WO2021225113A1 - Method for concentrating ionic chemical species - Google Patents

Abstract

This method for concentrating an ionic chemical species is a method for concentrating a cation inside liposomes (R) formed from phospholipid bilayers (BLMs). The method for concentrating an ionic chemical species includes a step for dispersing liposomes in an ionic chemical species solution that includes both an anion and a cation and thereby trapping the anion and the cation inside the liposomes (R). The molar concentration of the anion in the ionic chemical species solution is at least two times the molar concentration of the cation, and the cation is concentrated inside the liposomes (R).

Description

Ionic species concentration method

The present invention relates to a method for concentrating ionic species.

A method for producing a nanoparticle composition in which a metal cation is encapsulated inside nanoparticles composed of a lipid bilayer such as a liposome has been proposed (see, for example, Patent Document 1). In this production method, a step of preparing a nanoparticle composition containing a vesicle-forming component and a water-soluble and non-lipophilic chelating agent surrounded by the vesicle-forming component, and a step of preparing the nanoparticle composition in a solution containing a metal cation are performed. By culturing, the step of encapsulating the metal cation inside the nanoparticle composition by allowing the movement of the metal cation through the film formed by the vesicle-forming component is included. According to this production method, metal cations can be encapsulated in nanoparticles without using an ionophore.

International Publication No. 2012/079582

By the way, there is a demand for a technique for concentrating ionic chemical species such as metal cations at a high concentration inside particles composed of a lipid bilayer such as liposomes.

The present invention has been made in view of the above reasons, and an object of the present invention is to provide a method for concentrating ionic species in particles composed of lipids, which can concentrate ionic species at a high concentration.

In order to achieve the above object, the ionic chemical species concentration method according to the present invention is used.
It is a method for concentrating ionic species by encapsulating ionic species in lipid particles formed from lipids.
It comprises a step of encapsulating the anion and the cation in the lipid particle by dispersing the lipid particle in an ionic species solution in which the anion and the cation coexist.
The molar concentration of either the anion or the cation in the ionic species solution is at least twice the molar concentration of the other, and the other is concentrated in the lipid particles.

According to the present invention, the molar concentration of either anion or cation in the ionic species solution is at least twice the molar concentration of the other, and the other is concentrated in the lipid particles. As a result, by increasing the molar concentration of one of the ionic species contained in the ionic species solution existing outside the lipid particles, the distribution of cations and anions to the lipid particles can be increased accordingly. can. Therefore, the distribution of one ionic species outside the lipid particle into the lipid particle with the other ionic species is promoted, and as a result, the lipid particle together with one ionic species. The concentration of the other ionic species enclosed therein can be increased.

It is a figure for demonstrating the encapsulation method of an ionic chemical species which concerns on embodiment of this invention. Is a diagram for explaining the permeation mechanism of the lipid bilayer of the ionic species according to the embodiment. It is a fluorescence image (left side) and a bright field image (right side) of the state of the evaluation sample according to Example 1 before addition of epirubicin. It is a fluorescence image (left side) and a bright field image (right side) of the state of the evaluation sample according to Example 1 immediately after addition of epirubicin. It is a fluorescence image (left side) and a bright field image (right side) of the state 15 minutes after the addition of epirubicin of the evaluation sample according to Example 1. It is a figure which shows the distribution of the fluorescence intensity in line AA of FIG. 2B of the evaluation sample which concerns on Example 1. FIG. It is a figure which shows the distribution of the fluorescence intensity in line AA of FIG. 2C of the evaluation sample which concerns on Example 1. FIG. Example 1 ClO the evaluation sample according to ₄ ^- is a 1.0 × ¹⁰ -3 mol / dm ³ fluorescence image in a state immediately after addition (left) and bright-field image (right). ClO the evaluation sample according to Example 1 ₄ ^- is a 1.0 × ¹⁰ -3 mol / dm ³ fluorescence images of the addition to from 15 minutes after the state (left) and bright-field image (right). Example 1 ClO the evaluation sample according to ₄ ^- is a 1.0 × ¹⁰ -3 mol / dm ³ after the addition of the after 40 minutes state of the fluorescent image (left) and bright-field image (right). It is a figure which shows the distribution of the fluorescence intensity in line AA of FIG. 4A of the evaluation sample which concerns on Example 1. FIG. It is a figure which shows the distribution of the fluorescence intensity in line AA of FIG. 4B of the evaluation sample which concerns on Example 1. FIG. It is a figure which shows the distribution of the fluorescence intensity in line AA of FIG. 4C of the evaluation sample which concerns on Example 1. FIG. It is a fluorescence image (left side) and a bright-field image (right side) of the state of the evaluation sample according to Example 2 before addition of epirubicin. It is a fluorescence image (left side) and a bright field image (right side) of the state of the evaluation sample according to Example 2 immediately after the addition of epirubicin. It is a fluorescence image (left side) and a bright field image (right side) of the state 15 minutes after the addition of epirubicin of the evaluation sample according to Example 2. It is a figure which shows the distribution of the fluorescence intensity in line BB of FIG. 6B of the evaluation sample which concerns on Example 2. FIG. It is a figure which shows the distribution of the fluorescence intensity in line BB of FIG. 6C of the evaluation sample which concerns on Example 2. FIG. Example 2 ClO the evaluation sample according to ₄ ^- is a 5.0 × ¹⁰ -3 mol / dm ³ fluorescence image in a state immediately after addition (left) and bright-field image (right). Example 2 ClO the evaluation sample according to ₄ ^- is a 5.0 × ¹⁰ -3 mol / dm ³ fluorescence images of the addition to from 15 minutes after the state (left) and bright-field image (right). Example 2 ClO the evaluation sample according to ₄ ^- is a 5.0 × ¹⁰ -3 mol / dm ³ fluorescence images of the addition to the after lapse of 40 minutes state (left) and bright-field image (right). It is a figure which shows the distribution of the fluorescence intensity in line BB of FIG. 8A of the evaluation sample which concerns on Example 2. FIG. It is a figure which shows the distribution of the fluorescence intensity in line BB of FIG. 8B of the evaluation sample which concerns on Example 2. FIG. It is a figure which shows the distribution of the fluorescence intensity in line BB of FIG. 8C of the evaluation sample which concerns on Example 2. FIG. It is a figure which shows the elapse time dependence after the addition of epirubicin of the ratio (fluorescence intensity ratio) of the fluorescence intensity on the outside of a liposome and the fluorescence intensity on the inside of a liposome according to Examples 1 and 2. It is a fluorescence image (left side) and a bright field image (right side) of the evaluation sample according to Example 3 before the addition of Rhodamine 6G. It is a fluorescence image (left side) and a bright field image (right side) of the evaluation sample according to Example 3 immediately after the addition of Rhodamine 6G. It is a fluorescence image (left side) and a bright field image (right side) of the state 15 minutes after the addition of the evaluation sample Rhodamine 6G according to Example 3. It is a figure which shows the distribution of the fluorescence intensity in the CC line of FIG. 11B of the evaluation sample which concerns on Example 3. FIG. It is a figure which shows the distribution of the fluorescence intensity in the CC line of FIG. 11C of the evaluation sample which concerns on Example 3. FIG. Example of evaluation samples of 3 BF ₄ ^- is a 1.0 × ¹⁰ -2 mol / dm ³ fluorescence image in a state immediately after addition (left) and bright-field image (right). Example of evaluation samples of 3 BF ₄ ^- is a 1.0 × ¹⁰ -2 mol / dm ³ fluorescence images of the addition to the after lapse 35 minutes state (left) and bright-field image (right). It is a figure which shows the distribution of the fluorescence intensity in the CC line of FIG. 13A of the evaluation sample which concerns on Example 3. FIG. It is a figure which shows the distribution of the fluorescence intensity in the CC line of FIG. 13B of the evaluation sample which concerns on Example 3. FIG. It is a fluorescence image (left side) and a bright field image (right side) of the evaluation sample according to Example 4 before the addition of Rhodamine 6G. It is a fluorescence image (left side) and a bright field image (right side) of the evaluation sample according to Example 4 immediately after the addition of Rhodamine 6G. It is a fluorescence image (left side) and a bright field image (right side) of the state 15 minutes after the addition of the evaluation sample Rhodamine 6G according to Example 4. It is a figure which shows the distribution of the fluorescence intensity in the DD line of FIG. 15B of the evaluation sample which concerns on Example 4. FIG. It is a figure which shows the distribution of the fluorescence intensity in the DD line of FIG. 15C of the evaluation sample which concerns on Example 4. FIG. Example 4 ClO the evaluation sample according to ₄ ^- is a 1.0 × ¹⁰ -2 mol / dm ³ fluorescence image in a state immediately after addition (left) and bright-field image (right). Example 4 ClO the evaluation sample according to ₄ ^- is a 1.0 × ¹⁰ -2 mol / dm ³ fluorescence images added to the post after 20 minutes state (left) and bright-field image (right). It is a figure which shows the distribution of the fluorescence intensity in the DD line of FIG. 17A of the evaluation sample which concerns on Example 4. FIG. It is a figure which shows the distribution of the fluorescence intensity in the DD line of FIG. 17B of the evaluation sample which concerns on Example 4. FIG. It is a figure which shows the elapse time dependence after addition of rhodamine 6G of the ratio (fluorescence intensity ratio) of the fluorescence intensity outside the liposome and the fluorescence intensity inside the liposome according to Examples 3 and 4. Is a fluorescence image of the state after the lapse of 15 minutes the ^{FAM 2-evaluation} sample according to Example 5 was added ^{1.0 × 10 -6 mol / dm 3} ( left) and bright-field image (right). It is a fluorescence image (left side) and a bright field image (right side) of the state 20 minutes after the addition of 1.0 × 10 ^-3 mol / dm ^{3 of BTPPA + of the} evaluation sample according to Example 5. It is a figure which shows the distribution of the fluorescence intensity in the EE line of FIG. 20A of the evaluation sample which concerns on Example 5. FIG. It is a figure which shows the distribution of the fluorescence intensity in the EE line of FIG. 20B of the evaluation sample which concerns on Example 5. FIG. It is a figure which shows the time dependence of the fluorescence intensity inside the liposome according to Examples 6 to 8 ^{after the addition of FAM 2-.} 6 is a fluorescence image of a state 15 minutes after the addition of 5.0 × ^10-7 mol / dm ^{3 of FAM-AAA 5-} of the evaluation sample according to Example 9. 6 is a fluorescence image of a state 10 minutes after the addition of 5.0 × 10 ^-4 mol / dm ^{3 of BTPPA + of the} evaluation sample according to Example 9. It is a figure which shows the distribution of the fluorescence intensity in the FF line of FIG. 23A of the evaluation sample which concerns on Example 9. FIG. It is a figure which shows the distribution of the fluorescence intensity in the FF line of FIG. 23B of the evaluation sample which concerns on Example 9. FIG. It is a figure which shows the time dependence of the fluorescence intensity inside the liposome according to Examples 9 to 11 ^{after the addition of FAM-AAA 5-.}

Hereinafter, the method for concentrating ionic chemical species according to the embodiment of the present invention will be described with reference to the drawings. The method for concentrating ionic species according to the present embodiment is a method for concentrating ionic species in lipid particles formed from lipids. Here, the ionic species are anions and cations. In addition, lipid particles represent liposomes or micelles, and liposomes represent unilamella vesicles or multilamella vesicles (multilamella vesicles) composed of a single layer of lipid bilayer.

This ionic species concentration method includes a step of encapsulating anions and cations in the lipid particles by dispersing the lipid particles in an ionic species solution in which anions and cations coexist. The molar concentration of either the anion or the cation in the ionic species solution is at least twice the molar concentration of the other, and the other ionic species is concentrated in the lipid particles.

In the method for concentrating an ionic species according to the present embodiment, for example, as shown in FIG. 1A, a cation or an anion which is an ionic species is encapsulated in a liposome R which is a unilamella vesicle formed from a phospholipid bilayer BLM. do.

Examples of the lymphocytes forming the liposome R include PC (1,2-dioleoil-sn-glycero-phosphocholine), 1,2-dioreoil phosphatidylcholine, 1,2-dipalmitylphosphatidylcholine, 1,2-dimylistylphosphatidylcholine, and the like. 1,2-Dystearoylphosphatidylcholine, 1-oleoil-2-palmitoylphosphatidylcholine, 1-oleoil-2-stearoylphosphatidylcholine, 1-palmitoyle-2-oleoil phosphatidylcholine, 1-stearoyl-2-oleoil phosphatidylcholine and the like , 1,2-diore oil phosphatidylethanolamine, 1,2-dipalmityl phosphatidylethanolamine, 1,2-dimylistyl phosphatidylethanolamine, 1,2-dystearoyl phosphatidylethanolamine, 1-oleyl-2-palmityl Phosphatidylethanolamine, 1-oleoil-2-stearoylphosphatidylethanolamine, 1-palmityl-2-oleoil phosphatidylethanolamine, 1-stearoyl2-oleoil phosphatidylethanolamine, N-succinyldiore oil phosphatidylethanolamine, etc. Phosphatidylethanolamine, 1,2-dioreoil phosphatidylserine, 1,2-dipalmityl phosphatidylserine, 1,2-dimylistyl phosphatidylserine, 1,2-dystearoyl phosphatidylserine, 1-oleyl-2-palmityl phosphatidyl Serine, 1-oleoil-2-stearoylphosphatidylserine, 1-palmitoyle 2-oleoil phosphatidylserine, 1-stearoyl-2-oleoil phosphatidylserine and other phosphatidylserine, 1,2-dioreoil phosphatidylglycerin, 1, 2-Dipalmityl phosphatidyl glycerin, 1,2-dimylistyl phosphatidyl glycerin, 1,2-dystearoyl phosphatidyl glycerin, 1-oleoil-2-palmityl phosphatidyl glycerin, 1-oleoil-2-stearoyl phosphatidyl glycerin, 1-palmityl -2-Ole oil phosphatidyl glycerin, 1-stearoyl-2-ole oil phosphatidyl glycerin such as phosphatidyl glycerin, phosphatidyl ethanolamine-N- [methoxy (polyethylene) Recall) -1000], phosphatidylethanolamine-N- [methoxy (polyethylene glycol) -2000], phosphatidylethanolamine-N- [methoxy (polyethylene glycol) -3000], phosphatidylethanolamine-N- [methoxy (polyethylene glycol)) -5000] and the like, PEGylated phospholipids and the like.

The phospholipids that form liposome R include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), COOL (cholesterol), and DSPE-PEG-2000 (1,2-distearoyl-n-). Glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000]), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl- sn-glycero-3-phosphocholine), DSPE-PEG2000-TATE, (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] -TATE), HSPC ( Contains compounds selected from the group consisting of purified hydrogenated soybean phosphatidylcholine).

Further, as the cation, at least one selected from a group of a plurality of types of anthracycline antibiotics including the hydrophobic cations epirubicin, daunorubicin, doxorubicin, amrubicin, idarubicin, balrubicin, acralvisi, pyrarubicin, and mitoxantrone. Anthracycline antibiotics can be adopted. The cation is selected from hydrophobic cations such as amines, rhodamines, cyanines, phosphonium cations, arsonium cations, imidazoliums, primary, secondary, tertiary or quaternary ammoniums. At least one ion can be employed. Further, as the cation, a local anesthetic having an amine structure, specifically, dibucaine, mepivacaine, bupivacaine, levobupivacaine, ropivacaine, procaine, tetracaine, prlocaine, cocaine, ambroxol, phenylpiperidin derivative, morphinan derivative At least one selected from the group can be adopted. Furthermore, as cations, antiallergic agents having an amine structure, antiarrhythmic agents, antidepressants, antihypertensive agents, cardiotonics, muscle relaxants, analgesics, antimalaria agents, hypotensive agents, nerve blockers, centralized agents Antihypertensive drugs, ovulation inducers, neuropsychiatric drugs, antidigestive ulcer drugs, vitamin replacement drugs, antihypertensive drugs, metabolic enzyme matrix drugs, cardiovascular drugs, antineoplastic drugs, specifically, azelastin, aprinzin, Amiodaron, amitriptilin, amlogipin, alprenolol, bopindolol, pindrol, bisopranolol, ambinonium, isoxpurin, imibramine, indenolol, ethylmorphine, ethyrefrin, edrophonium, ephedrine, emerison, oxycodon, oxybuprokine, orsibrenerol, carteol Guanabenz, clocaplamin, chronidine, clofedalol, clopelastin, tamoxiphene derivative, chromipramine, chromiferamine, ketamine, ketotiphen, codeine, phenethylamine derivative, dystigmine, ahen alkaloid derivative, diphenhydramine derivative, cyproheptazine derivative, sibenzoline, dilazep, benzodiazep Setraxate, tamthrosin, thiaprid, thiamine, tizanidine, tipepidin, thymepidium, thymorol, temocapril, tervinafin, prazosin derivative, doxaplum, hydradinophthalazine derivative, donepezil, dopamine, trimetadidin, trimetokinol, trimebtin, nafamostat , Dihydropyridine derivative, phizostigmine derivative, tryptyrin derivative, 2-amino-1-phenylethanol derivative, thiazolidinedione derivative, hydroxydine, biperidene, phyzostigmine derivative, pyrenzepine, binca alkaloid antineoplastic drug, hexofenazine, propranolol derivative, butyl Scobolamine, butenafin, butropium, morphine derivative, prazosin derivative, flavoxate, progalvazine, propaphenone, propiverine, propranolol derivative, bromhexin derivative, phenothiazine derivative, propranolol derivative, betahistin, phenylpiperidin derivative, phenylalkylamine derivative, perphenazine, velverin Benzetonium, benzerazide, homochlorcyclidine, phenethylamine derivative, mabrochili At least one selected from mosapride, imidazole derivative, mexiletine, meclophenoxate, methylephedrine, methylergometrine, mefloquine, mepenzolate, mosapride, ranitidine, labetalol, and ritodrine can be employed. Alternatively, the cation may be at least one dye selected from a group of dyes having a rhodamine skeleton, which is a hydrophobic cation. Further, as the cation, at least one selected from diarylmethane, triarylmethane, an azo compound and a nitrogen-containing heterocyclic compound may be adopted.

Further, as cations, metal ions (for example, alkali metal ions such as ^{Li +} , Na ⁺ , K ⁺ , etc., alkaline earth metal ions such as ^{Mg 2+} , Ca ^{2+ , Cu 2+} , Mn ²⁺ , Fe ^{2+, which are hydrophilic cations, are used.} Etc.), ammonium ions, H ⁺ , basic amino acids (lysine, arginine, etc.) or at least one selected from cationic oligopeptides comprising them may be adopted.

The ionic species concentration method according to the present embodiment includes a step of encapsulating the cation or anion in the liposome R by dispersing the liposome R in an ionic species solution in which an anion and a cation coexist.

Anions include hydrophilic anions such as halide ion, sulfate ion, nitrate ion, phosphate ion, nucleic acid consisting of 3 or less nucleic acid bases, sulfonic acids, carboxylic acids, nucleic acids or acidic amino acids (aspartic acid, glutamate). Alternatively, at least one selected from anionic oligopeptides comprising fluoresceins and fluoresceins can be adopted. As the halide ion, at least one selected from the group of ^{Cl −} , Br ⁻ , and I ^{− can be adopted.} Further, as an anion, a hydrophobic anion, borate anions of the _{^{ion, ClO 4 -, PF 6 -}} , BF 4 -, is selected aromatic sulfonic acids carboxylic acids, alkyl sulfonic acids, from ions of alkyl carboxylic acids At least one can be adopted. Further, as the anion, at least one selected from a sulfonic acid derivative / carboxylic acid derivative having an alkyl group and an aryl group, and tetraphenylborate may be adopted.

Here, the process of encapsulation of anions and cations existing in the outer side S2 of the liposome R into the inner side S1 of the liposome R will be described. First, between the ionic species solution and the phospholipid bilayer BLM of liposome R, as shown in FIG. 1B, the adsorption equilibrium of the cation to the phospholipid bilayer BLM surface and the anion and cation phospholipid bilayer There are three equilibrium states: a distribution equilibrium within the BLM and an ion pair formation equilibrium from anions and cations within the phospholipid bilayer BLM. The adsorption equilibrium state on the surface of the phospholipid bilayer BLM and the ion distribution equilibrium state in the phospholipid bilayer BLM are independently generated. That is, the cations adsorbed on the surface of the phospholipid bilayer BLM do not invade the inner S1 of the liposome R. On the other hand, equal amounts of cations and anions are distributed from the ionic species solution W existing on the outer side S2 of the liposome R into the phospholipid bilayer BLM of the liposome R so as to maintain electrical neutrality. Then, the cations and anions distributed to the phospholipid bilayer BLM are distributed to the inner S1 of the phospholipid bilayer BLM. Here, the distribution of cations and anions from the outer S2 of the liposome R into the phospholipid bilayer BLM is based on the cation concentration and anion concentration in the outer S2 of the liposome R and the cation concentration and anion concentration in the phospholipid bilayer BLM. It is determined by the distribution constant represented. Therefore, when the cation concentration and the anion concentration in the outer S2 of the liposome R are increased, the cation and anion concentrations in the phospholipid bilayer BLM increase, and the cation from the phospholipid bilayer BLM to the inner S1 of the liposome R becomes The amount of anions enclosed also increases.

Therefore, in the present embodiment, the molar concentration of the anion in the ionic species solution is set to be at least twice the molar concentration of the cation. As a result, the distribution of cations and anions between S2 on the outer side of the liposome R and the phospholipid bilayer BLM becomes larger than in the case where the molar concentration of the anions is the same as the molar concentration of the cations. Is promoted in the phospholipid bilayer BLM. As a result, the distribution of the cation and anion from the phospholipid bilayer BLM to the inner S1 of the lipome R is increased, and the cation is encapsulated in the inner S1 of the liposome R at a high concentration. The anion concentration added to the ionic species solution W at a high concentration is a molar amount of the salt contained in the buffer solution added to the ionic species solution W in order to avoid bursting or contraction of the liposome R due to osmotic pressure. It is preferable that the concentration is (for example, 0.1 mol / dm ^{3) or less.}

Further, in the present embodiment, the molar concentration of the cation in the ionic species solution may be twice or more the molar concentration of the anion. As a result, the distribution of cations and anions between S2 on the outer side of the liposome R and the phospholipid bilayer BLM becomes larger than in the case where the molar concentration of the cation is the same as the molar concentration of the anion, and the anion Is promoted in the phospholipid bilayer BLM. As a result, the distribution of cations and anions from the phospholipid bilayer BLM to the inner S1 of the lipome R is increased, and the anions are encapsulated in the inner S1 of the liposome R at a high concentration.

As described above, according to the ionic species concentration method according to the present embodiment, the molar concentration of anions in the ionic species solution is at least twice the molar concentration of cations. As a result, by increasing the molar concentration of the anion contained in the ionic species solution existing outside the liposome R, the distribution of the cation and the anion to the phospholipid bilayer BLM can be increased accordingly. Therefore, the distribution of the anion present on the outer side S2 of the liposome R to the phospholipid bilayer BLM accompanied by the cation is promoted, and as a result, the concentration of the cation enclosed in the liposome R inner side S1 together with the anion can be increased. can.

Although the embodiments of the present invention have been described above, the present invention is not limited to the configuration of the above-described embodiments. For example, the lipid particles may be micelles formed from phospholipids. In this case, the micelle formed from the phospholipid may be dispersed in the ionic species solution in which the anion and the cation coexist to enclose the cation in the micelle. In this case, the molar concentration of the anion in the ionic species solution may be at least twice the molar concentration of the cation.

The present invention enables various embodiments and modifications without departing from the broad spirit and scope of the present invention. Moreover, the above-described embodiment is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is indicated not by the embodiment but by the claims. And various modifications made within the scope of the claims and within the equivalent meaning of the invention are considered to be within the scope of the invention.

Hereinafter, the present invention will be specifically described with reference to Examples 1 to 11. First, a method for producing liposomes used in each example will be described. First, a lipid thin film was formed on the agarose film, and then a phosphate buffer solution (pH: 7.0) was added and hydrated. Here, the agarose film is prepared at about 40 ° C. after dropping a 1 wt% agarose aqueous solution onto a disk-shaped cover glass (manufactured by Matsunami Glass Ind. Co., Ltd.) having a diameter of 22 mm and a thickness of 0.12 to 0.17 mm. It was produced on a cover glass by leaving it in a heated state for 1 hour. For the lipid thin film, 15 μL of a chloroform solution of lipid was dropped onto the above-mentioned agarose film using a microdispenser (25 μL, manufactured by DRUMMOND SCIENTIFIC CO.), And then the cover glass was placed in a desiccator in a negative pressure environment. It was prepared by leaving it for a while to dry. Here, the lipid solution contains 126.8 mg of PC (1,2-dioleoil-sn-glycero-phosphocholine) (manufactured by Tokyo Chemical Industry Co., Ltd.) having a purity of more than 97.0% and cholesterol (manufactured by Nacalai Tesque, Inc.) 63. .4 mg was prepared by dissolving in 20 ml of chloroform (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). The molar ratio of PC to cholesterol in this lipid solution is approximately 1: 1. ^{Then, in Examples 1 and 2, 160 μL of a phosphate buffer solution having a molar concentration of 0.01 mol / dm 3} was added to the lipid thin film formed on the agarose film, and then the liposome was left in a dark place for 3 hours to form the liposome. Made. ^{Further, in Examples 3 and 4, 160 μL of a phosphate buffer solution having a molar concentration of 0.1 mol / dm 3} was added to the lipid thin film formed on the agarose film, and then the liposomes were left in a dark place for 3 hours to disperse the liposomes. Made.

Next, a method for preparing a sample for evaluation by immobilizing the liposome on a cover glass coated with a cell membrane modifier (BAM: Biocompatible Anchor for cell Membrane) will be described. First, a disk-shaped cover glass (manufactured by Matsunami Glass Ind. Co., Ltd.) having a diameter of 22 mm and a thickness of 0.12 to 0.17 mm was subjected to silane treatment. Silane treatment is obtained by mixing APTS (3-aminopropyltriethoxysilane) (manufactured by Shin-Etsu Chemical Industries, Ltd.) and ethanol (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) in a volume ratio of 1: 5. This was done by immersing the cover glass in the resulting solution for 2 hours. After the silane treatment, the cover glass was washed with methanol and dried. Then, a dimethylsulfoxide solution of a polyethylene glycol modifier was dropped onto a 200 μL cover glass, left to stand for 1 hour, and then the surface of the cover glass was washed with distilled water to prepare a cover glass coated with BAM. Here, dimethyl sulfoxide solutions, polyethylene glycol modifier (SUNBRIGHT (R) OE-040CS: NOF Co., Ltd.), dimethylsulfoxide such that the molar concentration of 10 mmol / dm ³ dimethylsulfoxide (Fuji Film Wako Pure Chemical stock Made by dissolving in (manufactured by the company).

Next, the cover glass coated with BAM was made into a hole of Aznol Petri dish (manufactured by AS ONE Corporation) with a diameter of 40 mm and a height of 13.5 mm in which a hole with a diameter of 18 m was drilled in the bottom wall. It was adhered to the Aznol petri dish so as to cover it from the lower side. Subsequently, using a micropipette, 100 μL of a phosphate buffer solution containing the liposomes formed on the agarose film was weighed and dropped onto a cover glass coated with BAM to coat the liposomes with BAM. Fixed to the cover glass.

Next, the evaluation method of the evaluation sample according to each example will be described. In each example, a confocal laser scanning microscope (FLOUVIEW (registered trademark) FV10i: manufactured by Olympus Corporation) was used to observe the change over time in the fluorescence intensity of the evaluation sample. Here, as the excitation light source of the evaluation sample, a laser light source of a laser microscope having an oscillation wavelength of 473 nm was used. Further, an optical filter having a transmission wavelength band of 490 nm to 590 nm was used, and the intensity of the light emitted from the evaluation sample and transmitted through the optical filter was measured as the fluorescence intensity.

In Example 1, 50 μL of a concentration of 0.01 mol / dm ³ phosphate buffer containing epirubicin hydrochloride was added to the evaluation sample so that the molar concentration of epirubicin was 1.7 × 10 ^-5 mol / dm ^3. bottom. Then, in a state of focusing on the liposomes contained in the evaluation sample, the fluorescence intensities were observed immediately after the addition and 15 minutes after the addition. As shown in FIGS. 2A and 2B, when epirubicin hydrochloride was added, fluorescence from the cation epirubicin was observed on the outside of the liposome and near the surface of the liposome. In particular, as shown in FIG. 3A, it was found that the fluorescence intensity near the surface of the liposome was increased as compared with other regions. This is considered to be due to the adsorption equilibrium state between the liposome surface and the phosphate buffer solution containing epirubicin. On the other hand, as shown in FIG. 2B, almost no fluorescence from epirubicin was observed inside the liposome. Then, even after 15 minutes had passed since the addition of epirubicin hydrochloride, as shown in FIGS. 2C and 3B, almost no increase in fluorescence intensity from epirubicin was observed inside the liposome.

Then, in Example 1, after a lapse 17min after the addition of epirubicin hydrochloride, ClO ₄ ^- so that the molar concentration of 1.0 × ¹⁰ -3 mol / dm ³ of, 0 containing NaClO _4. Only 50 μL of 01 mol / dm ³ phosphate buffer was added to the evaluation sample. Then, the fluorescence intensities were observed immediately after the addition, 15 minutes after the addition, and 40 minutes after the addition. As shown in FIGS. 4A to 4C and 5A to 5C, it was observed that after the addition of epirubicin hydrochloride, the fluorescence from epirubicin inside the liposome increased with the passage of time. This is outside of the liposome ClO ₄ is an anion ^- by is added, epirubicin into liposomes lipid bilayer and ClO ₄ ^- epirubicin distributed to the distribution and lipid bilayer and ClO ₄ ^- of liposomes It is probable that the invasion was promoted to the inside.

In Example 2, 50 μL of 0.01 mol / dm ³ phosphate buffer containing epirubicin hydrochloride was prepared so that the molar concentration of epirubicin was 1.7 × 10 ^-5 mol / dm ^{3, as in Example 1.} Was added to the evaluation sample. Then, in a state of focusing on the liposomes contained in the evaluation sample, the fluorescence intensities were observed immediately after the addition and 15 minutes after the addition. As shown in FIGS. 6A, 6B and 7A, when epirubicin hydrochloride was added, fluorescence from the cation epirubicin was observed on the outside of the liposome and near the surface of the liposome. On the other hand, as shown in FIGS. 6B and 7A, almost no fluorescence from epirubicin was observed inside the liposome. Then, even after 15 minutes had passed since the addition of epirubicin hydrochloride, as shown in FIGS. 6C and 7B, almost no increase in fluorescence intensity from epirubicin was observed inside the liposome.

Next, in Example 2, after a lapse 17min after the addition of epirubicin chloride, ClO ₄ ^- so that the molar concentration of 5.0 × ¹⁰ -3 mol / dm ³ of, 0 containing NaClO ₄ Only 50 μL of 0.01 mol / dm ³ phosphate buffer was added to the evaluation sample. That is, phosphate buffer ClO ₄ outside the liposomes as compared to Example 1 ^- molar concentration of was set to be 5 times. Then, the fluorescence intensities were observed immediately after the addition, 15 minutes after the addition, and 40 minutes after the addition. As shown in FIGS. 8A to 8C and FIGS. 9A to 9C, as in Example 1, it was observed that the fluorescence from epirubicin inside the liposome increased with the passage of time after the addition of epirubicin hydrochloride. Was done. FIG. 10 shows the elapsed time dependence of the ratio of the fluorescence intensity outside the liposome to the fluorescence intensity inside the liposome (hereinafter referred to as “fluorescence intensity ratio”) for the evaluation samples according to Examples 1 and 2. The result is shown. Fig As shown in 10, the second embodiment according to the ClO ₄ ^- increase of the fluorescence intensity inside the liposomes after the addition, Example 2 ClO ₄ as compared with ^- compared to that of the added amount is less Example 1 It turned out to be bigger. Therefore, ClO ₄ is an anion ^- A greater amount of it can be seen that encapsulation in inner liposome epirubicin a cation is promoted.

In Example 3, 50 μL of 0.1 mol / dm ³ phosphate buffer containing Rhodamine 6G was added to the evaluation sample so that the molar concentration of Rhodamine 6G was 1.0 × 10 ^-5 mol / dm ^3. Then, in a state of focusing on the liposomes contained in the evaluation sample, the fluorescence intensities were observed immediately after the addition and 15 minutes after the addition. As shown in FIGS. 11A and 11B, when rhodamine 6G chloride was added, fluorescence from the cation rhodamine 6G was observed on the outside of the liposome and near the surface of the liposome. In particular, as shown in FIG. 12A, it is considered that this is due to the adsorption equilibrium state in which the fluorescence intensity near the surface of the liposome is increased as compared with other regions. On the other hand, as shown in FIG. 11B, almost no fluorescence from rhodamine 6G was observed inside the liposome. Then, even after 15 minutes had passed since the addition of rhodamine 6G chloride, as shown in FIGS. 11C and 12B, almost no increase in fluorescence intensity from rhodamine 6G was observed inside the liposome.

Then, in the third embodiment, after a lapse 17min after the addition of Rhodamine 6G chloride, BF ₄ ^- as the molar concentration of the ^{1.0 × 10 -2 mol / dm 3} , 0 comprising NaBF ₄ . 50 μL of 1 mol / dm ³ phosphate buffer was added to the evaluation sample. Then, the fluorescence intensity was observed immediately after the addition and 35 minutes after the addition. As shown in FIGS. 13A and 13B and FIGS. 14A and 14B, after the addition of rhodamine 6G chloride, it was observed that the fluorescence from the rhodamine 6G inside the liposome increased with the passage of time. This is outside of the liposome BF ₄ an anion ^- by was added, Rhodamine 6G and BF into liposomes of lipid bilayer ₄ ^- Rhodamine 6G distributed and distributed in the lipid bilayer and BF ₄ ^- of It is considered that the invasion into the inside of the liposome was promoted.

In Example 4, as in Example 3, a phosphate buffer containing Rhodamine 6G chloride at a concentration of 0.1 mol / dm ^{3 so that the molar concentration of Rhodamine 6G is 1.0 × 10-5} mol / dm ^3. Only 50 μL of the solution was added to the evaluation sample. Then, in a state of focusing on the liposomes contained in the evaluation sample, the fluorescence intensities were observed immediately after the addition and 15 minutes after the addition. As shown in FIGS. 15A, 15B and 16A, almost no fluorescence from rhodamine 6G was observed immediately after the addition. Then, as shown in FIGS. 15C and 16B, fluorescence from rhodamine 6G, which is a cation, was observed on the outside of the liposome and near the surface of the liposome 15 minutes after the addition of rhodamine 6G chloride. However, the fluorescence from rhodamine 6G hardly increased inside the liposome even 15 minutes after the addition of rhodamine 6G chloride.

Next, in Example 4, after a lapse 17min after the addition of Rhodamine 6G chloride, ClO ₄ ^- so that the molar concentration of 1.0 × ¹⁰ -2 mol / dm ³ of, including NaClO ₄ Only 50 μL of 0.1 mol / dm ³ phosphate buffer was added to the evaluation sample. That is, as an anion, BF ₄ in Example 3 ^- in the same concentration as ClO ₄ ^- was added to the outside of the liposomes. Then, the fluorescence intensity was observed immediately after the addition and 20 minutes after the addition. As shown in FIGS. 17A and 17B and FIGS. 18A and 18B, as in Example 3, after adding rhodamine 6G chloride, the fluorescence from rhodamine 6G inside the liposome increases with the passage of time. The situation was observed. FIG. 19 shows the results of measuring the elapsed time dependence of the fluorescence intensity inside the liposomes for the evaluation samples according to Examples 3 and 4. As shown in FIG. 19, it was found that the rate of increase in fluorescence intensity inside the liposome after _{ClO 4} ^- _{addition according to Example 4 was larger than that after BF 4} ^{-addition in Example 3.} Here, rhodamine 6G and ClO ₄ ^- are distributed equilibrium constant K _D = when dispensed from the outside of the liposome to the membrane of liposomes _{(C R6G, BLM C X,} BLM) / (C R6G, W C X, W) ^{_{(X = BF 4 -, ClO}} 4 -) whereas 1.1, rhodamine 6G and BF ₄ ^- the distribution equilibrium constant, K _D, when the dispensing from the outside of the liposome to the membrane of liposomes 0. 31. That is, the results shown in FIG. 19, in the lipid bilayer of the liposome, rhodamine 6G and BF ₄ ^- as compared to the combination of a rhodamine 6G ClO ₄ ^- towards the combination of is partitioned phospholipid bilayer BLM It is thought that the ease is reflected. That is, if the cation is rhodamine 6G, ClO ₄ ^- as the anion rather adopting it, it can be seen that the encapsulation in the inside of the liposomes rhodamine 6G is promoted.

In Example 5, fluorescein molar concentration of ^{(FAM 2-)} are formed so that ^{1.0 × 10 -6 mol / dm 3} , sodium fluorescein ^{(FAM 2- 2Na +) 1.0 ×} 10 -4 including 50 μL of mol / dm ³ phosphate buffer was added to the evaluation sample. Then, in a state of focusing on the liposomes contained in the evaluation sample, the fluorescence intensity 15 minutes after the addition was observed. As shown in FIGS. 20A and 21A, almost no fluorescence from ^{FAM 2-was observed inside the liposome.}

Then, in Example ^{5, FAM} 2-2Na ⁺ after the addition of the after a lapse 17Min, bis molarity 1.0 × ¹⁰ -3 mol / dm of (triphenylphosphoranylidene) ammonium (BTPPA ⁺⁾ at ^3, bis (triphenylphosphoranylidene) ammonium chloride ^- was added 1.0 × ¹⁰ -4 mol / dm ³ phosphate buffer 50μL containing the evaluation sample (BTPPA ⁺ Cl). Then, the fluorescence intensity 20 minutes after the addition was observed. As shown in FIGS. 20B and 21B, ^{fluorescence from FAM 2-} inside the liposome was observed. This is because the hydrophobic cation BTPPA ⁺ was added to the outside of the liposome, to the liposome lipid bilayer ^{FAM 2} and BTPPA ⁺ distribution and ^FAM were distributed into the lipid bilayer ² and BTPPA ⁺ It is considered that the invasion into the inside of the liposome was promoted.

FIG. 22 shows the results of measuring the elapsed time dependence of the fluorescence intensity inside the liposomes for the evaluation samples according to Examples 6 to 8. In Examples 6 to 8, ^{the molar concentration of FAM 2- and} the molar concentration of the phosphate buffer were 1.0 × ^10-6 mol / dm ³ , 1.0 ×, respectively, as in Example 5. It was ^{adjusted to} 10 -4 mol / dm ³ . However, in Example ^6-8, the molar concentration of BTPPA ⁺ added after a lapse 17min after the addition of ^FAM 2-2Na ^{+, respectively, 5.0 × 10 -4 mol / dm} 3, 1.0 × It was ^{adjusted to be} 10 -4 mol / dm ³ and 5.0 × 10 ^-5 mol / dm ³ . As shown in FIG. 22, in Examples 6 to 8, an increase in fluorescence intensity from ^{FAM 2-} inside the liposome was observed after the addition of ^{BTPPA +.}

In Example 9, so that the molar concentration of fluorescein ^{(FAM 2-)} nucleic acid composed of modified three adenine at ^(FAM-AAA 5- 5Na ⁺⁾ is 5.0 × ¹⁰ -7 mol / dm ³ in, it was added 1.0 × ¹⁰ -4 mol / dm ³ phosphate buffer 50μL containing a nucleic acid-sodium ^(FAM-AAA 5- 5Na ⁺⁾ in the evaluation sample. Then, in a state of focusing on the liposomes contained in the evaluation sample, the fluorescence intensity 15 minutes after the addition was observed. As shown in FIGS. 23A and 24A, almost no fluorescence from ^{FAM 2-was observed inside the liposome.}

Then, in Example 9, in after 17min elapsed since the addition of ^FAM-AAA 5- 5Na ^+, so that the molar concentration of BTPPA ⁺ is ^{1.0 × 10 -3 mol / dm 3} , BTPPA + Cl 50 μL of 1.0 × 10 ^-4 mol / dm ^{3 phosphate buffer containing −} was added to the evaluation sample. Then, the fluorescence intensity 10 minutes after the addition was observed. As shown in FIGS. 23B and 24B, ^{fluorescence from FAM 2-} inside the liposome was observed. This is because the BTPPA ⁺ a hydrophobic cation to the outside of the liposomes was added, FAM-AAA ⁵ distributed to ^{FAM-AAA 5-and} BTPPA ⁺ distribution and lipid bilayer of the liposome lipid bilayer ^- and BTPPA ⁺ intrusion into the inside of the liposome is considered because it was promoted. Then, it was found that even a molecule having a relatively large molecular weight such as FAM-AAA ^5- can be introduced into the liposome by utilizing the concentration gradient of the hydrophobic cation.

FIG. 25 shows the results of measuring the elapsed time dependence of the fluorescence intensity inside the liposomes for the evaluation samples according to Examples 9 to 11. In Examples 10 to 11, ^{the molar concentration of FAM-AAA 5- and} the molar concentration of the phosphate buffer were 5.0 × ^10-7 mol / dm ³ , 1. It was set to 0 × 10 ^-4 mol / dm ³ . However, in Examples 10 and 11, the molar concentration of BTPPA ⁺ added after a lapse 17min after the addition of ^FAM-AAA 5- 5Na ^{+, respectively, 2.5 × 10 -4 mol / dm} 3, 1. It was set to 0 × 10 ^-4 mol / dm ³ . As shown in FIG. 25, in all of Examples 9 to 11, an increase in fluorescence intensity from ^{FAM 2-} inside the liposome was observed after the addition of ^{BTPPA +.} In addition, no difference in the rate of increase in fluorescence intensity from ^{FAM 2-} could be confirmed due to the difference in the molar concentration of ^{BTPPA +.}

This application is based on Japanese Patent Application No. 2020-082512, which was filed on May 8, 2020. The specification, claims and the entire drawings of Japanese Patent Application No. 2020-082512 are incorporated herein by reference.

The present invention is suitable for introducing ionic drugs, nucleic acid drugs, etc. into cells, and for producing exosomes for drug delivery containing ionic drugs, nucleic acid drugs, etc.

BLM: phospholipid bilayer, R: liposome, S1: inside of liposome, S2: outside of liposome, W: ionic species solution

Claims (11)

It is a method for concentrating ionic species by encapsulating ionic species in lipid particles formed from lipids.
It comprises a step of encapsulating the anion and the cation in the lipid particle by dispersing the lipid particle in an ionic species solution in which the anion and the cation coexist.
The molar concentration of either the anion or the cation in the ionic species solution is at least twice the molar concentration of the other, and the other is concentrated in the lipid particles.
Ionic species concentration method. The lipid particles are lamella vesicles formed from a phospholipid bilayer.
The cation is a hydrophobic cation and
The molar concentration of the anion in the ionic species solution is 100 times or more the molar concentration of the cation.
The method for concentrating an ionic chemical species according to claim 1. The molar concentration of the anion contained in the ionic species solution is equal to or less than the molar concentration of the salt contained in the buffer solution added to the ionic species solution.
The method for concentrating an ionic chemical species according to claim 2. The cation is at least one anthracycline antibiotic selected from the group of a plurality of anthracycline antibiotics including epirubicin, daunorubicin, doxorubicin, amrubicin, idarubicin, barrubicin, aclarubicin, pirarubicin, and mitoxantrone.
The method for concentrating an ionic chemical species according to any one of claims 1 to 3. The cation is at least one dye selected from the group of dyes having a rhodamine skeleton.
The method for concentrating an ionic chemical species according to any one of claims 1 to 3. The _{^{anion, Cl -, Br -, BF}} 4 -, ClO 4 - is at least one selected from the group of,
The method for concentrating an ionic chemical species according to any one of claims 1 to 5. The lipid particles are lamella vesicles formed from a phospholipid bilayer.
The anion is a hydrophilic anion and
The cation is a hydrophobic cation and
The molar concentration of the cation in the ionic species solution is 100 times or more the molar concentration of the anion.
The method for concentrating an ionic chemical species according to claim 1. The molar concentration of the anion contained in the ionic species solution is equal to or less than the molar concentration of the salt contained in the buffer solution added to the ionic species solution.
The method for concentrating an ionic chemical species according to claim 7. The anion is such as a halide ion, a sulfate ion, a nitrate ion, a phosphate ion, a nucleic acid consisting of three or less nucleic acid bases, sulfonic acids, carboxylic acids or anionic oligopeptides such as polyaspartic acid and polyglutamic acid. At least one selected from polyanions and fluoresceins,
The method for concentrating an ionic chemical species according to any one of claims 1, 7, and 8. The cation is at least one selected from the group of amines, rhodamines, cyanines, phosphonium cations, arsonium cations, imidazoliums, primary, secondary, tertiary or quaternary ammoniums.
The method for concentrating an ionic chemical species according to any one of claims 1, 7 to 9. The lipid particles are micelles formed from phospholipids.
The method for concentrating an ionic chemical species according to claim 1.

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