WO2021225113A1 - Procédé de concentration d'espèces chimiques ioniques - Google Patents

Procédé de concentration d'espèces chimiques ioniques Download PDF

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WO2021225113A1
WO2021225113A1 PCT/JP2021/017165 JP2021017165W WO2021225113A1 WO 2021225113 A1 WO2021225113 A1 WO 2021225113A1 JP 2021017165 W JP2021017165 W JP 2021017165W WO 2021225113 A1 WO2021225113 A1 WO 2021225113A1
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cation
ionic
anion
molar concentration
concentrating
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PCT/JP2021/017165
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Japanese (ja)
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裕美 吉田
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国立大学法人京都工芸繊維大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes

Definitions

  • the present invention relates to a method for concentrating ionic species.
  • a method for producing a nanoparticle composition in which a metal cation is encapsulated inside nanoparticles composed of a lipid bilayer such as a liposome has been proposed (see, for example, Patent Document 1).
  • a step of preparing a nanoparticle composition containing a vesicle-forming component and a water-soluble and non-lipophilic chelating agent surrounded by the vesicle-forming component, and a step of preparing the nanoparticle composition in a solution containing a metal cation are performed.
  • the step of encapsulating the metal cation inside the nanoparticle composition by allowing the movement of the metal cation through the film formed by the vesicle-forming component is included. According to this production method, metal cations can be encapsulated in nanoparticles without using an ionophore.
  • the present invention has been made in view of the above reasons, and an object of the present invention is to provide a method for concentrating ionic species in particles composed of lipids, which can concentrate ionic species at a high concentration.
  • the ionic chemical species concentration method is used. It is a method for concentrating ionic species by encapsulating ionic species in lipid particles formed from lipids. It comprises a step of encapsulating the anion and the cation in the lipid particle by dispersing the lipid particle in an ionic species solution in which the anion and the cation coexist.
  • the molar concentration of either the anion or the cation in the ionic species solution is at least twice the molar concentration of the other, and the other is concentrated in the lipid particles.
  • the molar concentration of either anion or cation in the ionic species solution is at least twice the molar concentration of the other, and the other is concentrated in the lipid particles.
  • the distribution of cations and anions to the lipid particles can be increased accordingly. can. Therefore, the distribution of one ionic species outside the lipid particle into the lipid particle with the other ionic species is promoted, and as a result, the lipid particle together with one ionic species.
  • the concentration of the other ionic species enclosed therein can be increased.
  • FIG. Example 1 ClO the evaluation sample according to 4 - is a 1.0 ⁇ 10 -3 mol / dm 3 fluorescence image in a state immediately after addition (left) and bright-field image (right). ClO the evaluation sample according to Example 1 4 - is a 1.0 ⁇ 10 -3 mol / dm 3 fluorescence images of the addition to from 15 minutes after the state (left) and bright-field image (right).
  • Example 1 ClO the evaluation sample according to 4 - is a 1.0 ⁇ 10 -3 mol / dm 3 after the addition of the after 40 minutes state of the fluorescent image (left) and bright-field image (right). It is a figure which shows the distribution of the fluorescence intensity in line AA of FIG. 4A of the evaluation sample which concerns on Example 1. FIG. It is a figure which shows the distribution of the fluorescence intensity in line AA of FIG. 4B of the evaluation sample which concerns on Example 1. FIG. It is a figure which shows the distribution of the fluorescence intensity in line AA of FIG. 4C of the evaluation sample which concerns on Example 1.
  • Example 2 ClO the evaluation sample according to 4 - is a 5.0 ⁇ 10 -3 mol / dm 3 fluorescence image in a state immediately after addition (left) and bright-field image (right).
  • Example 2 ClO the evaluation sample according to 4 - is a 5.0 ⁇ 10 -3 mol / dm 3 fluorescence images of the addition to from 15 minutes after the state (left) and bright-field image (right).
  • Example 2 ClO the evaluation sample according to 4 - is a 5.0 ⁇ 10 -3 mol / dm 3 fluorescence images of the addition to the after lapse of 40 minutes state (left) and bright-field image (right).
  • FIG. 8A It is a figure which shows the distribution of the fluorescence intensity in line BB of FIG. 8A of the evaluation sample which concerns on Example 2.
  • FIG. 8B shows the distribution of the fluorescence intensity in line BB of FIG. 8B of the evaluation sample which concerns on Example 2.
  • FIG. 8C shows the distribution of the fluorescence intensity in line BB of FIG. 8C of the evaluation sample which concerns on Example 2.
  • FIG. It is a figure which shows the elapse time dependence after the addition of epirubicin of the ratio (fluorescence intensity ratio) of the fluorescence intensity on the outside of a liposome and the fluorescence intensity on the inside of a liposome according to Examples 1 and 2.
  • FIG. Example of evaluation samples of 3 BF 4 - is a 1.0 ⁇ 10 -2 mol / dm 3 fluorescence image in a state immediately after addition (left) and bright-field image (right).
  • Example of evaluation samples of 3 BF 4 - is a 1.0 ⁇ 10 -2 mol / dm 3 fluorescence images of the addition to the after lapse 35 minutes state (left) and bright-field image (right). It is a figure which shows the distribution of the fluorescence intensity in the CC line of FIG. 13A of the evaluation sample which concerns on Example 3.
  • FIG. It is a figure which shows the distribution of the fluorescence intensity in the CC line of FIG. 13B of the evaluation sample which concerns on Example 3.
  • Example 4 ClO the evaluation sample according to 4 - is a 1.0 ⁇ 10 -2 mol / dm 3 fluorescence image in a state immediately after addition (left) and bright-field image (right).
  • Example 4 ClO the evaluation sample according to 4 - is a 1.0 ⁇ 10 -2 mol / dm 3 fluorescence images added to the post after 20 minutes state (left) and bright-field image (right). It is a figure which shows the distribution of the fluorescence intensity in the DD line of FIG. 17A of the evaluation sample which concerns on Example 4.
  • FIG. It is a figure which shows the distribution of the fluorescence intensity in the DD line of FIG. 17B of the evaluation sample which concerns on Example 4.
  • FIG. 20A shows the distribution of the fluorescence intensity in the EE line of FIG. 20A of the evaluation sample which concerns on Example 5.
  • FIG. 20B shows the distribution of the fluorescence intensity in the EE line of FIG. 20B of the evaluation sample which concerns on Example 5.
  • FIG. It is a figure which shows the time dependence of the fluorescence intensity inside the liposome according to Examples 6 to 8 after the addition of FAM 2-.
  • 6 is a fluorescence image of a state 15 minutes after the addition of 5.0 ⁇ 10-7 mol / dm 3 of FAM-AAA 5- of the evaluation sample according to Example 9.
  • FIG. 6 is a fluorescence image of a state 10 minutes after the addition of 5.0 ⁇ 10 -4 mol / dm 3 of BTPPA + of the evaluation sample according to Example 9. It is a figure which shows the distribution of the fluorescence intensity in the FF line of FIG. 23A of the evaluation sample which concerns on Example 9. FIG. It is a figure which shows the distribution of the fluorescence intensity in the FF line of FIG. 23B of the evaluation sample which concerns on Example 9. FIG. It is a figure which shows the time dependence of the fluorescence intensity inside the liposome according to Examples 9 to 11 after the addition of FAM-AAA 5-.
  • the method for concentrating ionic species according to the present embodiment is a method for concentrating ionic species in lipid particles formed from lipids.
  • the ionic species are anions and cations.
  • lipid particles represent liposomes or micelles, and liposomes represent unilamella vesicles or multilamella vesicles (multilamella vesicles) composed of a single layer of lipid bilayer.
  • This ionic species concentration method includes a step of encapsulating anions and cations in the lipid particles by dispersing the lipid particles in an ionic species solution in which anions and cations coexist.
  • the molar concentration of either the anion or the cation in the ionic species solution is at least twice the molar concentration of the other, and the other ionic species is concentrated in the lipid particles.
  • a cation or an anion which is an ionic species is encapsulated in a liposome R which is a unilamella vesicle formed from a phospholipid bilayer BLM. do.
  • lymphocytes forming the liposome R examples include PC (1,2-dioleoil-sn-glycero-phosphocholine), 1,2-dioreoil phosphatidylcholine, 1,2-dipalmitylphosphatidylcholine, 1,2-dimylistylphosphatidylcholine, and the like.
  • Phosphatidylethanolamine 1,2-dioreoil phosphatidylserine, 1,2-dipalmityl phosphatidylserine, 1,2-dimylistyl phosphatidylserine, 1,2-dystearoyl phosphatidylserine, 1-oleyl-2-palmityl phosphatidyl Serine, 1-oleoil-2-stearoylphosphatidylserine, 1-palmitoyle 2-oleoil phosphatidylserine, 1-stearoyl-2-oleoil phosphatidylserine and other phosphatidylserine, 1,2-dioreoil phosphatidylglycerin, 1, 2-Dipalmityl phosphatidyl glycerin, 1,2-dimylistyl phosphatidyl glycerin, 1,2-dystearoyl phosphatidyl glycerin, 1-o
  • the phospholipids that form liposome R include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), COOL (cholesterol), and DSPE-PEG-2000 (1,2-distearoyl-n-).
  • anthracycline antibiotics including the hydrophobic cations epirubicin, daunorubicin, doxorubicin, amrubicin, idarubicin, balrubicin, acralvisi, pyrarubicin, and mitoxantrone.
  • Anthracycline antibiotics can be adopted.
  • the cation is selected from hydrophobic cations such as amines, rhodamines, cyanines, phosphonium cations, arsonium cations, imidazoliums, primary, secondary, tertiary or quaternary ammoniums. At least one ion can be employed.
  • a local anesthetic having an amine structure specifically, dibucaine, mepivacaine, bupivacaine, levobupivacaine, ropivacaine, procaine, tetracaine, prlocaine, cocaine, ambroxol, phenylpiperidin derivative, morphinan derivative At least one selected from the group can be adopted.
  • antiallergic agents having an amine structure antiarrhythmic agents, antidepressants, antihypertensive agents, cardiotonics, muscle relaxants, analgesics, antimalaria agents, hypotensive agents, nerve blockers, centralized agents
  • Antihypertensive drugs, ovulation inducers neuropsychiatric drugs, antidigestive ulcer drugs, vitamin replacement drugs, antihypertensive drugs, metabolic enzyme matrix drugs, cardiovascular drugs, antineoplastic drugs, specifically, azelastin, abluin, Amiodaron, amitriptilin, amlogipin, alprenolol, bopindolol, pindrol, bisopranolol, ambinonium, isoxpurin, imibramine, indenolol, ethylmorphine, ethyrefrin, edrophonium, ephedrine, emerison, oxycodon, oxybuprokine,
  • the cation may be at least one dye selected from a group of dyes having a rhodamine skeleton, which is a hydrophobic cation. Further, as the cation, at least one selected from diarylmethane, triarylmethane, an azo compound and a nitrogen-containing heterocyclic compound may be adopted.
  • metal ions for example, alkali metal ions such as Li + , Na + , K + , etc., alkaline earth metal ions such as Mg 2+ , Ca 2+ , Cu 2+ , Mn 2+ , Fe 2+, which are hydrophilic cations, are used.
  • Etc. alkali metal ions such as Li + , Na + , K + , etc., alkaline earth metal ions such as Mg 2+ , Ca 2+ , Cu 2+ , Mn 2+ , Fe 2+, which are hydrophilic cations
  • ammonium ions H +
  • basic amino acids lysine, arginine, etc.
  • at least one selected from cationic oligopeptides comprising them may be adopted.
  • the ionic species concentration method according to the present embodiment includes a step of encapsulating the cation or anion in the liposome R by dispersing the liposome R in an ionic species solution in which an anion and a cation coexist.
  • Anions include hydrophilic anions such as halide ion, sulfate ion, nitrate ion, phosphate ion, nucleic acid consisting of 3 or less nucleic acid bases, sulfonic acids, carboxylic acids, nucleic acids or acidic amino acids (aspartic acid, glutamate).
  • at least one selected from anionic oligopeptides comprising fluoresceins and fluoresceins can be adopted.
  • the halide ion at least one selected from the group of Cl ⁇ , Br ⁇ , and I ⁇ can be adopted.
  • a hydrophobic anion, borate anions of the ion, ClO 4 -, PF 6 - , BF 4 - is selected aromatic sulfonic acids carboxylic acids, alkyl sulfonic acids, from ions of alkyl carboxylic acids At least one can be adopted. Further, as the anion, at least one selected from a sulfonic acid derivative / carboxylic acid derivative having an alkyl group and an aryl group, and tetraphenylborate may be adopted.
  • the cations adsorbed on the surface of the phospholipid bilayer BLM do not invade the inner S1 of the liposome R.
  • equal amounts of cations and anions are distributed from the ionic species solution W existing on the outer side S2 of the liposome R into the phospholipid bilayer BLM of the liposome R so as to maintain electrical neutrality.
  • the cations and anions distributed to the phospholipid bilayer BLM are distributed to the inner S1 of the phospholipid bilayer BLM.
  • the distribution of cations and anions from the outer S2 of the liposome R into the phospholipid bilayer BLM is based on the cation concentration and anion concentration in the outer S2 of the liposome R and the cation concentration and anion concentration in the phospholipid bilayer BLM. It is determined by the distribution constant represented. Therefore, when the cation concentration and the anion concentration in the outer S2 of the liposome R are increased, the cation and anion concentrations in the phospholipid bilayer BLM increase, and the cation from the phospholipid bilayer BLM to the inner S1 of the liposome R becomes The amount of anions enclosed also increases.
  • the molar concentration of the anion in the ionic species solution is set to be at least twice the molar concentration of the cation.
  • the distribution of cations and anions between S2 on the outer side of the liposome R and the phospholipid bilayer BLM becomes larger than in the case where the molar concentration of the anions is the same as the molar concentration of the cations. Is promoted in the phospholipid bilayer BLM.
  • the distribution of the cation and anion from the phospholipid bilayer BLM to the inner S1 of the lipome R is increased, and the cation is encapsulated in the inner S1 of the liposome R at a high concentration.
  • the anion concentration added to the ionic species solution W at a high concentration is a molar amount of the salt contained in the buffer solution added to the ionic species solution W in order to avoid bursting or contraction of the liposome R due to osmotic pressure. It is preferable that the concentration is (for example, 0.1 mol / dm 3) or less.
  • the molar concentration of the cation in the ionic species solution may be twice or more the molar concentration of the anion.
  • the distribution of cations and anions between S2 on the outer side of the liposome R and the phospholipid bilayer BLM becomes larger than in the case where the molar concentration of the cation is the same as the molar concentration of the anion, and the anion Is promoted in the phospholipid bilayer BLM.
  • the distribution of cations and anions from the phospholipid bilayer BLM to the inner S1 of the lipome R is increased, and the anions are encapsulated in the inner S1 of the liposome R at a high concentration.
  • the molar concentration of anions in the ionic species solution is at least twice the molar concentration of cations.
  • the distribution of the cation and the anion to the phospholipid bilayer BLM can be increased accordingly. Therefore, the distribution of the anion present on the outer side S2 of the liposome R to the phospholipid bilayer BLM accompanied by the cation is promoted, and as a result, the concentration of the cation enclosed in the liposome R inner side S1 together with the anion can be increased. can.
  • the lipid particles may be micelles formed from phospholipids.
  • the micelle formed from the phospholipid may be dispersed in the ionic species solution in which the anion and the cation coexist to enclose the cation in the micelle.
  • the molar concentration of the anion in the ionic species solution may be at least twice the molar concentration of the cation.
  • a method for producing liposomes used in each example will be described.
  • a lipid thin film was formed on the agarose film, and then a phosphate buffer solution (pH: 7.0) was added and hydrated.
  • the agarose film is prepared at about 40 ° C. after dropping a 1 wt% agarose aqueous solution onto a disk-shaped cover glass (manufactured by Matsunami Glass Ind. Co., Ltd.) having a diameter of 22 mm and a thickness of 0.12 to 0.17 mm. It was produced on a cover glass by leaving it in a heated state for 1 hour.
  • the lipid thin film 15 ⁇ L of a chloroform solution of lipid was dropped onto the above-mentioned agarose film using a microdispenser (25 ⁇ L, manufactured by DRUMMOND SCIENTIFIC CO.), And then the cover glass was placed in a desiccator in a negative pressure environment. It was prepared by leaving it for a while to dry.
  • the lipid solution contains 126.8 mg of PC (1,2-dioleoil-sn-glycero-phosphocholine) (manufactured by Tokyo Chemical Industry Co., Ltd.) having a purity of more than 97.0% and cholesterol (manufactured by Nacalai Tesque, Inc.) 63.
  • a phosphate buffer solution having a molar concentration of 0.01 mol / dm 3 was added to the lipid thin film formed on the agarose film, and then the liposome was left in a dark place for 3 hours to form the liposome. Made.
  • Example 3 160 ⁇ L of a phosphate buffer solution having a molar concentration of 0.1 mol / dm 3 was added to the lipid thin film formed on the agarose film, and then the liposomes were left in a dark place for 3 hours to disperse the liposomes. Made.
  • a method for preparing a sample for evaluation by immobilizing the liposome on a cover glass coated with a cell membrane modifier (BAM: Biocompatible Anchor for cell Membrane) will be described.
  • a disk-shaped cover glass manufactured by Matsunami Glass Ind. Co., Ltd.
  • Silane treatment is obtained by mixing APTS (3-aminopropyltriethoxysilane) (manufactured by Shin-Etsu Chemical Industries, Ltd.) and ethanol (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) in a volume ratio of 1: 5.
  • dimethyl sulfoxide solutions polyethylene glycol modifier (SUNBRIGHT (R) OE-040CS: NOF Co., Ltd.), dimethylsulfoxide such that the molar concentration of 10 mmol / dm 3 dimethylsulfoxide (Fuji Film Wako Pure Chemical stock Made by dissolving in (manufactured by the company).
  • the cover glass coated with BAM was made into a hole of Aznol Petri dish (manufactured by AS ONE Corporation) with a diameter of 40 mm and a height of 13.5 mm in which a hole with a diameter of 18 m was drilled in the bottom wall. It was adhered to the Aznol petri dish so as to cover it from the lower side. Subsequently, using a micropipette, 100 ⁇ L of a phosphate buffer solution containing the liposomes formed on the agarose film was weighed and dropped onto a cover glass coated with BAM to coat the liposomes with BAM. Fixed to the cover glass.
  • a confocal laser scanning microscope FLOUVIEW (registered trademark) FV10i: manufactured by Olympus Corporation
  • FV10i a confocal laser scanning microscope
  • the excitation light source of the evaluation sample a laser light source of a laser microscope having an oscillation wavelength of 473 nm was used.
  • Example 1 50 ⁇ L of a concentration of 0.01 mol / dm 3 phosphate buffer containing epirubicin hydrochloride was added to the evaluation sample so that the molar concentration of epirubicin was 1.7 ⁇ 10 -5 mol / dm 3. bottom. Then, in a state of focusing on the liposomes contained in the evaluation sample, the fluorescence intensities were observed immediately after the addition and 15 minutes after the addition. As shown in FIGS. 2A and 2B, when epirubicin hydrochloride was added, fluorescence from the cation epirubicin was observed on the outside of the liposome and near the surface of the liposome. In particular, as shown in FIG.
  • Example 1 after a lapse 17min after the addition of epirubicin hydrochloride, ClO 4 - so that the molar concentration of 1.0 ⁇ 10 -3 mol / dm 3 of, 0 containing NaClO 4. Only 50 ⁇ L of 01 mol / dm 3 phosphate buffer was added to the evaluation sample. Then, the fluorescence intensities were observed immediately after the addition, 15 minutes after the addition, and 40 minutes after the addition. As shown in FIGS. 4A to 4C and 5A to 5C, it was observed that after the addition of epirubicin hydrochloride, the fluorescence from epirubicin inside the liposome increased with the passage of time.
  • Example 2 50 ⁇ L of 0.01 mol / dm 3 phosphate buffer containing epirubicin hydrochloride was prepared so that the molar concentration of epirubicin was 1.7 ⁇ 10 -5 mol / dm 3, as in Example 1. was added to the evaluation sample. Then, in a state of focusing on the liposomes contained in the evaluation sample, the fluorescence intensities were observed immediately after the addition and 15 minutes after the addition. As shown in FIGS. 6A, 6B and 7A, when epirubicin hydrochloride was added, fluorescence from the cation epirubicin was observed on the outside of the liposome and near the surface of the liposome. On the other hand, as shown in FIGS.
  • Example 2 After a lapse 17min after the addition of epirubicin chloride, ClO 4 - so that the molar concentration of 5.0 ⁇ 10 -3 mol / dm 3 of, 0 containing NaClO 4 Only 50 ⁇ L of 0.01 mol / dm 3 phosphate buffer was added to the evaluation sample. That is, phosphate buffer ClO 4 outside the liposomes as compared to Example 1 - molar concentration of was set to be 5 times. Then, the fluorescence intensities were observed immediately after the addition, 15 minutes after the addition, and 40 minutes after the addition. As shown in FIGS. 8A to 8C and FIGS.
  • FIG. 10 shows the elapsed time dependence of the ratio of the fluorescence intensity outside the liposome to the fluorescence intensity inside the liposome (hereinafter referred to as “fluorescence intensity ratio”) for the evaluation samples according to Examples 1 and 2. The result is shown.
  • Fig As shown in 10 the second embodiment according to the ClO 4 - increase of the fluorescence intensity inside the liposomes after the addition, Example 2 ClO 4 as compared with - compared to that of the added amount is less Example 1 It turned out to be bigger. Therefore, ClO 4 is an anion - A greater amount of it can be seen that encapsulation in inner liposome epirubicin a cation is promoted.
  • Example 3 50 ⁇ L of 0.1 mol / dm 3 phosphate buffer containing Rhodamine 6G was added to the evaluation sample so that the molar concentration of Rhodamine 6G was 1.0 ⁇ 10 -5 mol / dm 3. Then, in a state of focusing on the liposomes contained in the evaluation sample, the fluorescence intensities were observed immediately after the addition and 15 minutes after the addition. As shown in FIGS. 11A and 11B, when rhodamine 6G chloride was added, fluorescence from the cation rhodamine 6G was observed on the outside of the liposome and near the surface of the liposome. In particular, as shown in FIG.
  • BF 4 - as the molar concentration of the 1.0 ⁇ 10 -2 mol / dm 3 , 0 comprising NaBF 4 . 50 ⁇ L of 1 mol / dm 3 phosphate buffer was added to the evaluation sample. Then, the fluorescence intensity was observed immediately after the addition and 35 minutes after the addition. As shown in FIGS. 13A and 13B and FIGS. 14A and 14B, after the addition of rhodamine 6G chloride, it was observed that the fluorescence from the rhodamine 6G inside the liposome increased with the passage of time.
  • Example 4 As in Example 3, a phosphate buffer containing Rhodamine 6G chloride at a concentration of 0.1 mol / dm 3 so that the molar concentration of Rhodamine 6G is 1.0 ⁇ 10-5 mol / dm 3. Only 50 ⁇ L of the solution was added to the evaluation sample. Then, in a state of focusing on the liposomes contained in the evaluation sample, the fluorescence intensities were observed immediately after the addition and 15 minutes after the addition. As shown in FIGS. 15A, 15B and 16A, almost no fluorescence from rhodamine 6G was observed immediately after the addition. Then, as shown in FIGS.
  • Example 4 After a lapse 17min after the addition of Rhodamine 6G chloride, ClO 4 - so that the molar concentration of 1.0 ⁇ 10 -2 mol / dm 3 of, including NaClO 4 Only 50 ⁇ L of 0.1 mol / dm 3 phosphate buffer was added to the evaluation sample. That is, as an anion, BF 4 in Example 3 - in the same concentration as ClO 4 - was added to the outside of the liposomes. Then, the fluorescence intensity was observed immediately after the addition and 20 minutes after the addition. As shown in FIGS. 17A and 17B and FIGS.
  • FIG. 19 shows the results of measuring the elapsed time dependence of the fluorescence intensity inside the liposomes for the evaluation samples according to Examples 3 and 4. As shown in FIG. 19, it was found that the rate of increase in fluorescence intensity inside the liposome after ClO 4 - addition according to Example 4 was larger than that after BF 4 -addition in Example 3.
  • Example 5 fluorescein molar concentration of (FAM 2-) are formed so that 1.0 ⁇ 10 -6 mol / dm 3 , sodium fluorescein (FAM 2- 2Na +) 1.0 ⁇ 10 -4 including 50 ⁇ L of mol / dm 3 phosphate buffer was added to the evaluation sample. Then, in a state of focusing on the liposomes contained in the evaluation sample, the fluorescence intensity 15 minutes after the addition was observed. As shown in FIGS. 20A and 21A, almost no fluorescence from FAM 2-was observed inside the liposome.
  • Example 5 FAM 2-2Na + after the addition of the after a lapse 17Min, bis molarity 1.0 ⁇ 10 -3 mol / dm of (triphenylphosphoranylidene) ammonium (BTPPA +) at 3, bis (triphenylphosphoranylidene) ammonium chloride - was added 1.0 ⁇ 10 -4 mol / dm 3 phosphate buffer 50 ⁇ L containing the evaluation sample (BTPPA + Cl). Then, the fluorescence intensity 20 minutes after the addition was observed. As shown in FIGS. 20B and 21B, fluorescence from FAM 2- inside the liposome was observed.
  • FIG. 22 shows the results of measuring the elapsed time dependence of the fluorescence intensity inside the liposomes for the evaluation samples according to Examples 6 to 8.
  • the molar concentration of FAM 2- and the molar concentration of the phosphate buffer were 1.0 ⁇ 10-6 mol / dm 3 , 1.0 ⁇ , respectively, as in Example 5. It was adjusted to 10 -4 mol / dm 3 .
  • Example 6-8 the molar concentration of BTPPA + added after a lapse 17min after the addition of FAM 2-2Na +, respectively, 5.0 ⁇ 10 -4 mol / dm 3, 1.0 ⁇ It was adjusted to be 10 -4 mol / dm 3 and 5.0 ⁇ 10 -5 mol / dm 3 .
  • an increase in fluorescence intensity from FAM 2- inside the liposome was observed after the addition of BTPPA +.
  • Example 9 so that the molar concentration of fluorescein (FAM 2-) nucleic acid composed of modified three adenine at (FAM-AAA 5- 5Na +) is 5.0 ⁇ 10 -7 mol / dm 3 in, it was added 1.0 ⁇ 10 -4 mol / dm 3 phosphate buffer 50 ⁇ L containing a nucleic acid-sodium (FAM-AAA 5- 5Na +) in the evaluation sample. Then, in a state of focusing on the liposomes contained in the evaluation sample, the fluorescence intensity 15 minutes after the addition was observed. As shown in FIGS. 23A and 24A, almost no fluorescence from FAM 2-was observed inside the liposome.
  • Example 9 in after 17min elapsed since the addition of FAM-AAA 5- 5Na +, so that the molar concentration of BTPPA + is 1.0 ⁇ 10 -3 mol / dm 3 , BTPPA + Cl 50 ⁇ L of 1.0 ⁇ 10 -4 mol / dm 3 phosphate buffer containing ⁇ was added to the evaluation sample. Then, the fluorescence intensity 10 minutes after the addition was observed. As shown in FIGS. 23B and 24B, fluorescence from FAM 2- inside the liposome was observed.
  • FIG. 25 shows the results of measuring the elapsed time dependence of the fluorescence intensity inside the liposomes for the evaluation samples according to Examples 9 to 11.
  • the molar concentration of FAM-AAA 5- and the molar concentration of the phosphate buffer were 5.0 ⁇ 10-7 mol / dm 3 , 1. It was set to 0 ⁇ 10 -4 mol / dm 3 .
  • FIG. 25 shows the results of measuring the elapsed time dependence of the fluorescence intensity inside the liposomes for the evaluation samples according to Examples 9 to 11.
  • the molar concentration of FAM-AAA 5- and the molar concentration of the phosphate buffer were 5.0 ⁇ 10-7 mol / dm 3
  • the present invention is suitable for introducing ionic drugs, nucleic acid drugs, etc. into cells, and for producing exosomes for drug delivery containing ionic drugs, nucleic acid drugs, etc.
  • BLM phospholipid bilayer
  • R liposome
  • S1 inside of liposome
  • S2 outside of liposome
  • W ionic species solution

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Abstract

Le procédé de concentration d'espèces chimiques ioniques de l'invention consiste en un procédé de concentration d'un cation à l'intérieur de liposomes (R) formés à partir de bicouches de phospholipides (BLM). Le procédé de concentration d'espèces chimiques ioniques comprend une étape consistant à disperser des liposomes dans une solution d'espèces chimiques ioniques qui contient à la fois un anion et un cation, et à piéger ainsi l'anion et le cation à l'intérieur des liposomes (R). La concentration molaire de l'anion dans la solution d'espèces chimiques ioniques est au moins deux fois supérieure à la concentration molaire du cation, et le cation est concentré à l'intérieur des liposomes (R).
PCT/JP2021/017165 2020-05-08 2021-04-30 Procédé de concentration d'espèces chimiques ioniques WO2021225113A1 (fr)

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JP2018530623A (ja) * 2015-10-15 2018-10-18 リポメディックス・ファーマシューティカルズ・リミテッドＬｉｐｏｍｅｄｉｘ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ Ｌｔｄ． ドキソルビシンおよびマイトマイシンｃプロドラッグを共封入したリポソーム組成物

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Publication number Priority date Publication date Assignee Title
JP2018530623A (ja) * 2015-10-15 2018-10-18 リポメディックス・ファーマシューティカルズ・リミテッドＬｉｐｏｍｅｄｉｘ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ Ｌｔｄ． ドキソルビシンおよびマイトマイシンｃプロドラッグを共封入したリポソーム組成物

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MURAKAMI, KOJI ET AL.: "Adsorption and Distribution of Ions to a Bilayer Lipid Membrane", THE JOURNAL SOCIETY FOR ANALYTICAL CHEMISTRY, vol. 67, no. 10, 2018, pages 581 - 588, XP055873108 *
MURAKAMI, KOJI ET AL.: "Distribution and Adsorption of Ionic Species into a Liposome Membrane and Their Dependence upon the Species and Concentration of a Coexisting Counterion", LANGMUIR, vol. 32, 29 September 2016 (2016-09-29), pages 10678 - 10684, XP055873109, DOI: 10.1021/acs.langmuir.6b03162 *
OMATSU, TERUMASA ET AL.: "Dynamic behavior analysis of ion transport through a bilayer lipid membrane by an electrochemical method combined with fluorometr y", ANALYST, vol. 145, 27 March 2020 (2020-03-27), pages 3839 - 3845, XP055873110, DOI: 10.1039/d0an00222d *

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