Method for concentrating ionic chemical species

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

[Technical Field]

[0001] The present invention relates to a method for concentrating ionic chemical species. [Background technology]

[0002] A method has been proposed for producing nanoparticle compositions in which metal cations are encapsulated inside nanoparticles consisting of a lipid bilayer, such as liposomes (see, for example, Patent Document 1). This method includes the steps 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 culturing the nanoparticle composition in a solution containing metal cations, thereby enabling the movement of metal cations that permeate the membrane formed by the vesicle-forming component and encapsulating the metal cations inside the nanoparticle composition. According to this method, metal cations can be encapsulated in nanoparticles without using ionophores. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2012 / 079582 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Incidentally, there is a need for technology to concentrate ionic chemical species, such as metal cations, to a high concentration inside particles made of lipid bilayers, such as liposomes.

[0005] This invention has been made in view of the above-mentioned reasons, and aims to provide a method for concentrating ionic chemical species in lipid particles to a high concentration. [Means for solving the problem]

[0006] To achieve the above objective, the ionic chemical species concentration method according to the present invention is A method for concentrating ionic chemical species, comprising encapsulating ionic chemical species within lipid particles formed from lipids, The steps include adding a first buffer solution to the lipid particles, The first cation or first anion include 2nd The buffer solution is added to the lipid particles. Add The process, If the second buffer solution contains the first cation, a third buffer solution containing a second anion paired with the first cation is added to the lipid particles; if the second buffer solution contains the first anion, a third buffer solution containing a second cation paired with the first anion is added to the lipid particles. The aforementioned lipid particles The first cation and The second anion Ionic chemical species solution in which these coexist , or an ionic chemical species solution in which the first anion and the second cation coexist. By distributing it to The first cation contained in the second buffer, or the first Anio n The process includes encapsulating the lipid particles, The aforementioned lipid particles are lamellar vesicles formed from a phospholipid bilayer. The aforementioned 1 Cation and the second cation It is a hydrophobic cation, The aforementioned 1 Anion and the second anion It is a hydrophilic anion, In the aforementioned ionic chemical species solution When the first cation and the second anion are present together, the second anion Molar concentration is, The first cation The molar concentration is more than twice that of the above. First cation The lipid particles are concentrated within the lipid particles. Therefore, when the first anion and the second cation coexist in the ionic chemical species solution, the molar concentration of the second cation is at least twice the molar concentration of the first anion, and the first anion is concentrated within the lipid particles. . [Effects of the Invention]

[0007] According to the present invention, the molar concentration of either anion or cation in the ionic chemical species solution is at least twice the molar concentration of the other, and the other is concentrated within the lipid particles. This increases the molar concentration of one ionic chemical species in the ionic chemical species solution present outside the lipid particles, thereby increasing the distribution of cations and anions into the lipid particles. Consequently, the distribution of one ionic chemical species present outside the lipid particles into the lipid particles along with the other ionic chemical species is promoted, and as a result, the concentration of the other ionic chemical species encapsulated within the lipid particles along with the other ionic chemical species can be increased. [Brief explanation of the drawing]

[0008] [Figure 1A] This is a diagram illustrating a method for encapsulating ionic chemical species according to an embodiment of the present invention. [Figure 1B] This is a diagram illustrating the permeation mechanism of ionic chemical species in the lipid bilayer according to the embodiment. [Figure 2A] These are fluorescence images (left) and bright-field images (right) of the evaluation sample used in Example 1 before the addition of epirubicin. [Figure 2B] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 1 immediately after the addition of epirubicin. [Figure 2C] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 1 15 minutes after the addition of epirubicin. [Figure 3A] This figure shows the fluorescence intensity distribution in the AA line of the evaluation sample according to Example 1. [Figure 3B] This figure shows the fluorescence intensity distribution in the AA line of the evaluation sample according to Example 1. [Figure 4A] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 1 immediately after the addition of 1.0 × 10⁻³ mol / dm³ of ClO₄⁻. [Figure 4B] The images on the left and right show the fluorescence (fluorescence) and bright-field (bright) images of the evaluation sample according to Example 1 15 minutes after adding 1.0 × 10⁻³ mol / dm³ of ClO₄⁻. [Figure 4C] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 1 40 minutes after adding 1.0 × 10⁻³ mol / dm³ of ClO₄⁻. [Figure 5A] This figure shows the fluorescence intensity distribution in the AA line of the evaluation sample according to Example 1. [Figure 5B] This figure shows the fluorescence intensity distribution in the AA line of the evaluation sample according to Example 1. [Figure 5C]This figure shows the fluorescence intensity distribution in the AA line of the evaluation sample according to Example 1. [Figure 6A] These are fluorescence images (left) and bright-field images (right) of the evaluation sample used in Example 2 before the addition of epirubicin. [Figure 6B] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 2 immediately after the addition of epirubicin. [Figure 6C] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 2 15 minutes after the addition of epirubicin. [Figure 7A] This figure shows the fluorescence intensity distribution in the BB line of the evaluation sample according to Example 2. [Figure 7B] This figure shows the fluorescence intensity distribution in the BB line of the evaluation sample according to Example 2. [Figure 8A] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 2 immediately after the addition of 5.0 × 10⁻³ mol / dm³ of ClO₄⁻. [Figure 8B] The images on the left and right show the fluorescence (fluorescence) and bright-field (bright) images of the evaluation sample according to Example 2 15 minutes after adding 5.0 × 10⁻³ mol / dm³ of ClO₄⁻. [Figure 8C] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 2 40 minutes after adding 5.0 × 10⁻³ mol / dm³ of ClO₄⁻. [Figure 9A] This figure shows the fluorescence intensity distribution in the BB line of the evaluation sample according to Example 2. [Figure 9B] This figure shows the fluorescence intensity distribution in the BB line of the evaluation sample according to Example 2. [Figure 9C] This figure shows the fluorescence intensity distribution in the BB line of the evaluation sample according to Example 2. [Figure 10]This figure shows the time dependence of the ratio of fluorescence intensity on the outside of the liposome to the fluorescence intensity on the inside of the liposome (fluorescence intensity ratio) in Examples 1 and 2, after the addition of epirubicin. [Figure 11A] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 3 before the addition of rhodamine 6G. [Figure 11B] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 3 immediately after the addition of rhodamine 6G. [Figure 11C] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 3 15 minutes after the addition of rhodamine 6G. [Figure 12A] This figure shows the fluorescence intensity distribution in the CC line of the evaluation sample according to Example 3. [Figure 12B] This figure shows the fluorescence intensity distribution along the CC line in Figure 11C for the evaluation sample according to Example 3. [Figure 13A] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 3 immediately after adding 1.0 × 10⁻² mol / dm³ of BF4-. [Figure 13B] The images on the left and right show the fluorescence (fluorescence) and bright-field (bright) images of the evaluation sample according to Example 3 35 minutes after adding 1.0 × 10⁻² mol / dm³ of BF4-. [Figure 14A] This figure shows the fluorescence intensity distribution in the CC line of the evaluation sample according to Example 3. [Figure 14B] This figure shows the fluorescence intensity distribution in the CC line of the evaluation sample according to Example 3. [Figure 15A] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 4 before the addition of rhodamine 6G. [Figure 15B] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 4 immediately after the addition of rhodamine 6G. [Figure 15C]These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 4 15 minutes after the addition of rhodamine 6G. [Figure 16A] This figure shows the fluorescence intensity distribution in the DD line of the evaluation sample according to Example 4. [Figure 16B] This figure shows the fluorescence intensity distribution in the DD line of the evaluation sample according to Example 4. [Figure 17A] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 4 immediately after adding 1.0 × 10⁻² mol / dm³ of ClO₄⁻. [Figure 17B] These are fluorescence images (left) and bright-field images (right) of the evaluation sample according to Example 4, 20 minutes after adding 1.0 × 10⁻² mol / dm³ of ClO₄⁻. [Figure 18A] This figure shows the fluorescence intensity distribution in the DD line of the evaluation sample according to Example 4. [Figure 18B] This figure shows the fluorescence intensity distribution in the DD line of the evaluation sample according to Example 4. [Figure 19] This figure shows the time dependence of the ratio of fluorescence intensity on the outside of the liposome to the fluorescence intensity on the inside of the liposome (fluorescence intensity ratio) in Examples 3 and 4, after the addition of rhodamine 6G. [Figure 20A] The images on the left and right show the fluorescence (fluorescence) and bright-field (bright) of the evaluation sample according to Example 5 15 minutes after adding 1.0 × 10⁻⁶ mol / dm³ of FAM2-. [Figure 20B] The images on the left and right show the fluorescence (fluorescence) and bright-field (bright) images of the evaluation sample according to Example 5 20 minutes after the addition of 1.0 × 10⁻³ mol / dm³ of BTPPA+. [Figure 21A] This figure shows the fluorescence intensity distribution in the EE line of the evaluation sample according to Example 5 (Figure 20A). [Figure 21B] This figure shows the fluorescence intensity distribution in the EE line of the evaluation sample according to Example 5 (Figure 20B). [Figure 22]This figure shows the time dependence of the fluorescence intensity inside liposomes in Examples 6 to 8 after the addition of FAM2. [Figure 23A] This is a fluorescence image of the evaluation sample according to Example 9, 15 minutes after adding 5.0 × 10⁻⁷ mol / dm³ of FAM-AAA5-. [Figure 23B] This is a fluorescence image of the evaluation sample according to Example 9, 10 minutes after the addition of 5.0 × 10⁻⁴ mol / dm³ of BTPPA+. [Figure 24A] This figure shows the fluorescence intensity distribution in the FF line of the evaluation sample according to Example 9. [Figure 24B] This figure shows the fluorescence intensity distribution in the FF line of the evaluation sample according to Example 9 (Figure 23B). [Figure 25] This figure shows the time dependence of the fluorescence intensity inside liposomes in Examples 9 to 11 after the addition of FAM-AAA5. [Modes for carrying out the invention]

[0009] The following describes an ionic species concentration method according to an embodiment of the present invention with reference to the drawings. The ionic species concentration method according to this embodiment is a method for concentrating ionic species within lipid particles formed from lipids. Here, ionic species refer to anions and cations. Lipid particles refer to liposomes or micelles, and liposomes refer to unilamellar vesicles or multilamellar vesicles (multilamellar vesicles) consisting of a single layer of lipid bilayer.

[0010] This method for concentrating ionic chemical species includes a step of encapsulating anions and cations within lipid particles by dispersing lipid particles in an ionic chemical species solution in which anions and cations coexist. In this method, the molar concentration of either the anion or the cation in the ionic chemical species solution is at least twice the molar concentration of the other, and the other ionic chemical species is concentrated within the lipid particles.

[0011] In the ionic species concentration method according to this embodiment, for example, as shown in Figure 1A, an ionic species, such as a cation or anion, is encapsulated within a liposome R, which is a unilamela vesicle formed from a phospholipid bilayer BLM.

[0012] The phospholipids that form liposomes R include PC (1,2-dioleoyl-sn-glycero-phosphocholine), 1,2-dioleoylphosphatidylcholine, 1,2-dipalmitoylphosphatidylcholine, 1,2-dimiristoylphosphatidylcholine, 1,2-distearoylphosphatidylcholine, 1-oleoyl-2-palmitoylphosphatidylcholine, 1-oleoyl-2-stearoylphosphatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylcholine. Phosphatidylcholines such as oil phosphatidylcholine, 1-stearoyl-2-oleoylphosphatidylcholine, 1,2-dioleoylphosphatidylethanolamine, 1,2-dipalmitoylphosphatidylethanolamine, 1,2-dimyristoylphosphatidylethanolamine, 1,2-distearoylphosphatidylethanolamine, 1-oleoyl-2-palmitoylphosphatidylethanolamine, 1-oleoyl-2-stearoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine, N-succinyldioleoylphosphatidylethanolamine, 1,2-dioleoylphosphatidylserine, 1,2-dipalmitoylphosphatidylserine, 1,2-dimyristoylphosphatidylserine, 1,2-distearoyl Phosphatidylserine such as 1,2-oleoylphosphatidylserine, 1,2-oleoylphosphatidylserine, 1,2-oleoylphosphatidylglycerin, 1,2-dipalmitoylphosphatidylglycerin, 1,2-dimyristoylphosphatidylglycerin, 1,Examples include phosphatidylglycerins such as 2-distearoylphosphatidylglycerin, 1-oleoyl-2-palmitoylphosphatidylglycerin, 1-oleoyl-2-stearoylphosphatidylglycerin, 1-palmitoyl-2-oleoylphosphatidylglycerin, and 1-stearoyl-2-oleoylphosphatidylglycerin, as well as PEGylated phospholipids such as phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-1000], phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000], phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-3000], and phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-5000].

[0013] Furthermore, the phospholipids that form liposomes R include compounds selected from the group consisting of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), CHOL (cholesterol), 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), and HSPC (purified hydrogenated soybean phosphatidylcholine).

[0014] Furthermore, as the cation, at least one anthracycline antibiotic can be selected from the group of multiple anthracycline antibiotics, including epirubicin, daunorubicin, doxorubicin, amrubicin, idarubicin, barurubicin, akurarubicin, pirarubicin, and mitoxantrone, which are hydrophobic cations. Additionally, as the cation, at least one ion can be selected from the group of hydrophobic cations, including amines, rhodamines, cyanines, phosphonium cations, arsonium cations, imidazoliums, and primary, secondary, tertiary, or quaternary ammonium compounds. Moreover, as the cation, at least one local anesthetic having an amine structure can be selected from the group of dibucaine, mepivacaine, bupivacaine, levobupivacaine, ropivacaine, procaine, tetracaine, prilocaine, cocaine, ambroxol, phenylpiperidine derivatives, and morphinan derivatives. Furthermore, as cations, antiallergic drugs, antiarrhythmic drugs, antidepressants, antihypertensive drugs, cardiac drugs, muscle relaxants, analgesics, antimalarial drugs, antihypertensive drugs, nerve blockers, central antitussives, ovulation inducers, psychotropic drugs, anti-peptic ulcer drugs, vitamin supplements, antibacterial drugs, metabolic enzyme substrates, cardiovascular drugs, and anti-cancer drugs, specifically azelastine, aprindine, amiodarone, amitriptyline, amlodipine, alprenolol, bopindolol, pindolol, bisoprolol, ambenonium, isoxuprine, imibramin, indenolol, ethylmorphine, etilefrine, edrophonium, ephedrine, eperisone, oxymethyl Cycodone, oxybuprocaine, orcibrenaline, carteolol, quinabril, quinine, guanabenz, clocapramine, clonidine, clofedalol, cloperastin, tamoxifen derivatives, clomipramine, clomiferamine, ketamine, ketotifen, codeine, phenethylamine derivatives, distigmine, opium alkaloid derivatives, diphenhydramine derivatives, cyproheptadine, cibenzoline, dilazep, benzodiazepine derivatives, cetirizine, cetraxate, tamsulosin, tiapride, thiamine, tizanidine, tipepidine, thimepidium, timolol, temocapril, terbinafine, prazosin derivatives,At least one selected from doxapram, hydrazinophthalazine derivatives, donepezil, dopamine, trimetazidine, trimethokinol, trimebutine, nafamostat mesilate, normorphine derivatives, dihydropyridine derivatives, physostigmine derivatives, tricyclic antidepressant derivatives, 2-amino-1-phenylethanol derivatives, thiazolidinedione derivatives, hydroxyzine, biperiden, physostigmine derivatives, pirenzepine, vinca alkaloid anti-cancer drugs, fexofenadine, propranolol derivatives, butylscopolamine, butenafine, butropium, morphinan derivatives, prazosin derivatives, flavoxate, progabide, propafenone, propiverine, propranolol derivatives, bromhexine derivatives, phenothiazine derivatives, propranolol derivatives, betahistine, phenylpiperidine derivatives, phenylalkylamine derivatives, perphenazine, berberine, benzethonium, benserazide, homochlorcyclizine, phenethylamine derivatives, mabrotoline, imidazole derivatives, mexiletine, meclofenoxate, methyl ephedrine, methylergometrine, mefloquine, mepenzolate, mosapride, ranitidine, labetalol, ritodrine can be adopted. Alternatively, as the cation, at least one dye selected from a group of dyes having a hydrophobic cation and a rhodamine skeleton may be used. Also, at least one selected from diarylmethane, triarylmethane, azo-based and nitrogen-containing heterocyclic compounds may be adopted as the cation.

[0015] Also, as the cation, a hydrophilic cation, a metal ion (e.g., Li + , Na + , K + and other alkali metal ions, Mg 2+ , Ca 2+ and other alkaline earth metal ions, Cu 2+ , Mn 2+ , Fe 2+ etc.), ammonium ions, H + , basic amino acids (lysine, arginine, etc.) or at least one selected from cationic oligopeptides composed of them may be adopted.

[0016] The ionic species concentration method according to this embodiment includes a step of encapsulating cations or anions within the liposomes R by dispersing the liposomes R in an ionic species solution in which anions and cations coexist.

[0017] As anions, at least one can be selected from hydrophilic anions such as halide ions, sulfate ions, nitrate ions, phosphate ions, nucleic acids consisting of three or fewer nucleic acid bases, sulfonic acids, carboxylic acids, nucleic acids or acidic amino acids (aspartic acid, glutamic acid) or anionic oligopeptides made therefrom, and fluoresceins. As halide ions, Cl - , Br - , I - At least one selected from the group can be adopted. Also, as anions, hydrophobic anions such as borate anions and ClO4 - PF6 - BF4 - At least one ion can be selected from aromatic sulfonic acids / carboxylic acids, alkyl sulfonic acids, and alkyl carboxylic acids. Furthermore, at least one anion can be selected from sulfonic acid derivatives / carboxylic acid derivatives having alkyl and aryl groups, and tetraphenyl borate.

[0018] Here, we will explain the process by which anions and cations present in the outer S2 of liposome R are encapsulated in the inner S1 of liposome R. First, as shown in Figure 1B, three equilibrium states exist between the ionic chemical species solution and the phospholipid bilayer BLM of liposome R: an adsorption equilibrium of cations to the surface of the phospholipid bilayer BLM, a distribution equilibrium of anions and cations into the phospholipid bilayer BLM, and an ion pair production equilibrium from anions and cations within the phospholipid bilayer BLM. The adsorption equilibrium state to the surface of the phospholipid bilayer BLM and the ion distribution equilibrium state into the phospholipid bilayer BLM occur independently of each other. That is, cations adsorbed on the surface of the phospholipid bilayer BLM do not penetrate into the inner S1 of liposome R. On the other hand, cations and anions are distributed in equal amounts from the ionic chemical species solution W present in the outer S2 of liposome R into the phospholipid bilayer BLM of liposome R, maintaining electrical neutrality. The cations and anions distributed to the phospholipid bilayer (BLM) are then 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 to the phospholipid bilayer (BLM) is determined by the distribution constants, which are expressed by the cation and anion concentrations in the outer S2 of the liposome R and the cation and anion concentrations in the phospholipid bilayer (BLM). Therefore, increasing the cation and anion concentrations in the outer S2 of the liposome R increases the concentration of cations and anions in the phospholipid bilayer (BLM), and also increases the amount of cations and anions encapsulated from the phospholipid bilayer (BLM) to the inner S1 of the liposome R.

[0019] Therefore, in this embodiment, the molar concentration of anions in the ionic chemical species solution is set to at least twice the molar concentration of cations. As a result, the distribution of cations and anions between S2 on the outside of the liposome R and the phospholipid bilayer BLM is greater compared to when the molar concentration of anions is the same as the molar concentration of cations, and the distribution of cations into the phospholipid bilayer BLM is promoted. Consequently, the distribution of cations and anions from the phospholipid bilayer BLM to S1 on the inside of the liposome R increases, and cations are encapsulated at a high concentration in S1 on the inside of the liposome R. Note that the high concentration of anions added to the ionic chemical species solution W is set to avoid bursting or contraction of the liposome R due to osmotic pressure, and is equal to the molar concentration of the salt contained in the buffer added to the ionic chemical species solution W (e.g., 0.1 mol / dm³). 3 It is preferable to keep it below the following:

[0020] Furthermore, in this embodiment, the molar concentration of cations in the ionic chemical species solution may be twice or more the molar concentration of anions. This increases the distribution of cations and anions between S2 on the outside of the liposome R and the phospholipid bilayer BLM compared to the case where the molar concentration of cations is the same as the molar concentration of anions, thereby promoting the distribution of anions into the phospholipid bilayer BLM. As a result, the distribution of cations and anions from the phospholipid bilayer BLM to S1 on the inside of the liposome R increases, and anions are encapsulated at a high concentration in S1 on the inside of the liposome R.

[0021] As described above, according to the ionic species concentration method of this embodiment, the molar concentration of anions in the ionic species solution is at least twice the molar concentration of cations. This increases the molar concentration of anions in the ionic species solution present outside the liposome R, thereby increasing the distribution of cations and anions to the phospholipid bilayer BLM. Consequently, the distribution of anions present in S2 on the outside of the liposome R to the phospholipid bilayer BLM along with cations is promoted, and as a result, the concentration of cations encapsulated together with anions in S1 on the inside of the liposome R can be increased.

[0022] Although embodiments of the present invention have been described above, the present invention is not limited to the configuration of the embodiments described above. For example, the lipid particles may be micelles formed from phospholipids. In this case, the micelles formed from phospholipids can be dispersed in an ionic chemical species solution in which anions and cations coexist, thereby encapsulating the cations within the micelles. In this case, the molar concentration of anions in the ionic chemical species solution should be at least twice the molar concentration of cations.

[0023] This invention allows for various embodiments and modifications without departing from the broad spirit and scope of the invention. Furthermore, the embodiments described above are for illustrative purposes only and do not limit the scope of the invention. In other words, the scope of the invention is indicated by the claims, not by the embodiments. Various modifications made within the scope of the claims and the equivalent scope of the meaning of the invention are considered to be within the scope of this invention. [Examples]

[0024] The present invention will now be described in detail based on Examples 1 to 11. First, the method for preparing the liposomes used in each example will be explained. First, a lipid thin film was formed on an agarose film, and then hydrated by adding phosphate buffer (pH: 7.0). The agarose film was prepared on a disc-shaped coverslip (manufactured by Matsunami Glass Industry Co., Ltd.) with a diameter of 22 mm and a thickness of 0.12 to 0.17 mm by dropping a 1 wt% agarose aqueous solution onto the coverslip and then leaving it for 1 hour while heated at approximately 40°C. The lipid thin film was prepared by dropping only 15 μL of a lipid chloroform solution onto the aforementioned agarose film using a microdispenser (25 μL, manufactured by DRUMMOND SCIENTIFIC CO.), and then drying the coverslip in a desiccator under negative pressure for 1 hour. Here, the lipid solution was prepared by dissolving 126.8 mg of PC (1,2-dioleoyl-sn-glycerophosphocholine) (manufactured by Tokyo Chemical Industry Co., Ltd.) with a purity of over 97.0% and 63.4 mg of cholesterol (manufactured by Nacalai Tesque Co., Ltd.) in 20 ml of chloroform (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The molar ratio of PC to cholesterol in this lipid solution was approximately 1:1. Then, in Examples 1 and 2, a lipid thin film formed on an agarose film was subjected to a molar concentration of 0.01 mol / dm³. 3 Liposomes were prepared by adding 160 μL of phosphate buffer and leaving it in the dark for 3 hours. In Examples 3 and 4, a lipid thin film formed on an agarose film was treated with a molar concentration of 0.1 mol / dm³. 3 Liposomes were prepared by adding 160 μL of phosphate buffer and leaving the mixture in the dark for 3 hours.

[0025] Next, we will describe a method for preparing evaluation samples by immobilizing liposomes on coverslips coated with a cell membrane modifier (BAM: Biocompatible Anchor for cell Membrane). First, a disc-shaped coverslip (manufactured by Matsunami Glass Industry Co., Ltd.) with a diameter of 22 mm and a thickness of 0.12 to 0.17 mm was subjected to silane treatment. The silane treatment was performed by immersing the coverslip in a solution obtained by mixing APTS (3-aminopropyltriethoxysilane) (manufactured by Shin-Etsu Chemical Co., Ltd.) and ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) in a volume ratio of 1:5 for 2 hours. After the silane treatment, the coverslip was washed with methanol and dried. Then, 200 μL of a dimethyl sulfoxide solution of a polyethylene glycol modifier was dropped onto the coverslip, left for 1 hour, and then the surface of the coverslip was washed with distilled water to prepare a coverslip coated with BAM. Here, the dimethyl sulfoxide solution contains a polyethylene glycol modifier (SUNBRIGHT® OE-040CS: manufactured by NOF Corporation) at a molar concentration of 10 mmol / dm³. 3 It was prepared by dissolving it in dimethyl sulfoxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to achieve the desired result.

[0026] Next, a BAM-coated coverslip was attached to an Azunol Petri dish (manufactured by AS ONE Corporation), which had a 40 mm diameter, 13.5 mm high, and a 18 mm diameter hole perforated in its bottom wall, so that the coverslip covered the hole from the vertically downward side when the Azunol Petri dish was in use. Subsequently, 100 μL of phosphate buffer containing liposomes formed on an agarose film was measured using a micropipette and dropped onto the BAM-coated coverslip to fix the liposomes to the BAM-coated coverslip.

[0027] Next, the evaluation method for the evaluation samples in each embodiment will be described. In each embodiment, a confocal laser microscope (FLUOVIEW® FV10i: manufactured by Olympus Corporation) was used to observe the change in fluorescence intensity of the evaluation sample over time. Here, a laser light source of a laser microscope with an oscillation wavelength of 473 nm was used as the excitation light source for the evaluation sample. In addition, an optical filter with 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 fluorescence intensity.

[0028] In Example 1, the molar concentration of epirubicin was 1.7 × 10⁻⁶. -5 mol / dm 3 Therefore, the concentration containing epirubicin hydrochloride is 0.01 mol / dm³. 3 Only 50 μL of phosphate buffer was added to the evaluation sample. The fluorescence intensity was then observed immediately after addition and 15 minutes later, with the focus on the liposomes contained in the evaluation sample. As shown in Figures 2A and 2B, when epirubicin hydrochloride was added, fluorescence from the cation epirubicin was observed on the outside of the liposomes and near the surface of the liposomes. In particular, as shown in Figure 3A, the fluorescence intensity near the surface of the liposomes was found to be increased compared to other regions. This is thought to be due to the adsorption equilibrium between the liposome surface and the phosphate buffer containing epirubicin. On the other hand, as shown in Figure 2B, almost no fluorescence from epirubicin was observed inside the liposomes. Furthermore, even 15 minutes after the addition of epirubicin hydrochloride, as shown in Figures 2C and 3B, there was almost no increase in the fluorescence intensity from epirubicin inside the liposomes.

[0029] In Example 1, after 17 minutes had elapsed since the addition of epirubicin hydrochloride, ClO4 - The molar concentration is 1.0 × 10 -3 mol / dm 3 To achieve this, a solution containing NaClO4 at a concentration of 0.01 mol / dm³ 3Only 50 μL of phosphate buffer was added to the evaluation sample. Fluorescence intensity was observed immediately after addition, and 15 and 40 minutes after addition. As shown in Figures 4A to 4C and 5A to 5C, after the addition of epirubicin hydrochloride, an increase in fluorescence from epirubicin inside the liposome was observed over time. This is because the anion ClO4 is present on the outside of the liposome. - The addition of epirubicin and ClO4 to the lipid bilayer of liposomes - The distribution of epirubicin and ClO4 distributed to the lipid bilayer - This is thought to be because the entry into the inside of the liposome was facilitated.

[0030] In Example 2, similar to Example 1, the molar concentration of epirubicin was 1.7 × 10⁻⁶. -5 mol / dm 3 Therefore, 0.01 mol / dm³ containing epirubicin hydrochloride 3 Only 50 μL of phosphate buffer was added to the evaluation sample. The fluorescence intensity was then observed immediately after addition and 15 minutes after addition, with the focus on the liposomes contained in the evaluation sample. As shown in Figures 6A, 6B, and 7A, when epirubicin hydrochloride was added, fluorescence from the cation epirubicin was observed on the outside of the liposomes and near the surface of the liposomes. On the other hand, as shown in Figures 6B and 7A, almost no fluorescence from epirubicin was observed inside the liposomes. Furthermore, even 15 minutes after the addition of epirubicin hydrochloride, as shown in Figures 6C and 7B, there was almost no increase in the fluorescence intensity from epirubicin inside the liposomes.

[0031] Next, in Example 2, after 17 minutes had elapsed since the addition of epirubicin chloride, ClO4 - The molar concentration is 5.0 × 10 -3 mol / dm 3 To achieve this, a solution containing NaClO4 at a concentration of 0.01 mol / dm³ 3Only 50 μL of phosphate buffer was added to the evaluation sample. That is, compared to Example 1, the phosphate buffer ClO4 outside the liposome was - The molar concentration was increased fivefold. The fluorescence intensity was observed immediately after addition, and 15 minutes and 40 minutes after addition. As shown in Figures 8A to 8C and Figures 9A to 9C, similar to Example 1, after adding epirubicin hydrochloride, an increase in fluorescence from epirubicin inside the liposome was observed over time. Figure 10 shows the results of evaluating the time dependence of the ratio of fluorescence intensity outside the liposome to fluorescence intensity inside the liposome (hereinafter referred to as the "fluorescence intensity ratio") for the evaluation samples related to Examples 1 and 2. As shown in Figure 10, the ClO4 related to Example 2 - The rate of increase in fluorescence intensity inside the liposome after addition was compared to Example 2 with ClO4 - It was found that the amount of added ClO4 was larger compared to that in Example 1, where the amount added was small. From this, it was found that the anion ClO4 - It was found that a higher amount of additive promotes the encapsulation of the cation epirubicin into the liposome.

[0032] In Example 3, the molar concentration of rhodamine 6G was 1.0 × 10⁻⁶. -5 mol / dm 3 To achieve this, 0.1 mol / dm³ containing rhodamine 6G 350 μL of phosphate buffer was added to the evaluation sample. The fluorescence intensity was then observed immediately after addition and 15 minutes later, with the focus on the liposomes contained in the evaluation sample. As shown in Figures 11A and 11B, when rhodamine 6G chloride was added, fluorescence from the cation rhodamine 6G was observed on the outside of the liposomes and near the surface of the liposomes. In particular, as shown in Figure 12A, the increased fluorescence intensity near the surface of the liposomes compared to other regions is thought to be due to an adsorption equilibrium state. On the other hand, as shown in Figure 11B, almost no fluorescence from rhodamine 6G was observed inside the liposomes. Furthermore, even 15 minutes after the addition of rhodamine 6G chloride, as shown in Figures 11C and 12B, there was almost no increase in fluorescence intensity from rhodamine 6G inside the liposomes.

[0033] In Example 3, after 17 minutes had elapsed since the addition of rhodamine 6G chloride, BF4 - The molar concentration is 1.0 × 10 -2 mol / dm 3 To achieve this, 0.1 mol / dm³ containing NaBF4 3 50 μL of phosphate buffer was added to the evaluation sample. Fluorescence intensity was observed immediately after addition and 35 minutes later. As shown in Figures 13A, 13B, 14A, and 14B, after the addition of rhodamine 6G chloride, an increase in fluorescence from rhodamine 6G inside the liposome was observed over time. This is due to the presence of the anion BF4 on the outside of the liposome. - The addition of rhodamine 6G and BF4 to the lipid bilayer of liposomes - The distribution of rhodamine 6G and BF4 distributed to the lipid bilayer - This is thought to be because the entry into the inside of the liposome was facilitated.

[0034] In Example 4, similar to Example 3, the molar concentration of rhodamine 6G was 1.0 × 10⁻⁶. -5 mol / dm 3 To achieve this, rhodamine 6G chloride is administered at a concentration of 0.1 mol / dm³.3 Only 50 μL of phosphate buffer containing rhodamine 6G was added to the evaluation sample. The fluorescence intensity was then observed immediately after addition and 15 minutes after addition, with the focus on the liposomes contained in the evaluation sample. As shown in Figures 15A, 15B, and 16A, almost no fluorescence from rhodamine 6G was observed immediately after addition. Then, as shown in Figures 15C and 16B, fluorescence from the rhodamine 6G cation was observed on the outside of the liposomes and near the surface of the liposomes 15 minutes after the addition of rhodamine 6G chloride. However, even after 15 minutes, there was almost no increase in fluorescence from rhodamine 6G inside the liposomes.

[0035] Next, in Example 4, after 17 minutes had elapsed since the addition of rhodamine 6G chloride, ClO4 - The molar concentration is 1.0 × 10 -2 mol / dm 3 To achieve this, 0.1 mol / dm³ containing NaClO4 3 Only 50 μL of phosphate buffer was added to the evaluation sample. That is, as the anion, BF4 in Example 3 was used. - ClO4 at the same concentration - The rhodamine 6G was added to the outside of the liposome. The fluorescence intensity was observed immediately after addition and 20 minutes after addition. As shown in Figures 17A and 17B and Figures 18A and 18B, similar to Example 3, after adding rhodamine 6G chloride, an increase in fluorescence from rhodamine 6G inside the liposome was observed over time. Figure 19 shows the results of measuring the time dependence of fluorescence intensity inside the liposome for evaluation samples from Examples 3 and 4. As shown in Figure 19, the ClO4 from Example 4 - The rate of increase in fluorescence intensity inside the liposome after addition is the same as that of BF4 in Example 3. - It was found that the amount increased compared to the amount after addition. Here, rhodamine 6G and ClO4 - The partition equilibrium constant K when the substance is distributed from the outside of the liposome to the membrane of the liposome. D =(C R6G,BLM CX,BLM ) / (C R6G,W C X,W )(X=BF4 - ,ClO4 - ) is 1.1, whereas Rhodamine 6G and BF4 - The partition equilibrium constant K when the substance is distributed from the outside of the liposome to the membrane of the liposome. D The ratio is 0.31. In other words, the results shown in Figure 19 indicate that within the lipid bilayer of liposomes, rhodamine 6G and BF4 - Compared to the combination of Rhodamine 6G and ClO4 - This suggests that the combination is more easily distributed to the phospholipid bilayer BLM. In other words, when the cation is rhodamine 6G, the anion is ClO4 - It was found that using this method promotes the encapsulation of rhodamine 6G inside the liposome.

[0036] In Example 5, fluorescein (FAM) 2- The molar concentration of ) is 1.0 × 10 -6 mol / dm 3 Therefore, fluorescein sodium (FAM 2- 2Na + ) including 1.0 × 10 -4 mol / dm 3 50 μL of phosphate buffer was added to the evaluation sample. The fluorescence intensity was then observed 15 minutes after addition, with the focus on the liposomes in the evaluation sample. As shown in Figures 20A and 21A, FAM was present inside the liposomes. 2- Almost no fluorescence was observed from it.

[0037] And in Example 5, FAM 2- 2Na + After 17 minutes have elapsed since the addition of bis(triphenylphosphoranylidene)ammonium (BTPPA) + The molar concentration of ) is 1.0 × 10 -3 mol / dm 3 Therefore, bis(triphenylphosphoranylidene)ammonium chloride (BTPPA) + Cl- ) containing 1.0×10 -4 mol / dm 3 50 μL of phosphate buffer was added to the sample for evaluation. Then, the fluorescence intensity 20 min after the addition was observed. As shown in FIGS. 20B and 21B, fluorescence from FAM inside the liposome was observed. This is because hydrophobic cation BTPPA was added to the outside of the liposome, promoting the distribution of FAM and BTPPA into the lipid bilayer of the liposome and the penetration of FAM and BTPPA distributed in the lipid bilayer into the inside of the liposome.

[0038] FIG. 22 shows the results of measuring the time-dependence of the fluorescence intensity inside the liposome for the samples for evaluation according to Examples 6 to 8. In Examples 6 to 8, the molar concentration of FAM and the molar concentration of the phosphate buffer were 1.0×10 2- mol / dm -6 and 1.0×10 3 mol / dm -4 , respectively, as in Example 5. However, in Examples 6 to 8, the molar concentration of BTPPA added 17 min after the addition of FAM 2- 2Na + was 5.0×10 + mol / dm -4 , 1.0×10 3 mol / dm -4 , and 5.0×10 3 mol / dm -5 , respectively. As shown in FIG. 22, in Examples 6 to 8, an increase in the fluorescence intensity from FAM inside the liposome was observed after the addition of BTPPA.

[0039] In Example 9, fluorescein (FAM 2- ​​​​​​​​​​​​​​​​​​​​Nucleic acid (FAM-AAA) composed of three adenines modified with ) 5- 5Na + The molar concentration of ) is 5.0 × 10 -7 mol / dm 3 Therefore, nucleic acid sodium (FAM-AAA 5- 5Na + ) including 1.0 × 10 -4 mol / dm 3 50 μL of phosphate buffer was added to the evaluation sample. The fluorescence intensity was then observed 15 minutes after addition, with the focus on the liposomes in the evaluation sample. As shown in Figures 23A and 24A, FAM was present inside the liposomes. 2- Almost no fluorescence was observed from it.

[0040] And in Example 9, FAM-AAA 5- 5Na + After 17 minutes have elapsed since the addition of BTPPA + The molar concentration is 1.0 × 10 -3 mol / dm 3 So that BTPPA + Cl - 1.0 × 10 -4 mol / dm 3 50 μL of phosphate buffer was added to the evaluation sample. The fluorescence intensity was then observed 10 minutes after addition. As shown in Figures 23B and 24B, the FAM inside the liposome 2- Fluorescence was observed from the outside of the liposome, which is a hydrophobic cation called BTPPA. + The addition of FAM-AAA to the lipid bilayer of liposomes resulted in 5- and BTPPA + The distribution of FAM-AAA distributed to the lipid bilayer 5- and BTPPA + This is thought to be because the intrusion into the inside of the liposome was promoted. And FAM-AAA 5- It was found that even molecules with relatively large molecular weights, such as those mentioned above, can be introduced into the inside of liposomes by utilizing the concentration gradient of hydrophobic cations.

[0041] Figure 25 shows the results of measuring the time dependence of fluorescence intensity inside liposomes for evaluation samples according to Examples 9 to 11. In Examples 10 to 11, FAM-AAA 5- The molar concentrations of the solution and the phosphate buffer were 5.0 × 10⁻⁶, respectively, as in Example 9. -7 mol / dm 3 , 1.0 × 10 -4 mol / dm 3 This was done so. However, in Examples 10 and 11, FAM-AAA 5- 5Na + Add BTPPA 17 minutes after adding the other ingredient. + The molar concentrations are 2.5 × 10⁻⁶ each. -4 mol / dm 3 , 1.0 × 10 -4 mol / dm 3 This was done so. As shown in Figure 25, in all of Examples 9 to 11, BTPPA + After the addition of FAM inside the liposome 2- An increase in fluorescence intensity was observed from BTPPA. + FAM due to differences in molar concentration 2- No difference in the rate of increase in fluorescence intensity was observed.

[0042] This application is based on Japanese Patent Application No. 2020-082512, filed on 8 May 2020. The entire specification, claims, and drawings of Japanese Patent Application No. 2020-082512 are incorporated herein by reference. [Industrial applicability]

[0043] The present invention is suitable for introducing ionic drugs, nucleic acid drugs, etc., into cells, and for producing drug delivery exosomes containing ionic drugs, nucleic acid drugs, etc. [Explanation of symbols]

[0044] BLM: Phospholipid bilayer, R: Liposome, S1: Inside of liposome, S2: Outside of liposome, W: Ionic chemical species solution

Claims

1. A method for concentrating ionic chemical species, comprising encapsulating ionic chemical species within lipid particles formed from lipids, The steps include adding a first buffer solution to the lipid particles, A step of adding a second buffer containing a first cation or a first anion to the lipid particles, The process includes the steps of: if the second buffer solution contains the first cation, adding a third buffer solution containing a second anion paired with the first cation to the lipid particles; and if the second buffer solution contains the first anion, adding a third buffer solution containing a second cation paired with the first anion to the lipid particles to disperse the lipid particles in an ionic chemical species solution in which the first cation and the second anion coexist, or in an ionic chemical species solution in which the first anion and the second cation coexist, thereby encapsulating the first cation or the first anion contained in the second buffer solution within the lipid particles; The aforementioned lipid particles are lamellar vesicles formed from a phospholipid bilayer. The first cation and the second cation are hydrophobic cations, The first and second anions are hydrophilic anions. When the first cation and the second anion coexist in the ionic chemical species solution, the molar concentration of the second anion is at least twice the molar concentration of the first cation, and the first cation is concentrated within the lipid particles. Method for concentrating ionic chemical species.

2. When the first cation and the second anion are present together in the ionic chemical species solution, The molar concentration of the second anion in the ionic chemical species solution is 100 times or more the molar concentration of the first cation. The method for concentrating ionic chemical species according to claim 1.

3. The molar concentration of the second anion contained in the ionic chemical species solution is less than or equal to the molar concentration of the salt contained in the buffer solution added to the ionic chemical species solution. The method for concentrating ionic chemical species according to claim 2.

4. The first cation is at least one anthracycline antibiotic selected from the group of multiple anthracycline antibiotics, including epirubicin, daunorubicin, doxorubicin, amrubicin, idarubicin, barurubicin, akurarubicin, pirarubicin, and mitoxantrone. A method for concentrating ionic chemical species according to any one of claims 1 to 3.

5. The first cation is at least one dye selected from the group of dyes having a rhodamine skeleton. A method for concentrating ionic chemical species according to any one of claims 1 to 3.

6. The aforementioned second anion is Cl － , Br － , I － At least one selected from the group A method for concentrating ionic chemical species according to any one of claims 1 to 5.

7. When the first anion and the second cation coexist in the ionic chemical species solution, The molar concentration of the second cation in the ionic chemical species solution is 100 times or more the molar concentration of the first anion. The method for concentrating ionic chemical species according to claim 1.

8. The molar concentration of the second cation contained in the ionic chemical species solution is less than or equal to the molar concentration of the salt contained in the buffer solution added to the ionic chemical species solution. The method for concentrating ionic chemical species according to claim 7.

9. The first anion is at least one selected from halide ions, sulfate ions, nitrate ions, phosphate ions, nucleic acids consisting of three or fewer nucleic acid bases, aspartic acid, and glutamic acid. A method for concentrating ionic chemical species according to any one of claims 1, 7, or 8.

10. The first cation is dibucaine, mepivacaine, bupivacaine, levobupivacaine, ropivacaine, procaine, tetracaine, prilocaine, cocaine, ambroxol, phenylpiperidine derivatives, morphinan derivatives, azelastine, aprindine, amiodarone, amitriptyline, amlodipine, alprenolol, bopindolol, pindolol, bisoprolol, ambenonium, isoxuprine, imibramin, indenolol, ethylmorphine, etilephrine, edrophonium, ephedrine, eperisone, oxycodone, oxy Buprocaine, orcibrenaline, carteolol, quinabril, quinine, guanabenz, clocapramine, clonidine, clofedalol, cloperastin, tamoxifen derivatives, clomipramine, clomiferamine, ketamine, ketotifen, codeine, phenethylamine derivatives, distigmine, opium alkaloid derivatives, diphenhydramine derivatives, cyproheptadine, cibenzoline, dilazep, benzodiazepine derivatives, cetirizine, cetraxate, tamsulosin, tiapride, thiamine, tizanidine, tipepidine, thimepidium, thymo Lol, temocapril, terbinafine, prazosin derivatives, doxapram, hydrazinophthalazine derivatives, donepezil, dopamine, trimetazidine, trimethoquinol, trimebutine, nafamostat mesil, normorphine derivatives, dihydropyridine derivatives, physostigmine derivatives, triptyline derivatives, 2-amino-1-phenylethanol derivatives, thiazolidinedione derivatives, hydroxyzine, biperiden, physostigmine derivatives, pirenzepine, vinca alkaloid anti-cancer drugs, fexofenadine, propranolol derivatives, buty Ruscobolamine, butenafine, buttropium, morphinan derivatives, prazosin derivatives, flavoxate, progalvazine, propafenone, propiverine, propranolol derivatives, bromhexine derivatives, phenothiazine derivatives, propranolol derivatives, betahistine, phenylpiperidine derivatives, phenylalkylamine derivatives, perphenazine, berberine, benzethonium, benserazide, homochlorcyclidine, phenethylamine derivatives, mabrotiline, imidazole derivatives, mexiletine, meclofenoxate, methyl ephedrine,At least one selected from the group consisting of methylergometrine, mefloquine, mepenzolate, mosapride, ranitidine, labetalol, ritodrine, diarylmethane, and triarylmethane. A method for concentrating ionic chemical species according to any one of claims 1 to 3.

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