Ionic chemical species concentration method and lipid particle

Abstract

To provide an ionic chemical species concentration method capable of concentrating an ionic chemical species at a high concentration in a lipid particle formed of a lipid bilayer membrane, and to provide a lipid particle.SOLUTION: An ionic chemical species concentration method includes encapsulating an ionic chemical species in a liposome R formed of a lipid bilayer membrane BLM that contains neutral phospholipid L and cationic lipid molecules CL, the cationic lipid molecules CL being present in at least an outer monolayer. The ionic chemical species concentration method includes a step of encapsulating a cation in the liposome R by utilizing the flip-flop phenomenon of the cationic lipid molecules CL in the lipid bilayer membrane BLM by dispersing the liposome R in an ionic chemical species solution in which cations and anions coexist.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to methods for concentrating ionic species and lipid particles. [Background technology]

[0002] A method has been proposed for concentrating an ionic drug inside a liposome formed from a lipid bilayer membrane (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Pat. No. 5,736,155 Summary of the Invention [Problem to be solved by the invention]

[0004] Meanwhile, as described in Patent Document 1, there is a demand for utilizing a technique for concentrating an ionic drug at a high concentration inside a liposome, and for efficiently delivering the drug to a target lesion via a liposome having an ionic drug concentrated inside.

[0005] The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide a method for concentrating an ionic species and lipid particles capable of concentrating an ionic species to a high concentration within lipid particles formed from a lipid bilayer membrane. [Means for solving the problem]

[0006] In order to achieve the above object, a method for concentrating an ionic chemical species according to the present invention comprises: 1. A method for concentrating ionic species, comprising encapsulating the ionic species within lipid particles formed from a lipid bilayer comprising a first lipid molecule, which is neutral, and a second lipid molecule, which is anionic or cationic, with the second lipid molecule present in an outer monolayer, the method comprising: The method includes a step of dispersing the lipid particles in an ionic species solution in which a first ionic species having the same polarity as the second lipid molecule and a second ionic species having a different polarity from the second lipid molecule coexist, thereby encapsulating the first ionic species and the second ionic species within the lipid particles by utilizing the flip-flop phenomenon of the second lipid molecules in the lipid bilayer membrane. Effect of the Invention

[0007] According to the present invention, the lipid particles formed from the lipid bilayer membrane in which the second lipid molecules are present in the outer monolayer are dispersed in an ionic species solution in which the first ionic species and the second ionic species coexist, so that the second lipid molecules present in the outer monolayer trap the second ionic species having a different polarity from that of the second lipid molecules in the ionic species solution. Then, as the second lipid molecules that have trapped the second ionic species diffuse and move to the inner monolayer of the lipid bilayer membrane due to the flip-flop phenomenon, the second ionic species trapped in the second lipid molecule and the first ionic species having the same polarity as that of the second lipid molecule attracted to the second ionic species are encapsulated in the lipid particle, and the first ionic species is concentrated in the lipid particle. As a result, even if the molar concentration of the first ionic species contained in the ionic species solution present outside the lipid particle is low, the efficiency of concentration of the first ionic species in the lipid particle can be increased accordingly if the molar concentration of the second ionic species is high. Thus, for example, the molar concentration of the second ionic species in the ionic species solution can be increased, but the concentration of the first ionic species concentrated within the lipid particles can be increased, subject to the constraint that the molar concentration of the first ionic species must be kept low. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic diagram showing the structure of a lipid particle according to an embodiment of the present invention. [Diagram 2]FIG. 1 shows the mechanism by which ionic chemical species permeate through a lipid bilayer membrane in an embodiment, in which (A) is a diagram showing a state in which an anion is trapped by a cationic lipid molecule, (B) is a diagram showing a state in which a cation is attracted to an anion, and (C) is a diagram showing a state in which a lipid molecule that has trapped an anion present in the outer monolayer of the lipid bilayer membrane has moved to the inner monolayer of the lipid bilayer membrane due to the flip-flop phenomenon. [Diagram 3] 1A and 1B show fluorescent images of evaluation samples of Example 1, where (A) shows the case where the Cl- concentration is 100 mM, (B) shows the case where the Cl- concentration is 10 mM, and (C) shows the case where the Cl- concentration is 1 mM. [Figure 4] FIG. 3(A) is a diagram showing the distribution of fluorescence intensity at line AA in FIG. 3(A) for the evaluation sample of Example 1; FIG. 3(B) is a diagram showing the distribution of fluorescence intensity at line BB in FIG. 3(B) for the evaluation sample of Example 1; and FIG. 3(C) is a diagram showing the distribution of fluorescence intensity at line CC in FIG. 3(C) for the evaluation sample of Example 1. [Diagram 5] FIG. 2 is a graph showing the relationship between the diameter of lipid particles and the concentration ratio of an evaluation sample in Example 1. [Figure 6] FIG. 2A is a diagram showing a fluorescence image of an evaluation sample according to Comparative Example 1, and FIG. 2B is a diagram showing the relationship between the diameter of lipid particles and the concentration ratio of the evaluation sample according to Comparative Example 1. [Figure 7] FIG. 2A is a diagram showing a fluorescence image of an evaluation sample according to Example 2, and FIG. 2B is a diagram showing the relationship between the diameter of lipid particles and the concentration ratio of the evaluation sample according to Example 2. [Figure 8] FIG. 1A is a diagram showing a fluorescence image of an evaluation sample according to Example 3, and FIG. 1B is a diagram showing the relationship between the diameter of lipid particles and the concentration ratio of the evaluation sample according to Example 3. [Figure 9] FIG. 1A is a diagram showing a fluorescence image of an evaluation sample according to Example 4, and FIG. 1B is a diagram showing the relationship between the diameter of lipid particles and the concentration ratio of the evaluation sample according to Example 4. [Figure 10](A-1) is a fluorescent image showing the state of the evaluation sample of Example 5 15 minutes after the addition of epirubicin and before the addition of Cl-; (A-2) is a diagram showing the distribution of fluorescent intensity at the DD line of (A-1); (B-1) is a fluorescent image showing the state of the evaluation sample of Example 5 15 minutes after the addition of Cl-; (B-2) is a diagram showing the distribution of fluorescent intensity at the EE line of (B-1); and (C) is a diagram showing the relationship between the diameter of lipid particles and the concentration ratio of the evaluation sample of Example 5. [Figure 11] (A-1) is a fluorescent image showing the state of the evaluation sample of Example 6 16 minutes after the addition of daunorubicin and before the addition of Cl-, (A-2) is a diagram showing the distribution of fluorescent intensity at the FF line of (A-1), (B-1) is a fluorescent image showing the state of the evaluation sample of Example 6 20 minutes after the addition of Cl-, (B-2) is a diagram showing the distribution of fluorescent intensity at the GG line of (B-1), and (C) is a diagram showing the relationship between the diameter of lipid particles and the concentration ratio of the evaluation sample of Example 6. [Figure 12] (A-1) is a fluorescent image showing the state of the evaluation sample of Example 7 15 minutes after the addition of idarubicin and before the addition of Cl-, (A-2) is a diagram showing the distribution of fluorescent intensity at the HH line of (A-1), (B-1) is a fluorescent image showing the state of the evaluation sample of Example 7 20 minutes after the addition of Cl-, (B-2) is a diagram showing the distribution of fluorescent intensity at the II line of (B-1), and (C) is a diagram showing the relationship between the diameter of lipid particles and the concentration ratio of the evaluation sample of Example 7. [Figure 13] (A-1) is a fluorescent image showing the state of the evaluation sample of Example 8 before Cl- addition, (A-2) is a bright field image showing the state of the evaluation sample of Example 8 before Cl- addition, (B-1) is a fluorescent image showing the state of the evaluation sample of Example 8 20 minutes after Cl- addition, and (B-2) is a bright field image showing the state of the evaluation sample of Example 8 20 minutes after Cl- addition. [Figure 14](A-1) is a figure showing a fluorescence image of the evaluation sample of Comparative Example 2, 20 minutes after the addition of Cl-, (A-2) is a figure showing the distribution of fluorescence intensity at the JJ line of (A-1), and (B) is a figure showing the relationship between the diameter of lipid particles and the concentration ratio of the evaluation sample of Comparative Example 2. [Figure 15] (A-1) is a figure showing a fluorescence image of the evaluation sample of Comparative Example 3, 20 minutes after the addition of ClO4-, (A-2) is a figure showing the distribution of fluorescence intensity at the KK line of (A-1), and (B) is a figure showing the relationship between the diameter of lipid particles and the concentration ratio of the evaluation sample of Comparative Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, an ionic species concentration method according to an embodiment of the present invention will be described with reference to the drawings. The ionic species concentration method 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. The lipid particles refer to liposomes, which refer to unilamellar vesicles consisting of a single lipid bilayer membrane. Furthermore, the lipid particles are formed from a lipid bilayer membrane that includes neutral first lipid molecules and anionic or cationic second lipid molecules, with the second lipid molecules present in at least the outer monolayer.

[0010] This method for concentrating an ionic species includes a step of dispersing the lipid particles described above in an ionic species solution in which a first ionic species having the same polarity as the second lipid molecule and a second ionic species having a different polarity from the second lipid molecule coexist, thereby encapsulating the first ionic species and the second ionic species within the lipid particles by utilizing the flip-flop phenomenon of the second lipid molecules in the lipid bilayer membrane.

[0011] In the ionic species concentration method according to the present embodiment, for example as shown in FIG. 1, a cation, which is a first ionic species contained in an ionic species solution, is encapsulated in a liposome R, which is a unilamellar vesicle formed from a lipid bilayer membrane BLM containing a phospholipid L, which is a neutral first lipid molecule, and a lipid molecule CL, which is a cationic second lipid molecule.

[0012] Neutral phospholipids constituting the lipid bilayer BLM include phosphatidylcholines such as PC (1,2-dioleoyl-sn-glycero-phosphocholine), 1,2-dioleoylphosphatidylcholine, 1,2-dipalmitoylphosphatidylcholine, 1,2-dimyristoylphosphatidylcholine, 1,2-distearoylphosphatidylcholine, 1-oleoyl-2-palmitoylphosphatidylcholine, 1-oleoyl-2-stearoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, 1-stearoyl-2-oleoylphosphatidylcholine, 1,2-dioleoylphosphatidylethanolamine, 1,2-dipalmitoylphosphatidylethanolamine, 1,2-dimyristoylphosphatidylethanolamine, 1,2-distearo ... amines, phosphatidylethanolamines such as 1-oleoyl-2-palmitoylphosphatidylethanolamine, 1-oleoyl-2-stearoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine, and N-succinyldioleoylphosphatidylethanolamine; and 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] In addition, the neutral phospholipids constituting the lipid bilayer BLM 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] Examples of cationic lipid molecules constituting the lipid bilayer membrane BLM include tertiary amines such as N-[(2,3-dioleoyloxypropyl)-1-trimethylammonium), stearamidopropyl dimethylamine, behenamidopropyl dimethylamine, etc., and quaternary ammoniums such as cetrimonium, steartrimonium, behentrimonium, stearoxypropyltrimonium, behenyl PG trimonium, distearyldimonium, dicocodimonium, dialkyl (C12-18) dimonium, quaternium-18, dicocoyl ethyl hydroxyethylmonium, distearoyl ethyl hydroxyethylmonium, and benzalkonium. Examples of cationic lipid molecules include transition metal ions, alkaline earth metal ions, and alkali metal ions, and positively charged complex molecules formed by neutral phospholipids.

[0015] As the cation, at least one anthracycline antibiotic selected from a group of hydrophobic cations including epirubicin, daunorubicin, doxorubicin, amrubicin, idarubicin, valrubicin, aclarubicin, pirarubicin, and mitoxantrone can be used. As the cation, at least one selected from a group of local anesthetic drugs having an amine structure, specifically, dibucaine, mepivacaine, bupivacaine, bupivacaine, levobupivacaine, ropivacaine, procaine, tetracaine, dibucaine, prilocaine, cocaine, ambroxol, phenylpiperidine derivatives, and morphinan derivatives can be used. Further, examples of cations include antiallergic drugs having an amine structure, antiarrhythmic drugs, antidepressants, antihypertensive drugs, cardiac stimulants, muscle relaxants, analgesics, antimalarials, antihypertensive drugs, neuroleptics, central antitussives, ovulation inducers, psychoneurotic drugs, anti-peptic ulcer drugs, vitamin supplements, antibacterial drugs, metabolic enzyme substrate drugs, circulatory system drugs, and antineoplastic drugs, specifically, azelastine, aprindine, amiodarone, amitriptyline, amlodipine, alprenolol, bopindolol, pindolol, bisoprolol, ambenonium, isoxsuprine, imipramine, indenolol, ethylmorphine, etilefrine, edrophonium, ephedrine, eperisone, oxycodone, oxybuprocaine, orsiverenaline, carteolol, quinabryl, quinine, guanabenz, chloramphenicol ... Rocapramine, clonidine, clofedalol, cloperastine, 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, timepidium, timolol, temocapril, terbinafine, prazosin derivatives, doxapram, hydrazinophthalazine derivatives, donepezil, dopamine, trimetazidine, trimetoquinol, trimebutine, nafamostat mesyl, normorphine derivatives, dihydropyridine derivatives, physostigmine derivatives,Triptyline derivatives, 2-amino-1-phenylethanol derivatives, thiazolidinedione derivatives, hydroxyzine, biperiden, physostigmine derivatives, pirenzepine, vinca alkaloid anticancer drugs, fexofenadine, propranolol derivatives, butylscovoramine, butenafine, butropium, morphinan derivatives, prazosin derivatives, flavoxate, progalvazine, propafenone, propiverine, propranolol derivatives, bromhexine derivatives, phenothiazine derivatives At least one selected from the group consisting of benzodiazepine, propranolol derivatives, betahistine, phenylpiperidine derivatives, phenylalkylamine derivatives, perphenazine, berberine, benzethonium, benserazide, homochlorcyclidine, phenethylamine derivatives, mabrotiline, imidazole derivatives, mexiletine, meclofenoxate, methylephedrine, methylergometrine, mefloquine, mepenzolate, mosapride, ranitidine, labetalol, and ritodrine can be used. Alternatively, the cation may be at least one dye selected from the group consisting of hydrophobic cations rhodamine, fluorescein, and eosin. In addition, at least one selected from the group consisting of diarylmethane, triarylmethane, azo-based, and nitrogen-containing heterocyclic compounds may be used as the cation.

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

[0017] The anion may be at least one selected from hydrophilic ions such as chloride ions, bromide ions, various phosphate ions, nitrate ions, and sulfate ions, carboxylic acid derivatives such as acetate, sulfonic acid derivatives, and polyaniline such as oligonucleic acid.

[0018] Here, the process of encapsulating anions and cations present on the outside S2 of the liposome R into the inside S1 of the liposome R will be described. First, as shown in FIG. 2(A), the anion AN, which is the second ionic species in the ionic species solution, is trapped by the cationic lipid molecule CL contained in the outer monolayer of the lipid bilayer BLM of the liposome R. At this time, as shown in FIG. 2(B), the cation CA in the ionic species solution is attracted to the anion AN trapped in the cationic lipid molecule CL contained in the outer monolayer of the lipid bilayer BLM. Then, as shown by the arrow AR1, the lipid molecule CL that has trapped the anion AN moves to the inner monolayer of the lipid bilayer BLM due to the flip-flop phenomenon. At this time, as the lipid molecule CL diffuses and moves to the inner monolayer of the lipid bilayer BLM, the anion AN trapped in the lipid molecule CL and the cation CA having the same polarity as the polarity of the lipid molecule CL attracted to the anion are encapsulated in the liposome R.

[0019] In the method for concentrating an ionic species according to the present embodiment, the concentration of anions present outside the liposome R is 10 mM or more. The content of cationic lipid molecules contained in the lipid bilayer membrane of the liposome R is 5% or more and 37% or less.

[0020] As described above, according to the ionic species concentration method of the present embodiment, the liposome R formed from the lipid bilayer BLM in which cationic lipid molecules exist in the outer monolayer is dispersed in an ionic species solution in which anions and cations coexist, so that the cationic lipid molecules present in the outer monolayer of the lipid bilayer BLM trap the anions in the ionic species solution. Then, as the cationic lipid molecules that have trapped the anions diffuse and move to the inner monolayer of the lipid bilayer BLM due to the flip-flop phenomenon, the anions trapped in the cationic lipid molecules and the cations attracted to the anions are encapsulated in the liposome R, and the cations are concentrated in the liposome R. As a result, even if the molar concentration of the cations contained in the ionic species solution present outside the liposome R is low, if the molar concentration of the anions is high, the concentration efficiency of the cations in the liposome R can be increased accordingly. Thus, for example, the molar concentration of anions in a solution of ionic species can be increased, but the molar concentration of cations must be kept low, thereby allowing the concentration of cations concentrated within liposomes R to be increased.

[0021] Although the embodiments of the present invention have been described above, the present invention is not limited to the configuration of the above-mentioned embodiments. For example, the liposome may be a unilamellar vesicle formed of a lipid bilayer membrane containing neutral phospholipids and anionic phospholipids, with the anionic phospholipid present at least in the outer monolayer. Here, the anionic phospholipid may be a phosphatidylserine such as 1,2-dioleoylphosphatidylserine, 1,2-dipalmitoylphosphatidylserine, 1,2-dimyristoylphosphatidylserine, 1,2-distearoylphosphatidylserine, 1-oleoyl-2-palmitoylphosphatidylserine, 1-oleoyl-2-stearoylphosphatidylserine, 1-palmitoyl-2-oleoylphosphatidylserine, 1-stearoyl-2-oleoylphosphatidylserine, 1,2 Examples of phosphatidylglycerin include phosphatidylglycerin such as 1-dioleoylphosphatidylglycerin, 1,2-dipalmitoylphosphatidylglycerin, 1,2-dimyristoylphosphatidylglycerin, 1,2-distearoylphosphatidylglycerin, 1-oleoyl-2-palmitoylphosphatidylglycerin, 1-oleoyl-2-stearoylphosphatidylglycerin, 1-palmitoyl-2-oleoylphosphatidylglycerin, and 1-stearoyl-2-oleoylphosphatidylglycerin. In this case, liposome R is dispersed in an ionic species solution in which anions and cations coexist, and cations in the ionic species solution are trapped in the anionic phospholipids present in the monolayer outside the lipid bilayer BLM. Then, as the anionic lipid molecules that have trapped the cations diffuse and move to the inner monolayer of the lipid bilayer due to the flip-flop phenomenon, the cations trapped in the anionic phospholipids and the anions attracted to the cations are encapsulated in the liposome.

[0022] Although the embodiment and the modified examples of the present invention have been described above, the present invention is not limited to these. The present invention includes appropriate combinations of the embodiment and the modified examples, and appropriate modifications thereto. EXAMPLES

[0023] The present invention will be specifically described below based on Examples 1 to 8 and Comparative Examples 1 to 3. First, the method of preparing the liposomes used in each Example and Comparative Example will be described. First, a lipid thin film containing neutral phospholipids and cationic lipid molecules was formed on an agarose film, and then phosphate buffer (pH: 7.0) was added to hydrate it. Here, the agarose film was prepared on a disk-shaped cover glass (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 cover glass, and then leaving it in a heated state at about 40°C for 1 hour. The lipid thin film was also prepared by dropping only 15 μL of a lipid chloroform solution onto the above-mentioned agarose film using a microdispenser (25 μL, manufactured by DRUMMOND SCIENTIFIC CO.), and then leaving the cover glass in a desiccator in a negative pressure environment for 1 hour to dry it.

[0024] Here, the lipid solution according to each Example was prepared by dissolving DOPC (1,2-dioleoyl-sn-glycero-phosphocholine) (Tokyo Chemical Industry Co., Ltd.) with a purity of more than 97.0%, DOTAP chloride (N-[(2,3-dioleoyloxypropyl)-1-trimethylammonium) chloride) (CAYMAN CHEMICAL Co., Ltd.), and cholesterol (Nacalai Tesque Co., Ltd.) in chloroform (Wako Pure Chemical Industries, Ltd.) to a total volume of 2 mL. Here, the lipid solutions of Examples 1, 6 to 9 were prepared by mixing 8 mg of DOPC, 2 mg of DOTAP, and 2 mg of cholesterol, so that the molar ratio of DOPC, DOTAP, and cholesterol was 58:15:27. The lipid solution of Example 2 was prepared by mixing 10 mg of DOPC, 0.7 mg of DOTAP, and 2 mg of cholesterol, so that the molar ratio of DOPC, DOTAP, and cholesterol was 67:5:27. The lipid solution of Example 3 was prepared by mixing 8 mg DOPC, 2 mg DOTAP, and 2 mg cholesterol, so that the molar ratio of DOPC, DOTAP, and cholesterol was 65:8:27. The lipid solution of Example 4 was prepared by mixing 5 mg DOPC, 5 mg DOTAP, and 2 mg cholesterol, so that the molar ratio of DOPC, DOTAP, and cholesterol was 37:37:25. The lipid solutions according to each comparative example were prepared by dissolving the above-mentioned DOPC (1,2-dioleoyl-sn-glycero-phosphocholine) (manufactured by Tokyo Chemical Industry Co., Ltd.) and cholesterol (manufactured by Nacalai Tesque Co., Ltd.) in chloroform (manufactured by Wako Pure Chemical Industries, Ltd.) to a total volume of 2 mL. The lipid solution of the comparative example was prepared by mixing 10 mg DOPC and 2 mg cholesterol, so that the molar ratio of DOPC and cholesterol was 73:27.

[0025] In Examples 1 to 7 and Comparative Examples, 800 μL of 1 M sucrose solution was added to the lipid thin film formed on the agarose film, and the thin film was left in a dark place for 3 hours to prepare liposomes. In Example 8, 800 μL of 1 M sucrose solution with acetate buffer was added to the lipid thin film formed on the agarose film, and the thin film was left in a dark place for 3 hours to prepare liposomes.

[0026] Next, a method for preparing an evaluation sample by placing liposomes on a cover glass that has been treated with a strong acid (piranha acid) and washed will be described. First, a disk-shaped cover glass (manufactured by Matsunami Glass Industry Co., Ltd.) with a diameter of 22 mm and a thickness of 0.12 to 0.17 mm was silane-treated. The silane treatment was performed by immersing the cover glass in a solution obtained by mixing APTS (3-aminopropyltriethoxysilane) (manufactured by Shin-Etsu Chemical Co., Ltd.) and ethanol (manufactured by Wako Pure Chemical Industries Co., Ltd.) in a volume ratio of 1:5 for 2 hours. After the silane treatment, the cover glass was washed with methanol and dried. Then, 200 μL of a dimethyl sulfoxide solution of a polyethylene glycol modifier was dropped onto the cover glass, and the cover glass was left for 1 hour, and then the cover glass surface was washed with distilled water to prepare a cover glass coated with BAM. The dimethyl sulfoxide solution was prepared by dissolving a polyethylene glycol modifying agent (SUNBRIGHT (registered trademark) OE-040CS, manufactured by NOF Corporation) in a molar concentration of 10 mmol / dm 3 The solution was prepared by dissolving the solution in dimethyl sulfoxide (manufactured by Wako Pure Chemical Industries, Ltd.) so that the solution became

[0027] Next, the cover glass was attached to an Azunol Petri dish (manufactured by Azunol Corporation) with a diameter of 40 mm and a height of 13.5 mm, which had a hole with a diameter of 18 μm drilled in the bottom wall, so as to cover the hole from the vertically lower side when the Azunol Petri dish is in use. Next, 160 μL of the sucrose solution containing the liposomes formed on the agarose film was measured using a micropipette and dropped onto the cover glass, thereby placing the liposomes on the cover glass.

[0028] Next, the evaluation method of the evaluation samples according to each example and each comparative example will be described. In each example, a confocal laser microscope (FLUOVIEW (registered trademark) FV10i: manufactured by Olympus Corporation) was used to observe the change over time in the fluorescence intensity of the evaluation sample. 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 the fluorescence intensity.

[0029] In Example 1, the molar concentration of rhodamine 6G was 2.5×10 -6 M, Cl - The molar concentrations of rhodamine 6G and Cl were adjusted to 100 mM, 10 mM, and 1 mM, and the molar concentration of sucrose was adjusted to 1 M. The aqueous solutions were added to the evaluation sample in an amount of 160 μL. - The sucrose solution containing Cl and Cl2 was added to the evaluation sample in two separate aliquots of 80 μL each. Then, the fluorescence intensity was observed 20 min after addition, with the focus on the liposomes contained in the evaluation sample. As shown in Figs. 3(A) to (C) and 4(A) to (C), the Cl and Cl2 of Example 1 were - Fluorescence from rhodamine 6G was observed inside the liposomes for all of the evaluation samples with molar concentrations of 100 mM, 10 mM, and 1 mM. - The fluorescence intensity increased with increasing molar concentration of Cl. - When the molar concentrations of rhodamine 6G were 100 mM and 10 mM, the fluorescence from rhodamine 6G inside the liposome was observed relatively prominently. - The concentration of liposomes increased with increasing molar concentration. The concentration ratio corresponds to the ratio of the average fluorescence intensity inside the liposome to the average fluorescence intensity outside the liposome in the fluorescence image of the liposome. The diameter of the liposome was estimated from the bright field image.

[0030] In Comparative Example 1 and Examples 2 to 4, the molar concentration of rhodamine 6G was 2.5×10-6 M, Cl - The molar concentration of rhodamine 6G and Cl was 100 mM, and the combined molar concentration of sucrose and glucose was 1.0 M. 160 μL of the aqueous solution was added to the evaluation sample. - After adding 80 μL of sucrose solution containing glucose and Cl to the evaluation sample, - A sucrose solution containing 80 μL of sucrose was added to the evaluation sample. Then, the fluorescence intensity was observed 20 min after the addition, with the focus on the liposomes contained in the evaluation sample. As shown in FIG. 6(A), the fluorescence from rhodamine 6G inside the liposomes was hardly observed in the evaluation sample according to Comparative Example 1. As shown in FIG. 6(B), many liposomes with a relatively low level of rhodamine 6G concentration ratio to the inside of 2 or less were observed in the evaluation sample according to Comparative Example 1. On the other hand, as shown in FIG. 7(A), FIG. 8(A), and FIG. 9(A), the fluorescence from rhodamine 6G inside the liposomes was observed in the evaluation samples according to Examples 2 to 4. As shown in FIG. 7(B), FIG. 8(B), and FIG. 9(B), it was found that the concentration ratio of rhodamine 6G to the inside of the liposomes was distributed to a relatively high level of 2 or more in the evaluation sample according to Comparative Example 1. This indicates that the concentration ratio of rhodamine 6G increases due to the inclusion of cationic lipid molecules in the lipid bilayer membrane of the liposome.

[0031] In Example 5, the molar concentration of epirubicin was 1.0×10 -5 M, Cl - The 5 mM acetate buffer solution was added to the evaluation sample in an amount of 160 μL, so that the molar concentration of sucrose was 100 mM and the combined molar concentration of sucrose and glucose was 1.0 M. In this case, the acetate buffer solution was present only on the outside of the liposome, and sucrose was present inside the liposome but glucose was not present. Then, with the focus on the liposome contained in the evaluation sample, 15 minutes after the addition of epirubicin, Cl was further added. -The fluorescence intensity was observed 20 min after the addition of epirubicin. As shown in Figures 10(A-1) and (A-2), the fluorescence intensity was observed 15 min after the addition of epirubicin and 20 min after the addition of Cl - In the evaluation sample before the addition of Cl, almost no fluorescence from epirubicin inside the liposome was observed. - In the evaluation sample 20 minutes after the addition of Cl, slight fluorescence from epirubicin inside the liposome was observed. In the evaluation sample of Example 5, as shown in FIG. 10(C), it was found that the concentration ratio of epirubicin inside the liposome was distributed at a relatively low level of 2 or less. From this, it can be seen that in the case of epirubicin, Cl - If Cl is not added, no concentration occurs inside the liposomes. - It can be seen that the addition of causes the liposomes to be concentrated toward the inside.

[0032] In Example 6, the molar concentration of daunorubicin was 1.0×10 -5 M, Cl - Daunorubicin, Cl, and sucrose and glucose were prepared to a combined molar concentration of 1.0M. - Then, 80 μL of 5 mM acetate buffer containing sucrose and glucose was added to the evaluation sample. In this case, the acetate buffer was present only on the outside of the liposome, and sucrose was present inside the liposome but not glucose. Then, with the focus on the liposome contained in the evaluation sample, 16 minutes after the addition of daunorubicin, Cl was further added. - The fluorescence intensity was observed 20 min after the addition of daunorubicin. As shown in Figures 11(A-1) and (A-2), the fluorescence intensity was observed 16 min after the addition of daunorubicin and 20 min after the addition of Cl. - Fluorescence from daunorubicin inside the liposome was observed even in the evaluation sample before the addition of Cl. -In the evaluation sample 20 minutes after the addition of Cl, it was observed that the fluorescence from daunorubicin inside the liposomes became stronger. In the evaluation sample of Example 6, as shown in FIG. 11(C), it was found that the concentration ratio of daunorubicin inside the liposomes was distributed at a level of 2 to 5. From this, it was found that in the case of daunorubicin, Cl - Although the concentration of liposomes in the inside occurs without the addition of Cl - It can be seen that the degree of concentration inside the liposome increases by adding 100 mg of daunorubicin. It can also be seen that daunorubicin is more easily concentrated inside the liposome than epirubicin.

[0033] In Example 7, the molar concentration of idarubicin was 1.0×10 -5 M, Cl - The molarity of idarubicin, Cl was adjusted to 100 mM, and the combined molarity of sucrose and glucose was adjusted to 1.0 M. - Then, 80 μL of 5 mM acetate buffer containing sucrose and glucose was added to the evaluation sample. In this case, the acetate buffer was present only on the outside of the liposome, and sucrose was present inside the liposome but not glucose. Then, with the focus on the liposome contained in the evaluation sample, 15 minutes after the addition of idarubicin, Cl was further added. - The fluorescence intensity was observed 20 min after the addition of idarubicin. As shown in Figures 12(A-1) and (A-2), the fluorescence intensity was observed 15 min after the addition of idarubicin and - Fluorescence from idarubicin inside the liposome was observed even in the evaluation sample before the addition of Cl. - In the evaluation sample 20 minutes after the addition of Cl, it was observed that the fluorescence from idarubicin inside the liposomes became stronger. As shown in FIG. 12(C), it was found that the concentration ratio of idarubicin inside the liposomes was distributed at a level of 2 to 10 in the evaluation sample of Example 7. From this, it was found that in the case of idarubicin, Cl - Although the concentration of liposomes in the inside occurs without the addition of Cl- It can be seen that the degree of concentration inside the liposome increases by adding 100% EDTA. It can also be seen that idarubicin is more easily concentrated inside the liposome than epirubicin.

[0034] In Example 8, the molar concentration of daunorubicin was 1.0×10 -5 M, Cl - Daunorubicin, Cl, and sucrose were added to make the total molar concentration 1.0M. - Then, 80 μL of acetate buffer containing sucrose and glucose at a concentration of 5 mM was added to the evaluation sample. Here, acetate ions were present not only on the outside but also on the inside of the liposome. Here, the acetate ions on the inside of the liposome function to adjust the pH so that daunorubicin becomes a cation. Also, sucrose was present on the inside of the liposome, but glucose was not present. Then, with the focus focused on the liposome contained in the evaluation sample, 16 minutes after the addition of daunorubicin, Cl was further added. - The fluorescence intensity was observed 20 min after the addition of daunorubicin. As shown in Figures 13(A-1) and (A-2), the fluorescence intensity was observed 15 min after the addition of daunorubicin and 20 min after the addition of Cl. - In the evaluation sample before the addition of Cl, no fluorescence from daunorubicin inside the liposome was observed. - Even in the evaluation sample 20 minutes after addition of daunorubicin, no fluorescence from the inside of the liposome was observed. This shows that when ions such as acetate ions are contained inside the liposome, concentration inside the liposome does not occur.

[0035] In Comparative Example 2, the molar concentration of daunorubicin was 1.0×10 -5 M, Cl - Daunorubicin, Cl, and sucrose were added to make the total molar concentration 1.0M. -Then, 80 μL of 5 mM acetate buffer containing sucrose and glucose was added to the evaluation sample. Then, with the focus on the liposomes contained in the evaluation sample, Cl - The fluorescence intensity was observed 20 minutes after the addition of daunorubicin. As shown in Figs. 14(A-1) and (A-2), no fluorescence from daunorubicin inside the liposome was observed. As shown in Fig. 14(B), it was found that the concentration ratio of daunorubicin inside the liposome was relatively low, being 2 or less, in the evaluation sample according to Comparative Example 2. In Comparative Example 3, the molar concentration of daunorubicin was 1.0 x 10 -5 M, ClO4 - Daunorubicin, ClO4 were added so that the molar concentration of 100 mM, sucrose and glucose were added so that the combined molar concentration was 1.0 M. - Then, 80 μL of 5 mM acetate buffer containing sucrose and glucose was added to the evaluation sample. Then, with the focus on the liposomes contained in the evaluation sample, ClO4 - The fluorescence intensity was observed 20 minutes after the addition of Daunorubicin. As shown in Fig. 15 (A-1) and (A-2), no fluorescence from Daunorubicin inside the liposome was observed. As shown in Fig. 15 (B), it was found that the concentration ratio of Daunorubicin inside the liposome was relatively low, being 2 or less, for the evaluation sample according to Comparative Example 3. From these results, it is found that in the case of liposomes formed from a lipid bilayer membrane that does not contain cationic lipid molecules, no concentration occurs inside the liposome. [Industrial Applicability]

[0036] The present invention is suitable for introducing ionic drugs, nucleic acid medicines, and the like into cells, and for producing exosomes for drug delivery containing ionic drugs, nucleic acid medicines, and the like. [Explanation of symbols]

[0037] AN: anion, BLM: lipid bilayer, CA: cation, CL: lipid molecule, L: phospholipid, R: liposome, S1: inside of liposome, S2: outside of liposome

Claims

1. 1. A method for concentrating ionic species, comprising encapsulating the ionic species within lipid particles formed from a lipid bilayer comprising a first lipid molecule, which is neutral, and a second lipid molecule, which is either anionic or cationic, with the second lipid molecule present in an outer monolayer, the method comprising: The method includes a step of dispersing the lipid particles in an ionic species solution in which a first ionic species having the same polarity as the second lipid molecules and a second ionic species having a different polarity from the second lipid molecules coexist, thereby encapsulating the first ionic species and the second ionic species in the lipid particles by utilizing the flip-flop phenomenon of the second lipid molecules in the lipid bilayer membrane. Methods for concentrating ionic species.

2. the second ionic species is an anion; 2. The method of claim 1 .

3. The molar concentration of the second ionic species in the ionic species solution is 10 mM or more. The method of concentrating ionic species according to claim 2.

4. the second lipid molecule is a cationic lipid molecule; The content of the second lipid molecule contained in the lipid bilayer is 5% or more and 37% or less. A method for concentrating an ionic species according to any one of claims 1 to 3.

5. The first ionic species is at least one anthracycline antibiotic selected from the group of anthracycline antibiotics including epirubicin, daunorubicin, doxorubicin, amrubicin, idarubicin, valrubicin, aclarubicin, pirarubicin, and mitoxantrone; 5. The method of claim 4 for concentrating an ionic species.

6. The first ionic species is at least one dye selected from the group consisting of rhodamine, fluorescein, and eosin; 5. The method of claim 4 for concentrating an ionic species.

7. The anion is Cl － , B － , B.F. ４ － , ClO ４ － At least one selected from the group consisting of The method of concentrating ionic species according to claim 2.

8. A lipid particle formed from a lipid bilayer comprising neutral first lipid molecules and anionic or cationic second lipid molecules, the second lipid molecules being present in an outer monolayer, The content of the second lipid molecule contained in the lipid bilayer is 5% or more and 37% or less. lipid particles.

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