compound 1a was measured. For comparison, only phosphate buffer solution (Only (-)-PBS), supernatant solution after adding pyrene to phosphate buffer solution (Pyrene / (-)-PBS), micelle containing pyrene (Pyrene / PBS) 5 / (−)-PBS) was measured.
The maximum absorption wavelength was estimated from the absorption spectrum of FIG. 23, and the excitation wavelength (λex = 320 ± 10 nm) was determined. The results are shown in FIGS.
For nanoemulsion particles (Pyrene / (±) -10c / 5 / (−)-PBS), an enlarged view of the fluorescence spectrum in the range of 425 to 500 nm is shown in FIG.

From FIG. 28, no luminescence phenomenon with high intensity was observed for the nanoemulsion particles encapsulating Compound 1a and pyrene. This is probably because the nitroxide radical (N—O.) In the compound 1a has an n-π ^* transition (450 nm) (see FIG. 24), and thus quenches fluorescence.
As evidence, when the fluorescence spectrum of nanoemulsion particles (Pyrene / (±) -10c / 5 / (−)-PBS) encapsulating compound 1a and pyrene in FIG. 28 is enlarged, a slight emission is seen at around 450 nm. (FIG. 29).

Example 4
Evaluation of MRI imaging ability of magnetic nanoemulsion and cytotoxicity evaluation of magnetic nanoemulsion containing anticancer drug

(1) Synthesis of organic nitroxide radical compound (±) -2 (n = 18) In the formula (1), an organic nitroxide radical compound (compound 1b) in which R ¹ is a linear alkyl group having 18 carbon atoms is In this way, it was synthesized.

  Sharpened magnesium (0.253 g, 10 mmol) was placed in a two-necked flask and dried under reduced pressure, and the flask was filled with argon. To this was added tetrahydrofuran (10 mL) and a small amount of 1,2-dibromoethane and allowed to stir for 10 minutes. To this was added a tetrahydrofuran solution (40 mL) in which 1-bromo-4-octadecyloxybenzene (4.255 g, 10 mmol) previously dried by benzene azeotropy was dissolved. The mixture was stirred at room temperature for 2 hours to prepare a Grignard reagent.

Meanwhile, tetramethylnitrone (0.635 g, 5.00 mmol) synthesized in Production Example 1 was placed in a two-necked flask and azeotroped with several drops of benzene. The inside of the two-necked flask was evacuated and then filled with argon. Tetrahydrofuran (20 mL) was added thereto, cooled to −78 ^° C., and Grignard reagent was added dropwise. The mixture was slowly warmed and then stirred at room temperature overnight. The reaction mixture was poured into a saturated aqueous ammonium chloride solution (100 mL) and extracted with dichloromethane (100 mL). After drying over anhydrous magnesium sulfate, the solvent was distilled off.

  The residue was dissolved in a mixed solution of methanol (30 mL) and dichloromethane (30 mL), 25% concentrated aqueous ammonia (2 mL) and copper acetate (0.805 g, 4.00 mmol) were added, and oxygen was blown in for 1 minute. After changing to dark blue, the reaction solution was concentrated under reduced pressure to distill off methanol, and saturated aqueous sodium hydrogen carbonate solution (100 mL) was added to the residue, followed by extraction with dichloromethane (100 mL).

The obtained organic layer was dried over anhydrous magnesium sulfate, and then the solvent was distilled off. Finally, it was purified by silica gel column chromatography (n-hexane / diethyl ether = 90/10 (volume ratio)), and organic nitroxide radical compound (±) -2 (n = 18) (0.440 g, 0.93 mmol, yield) 18.6%).
Elemental Analysis Calculated (%): C, 78.76; H, 11.51; N, 2.96. Found (%): C, 78.89; H, 11.49; N, 2.78.
EPR (0.1 mM in THF, 25 ^o C): g = 2.0068, aN = 1.39 mT

(2) Magnetic properties of surfactant 1 containing (±) -2 (n = 18) in (-)-PBS ( Preparation of nanoemulsion particles)
As a surfactant, Brij (registered trademark) 58 (polyoxyethylene cetyl ether, n = 20) (0.225 g, 200 μmol) manufactured by Sigma-Aldrich was put in a vial, and a necessary amount of (−)-PBS was added, Heating (90 ^° C., 2 minutes) and ultrasonic irradiation (1 minute) were performed to prepare 20 mL of a 10 mM surfactant aqueous solution.
Compound (±) -2 (n = 18) (7.06 mg, 15 μmol) was dissolved in a small amount of diethyl ether. A surfactant aqueous solution (1.5 mL) was added dropwise thereto, and the mixture was stirred for 1 minute. Thereafter, a magnetic nanoemulsion of Surfactant 1 containing (±) -2 (n = 18) was prepared by heating at 90 ^° C. for 10 minutes.

(3) Reduction resistance of surfactant 1 magnetic nanoemulsion containing (±) -2 (n = 18)

  A magnetic nanoemulsion of Surfactant 1 containing (±) -2 (n = 18) was prepared in Dulbecco's phosphate buffer (-)-PBS (Wako, Lot. DSJ7024), and the reducing agent for magnetic nanoemulsion The stability against was evaluated. Specifically, 1.75 times ascorbic acid is added to the aqueous solution of surfactant 1 magnetic nanoemulsion containing (±) -2 (n = 18) to reduce the ascorbic acid resistance of the magnetic nanoemulsion. Evaluation was made using DLS and EPR spectroscopy.

The concentration ratio of surfactant 1 and (±) -2 (n = 18) to ascorbic acid is [1 / (±) -2 (n = 18) / Ascorbic acid = 1.0 × 10 ^-2 mM / 1.0 × 10 ⁻² mM / 1.75 × 10 ⁻² mM]. In the preparation method, 1.5 mL of a surfactant aqueous solution was added to a solution of (±) -2 (n = 18) dissolved in a small amount of ether, and then heated at 90 ° C. to remove the ether from the system. A magnetic nanoemulsion was prepared. Ascorbic acid was added to the obtained magnetic nanoemulsion, and EPR measurement and DLS measurement were immediately performed.

The change in radical concentration was calculated by integrating the EPR spectrum twice and setting the double integration value of the EPR spectrum immediately after the start to 100 (FIG. 30).
As a result, it was found that this magnetic nanoemulsion undergoes reduction more slowly than water-soluble radicals such as TEMPOL. This result indicated that the magnetic nanoemulsion has excellent reduction resistance against ascorbic acid.
In this measurement, JEOL RE-2X was used as the EPR device.

(4) MRI phantom measurement A magnetic nanoemulsion of surfactant 1 encapsulating (±) -2 (n = 18) was considered to be effective as an MRI contrast agent instead of a gadolinium preparation.
Therefore, MRI phantom measurement was performed under a 4.7 T magnetic field application, and the imaging capability of MRI of magnetic emulsion containing magnetic nanoemulsion and hydrophobic pseudo-drug or drug was evaluated.
Pyrene 6 (SIGMA-ALDRICH, Lot. S22012) and tetraphenylporphyrin 8 were used as pseudo-drugs. Moreover, hydrocortisone 9 (TCI, LOT. SZ6YA) and paclitaxel 7 (Sawai, paclitaxel injection solution 30 mg) were used as hydrophobic drugs. 7 is an anticancer agent actually used in the medical field.

Measurement method In this measurement, Bruker 4.7 T machine (BioSpec 47 USR) was used. Preparation of surfactant 1 magnetic nanoemulsion [1 / (±) -2 (n = 18) = 10 mM / 10 mM] containing (±) -2 (n = 18) in (-)-PBS did. Further, this solution was diluted 1/2, 1/8, 1/16 to prepare a total of 4 types of magnetic nanoemulsions containing (±) -2 (n = 18) of 1.25 mM to 10 mM.

  For magnetic emulsions containing pseudo-drugs or drugs, magnetic nanoemulsions containing 10 mol% of hydrophobic compound 6, 7, 8 or 9 with respect to (±) -2 (n = 18) [1 and Final concentrations of (±) -2 (n = 18): 10 mM each were prepared. However, for the magnetic nanoemulsion composed of surfactant 1 encapsulating 7 and (±) -2 (n = 18), the aqueous solution was diluted as before, and 1.25 mM to 10 mM (±) − Four types of magnetic nanoemulsions containing 2 (n = 18) were prepared.

  1 mL from these samples was transferred to a plastic tube and placed in a sample holder. Subsequently, this holder was inserted into the MR apparatus. Finally, after positioning the sample with a laser, measurement was performed under room temperature conditions. The spin echo method was used as a measurement method. Measurement conditions for images and r1 value calculation are different, and are as follows.

[Conditions for image; Echo time (TE) = 20 ms, Repetition time (TR) = 200 ms, r Measurement conditions for calculating ₁ value; Echo time (TE) = 10 ms, Repetition time (TR) = 5000, 3000, 1500, 800, 400 and 200 ms].

Measurement results FIG. 31a shows an MR image obtained.
(a) Panel (AD): the final concentration of (±) -2 (n = 18 ) included the magnetic nanoemulsion surfactant 1 T ₁ -weighted images [1 a (±) -2 (n = 18 ) : 10, 5, 2.5, 1.25 mM each] Panel (EH): T ₁ enhancement of magnetic nanoemulsion composed of surfactant 1 and (±) -2 (n = 18) containing 10 mol% 7 Image [final concentrations of 1 and (±) -2 (n = 18): 10, 5, 2.5, 1.25 mM respectively] Panel (IK): 10 mol% (I) 6, (J) 8, (K) the final concentration of 9 containing the surfactant 1 and a (±) -2 (n = 18 ) configured magnetic nanoemulsion T ₁ -weighted images [1 from (±) -2 (n = 18 ): each 10 mM] Panel (L): Control (-)-PBS.
From FIG. 31a, an increase in contrast was observed in any sample as compared with control [(−)-PBS], but the surfactant containing 10 mM (±) -2 (n = 18) was included. One magnetic nanoemulsion showed an excellent contrast effect.
Furthermore, it was found that a magnetic nanoemulsion composed of surfactant 1 encapsulating 10 mol% of 7 and (±) -2 (n = 18) showed a better contrast effect.
Regarding magnetic nanoemulsion composed of surfactant 1 encapsulating (±) -2 (n = 18) and surfactant 1 encapsulating 7 and (±) -2 (n = 18) Then, the T ₁ relaxation time at each concentration was calculated, and the r ₁ value serving as an index of the imaging ability of (±) -2 (n = 18) was calculated. The r ₁ value is obtained as the slope of an approximate straight line drawn with the reciprocal of the T ₁ relaxation time on the y axis and the concentration of (±) -2 (n = 18) on the x axis.

FIG. 31b and FIG. 31c respectively show the magnetic nanoemulsion of surfactant 1 encapsulating (±) -2 (n = 18) and surfactant 1 encapsulating 7 and (±) -2 (n = 18) 3 shows the relationship between the concentration of (±) -2 (n = 18) contained in the magnetic nanoemulsion composed of T and the T ₁ relaxation time.
(b) It is composed of a surfactant 1 magnetic nanoemulsion containing (±) -2 (n = 18) and (c) 7 containing surfactant 1 and (±) -2 (n = 18). Between (±) -2 (n = 18) concentration and T ₁ relaxation time in magnetic nanoemulsions.
When the r ₁ value was calculated from the slope of the graph, it was calculated as 0.12 s ⁻¹ / mM and 0.16 s ⁻¹ / mM, respectively. This result shows that a magnetic nanoemulsion composed of surfactant 1 encapsulating 7 and (±) -2 (n = 18) is a magnetic nanoemulsion encapsulating only (±) -2 (n = 18). This suggests that the relaxation of water can be shortened more effectively.
The particle size of the magnetic nanoemulsion composed of surfactant 1 encapsulating 7 and (±) -2 (n = 18) is 12 nm, and the interface encapsulating only (±) -2 (n = 18) It is smaller than the magnetic nanoemulsion (particle size 16 nm) of Activator 1. In addition, a triplet component was observed in the EPR spectrum.
Considering these facts, in the magnetic nanoemulsion composed of surfactant 1 encapsulating 7 and (±) -2 (n = 18), the water molecules present inside and the outside exist. It is thought that fast exchange is taking place with water molecules. Furthermore, it is suggested that the rotational correlation time of (±) -2 (n = 18) is increased.

(5) In vitro cytotoxicity assessment
(1) Magnetic nanoemulsion of surfactant 1 encapsulating (±) -2 (n = 18), and magnetic nanoemulsion composed of surfactant 1 encapsulating 7 and (±) -2 (n = 18) Toxicity evaluation for HeLa cells was performed.

Measuring method
Cultured in Dulbecco's modified Eagle medium (Wako, LOT DSG7015) containing 10% fetal bovine serum (SIGMA-ALDRICH, LOT 14A189) and 1% penicillin / streptomycin solution (Wako, Penicillin-Streptomycin Solution (× 100)) HeLa cells were seeded in 9,000 / 100 μL HeLa cells in each well of a 96-well microplate (ThermoFisher SCIENTIFIC, Nunclon ™ Delta 96-Well MicroWell ™ Plates, Sterile).
Subsequently, surfactant 1 micelle aqueous solution, surfactant 1 magnetic nanoemulsion containing (±) -2 (n = 18), surfactant 1 containing 0.001 mol% of 7 and (±) -2 Add 10 μL each of the magnetic nanoemulsion composed of (n = 18) and 7 (-)-PBS solution to each well, and incubate for 24 hours in an incubator (Thermo SCIENTIFIC, HERACELL 150i) (temperature: 37 ^° C, 2 Oxygen concentration: 5%).
Micellar aqueous solution [1 concentration: 10 mM], magnetic nanoemulsion of surfactant 1 containing (±) -2 (n = 18) [concentration of 1 and (±) -2 (n = 18): 10 each mM] and 7 (-)-PBS solution [7 concentration: 10 μM] was added 1/32 [1 and (±) -2 (n = 18) in (-)-PBS before being added to the wells Final concentrations of: 0.313 mM], 1/64 [0.156 mM], 1/128 [0.078 mM] and 1/256 [0.039 mM], respectively.
Subsequently, these aqueous solutions were added to each well. After culturing for 24 hours, each well was washed twice with (-)-PBS and evaluated by the ckk-8 evaluation method (Dojindo Laboratories, LOT JH159).
In this measurement, the ckk-8 reagent was added, followed by incubation for 30 minutes, and the absorbance (wavelength: 450 nm) corresponding to the number of cells was measured with a microplate reader (TECAN, infinite M200). In the analysis, the cell viability in each sample was calculated with the cell viability obtained when (-)-PBS alone was added as a control as 100%. In addition, 4 wells were used for each sample, and the average cell viability obtained from each well was calculated.
The micellar aqueous solution and all magnetic emulsions were prepared in (−)-PBS.

Measurement result FIG. 32a: cytotoxicity of surfactant 1 micelle aqueous solution and surfactant 1 magnetic nanoemulsion containing (±) -2 (n = 18).
FIG. 32b: Cytotoxicity of magnetic nanoemulsion composed of surfactant 1 encapsulating 7 and (±) -2 (n = 18) and 7 (−)-PBS solution.
From FIG. 32a, it was found that the dilution condition of 32 times or more shows little toxicity to HeLa cells.
On the other hand, from FIG. 32b, the magnetic nanoemulsion composed of surfactant 1 encapsulating 7 and (±) -2 (n = 18) and 7 (−) PBS solution are diluted by a factor of 32 or more. Was also toxic to HeLa cells.
This result shows that even when an anticancer agent is encapsulated in a magnetic nanoemulsion, it works effectively as an anticancer agent. Furthermore, when the cell killing effect under the 64-fold and 128-fold dilution conditions was observed, it was found that 7 encapsulated in the magnetic nanoemulsion showed twice as much cytotoxicity as 7 dispersed in the solution.
This indicates that a magnetic nanoemulsion composed of surfactant 1 encapsulating 7 and (±) -2 (n = 18) is effectively taken up into HeLa cells under relatively low concentration conditions. ing. The fact that 7 released from the inside of the magnetic nanoemulsion is drug-active is that the magnetic nanoemulsion composed of surfactant 1 encapsulating 7 and (±) -2 (n = 18) is the carrier of the drug delivery system. As effective.

界面活性剤1とそれぞれ長さの異なるアルキル側鎖を有する疎水性のラジカルを用いて、リン酸緩衝液(-)-PBS中で純有機磁性ナノエマルションを作製し、その安定性を評価した。
具体的には(-)-PBS中で作製した磁性ナノエマルションの安定性をEPR分光法とDLS法により評価した。
磁性ナノエマルションの安定化条件の結果を示す。

Example 5
Data showing the process of compound optimization

Pure organic magnetic nanoemulsions were prepared in phosphate buffer (-)-PBS using surfactant 1 and hydrophobic radicals having different alkyl side chains, and their stability was evaluated.
Specifically, the stability of the magnetic nanoemulsion prepared in (-)-PBS was evaluated by EPR spectroscopy and DLS method.
The result of the stabilization conditions of a magnetic nanoemulsion is shown.

(±)-3をエーテルに溶解させて、この溶液を界面活性剤水溶液で分散させることにより磁性ナノエマルションの調製を試み、磁性ナノエマルションの安定性をEPR分光法とDLS法により評価した。
界面活性剤1と(±)-3の濃度比は、[1 /(±)-3 = 10 mM / 10 mM]とした。調製方法は、少量のエーテルに溶解させた(±)-3の溶液に、界面活性剤水溶液を1 mL加えた後、90 ℃で加熱し、エーテルを系から除去することで、ナノエマルションを調製した。系からエーテルが除去されると、溶液の色が黄濁色から黄色透明に変化した。
図３３は調製後のEPRスペクトルと平均粒径である。EPR測定の結果から、三本線成分が確認された。これは、ラジカルが(-)-PBS中で分散していることを示している。 (1) Surfactant 1 magnetic nanoemulsion containing (±) -3

Preparation of a magnetic nanoemulsion was attempted by dissolving (±) -3 in ether and dispersing this solution in an aqueous surfactant solution, and the stability of the magnetic nanoemulsion was evaluated by EPR spectroscopy and DLS method.
The concentration ratio between the surfactant 1 and (±) -3 was [1 / (±) -3 = 10 mM / 10 mM]. The preparation method is to prepare a nanoemulsion by adding 1 mL of a surfactant aqueous solution to a solution of (±) -3 dissolved in a small amount of ether and then heating at 90 ° C to remove the ether from the system. did. When ether was removed from the system, the color of the solution changed from yellow to clear.
FIG. 33 shows the EPR spectrum and average particle diameter after preparation. From the EPR measurement results, a triple line component was confirmed. This indicates that the radicals are dispersed in (−)-PBS.

(2) Like the magnetic nanoemulsion of Surfactant 1 containing (±) -2 (n = 4), (±) -2 (n = 4) is dissolved in ether, and this solution is added to the surfactant. Attempts were made to prepare magnetic nanoemulsions by dispersing them in aqueous solution, and the stability of magnetic nanoemulsions was evaluated by EPR spectroscopy and DLS methods.
The concentration ratio between surfactant 1 and (±) -2 (n = 4) was [1 / (±) -2 (n = 4) = 10 mM / 5.0 mM]. The amount of radicals is half that of the surfactant. In the preparation method, 1 mL of a surfactant aqueous solution was added to a solution of (±) -2 (n = 4) dissolved in a small amount of ether, and then heated at 90 ° C. to remove the ether from the system. A nanoemulsion was prepared.
When ether was removed from the system, the color of the solution changed from yellow to clear. FIG. 34 shows the EPR spectrum and average particle diameter after preparation. As in the case of (±) -3, when the EPR spectrum was measured, a triple line component was confirmed.
This indicates that the radicals are dispersed in (−)-PBS.

(3) Surfactant 1 magnetic nanoemulsion containing (±) -2 (n = 8)
Attempt to prepare magnetic nanoemulsion by dissolving (±) -2 (n = 8) in ether and dispersing this solution in aqueous surfactant solution. The stability of magnetic nanoemulsion was evaluated by EPR spectroscopy and DLS method. (FIGS. 35 and 36).
The concentration ratio of surfactant 1 and (±) -2 (n = 8) was [1 / (±) -2 (n = 8) = 10 mM / 5.0 mM]. The amount of radicals is half that of the surfactant. In the preparation method, 1 mL of a surfactant aqueous solution was added to a solution of (±) -2 (n = 8) dissolved in a small amount of ether, and then heated at 90 ° C. to remove the ether from the system. A magnetic nanoemulsion was prepared.
When ether was removed from the system, the color of the solution changed from yellow to clear. The solution became cloudy within 20 minutes after preparation. From the result of DLS measurement, a peak derived from a large aggregate was observed at around 1000 nm 3 days after preparation.
These results indicate that the magnetic nanoemulsion of surfactant 1 encapsulating (±) -2 (n = 8) is very unstable in (−)-PBS.

(4) Magnetic nanoemulsion of surfactant 1 containing (±) -2 (n = 14)
(±) -2 (n = 14) is hardly soluble in water. Therefore, (±) -2 (n = 14) was dissolved in ether and this solution was dispersed in a surfactant aqueous solution to try to prepare a magnetic nanoemulsion. The stability of the magnetic nanoemulsion was compared with that of EPR spectroscopy. Evaluation was performed by the DLS method (FIGS. 37 and 38).
The concentration ratio between surfactant 1 and (±) -2 (n = 14) was [1 / (±) -2 (n = 14) = 1.0 × 10 ⁻² M / 1.0 × 10 ⁻² M] . In the preparation method, 1 mL of a surfactant aqueous solution was added to a solution of (±) -2 (n = 14) dissolved in a small amount of ether, and then heated at 90 ° C. to remove the ether from the system. A nanoemulsion was prepared.
When the ether was removed, the color of the solution changed from yellow to clear. From the results of DLS measurement, it was found that the particle size of the magnetic nanoemulsion after 2 days was increased compared with that immediately after preparation. Further, the color of the aqueous solution also changed from yellow transparent to cloudy. After 6 days, suspended matter was confirmed in the aqueous solution.
Since this aqueous solution could not pass through a 0.45 μmφ membrane filter, DLS measurement or the like was not performed.
From the above results, it is considered that the magnetic nanoemulsion of surfactant 1 encapsulating (±) -2 (n = 14) is unstable in (−)-PBS.

(5) Magnetic nanoemulsion of surfactant 1 containing (±) -2 (n = 16)
(±) -2 (n = 16) was dissolved in ether, and this solution was dispersed in a surfactant aqueous solution to try to prepare a yellow transparent magnetic nanoemulsion. The stability of the magnetic nanoemulsion was analyzed by EPR spectroscopy. And the DLS method (FIGS. 39 and 40).
As a result of EPR measurement, only a broad single line derived from aggregate formation was observed in (−)-PBS. In addition, the time course of the particle shape was 16 nm on the first day, 27 nm on the second day, and 37 nm on the sixth day, and gradually increased.
From this result, it was found that the magnetic nanoemulsion existed stably for 7 days.

(6) Surfactant 1 magnetic nanoemulsion containing (±) -2 (n = 18)
(±) -2 (n = 18) was dissolved in ether, and this solution was dispersed with an aqueous surfactant solution to prepare a yellow transparent magnetic nanoemulsion. The stability of the magnetic nanoemulsion was analyzed by EPR spectroscopy. And the DLS method (FIGS. 41 and 42).
As a result of EPR measurement, only a broad single line derived from aggregate formation was observed in (−)-PBS. In addition, the time course of the particle shape was 16 nm on the first day, 17 nm on the second day, and 24 nm on the 14th day, and gradually increased.
From this result, it was found that the magnetic nanoemulsion was stably present for 14 days. In addition, it was found from the results of DLS measurement that the association was difficult compared with the magnetic nanoemulsion of surfactant 1 encapsulating (±) -2 (n = 16).
This is because the surfactant 1 magnetic nanoemulsion containing (±) -2 (n = 18) is more than the magnetic nanoemulsion of surfactant 1 containing (±) -2 (n = 16). (-) Stable in PBS.

(7) Like the magnetic nanoemulsion of surfactant 1 encapsulating (±) -2 (n = 20), (±) -2 (n = 20) is dissolved in ether and this solution is added to the surfactant. An attempt was made to prepare a yellow transparent magnetic nanoemulsion by dispersing in an aqueous solution, and the stability of the magnetic nanoemulsion was evaluated by EPR spectroscopy and DLS method (FIGS. 43 and 44).
As a result of EPR measurement, only a broad single line derived from aggregate formation was observed in (−)-PBS. In addition, the time course of the particle shape was constant at 17 nm over 7 days.
From this result, the magnetic nanoemulsion of surfactant 1 encapsulating (±) -2 (n = 20) is similar to the magnetic nanoemulsion of surfactant 1 encapsulating (±) -2 (n = 18). In addition, it was found to be stable in (−)-PBS for 7 days or more.

界面活性剤4と界面活性剤5を用いて、(-)-PBS中で安定な(±)-2 (n = 12, 14, 16, 18 or 20)を内包した磁性ナノエマルションを調製することはできなかった。この理由としては、入れ子構造を形成するには、4と5のポリエチレングリコール鎖或いは、アルキル鎖の長さが短すぎると考えられる。 (8) Magnetic nanoemulsion composed of similar PEG surfactants

Prepare a magnetic nanoemulsion containing (±) -2 (n = 12, 14, 16, 18 or 20) that is stable in (-)-PBS using surfactant 4 and surfactant 5. I couldn't. The reason for this is considered that the lengths of polyethylene glycol chains 4 and 5 or alkyl chains are too short to form a nested structure.

(9) Conclusion
The average particle size of the surfactant 1 magnetic nanoemulsion containing (±) -2 (n = 18) was 16 nm (immediately after preparation). This value is the sum of the particle size (9.0 nm) of the micelle aqueous solution of Surfactant 1 and the molecular length (3.3 nm, calculated from MM2) of (±) -2 (n = 18) (6.6 nm). The value is almost equal to (15.6 nm).
In the case of surfactant 1 magnetic nanoemulsion containing (±) -2 (n = 20), the molecular length of (±) -2 (n = 20) is 3.4 nm, so the total (15.8 nm) It becomes. This value also agrees well with the result of DLS measurement.
From the above, we conclude that the combination of (±) -2 (n = 18) and 1 or (±) -2 (n = 20) and 1 is the best choice for producing a stable magnetic nanoemulsion. Attached.

1 Nanoemulsion particles 2 Organic nitroxide radical compound 3 Nonionic surfactant

Claims (7)

Formula (1):

(In the formula, R ¹ represents a linear alkyl group having 10 to 26 carbon atoms.)
An organic nitroxide radical compound represented by: It is a manufacturing method of the organic nitroxide radical compound of Claim 1, Comprising: Formula (2):

(In the formula, R ¹ represents a linear alkyl group having 10 to 26 carbon atoms.)
The manufacturing method which oxidizes the compound represented by these in presence of a bivalent copper compound.   Nanoemulsion particles comprising the organic nitroxide radical compound according to claim 1 and a nonionic surfactant in a molar ratio of 0.8: 1 to 1.2: 1, wherein nitroxide radicals are encapsulated.   The nanoemulsion particle according to claim 3, wherein the nonionic surfactant is a nonionic surfactant having a linear alkyl group having 10 to 26 carbon atoms. The nonionic surfactant is represented by the formula (3):

(In the formula, R ² represents a linear alkyl group having 10 to 26 carbon atoms. N is an integer of 2 or more.)
The nanoemulsion particles according to claim 3 or 4, which are polyoxyethylene alkyl ethers represented by:   The contrast agent for MRI containing the nanoemulsion particle of any one of Claims 3-5.   A drug delivery system carrier comprising the nanoemulsion particles according to any one of claims 3 to 5.

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