WO2014133172A1 - 物質内包ベシクル及びその製造方法 - Google Patents
物質内包ベシクル及びその製造方法 Download PDFInfo
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- WO2014133172A1 WO2014133172A1 PCT/JP2014/055186 JP2014055186W WO2014133172A1 WO 2014133172 A1 WO2014133172 A1 WO 2014133172A1 JP 2014055186 W JP2014055186 W JP 2014055186W WO 2014133172 A1 WO2014133172 A1 WO 2014133172A1
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- 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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
-
- 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
-
- 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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/06—Making microcapsules or microballoons by phase separation
- B01J13/14—Polymerisation; cross-linking
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
- C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
- C08G69/10—Alpha-amino-carboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/40—Polyamides containing oxygen in the form of ether groups
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L77/00—Compositions of polyamides obtained by reactions forming a carboxylic amide link in the main chain; Compositions of derivatives of such polymers
- C08L77/04—Polyamides derived from alpha-amino carboxylic acids
Definitions
- the present invention relates to a novel substance-encapsulating vesicle and a method for producing the same.
- vesicles can be formed by self-organizing a polymer whose primary structure is precisely controlled. Since such vesicles can be designed in various ways and may exhibit new functions in addition to the properties inherent in macromolecules, they can be used for drug delivery systems (DDS) carriers, Use as materials and functional materials is under consideration.
- DDS drug delivery systems
- Patent Document 1 Japanese Patent Application Laid-Open No. 8-188541 discloses an electrostatically coupled polymer micelle drug carrier obtained by self-organizing a block copolymer having an uncharged segment and a charged segment. Disclosed by some of them.
- Non-Patent Document 1 (Schlaad H. et al., Macromolecules, 2003, 36 (5), 1417-1420) describes a block copolymer comprising a poly (1,2-butadiene) block and a poly (cesium methacrylate) block. And a vesicle called a polymersome, which is obtained by self-organizing a block copolymer composed of a polystyrene block and a poly (1-methyl-4-vinylpyridinium iodide) block.
- Patent Document 2 International Publication No. 2006/118260 discloses a first block copolymer (for example, PEG-polycation) having an uncharged hydrophilic segment and a cationic segment, and an uncharged hydrophilic segment.
- a vesicle obtained by self-assembling a second block copolymer (for example, PEG-polyanion) having an anionic segment and an anionic segment has been disclosed by some of the present inventors.
- Non-Patent Document 2 (Anraku Y. et al., J. Am. Chem. Soc., 2010, 132 (5), 1631-1636) describes a block co-polymer with an uncharged hydrophilic segment and a charged segment.
- a vesicle obtained by self-assembling a polymer (for example, PEG-polycation) and a copolymer (for example, polyanion) charged to a charge opposite to that of the chargeable segment is a part of the present inventors. It is disclosed.
- Non-Patent Document 3 H. Nyin et al. Soft Matter , 2006, 2, 940-949
- Non-Patent Document 4 New Development of Liposome Application", supervised by Kazunari Akiyoshi, NTS, 2005
- a substance to be included (hereinafter, sometimes referred to as “substance vesicle”)
- encapsulated substance A typical method is to form a vesicle by self-organization and enclose a substance in the void simultaneously by mixing with a polymer that is a component of the membrane or a polymer membrane that has been formed in advance (hereinafter referred to as “simultaneous”. May be labeled "mixing method”).
- Specific examples include the emulsion method (see Non-Patent Document 5 (F. Szoka, Jr et al., Proc. Natl.
- Non-Patent Document 6 (Batzri, S. et al., Biochim. Biophys Acta 1973, 298, 1015-1019)).
- the presence of the encapsulated substance affects the formation of vesicles by self-organization, and the formation of vesicles may be inhibited, or even if vesicles are formed, the substance may not be encapsulated in the voids. is there.
- an organic solvent harmful to film formation is often used, and the process becomes complicated, and there is a problem that the encapsulated material is easily damaged by the organic solvent.
- it is difficult to form vesicles having a uniform particle size and structure and in order to ensure such a uniform particle size and structure, another process must be added, and the process tends to be complicated. There are also challenges. Therefore, this method is not versatile and is not practical as a method for producing various substance-encapsulating vesicles.
- a method mainly used for encapsulating a substance in a hollow particle a method of introducing an inclusion substance into a void portion of an existing hollow particle and including and supporting it (hereinafter referred to as “post-supporting method”) (See, for example, Non-Patent Document 7 (W. Tong et al. J. Phys. Chem. B, 2005, 109, 13159-13165), etc.) and such a method may be applied to a vesicle.
- lipid bilayer vesicles such as liposomes
- a technique such as embedding a channel protein in the lipid bilayer (non-patent document 8 (Ranquin A, Versees W, Miere W, Steyaert J, Gelder PV. Therapeutic Nanoreactors: Combining Chemistry and Biology in a Novel Triblock Copolymer Drug Delivery System. Nano Lett. 2005; 5: 2220-4)) has also been reported, but again the process is extremely complicated and the versatility is also very low and practical. Not right.
- the present inventors are opposite to the first polymer that is a block copolymer having an uncharged hydrophilic segment and a first charged segment, and the first charged segment. And an empty vesicle having a void portion surrounded by the membrane, and a target substance to be encapsulated in the empty vesicle. Even in the presence of mixing in an aqueous medium (corresponding to the “post-loading method”), the target substance is encapsulated in the void (inner aqueous phase) by self-organization of the first and second polymers.
- Patent Document 3 International Publication No. 2011/145745 pamphlet.
- the concentration of the target substance existing in the aqueous medium exceeds a certain concentration
- the target substance is enclosed in the monodisperse aggregate of empty vesicles while maintaining the monodispersity. Therefore, the substance-containing vesicle formed is a polydisperse aggregate.
- the encapsulation of the target substance in the empty vesicle occurs as a probabilistic event depending on the concentration of the target substance present in the aqueous medium
- two or more objectives are included in the empty vesicle. It is difficult to enclose a substance by controlling the amount of the substance.
- the electrostatic interaction type vesicle manufactured by the conventional method has a limitation in the size of a substance that can be included. Specifically, the upper limit of the diameter of the encapsulated substance is limited to about 2 to 30 nm at the maximum, and a vesicle that encloses particles of a larger size (for example, more than 30 nm) has not been obtained. In addition, since normal electrostatic interaction type vesicles have a semipermeable membrane property, the lower limit of the diameter of the encapsulated substance is limited to about several thousand molecular weights, and low molecular weight compounds having smaller molecular weights are included. It was extremely difficult to do.
- the inclusion rate of substances in conventional electrostatic interaction vesicles depends on the physicochemical characteristics of the inclusion substance, but the probability theory depends on the concentration in the solution when the vesicle and inclusion substance are mixed. It was extremely difficult to encapsulate the substance in the vesicle with higher efficiency.
- the ratio of the size of the encapsulated substance to the vesicle is large (for example, when encapsulating particles having a diameter of 10 nm (tenths) or more with respect to a vesicle having a diameter of about 100 nm)
- the encapsulation efficiency is extremely low and usually a large amount
- the encapsulated substance remains unencapsulated and a large amount of empty vesicles remain.
- the vesicle is included in the vesicle by mixing the vesicle (or a polymer constituting the component) and the inclusion material in an aqueous medium. Therefore, it has been extremely difficult to efficiently encapsulate a vesicle with a substance having low solubility in a water-soluble medium (low water-soluble substance).
- the present invention firstly provides a substance-encapsulating vesicle in which a target substance having a higher concentration than the conventional method (for example, a concentration at which formation of a monodisperse aggregate of substance-encapsulating vesicles is inhibited in the post-loading method) is encapsulated. Even if the concentration of the monodisperse aggregate and the target substance existing in the aqueous medium is high (for example, the concentration at which formation of the monodisperse aggregate of the substance-encapsulating vesicle is inhibited in the post-loading method) It is an object of the present invention to provide a method for producing a substance-encapsulating vesicle that can form a monodisperse aggregate of vesicles.
- the present invention secondly includes a substance-encapsulating vesicle in which two or more kinds of target substances are encapsulated by controlling the amount of encapsulation, and two or more kinds of target substances are encapsulated by controlling the amount of encapsulation. It is an object of the present invention to provide a method for producing a substance-containing vesicle that can be used.
- the third aspect of the present invention is that, in the electrostatic interaction type vesicle based on the self-organization of the polymer, particles having a larger diameter and a smaller low molecular weight compound can be efficiently encapsulated than before, It is an object of the present invention to provide a method for producing a substance-encapsulating vesicle capable of improving the substance-encapsulating efficiency as compared with the prior art.
- the present invention fourthly relates to an electrostatic interaction type vesicle by self-organization of a polymer, in which an insoluble or low water-soluble substance can be efficiently encapsulated in the vesicle.
- An object is to provide a method for producing a vesicle.
- the gist of the present invention is as follows.
- a first polymer which is a block copolymer having an uncharged hydrophilic segment and a first charged segment, and a second charged property charged to a charge opposite to that of the first charged segment
- a second polymer having a segment, and a crosslinked film in which the first and / or the second polymer is crosslinked
- a monodisperse aggregate of substance-encapsulated crosslinked vesicles comprising a target substance encapsulated in the inner aqueous phase, the inner aqueous phase surrounded by the crosslinked membrane,
- the concentration of the target substance contained in the inner aqueous phase is Monodisperse set of empty non-crosslinked vesicles different from the monodisperse aggregate of the substance-encapsulated crosslinked vesicles in that the first and / or the second polymer is not crosslinked and the target substance is not encapsulated.
- the first and / or the second polymer is formed between a cation group and a cross-link bond formed between cation groups, a cross-link bond formed between anion groups, and a cation group and an anion group.
- Crosslinked by one or more types of crosslinks selected from the group consisting of crosslinks, and the proportion of the crosslinks formed is the total moles of cation groups and / or anion groups contained in the cross-linked film.
- a second polymer having a segment comprising a crosslinked film in which the first and / or the second polymer are crosslinked, and an inner aqueous phase surrounded by the crosslinked film,
- a substance-containing cross-linked vesicle in which a first target substance and a second target substance having a molecular weight smaller than that of the first target substance are included in the inner aqueous phase The substance-encapsulated cross-linked vesicle, wherein the first target substance is more stabilized than the case where the first target substance is contained in the inner aqueous phase in the absence of the second target substance.
- a method for producing a substance-encapsulating vesicle A first polymer which is a block copolymer having an uncharged hydrophilic segment and a first charged segment, and a second charged segment charged to a charge opposite to that of the first charged segment
- a crosslinked film comprising a second polymer, wherein the first and / or the second polymer is crosslinked,
- a monodisperse aggregate of empty cross-linked vesicles comprising an inner aqueous phase surrounded by the cross-linked membrane, wherein the target substance is not encapsulated in the inner aqueous phase, Mixing in a mixture containing the target substance with an aqueous medium;
- a crosslinked film comprising the first and second polymers, wherein the first and / or second polymers are crosslinked;
- a monodispersed assembly of the empty crosslinked vesicles, a monodispersed assembly of the substance-containing crosslinked vesicles, a monodispersed assembly of the empty non-crosslinked vesicles, and a monodispersed assembly of the substance-encapsulated uncrosslinked vesicles The method according to [7] above, having a polydispersity index of 0.2 or less. [9] The method according to [7] or [8] above, wherein the target substance has a weight average molecular weight of 10,000 to 40,000 and the concentration of the target substance contained in the mixed solution exceeds 5 mg / mL.
- the first and / or the second polymer are cross-linked bonds formed between cationic groups, cross-linked bonds formed between anionic groups, And a cross-linked film formed by one or more cross-links selected from the group consisting of a cross-link bond formed between a cation group and an anion group, and the ratio of the cross-link bond formed is the cross-linked film.
- a method for producing a substance-encapsulating vesicle A first polymer which is a block copolymer having an uncharged hydrophilic segment and a first charged segment, and a second charged segment charged to a charge opposite to that of the first charged segment
- a crosslinked film comprising a second polymer, wherein the first and / or the second polymer is crosslinked, An inner aqueous phase surrounded by the crosslinked membrane, and a first substance-encapsulated crosslinked vesicle in which the first target substance is encapsulated in the inner aqueous phase, Mixing a second target substance having a molecular weight smaller than that of the first target substance in a mixed solution containing an aqueous medium; A crosslinked film comprising the first and second polymers, wherein the first and / or second polymers are crosslinked; An inner aqueous phase surrounded by the crosslinked membrane, and forming a second substance-encapsulated crosslinked vesicle in which the first and second target substances are en
- a crosslinked film comprising the first and second polymers, wherein the first and / or the second polymer is crosslinked;
- An empty cross-linked vesicle comprising an inner aqueous phase surrounded by the cross-linked membrane, wherein the inner aqueous phase does not contain any of the first and second target substances, Mixing in a liquid mixture containing the first target substance together with an aqueous medium, and if necessary, reacting with a crosslinking agent capable of reacting with the first and / or the second polymer;
- the method according to any one of [12] to [14] above, further comprising the step of forming the first substance-encapsulated crosslinked vesicle.
- the empty crosslinked vesicle is a monodispersed aggregate thereof
- the first substance-containing crosslinked vesicle is a monodispersed aggregate thereof.
- the concentration of the first target substance contained in the mixed solution is When a monodispersed aggregate of empty non-crosslinked vesicles different from the monodispersed aggregate of empty crosslinked vesicles is mixed in the mixed solution in that the first and / or the second polymer is not crosslinked.
- the concentration is a concentration that inhibits formation of a monodisperse aggregate of a substance-encapsulated non-crosslinked vesicle in which the first target substance is encapsulated.
- a first polymer which is a block copolymer having an uncharged hydrophilic segment and a first charged segment, and a second charge charged to a charge opposite to the first charged segment A vesicle comprising a film containing a second polymer having a sex segment and adsorbent particles encapsulated in the vesicle, wherein at least one of the first and second polymers is adsorbed on the adsorbent particles.
- a first polymer which is a block copolymer having an uncharged hydrophilic segment and a first charged segment, and a second charge charged to a charge opposite to that of the first charged segment A method for producing an adsorbent-encapsulating vesicle in which adsorbent particles are encapsulated in a vesicle comprising a film containing a second polymer having a sex segment, (A) mixing one of the first and second polymers with the adsorbent particles and adsorbing the adsorbent particles; and (B) A vesicle composed of a film containing the first and second polymers around the adsorbent particles by further mixing the mixture of the step (a) with the other of the first and second polymers.
- a first polymer which is a block copolymer having an uncharged hydrophilic segment and a first charged segment, and a second charge charged to a charge opposite to that of the first charged segment A method for producing a substance-encapsulating vesicle in which a target substance is encapsulated in a vesicle comprising a film containing a second polymer having a sex segment, (A) An enzyme-encapsulating vesicle is prepared in which an enzyme capable of converting a precursor having a higher water solubility than the target substance into the target substance is encapsulated in a vesicle composed of a film containing the first and second polymers.
- a low water-soluble substance that can be converted from a precursor having a higher water solubility than the target substance into a vesicle comprising a film containing a second polymer having a sex segment converts the precursor into the low water-soluble substance.
- a low water-soluble substance-encapsulating vesicle, which is encapsulated together with the enzyme obtained.
- a method for delivering a drug to a subject comprising: (A) a first polymer which is a block copolymer having an uncharged hydrophilic segment and a first charged segment, and a second charge charged to a charge opposite to the first charged segment Preparing an enzyme-encapsulating vesicle in which an enzyme capable of converting a precursor of the drug into the drug is encapsulated in a vesicle comprising a film containing a second polymer having a sex segment; and (B) A method comprising forming the drug by allowing the precursor to penetrate into the enzyme-encapsulating vesicle at a predetermined site of interest and converting the precursor into the drug by the enzyme.
- the water solubility of the precursor is lower than that of the drug, and the solubility in the drug is lower than that of the precursor in the step (b).
- a substance-encapsulating vesicle in which a target substance having a higher concentration than the conventional method for example, a concentration at which formation of a monodisperse aggregate of the substance-encapsulating vesicle is inhibited in the post-loading method
- a concentration at which formation of a monodisperse aggregate of the substance-encapsulating vesicle is inhibited in the post-loading method is encapsulated. Even if the concentration of the monodisperse aggregate and the target substance existing in the aqueous medium is high (for example, the concentration at which formation of the monodisperse aggregate of the substance-encapsulating vesicle is inhibited in the post-loading method)
- a method for producing a substance-encapsulating vesicle capable of forming a monodisperse aggregate of vesicles is provided.
- a substance-encapsulating vesicle in which two or more kinds of target substances are encapsulated by controlling the amount of encapsulation, and two or more kinds of target substances are encapsulated by controlling the amount of encapsulation.
- a method for producing a substance-encapsulating vesicle that can be provided is provided.
- a substance-encapsulating vesicle (adsorbent-encapsulating vesicle) having a new structure that has not been heretofore encapsulated with relatively large adsorbent particles relative to the size of the vesicle.
- a method capable of extremely efficiently producing such an adsorbent-containing vesicle is provided.
- a vesicle encapsulating a target substance with low water solubility which was difficult with the conventional method, can be efficiently produced.
- FIGS. 1A and 1B are diagrams for explaining the method of the present invention.
- FIG. 2 is a diagram for explaining the structure of an empty vesicle.
- FIGS. 3A and 3B are diagrams for explaining one mode of the film structure of the empty vesicle.
- FIGS. 4A and 4B are diagrams for explaining another aspect of the film structure of the empty vesicle.
- FIG. 5A is a diagram showing a transmission electron microscope (TEM) image of the FITC-Dex40k-encapsulated crosslinked vesicle obtained in Example I-1.
- TEM transmission electron microscope
- FIG. 5B is a view showing a transmission electron microscope (TEM) image of the FITC-Dex40k-containing non-crosslinked vesicle obtained in Comparative Example I-1 (simultaneous mixing method).
- FIG. 5C is a view showing a transmission electron microscope (TEM) image of the FITC-Dex40k-containing non-crosslinked vesicle obtained in Comparative Example I-2 (post-supporting method).
- FIG. 6 is a diagram showing the measurement results of GPC (gel permeation chromatography) carried out to confirm the release behavior of FITC-Dex40k enclosed in the vesicle.
- FIG. 7 (a) is a diagram showing the GPC measurement results of empty vesicles, and FIG.
- FIG. 7 (b) is a diagram showing the GPC measurement results of the purified cytochrome c-encapsulated crosslinked vesicles obtained in Example I-2. It is.
- FIG. 8 (a) shows the absorption spectrum of the cytochrome c-encapsulated crosslinked vesicle obtained in Example I-2
- FIG. 8 (b) shows the cytochrome c and FITC obtained in Example I-2.
- FIG. 9 is a diagram showing measurement results of a circular dichroism spectrum.
- FIG. 8 (a) shows the absorption spectrum of the cytochrome c-encapsulated crosslinked vesicle obtained in Example I-2
- FIG. 8 (b) shows the cytochrome c and FITC obtained in Example I-2.
- -Dex4k inclusion cross-linked vesicles showing absorption spectra.
- FIG. 9 is a diagram showing measurement results of
- FIG. 10 (A) is a diagram showing the results of a colorimetric test based on the absorption color of the vesicle AlPcS2a obtained in Example I-3 and Comparative Example I-3.
- FIG. 10B shows the absorption spectra of the vesicles obtained in Example I-3 and Comparative Example I-3.
- FIG. 11 is a transmission electron microscope (TEM) image of vesicles after addition of 5 mM, 10 mM, or 20 mM NaCl.
- FIG. 12 is a transmission electron microscope (TEM) image of the CD-encapsulated crosslinked vesicle obtained in Example I-4.
- FIG. 13 is a graph showing the conversion rate from 5-FC to 5-FU by the CD-encapsulated crosslinked vesicle obtained in Example I-4.
- FIG. 14 is a graph showing the stability of 5-FC to 5-FU converting enzyme activity by the CD-encapsulated cross-linked vesicle obtained in Example I-4.
- FIGS. 15 (a) to 15 (d) are graphs showing the tumor growth inhibitory effect by treatment with the CD-encapsulated crosslinked vesicle obtained in Example I-4.
- FIGS. 16A to 16D are graphs showing side effects (weight loss) due to treatment with the CD-encapsulated cross-linked vesicle obtained in Example I-4.
- FIGS. 17 (a) to (d) are graphs showing the number of survival by treatment using the CD-encapsulated cross-linked vesicle obtained in Example I-4.
- FIG. 18 is a graph showing the retention in blood of the CD-encapsulated crosslinked vesicle obtained in Example I-4.
- FIG. 19 is a graph showing the particle size distribution of the crosslinked MSN-containing vesicle-containing solution obtained in Example II-1.
- FIG. 20 is a TEM photograph of the crosslinked MSN-containing vesicle obtained in Example II-1.
- FIG. 21 is a chromatogram obtained by capillary electrophoresis of the crosslinked MSN-encapsulating vesicle obtained in Example II-1.
- FIG. 22 is a graph showing the particle size distribution of a solution containing a crosslinked MSN-containing vesicle obtained in Example II-2.
- FIG. 23 is a TEM photograph of the crosslinked MSN-containing vesicle obtained in Example II-2.
- FIG. 24 is a chromatogram obtained by capillary electrophoresis of the crosslinked MSN-encapsulating vesicle obtained in Example II-2.
- FIG. 25 is a graph showing the particle size distribution of a solution containing a crosslinked MSN-containing vesicle obtained in Reference Example II-1.
- FIG. 26 is a TEM photograph of the crosslinked MSN-containing vesicle obtained in Reference Example II-1.
- FIG. 27 is a chromatogram obtained by capillary electrophoresis of the crosslinked MSN-encapsulating vesicle obtained in Reference Example II-1.
- FIG. 28 (a) is a TEM photograph of an untreated MSN-encapsulating vesicle used in Example II-3A
- FIG. 28 (b) is a TEM photograph of an aminated MSN-encapsulating vesicle obtained in Example II-3A. It is.
- FIG. 29 (a) is a TEM photograph of the untreated MSN-encapsulating vesicle used in Example II-3B
- FIG. 29 (b) is a TEM photograph of the sulfonylated MSN-encapsulating vesicle obtained in Example II-3B. It is.
- FIG. 30 is an X-ray analysis spectrum of an untreated MSN-encapsulating vesicle and the obtained sulfonylated MSN-encapsulating vesicle used in Example II-3B.
- FIG. 31 is a graph showing the release characteristics of rose bengal by the rose bengal adsorption aminated MSN-encapsulated vesicle obtained in Example II-4A.
- FIG. 32 is a graph showing the release characteristics of gemcitabine by the gemcitabine-adsorbed sulfonylated MSN-encapsulating vesicle obtained in Example II-4B.
- FIGS. 34 (a) and (b) are graphs showing the cell-killing effect on A549 cells by the gemcitabine-adsorbed sulfonylated MSN-encapsulating vesicle obtained in Example II-4B.
- FIG. 33 (a) shows the measurement results of Cy3 fluorescence in all cells in the absence of trypan blue
- FIG. 33 (b) shows the measurement results of Cy3 fluorescence in living cells under trypan blue staining.
- FIGS. 34 (a) and (b) are graphs showing the cell-killing effect on A549 cells by the gemcitabine-adsorbed sulfonylated MSN-encapsulating vesicle obtained in Example II-4B.
- FIG. 34 (a) and (b) are graphs showing the cell-killing effect on A549 cells by the gemcitabine-adsorbed sulfonylated MSN-encapsulating vesicle obtained in Example II-4B.
- FIGS. 35 (a) and (b) are graphs showing the cell uptake characteristics of C26 cells by the gemcitabine-adsorbed sulfonylated MSN-encapsulating vesicle obtained in Example II-4B.
- FIG. 35 (a) shows the measurement result of Cy3 fluorescence amount in all cells in the absence of trypan blue
- FIG. 35 (b) shows the measurement result of Cy3 fluorescence amount in living cells under trypan blue staining.
- FIGS. 37 (a) and (b) are graphs showing the tumor therapeutic effect using the gemcitabine-adsorbed sulfonylated MSN-encapsulating vesicle obtained in Example II-4B.
- A shows the tumor growth inhibitory effect
- (b) shows the side effect (weight loss) reducing effect.
- FIG. 38 is a graph showing the retention in blood of gemcitabine-adsorbed sulfonylated MSN-encapsulating vesicles obtained in Example II-4B.
- FIG. 39 is a graph showing the tumor accumulation property of gemcitabine-adsorbed sulfonylated MSN-encapsulating vesicles obtained in Example II-4B.
- FIG. 40 is a graph showing the particle size distribution of the ⁇ -galactosidase-containing cross-linked vesicle-containing solution obtained in Example III-1.
- FIG. 41 is a graph showing the particle size distribution of the indigo dye-containing vesicle-containing solution obtained in Example III-1.
- FIG. 42 is a TEM photograph of the indigo dye-containing vesicle obtained in Example III-1.
- FIG. 43 is a high-resolution TEM photograph of the indigo dye-containing vesicle obtained in Example III-1.
- FIG. 44 is a graph showing the particle size distribution of the crosslinked empty vesicle-containing solution obtained in Comparative Example III-1.
- FIG. 45 is a graph showing the particle size distribution of an empty vesicle-containing solution after production of the indigo dye obtained in Comparative Example III-1.
- FIG. 46 is a TEM photograph of the empty vesicle obtained after the indigo dye was obtained in Comparative Example III-1.
- vesicle means a basic structure having a single lamellar structure film and a void (inner water phase) surrounded by the film.
- alkyl as a group name or a part thereof represents a monovalent aliphatic saturated hydrocarbon group.
- alkyl may be linear or cyclic, and may be a combination of linear and cyclic.
- the chain alkyl may be linear or branched.
- Cyclic alkyl may be monocyclic or bicyclic, and in the case of polycyclic, it may be a linked ring, a condensed ring or a spiro ring.
- alkoxy as a group name or a part thereof represents a group in which the alkyl is bonded to one bond of a divalent oxygen atom.
- aryl as a group name or a part thereof represents a monovalent aromatic hydrocarbon group. Unless otherwise specified, aryl may be monocyclic or polycyclic, and in the case of polycyclic, aryl may be a linked ring, a condensed ring, or a spiro ring.
- the number of carbon atoms of the group is represented as “C 1-12 alkyl”, for example.
- C 1-12 means that the alkyl has 1 to 12 carbon atoms.
- halogen atom means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
- a certain group may be “substituted” in which one or two or more hydrogen atoms of the group have one or more substituents (in the case of two or more, they may be the same or different). It may be substituted by (good).
- the maximum number of substituents can be easily determined by those skilled in the art depending on the structure of the group and the number of hydrogen atoms that can be substituted.
- the “substituent” is a halogen atom, aryl group, hydroxyl group, amino group, carboxyl group, cyano group, formyl group, dimethylacetalized formyl group, diethylacetalized formyl group, C 1. -6 alkoxycarbonyl group, C 2-7 acylamide group, siloxy group, tri (C 1-6 alkyl) siloxy group (wherein C 1-6 alkyl may be the same or different) and silylamino group Selected.
- the term “monodispersed aggregate” used for various vesicles means a vesicle aggregate having a narrow particle size distribution.
- the width of the particle size distribution is preferably determined based on a polydispersity index (PDI), and the monodisperse aggregate is preferably 0.2 or less, more preferably 0.15 or less, and still more preferably It has a polydispersity index of 0.1 or less.
- PDI polydispersity index
- polydispersity index used in the present specification for various vesicles means a dimensionless index indicating the spread of the particle size distribution
- average particle size refers to the harmonic mean particle size according to the scattered light intensity standard ( Diameter). Both the polydispersity index and the average particle size are measured by dynamic light scattering.
- the dynamic light scattering method can be carried out in accordance with JIS Z 8826: 2005 (Particle Size Analysis-Photon Correlation Spectroscopy). JIS Z 8826: 2005 is a standard corresponding to a dispersion having a dilute particle concentration. Therefore, even in a high concentration sample, the polydispersity index and the average particle diameter can be measured. Appropriate changes can be made to 2005.
- backscatter detection can be employed instead of side scatter detection.
- backscattering detection it is possible to eliminate or reduce the influence of multiple scattering, thereby enabling measurement of the polydispersity index and average particle diameter even in a high concentration sample.
- examples of commercially available instruments for measuring the polydispersity index and the average particle diameter by the dynamic light scattering method include Zetasizer Nano-ZS manufactured by Malvern. This instrument employs backscatter detection, and can measure the polydispersity index and the average particle diameter of samples in a wide concentration range from low concentration to high concentration.
- the main method for producing a substance-encapsulating vesicle is as follows: (i) Formation of a vesicle by self-organization by mixing an encapsulated substance with a polymer that is a constituent element of a film or a pre-formed polymer film And (ii) mixing an empty vesicle formed in advance with the encapsulated substance, introducing the encapsulated substance into the void of the empty vesicle, And a method of including and supporting (post-supporting method).
- any method including a simultaneous mixing method and a post-supporting method can be used unless the manufacturing conditions of each vesicle are described separately.
- the post-carrying method developed by the inventors of the present application for details, see Patent Document 3 (International Publication No. 2011/145745 pamphlet)
- the inclusion substance is efficiently introduced into the vesicle by a simple method.
- the structure of the vesicle is hardly damaged before and after the introduction, and it is preferable because it has various advantages such as being able to encapsulate a charged substance that was difficult to encapsulate by the simultaneous mixing method.
- the method for producing a substance-encapsulating vesicle will be described on the premise of the post-loading method.
- the method for producing a substance-encapsulating vesicle is not limited to the post-loading method, and is contrary to the characteristics of each substance-encapsulating vesicle described below. As long as this is not the case, any other method including the simultaneous mixing method can be used. Further, when the substance is included in the vesicle using a method other than the post-supporting method, the conditions of the post-supporting method described later can be applied mutatis mutandis.
- an empty vesicle is not prepared in advance as in the post-loading method, but is basically the same as the post-loading method, except that the polymer or the like that is the material of the empty vesicle is mixed with the encapsulated substance.
- the same conditions can be used.
- FIG. 1 is a schematic diagram, and the present invention is not limited to FIG.
- an empty vesicle 1 having a predetermined structure having a void 1 b surrounded by a film 1 a is prepared, and this is mixed with an encapsulated substance 9 in an aqueous medium.
- the substance to be encapsulated 9 is introduced into the gap 1b beyond the membrane 1a of the empty vesicle 1, and the substance-encapsulating vesicle 1 in which the substance to be encapsulated 9 is encapsulated in the gap 1b.
- the post-loading method will be described in detail, but the details of the empty vesicle and the encapsulated substance will be described later again, and other conditions and procedures will be described here.
- the post-loading method includes a step of mixing an empty vesicle and an encapsulated substance in an aqueous medium (note that a liquid containing an empty vesicle and an encapsulated substance in an aqueous medium, which is an object of mixing, is hereinafter referred to as “Often referred to as “mixed liquid”).
- a liquid containing an empty vesicle and an encapsulated substance in an aqueous medium which is an object of mixing, is hereinafter referred to as “Usually referred to as “mixed liquid”).
- the method for mixing is not particularly limited, the mixing is performed by applying an external force to the aqueous medium.
- a method of adding an empty vesicle and an encapsulated substance to an aqueous medium, allowing the mixture to stand, and naturally diffusing and mixing (hereinafter sometimes referred to as “stationary / diffusion mixing”) is excluded.
- Examples of the mixing method in which an external force is applied to the aqueous medium include stirring, shaking, impact and the like.
- Examples of the technique by stirring include a technique in which a container containing the liquid to be mixed is swirled by a vortex mixer or the like, a technique in which the solution is directly stirred by a stirring blade, or the like.
- As an example of the technique by shaking there is a technique of shaking a container containing the liquid to be mixed with a shaker or the like.
- Examples of the technique using impact include a technique of applying various impacts including vibration to the liquid to be mixed by ultrasonic irradiation or the like.
- the substance is included in the vesicle space, and the substance-containing vesicle is manufactured.
- a shear stress acts on the empty vesicle (so that mixing that applies an external force to the aqueous medium is performed under the shear stress. It can be paraphrased as mixing.) Due to such shear stress, the structure of the empty vesicle is disturbed and decomposed into substantially uniform small aggregates, which are self-assembled again to regenerate the vesicle uniformly, and the encapsulated substance present in the aqueous medium is also recovered.
- the mixing conditions are not limited, but considering the above mechanism, it is preferable to select conditions such that the structure of the empty vesicles in the aqueous medium is sufficiently disturbed and the structure of the vesicles can be regenerated after the disturbance.
- mixing may be performed to such an extent that force acts on the entire liquid to be mixed, but it is preferable to perform mixing so that the entire liquid to be mixed becomes substantially uniform.
- mixing conditions vary depending on the mixing method, for example, in the case of stirring, it is usually performed at a rotation speed of 500 rpm or more, preferably 1000 rpm or more, and usually 10,000 rpm or less, preferably 5000 rpm or less. If the rotational speed is too low, it may be difficult to form a homogeneous substance-containing vesicle. If the rotational speed is too high, vesicles and encapsulated materials may be damaged or destroyed. Further, the stirring time by the vortex mixer varies depending on the number of rotations, but is usually 60 seconds or longer, preferably 120 seconds or longer, and usually within 10 minutes, preferably within 5 minutes.
- stirring time is too short, it may be difficult to form a homogeneous substance-encapsulating vesicle. If the stirring time is too long, the vesicle and the encapsulated substance may be damaged or destroyed.
- stirring with a vortex mixer is performed under the above-described rotation speed and stirring time. The conditions may be appropriately adjusted so that the same level of force as that obtained by the above acts on the liquid to be mixed.
- the mixing target liquid is allowed to stand for a certain period of time after the mixing target liquid is mixed to ensure a time for the vesicles to be uniformly regenerated.
- standing time is not limited, but is, for example, 1 minute or longer, preferably 3 minutes or longer.
- the inclusion of the substance in the void of the vesicle means that the diffusion coefficient is detected by fluorescence correlation spectroscopy (FCS), the separation by size exclusion chromatography, and the direct transmission electron microscope. It can be confirmed by a method such as observation.
- FCS fluorescence correlation spectroscopy
- a fluorescent material is used as the inclusion substance, and the change in the diffusion coefficient of the fluorescent material is measured, whereby the inclusion substance is unevenly distributed in the vesicle (that is, the substance inclusion vesicle is Obtained).
- a liquid (a liquid to be mixed) containing empty vesicles and inclusion substances in an aqueous medium is prepared and subjected to the above-described mixing.
- the kind of aqueous medium (aqueous solvent) is not limited. Water is preferable, but a solvent in which other components are mixed with water (for example, physiological saline, aqueous solution, as long as it does not unfavorably affect the structure of the empty vesicles and does not prevent introduction of the encapsulated substance into the interior) Buffer solutions, mixed solvents of water and water-soluble organic solvents, etc.) can also be used.
- the aqueous buffer include 10 mM HEPES buffer.
- water-soluble organic solvent examples include alcohols such as methanol and ethanol, ketones such as acetone, chlorine-based organic solvents such as chloroform, ether-based organic solvents such as dimethyl ether, and ester-based organic solvents such as ethyl acetate.
- the procedure for preparing the liquid to be mixed is also arbitrary. However, since empty vesicles are usually prepared in an aqueous medium as described later, an encapsulated substance is added to the prepared empty vesicle-containing liquid and used for mixing. Is preferred. The encapsulated substance may be added to the empty vesicle-containing liquid as it is, or may be added in the form of a solution or suspension in an aqueous medium.
- the concentration of the empty vesicle and the encapsulated substance in the mixed liquid is not particularly limited, and is determined in consideration of conditions such as the structure of the empty vesicle, the type of the encapsulated substance, and the desired inclusion ratio of the encapsulated substance to the empty vesicle. do it.
- the concentration of the empty vesicle with respect to the aqueous medium is usually 0.1 mg / mL or more, particularly 1 mg / mL or more, and usually 100 mg / mL or less, It is preferable to be 10 mg / mL or less.
- the particle size of the obtained substance-encapsulating vesicle is considered to depend on the concentration of the empty vesicle, and therefore the concentration of the empty vesicle should be determined according to the desired particle size of the substance-encapsulating vesicle.
- the concentration of the encapsulated substance with respect to the aqueous medium varies depending on the nature of the encapsulated substance, but is usually 0.1 mg / mL or more, particularly 1 mg / mL or more, and usually 100 mg / mL or less, especially 50 mg / mL or less. It is preferable to do. In particular, if the concentration of the inclusion substance is too low, the substance-encapsulating vesicle may not be formed.
- the pH of the liquid to be mixed is not particularly limited, and can be appropriately adjusted in consideration of conditions such as the structure of the empty vesicle, the type of the encapsulated substance, and the concentrations of the empty vesicle and the encapsulated substance in the mixed liquid.
- the pH is preferably 5 or more, more preferably 6.5 or more, and preferably 9 or less, more preferably 7.5 or less.
- the pH can be easily adjusted by using a buffer as a solvent. Adjusting the pH of the liquid mixture to be used is advantageous in maintaining the structure of the empty vesicle and efficiently including the encapsulated substance in the empty vesicle.
- the ionic strength of the liquid mixture can be adjusted as appropriate as long as it does not destroy the structure of the empty vesicle or inhibit the inclusion of the inclusion substance in the empty vesicle, but is preferably 0 mM or more, more preferably Is 10 mM or more, preferably 200 mM or less, more preferably 50 mM or less.
- the temperature at the time of mixing of the liquid mixture is not limited as long as it does not destroy the structure of the empty vesicle or inhibit the inclusion of the inclusion substance in the empty vesicle, but preferably 10 ° C. or more, more preferably It is 20 ° C or higher, preferably 80 ° C or lower, more preferably 50 ° C or lower.
- the formed substance-encapsulating vesicle may be immediately used for a desired use, but in order to equilibrate the system, a time for allowing the mixed solution to stand may be provided.
- the time for which the mixed solution is allowed to stand varies depending on conditions such as the formation efficiency of the substance-encapsulating vesicle, but is preferably 50 hours or less, more preferably 30 hours or less.
- a crosslinking agent is not used as described later, since the diameter of the formed substance-encapsulating vesicle tends to increase with time, it is preferable not to provide a standing time longer than the time required for uniform regeneration of the vesicle. In some cases.
- the cross-linking agent may be added and mixed with the liquid to be mixed containing the formed substance-encapsulating vesicle.
- the cross-linking agent may be added as it is, but an aqueous solution containing the cross-linking agent may be prepared and added.
- Preparation conditions such as an aqueous solvent, pH, temperature, and ionic strength in the preparation of the aqueous solution of the crosslinking agent are the same as those described above for the liquid mixture.
- operations such as dialysis, dilution, concentration, and stirring may be added as appropriate.
- [B2: Empty vesicle] (B2-1: Structure of empty vesicles)
- a first polymer that is a block copolymer having an uncharged hydrophilic segment and a first charged segment, and a second polymer charged to a charge opposite to the first charged segment.
- a vesicle having a film formed from a second polymer having a chargeable segment and having a void surrounded by the film is used as an empty vesicle.
- FIG. 2 is a partially cutaway view of the vesicle 1. As shown in FIG. 2, the vesicle 1 has a film 1a and a void 1b surrounded by the film 1a.
- FIG. 3A is an enlarged partial cross-sectional view of the film 1a of the vesicle 1 according to an aspect of the present invention.
- Film 1a illustrated in FIG. 3 (a), the outer layer 1a o has a three-layer structure consisting of an intermediate layer 1a m and the inner layer 1a i, mainly the first polymer 2, the second polymer 3 which It is formed.
- FIG. 3B is an enlarged view of the first polymer 2 and the second polymer 3 shown in FIG. As shown in FIG.
- the first polymer 2 is a block copolymer having an uncharged hydrophilic segment 2a and a first charged segment 2b
- the second polymer 3 is This is a polymer composed of the second chargeable segment 3 charged to the opposite charge to the first chargeable segment 2b.
- the non-charged hydrophilic segment 2a forms the outer layer 1a o of the membrane 1a
- the first charged segment 2b and the second charged segment 3 are electrostatic. combine to form an intermediate layer 1a m.
- predominantly uncharged hydrophilic segments 2a to form an inner layer 1a i of film 1a.
- FIG. 4A is an enlarged partial cross-sectional view of the film 1a of the vesicle 1 according to another aspect of the present invention.
- film 1a illustrated in FIG. 4 (a) the outer layer 1a o, has a three-layer structure consisting of an intermediate layer 1a m and the inner layer 1a i, a main first polymer 2, since the second polymer 3 ' It is formed.
- FIG. 4B is an enlarged view of the first polymer 2 and the second polymer 3 ′ shown in FIG. As shown in FIG.
- the first polymer 2 is a block copolymer having an uncharged hydrophilic segment 2a and a first charged segment 2b
- the second polymer 3 ' The polymer is composed of the non-charged hydrophilic segment 3a and the second charged segment 3b charged to the opposite charge to the first charged segment 2b.
- one or both of the uncharged hydrophilic segments 2a, 3a form the outer layer 1a o of the membrane 1a
- the first charged segment 2b and the second charged property segment 3b transgressions electrostatically bonded to form an intermediate layer 1a m.
- mainly one or both of the uncharged hydrophilic segments 2a, 3a form the inner layer 1a i of the membrane 1a.
- the mechanism by which the vesicle 1 is formed from the first polymer 2 and the second polymer 3, 3 ′ is considered as follows. That is, the first polymer 2 and the second polymer 3, 3 ′ shown in FIGS. 3B and 4B are placed in a system (for example, in an aqueous medium) where charge interaction can occur. Then, as shown in FIGS. 3 (a) and 4 (a), the first charged segment 2b and the second charged segments 3, 3b that are oppositely charged are electrostatically coupled with each other. to form the intermediate layer 1a m Te, uncharged hydrophilic segment 2a on the outside, 3a is arranged to form an outer layer 1a o.
- primarily uncharged hydrophilic segments 2a, 3a is arranged to form an inner layer 1a i.
- the film 1a having the three-layer structure shown in FIGS. 3A and 4A is formed, and as a result, the vesicle 1 shown in FIG. 2 is formed.
- the film 1a of the vesicle 1 may be composed only of the first polymer 2 and the second polymer 3, 3 ′, but contains other components as long as the above structure is roughly maintained. You may do it.
- Other components are not limited, but examples include a crosslinking agent, a charged polymer, a charged molecule, and the like. The crosslinking agent will be described in detail later.
- vesicle 1 as described later are usually prepared in aqueous medium, also the inner layer 1a i of film 1a primarily uncharged hydrophilic segments 2a, since it is composed of 3a, the gap portion 1b of the vesicle 1
- an aqueous medium is present (thus, in this specification, the void 1b may be indicated as “inner aqueous phase”).
- another substance may be present in the gap 1b.
- the shape of the vesicle 1 is not limited, but is usually spherical or substantially spherical.
- the particle size of the vesicle 1 varies depending on the type and amount ratio of the first polymer 2 and the second polymer 3, 3 ′, the presence or absence of a crosslinking agent, the surrounding environment of the vesicle 1 (type of aqueous medium), and the like. However, it is preferably 10 nm or more, more preferably 50 nm or more, preferably 1000 nm or less, more preferably 400 nm or less, and still more preferably 200 nm or less.
- the film thickness of the film 1a of the vesicle 1 depends on the type and amount ratio of the first polymer 2 and the second polymer 3, 3 ′, the presence or absence of a crosslinking agent, the surrounding environment of the vesicle 1 (type of aqueous medium), and the like. Although depending on the case, it is preferably 5 nm or more, more preferably 10 nm or more, and preferably 30 nm or less, more preferably 15 nm or less.
- An empty vesicle used for the post-supporting method has a film composed of a first polymer and a second polymer.
- the first polymer is a block copolymer having an uncharged hydrophilic segment and a first charged segment. Only 1 type may be sufficient as a 1st polymer, and arbitrary combinations and ratios may use 2 or more types together.
- the second polymer is a polymer having a second charged segment charged to a charge opposite to that of the first charged segment. Although it may be a polymer composed only of the second charged segment, it may be a block copolymer having an uncharged hydrophilic segment in addition to the second charged segment.
- the second polymer Only one type of the second polymer may be used, or two or more types may be used in any combination and ratio. In the case of two or more types, a second polymer consisting only of the second charged segment and a second polymer having an uncharged hydrophilic segment in addition to the second charged segment are used in combination. Also good. Each of the first polymer and the second polymer may have another segment in addition to the segments described above.
- Uncharged hydrophilic segment The first polymer has uncharged hydrophilic segments.
- the second polymer may also have an uncharged hydrophilic segment.
- Uncharged hydrophilic segments are polymer segments that have uncharged and hydrophilic properties.
- “uncharged” means that the segment is neutral as a whole. An example is when the segment has no positive or negative charge. Even if the segment has positive and negative charges in the molecule, the local effective charge density is not high, and the charge of the entire segment is neutralized to the extent that it does not interfere with the formation of vesicles due to self-assembly. If so, it falls under “uncharged”.
- Hydrophilic means to be soluble in an aqueous medium.
- the type of uncharged hydrophilic segment is not limited.
- the segment which consists of a single repeating unit may be sufficient, and the segment which contains 2 or more types of repeating units by arbitrary combinations and a ratio may be sufficient.
- Specific examples of the uncharged hydrophilic segment include polyalkylene glycol, poly (2-oxazoline), polysaccharide, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylamide, polymethacrylamide, polyacrylate ester, polymethacrylate ester, poly ( 2-methacryloyloxyethyl phosphorylcholine), peptides having isoelectric point around 7, proteins and derivatives thereof.
- polyalkylene glycol poly (2-oxazoline) and the like are preferable, and polyalkylene glycol is particularly preferable.
- examples of the polyalkylene glycol include polyethylene glycol and polypropylene glycol, and polyethylene glycol is preferable.
- the molecular weight of the uncharged hydrophilic segment is not limited, but it is predetermined from the viewpoint of promoting the self-assembly of the first polymer and the second polymer and efficiently producing a homogeneous vesicle. It is preferable to have a molecular weight within the range.
- the specific molecular weight range varies depending on the type of the uncharged hydrophilic segment and the combination with the charged segment, but when polyethylene glycol is used as the uncharged hydrophilic segment, the molecular weight (Mw) is preferably 500. Above, more preferably 1000 or more, preferably 15000 or less, more preferably 5000 or less.
- the number of repeating units of the uncharged hydrophilic segment is not limited, but is usually determined according to the type of the repeating unit so that the molecular weight of the uncharged hydrophilic segment satisfies the molecular weight range.
- the first charged segment of the first polymer and the second charged segment of the second polymer are charged segments charged to opposite charges. That is, if the first charged segment is a cationic segment, the second charged segment is an anionic segment, and if the first charged segment is an anionic segment, the second charged segment is a cation segment. Sex segment.
- the cationic segment is a polymer segment having a cationic group and exhibiting a cationic property (cationic property).
- the cationic segment may have some anionic groups as long as the formation of vesicles by self-assembly of the first polymer and the second polymer is not hindered.
- the kind of the cationic segment is not limited.
- the segment which consists of a single repeating unit may be sufficient, and the segment which contains 2 or more types of repeating units by arbitrary combinations and a ratio may be sufficient.
- a polyamine or the like is preferable, and a polyamino acid having an amino group in a side chain or a derivative thereof is particularly preferable.
- polyamino acid having an amino group in the side chain or a derivative thereof examples include polyaspartamide, polyglutamide, polylysine, polyarginine, polyhistidine, and derivatives thereof. Particularly preferred are polyaspartamide derivatives and polyglutamide derivatives.
- the molecular weight of the cationic segment is not limited, but from the viewpoint of facilitating the self-assembly of the first polymer and the second polymer and efficiently producing a homogeneous vesicle, a predetermined range.
- a molecular weight of The number of repeating units of the cationic segment is not limited, but is usually determined according to the type of the repeating unit so that the molecular weight of the cationic segment satisfies a predetermined range.
- the number of repeating units is preferably 10 or more, more preferably 50 or more, and preferably 200 or less, more preferably 100 or less.
- anionic segment is a polymer segment which has an anionic group and shows anionicity (anionic property).
- the anionic segment may have some cationic groups as long as it does not hinder the formation of vesicles by self-assembly of the first polymer and the second polymer.
- the kind of anionic segment is not limited.
- the segment which consists of a single repeating unit may be sufficient, and the segment which contains 2 or more types of repeating units by arbitrary combinations and a ratio may be sufficient.
- polycarboxylic acid polysulfonic acid, polyphosphoric acid (nucleic acid and the like) and the like are preferable, and a polyamino acid having a carboxyl group in the side chain, a derivative thereof, and a nucleic acid are particularly preferable.
- polyamino acid having a carboxyl group in the side chain or a derivative thereof examples include polyaspartic acid, polyglutamic acid, and the amino group of the polyamino acid having an amino group in the side chain with the above polycation or a derivative thereof, an aconitic acid anhydride or citraconic acid.
- Polycarboxylic acid obtained by allowing an appropriate amount of an anhydride to act, and derivatives thereof are exemplified, and polyaspartic acid and polyglutamic acid are particularly preferable.
- Nucleic acids include single-stranded or double-stranded DNA or RNA.
- the nucleic acid may be a functional nucleic acid according to the use of the vesicle.
- functional nucleic acids include siRNA, miRNA (microRNA), antisense RNA, antisense DNA, ribozyme, DNA enzyme and the like. These are selected according to the use of the vesicle. For example, when a vesicle is used for DDS for RNAi, siRNA is used as the nucleic acid.
- the nucleic acid may be modified. Examples of the modified nucleic acid include a nucleic acid to which a hydrophobic functional group such as cholesterol or vitamin E is bound for uses such as vesicle stabilization.
- the molecular weight of the anionic segment is not limited, but from the viewpoint of promoting the self-assembly of the first polymer and the second polymer and efficiently producing a homogeneous vesicle, the molecular weight of the anionic segment is within a predetermined range.
- Preferably having a molecular weight of The number of repeating units of the anionic segment is not limited, but is usually determined according to the type of the repeating unit so that the molecular weight of the anionic segment satisfies a predetermined range.
- the number of repeating units is preferably 10 or more, more preferably 50 or more, and preferably 200 or less, more preferably 100 or less. Range.
- B2-2c Combination of uncharged hydrophilic segment and charged segment
- Any combination of the non-charged hydrophilic segment and the second charged segment is not limited, and any uncharged hydrophilic segment and any charged segment can be combined (in the following description, , The first charged segment and the second charged segment may be collectively displayed as “charged segment”).
- the number of uncharged hydrophilic segments and charged segments is also arbitrary, and may be one or two or more, respectively, and in the case of two or more, they may be the same or different.
- the bonding form between the uncharged hydrophilic segment and the charged segment is not limited, and may be directly bonded, or may be bonded via a linking group.
- linking groups include hydrocarbon groups having a valence corresponding to the total number of uncharged hydrophilic segments and charged segments.
- the hydrocarbon group as the linking group may be aliphatic, aromatic, or a group in which they are linked. In the case of aliphatic, the hydrocarbon group may be saturated or unsaturated, and may be linear, branched or cyclic.
- the molecular weight of the hydrocarbon group as the linking group is not limited, but is usually 5000 or less, preferably 1000 or less.
- hydrocarbon group as the linking group examples include gallic acid derivatives, 3,5-dihydroxybenzoic acid derivatives, glycerin derivatives, cyclohexane derivatives, L-lysine, etc., and 3,5-dihydroxybenzoic acid derivatives, etc. preferable.
- the linking group is a disulfide group.
- the disulfide group is used to link one uncharged hydrophilic segment and one charged segment. By connecting the uncharged hydrophilic segment and the charged segment via a disulfide group, it is possible to cleave the disulfide group by the environment where the vesicle is placed or from the outside to change the shape and properties of the vesicle. become. If this is utilized, for example, when a drug is encapsulated in a vesicle and the obtained substance-encapsulated vesicle is used for DDS for drug delivery, the substance encapsulated in the vesicle is released by cleaving the disulfide group in vivo. It is also possible to encourage
- the ratio between the first charged segment and the second charged segment (the ratio between the cationic segment and the anionic segment) and the ratio between the uncharged hydrophilic segment and the charged segment are also arbitrary. From the viewpoint of promoting the self-organization of the first polymer and the second polymer and efficiently producing a homogeneous vesicle, the following criteria are preferred.
- the C / A ratio defined in the following formula (i) is usually 0.3, preferably 0.5 or more, more preferably 0.6 or more, Usually, it is desirable to adjust so that it may be less than 3.0, preferably 2.0 or less, more preferably 1.7 or less.
- the number of moles of the cationic group and the anionic group in the first and second polymers is a value depending on the structures of the cationic segment and the anionic segment, but a general potential difference (acid / base) titration. It is possible to obtain by
- the ratio of the uncharged hydrophilic segment to the charged segment in the first and second polymers is determined in consideration of the ratio of the cationic segment and the anionic segment that satisfy the range of the C / A ratio. It is preferable.
- the molecular weight ratio X of the uncharged hydrophilic segment defined by the following formula (ii) is usually 0.01 or more, preferably 0.05 or more, and usually 0.35 or less, preferably 0. It is desirable to be within the range of 1 or less.
- cationic segment assuming one positive charge per monomer
- anionic segment assuming one negative charge per monomer
- at least one uncharged hydrophilic segment Ie, the first polymer is a block copolymer composed of a cationic or anionic segment and an uncharged hydrophilic segment, and the second polymer is an anionic or cationic segment
- X is defined by the following formula: or a block copolymer consisting of it and an uncharged hydrophilic segment).
- Specific examples of the first and second polymers include the following [Example 1] and [Example 2].
- Example 1 The following (A1) is used as the first polymer, and the following (B1) is used as the second polymer.
- A1 A block copolymer containing an uncharged hydrophilic segment and an anionic segment.
- B1 The following block copolymer (i) and / or the following polymer (ii).
- i) A block copolymer comprising an uncharged hydrophilic segment and a cationic segment.
- a polymer containing a cationic segment but not including an uncharged hydrophilic segment).
- Example 2 The following (A2) is used as the first polymer, and the following (B2) is used as the second polymer.
- A2) A block copolymer containing an uncharged hydrophilic segment and a cationic segment.
- B2 The following block copolymer (iii) and / or the following polymer (iv).
- iii) A block copolymer comprising an uncharged hydrophilic segment and an anionic segment.
- a polymer containing an anionic segment but not including an uncharged hydrophilic segment).
- a polymer that does not contain an uncharged hydrophilic segment such as the polymers of (B1) (ii) and (B2) (iv) may be referred to as a homopolymer for convenience.
- the cationic segment in each of the polymers (B1) (i), (ii), and (A2) is not limited. For example, those derived from a polypeptide having a cationic group in the side chain are preferable. It is done.
- the anionic segment is not limited. For example, it may be a polypeptide or nucleic acid having an anionic group in the side chain. Preferred are those derived.
- block copolymers (A1) and (B2) (iii) include those represented by the following general formulas (I) and / or (II).
- the segment is represented as a non-charged hydrophilic segment derived from PEG (hereinafter “PEG segment”)
- PEG segment a non-charged hydrophilic segment derived from PEG
- polyanion segment an anionic segment derived from polyanion
- R 1a and R 1b each independently represent a hydrogen atom or an unsubstituted or substituted linear or branched C 1-12 alkyl group.
- linear or branched C 1-12 include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, decyl, undecyl Etc.
- examples of the substituent include an acetalized formyl group, cyano group, formyl group, carboxyl group, amino group, C 1-6 alkoxycarbonyl group, C 2-7 acylamide group, the same or different tri-C 1 -6 alkylsiloxy group, siloxy group or silylamino group.
- the acetalization means that the acetal part formed by the reaction of a carbonyl of formyl with, for example, two molecules of an alkanol having 1 to 6 carbon atoms or an alkylene diol having 2 to 6 carbon atoms which may be branched. Meaning formation, and also a method for protecting the carbonyl group.
- the substituent when the substituent is an acetalized formyl group, it can be converted into another substituent, a formyl group (—CHO) (or an aldehyde group) by hydrolysis under acidic mild conditions.
- L 1 and L 2 represent a linking group. Specifically, L 1 is preferably — (CH 2 ) b —NH— (where b is an integer of 1 to 5), and L 2 is — (CH 2 ) c —CO— (here And c is an integer of 1 to 5.
- R 2a , R 2b , R 2c and R 2d each independently represents a methylene group or an ethylene group.
- R 2a and R 2b are both methylene groups, they correspond to poly (aspartic acid derivatives), and when they are ethylene groups, they correspond to poly (glutamic acid derivatives), and both R 2c and R 2d are methylene groups.
- the group corresponds to poly (aspartic acid derivative), and the ethylene group corresponds to poly (glutamic acid derivative).
- R 2a and R 2b represent both a methylene group and an ethylene group
- R 2c and R 2d represent a methylene group and an ethylene group.
- the repeating units of the aspartic acid derivative and glutamic acid derivative may be present in the form of blocks, respectively, or randomly.
- R 3 represents a hydrogen atom, a protecting group, a hydrophobic group or a polymerizable group. Specifically, R 3 is preferably an acetyl group, an acryloyl group, or a methacryloyl group.
- R 4 represents a hydroxyl group, an oxybenzyl group, an —NH— (CH 2 ) a —X group or an initiator residue.
- a is an integer of 1 to 5
- X is an amine compound residue containing one or more of primary, secondary, tertiary amine or quaternary ammonium salt, or a compound which is not an amine It is preferably a residue.
- R 4 is —NH—R 9 (wherein R 9 represents an unsubstituted or substituted linear or branched C 1-20 alkyl group).
- m is an integer of 5 to 2,000, preferably an integer of 5 to 270, more preferably an integer of 10 to 100.
- N is an integer of 2 to 5,000
- y is an integer of 0 to 5,000
- n and y are preferably integers of 5 to 300, more preferably an integer of 10 to 100 It is. However, y is not larger than n.
- the molecular weight (Mw) of the block copolymers represented by the general formulas (I) and (II) is not limited, but is preferably 3,000 to 30,000, more preferably 5,000 to 20,000. is there.
- the molecular weight (Mw) of the PEG segment is preferably 500 to 15,000, more preferably 1,000 to 5,000, and the molecular weight (Mw) of the polyanion segment is The total is preferably 500 to 50,000, more preferably 1,000 to 20,000.
- the method for producing the block copolymer represented by the general formulas (I) and (II) is not limited, but includes, for example, a segment (PEG segment) containing R 1a O— or R 1b O— and a block part of the PEG chain.
- a predetermined monomer is polymerized in order on one end of the PEG segment (the end opposite to R 1a O— or R 1b O—), and then the side chain contains an anionic group as necessary.
- the PEG segment can be prepared using, for example, a method for producing a PEG segment portion of a block copolymer described in WO96 / 32434, WO96 / 33233, WO97 / 06202, and the like.
- block copolymers represented by the general formulas (I) and (II) for example, using a PEG segment derivative having an amino group at the terminal, ⁇ -benzyl-L at the amino terminal is used.
- -Block copolymers were synthesized by polymerizing N-carboxylic anhydride (NCA) of protected amino acids such as aspartate (BLA) and N ⁇ -ZL-lysine, and then the side chain of each segment was anionic as described above A method of substitution or conversion so as to be a side chain having a group is preferred.
- NCA N-carboxylic anhydride
- BLA aspartate
- N ⁇ -ZL-lysine N ⁇ -ZL-lysine
- block copolymers represented by the general formulas (I) and (II) include, for example, an uncharged hydrophilic segment polyethylene glycol (hereinafter sometimes referred to as “PEG”), an anion An anionic block copolymer of the following formula (hereinafter, sometimes referred to as “PEG-P (Asp)”) composed of polyaspartic acid (hereinafter sometimes referred to as “P (Asp)”), which is an ionic segment, etc.
- PEG-P (Asp) an anionic block copolymer of the following formula
- P (Asp) an anionic block copolymer of the following formula
- P (Asp) an anionic block copolymer of the following formula
- Na + may be shown as an example of the counter cation, but the counter cation is not limited thereto).
- m is an integer representing the degree of polymerization of PEG.
- n is an integer representing the degree of polymerization of P (Asp).
- PEG-P (Asp) those in which the molecular weight (Mw) of the PEG segment is 2,000 and the number of P (Asp) units representing the polyanion segment (n in the above formula) is 70 or 75 are particularly preferable.
- Preferred examples of the block copolymers (A2) and (B1) (i) include those represented by the following general formulas (III) and / or (IV).
- the segment whose number of repeating units (degree of polymerization) is “m” is a non-charged hydrophilic segment (PEG segment) derived from PEG, and the number of repeating units Is a segment obtained by combining the “nyz” portion, the “y” portion, and the “z” portion is a polycation-derived cationic segment (hereinafter, polycation segment).
- R 1a and R 1b each independently represent a hydrogen atom or an unsubstituted or substituted linear or branched C 1-12 alkyl group.
- linear or branched C 1-12 include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, decyl, undecyl Etc.
- examples of the substituent include an acetalized formyl group, cyano group, formyl group, carboxyl group, amino group, C 1-6 alkoxycarbonyl group, C 2-7 acylamide group, the same or different tri-C 1 -6 alkylsiloxy group, siloxy group or silylamino group.
- the acetalization means that the acetal part formed by the reaction of a carbonyl of formyl with, for example, two molecules of an alkanol having 1 to 6 carbon atoms or an alkylene diol having 2 to 6 carbon atoms which may be branched. Meaning formation, and also a method for protecting the carbonyl group.
- the substituent when the substituent is an acetalized formyl group, it can be converted to another formyl group (—CHO: or aldehyde group) by hydrolysis under acidic mild conditions.
- L 1 and L 2 represent a linking group.
- L 1 is preferably — (CH 2 ) b —NH— (where b is an integer of 1 to 5), and L 2 is — (CH 2 ) c —CO— (
- c is preferably an integer of 1 to 5.
- R 2a , R 2b , R 2c and R 2d each independently represent a methylene group or an ethylene group.
- R 2a and R 2b are both methylene groups, they correspond to poly (aspartic acid derivatives), and when they are ethylene groups, they correspond to poly (glutamic acid derivatives), and both R 2c and R 2d are methylene groups.
- the group corresponds to poly (aspartic acid derivative), and the ethylene group corresponds to poly (glutamic acid derivative).
- R 2a and R 2b represent both a methylene group and an ethylene group
- R 2c and R 2d represent a methylene group and an ethylene group.
- the repeating units of the aspartic acid derivative and glutamic acid derivative may be present in the form of blocks, respectively, or randomly.
- R 3 represents a hydrogen atom, a protecting group, a hydrophobic group or a polymerizable group. Specifically, R 3 is preferably an acetyl group, an acryloyl group, or a methacryloyl group.
- R 4 represents a hydroxyl group, an oxybenzyl group, a —NH— (CH 2 ) a —X group or an initiator residue.
- a is an integer of 1 to 5
- X is an amine compound residue containing one or more of primary, secondary, tertiary amine, quaternary ammonium salt or guanidino group, or A compound residue that is not an amine is preferred.
- R 4 is —NH—R 9 (wherein R 9 represents an unsubstituted or substituted linear or branched C 1-20 alkyl group).
- R 5a , R 5b , R 5c and R 5d each independently represent a hydroxyl group, an oxybenzyl group, or a —NH— (CH 2 ) a —X group.
- a is an integer of 1 to 5
- X is an amine compound residue containing one or more of primary, secondary, tertiary amine, quaternary ammonium salt or guanidino group, or A compound residue that is not an amine is preferred.
- R 5a and R 5b and the total number of R 5c and R 5d the —NH— (CH 2 ) a —X group (where X is (NH (CH 2 ) 2 ) e —NH 2 ( However, e is preferably an integer of 0 to 5), preferably at least 2 or more, more preferably 50% or more of the total number, and 85% or more of the total number. Further preferred.
- all or part of R 5a , R 5b , R 5c and R 5d is a —NH— (CH 2 ) a —X group (where a is 2 and X is (NH (CH 2 ) 2 E ) -NH 2 (where e is 1)).
- X is selected from the groups represented by the following formulas: Some cases are particularly preferred.
- X 2 represents a hydrogen atom, a C 1-6 alkyl group or an amino C 1-6 alkyl group
- R 7a , R 7b and R 7c are each independently a hydrogen atom or Represents a methyl group
- d1, d2 and d3 each independently represents an integer of 1 to 5
- e1, e2 and e3 each independently represents an integer of 1 to 5
- f represents 0 to 15
- g represents an integer of 0 to 15
- R 8a and R 8b each independently represents a hydrogen atom or a protecting group.
- the protecting group is preferably a group selected from the group consisting of a Z group, a Boc group, an acetyl group, and a trifluoroacetyl group, which are usually used as a protecting group for an amino group.
- R 6a and R 6b are each independently a hydrogen atom, —C ( ⁇ NH) NH 2 , or a protecting group, where the protecting group is usually an amino group protected. It is preferably a group selected from the group consisting of Z group, Boc group, acetyl group, and trifluoroacetyl group used as a group.
- t is preferably an integer of 2 to 6, more preferably 3 or 4.
- m is an integer of 5 to 2,000, preferably an integer of 5 to 270, more preferably an integer of 10 to 100.
- N is an integer of 2 to 5,000, y is an integer of 0 to 5,000, and z is an integer of 0 to 5,000.
- n is preferably an integer of 5 to 300, more preferably 0 or an integer of 10 to 100.
- y and z are preferably 0 or an integer of 5 to 300, more preferably 0 or an integer of 10 to 100.
- the sum (y + z) of y and z is not larger than n.
- each repeating unit in the general formulas (III) and (IV) is shown in the order specified for convenience of description, each repeating unit can exist in a random order. In particular, it is preferable that only the repeating units in the polycation segment can be present in a random order as described above.
- the molecular weight (Mw) of the block copolymers represented by the general formulas (III) and (IV) is not limited, but is preferably 23,000 to 45,000, more preferably 28,000 to 34,000. is there.
- the molecular weight (Mw) of the PEG segment is preferably 500 to 15,000, more preferably 1,000 to 5,000
- the molecular weight (Mw) of the polycation segment is The total amount is preferably 500 to 50,000, more preferably 1,000 to 30,000.
- the method for producing the block copolymer represented by the general formulas (III) and (IV) is not limited, but includes, for example, a segment (PEG segment) containing R 1a O— or R 1b O— and a block part of the PEG chain.
- a predetermined monomer is polymerized in order at one end of the PEG segment (the end opposite to R 1a O— or R 1b O—), and then the side chain contains a cationic group as necessary.
- the PEG segment can be prepared using, for example, a method for producing a PEG segment portion of a block copolymer described in WO96 / 32434, WO96 / 33233, WO97 / 06202, and the like.
- block copolymers represented by the general formulas (III) and (IV) for example, using a PEG segment derivative having an amino group at the terminal, ⁇ -benzyl-L at the amino terminal is used.
- -Block copolymer was synthesized by polymerizing N-carboxylic anhydride (NCA) of protected amino acids such as aspartate (BLA) and N ⁇ -ZL-lysine, and then the side chain of each segment was cationic as described above
- NCA N-carboxylic anhydride
- BLA aspartate
- N ⁇ -ZL-lysine N ⁇ -ZL-lysine
- block copolymer represented by the general formulas (III) and (IV) include, for example, an uncharged hydrophilic segment polyethylene glycol (hereinafter sometimes referred to as “PEG”) and a cationic segment.
- PEG polyethylene glycol
- a cationic segment a Cationic block copolymer (hereinafter referred to as “PEG-P (Asp-AP)”) composed of poly (diaminopentane structure-containing asparagine derivative) (hereinafter sometimes referred to as “P (Asp-AP)”)
- P (Asp-AP) Cationic block copolymer
- Cl ⁇ is shown as an example of the counter anion in the following formulas, but the counter anion is not limited to this).
- m is an integer representing the degree of polymerization of PEG.
- PEG-P (Asp-AP) the molecular weight of PEG segment (Mw): 2,000, the number of P (Asp-AP) units indicating a polycation segment (n in the above formula): 70 or 75 Is particularly preferred.
- Preferred examples of the polymer of (B2) (iv) include those represented by the following general formula (V) and / or (VI).
- general formula (V) and / or (VI) As for the explanation about the general formulas (V) and (VI), the explanation about the general formulas (I) and (II) mentioned above (except for the explanation about the PEG segment) can be applied as appropriate.
- polymers represented by the general formulas (V) and (VI) include, for example, an anionic homopolymer (hereinafter referred to as an anionic segment) composed of polyaspartic acid (P (Asp)) which is an anionic segment.
- Preferred examples include “Homo-P (Asp)”.
- n is an integer representing the degree of polymerization of P (Asp).
- a and b are both greater than 0 and less than 1.
- a + b 1.
- Homo-P (Asp) is particularly preferably a P (Asp) unit number (n in the above formula) representing a polyanion segment: 70 or 82.
- Preferred examples of the polymer (B1) (ii) include those represented by the following general formula (VII) and / or (VIII).
- general formulas (VII) and (VIII) the explanations regarding the general formulas (III) and (IV) described above can be similarly applied as appropriate.
- polymers represented by the general formulas (VII) and (VIII) include, for example, a cationic segment poly (diaminopentane structure-containing asparagine derivative) (P (Asp-AP)) Preferred examples include a cationic homopolymer of the formula (hereinafter, sometimes referred to as “Homo-P (Asp-AP)”).
- Homo-P (Asp-AP) a cationic homopolymer of the formula
- n is an integer representing the degree of polymerization of P (Asp-AP).
- Homo-P (Asp-AP) those having the number of P (Asp-AP) units representing the polycation segment (n in the above formula): 70 or 82 are particularly preferred.
- the chargeable polymer is a polymer having one or more of the above-described chargeable segments (cationic segments or anionic segments), and any one not corresponding to the first polymer and the second polymer. And a chargeable polymer.
- the chargeable nanoparticles include metal-based nanoparticles having a charge on the surface.
- One of these other film components may be used alone, or two or more thereof may be used in any combination and ratio.
- the amount of the other film component used is not limited, but it is preferable to suppress the vesicle formation by self-organization of the first polymer and the second polymer. Specifically, it is desirable that it is usually 30% or less, preferably 20% or less, more preferably 10% or less with respect to the total weight of the vesicle.
- a first aqueous solution containing a first polymer and a second aqueous solution containing a second polymer are prepared.
- the first and second aqueous solutions may be purified by filtration if desired.
- the concentration of the first polymer in the first aqueous solution and the concentration of the second polymer in the second aqueous solution are not limited, and the total number of charges of the first polymer and the second polymer is not limited.
- the ratio is appropriately determined in consideration of conditions such as the ratio of the first polymer and the second polymer in an aqueous solution, and the efficiency of vesicle formation.
- the solvent of the 1st and 2nd aqueous solution is an aqueous solvent
- the kind will not be limited.
- Water is preferable, but a solvent in which other components are mixed with water, for example, physiological saline, aqueous buffer, a mixed solvent of water and a water-soluble organic solvent, or the like may be used as long as the formation of vesicles is not hindered. it can.
- the aqueous buffer include 10 mM HEPES buffer.
- the pH of the first and second aqueous solutions can be appropriately adjusted within a range that does not interfere with the formation of vesicles, but is preferably pH 5 or higher, more preferably pH 6.5 or higher, and preferably pH 9 Below, more preferably pH 7.5 or less.
- the pH can be easily adjusted by using a buffer as a solvent. Adjusting and using the pH of the first and second aqueous solutions is advantageous for maintaining the charged state of the first polymer and the second polymer and efficiently forming vesic
- the temperature of the first and second aqueous solutions is appropriately determined according to the solubility of the first polymer and the second polymer in the solvent, but is preferably 10 ° C or higher, more preferably 20 ° C or higher. Moreover, it is preferably 80 ° C. or lower, more preferably 50 ° C. or lower.
- the ionic strength of the first and second aqueous solutions can be appropriately adjusted within a range that does not interfere with the formation of vesicles, but is preferably 0 mM or more, more preferably 10 mM or more, and preferably 200 mM or less. More preferably, it is 50 mM or less.
- a vesicle is prepared by mixing the above first and second aqueous solutions.
- the mixing method is not limited, and the second aqueous solution may be added to the first aqueous solution, or the first aqueous solution may be added to the second aqueous solution.
- you may mix the 1st and 2nd aqueous solution simultaneously in a container. You may stir suitably the liquid mixture of the obtained 1st and 2nd aqueous solution.
- the temperature at the time of mixing the first and second aqueous solutions is not limited as long as it does not interfere with the formation of vesicles, but considering the solubility according to the temperature of the first polymer and the second polymer. It is preferable to set. Specifically, it is preferably 10 ° C or higher, more preferably 20 ° C or higher, and preferably 60 ° C or lower, more preferably 50 ° C or lower.
- the formed empty vesicle may be immediately subjected to the post-loading method, but in order to equilibrate the system, a time for allowing the mixed solution to stand may be provided.
- a time for allowing the mixed solution to stand may be provided.
- the diameter of the formed vesicle tends to increase with time, it is preferable that the formed empty vesicle is immediately subjected to the post-carrying method without providing a standing time.
- the first and second aqueous solutions and the membrane components may be mixed.
- the membrane component may be added to and mixed with the first or second aqueous solution before mixing, but there is an association between the membrane component and the first or second aqueous solution that prevents vesicle formation. It is desirable that there is no interaction.
- the membrane component may be added and mixed together at the time of mixing the first and second aqueous solutions, and the membrane component may be added and further mixed after the mixing of the first and second aqueous solutions. .
- Other membrane components may be used for mixing as they are, but an aqueous solution containing the membrane component may be prepared and used for mixing.
- the preparation conditions such as the aqueous solvent, pH, temperature, and ionic strength in the preparation of the aqueous solution of the membrane component are the same as those described above for the first and second aqueous solutions. Furthermore, operations such as dialysis, dilution, concentration, and stirring may be added as appropriate.
- the encapsulated substance to be included in the vesicle is not limited, and can be arbitrarily selected as appropriate according to the use and properties of the substance-encapsulating vesicle.
- the simultaneous mixing method which is one of the conventional manufacturing methods, when a charged substance is used as the encapsulated substance, the formation of vesicles by the self-organization of the polymer that is a film constituent component is the encapsulated substance.
- an appropriate substance-encapsulating vesicle could not be obtained.
- the substance-encapsulated vesicles can be used regardless of whether a charged substance or an uncharged substance is used as the encapsulated substance. Can be formed efficiently.
- examples of the encapsulated substance include biomolecules, organic compounds, and inorganic substances.
- Biomolecules include proteins, polypeptides, amino acids, nucleic acids (DNA, RNA), lipids (fatty acids, glycerides, steroids, etc.), carbohydrates (monosaccharides, polysaccharides), and derivatives thereof, and two or more of these. Examples thereof include glycoproteins and glycolipids. Of these, proteins, carbohydrates and the like are preferable.
- organic compounds include luminescent (fluorescent, phosphorescent, etc.) molecules, water-soluble drugs, water-soluble polymers, water-soluble molecular aggregates having an average particle size of 100 nm or less (such as micelles, vesicles, and nanogels), and emulsions having an average particle size of 100 nm or less. Etc.
- polymer micelles having an average particle size of 50 nm or less and water-soluble polymers having a molecular weight of 100,000 or less are preferable.
- Inorganic substances include water-dispersible metal nanoparticles, oxide nanoparticles (silica nanoparticles, titania nanoparticles, iron oxide nanoparticles, etc.), semiconductor nanoparticles (quantum dots, etc.), water-soluble carbon clusters, boron clusters, metals A complex etc. are mentioned. Among these, quantum dots having an average particle size of 20 nm or less are preferable.
- encapsulated substances when classified by use, include anticancer agents (for example, hydrophobic anticancer agents such as doxorubicin and paclitaxel, metal complex anticancer agents such as cisplatin, and the like, and their polymer micelles. ), Gadolinium and iron compounds used for diagnosis such as MRI, organic light emitting (fluorescence, phosphorescence, etc.) dyes, quantum dots, and the like.
- anticancer agents for example, hydrophobic anticancer agents such as doxorubicin and paclitaxel, metal complex anticancer agents such as cisplatin, and the like, and their polymer micelles.
- Gadolinium and iron compounds used for diagnosis such as MRI, organic light emitting (fluorescence, phosphorescence, etc.) dyes, quantum dots, and the like.
- the molecular weight and particle size of the encapsulated substance are not limited, but from the viewpoint of efficiently introducing the encapsulated substance into the empty vesicle, the molecular weight of the encapsulated substance is usually 200,000 or less, preferably 100,000 or less.
- the particle size of the encapsulated substance is usually 100 nm or less, preferably 50 nm or less.
- the ratio of the inclusion substance to the empty vesicle is also adjusted according to the amount of inclusion substance desired, as long as it does not destroy the structure of the empty vesicle or inhibit the inclusion of the inclusion substance in the empty vesicle. That's fine.
- the encapsulated substance may be used alone or in combination of two or more in any ratio and combination.
- the post-carrying method may include at least a step of preparing an empty vesicle having a predetermined structure and a step of mixing the empty vesicle and the encapsulated substance in an aqueous medium.
- a salt such as physiological saline (for example, when used as DDS)
- it is formed from the viewpoint of preventing the increase in particle size over time.
- the substance-encapsulating vesicle is subjected to a crosslinking agent treatment as a post-treatment.
- a crosslinking agent treatment As a post-treatment, the particle size of vesicles that do not contain a crosslinking agent tends to increase over time under physiological conditions or in the presence of salt, such as saline, but the increase in particle size is prevented by applying a crosslinking agent treatment. can do.
- the type of the crosslinking agent is not limited and can be appropriately selected according to the use of the vesicle, the types of the first polymer and the second polymer, the types of other film components, etc. From the viewpoint of improving the stability of the substance-encapsulating vesicle, the charged group of the charged segment of the first polymer and the second polymer (for example, a cationic group such as an amino group or an anionic group such as a carboxyl group) ) And a crosslinking agent that does not react with the encapsulated substance is preferable.
- cross-linking agent examples include a cross-linking agent that cross-links an amino group (for example, glutaraldehyde, dimethyl-suberimidate dihydrochloride (DMS), dimethyl 3,3′-dithiobispropionimidate (dimethyl 3,3).
- an amino group for example, glutaraldehyde, dimethyl-suberimidate dihydrochloride (DMS), dimethyl 3,3′-dithiobispropionimidate (dimethyl 3,3).
- DTBP '-dithiobispropionimidate
- a crosslinking agent that crosslinks by condensing an amino group and a carboxyl group for example, 1-ethyl-3- (3-dimethylaminopropy) carbodiimide (EDC)) and the like
- crosslinking agents that crosslink phosphoric acid groups for example, metal ions such as calcium ions
- glutaraldehyde, EDC, etc. are preferred, and EDC is particularly preferred.
- one type of cross-linking agent may be used alone, but two or more types of cross-linking agents may be used in any combination and ratio.
- the amount of the crosslinking agent is not limited, and may be appropriately determined in consideration of the type of crosslinking agent, the number of crosslinking points, the amount of components to be crosslinked, and the like.
- a crosslinking agent that crosslinks an amino group and a carboxyl group In this case, it is desirable to select the usage amount so that the CL ratio defined in the following formula (iii) satisfies the condition described below.
- the crosslinking agent and the first and second weights are adjusted so that the CL ratio is usually 0.1 or more, preferably 0.5 or more. It is desirable to adjust the amount ratio with the coalescence.
- the amount of the crosslinking agent used is not too large. It is desirable to adjust the quantitative ratio between the crosslinking agent and the first and second polymers so that the CL ratio is usually 10 or less, preferably 5 or less.
- the range of the CL ratio described above is only a guideline.
- the CL ratio is set according to the use of the vesicle, the types of the first polymer and the second polymer, the types of other film components, and the like. It is preferable to adjust appropriately.
- the substance-encapsulating vesicle in which the inclusion substance is included in the void portion of the empty vesicle is obtained.
- a substance-encapsulating vesicle includes a first polymer which is a block copolymer having an uncharged hydrophilic segment and a first charged segment, and a first polymer charged to a charge opposite to that of the first charged segment.
- the structure of the substance-encapsulating vesicle film is basically the same as the structure of the above-described empty vesicle film. That is, the substance-encapsulating vesicle preferably has a three-layer structure film similar to the structure film of the empty vesicle described with reference to FIGS. 2 to 4, and the shape is usually spherical or substantially spherical.
- the particle size of the substance-encapsulated vesicle obtained by the post-loading method varies depending on the structure of the empty vesicle, the type of the substance to be encapsulated, the surrounding environment of the vesicle (type of aqueous medium), mixing conditions, etc. Is approximately equivalent to the particle size of
- the particle size of the substance-encapsulating vesicle obtained is preferably 10 nm or more, more preferably 50 nm or more, and preferably 1000 nm or less, more preferably 400 nm or less, more preferably, regardless of the post-loading method or other production methods. Is 200 nm or less.
- the particle size of each substance-encapsulating vesicle of the present invention, which will be described later, is about the same unless otherwise specified.
- the film thickness of the substance-encapsulated vesicles obtained by the post-loading method also varies depending on the structure of the empty vesicle, the type of the encapsulated substance, the surrounding environment of the vesicle (type of aqueous medium), the mixing conditions, etc.
- the particle size and the film thickness are substantially the same.
- the film thickness of the substance-encapsulating vesicle obtained is preferably 5 nm or more, more preferably 10 nm or more, and preferably 30 nm or less, more preferably 15 nm or less, regardless of the post-loading method or other production methods.
- the film thickness of each substance-encapsulating vesicle of the present invention, which will be described later, is about the same unless otherwise specified.
- Substance-encapsulating vesicles have various substances stably held in the voids of vesicles formed by polymer self-organization, so that DDS for delivering drugs, functional materials carrying active ingredients, etc. It can be effectively used for various applications.
- siRNA small interfering RNA
- the resulting substance-encapsulating vesicle can be used for DDS for RNAi (RNA interference).
- RNAi RNA interference
- it can be used also for the combined therapy of a chemical
- ⁇ C-1 First embodiment / monodisperse aggregate of substance-incorporated crosslinked vesicles and production method thereof> [Overview]
- a monodispersed aggregate of empty cross-linked vesicles is mixed in a mixed solution containing the target substance together with an aqueous medium, and the substance-containing cross-linked vesicle in which the target substance is encapsulated in the inner aqueous phase.
- a method for producing a substance-incorporated crosslinked vesicle comprising a step of forming a monodisperse aggregate.
- the concentration of the target substance contained in the mixed solution is the same as that of an empty non-crosslinked vesicle monodisperse assembly different from that of an empty crosslinked vesicle in that it is non-crosslinked.
- the concentration is such that the formation of a monodisperse aggregate of a substance-containing non-crosslinked vesicle in which the target substance is included in the inner aqueous phase is inhibited.
- a monodisperse aggregate of empty non-crosslinked vesicles When a monodisperse aggregate of empty non-crosslinked vesicles is mixed in an aqueous medium in the presence of the target substance, if the concentration of the target substance exceeds a certain level, a monodisperse aggregate of the substance-encapsulated non-crosslinked vesicles is formed. It may be inhibited (ie, a polydisperse aggregate is formed). On the other hand, when a monodisperse aggregate of empty crosslinked vesicles is mixed in an aqueous medium in the presence of the target substance, the monodisperse aggregate of the substance-encapsulated crosslinked vesicles is exceeded even if this certain concentration is exceeded. Formation is not inhibited.
- the concentration of the target substance present in the aqueous medium is higher than that in the conventional method (for example, the formation of a monodisperse aggregate of substance-encapsulating vesicles is inhibited in the post-loading method.
- the target substance can be encapsulated in the empty cross-linked vesicles while maintaining its monodispersity, so that a substance with a higher concentration of the target substance than the conventional method can be encapsulated.
- a monodisperse assembly of an encapsulated vesicle can be produced.
- the average particle size of the monodispersed aggregate of empty cross-linked vesicles is usually 30 nm or more, preferably 50 nm or more, more preferably 70 nm or more, and usually 10,000 nm or less, preferably 1000 nm or less, more preferably 400 nm or less.
- Each vesicle constituting a monodisperse aggregate of empty crosslinked vesicles comprises a first polymer and a second polymer, and a crosslinked film in which the first and / or second polymer is crosslinked, and a crosslinked film.
- a vesicle that contains an enclosed inner aqueous phase, and the target substance is not encapsulated in the inner aqueous phase.
- the first and second polymers in the empty crosslinked vesicle are as described in [B2-2: First and second polymers] above.
- the first and / or second polymer contained in the crosslinked film of the empty crosslinked vesicle is crosslinked. That is, the cross-linked film of the empty cross-linked vesicle is formed from a cross-linked bond between the first polymers, a cross-linked bond between the second polymers, and a cross-linked bond between the first polymer and the second polymer. 1 type, or 2 or more types of cross-linking selected from the group consisting of
- the crosslinked film of the empty crosslinked vesicle may contain a crosslinked bond formed between the first polymer and the second polymer from the viewpoint of improving the monodispersity of the substance-containing crosslinked vesicle. preferable.
- the first and / or second polymer contained in the crosslinked film of the empty crosslinked vesicle includes a crosslinked bond formed between cationic groups, a crosslinked bond formed between anionic groups, and a cation group and an anionic group. It is preferably crosslinked by one or two or more kinds of crosslinking bonds selected from the group consisting of crosslinking bonds formed between them, and is crosslinked by a crosslinking bond formed between a cation group and an anion group. More preferably.
- the cationic group and the anionic group are charged groups that the first polymer (first charged segment) and the second polymer (second charged segment) have.
- the crosslinking ratio is the ratio of the total number of moles of cationic groups and / or anionic groups contained in the crosslinked film, that is, when the cationic groups are crosslinked, Of the total number of moles of cation groups contained in the membrane, this is the ratio at which cross-linking bonds between cation groups are formed, and when the anion groups are cross-linked, the total number of moles of anionic groups contained in the cross-linked membrane Of these, it is a ratio in which a cross-linked bond between anionic groups is formed, and in the case where the cation group and the anionic group are cross-linked, the cation out of the total number of moles of the cation group and the anionic group contained in the cross-linked film This is the ratio at which a cross-linked bond between anionic groups is formed, and in the case where the cation group and the anionic group are cross-linked, the cation out of the total number of moles of the cation group and the anionic group contained in the cross-linked film This
- the maximum external force that can be maintained is reduced. Therefore, the higher the external force applied to the empty cross-linking vesicle, the higher the lower limit of the cross-linking rate, while the lower the external force applied to the empty cross-linking vesicle, the lower the lower limit of the cross-linking rate.
- the lower limit of the crosslinking rate is high. From the viewpoint of expanding the range of mixing methods that can be used when enclosing a target substance in an empty cross-linked vesicle and improving the versatility of the method according to the first aspect, it is possible to increase the cross-linking rate of the empty cross-linked vesicle. preferable.
- the lower limit of the cross-linking rate is preferably 35%, more preferably 37%, and still more preferably Is 38%, and when ultrasonic irradiation is used as a mixing method, the lower limit of the crosslinking rate is preferably 50%, more preferably 55%.
- the upper limit of the crosslinking rate is preferably 80%, more preferably 75%.
- the method for measuring the crosslinking rate can be appropriately selected according to the type of the crosslinking agent.
- an amide condensation type crosslinking agent such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC)
- EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
- an example of a method for calculating the crosslinking rate is a carboxyl group (—COO ⁇ ) determined from an infrared absorption spectrum. ) On the basis of the amount of consumption).
- Another method is to measure the crosslinking rate based on the amount of increase in amide bond (—CONH—).
- the first and second charged segments are polyamino acids or derivatives thereof, the first and second charged segments contain a large amount of amide bonds, which may reduce the measurement accuracy of the crosslinking rate. . Therefore, in this case, it is preferable to use in combination with another measurement method (for example, a measurement method based on the consumption of carboxyl groups). Furthermore, as another method, there is a method of calculating the crosslinking rate based on the remaining amount of amino group determined by the amino group coloring reagent.
- the amino group coloring reagent include trinitrobenzenesulfonic acid and fluorescamine.
- the space (void) surrounded by the crosslinked membrane usually contains an aqueous medium used as an inner aqueous phase when producing a monodisperse aggregate of empty crosslinked vesicles.
- Examples of a method for producing a monodisperse aggregate of empty crosslinked vesicles include a method of reacting a monodisperse aggregate of empty non-crosslinked vesicles with a crosslinking agent capable of reacting with the first and / or second polymer. Can be mentioned.
- Each vesicle constituting the monodisperse aggregate of empty non-crosslinked vesicles comprises a first and second polymer, and a non-crosslinked film in which neither of the first and second polymers is crosslinked;
- a vesicle comprising an inner aqueous phase surrounded by a non-crosslinked membrane, and the target substance is not encapsulated in the inner aqueous phase.
- each vesicle constituting the monodisperse aggregate of empty non-crosslinked vesicles is as described in [B2-1: Structure of empty vesicle].
- the average particle diameter of the monodisperse aggregate of empty non-crosslinked vesicles is usually 30 nm or more, preferably 50 nm or more, more preferably 70 nm or more, and usually 10,000 nm or less, preferably 1000 nm or less, more preferably 400 nm or less.
- the crosslinking agent is not particularly limited as long as it can react with the first and / or second polymer.
- the crosslinking agent one kind may be used alone, or two or more kinds may be used in combination.
- the crosslinking bond between the first polymers, the first One or two or more types of crosslinks selected from the group consisting of a crosslink between two polymers and a crosslink between the first polymer and the second polymer are formed.
- the crosslinking agent involved in the crosslinking reaction may or may not remain during the crosslinking.
- 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) does not remain in the cross-linking as described below.
- the crosslinking agent capable of reacting with the first and / or second polymer has two or more functional groups capable of reacting with the functional group possessed by the first polymer, and crosslinks between the first polymers.
- a crosslinking agent hereinafter referred to as “first crosslinking agent”
- first crosslinking agent a crosslinking agent having two or more functional groups capable of reacting with the functional group of the second polymer, and capable of crosslinking between the second polymers.
- second cross-linking agent and one or more functional groups capable of reacting with the functional group possessed by the first polymer, and one or more functional groups capable of reacting with the functional group possessed by the second polymer.
- a functional group, and a cross-linking agent capable of cross-linking between the first polymer and the second polymer (hereinafter referred to as “third cross-linking agent”).
- the third crosslinking agent is preferably used alone or in combination with other crosslinking agents.
- the first to third crosslinking agents are preferably capable of reacting with the chargeable group of the chargeable segment among the functional groups of the first and / or second polymer. That is, it is preferable that the first cross-linking agent can cross-link between the chargeable groups of the first chargeable segment, and the second cross-linker cross-links between the chargeable groups of the second chargeable segment.
- the third cross-linking agent is preferably capable of cross-linking between the charged group of the first charged segment and the charged group of the second charged segment.
- the cationic group that can react with the first to third crosslinking agents is preferably an amino group.
- the anionic group capable of reacting with the first to third crosslinking agents is, for example, a carboxyl group, a phosphate group or the like, preferably a carboxyl group.
- the functional group capable of reacting with the first to third crosslinking agents has no charge under neutral conditions, but may be a functional group with charge under predetermined conditions. Examples thereof include phenol derivatives, pyridine derivatives, imidazole derivatives, and thiol derivatives. These functional groups can be molecularly designed to have a charge under neutral conditions.
- crosslinking agent that crosslinks the amino group
- examples of the crosslinking agent that crosslinks the amino group include glutaraldehyde, dimethyl suberimidate dihydrochloride (DMS), dimethyl 3,3′-dithiobispropionimidate (DTBP), disuccinimidyl glutarate (DSG), Disuccinimide derivatives such as disuccinimidyl suberate (DSS), difluorobenzene derivatives such as 1,5-difluoro-2,4-dinitrobenzene (DFDNB), 3- [tris (hydroxymethyl) phosphonio] propionate (THPP) And bismaleimide derivatives.
- cross-linking agent that condenses the amino group and the carboxyl group to cross-link (therefore, the cross-linking agent does not remain in the cross-linking)
- examples of the cross-linking agent that condenses the amino group and the carboxyl group to cross-link include, for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), N, N Carbodiimide condensing agents such as' -dicyclohexanecarbodiimide (DCC), diisopropylcarbodiimide, N, N'-carbonyldiimidazole (CDI), 4- (4,6-dimethoxy-1,3,5-triazin-2-yl ) -4-methylmorpholinium chloride n hydrate (DMT-MM).
- EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
- DCC diisopropylcarbodiimide
- crosslinking agent for crosslinking the phosphate group examples include metal ions such as calcium ions.
- a preferred cross-linking agent is EDC.
- EDC does not remain in the cross-linking. Therefore, when a vesicle is administered in vivo (for example, when administered as a drug delivery vesicle in vivo), even if the vesicle is degraded in vivo, the crosslinking agent is not released and exhibits cytotoxicity.
- the amount of cross-linking agent used is the mixing method used when enclosing the target substance in an empty cross-linking vesicle so that the desired cross-linking rate is achieved. It can be appropriately adjusted according to the type of the material.
- the amount of crosslinking agent used is preferably in the following range. That is, when mixing by stirring at the time of encapsulation, the CL ratio defined by the above formula (iii) is preferably 0.3 or more, more preferably 0.5 or more, and preferably The amount of the crosslinking agent used is adjusted so as to be 1.2 or less, more preferably 1.0 or less.
- the CL ratio defined by the above formula (iii) is preferably 0.8 or more, more preferably 1.0 or more, and preferably 4
- the amount of the crosslinking agent used is adjusted so that it is 0.0 or less, more preferably 3.0 or less.
- the mixed solution contains the target substance together with an aqueous medium.
- the target substance is a substance to be included in the vesicle, and can be appropriately selected according to the use of the vesicle.
- the target substance is as described in [B3: Encapsulated substance], and the aqueous medium is as described in [B1-3: Other conditions for mixing].
- the concentration of the target substance contained in the mixed solution is different from the monodispersed aggregate of empty crosslinked vesicles in that the first and / or second polymer is not crosslinked. Is a concentration that inhibits the formation of a monodispersed aggregate of non-crosslinked vesicles containing a substance in which the target substance is encapsulated in the inner aqueous phase.
- Each vesicle constituting the monodisperse aggregate of empty non-crosslinked vesicles comprises a first and second polymer, and a non-crosslinked film in which neither of the first and second polymers is crosslinked;
- a vesicle comprising an inner aqueous phase surrounded by a non-crosslinked membrane, and the target substance is not encapsulated in the inner aqueous phase.
- Each vesicle constituting the monodispersed aggregate of substance-containing non-crosslinked vesicles comprises a first and a second polymer, and a non-crosslinked film in which neither of the first and second polymers is crosslinked;
- a vesicle comprising an inner aqueous phase surrounded by a non-crosslinked membrane, and the target substance is encapsulated in the inner aqueous phase.
- a monodisperse aggregate of empty uncrosslinked vesicles differs from an empty crosslinked vesicle in that the first and / or second polymer is not crosslinked.
- monodisperse aggregates of empty non-crosslinked vesicles are monodisperse aggregates of empty crosslinked vesicles at points other than crosslinking. It is preferably substantially the same as the body.
- the conditions for mixing an empty non-crosslinked vesicle monodisperse aggregate in a mixed solution so that whether or not a monodispersed aggregate of substance-encapsulating vesicles is formed depends only on the presence or absence of crosslinking (mixed solution).
- the composition, mixing method, etc. are preferably substantially the same as the conditions for mixing the monodispersed aggregate of empty crosslinked vesicles in the mixed solution.
- a monodisperse aggregate of empty non-crosslinked vesicles is mixed in an aqueous medium in the presence of the target substance, if the concentration of the target substance contained in the mixed solution exceeds a certain level, the substance-encapsulated vesicle single unit In some cases, the formation of a dispersed aggregate is inhibited (ie, a polydispersed aggregate is formed).
- This constant concentration varies depending on the type of target substance. For example, when the molecular weight of the target substance is 10,000 to 40,000, the concentration is usually 5 mg / mL, preferably 15 mg / mL, more preferably 40 mg / mL.
- the effect of the method according to the first aspect becomes more remarkable as the concentration of the hardly water-soluble substance contained in the mixed solution increases. That is, when mixing a monodisperse aggregate of empty non-crosslinked vesicles in a mixed solution, if the concentration of the poorly water-soluble substance contained in the mixed solution exceeds a certain concentration, the monodisperse of the substance-containing uncrosslinked vesicles Aggregate formation may be inhibited (ie, polydisperse aggregates are formed).
- the concentration of the poorly water-soluble substance contained in the mixed solution is a concentration at which formation of a monodisperse aggregate of the substance-containing non-crosslinked vesicles is inhibited when empty non-crosslinked vesicles are mixed in the same mixed solution.
- concentration is usually higher than the solubility of the poorly water-soluble substance (in this case, the poorly water-soluble substance is dispersed and suspended in the mixed solution).
- the “slightly water-soluble” means that it is hardly soluble or insoluble in an aqueous medium.
- the solubility in water at 25 ° C. is usually 1.0 mg / It means not more than mL, preferably not more than 0.1 mg / mL, more preferably not more than 0.02 mg / mL.
- the concentration of empty crosslinked vesicles to be mixed in the mixed solution can be adjusted as appropriate within a range in which the vesicles do not aggregate. If the concentration of empty cross-linked vesicles is too low, a substance-encapsulating vesicle may not be formed after mixing. Therefore, the concentration of empty cross-linked vesicles is preferably as high as possible within a range where vesicles do not aggregate.
- the concentration of empty cross-linked vesicles is usually 0.1 mg / mL or more, preferably 1 mg / mL or more, more preferably 10 mg / mL or more, and usually 100 mg / mL or less, preferably 70 mg / mL or less, more preferably 50 mg.
- the concentration of the empty crosslinked vesicle should be determined according to the particle diameter of the substance-encapsulating vesicle to be manufactured. .
- the pH of the mixed solution is not particularly limited and can be appropriately adjusted according to the structure of the empty crosslinked vesicle, the type of the target substance, the concentration of the empty crosslinked vesicle and the target substance in the mixed solution, and preferably
- the pH is 5 or more, more preferably 6.5 or more, and preferably 9 or less, more preferably 7.5 or less.
- the pH can be easily adjusted by using a buffer as a solvent. It is advantageous to adjust the pH of the mixed solution to maintain the structure of the empty cross-linked vesicle and efficiently encapsulate the target substance in the empty cross-linked vesicle.
- the salt concentration (ionic strength) of the mixed solution can be appropriately adjusted within a range that does not destroy the structure of the empty crosslinked vesicles or inhibit the inclusion of the target substance in the empty crosslinked vesicles.
- the effect of the method according to the first aspect becomes more remarkable as the salt concentration (ionic strength) of the mixed solution increases. That is, when a monodisperse aggregate of empty non-crosslinked vesicles is mixed in a mixed solution, if the salt concentration of the mixed solution exceeds a certain value, the formation of a monodisperse aggregate of substance-containing uncrosslinked vesicles is inhibited. (Ie, a polydisperse aggregate is formed).
- the salt concentration of the mixed solution is preferably a concentration at which formation of a monodisperse aggregate of substance-containing non-crosslinked vesicles is inhibited when empty non-crosslinked vesicles are mixed in the same mixed solution.
- the salt concentration of the mixed solution is preferably 20 mM or more, more preferably 75 mM or more, still more preferably 150 mM or more, in terms of sodium chloride concentration.
- the upper limit of the salt concentration of the mixed solution is usually 1.0M, preferably 0.5M.
- the viscosity of the mixed solution can be appropriately adjusted within a range that does not destroy the structure of the empty cross-linked vesicle or inhibit the inclusion of the target substance in the empty cross-linked vesicle.
- the effect of the method according to the first aspect becomes more remarkable as the viscosity of the mixed liquid increases. That is, when a monodisperse aggregate of empty non-crosslinked vesicles is mixed in a mixed solution, if the viscosity of the mixed solution exceeds a certain value, the formation of a monodisperse aggregate of substance-containing non-crosslinked vesicles is inhibited. (Ie, a polydisperse aggregate is formed).
- the viscosity of the mixed solution is preferably a viscosity at which formation of a monodisperse aggregate of substance-containing non-crosslinked vesicles is inhibited when empty non-crosslinked vesicles are mixed in the same mixed solution.
- the viscosity of the mixed solution is preferably 0.05 Pa ⁇ s or more, more preferably 0.1 Pa ⁇ s or more, and still more preferably 0.5 Pa ⁇ s or more.
- the upper limit of the viscosity of the mixed solution is usually 1.0 Pa ⁇ s, preferably 0.7 Pa ⁇ s.
- the temperature of the mixed solution is not limited as long as it does not destroy the structure of the empty crosslinked vesicle or inhibit the inclusion of the target substance in the empty crosslinked vesicle, but it is preferably 4 ° C. or higher, more preferably 20 It is 80 degreeC or more, Preferably it is 80 degrees C or less, More preferably, it is 50 degrees C or less.
- the mixing method is as described in [B1-2: Mixing of empty vesicle and inclusion substance].
- the target substance is a poorly water-soluble substance, it is preferable to use ultrasonic treatment as a mixing method.
- a monodispersed aggregate of empty cross-linked vesicles is mixed in a mixed solution containing the target substance together with an aqueous medium, and the substance-containing cross-linked vesicle in which the target substance is encapsulated in the inner aqueous phase.
- the post-process can include a cross-linking step for a substance-containing cross-linking vesicle obtained by mixing empty cross-linking vesicles in a mixed solution.
- the crosslinking agent used in the crosslinking step is a crosslinking agent that can react with the first and / or second polymer, and the details thereof are the same as described above.
- a crosslinked membrane comprising the first and second polymers, wherein the first and / or second polymer is crosslinked, and an inner aqueous phase surrounded by the crosslinked membrane
- a monodispersed aggregate of substance-encapsulated crosslinked vesicles containing the target substance in the inner aqueous phase is produced.
- the concentration of the target substance contained in the inner aqueous phase is that the first and / or second polymer is not crosslinked and the target substance is not encapsulated.
- a monodisperse aggregate of empty non-crosslinked vesicles different from the monodisperse aggregate of substance-embedded crosslinked vesicles is mixed in a mixed solution containing the target substance in the same concentration as the inner aqueous phase of the substance-embedded crosslinked vesicles together with an aqueous medium
- it is a concentration that inhibits the formation of a monodisperse aggregate of non-crosslinked vesicles containing a substance containing the target substance in the inner aqueous phase.
- the substance-incorporated crosslinked vesicle produced by the method according to the first aspect includes a monodisperse aggregate of empty non-crosslinked vesicles and a target substance having the same concentration as the inner aqueous phase of the substance-encapsulated crosslinked vesicle together with an aqueous medium. It has higher monodispersibility than the substance-containing non-crosslinked vesicle obtained by mixing in the mixed liquid.
- the inner aqueous phase of the substance-encapsulated crosslinked vesicle varies depending on the mixed solution used in the method according to the first aspect.
- the salt concentration of the mixed solution is 20 mM or more in terms of sodium chloride concentration
- the salt concentration of the inner aqueous phase is the same.
- the viscosity of a liquid mixture is 0.05 Pa.s or more
- the viscosity of an inner water phase is also the same.
- the mixed solution contains a target substance having a weight average molecular weight of 10,000 to 40,000 at a concentration exceeding 5 mg / mL, the same applies to the inner aqueous phase.
- the inner aqueous phase is the same.
- the cross-linking rate of the substance-encapsulated cross-linking vesicle varies depending on the cross-linking rate of the empty cross-linking vesicle used in the method according to the first aspect and the presence or absence of the cross-linking step performed as a subsequent step.
- the first and / or second polymer is formed between a cross-linked bond formed between cationic groups, a cross-linked bond formed between anionic groups, and between a cationic group and an anionic group.
- Crosslinks are formed by one or more types of crosslinks selected from the group consisting of the formed crosslinks, and the proportion of crosslinks formed is the total of the cation groups and / or anion groups contained in the cross-linked film.
- the number of moles is 35% or more
- the crosslinking rate of the substance-containing crosslinked vesicle obtained by mixing empty crosslinked vesicles in the mixed solution is the same as that of the empty crosslinked vesicle, and the crosslinking step is performed as a subsequent step. In some cases, the crosslinking rate is increased over empty crosslinking vesicles.
- the method according to the second aspect includes a mixed liquid containing a first substance-containing crosslinked vesicle encapsulating a first object substance and a second object substance having a molecular weight smaller than that of the first object substance together with an aqueous medium.
- a method for producing a multi-substance-encapsulated cross-linked vesicle comprising a step of forming a second substance-encapsulated cross-linked vesicle in which the first and second target substances are encapsulated.
- the first and second target substances can be encapsulated while controlling the amount of the encapsulated substances, whereby the first and second target substances are encapsulated. Can be manufactured.
- the first substance-encapsulated crosslinked vesicle includes a first and a second polymer, a crosslinked film in which the first and / or second polymer is crosslinked, and an inner aqueous phase surrounded by the crosslinked film. And a vesicle in which the first target substance is encapsulated in the inner aqueous phase.
- the configuration of the first substance-containing cross-linking vesicle is the same as that of the substance-containing cross-linking vesicle in the method according to the first aspect, except that the target substance is the first target substance.
- the first substance-encapsulated crosslinked vesicle is preferably a monodisperse aggregate.
- the second substance-encapsulated crosslinked vesicle can be produced as the monodispersed aggregate.
- the average particle size of the monodisperse aggregate of the first substance-encapsulated crosslinked vesicle is usually 30 nm or more, preferably 50 nm or more, more preferably 70 nm or more, and usually 10,000 nm or less, preferably 1000 nm or less, more preferably 400 nm or less. is there.
- Method 1 Method 1
- Method 1 a method of reacting with a crosslinking agent capable of reacting with the first and / or second polymer
- Method 1 can be performed in the same manner as the method according to the first aspect except that the first target substance is used as the target substance.
- the substance-containing non-crosslinked vesicle used in Method 2 can be produced by, for example, a simultaneous mixing method, a post-supporting method, or the like.
- the simultaneous mixing method is a method in which the first polymer, the second polymer, and the target substance are mixed in an aqueous medium
- the post-supporting method is a mixing in which an empty vesicle is contained together with the first substance together with the aqueous medium. It is a method of mixing in a liquid.
- the cross-linking agent used in Method 2 is the same as the cross-linking agent used in the method according to the first aspect.
- the first substance-encapsulated crosslinked vesicle can be produced as a monodisperse aggregate thereof.
- the empty crosslinked vesicle is a monodispersed aggregate thereof, and the first substance-containing crosslinked vesicle is the monodispersed aggregate.
- the concentration of the first target substance contained in the mixed solution is such that the first and / or second polymer is not crosslinked.
- the mixed solution contains a second target substance having a molecular weight smaller than that of the first target substance together with an aqueous medium.
- the second target substance is not particularly limited as long as the molecular weight is smaller than that of the first target substance, and can be appropriately selected according to the use of the vesicle.
- Specific examples of the first and second target substances include the same specific examples as the target substance used in the method according to the first aspect.
- a preferred example of the second target substance is a crowding agent.
- the crowding agent provides a molecular crowding environment (an environment in which molecules are crowded).
- the crowding agent is a water-soluble molecule that does not interact with the first target substance and has sufficient solubility to provide a molecular crowning environment.
- Specific examples thereof include polyethylene glycol and sugar polymers ( Dextran, Ficoll (registered trademark) ), hydrophilic polymers such as albumin protein, and hydrophilic low molecules such as glycerol, ethylene glycol, and diethylene glycol.
- the aqueous solvent is the same as the aqueous medium used in the method according to the first aspect.
- the composition, pH, salt concentration (ionic strength), viscosity and the like of the mixed solution are the same as those of the mixed solution used in the method according to the first aspect.
- the concentration of the second target substance contained in the mixed solution is such that the first and / or second polymer is not cross-linked.
- the monodispersed aggregate of the first substance-encapsulated non-crosslinked vesicle different from the monodispersed aggregate of the substance-encapsulated crosslinked vesicle is mixed in the mixed solution, the first and second target substances are encapsulated in the inner aqueous phase.
- the concentration is such that the formation of the monodisperse aggregate of the second substance-containing non-crosslinked vesicle is inhibited, the same effect as the method according to the first aspect can be obtained.
- the mixing method is the same as the method according to the first aspect.
- a crosslinked membrane comprising the first and second polymers, wherein the first and / or second polymer is crosslinked, and an inner aqueous phase surrounded by the crosslinked membrane And a substance-encapsulated crosslinked vesicle in which the first and second target substances are encapsulated in the inner aqueous phase.
- the first target substance is more stabilized than the case where it is contained in the inner aqueous phase in the absence of the second target substance.
- Such stabilization of the first target substance can be realized by controlling the amount of the second target substance enclosed.
- Adsorbent-encapsulating vesicle and production method thereof Another aspect of the present invention relates to a vesicle that encapsulates adsorbent particles (referred to as “adsorbent-encapsulating vesicle” as appropriate) and a production method thereof.
- encapsulated particles having a substance adsorbing ability in electrostatic interaction type vesicles by self-organization of two kinds of polymers having opposite charges. It has been found that a new substance-encapsulating vesicle (adsorbent-encapsulating vesicle) containing relatively large particles relative to the size of the vesicle can be obtained. Furthermore, it has been found that such adsorbent-containing vesicles can be obtained very efficiently by mixing one of the two polymers with adsorbent particles and then mixing with the other polymer. This aspect is based on such knowledge.
- the adsorbent-containing vesicle of the present invention has a first polymer that is a block copolymer having an uncharged hydrophilic segment and a first charged segment, and a charge opposite to that of the first charged segment.
- a vesicle comprising a membrane containing a second polymer having a charged second charged segment; and adsorbent particles encapsulated in the vesicle, wherein at least one of the first and second polymers is the It is adsorbed on the adsorbent particles.
- the first and second polymers are as described in the section [B].
- the adsorbent particles are particles having an action of adsorbing a substance.
- Adsorption herein refers to a phenomenon in which the concentration of a substance is higher than the surroundings at the interface of two phases (here, solid particles and the surrounding liquid phase).
- the kind of the adsorption action that the adsorbent particles have is not limited, and may be physical adsorption (adsorption by physical interaction), chemical adsorption (adsorption accompanied by chemical bonds), or a combination thereof.
- the basic physical interactions include interionic interactions, dipole interactions, van der Waals, and combinations of two or more of these, Either may be sufficient.
- the chemical bond serving as the group may be a hydrogen bond, a covalent bond, a coordination bond, an ionic bond, or a combination of two or more of these.
- the adsorbent particles are preferably particles having a charge on the surface and exhibiting dispersibility in an aqueous medium. Since the adsorbent particles have these properties, the first and / or second polymer, which is a constituent element of the vesicle, is easily adsorbed by the adsorbent particles by electrostatic interaction, and near the adsorbent particle surface. It is thought that the formation of vesicles is promoted.
- the absolute value of the zeta potential on the particle surface is usually 10 mV or more, preferably 20 mV or more, more preferably 30 mV or more.
- the zeta potential on the particle surface can be measured by using an electrophoresis method such as light scattering electrophoresis.
- the adsorbent particles are preferably particles having a large specific surface area.
- the particles having a large specific surface area include various porous particles.
- the pore volume of the particles is preferably 0.1 cm 3 / g or more, and more preferably 0.5 cm 3 / g or more.
- the pore characteristics such as the specific surface area of the particle surface and the pore volume of the porous particle can be measured using, for example, the BET method.
- adsorbent particles is not limited, but examples include various metal oxides such as silica, calcium phosphate, calcium carbonate, alumina, zeolite and iron oxide; simple metals such as gold, platinum and palladium; polystyrene resin, styrene- Examples thereof include particles made of a material such as a synthetic or natural resin such as divinylbenzene resin, ion exchange resin, polyacrylic acid fine particles, polylactic acid fine particles, and lactic acid-glycolic acid copolymer fine particles. Of these, silica, calcium phosphate, gold, polyacrylic acid fine particles and the like are preferable.
- the properties of the adsorbent particles are not limited and may be solid particles, but may form colloids or gels in an aqueous medium.
- the material may not be an adsorbent or a particulate substance at the beginning, but may be a substance that can become adsorbent particles after being included in a vesicle. An example of such an embodiment will be described later.
- adsorbent particles having a large particle size for example, more than 30 nm
- particles having a large particle size for example, more than 30 nm
- adsorbent particles having a diameter of usually 40 nm or more, particularly 50 nm or more, and more preferably 60 nm or more are preferable.
- limiting in the upper limit of an average particle diameter Usually, it is about 10 micrometers or less.
- the average particle size of the adsorbent particles depends on the type of adsorbent, but is usually dynamic light scattering (DLS), transmission electron microscope (TEM), scanning electron microscope. (Scanning ⁇ electron microscope: SEM), laser diffraction method, Coulter counter method, etc. can be used for measurement.
- DLS dynamic light scattering
- TEM transmission electron microscope
- SEM scanning electron microscope
- laser diffraction method laser diffraction method
- Coulter counter method etc.
- the adsorbent-encapsulating vesicle of the present invention it is possible to enclose adsorbent particles that are relatively large with respect to the size of the vesicle.
- the average particle diameter of the adsorbent particles (mesoporous silica nanoparticles: MSN) to be included is about 80 nm, whereas the average particle diameter of the obtained adsorbent-encapsulated vesicles is about Despite the extremely large particle size of the adsorbent particles relative to the vesicle particle size of 100 nm, an adsorbent-encapsulating vesicle that encloses the adsorbent particles with a high encapsulation rate is obtained.
- first and / or second polymer is self-assembled in a state of being adsorbed on the adsorbent particles and efficiently forms vesicles around the adsorbent particles. It is done.
- the adsorbent-encapsulating vesicle of the present invention can be produced by mixing the first and second polymers together with adsorbent particles, usually in an aqueous medium, in any order.
- either one of the first and second polymers is first mixed with the adsorbent particles in the aqueous medium, and the adsorbent particles
- the other polymer is added to the mixture containing the aqueous medium and mixed to form a vesicle comprising a film containing the first and second polymers around the adsorbent particles.
- It is preferably produced by a method comprising at least a step of producing (a method for producing an adsorbent-containing vesicle of the present invention).
- the polymer (first or second polymer) to be adsorbed first on the adsorbent particles in the step (a) may be any.
- a polymer having a charge opposite to the surface charge of the adsorbent particles is first brought into contact with the adsorbent particles.
- the surface charge of the adsorbent particles is positive, it is preferably a polymer having a negative charge (that is, a polymer having an anionic segment), and the surface charge of the adsorbent particles is negative.
- a polymer having a positive charge that is, a polymer having a cationic segment
- positive and negative charges are mixed on the surface of the adsorbent particles, it is only necessary to comprehensively consider the surface charge of the entire adsorbent particles to determine the positive or negative.
- the concentrations of the first and second polymers and the adsorbent particles, the mixing method, and other various manufacturing conditions are used as they are or as appropriate. It can be used with modification.
- a vesicle composed of a film containing the first and second polymers is formed around the adsorbent particles, and an adsorbent-encapsulating vesicle is obtained.
- the adsorbent-encapsulating vesicles and the adsorbent particles that are not encapsulated in the vesicles can be identified by, for example, the surface zeta potential.
- the electrostatic interaction between the first and second polymers causes The charge is canceled and the absolute value of the surface zeta potential is reduced. Therefore, it can be confirmed that the absolute value of the surface zeta potential has decreased, and further, the formation of adsorbent-encapsulating vesicles can be confirmed in consideration of the particle physical properties by DLS measurement, TEM observation, and the like.
- the entrapment rate of the adsorbent particles is improved by the above production method. That is, in this production method, the adsorbent particles are not encapsulated in the self-organized vesicle, but one of the first and second polymers is adsorbed to the adsorbent particles to be encapsulated and then the other is added. As a result, a vesicle film is formed in the vicinity of the surface of the adsorbent particles. In other words, by using the adsorbent particles as a template when forming the vesicles, by selectively increasing the concentration of the charged polymer (one of the first and second polymers) in the microenvironment near the adsorbent particles.
- the manufacturing method of the adsorbent inclusion vesicle of this invention you may implement by adding other processes other than the above.
- Cross-linking makes it possible to increase the stability of the vesicles.
- the polymer for example, when the adsorbent-containing vesicle is used for DDS or the like, the vesicle can be stably maintained even under physiological conditions.
- a method for crosslinking the first and / or second polymer will be described separately.
- a step of surface-treating the adsorbent may be performed before, during or after the formation of the adsorbent-encapsulating vesicle.
- various functional groups can be imparted to the surface of the adsorbent, and various characteristics of the adsorbent surface can be modified.
- the type of surface treatment is not particularly limited. For example, when silica particles are used as the adsorbent, treatment with various silane coupling agents can be mentioned. The type of the silane coupling agent is not particularly limited.
- a cationic group for example, an aminated silane coupling such as 3-aminopropyltrimethoxysilane or 3-aminopropyltriethoxysilane.
- an agent for example, when an anionic group is added to the surface, the mercapto group is oxidized after treatment with a mercapto silane coupling agent such as 3-mercaptopropyltrimethoxysilane or 3-mercaptopropylmethyldimethoxysilane. What is necessary is just to convert into a sulfonyl group.
- p-styryltrimethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, 3-methacryloxypropyltrimethoxysilane that can be expected to have hydrophobicity of aromatic rings and ⁇ -electron interactions The surface treatment by etc. is mentioned. Of course, the surface treatment is not limited to these treatments, and any surface treatment can be performed depending on the desired characteristics and application, the type of adsorbent used, the type of substance adsorbed on the adsorbent (described later), and the like. It can be selected appropriately.
- another substance can be adsorbed on the adsorbent particles encapsulated in the vesicle.
- a compound having a low molecular weight for example, 5000 Da or less
- adsorbent-encapsulating vesicle of the present invention can be suitably used for applications such as carrying low-molecular compounds such as various drugs and releasing them slowly.
- particles having a charge on the surface as adsorbent particles and exhibiting dispersibility in an aqueous medium can be stably present under physiological conditions and are supported on adsorbent particles. It is possible to realize an excellent DDS that can release low molecular compounds such as various drugs under physiological conditions.
- the adsorption / support characteristics can be improved by subjecting the adsorbent to a surface treatment as described above.
- Adsorption and loading of low molecular weight compounds on adsorbent particles can be performed before or after the adsorbent particles are encapsulated in vesicles, but the physicochemical properties of the adsorbent particles depend on the physicochemical properties of the low molecular weight compounds to be adsorbed. Therefore, after adsorbent particles are encapsulated in vesicles, it is considered desirable.
- a substance-encapsulating vesicle into which a substance capable of forming an adsorbent (adsorbent precursor) is introduced, and then converting the adsorbent precursor to the adsorbent, the adsorbent inclusion It is also possible to form vesicles.
- the adsorbent precursor include a chargeable polymer that can form a matrix such as a gel or a precipitate / micelle.
- a substance-encapsulating vesicle into which a charged polymer is introduced is prepared, and then an oppositely charged drug or the like is introduced into the vesicle.
- the electrostatic interaction is moderately canceled and the charged polymer becomes hydrophobic, and a matrix such as a gel, an adsorbent such as a precipitate and a micelle is generated inside the vesicle, and an adsorbent such as the matrix, the precipitate and the micelle is formed in the adsorbent.
- a drug-carrying adsorbent-containing vesicle in which the drug is trapped is formed.
- Such drug-loaded adsorbent-encapsulating vesicles are also useful as sustained-release agents capable of sustained-release of the drug.
- Low-water-soluble substance-encapsulating vesicle and method for producing the same
- a substance capable of producing a vesicle encapsulating a low-water-soluble substance referred to as “low-water-soluble substance-encapsulating vesicle” as appropriate.
- the present invention relates to a method for producing an encapsulated vesicle and a novel low water-soluble substance-encapsulating vesicle produced thereby.
- the present inventors prepared an enzyme-encapsulated vesicle in which an enzyme capable of converting a precursor having a higher water solubility than the target substance into the target substance is encapsulated in the vesicle, and then the precursor is contained in the enzyme-encapsulated vesicle. It has been found that efficient encapsulation is possible by infiltrating into the vesicles, converting into the target substance by the enzyme in the vesicle, precipitating the target substance and encapsulating it in the vesicle. This aspect is based on such knowledge.
- the method for producing a substance-encapsulating vesicle according to this aspect includes a first polymer which is a block copolymer having an uncharged hydrophilic segment and a first charged segment, and is opposite to the first charged segment.
- a method for producing a substance-encapsulating vesicle in which a target substance is encapsulated in a vesicle comprising a film containing a second polymer having a second chargeable segment charged to a charge comprising the following steps.
- An enzyme-encapsulating vesicle is prepared in which an enzyme capable of converting a precursor having a higher water solubility than the target substance into the target substance is encapsulated in a vesicle composed of a film containing the first and second polymers. Process. (B) allowing the precursor to penetrate into the enzyme-encapsulating vesicle under conditions that exhibit lower solubility in the target substance than the precursor, and converting the precursor to the target substance by the enzyme; The step of precipitating the target substance and encapsulating it in the enzyme-encapsulating vesicle to form a low-water-soluble substance-encapsulating vesicle.
- the target substance to be included in the vesicle is a substance that can be converted from a highly water-soluble precursor by an enzyme. Any kind of substance can be used as the target substance as long as a precursor having a higher water solubility than the target substance exists and an enzyme capable of converting the precursor to the target substance exists.
- the target substance to be included in the vesicle is a substance whose solubility in water is reduced by the enzymatic action.
- a substance having a relatively high solubility in water before being subjected to the enzyme action is a precursor
- a substance having a relatively low solubility in water after being subjected to the enzyme action is a target substance. Therefore, as a combination of the precursor and the target substance, not only a combination of substances having different chemical formulas and chemical compositions, but also a substance whose solubility in water changes due to a change in steric structure etc. even if the chemical formulas and chemical compositions are the same. Can also be a target.
- the ratio of the solubility of the target substance to the solubility of the precursor is preferably small. Specifically, although not limited, when the solubility in water at 25 ° C. is compared, the ratio of the solubility of the target substance to the solubility of the precursor is usually 90% or less, particularly 80% or less, Is preferably 70% or less.
- the difference in water solubility between the precursor and the target substance is large.
- the precursor has a higher solubility than the target substance, for example, usually 10 mg / mL or more, especially 20 mg / mL or more, more preferably 30 mg / mL or more. It may be preferable to have.
- the target substance is preferably low water soluble.
- the solubility in water at 25 ° C. is usually 1.0 mg / mL or less, preferably 0.3 mg / mL or less, more preferably 0.1 mg / mL or less.
- an enzyme-encapsulating vesicle in which an enzyme is encapsulated in a vesicle composed of a film containing the first and second polymers is prepared.
- Such enzyme-encapsulating vesicles include methods for producing various substance-encapsulating vesicles described in the above [B] column (post-loading method, simultaneous mixing method, etc.), methods for producing the substance-encapsulating vesicles described in the above [C] column, etc. It is prepared by encapsulating an enzyme in a vesicle using any technique.
- the precursor is infiltrated into the enzyme-encapsulated vesicle under the condition that the solubility to the target substance is lower than that of the precursor.
- the method for allowing the precursor to penetrate into the enzyme-encapsulating vesicle is arbitrary, but it is usually performed by mixing the enzyme-encapsulating vesicle and the precursor in a solvent.
- a solution containing the enzyme-encapsulating vesicle and the precursor in a solvent may be prepared separately, and then mixed, and one of the enzyme-encapsulating vesicle and the precursor may be mixed in a solution containing the other in the solvent. It may be added directly.
- the enzyme-encapsulating vesicle and the precursor may be contacted in a continuous phase such as a liquid (for example, in vivo use described later corresponds to this embodiment).
- the conditions that exhibit lower solubility in the target substance than the precursor are preferably such that the precursor can exist in a state of being substantially dissolved in a solvent, but the target substance is partially or wholly a solvent. It is a condition that precipitates from. This can be achieved by selecting an appropriate solvent in consideration of the water solubility of each of the precursor and the target substance. For example, when the target substance is a low water-soluble substance as defined above and the precursor is a water-soluble substance as defined above, such a condition can be achieved by a general aqueous medium.
- the solubility may be adjusted by dissolving various organic or inorganic electrolytes in the solvent, or various environmental factors such as temperature may be adjusted to control the solubility of the solvent to achieve the conditions. .
- the precursor When the precursor is infiltrated into the enzyme-encapsulating vesicle under the above conditions, the precursor is converted into the target substance by the enzyme.
- the precursor is converted into the target substance by the enzyme, so that the target substance is precipitated from the solvent and encapsulated in the enzyme-encapsulating vesicle, thereby forming the substance-encapsulating vesicle.
- the manufacturing method of the substance inclusion vesicle of this aspect you may implement by adding other processes other than the above. Examples include adding a step of cross-linking the first and / or second polymer before and / or after the precursor has penetrated into the enzyme-encapsulating vesicle.
- the first and / or second in the enzyme-encapsulating vesicle is allowed to penetrate before the water-soluble precursor penetrates into the enzyme-encapsulating vesicle. It is preferred to crosslink the polymer.
- the vesicle can be stably maintained even under physiological conditions.
- crosslinking the 1st and / or 2nd polymer it is possible to use the conditions demonstrated in the said [C] column as it is, or changing suitably.
- the production site of the target substance can be limited only to the inside of the vesicle, thereby efficiently It is considered that the target substance can be included in the vesicle.
- the amount of the target substance to be included in the vesicle can be controlled by changing the amount of the precursor added to the enzyme-encapsulated vesicle.
- the vesicle has a semipermeable membrane property, it is considered that the target substance encapsulated in the vesicle can be gradually released from the vesicle.
- a vesicle that encapsulates a low-water-soluble substance that has been extremely difficult or impossible to encapsulate in a vesicle.
- a low water-soluble substance-encapsulating vesicle in which a low-water-soluble substance is encapsulated (together with an enzyme) is obtained in a vesicle composed of a film containing the first and second polymers.
- Such a low water-soluble substance-encapsulating vesicle is also the gist of the present invention.
- the low water-soluble substance is included in a concentration exceeding the solubility of the low water-soluble substance in the inner aqueous phase.
- the first and / or the second polymer are preferably crosslinked.
- substance-encapsulating vesicles by the production method of this embodiment may be artificially achieved in vitro, but in vivo (by appropriately selecting the target substance / precursor / enzyme combination, etc.) It can also be designed to be achieved in in vivo. This aspect will be described later.
- Such pharmaceutical compositions and drug delivery systems containing the various substance-encapsulating vesicles of the present invention are also objects of the present invention.
- the subject to which such a pharmaceutical composition or drug delivery system is applied (any organism, but preferably an animal, more preferably a mammal, more preferably a human), a disease, a drug, and the like can be appropriately selected. it can.
- various conditions such as administration route, usage and dosage of such pharmaceutical composition and drug delivery system can be appropriately selected according to the subject, disease, drug and the like.
- substance-encapsulating vesicles by the production method described in [E] above may be accomplished artificially in vitro, but the target substance / precursor / enzyme combination should be selected appropriately.
- a substance-encapsulating vesicle containing the target substance can be formed in the body.
- the prodrug is converted into a drug at a site such as a desired organ or tissue in the body and converted into a vesicle. It is possible to achieve excellent DDS in which the drug is gradually released from the encapsulated and formed drug-encapsulating vesicle.
- a substance-encapsulated vesicle in which an enzyme having a function of converting a drug precursor (prodrug) into a drug is encapsulated as an encapsulated substance of the substance-encapsulated cross-linked vesicle of [C]
- a substance-encapsulating vesicle in which the enzyme is adsorbed and supported on the adsorbent of the adsorbent-encapsulating vesicle [D] may be prepared and used.
- the prodrug is introduced into the body, and these enzyme-encapsulated cross-linked vesicles and enzyme-supported adsorbent-encapsulated vesicles are separately introduced into the body, and the enzyme-encapsulated cross-linked vesicle and enzyme-supported adsorbent-encapsulated vesicle are contained at a desired site in the body. Is brought into contact with the prodrug and the vesicle is infiltrated with the prodrug. In this way, a drug can be produced from a prodrug in the body by allowing an enzyme to act on the prodrug and converting it to the drug.
- Such an aspect can be achieved using not only various substance-encapsulating vesicles of the present invention but also any substance-encapsulating vesicle. That is, an arbitrary substance-encapsulating vesicle (enzyme-supporting vesicle) in which an enzyme is encapsulated and supported is prepared and used.
- the prodrug is introduced into the body, the enzyme-supporting vesicle is separately introduced into the body, the enzyme-supporting vesicle is brought into contact with the prodrug at a desired site in the body, and the prodrug is infiltrated into the vesicle.
- a drug can be produced from a prodrug in the body by allowing an enzyme to act on the prodrug and converting it to the drug.
- the solubility relationship between the drug and its prodrug is not limited, and any drug / prodrug / enzyme combination can be used.
- Specific examples of such drugs / prodrugs / enzymes include the combinations shown in the following table, but these are merely examples, and the combinations of drugs / prodrugs / enzymes targeted by the present invention are those The combination is not limited.
- a method for delivering a drug to a subject comprising the following steps.
- Step (b) of the above method is achieved by allowing an enzyme-encapsulated vesicle and a precursor to coexist at a predetermined target site.
- the predetermined site of the target is arbitrary, and may be determined according to the target to which the drug is delivered, the type of the drug, the disease to be treated, and the like, but is usually the predetermined organ, tissue or cell of the target.
- the method for allowing the enzyme-encapsulating vesicle to be present at a predetermined site of the subject is arbitrary, but usually, the enzyme-encapsulating vesicle may be administered to the subject by various conventionally known administration methods to reach the predetermined site of the subject.
- the administration method may be oral or parenteral, and in the case of parenteral administration, it may be administered by any route such as intravenous, intramuscular, subcutaneous, transdermal, nasal, or pulmonary.
- the precursor may be converted into a drug by the enzyme encapsulated in the enzyme-encapsulated vesicle.
- the converted drug is released from the vesicle and delivered to a predetermined site of the subject.
- Various conditions such as other doses and usage can be appropriately determined depending on the subject to which the drug is delivered, the type of the drug, the disease to be treated, and the like.
- drug delivery systems including enzyme-encapsulated vesicles
- pharmaceutical compositions used in the above methods are also the subject of the present invention.
- solution or “dispersion” refers to a solution or “dispersion” using 10 mM phosphate buffer (pH 7.4) as a solvent or dispersion medium unless otherwise specified. To do.
- vortex mixer MixMate manufactured by Eppendorf was used unless otherwise specified.
- Example Group I Substance-encapsulating vesicle and its production method
- Example I-1 FITC-Dex40k encapsulation in empty crosslinked vesicle
- PEG-P Anionic block copolymer
- PEG-P composed of polyethylene glycol (weight average molecular weight of about 2000) (hereinafter sometimes referred to as “PEG”) and anionic segment polyaspartic acid (degree of polymerization of about 75) It was used.
- PEG-P polyethylene glycol (weight average molecular weight of about 2000)
- anionic segment polyaspartic acid degree of polymerization of about 75
- a cationic homopolymer Homo-P composed of poly (diaminopentane structure-containing asparagine derivative) (degree of polymerization of about 82) (hereinafter sometimes referred to as “P (Asp-AP)”). -AP was used.
- the first and second polymers were each dissolved in 10 mM phosphate buffer (pH 7.4) (aqueous medium) so that the polymer concentration was 1.0 mg / mL.
- the obtained empty vesicle-containing solution was measured by a dynamic light scattering method, and the particle size distribution, average particle size, and polydispersity index (PDI) were obtained. As a result, formation of monodisperse vesicle particles having an average particle diameter of 101.4 nm and a PDI of 0.070 was observed.
- FITC-Dex40k fluorescein isothiocyanate-dextran (manufactured by Sigma-Aldrich, hereinafter sometimes referred to as “FITC-Dex40k”) (weight average molecular weight 40,000) )It was used.
- a FITC-Dex40k solution concentration of FITC-Dex40k after mixing 5 to 50 mg / mL, charged polymer (first and second polymer) concentration 1.0 mg
- the mixture was stirred at 2000 rpm for 2 minutes using a vortex mixer, and FITC-Dex40k was enclosed in an empty crosslinked vesicle to obtain a FITC-Dex40k-encapsulated crosslinked vesicle-containing solution.
- the resulting solution was clear.
- Dex40k was encapsulated.
- the average particle diameter of FITC-Dex40k-containing crosslinked vesicles obtained by stirring and mixing with a 50 mg / mL FITC-Dex40k solution is 97.2 nm and PDI is 0.087 (the average particle diameter of empty vesicles is 101.10). 4 nm, PDI was 0.070), and a transmission electron microscope (TEM) image was as shown in FIG. 5A.
- FCS fluorescence correlation spectroscopy
- the slower the fluctuation that is, the slower the movement of the fluorescent object of the measurement reference.
- the fluctuation decreases as the number of molecules included in the observation field increases, the number of molecules can be estimated from the value of the correlation function.
- Table 2 shows the number of FITC-Dex40k per FITC-Dex40k-containing vesicle. As shown in Table 2, the number of FITC-Dex40k per FITC-Dex40k-encapsulating vesicle increased as the concentration of the FITC-Dex40k solution mixed with the empty cross-linked vesicle increased.
- Example I-1 Comparative Examples I-1 and I-2 (described later), even if the concentration of FITC-Dex40k present in the aqueous medium exceeds 5 mg / mL, FITC-Dex40k-encapsulated vesicles It was found that only Example I-1 was able to form a monodisperse assembly.
- Example I-1 FITC-Dex 40k enclosed by the simultaneous mixing method
- the first and second polymers were each 10 mM phosphoric acid so that the polymer concentration was 2.0 mg / mL. It was dissolved in a buffer solution (pH 7.4) (aqueous medium).
- the FITC-Dex40k final concentration after mixing with the second polymer solution is 5 to 50 mg / mL, and the charged polymer (first and second polymers) final concentration is included in the first polymer solution.
- the predetermined FITC-Dex40k was added so that the amount of the solution was 1.0 mg / mL.
- the self-assembled aggregate obtained when the final concentration of FITC-Dex 40k was 50 mg / mL was a unilamellar vesicle, but the particle size tended to increase remarkably.
- DLS results also showed an increase in size, loss of unimodality, and a significant increase in PDI (average particle size: about 630 nm, PDI: about 0.62). This is probably because the vesicle formation process (particularly stirring and mixing conditions) was affected by the solution viscosity, and thus gave a wide particle size distribution.
- Example I-2 FITC-Dex 40k Encapsulated by Post-Support Method
- the FITC-Dex 40k final concentration after mixing is 5 to 50 mg / mL, and the charged FITC (first and second polymer) concentration is 1.0 mg / mL.
- -Dex40k solution was added and again stirred for 2 minutes at 2000 rpm using a vortex mixer.
- a solution containing 10 equivalents of EDC with respect to the carboxyl group contained in PEG-P (Asp) was added, and the mixture was allowed to stand at room temperature for 12 hours. did.
- Dextran remaining in the solution in a free state was removed by centrifugal ultrafiltration in the same manner as in Example I-1, and the resulting self-assembled aggregate was evaluated by the DLS method and TEM.
- the self-assembled aggregate obtained when the final concentration of FITC-Dex40k was 50 mg / mL was a unilamellar vesicle, but the particle size tended to increase remarkably. DLS results also showed an increase in size, loss of unimodality, and a significant increase in PDI (average particle size: about 560 nm, PDI: about 0.57).
- the self-assembled aggregate obtained when the final concentration of FITC-Dex40k was 5 mg / mL monodispersed vesicle particles having a particle size of about 100 nm were formed, and the amount of encapsulation could be estimated by FCS ( Table 2).
- Example I-1 FITC-Dex40k release from FITC-Dex40k-encapsulating vesicles
- a predetermined FITC-Dex40k is added so that the final concentration of FITC-Dex40k after mixing is 4 mg / mL and the charged polymer (first and second polymer) concentration is 1.0 mg / mL.
- the mixture was again stirred with a vortex mixer at 2000 rpm for 2 minutes.
- a solution containing 10 equivalents of EDC in a 10 mM phosphate buffer (pH 7.4 NaCl) at pH 7.4 was added and allowed to stand at room temperature for 12 hours.
- FITC-Dex40k The release behavior of FITC-Dex40k enclosed in the vesicle was confirmed using GPC (gel permeation chromatography, manufactured by JASCO). As shown in FIG. 6, there was no significant difference in the chromatogram immediately after purification and after 3 days, and the release of FITC-Dex40k was not confirmed. From this result, it is presumed that FITC-Dex 40k does not enter and exit the vesicle when no external force is applied by mixing or the like.
- a rotary rheometer (Physica MCR302 manufactured by Anton Paar) was used for the measurement of the viscosity.
- Table 3 shows the measurement results of the viscosity (Pa ⁇ s).
- Example I-2 Encapsulation of FITC-Dex4k in cytochrome c-encapsulated vesicle (1) Preparation of cytochrome c-encapsulated vesicle Cytochrome c (Sigma-Aldrich) Manufactured, with a molecular weight of 12,327 Da). The specific method is as follows.
- the first and second polymers were each dissolved in 10 mM phosphate buffer (pH 7.4) (aqueous medium) so that the polymer concentration was 2.0 mg / mL.
- Containing vesicles empty vesicles formed by self-assembly of the first and second polymers by mixing in an Eppendorf tube and stirring for 2 minutes at 2000 rpm using a vortex mixer A solution was obtained. From the result of DLS, the average particle diameter was 102 nm, and PDI was 0.045.
- cytochrome c-encapsulated vesicle-containing solution To 0.96 mL of the obtained empty vesicle-containing solution, 0.5 mL of a 1 mg / mL cytochrome c solution was added and stirred again at 2000 rpm for 2 minutes using a vortex mixer to obtain a cytochrome c-encapsulated vesicle-containing solution.
- Cytochrome c remaining free in the solution without being encapsulated in the vesicle was removed by centrifugal ultrafiltration (fractionated molecular weight 300,000) in the same manner as in Example I-1, and the vesicle was purified. At this time, the average particle size was 99.5 nm and PDI was 0.032. From the GPC measurement, it was confirmed that cytochrome c (absorption maximum wavelength 409 nm) was enclosed in the vesicle (FIG. 7).
- FIG. 7 shows the GPC measurement results of empty vesicles
- (b) shows the GPC measurement results of cytochrome c-encapsulated crosslinked vesicles after purification.
- FITC-Dex4k that was not encapsulated in vesicles and remained in the solution in a free state was removed by centrifugal ultrafiltration in the same manner as in Example I-1. From the results of DLS measurement, the average particle size was 101 nm and PDI was 0.078. From the absorption spectrum of the obtained vesicle, when the cytochrome c and FITC-Dex4k are encapsulated in the vesicle, that is, when FITC-Dex4k is encapsulated in the cytochrome c-encapsulated crosslinked vesicle, the FITC-Dex4k is not released. Was confirmed to be enclosed (FIG. 8). In FIG.
- cytochrome c and FITC-Dex4k-encapsulated vesicles were prepared in the same manner as described above, and FCS measurement was performed in the same manner as in Example I-1.
- the number of FITC-Dex4k per one was estimated.
- Table 4 As shown in Table 4, the number of FITC-Dex4k per vesicle increased with increasing concentration of the FITC-Dex4k solution.
- 0 indicates the circular dichroism spectrum of cytochrome c-encapsulated crosslinked vesicles at pH 2.0
- (C) (CytC + FITC-Dex) @PICsome (pH 7.4)” indicates cytochrome c and pH 7.4
- the circular dichroism spectrum of FITC-Dex4k inclusion cross-linking vesicles is shown
- “(D) (CytC + FITC-Dex) @PICsome (pH 7.4)” is a circle of cytochrome c and FITC-Dex4k inclusion cross-linking vesicles at pH 7.4.
- the dichroic spectrum is shown.
- Example I-3 Encapsulation of AlPcS2a in empty crosslinked vesicles
- 1.0 equivalent of EDC with respect to the carboxyl group contained in PEG-P Asp
- the average particle diameter was 101 nm
- PDI was 0.084.
- Dispersions of sulfonated phthalocyanine (AlPcS2a) (Frontier Scientific) were prepared at various concentrations (0, 1, 2, 10, 20, 50 mg / mL), and empty crosslinked vesicles were prepared with a vesicle concentration of 1 mg / mL. And mixing with ultrasonic waves for 2 minutes. The vesicle concentration was quantified by fluorescent labeling Cy3.
- AlPcS2a is a drug (photosensitizer) used in photodynamic therapy and has the following structure.
- the purified vesicles had an average particle size of about 110 nm and a PDI of about 0.05 to 0.08, maintaining both particle size and monodispersity, regardless of the AlPcS2a concentration. Further, a colorimetric test based on the absorption color of AlPcS2a was performed on the purified vesicles. As shown in FIG. 10A, it was found that the blue color derived from AlPcS2a became stronger as the concentration of AlPcS2a to be contacted increased.
- a predetermined AlPcS2a solution is added to 0.5 mL of the obtained solution so that the AlPcS2a concentration after mixing is 2.0 mg / mL and the charged polymer concentration is 1.0 mg / mL, and again at 2000 rpm for 2 minutes using a vortex mixer. Stir. Subsequently, a solution containing 10 equivalents of EDC with respect to the carboxyl group contained in PEG-P (Asp) (in 10 mM phosphate buffer (0 mM NaCl) at pH 7.4) was added, and the mixture was allowed to stand at room temperature for 12 hours. did.
- Example I-1 AlPcS2a that was not encapsulated in vesicles and remained free in the solution was removed by centrifugal ultrafiltration in the same manner as in Example I-1. From the DLS measurement, the average particle size was 104 nm and the PDI was 0.062. Compared with the result of Example I-3, the amount of encapsulated AlPcS2a is compared with the result of Example I-3 by the colorimetric test shown in FIG. 10 (A). It was found that a large amount was enclosed. Moreover, when the AlPcS2a concentration after mixing was larger than 2.0 mg / mL, it was difficult to maintain the particle size and PDI.
- Example I-3 and Comparative Example I-3 both AlPcS2a concentration 20 mg / mL were diluted to a charged polymer concentration of 0.5 mg / mL, and the absorption spectrum was measured with a spectrophotometer. The result is shown in FIG. It was also found that Example I-3 contained a larger amount than that of Comparative Example I-3 even when the same concentration of AlPcS2a was contacted.
- the obtained empty vesicle-containing solution was measured by a dynamic light scattering method, and the particle size distribution, average particle size, polydispersity index (PDI), and zeta potential were determined.
- the empty vesicles having an average particle size of 126 nm before addition of NaCl and PDI of 0.049 showed the values shown in Table 5 after addition of NaCl.
- the PDI showed a large value of about 0.2
- the TEM image showed the formation of polydisperse vesicles containing many components having a particle diameter exceeding 200 nm in diameter (FIG. 11).
- the empty vesicle solution immediately became cloudy, suggesting an increase in size and polydispersity.
- Anionic block copolymer PEG-P (Asp) consisting of about 79) was used.
- a cationic homopolymer Homo-P (Asp) composed of poly (diaminopentane structure-containing asparagine derivative) (degree of polymerization of about 71) hereinafter sometimes referred to as “P (Asp-AP)”.
- P (Asp-AP) a cationic homopolymer Homo-P (Asp) composed of poly (diaminopentane structure-containing asparagine derivative) (degree of polymerization of about 71)
- Each of the first and second polymers is prepared in three types, 10 mM phosphate buffer (pH 7.4; no NaCl, containing 10,20 mM NaCl) so that the polymer concentration is 1.0 mg / mL; aqueous Medium).
- the mixture was stirred for 2 minutes at 2000 rpm using a vortex mixer, and particles were obtained by self-assembly of the first and second polymers.
- the particle size and its distribution were evaluated by dynamic light scattering, the result of the same tendency as the previous item was obtained (Table 6).
- PDI was given 0.2 and polydispersity was increased. It has been suggested.
- a cationic homopolymer Homo-P comprising poly (diaminopentane structure-containing asparagine derivative) (degree of polymerization of about 82) (hereinafter sometimes referred to as “P (Asp-AP)”).
- P (Asp-AP) degree of polymerization of about 82
- Total reflection infrared spectroscopy (manufactured by JASCO Corporation, FTIR-6300) was performed using 100 ⁇ L of an empty crosslinked vesicle-containing solution (solvent D 2 O) of 10 mg / mL.
- solvent D 2 O solvent used for total reflection infrared spectroscopy.
- the peak at 1550-1600 cm ⁇ 1 obtained by total reflection infrared spectroscopy was separated using analysis software (IGOR Pro), and the crosslinking rate was calculated by quantifying the decrease in area corresponding to COO ⁇ . (Table 7).
- Example I-6 Ultrasonic Treatment of Empty Non-Bridged Vesicles An empty vesicle prepared in the same manner as in Example I-1 was subjected to ultrasonic treatment (2 minutes). The particle size distribution, average particle size, and polydispersity index (PDI) were measured by a scattering method. As a result, when sonication was performed in an ice bath, the average particle size was 112.8 nm, PDI was 0.098, and no change was observed in the vesicle solution, but sonication was performed in an ice bath. When not carried out, the average particle size was 3606 nm, the PDI was 0.301, and the vesicle solution became cloudy.
- PDI polydispersity index
- the sonication of the vesicle should be performed so that the vesicle is not heated excessively (for example, when the sonication is performed discontinuously, or when the sonication is performed continuously, the vesicle is not used. Cooling) is considered desirable.
- CD Cytosine deaminase ammonium sulfate suspension, manufactured by CALZYME
- 5-FC 5-fluorocytosine
- 5-FU 5-fluorouracil
- An empty crosslinked vesicle was prepared by adding 1.0 equivalent of EDC to a carboxyl group contained in PEG-P (Asp) to an empty vesicle prepared in the same manner as in Example I-1.
- EDC electrospless polymer
- an empty crosslinked vesicle prepared in the same manner as in Example I-1.
- 500 ⁇ L of a dispersion of about 3 mg / mL (converted to PEG-P (Asp)) of an empty crosslinked vesicle and 500 ⁇ L of a dispersion of 1 mg of cytosine deaminase (CD), the mixture is stirred for 2 minutes with a vortex mixer (2000 rpm).
- CD was supported in cross-linked vesicles.
- the air-crosslinked vesicle and the CD-encapsulated crosslinked vesicle were measured by a dynamic light scattering (DLS) method, and an average particle diameter and a polydispersity index (PDI) were obtained.
- the results are shown in Table 8 below.
- a transmission electron micrograph of the CD-encapsulated crosslinked vesicle is shown in FIG.
- mice BALB / c mice (6 weeks old) transplanted with mouse colon cancer cell line C26 were prepared in 8 groups of 5 mice (groups 1 to 8), and the CD-encapsulated cross-linked vesicle (0 .5 U / mL), 5-FC, 5-FU, or PBS, 200 ⁇ L of each was intravenously administered at the timing shown in Table 9 below.
- 5-FC and 5-FU were adjusted to 10 mg / kg (body weight) or 80 mg / kg (body weight) with respect to the mouse body weight.
- the tumor volume was measured and the body weight of each mouse was measured over the maximum 28 days during the survival period of each mouse.
- the tumor volume was calculated by the following calculation formula by measuring the major axis and minor axis of the tumor using calipers.
- V (a 2 ⁇ b) / 2
- V tumor volume
- a major axis
- b minor axis
- the measurement result of the tumor volume is shown in FIG.
- the CD-encapsulated cross-linked vesicle and 5-FC administration group (Group 1: 10 mg / kg; Group 2: 80 mg / kg) were all significant, regardless of the 5-FC dose, compared to the untreated group (Group 8). An antitumor effect was observed (FIG. 15 (a)).
- CD-encapsulated cross-linked vesicle and 5-FC administration group (Group 1: 10 mg / kg; Group 2: 80 mg / kg) and 5-FU administration group (Group 3: 10 mg / kg; Group 4: 80 mg / kg)
- the 5-FU administration group (Group 3) showed a very weak antitumor effect
- the CD-encapsulated cross-linked vesicle and 5-FC administration group (Group 1) were significantly different.
- An excellent antitumor effect was observed (FIG. 15 (b)).
- the 5-FU administration group (Group 4) showed an antitumor effect that reduced the tumor, but due to its side effects, the total number died 17 days after the first administration, whereas CD inclusion In the cross-linked vesicle and 5-FC-administered group (Group 2), all the animals were alive even after 28 days of administration, without causing side effects while exhibiting an antitumor effect (FIG. 15 (c)). .
- the CD-encapsulated cross-linked vesicle single group administration group (Group 5) and the 5-FC single group administration group (Group 6: 10 mg / kg administration group; Group 7: 80 mg / kg administration group) compared with the non-treatment group (Group 8). Significant antitumor effects and toxicity were not observed (FIG. 15 (d)).
- FIG. 16 shows the measurement results of weight change.
- group 1 10 mg / kg
- group 2 80 mg / kg
- the relative body weight did not fall below 0.8 even 28 days after administration (FIG. 16 (a)).
- All other groups fell below 0.8 or died (FIGS. 16 (b) to (d)).
- Fig. 17 shows the results of counting the number of survivors.
- group 1 10 mg / kg
- group 2 80 mg / kg
- all animals remained alive even after 28 days of administration except for one in group 2 (80 mg / kg).
- 5-FU 10 mg / kg administration group Group 3
- 3 animals died by 28 days after administration (FIG. 17 (b) to ( d)).
- Adsorbent-encapsulating vesicle and its production method [Example II-1] MSN-encapsulating vesicle 1 ⁇ material: As the first polymer, an anionic block comprising polyethylene glycol (PEG; molecular weight of about 2000) as an uncharged hydrophilic segment and polyaspartic acid (P (Asp); degree of polymerization of about 75) as an anionic segment A copolymer (PEG-P (Asp); zeta potential-30.6 mV) was used.
- PEG polyethylene glycol
- a cationic homopolymer (Homo-P (Asp-AP)) comprising a cationic segment poly (diaminopentane structure-containing asparagine derivative) (P (Asp-AP); degree of polymerization of about 82); Zeta potential + 16.3 mV) was used.
- mesoporous silica nanoparticles produced according to the method described in Kim et al., Angew. Chem. Int. Ed. 2008, 47, 8438-8441 were used. According to this document, the total pore volume of the obtained mesoporous silica nanoparticles is 1.07 cm 3 / g. The average particle size was about 50 nm and the zeta potential was ⁇ 37 mV.
- the above first and second polymers were each dissolved in 10 mM phosphate buffer (pH 7.4) (aqueous medium) to prepare solutions having a concentration of each polymer of 1.0 mg / mL. Further, the above MSN was dispersed in 10 mM phosphate buffer (pH 7.4) (aqueous medium) by ultrasonic irradiation to prepare a dispersion having an MSN concentration of 1.0 mg / mL. In an Eppendorf tube, the MSN dispersion is added to the second polymer solution, and the MSN concentration becomes 20 w / w% of the total polymer concentration (total concentration of the first and second polymers).
- the obtained MSN-encapsulated vesicle-containing solution was subjected to DLS measurement to obtain a particle size distribution, an average particle size, and a PDI. Formation of monodisperse particles with an average particle diameter of 111 nm was observed.
- the PDI was 0.092.
- the obtained crosslinked MSN-encapsulated vesicle-containing solution was subjected to DLS measurement to obtain a particle size distribution, an average particle size, and a PDI.
- a particle size distribution graph is shown in FIG. Formation of monodisperse particles with an average particle diameter of 102 nm was observed.
- the PDI was 0.077 and the zeta potential was -17 mV.
- the above-mentioned crosslinked MSN-encapsulating vesicle-containing solution was also observed with a transmission electron microscope (TEM, JEOL JEM-1400, the same applies hereinafter) to confirm the validity of the DLS measurement results.
- the obtained TEM photograph is shown in FIG.
- the particle size of MSN-encapsulating vesicles (particles having high contrast in the figure) was 60 to 70 nm, and the particle size of empty vesicles (vesicles not including MSN) formed at the same time was about 100 nm. MSN not encapsulated in vesicles was not observed.
- a fluorescence detector was used as the detector, a capillary having an inner diameter of 75 ⁇ m (TSP075375 manufactured by polymicro Technology) was used, and a 200 mM glycine-sodium hydroxide buffer (pH 8.0) was used as the electrophoresis solution.
- MSN-encapsulated vesicles and MSN not encapsulated in vesicles are separated by capillary electrophoresis according to the difference in surface charge.
- the obtained chromatogram is shown in FIG. According to this result, MSN included in the vesicle was 85%.
- Cy5-labeled MSN sample a solution containing MSN adsorbed with the fluorescent dye Cy5 (the ratio of Cy5 to MSN is 0.4%).
- Cy5-labeled MSN-encapsulated vesicle sample MSN-encapsulated vesicle-containing solution prepared by the same procedure as described above except that Cy5-labeled MSN used in (i) above was used instead of MSN.
- Example II-2 MSN-containing vesicle 2
- the MSV-containing vesicle (MSN-encapsulating vesicle: adsorbent-encapsulating vesicle) was obtained by following the same procedure as in Example II-1, except that the resulting mixture was mixed with the second polymer solution. ) was obtained.
- the obtained crosslinked MSN-encapsulated vesicle-containing solution was subjected to DLS measurement to obtain a particle size distribution, an average particle size, and a PDI.
- a particle size distribution graph is shown in FIG. Formation of particles having an average particle diameter of 101 nm was observed.
- the PDI was 0.152.
- the above-mentioned cross-linked MSN-encapsulated vesicle-containing solution was also observed with a TEM to confirm the validity of the DLS measurement results.
- the obtained TEM photograph is shown in FIG.
- the particle size of MSN-encapsulating vesicles particles having high contrast in the figure
- the particle size of empty vesicles vesicles not including MSN
- a slight amount of MSN not encapsulated in vesicles was observed.
- Example II-1 Further, in order to examine the MSN inclusion rate, measurement by capillary electrophoresis was performed in the same procedure as in Example II-1. The obtained chromatogram is shown in FIG. According to this result, the MSN contained in the vesicle was 72%.
- the obtained MSN-encapsulated vesicle-containing solution was subjected to DLS measurement to obtain a particle size distribution, an average particle size, and a PDI. Formation of monodisperse particles with an average particle size of 104 nm was observed.
- the PDI was 0.049.
- the obtained crosslinked MSN-encapsulated vesicle-containing solution was subjected to DLS measurement to obtain a particle size distribution, an average particle size, and a PDI.
- a graph of the particle size distribution is shown in FIG. Formation of particles having an average particle diameter of 100 nm was observed.
- the PDI was 0.057.
- the above-mentioned cross-linked MSN-encapsulated vesicle-containing solution was also observed with a TEM to confirm the validity of the DLS measurement results.
- the obtained TEM photograph is shown in FIG.
- the particle size of MSN-encapsulating vesicles particles having high contrast in the figure
- the particle size of empty vesicles vesicles not including MSN
- some MSN not encapsulated in vesicles was observed.
- MSN included in the vesicle was 21%.
- Example II-3A Preparation of aminated MSN-encapsulated vesicles / aminated MSN-encapsulated vesicles: 50 ⁇ L of (3-aminopropyl) trimethoxysilane (APTS) was dissolved with 4 mL of distilled water at room temperature for 1 hour. Subsequently, 1 mL of a 10 mg / mL dispersion of MSN-encapsulated vesicles (adsorbent-encapsulated vesicles) obtained by the same procedure as in Example II-1 above was added, and the mixture was further stirred for 24 hours. Surface treatment with APTS was performed. Then, it was purified by ultrafiltration (fractionated molecular weight 300,000, 3 times with 20% ethanol, and then 5 times with 10 mM PBS (pH 7.4)) to obtain aminated MSN-encapsulated vesicles.
- APTS (3-aminopropyl) trimethoxysilane
- Example II-3B Preparation of mercapto- and sulfonylated MSN-encapsulating vesicles-mercapto- and sulfonylated MSN-encapsulating vesicles: 50 ⁇ L of (3-Mercaptopropyl) trimethoxysilane (MPTS) was dissolved with 4 mL of 1% aqueous acetic acid solution at room temperature for 1 hour.
- MPTS (3-Mercaptopropyl) trimethoxysilane
- MSN-encapsulated vesicles (adsorbent-encapsulated vesicles) obtained by the same procedure as in Example II-1 above was added, and the mixture was further stirred for 24 hours to encapsulate.
- MSN was surface treated with MPTS. Then, it refine
- Example II-4A Preparation of Rose Bengal Adsorbed Aminated MSN Encapsulated Vesicle / Rose Bengal Adsorbed Aminated MSN Encapsulated Vesicle: Rose bengal (hereinafter abbreviated as “RB” where appropriate) is added to the dispersion of the aminated MSN-encapsulating vesicle obtained in Example II-3A and mixed to adsorb the rose bengal to the aminated MSN-encapsulating vesicle. It was. Unadsorbed rose bengal was removed by ultrafiltration (fraction molecular weight 300,000). The RB content of the obtained RB-adsorbed aminated MSN-encapsulating vesicle was 3.2 w / w%. The chemical formula of Rose Bengal is shown below.
- Example II-4B Preparation of gemcitabine-adsorbed sulfonylated MSN-encapsulated vesicle / gemcitabine-adsorbed sulfonylated MSN-encapsulated vesicle: Gemcitabine (hereinafter abbreviated as “GEM” where appropriate) was added to the dispersion of the sulfonylated MSN-encapsulating vesicle obtained in Example II-3B and mixed to adsorb the gemcitabine to the sulfonylated MSN-encapsulating vesicle. Unadsorbed gemcitabine was removed by ultrafiltration (fractionated molecular weight 300,000).
- the GEM content of the obtained GEM-adsorbed sulfonylated MSN-encapsulating vesicles was 7.9 w / w% (sample for 10 mM PBS) and 8.1 w / w% (sample for simulated body fluid).
- Sample number 1 GEM-adsorbed sulfonylated MSN-encapsulated vesicle
- Sample number 2 Sulfonylated MSN-encapsulated vesicle
- Sample number 3 GEM-adsorbed sulfonylated MSN
- Sample number 4 sulfonylated MSN
- Sample number 5 Empty vesicle Sample number 6: GEM alone
- the empty vesicles (Example II-1) (Sample No. 5) were each labeled with Cy3 (25% of the number of anionic polymers was labeled), and A549 cells were 2.2 ⁇ 10 3 in RPMI-1640 medium. It added to each well of the plate which has the density of a cell / well, and it culture
- the amount of GEM-adsorbed sulfonylated MSN-encapsulated vesicles was such that gemcitabine had a concentration of 0.5 ⁇ g / mL in the medium, and sulfonylated MSN-encapsulated vesicles (sample number 2) and empty vesicles (sample number 5). ) was adjusted so as to be the same amount as the GEM-adsorbed sulfonylated MSN-encapsulated vesicle (sample number 1) in terms of MSN-encapsulated vesicles.
- sulfonylated MSN (sample No. 4) obtained by sulfonylating MSN used as a material for each MSN-encapsulating vesicle in the same procedure as Example II-3A (oxidized after mercaptolysis by MPTS surface treatment), and GEM-adsorbed sulfonylated MSN (GEM content 8.1 w / w%) (sample number 3) in which gemcitabine was adsorbed to MSN and gemcitabine alone (sample number 6) were added to each cell and cultured.
- GEM-adsorbed sulfonylated MSN (sample number 3) and gemcitabine alone (sample number 6) were added so that gemcitabine had a concentration of 0.5 ⁇ g / mL in the medium, and the amount of sulfonylated MSN (sample number 4) added was The amount was adjusted to be the same as that of GEM-adsorbed sulfonylated MSN (sample number 3) in terms of MSN.
- each MSN-encapsulated vesicle sample (sample numbers 1, 2, 5) at the time when 48 hours and 72 hours passed from the start of the culture was evaluated by the following procedure. That is, the cytoplasm of cultured cells was stained with calcein (Calcein, INVITROGEN, CA AM), and the cell nucleus was stained with Hoechst (INVITROGEN, Hoechst33342). 1000 (GE Healthcare Bioscience) was used and observed at an excitation wavelength of 550 nm. The obtained result is shown in FIG.
- sample numbers 1 to 6 the cell killing effect by each sample (sample numbers 1 to 6) at the time when 48 hours and 72 hours passed from the start of the culture was evaluated by the following procedure. That is, using Cell Counting Kit-8 (manufactured by Dojin Chemical Co., Ltd.), according to the manufacturer's instructions, the reagent was added to each well and further cultured for 1 hour, and then the number of viable cells was counted.
- FIG. 34 (a) The results after 48 hours of culture are shown in FIG. 34 (a), and the results after 72 hours of culture are shown in FIG. 34 (b).
- GEM-adsorbed sulfonylated MSN-encapsulated vesicles showed a strong cell killing effect similar to GEM-adsorbed sulfonylated MSN (sample number 3) and gemcitabine alone (sample number 6).
- the sulfonylated MSN (Sample No. 4) showed a weak cell-killing effect, but the sulfonylated MSN-encapsulated vesicle (Sample No. 2) and the empty vesicle (Sample No. 5) showed no cell-killing effect. .
- the addition amount of the sulfonylated MSN-encapsulating vesicle (sample number 2) and the empty vesicle (sample number 5) is the same amount as the GEM-adsorbed sulfonylated MSN-encapsulating vesicle (sample number 1) in terms of the MSN-encapsulating vesicle.
- the amount of sulfonylated MSN (Sample No. 4) added was adjusted so as to be the same as that of GEM-adsorbed sulfonylated MSN (Sample No. 3) in terms of MSN.
- FIG. 35 (a) shows the measurement result of Cy3 fluorescence in all cells in the absence of trypan blue
- FIG. 35 (b) shows the measurement result of Cy3 fluorescence in living cells under trypan blue staining.
- the amount of cell uptake was small in C26 cells compared to the case of A549 cells. Therefore, it is considered that the amount of cellular uptake varies depending on the cell type.
- the result after 48 hours of culture is shown in FIG. 36 (a), and the result after 72 hours of culture is shown in FIG. 36 (b).
- GEM-adsorbed sulfonylated MSN-encapsulated vesicles are as strong as GEM-adsorbed sulfonylated MSN (sample number 3) and gemcitabine alone (sample number 6).
- a cell killing effect was observed.
- the sulfonylated MSN (Sample No. 4) showed a weak cell-killing effect, but the sulfonylated MSN-encapsulated vesicle (Sample No. 2) and the empty vesicle (Sample No. 5) showed no cell-killing effect. .
- mice Seven groups of 5 BALB / c nude mice (7-week-old, each sample) transplanted with human lung cancer cell line A549 were prepared, of which 6 groups were intravenously administered with the above samples 1 to 6 (Group 1 to 6).
- each sample was 200 ⁇ L, and the concentration of each sample was as follows: GEM-adsorbed sulfonylated MSN-encapsulated vesicle (sample number 1), GEM-adsorbed sulfonylated MSN (sample number 3) and gemcitabine alone (sample number 6) Gemcitabine is adjusted to 5 mg / kg (body weight) with respect to body weight, and the addition amount of sulfonylated MSN-encapsulated vesicle (sample number 2) and empty vesicle (sample number 5) is GEM adsorption in terms of MSN-encapsulated vesicles.
- the amount of sulfonylated MSN-encapsulated vesicle (sample number 1) was adjusted to be the same amount, and the amount of sulfonylated MSN (sample number 4) added was the same amount as GEM-adsorbed sulfonylated MSN (sample number 3) in terms of MSN. It adjusted so that it might become.
- 200 ⁇ L of PBS was administered to the remaining one group (Group 7). Tumor volume was measured over 28 days after administration. The tumor volume was calculated by the following calculation formula by measuring the major axis and minor axis of the tumor using calipers.
- V (a 2 ⁇ b) / 2
- V tumor volume
- a major axis
- b minor axis
- the measurement result of the tumor volume is shown in the graph of FIG. PBS administration for sulfonylated MSN-encapsulated vesicle administration group (Group 2), GEM adsorption sulfonylated MSN administration group (Group 3), sulfonylated MSN administration group (Group 4), and empty vesicle administration group (Group 5)
- tumor growth was somewhat suppressed in the GEM alone administration group (Group 6)
- there was no significant difference from the PBS administration group (Group 7) there was no significant difference from the PBS administration group (Group 7), and no significant antitumor effect was observed.
- GEM-adsorbed sulfonylated MSN-encapsulated vesicle administration group Group 1
- PBS administration group Group 7
- tumor growth was significant compared to the GEM alone administration group (Group 6).
- a significant antitumor effect was observed. Therefore, according to the GEM-adsorbed sulfonylated MSN-encapsulating vesicle (sample 1) according to the present invention, GEM is effectively delivered to the tumor site, and has a high antitumor effect even at a low dose compared with a single GEM agent. It can be seen that it is possible to exhibit.
- mice Four groups of 3 BALB / c nude mice (7 weeks old, each sample) transplanted with human lung cancer cell line A549 were prepared. Two of these groups were administered intravenously with GEM-adsorbed sulfonylated MSN-encapsulated vesicles (sample No.
- the animals were sacrificed at 24 hours (groups A1 and B1) and 72 hours (groups A2 and B2) after administration, and GEM-adsorbed sulfonylated MSN-encapsulated vesicles and GEM were analyzed based on the amount of Cy5 fluorescence in the same manner as described above.
- the blood concentration of adsorbed sulfonylated MSN and the concentration in tumor were measured.
- the measurement result of the blood concentration of GEM adsorption sulfonylation MSN inclusion vesicle and GEM adsorption sulfonylation MSN is shown in the graph of FIG.
- groups A1 and A2 the blood concentration at 24 and 72 hours after administration was compared with the GEM-adsorbed sulfonylated MSN administration group (groups B1 and B2). Is much higher, and it is understood that the retention in blood is excellent.
- suction sulfonylation MSN inclusion vesicle and GEM adsorption sulfonylation MSN is shown in the graph of FIG.
- the concentration in the tumor at 24 hours and 72 hours after administration was compared with the GEM-adsorbed sulfonylated MSN administration group (groups B1 and B2). Is far higher, and it is understood that the tumor accumulation property is excellent.
- Example III Low-water-soluble substance-encapsulating vesicles and production method thereof
- Example III-1 Production and materials of indigo dye-encapsulating vesicles using ⁇ -galactosidase-encapsulating crosslinked vesicles:
- an anionic block comprising polyethylene glycol (PEG; molecular weight of about 2000) as an uncharged hydrophilic segment and polyaspartic acid (P (Asp); degree of polymerization of about 75) as an anionic segment A copolymer (PEG-P (Asp); zeta potential-30.6 mV) was used.
- a cationic homopolymer (Homo-P (Asp-AP)) comprising a cationic segment poly (diaminopentane structure-containing asparagine derivative) (P (Asp-AP); degree of polymerization of about 82); Zeta potential + 16.3 mV) was used.
- ⁇ -galactosidase was used as the encapsulating enzyme.
- Each of the first and second polymers was dissolved in 10 mM phosphate buffer (pH 7.4) (aqueous medium) to prepare a solution with each polymer concentration of 1.0 mg / mL. Further, ⁇ -galactosidase (enzyme) was dissolved in 10 mM phosphate buffer (pH 7.4, containing 150 mM sodium chloride) to a concentration of 1 mg / mL to prepare a solution.
- ⁇ -galactosidase solution having the same volume as that of the second polymer solution used was added, and the mixture was stirred with a vortex mixer at about 2000 rpm for 2 minutes.
- a solution containing ⁇ -galactosidase-encapsulating vesicle (enzyme-encapsulating vesicle) was obtained.
- the obtained ⁇ -galactosidase-encapsulating vesicle-containing solution was added to a solution containing 10 equivalents of EDC (manufactured by Dojindo Laboratories, WSC) with respect to the carboxyl group contained in PEG-P (Asp) and allowed to stand overnight. Then, crosslinking was performed by EDC reaction. By removing and purifying reaction by-products by centrifugal ultrafiltration (VIVASPIN-20, manufactured by sartorius-stedium-biotech, molecular weight cut off 300,000; 2000 rpm, 25 ° C.), containing cross-linked ⁇ -galactosidase-encapsulating vesicles A solution was obtained.
- VIVASPIN-20 manufactured by sartorius-stedium-biotech, molecular weight cut off 300,000; 2000 rpm, 25 ° C.
- the obtained ⁇ -galactosidase-containing cross-linked vesicle-containing solution was subjected to DLS measurement, and the particle size distribution, average particle size and PDI were determined.
- a particle size distribution graph is shown in FIG. Formation of monodisperse particles with an average particle size of 112 nm was observed.
- the PDI was 0.067.
- X-gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside: a water-soluble precursor) was added to a 10 mM phosphate buffer (pH 7.4) to a concentration of 5 mg / mL. , 150 mM sodium chloride solution) / dimethylformamide mixed solution (4: 1) to prepare a solution (note that X-gal is an indigo dye (5,5′-dibromo-) due to the enzymatic action of ⁇ -galactosidase. 4,4′-dichloro-indigo)).
- the mixture is filtered through a 0.45 ⁇ m PES filter and further purified by centrifugal ultrafiltration (VIVASPIN 20, manufactured by sartorius stedium biotech, using a molecular weight cut off of 300,000; 2000 rpm, 25 ° C.), and indigo dye-encapsulating vesicles
- VIVASPIN 20 manufactured by sartorius stedium biotech, using a molecular weight cut off of 300,000; 2000 rpm, 25 ° C.
- indigo dye-encapsulating vesicles A solution containing (the low-water-soluble substance-encapsulating vesicle of Example III-1) was obtained.
- the obtained indigo dye-containing vesicle-containing solution was subjected to DLS measurement, and the particle size distribution, average particle size, and PDI were obtained.
- a particle size distribution graph is shown in FIG. Monodispersed particles having an average particle size of 140 nm were observed.
- the PDI was 0.091.
- the indigo dye-containing vesicle-containing solution was observed for morphology by a transmission electron microscope (TEM).
- TEM transmission electron microscope
- FIG. 42 A TEM photograph obtained by TEM (JEM-1400 manufactured by JEOL Ltd.) is shown in FIG.
- the indigo dye low water-soluble substance
- X-gal water-soluble precursor
- ⁇ -galactosidase enzyme
- FIG. 43 A TEM photograph obtained by a high-resolution TEM (JEM-2100F manufactured by JEOL Ltd.) is shown in FIG.
- FIG. 43 diffraction lines considered to be derived from indigo dyes were observed. Therefore, it was confirmed that the indigo dye contained in the vesicle is crystalline.
- the indigo dye-encapsulating vesicles were allowed to stand overnight in a 90% dimethyl sulfoxide-containing aqueous solution, and the amount of indigo dye produced by absorptiometry was calculated. As a result, it was confirmed that 66.9% of indigo dye was included in the vesicle with respect to the weight of the total polymer (first and second polymers).
- Example III-1 12.5 ⁇ L of a 1-mg / mL ⁇ -galactosidase solution prepared in the same manner as in Example III-1 and 50 ⁇ L of the above-mentioned crosslinked empty vesicle-containing solution having a total polymer concentration of 0.5 mg / mL, and Example III-1 10 ⁇ L of a 1 mg / mL concentration X-gal solution prepared in the same manner as above was added, and 177.5 ⁇ L of 10 mM phosphate buffer was further added, and the mixture was allowed to stand at 37 ° C. for 24 hours.
- the mixture is filtered with a 0.45 ⁇ m PES filter and purified by centrifugal ultrafiltration (VIVASPIN-20, sartorius-stedium-biotech, molecular weight cut off 300,000; 2000 rpm, 25 ° C.), so that the empty after indigo dye production.
- VIVASPIN-20 centrifugal ultrafiltration
- sartorius-stedium-biotech molecular weight cut off 300,000; 2000 rpm, 25 ° C.
- the obtained vesicle-containing solution after the indigo dye was produced was subjected to DLS measurement, and the particle size distribution, average particle size and PDI were determined.
- a particle size distribution graph is shown in FIG. Particles having an average particle size of 104 nm were observed.
- the PDI was 0.131.
- the empty vesicle-containing solution after the indigo dye was produced was observed for morphology by a transmission electron microscope (TEM).
- FIG. 46 shows a TEM photograph obtained by TEM (JEM-1400 manufactured by JEOL). According to FIG. 46, although a low water-soluble indigo dye was produced by the reaction of ⁇ -galactosidase and X-gal, almost no indigo dye contained in the vesicle was observed.
- the empty vesicle after the production of the indigo dye was left overnight in a 90% dimethyl sulfoxide-containing aqueous solution, and the amount of the indigo dye produced by the absorptiometry was calculated. As a result, it was confirmed that 31.0% of the indigo dye was attached to the vesicle with respect to the weight of the total polymer (first and second polymers). Considering the results shown in FIG. 46, it is considered that the indigo dye adhering to the vesicle is not contained in the vesicle but mainly adhering to the outside of the empty vesicle.
- the substance-encapsulating vesicle obtained by the present invention is extremely useful in the fields of DDS for delivering drugs, biomaterials / functional materials, and the like.
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Abstract
Description
[1]非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体と、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体とを含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、
前記架橋膜によって包囲された内水相と
を含んでなり、前記内水相に目的物質が内包された物質内包架橋ベシクルの単分散集合体であって、
前記内水相に含有される前記目的物質の濃度が、
前記第1及び/又は前記第2の重合体が架橋されていない点及び前記目的物質が内包されていない点で前記物質内包架橋ベシクルの単分散集合体と異なる空の非架橋ベシクルの単分散集合体を、前記物質内包架橋ベシクルの内水相と同一濃度の前記目的物質を水性媒体とともに含有する混合液中で混合した場合に、
前記目的物質が内包された物質内包非架橋ベシクルの単分散集合体の形成を阻害する濃度である、前記物質内包架橋ベシクルの単分散集合体。
[2]0.2以下の多分散指数を有する、上記[1]に記載の物質内包架橋ベシクルの単分散集合体。
[3]前記目的物質の重量平均分子量が10000~40000であり、前記内水相に含有された前記目的物質の濃度が5mg/mLを上回る、上記[1]又は[2]に記載の物質内包架橋ベシクルの単分散集合体。
[4]前記第1及び/又は前記第2の重合体が、カチオン基間に形成された架橋結合、アニオン基間に形成された架橋結合、及びカチオン基とアニオン基との間に形成された架橋結合からなる群より選択された1種又は2種以上の架橋結合によって架橋されており、前記架橋結合が形成された割合が、前記架橋膜に含まれるカチオン基及び/又はアニオン基の総モル数の35%以上である、上記[3]に記載の物質内包架橋ベシクルの単分散集合体。
[5]非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体と、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体とを含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、前記架橋膜によって包囲された内水相とを含んでなり、前記内水相に第1の目的物質及び前記第1の目的物質よりも分子量の小さい第2の目的物質が内包された物質内包架橋ベシクルであって、
前記第1の目的物質が、前記第2の目的物質の不存在下で前記内水相に含有されている場合よりも安定化されている、前記物質内包架橋ベシクル。
[6]前記第2の目的物質がクラウディング剤である、上記[5]に記載の物質内包架橋ベシクル。
[7]物質内包ベシクルを製造する方法であって、
非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体と、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体とを含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、
前記架橋膜によって包囲された内水相と
を含んでなり、前記内水相に目的物質が内包されていない空の架橋ベシクルの単分散集合体を、
前記目的物質を水性媒体とともに含有する混合液中で混合し、
前記第1及び前記第2の重合体を含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、
前記架橋膜によって包囲された内水相と
を含んでなり、前記内水相に前記目的物質が内包された物質内包架橋ベシクルの単分散集合体を形成させる工程を含んでなる、前記方法。
[8]前記空の架橋ベシクルの単分散集合体、前記物質内包架橋ベシクルの単分散集合体、前記空の非架橋ベシクルの単分散集合体、及び前記物質内包非架橋ベシクルの単分散集合体が、0.2以下の多分散指数を有する、上記[7]に記載の方法。
[9]前記目的物質の重量平均分子量が10000~40000であり、前記混合液に含有される前記目的物質の濃度が5mg/mLを上回る、上記[7]又は[8]に記載の方法。
[10]前記空の架橋ベシクル及び前記物質内包架橋ベシクルにおいて、前記第1及び/又は前記第2の重合体が、カチオン基間に形成された架橋結合、アニオン基間に形成された架橋結合、及びカチオン基とアニオン基との間に形成された架橋結合からなる群より選択された1種又は2種以上の架橋結合によって架橋されており、前記架橋結合が形成された割合が、前記架橋膜に含まれるカチオン基及び/又はアニオン基の総モル数の35%以上である、上記[9]に記載の方法。
[11]前記物質内包架橋ベシクルの単分散集合体を、前記第1及び/又は前記第2の重合体と反応し得る架橋剤と反応させる工程をさらに含む、上記[7]~[10]のいずれか1項に記載の方法。
[12]物質内包ベシクルを製造する方法であって、
非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体と、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体とを含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、
前記架橋膜によって包囲された内水相と
を含んでなり、前記内水相に第1の目的物質が内包された第1の物質内包架橋ベシクルを、
前記第1の目的物質よりも分子量の小さい第2の目的物質を水性媒体とともに含有する混合液中で混合し、
前記第1及び前記第2の重合体を含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、
前記架橋膜によって包囲された内水相と
を含んでなり、前記内水相に前記第1及び前記第2の目的物質が内包された第2の物質内包架橋ベシクルを形成させる工程を含んでなる、前記方法。
[13]前記第1の物質内包架橋ベシクルが、その単分散集合体であり、前記第2の物質内包架橋ベシクルが、その単分散集合体である、上記[12]に記載の方法。
[14]前記混合液に含有される前記第2の目的物質の濃度が、
前記第1及び/又は前記第2の重合体が架橋されていない点で前記第1の物質内包架橋ベシクルの単分散集合体と異なる第1の物質内包非架橋ベシクルの単分散集合体を前記混合液中で混合した場合に、
前記第1及び前記第2の目的物質が内包された物質内包非架橋ベシクルの単分散集合体の形成を阻害する濃度である、上記[13]に記載の方法。
[15]前記第1及び前記第2の重合体を含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、
前記架橋膜によって包囲された内水相と
を含んでなり、前記内水相に前記第1及び前記第2の目的物質のいずれも内包されていない空の架橋ベシクルを、
前記第1の目的物質を水性媒体とともに含有する混合液中で混合し、必要に応じて、前記第1及び/又は前記第2の重合体と反応し得る架橋剤と反応させ、
前記第1の物質内包架橋ベシクルを形成させる工程をさらに含んでなる、上記[12]~[14]のいずれか1項に記載の方法。
[16]前記空の架橋ベシクルが、その単分散集合体であり、前記第1の物質内包架橋ベシクルが、その単分散集合体である、上記[15]に記載の方法。
[17]前記混合液に含有される前記第1の目的物質の濃度が、
前記第1及び/又は前記第2の重合体が架橋されていない点で前記空の架橋ベシクルの単分散集合体と異なる空の非架橋ベシクルの単分散集合体を前記混合液中で混合した場合に、
前記第1の目的物質が内包された物質内包非架橋ベシクルの単分散集合体の形成を阻害する濃度である、上記[16]に記載の方法。
[18]前記第2の目的物質がクラウディング剤である、上記[12]~[17]のいずれか1項に記載の方法。
[19]非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体、及び、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体を含む膜からなるベシクルと、当該ベシクルに内包された吸着材粒子とを含み、前記第1及び第2の重合体の少なくとも一方が前記吸着材粒子に吸着されてなる吸着材内包ベシクル。
[20]前記第1及び/又は第2の重合体が架橋されてなる、上記[19]に記載の吸着材内包ベシクル。
[21]前記吸着材粒子がシリカ粒子である、上記[19]又は[20]に記載の吸着材内包ベシクル。
[22]前記吸着材粒子の平均粒径が40nm~10μmである、上記[19]~[21]のいずれか1項に記載の吸着材内包ベシクル。
[23]前記吸着材粒子が表面処理されてなる、上記[19]~[22]のいずれか1項に記載の吸着材内包ベシクル。
[24]前記吸着材粒子に低分子化合物が吸着されてなる、上記[19]~[23]のいずれか1項に記載の吸着材内包ベシクル。
[25]非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体、及び、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体を含む膜からなるベシクルに、吸着材粒子が内包されてなる吸着材内包ベシクルを製造する方法であって、
(a)前記第1及び第2の重合体のうち一方を前記吸着材粒子と混合し、前記吸着材粒子に吸着させる工程、並びに、
(b)前記工程(a)の混合物を前記第1及び第2の重合体のうち他方と更に混合し、前記吸着材粒子の周囲に前記第1及び第2の重合体を含む膜からなるベシクルを形成させ、吸着材内包ベシクルとする工程
を含む方法。
[26](c)前記工程(b)のベシクル中の前記第1及び/又は前記第2の重合体を架橋する工程を更に含む、上記[25]に記載の方法。
[27]前記吸着材粒子がシリカ粒子である、上記[25]又は[26]に記載の方法。
[28]前記吸着材粒子の平均粒径が40nm~10μmである、上記[25]~[27]のいずれか1項に記載の方法。
[29]前記吸着材粒子を表面処理する工程を更に含む、上記[25]~[28]のいずれか1項に記載の方法。
[30]前記吸着材粒子に低分子化合物が吸着されてなる、上記[25]~[29]のいずれか1項に記載の方法。
[31]非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体、及び、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体を含む膜からなるベシクルに、目的物質が内包されてなる物質内包ベシクルを製造する方法であって、
(a)前記目的物質よりも水溶性の高い前駆体を前記目的物質に転換し得る酵素が、前記第1及び第2の重合体を含む膜からなるベシクルに内包されてなる酵素内包ベシクルを用意する工程、及び、
(b)前記前駆体よりも前記目的物質に対して低い溶解性を示す条件下で、前記酵素内包ベシクル内に前記前駆体を浸透させ、前記酵素によって前記前駆体を前記目的物質に転換することにより、前記目的物質を析出させて前記酵素内包ベシクルに内包させ、物質内包ベシクルとする工程
を含む方法。
[32]前記工程(b)において、前記酵素内包ベシクルを前記前駆体の水溶液と混合することにより、前記酵素内包ベシクル内に前記前駆体を浸透させる上記[31]に記載の方法。
[33]前記工程(b)の前に、前記酵素内包ベシクルの前記第1及び/又は前記第2の重合体を架橋する工程を更に含む、上記[32]に記載の方法。
[34]上記[31]~[33]のいずれか1項に記載の方法により製造される物質内包ベシクル。
[35]非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体、及び、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体を含む膜からなるベシクルに、前記目的物質よりも水溶性の高い前駆体から転換され得る低水溶性物質が、前記前駆体を前記低水溶性物質に転換し得る酵素と共に内包されてなる、低水溶性物質内包ベシクル。
[36]前記低水溶性物質が、内水相に対する前記低水溶性物質の溶解度を超える濃度で内包されてなる、上記[34]又は[35]に記載の低水溶性物質内包ベシクル。
[37]前記第1及び/又は前記第2の重合体が架橋されてなる、上記[34]~[36]のいずれか1項に記載の低水溶性物質内包ベシクル。
[38]上記[1]~[4]のいずれか1項に記載のベシクルの単分散集合体、及び/又は、上記[5]、[6]、[19]~[22]及び[34]~[37]のいずれか1項に記載のベシクルを含む薬物送達系。
[39]対象に薬物を送達するための方法であって、
(a)非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体、及び、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体を含む膜からなるベシクルに、前記薬物の前駆体を前記薬物に転換し得る酵素が内包されてなる酵素内包ベシクルを用意する工程、及び、
(b)対象の所定の部位で、前記酵素内包ベシクル内に前記前駆体を浸透させ、前記酵素によって前記前駆体を前記薬物に転換することにより、前記薬物を形成する工程
を含む方法。
[40]前記前駆体の水溶性がが前記薬物よりも低いとともに、前記工程(b)において、前記前駆体よりも前記薬物に対して低い溶解性を示す条件下で、前記酵素内包ベシクル内に前記前駆体を浸透させる、上記[39]に記載の方法。
[41]前記工程(b)において、前記薬物を析出させて前記酵素内包ベシクルに内包させ、薬物内包ベシクルを形成することを更に含む、上記[39]又は[40]に記載の方法。
本明細書において「ベシクル」とは、単ラメラ構造の膜と、前記膜により包囲された空隙部(内水相)とを有する基本構造体を意味する。
まず、本発明の基礎となる物質内包ベシクル及びその製法について説明する。本発明の各ベシクル及びその製法の特徴については、章を改めて[C]~[E]において説明するが、これらの章に別途記載なき事項については、本章の記載が適用されることとする。
上述の通り、物質内包ベシクルの主な製法としては、(i)被内包物質を、膜の構成要素となる高分子、或いは予め形成された高分子膜とともに混合し、自己組織化によるベシクルの形成と空隙部への物質の封入とを同時に行う方法(同時混合法)と、(ii)予め形成された空ベシクルを被内包物質と混合し、空ベシクルの空隙部に被内包物質を導入し、包含・担持させる方法(後担持法)とが挙げられる。
後担持法の概要について、以下、図1を用いて説明する。なお、図1は模式図であり、本発明は図1に限定されるものではない。
図1(a)に示すように、膜1aで包囲された空隙部1bを有する所定の構造の空ベシクル1を用意し、これを被内包物質9とともに、水性媒体中で混合する。これにより、図1(b)に示すように、被内包物質9が空ベシクル1の膜1aを越えて空隙部1bに導入され、被内包物質9が空隙部1bに内包された物質内包ベシクル1’が得られる。
以下、後担持法について詳述するが、空ベシクル及び被内包物質の詳細については章を改めて後述し、ここではその他の条件及び手順について説明する。
後担持法は、空ベシクル及び被内包物質を水性媒体中で混合する工程を含む(なお、ここで混合の対象となる、空ベシクル及び被内包物質を水性媒体中に含有する液を、以降「混合対象液」という場合がある。)。
混合を行う手法は特に制限されないが、水性媒体に外力を加える手法で行う。すなわち、空ベシクル及び被内包物質を水性媒体に加えて静置し、自然に拡散させて混合する手法(以降「静置・拡散混合」という場合がある。)は除外される。水性媒体に外力を加える混合法の例としては、撹拌、振盪、衝撃等が挙げられる。
振盪による手法の例としては、混合対象液を含む容器を振盪機等により振盪する手法が挙げられる。
衝撃による手法の例としては、混合対象液に対して超音波照射等により振動を含む各種の衝撃を与える手法等が挙げられる。
混合により物質内包ベシクルが形成される理由は定かではないが、水性媒体に外力を加えることによって、空ベシクルに剪断応力が作用する(よって、水性媒体に外力を加える混合を、剪断応力下での混合と言い換えることができる。)。斯かる剪断応力によって、空ベシクルの構造が撹乱されて概ね均一な小会合体に分解し、それが再度自己組織化してベシクルが均一に再生するとともに、水性媒体中に存在する被内包物質がベシクル再生時にベシクル内に封入されるものと考えられる(斯かるメカニズムは、後述する参考実験において、混合によるベシクルの小会合体への分解が確認されたことからも推測される。)。これは通常の状態のベシクルでは起こり難い現象であり、斯かる現象を利用した後担持法は、極めて斬新なものであるといえる。
また、ボルテックスミキサーによる撹拌時間は、回転数によっても異なるが、通常60秒以上、好ましくは120秒以上、また、通常10分以内、好ましくは5分以内である。攪拌時間が短過ぎると、均質な物質内包ベシクルが形成され難くなる場合がある。攪拌時間が長過ぎると、ベシクルや被内包物質が損傷・破壊されてしまう場合がある。
その他の混合法(撹拌翼による撹拌、振盪機による振盪、超音波照射による衝撃等)を用いる場合の具体的な条件としては、ボルテックスミキサーによる撹拌を上述した回転数及び攪拌時間のもとで行って得られるのと同程度の力が混合対象液に作用するように、適宜条件を調整すればよい。
通常は、水性媒体中に空ベシクル及び被内包物質を含有する液(混合対象液)を調製し、前述の混合に供する。
水性媒体(水性溶媒)の種類は限定されない。好ましくは水であるが、空ベシクルの構造に好ましからぬ影響を及ぼしたり、被内包物質の内部への導入を妨げたりしない範囲で、水に他の成分を混合した溶媒(例えば生理食塩水、水性緩衝液、水と水溶性有機溶媒との混合溶媒等)も用いることができる。水性緩衝液としては10mM HEPES緩衝液等が挙げられる。水溶性有機溶媒としては、メタノールやエタノール等のアルコール類、アセトン等のケトン類、クロロホルム等の塩素系有機溶媒、ジメチルエーテル等のエーテル系有機溶媒、酢酸エチル等のエステル系有機溶媒等が挙げられる。
但し、空ベシクルへの被内包物質の内包効率を高める観点からは、水性媒体に対する空ベシクルの濃度を、通常0.1mg/mL以上、中でも1mg/mL以上、また、通常100mg/mL以下、中でも10mg/mL以下とすることが好ましい。特に空ベシクルの濃度が低過ぎると、物質内包ベシクルが形成されない場合がある。なお、得られる物質内包ベシクルの粒経は空ベシクルの濃度に依存すると考えられるため、空ベシクルの濃度は所望の物質内包ベシクルの粒経に応じて決定すべきである。
また、水性媒体に対する被内包物質の濃度は、被内包物質の性質によっても異なるが、通常0.1mg/mL以上、中でも1mg/mL以上、また、通常100mg/mL以下、中でも50mg/mL以下とすることが好ましい。特に被内包物質の濃度が低過ぎると、物質内包ベシクルが形成されない場合がある。
被混合液の混合時の温度は、空ベシクルの構造を破壊したり、被内包物質の空ベシクルへの内包を阻害したりしない範囲であれば限定されないが、好ましくは10℃以上、より好ましくは20℃以上、また、好ましくは80℃以下、より好ましくは50℃以下である。
また、更に透析、希釈、濃縮、撹拌等の操作を適宜付加してもよい。
(B2-1:空ベシクルの構造)
後担持法では、非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体と、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体とから形成される膜を有するとともに、前記膜によって包囲された空隙部を有するベシクルを、空ベシクルとして用いる。
図2は、ベシクル1の部分破断図である。図2に示すように、ベシクル1は膜1aと、膜1aにより包囲される空隙部1bとを有する。
図3(b)は、図3(a)に示す第1の重合体2及び第2の重合体3の拡大図である。図3(b)に示すように、第1の重合体2は、非荷電親水性セグメント2aと第1の荷電性セグメント2bとを有するブロック共重合体であり、第2の重合体3は、第1の荷電性セグメント2bとは反対の電荷に帯電した第2の荷電性セグメント3からなる重合体である。そして好ましくは、図3(a)に示すように、非荷電親水性セグメント2aが膜1aの外層1aoを形成し、第1の荷電性セグメント2bと第2の荷電性セグメント3とが静電結合して中間層1amを形成する。そして好ましくは、主に非荷電親水性セグメント2aが膜1aの内層1aiを形成する。
図4(b)は、図4(a)に示す第1の重合体2及び第2の重合体3’の拡大図である。図4(b)に示すように、第1の重合体2は、非荷電親水性セグメント2aと第1の荷電性セグメント2bとを有するブロック共重合体であり、第2の重合体3’は、非荷電親水性セグメント3aと、第1の荷電性セグメント2bとは反対の電荷に帯電した第2の荷電性セグメント3bからなる重合体である。そして好ましくは、図4(a)に示すように、非荷電親水性セグメント2a、3aの一方又は両方が膜1aの外層1aoを形成し、第1の荷電性セグメント2bと第2の荷電性セグメント3bとが静電結合して中間層1amを形成する。そして好ましくは、主に非荷電親水性セグメント2a、3aの一方又は両方が、膜1aの内層1aiを形成する。
ベシクル1の粒径は、第1の重合体2及び第2の重合体3、3’の種類及び量比、架橋剤の有無、ベシクル1の周辺環境(水性媒体の種類)等に応じて異なるが、好ましくは10nm以上、より好ましくは50nm以上、また、好ましくは1000nm以下、より好ましくは400nm以下、更に好ましくは200nm以下である。
ベシクル1の膜1aの膜厚は、第1の重合体2及び第2の重合体3、3’の種類及び量比、架橋剤の有無、ベシクル1の周辺環境(水性媒体の種類)等に応じて異なるが、好ましくは5nm以上、より好ましくは10nm以上、また、好ましくは30nm以下、より好ましくは15nm以下である。
後担持法に用いられる空ベシクルは、第1の重合体及び第2の重合体から構成される膜を有する。
第1の重合体は、非荷電親水性セグメントと第1の荷電性セグメントとを有するブロック共重合体である。第1の重合体は1種類のみでもよく、2種類以上を任意の組み合わせ及び比率を併用してもよい。
第2の重合体は、第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する重合体である。第2の荷電性セグメントのみからなる重合体でもよいが、第2の荷電性セグメントに加えて非荷電親水性セグメントを有するブロック共重合体であってもよい。第2の重合体は1種類のみでもよく、2種類以上を任意の組み合わせ及び比率を併用してもよい。2種類以上の場合には、第2の荷電性セグメントのみからなる第2の重合体と、第2の荷電性セグメントに加えて非荷電親水性セグメントを有する第2の重合体とを併用してもよい。
第1の重合体及び第2の重合体は、それぞれ前述したセグメントに加えて、更に別のセグメントを有していてもよい。
第1の重合体は、非荷電親水性セグメントを有する。また、第2の重合体も、非荷電親水性セグメントを有していてもよい。
非荷電親水性セグメントは、非荷電且つ親水性の性質を有するポリマーセグメントである。ここで「非荷電」とは、セグメントが全体として中性であることをいう。例としてはセグメントが正・負の電荷を有さない場合が挙げられる。また、セグメントが正・負の荷電を分子内に有する場合であっても、局所的な実効電荷密度が高くなく、自己組織化によるベシクルの形成を妨げない程度にセグメント全体の荷電が中和されていれば、やはり「非荷電」に該当する。また、「親水性」とは水性媒体に対して溶解性を示すことをいう。
前記条件を満たす非荷電親水性セグメントを用いることにより、第1の重合体及び第2の重合体の水性溶液中での会合・沈殿を防止して安定化し、ベシクルを効率的に構築することが可能となる。
第1の重合体が有する第1の荷電性セグメントと、第2の重合体が有する第2の荷電性セグメントとは、互いに反対の電荷に帯電した荷電性セグメントである。即ち、第1の荷電性セグメントがカチオン性セグメントであれば、第2の荷電性セグメントはアニオン性セグメントとなり、第1の荷電性セグメントがアニオン性セグメントであれば、第2の荷電性セグメントはカチオン性セグメントとなる。
カチオン性セグメントは、カチオン基を有し、カチオン性(陽イオン性)を示すポリマーセグメントである。但し、カチオン性セグメントは、第1の重合体と第2の重合体との自己組織化によるベシクルの形成を妨げない範囲で、多少のアニオン基を有していてもよい。
カチオン性セグメントの種類も限定されない。単一の繰り返し単位からなるセグメントでもよく、二種以上の繰り返し単位を任意の組み合わせ及び比率で含有するセグメントでもよい。カチオン性セグメントとしては、ポリアミン等が好ましく、側鎖にアミノ基を有するポリアミノ酸又はその誘導体が特に好ましい。側鎖にアミノ基を有するポリアミノ酸又はその誘導体としては、ポリアスパルタミド、ポリグルタミド、ポリリシン、ポリアルギニン、ポリヒスチジン、及びこれらの誘導体等が挙げられるが、特にポリアスパルタミド誘導体及びポリグルタミド誘導体が好ましい。
前記条件を満たすカチオン性セグメントを用いることにより、第1の重合体と第2の重合体との水性溶液中での会合・沈殿を防止して安定化し、ベシクルを効率的に構築することが可能となる。
アニオン性セグメントは、アニオン基を有し、アニオン性(陰イオン性)を示すポリマーセグメントである。但し、アニオン性セグメントは、第1の重合体と第2の重合体との自己組織化によるベシクルの形成を妨げない範囲で、多少のカチオン基を有していてもよい。
アニオン性セグメントの種類も限定されない。単一の繰り返し単位からなるセグメントでもよく、二種以上の繰り返し単位を任意の組み合わせ及び比率で含有するセグメントでもよい。アニオン性セグメントとしては、ポリカルボン酸、ポリスルホン酸、ポリリン酸(核酸等)等が好ましく、側鎖にカルボキシル基を有するポリアミノ酸又はその誘導体、核酸が特に好ましい。
前記条件を満たすアニオン性セグメントを用いることにより、第1の重合体と第2の重合体との水性溶液中での会合・沈殿を防止して安定化し、ベシクルを効率的に構築することが可能となる。
第1の重合体が有する非荷電親水性セグメントと第1の荷電性セグメントとの組み合わせ、また、第2の重合体が第2の荷電性セグメントに加えて非荷電親水性セグメントを有する場合における、非荷電親水性セグメントと第2の荷電性セグメントとの組み合わせはいずれも制限されず、任意の非荷電親水性セグメントと任意の荷電性セグメントとを組み合わせることが可能である(なお、以降の記載では、第1の荷電性セグメント及び第2の荷電性セグメントを纏めて「荷電性セグメント」と表示する場合がある)。
非荷電親水性セグメントと荷電性セグメントとの結合形態も制限されず、直接結合していてもよいが、連結基を介して結合していてもよい。
第1及び第2の重合体の具体例としては、以下の[例1]、[例2]が挙げられる。
第1の重合体として下記(A1)を用い、第2の重合体として下記(B1)を用いる。
(A1)非荷電性親水性のセグメントとアニオン性のセグメントとを含むブロックコポリマー。
(B1)下記(i)のブロックコポリマー及び/又は下記(ii)のポリマー。
(i) 非荷電性親水性のセグメントとカチオン性のセグメントとを含むブロックコポリマー。
(ii) カチオン性のセグメントを含むポリマー(但し非荷電性親水性のセグメントを含まない)。
第1の重合体として下記(A2)を用い、第2の重合体として下記(B2)を用いる。
(A2)非荷電性親水性のセグメントとカチオン性のセグメントとを含むブロックコポリマー。
(B2)下記(iii)のブロックコポリマー及び/又は下記(iv)のポリマー。
(iii) 非荷電性親水性のセグメントとアニオン性のセグメントとを含むブロックコポリマー。
(iv) アニオン性のセグメントを含むポリマー(但し非荷電性親水性のセグメントを含まない)。
前記(B1)(i)、(ii)及び(A2)の各ポリマーにおけるカチオン性のセグメントとしては、限定はされないが、例えば、側鎖にカチオン性基を有するポリペプチドに由来するものが好ましく挙げられる。
同様に、前記(A1)及び(B2)(iii)、(iv)の各ポリマーにおいて、アニオン性のセグメントとしては、限定はされないが、例えば、側鎖にアニオン性基を有するポリペプチドや核酸に由来するものが好ましく挙げられる。
一般式(I)及び(II)中、R2a、R2b、R2c及びR2dは、それぞれ独立してメチレン基又はエチレン基を表す。R2a及びR2bのいずれもがメチレン基の場合はポリ(アスパラギン酸誘導体)に相当し、エチレン基の場合はポリ(グルタミン酸誘導体)に相当し、また、R2c及びR2dのいずれもがメチレン基の場合はポリ(アスパラギン酸誘導体)に相当し、エチレン基の場合はポリ(グルタミン酸誘導体)に相当する。これらの一般式中、R2a及びR2b(R2b及びR2a)がメチレン基及びエチレン基の両者を表す場合、及びR2c及びR2d(R2d及びR2c)がメチレン基及びエチレン基の両者を表す場合、アスパラギン酸誘導体およびグルタミン酸誘導体の反復単位は、それぞれブロックを形成して存在するか、あるいはランダムに存在できる。
一般式(I)及び(II)中、R4は水酸基、オキシベンジル基、-NH-(CH2)a-X基又は開始剤残基を表す。ここで、aは1~5の整数であり、Xは、一級、二級、三級アミン又は四級アンモニウム塩の内の1種類又は2種類以上を含むアミン化合物残基、又は、アミンでない化合物残基であることが好ましい。さらには場合により、R4が-NH-R9(ここで、R9は未置換又は置換された直鎖又は分枝のC1-20アルキル基を表す)であることが好ましい。
一般式(I)及び(II)中、mは5~2,000の整数であり、5~270の整数であることが好ましく、より好ましくは10~100の整数である。また、nは2~5,000の整数であり、yは0~5,000の整数であり、n及びyは、5~300の整数であることが好ましく、より好ましくは10~100の整数である。但し、yはnより大きくないものとする。
一般式(I)及び(II)で示されるブロックコポリマーの分子量(Mw)は、限定はされないが、3,000~30,000であることが好ましく、より好ましくは5,000~20,000である。また、個々のセグメントについては、PEGセグメントの分子量(Mw)は、500~15,000であることが好ましく、より好ましくは1,000~5,000であり、ポリアニオンセグメントの分子量(Mw)は、全体で500~50,000であることが好ましく、より好ましくは1,000~20,000である。
mはPEGの重合度を表す整数である。
nはP(Asp)の重合度を表す整数である。
a、bは何れも0より大きく、1未満の数である。但しa+b=1である。
PEG-P(Asp)としては、PEGセグメントの分子量(Mw):2,000、ポリアニオンセグメントを示すP(Asp)のユニット数(上記式中n):70又は75であるものが特に好ましい。
また、R5a、R5b、R5c及びR5dのすべて又は一部が、-NH-(CH2)a-X基(ここで、aは2であり、Xは(NH(CH2)2)e-NH2(但しeは1)である)ことが好ましい。
mはPEGの重合度を表す整数である。
nはP(Asp-AP)の重合度を表す整数である。
a、bは何れも0より大きく、1未満の数である。但しa+b=1である。
PEG-P(Asp-AP)としては、PEGセグメントの分子量(Mw):2,000,ポリカチオンセグメントを示すP(Asp-AP)のユニット数(上記式中n):70又は75であるものが特に好ましい。
nはP(Asp)の重合度を表す整数である。
a、bは何れも0より大きく、1未満の数である。但しa+b=1である。
Homo-P(Asp)としては、ポリアニオンセグメントを示すP(Asp)のユニット数(上記式中n):70又は82であるものが特に好ましい。
nはP(Asp-AP)の重合度を表す整数である。
a、bは何れも0より大きく、1未満の数である。但しa+b=1である。
Homo-P(Asp-AP)としては、ポリカチオンセグメントを示すP(Asp-AP)のユニット数(上記式中n):70又は82であるものが特に好ましい。
空ベシクルの形成時には、第1の重合体及び第2の重合体に加えて、ベシクルの形成を妨げない、或いは安定性を下げない範囲で、その他の膜成分を添加することができる。その他の膜成分に特に制限はないが、具体例としては荷電性重合体、荷電性ナノ粒子等が挙げられる。
荷電性重合体としては、前述した荷電性セグメント(カチオン性セグメント又はアニオン性セグメント)を1又は2以上有する重合体であって、前記の第1の重合体及び第2の重合体に該当しない任意の荷電性重合体が挙げられる。
荷電性ナノ粒子としては、表面に荷電を有する金属系ナノ粒子等が挙げられる。
前記その他の膜成分の使用量も制限されないが、第1の重合体と第2の重合体との自己組織化によるベシクル形成を妨げない程度に抑えることが好ましい。具体的には、ベシクルの総重量に対して通常30%以下、好ましくは20%以下、より好ましくは10%以下とすることが望ましい。
物質を内包させる空ベシクルは、第1の重合体と第2の重合体との静電相互作用を利用して形成されることから、第1の重合体と第2の重合体とを水性溶液中で混合することにより簡便に製造される。斯かる製造方法によれば、有機溶媒を用いなくともベシクルを製造し得るから、DDSやバイオマテリアル等の分野において有利である。
第1の水性溶液における第1の重合体の濃度、及び、第2の水性溶液における第2の重合体の濃度は限定されず、第1の重合体と第2の重合体との総電荷数の比率、第1の重合体及び第2の重合体の水性溶液への溶解度、ベシクルの形成効率等の条件を勘案して、適宜決定される。
第1及び第2の水性溶液のpHは、ベシクルの形成を妨げない範囲で適宜調整することが可能であるが、好ましくはpH5以上、より好ましくはpH6.5以上であり、また、好ましくはpH9以下、より好ましくはpH7.5以下である。pHの調整は、溶媒として緩衝液を用いることにより、容易に行うことができる。第1及び第2の水性溶液のpHを調整して用いることは、第1の重合体及び第2の重合体の荷電状態を保持し、効率的にベシクルを形成する上で有利である。
第1及び第2の水性溶液のイオン強度は、ベシクルの形成を妨げない範囲で適宜調整することが可能であるが、好ましくは0mM以上、より好ましくは10mM以上であり、また、好ましくは200mM以下、より好ましくは50mM以下である。
第1及び第2の水性溶液の混合時の温度は、ベシクルの形成を妨げない範囲であれば限定されないが、第1の重合体及び第2の重合体の温度に応じた溶解度を勘案して設定することが好ましい。具体的には、好ましくは10℃以上、より好ましくは20℃以上、また、好ましくは60℃以下、より好ましくは50℃以下である。
また、更に透析、希釈、濃縮、撹拌等の操作を適宜付加してもよい。
ベシクルに内包させる被内包物質は制限されず、物質内包ベシクルの用途や性質等に応じて適宜、任意に選択することができる。
特に、従来の製造方法の一つである同時混合法によれば、被内包物質として荷電性の物質を用いた場合、膜構成成分である重合体の自己組織化によるベシクルの形成が被内包物質の荷電によって阻害され、適切な物質内包ベシクルが得られないという問題があった。しかし、後担持法によれば、被内包物質の電気的性質に斯かる制限はなく、被内包物質として荷電性の物質を用いた場合でも非荷電性の物質を用いた場合でも、物質内包ベシクルを効率的に形成することが可能である。
生体分子としては、タンパク質、ポリペプチド、アミノ酸、核酸(DNA、RNA)、脂質(脂肪酸、グリセリド、ステロイド等)、炭水化物(単糖類、多糖類)、及びこれらの誘導体、並びにこれらの二種以上が結合したもの(糖タンパク質、糖脂質等)等が挙げられる。中でもタンパク質、炭水化物等が好ましい。
有機化合物としては、発光(蛍光、燐光等)分子、水溶性薬剤、水溶性高分子、平均粒径100nm以下の水溶性分子集合体(ミセル、ベシクル、ナノゲルなど)、平均粒径100nm以下のエマルション等が挙げられる。中でも平均粒径50nm以下の高分子ミセル、分子量10万以下の水溶性高分子が好ましい。
無機物質としては、水に分散可能な金属ナノ粒子、酸化物ナノ粒子(シリカナノ粒子、チタニアナノ粒子、酸化鉄ナノ粒子等)、半導体ナノ粒子(量子ドット等)、水溶性炭素クラスター、ホウ素クラスター、金属錯体等が挙げられる。中でも平均粒径20nm以下の量子ドットが好ましい。
空ベシクルに対する被内包物質の使用比率も、空ベシクルの構造を破壊したり、被内包物質の空ベシクルへの内包を阻害したりしない範囲で、所望の被内包物質の内包量に応じて調整すればよい。
被内包物質は1種を単独で使用しても、2種以上を任意の比率及び組み合わせで使用してもよい。
後担持法は、少なくとも、所定の構造を有する空ベシクルを用意する工程と、前記空ベシクル及び被内包物質を水性媒体中で混合する工程とを有していればよいが、更にその他の工程を有していてもよい。例としては、架橋剤処理、ろ過操作、透析操作、凍結乾燥操作等が挙げられる。
中でも、物質内包ベシクルを生理環境下や生理食塩水中等の塩存在条件下で使用する場合(例えばDDSとして使用する場合等)には、粒径の経時的な増大を防止する観点から、形成された物質内包ベシクルに対して、後処理として架橋剤処理を施すことが好ましい。すなわち、生理環境下や生理食塩水中等の塩存在条件下では、架橋剤を含有しないベシクルの粒径は経時的に増大する傾向があるが、架橋剤処理を施すことにより粒径の増大を防止することができる。
後担持法を含む各種の任意の手法で空ベシクルに物質を内包させることにより、前述の空ベシクルの空隙部内に前述の被内包物質が内包された、物質内包ベシクルが得られる。
斯かる物質内包ベシクルは、非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体と、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体とから形成される膜と、前記膜によって包囲された空隙部と、前記空隙部内に内包された物質とを含んでなる。
物質内包ベシクルの膜の構造は、上述した空ベシクルの膜の構造と基本的に同等である。即ち、物質内包ベシクルは、図2~4を用いて説明した空ベシクルの構造膜と同様の三層構造膜を有することが好ましく、その形状は通常は球状又は略球状である。
[概要]
第1の態様に係る方法は、空の架橋ベシクルの単分散集合体を、目的物質を水性媒体とともに含有する混合液中で混合し、内水相に目的物質が内包された物質内包架橋ベシクルの単分散集合体を形成させる工程を含んでなる、物質内包架橋ベシクルの製造方法である。
空の架橋ベシクルの単分散集合体の平均粒子径は、通常30nm以上、好ましくは50nm以上、さらに好ましくは70nm以上であり、通常10000nm以下、好ましくは1000nm以下、さらに好ましくは400nm以下である。
空の架橋ベシクルの単分散集合体の製造方法としては、例えば、空の非架橋ベシクルの単分散集合体を、第1及び/又は第2の重合体と反応し得る架橋剤と反応させる方法が挙げられる。
混合液は、目的物質を水性媒体とともに含有する。
目的物質は、ベシクルに内包すべき物質であり、ベシクルの用途等に応じて適宜選択することができる。
目的物質は、上記[B3:被内包物質]に記載した通りであり、水性媒体は、上記[B1-3:混合に関する他の条件]に記載した通りである。
混合方法は、上記[B1-2:空ベシクル及び被内包物質の混合]に記載した通りである。
目的物質が難水溶性物質である場合、混合方法として超音波処理を使用することが好ましい。
第1の態様に係る方法は、空の架橋ベシクルの単分散集合体を、目的物質を水性媒体とともに含有する混合液中で混合し、内水相に目的物質が内包された物質内包架橋ベシクルの単分散集合体を形成させる工程以外に、その他の工程を含むことができる。例えば、後工程として、空の架橋ベシクルを混合液中で混合して得られる物質内包架橋ベシクルに対する架橋工程を含むことができる。架橋工程で使用される架橋剤は、第1及び/又は第2の重合体と反応し得る架橋剤であり、その詳細は上記と同様である。
第1の態様に係る方法によって、第1及び第2の重合体を含んでなり、第1及び/又は第2の重合体が架橋された架橋膜と、架橋膜によって包囲された内水相とを含んでなり、内水相に目的物質が内包された物質内包架橋ベシクルの単分散集合体が製造される。
[概要]
第2の態様に係る方法は、第1の目的物質が内包された第1の物質内包架橋ベシクルを、第1の目的物質よりも分子量の小さい第2の目的物質を水性媒体とともに含有する混合液中で混合し、第1及び第2の目的物質が内包された第2の物質内包架橋ベシクルを形成させる工程を含んでなる、複数物質内包架橋ベシクルを製造する方法である。
第1の物質内包架橋ベシクルは、第1及び第2の重合体を含んでなり、第1及び/又は第2の重合体が架橋された架橋膜と、架橋膜によって包囲された内水相とを含んでなり、内水相に第1の目的物質が内包されたベシクルである。
第1の物質内包架橋ベシクルの製造方法としては、例えば、第1及び第2の重合体を含んでなり、第1及び/又は第2の重合体が架橋された架橋膜と、架橋膜によって包囲された内水相とを含んでなり、内水相に第1及び第2の目的物質のいずれも内包されていない空の架橋ベシクルを、第1の目的物質を水性媒体とともに含有する混合液中で混合し、必要に応じて、混合後、第1及び/又は第2の重合体と反応し得る架橋剤と反応させる方法(以下「方法1」という。)、第1及び第2の重合体を含んでなり、第1及び/又は第2の重合体のいずれも架橋されていない非架橋膜と、非架橋膜によって包囲された内水相とを含んでなり、内水相に第1の目的物質が内包された物質内包非架橋ベシクルを、第1及び/又は第2の重合体と反応し得る架橋剤と反応させる方法(以下「方法2」という。)等が挙げられる。
混合液は、第1の目的物質よりも分子量が小さい第2の目的物質を水性媒体とともに含有する。
混合液の組成、pH、塩濃度(イオン強度)、粘度等は、第1の態様に係る方法で使用される混合液と同様である。
混合方法は、第1の態様に係る方法と同様である。
第2の態様に係る方法によって、第1及び第2の重合体を含んでなり、第1及び/又は第2の重合体が架橋された架橋膜と、架橋膜によって包囲された内水相とを含んでなり、内水相に第1及び第2の目的物質が内包された物質内包架橋ベシクルが製造される。
本発明の別の態様は、吸着材粒子を内包するベシクル(適宜「吸着材内包ベシクル」という。)及びその製法に関する。
ここで、第1及び第2の重合体については、上記[B]欄で説明した通りである。
なお、本態様の変形例として、吸着材を形成し得る物質(吸着材前駆体)を導入した物質内包ベシクルを調製し、その後に吸着材前駆体を吸着材に変換することにより、吸着材内包ベシクルを形成することも可能である。吸着材前駆体の例としては、ゲル等のマトリックスや沈殿・ミセル等を形成し得る荷電性ポリマー等が挙げられる。この場合、荷電性ポリマーを導入した物質内包ベシクルを調製し、その後に反対荷電の薬剤等を当該ベシクル内に導入する。これにより、静電相互作用が適度にキャンセルされて荷電性ポリマーが疎水化し、ベシクル内部でゲル等のマトリックスや沈殿、ミセル等の吸着材が生じるとともに、当該マトリックス、沈殿、ミセル等の吸着材に薬剤がトラップされた薬物担持吸着材内包ベシクルが形成される。斯かる薬物担持吸着材内包ベシクルも、当該薬剤を徐放し得る徐放剤として有用である。
本発明の更に別の態様は、低水溶性物質を内包するベシクル(適宜「低水溶性物質内包ベシクル」という。)を製造することが可能な物質内包ベシクルの製造方法と、それにより製造される新規な低水溶性物質内包ベシクルに関する。
(b)前記前駆体よりも前記目的物質に対して低い溶解性を示す条件下で、前記酵素内包ベシクル内に前記前駆体を浸透させ、前記酵素によって前記前駆体を前記目的物質に転換することにより、前記目的物質を析出させて前記酵素内包ベシクルに内包させ、低水溶性物質内包ベシクルとする工程。
以上、本発明を各種の態様に即して説明したが、本発明は以上の態様に限定されるものではなく、任意の種々の変形を加えて実施することが可能である。例えば、上記<C-1>で説明した物質内包架橋ベシクルの単分散集合体、上記<C-2>で説明した複数物質内包架橋ベシクル、上記[D]で説明した吸着材内包架橋ベシクル、上記[E]で説明した低水溶性物質内包ベシクルは、薬剤を担持させることによって、医薬組成物や薬物送達系として用いることが可能である。斯かる本発明の各種の物質内包ベシクルを含む医薬組成物や薬物送達系も、本発明の対象となる。斯かる医薬組成物や薬物送達系を適用する対象(生物であれば任意であるが、好ましくは動物、より好ましくは哺乳動物、更に好ましくはヒト)、疾患、薬物等は、適宜選択することができる。また、斯かる医薬組成物や薬物送達系の投与経路、用法、用量等の各種条件は、対象、疾患、薬物等に応じて適宜選択することが可能である。
なお、斯かる態様は、本発明の各種の物質内包ベシクルのみならず、任意の物質内包ベシクルを用いて達成することが可能である。即ち、酵素を内包・担持させた任意の物質内包ベシクル(酵素担持ベシクル)を調製して用いる。この場合、プロドラッグを体内に導入するとともに、前記酵素担持ベシクルを別途体内に導入して、体内の所望の部位で酵素担持ベシクルをプロドラッグと接触させ、ベシクルにプロドラッグを浸透させる。こうしてプロドラッグに酵素を作用させ、薬物に転換することにより、体内でプロドラッグから薬物を生成することができる。
これらの態様では、薬物とそのプロドラッグとの溶解度の関係は制限されず、任意の薬物/プロドラッグ/酵素の組み合わせを用いることが可能である。
斯かる薬物/プロドラッグ/酵素の具体例としては、以下の表に示す組み合わせが挙げられるが、これらはあくまでも例示であり、本発明の対象となる薬物/プロドラッグ/酵素の組み合わせは、これらの組み合わせに限定されるものではない。
(a)非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体、及び、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体を含む膜からなるベシクルに、前記薬物の前駆体(プロドラッグ)を前記薬物に転換し得る酵素が内包されてなる酵素内包ベシクルを用意する工程。
(b)対象の所定の部位で、前記酵素内包ベシクル内に前記前駆体を浸透させ、前記酵素によって前記前駆体を前記薬物に転換することにより、前記薬物を形成する工程。
その他の用量や用法等の各種条件は、薬物を送達する対象、薬物の種類、治療対象となる疾患等に応じて、適宜決定することができる。
また、以上の方法に用いられる(酵素内包ベシクル等を含む)薬物送達系や医薬組成物も本発明の対象となる。
また、以下の記載において「ボルテックスミキサー」としては、別途記載しない限り、Eppendorf社製MixMateを使用した。
また、以下の記載において「透過型電子顕微鏡画像」は、別途記載しない限り、日本電子製JEM-1400を用いて取得した。
[実施例I-1]空の架橋ベシクルへのFITC-Dex40k封入
(1)空ベシクルの調製
第1の重合体として、非荷電親水性セグメントであるポリエチレングリコール(重量平均分子量約2000)(以降「PEG」と表示する場合がある)と、アニオン性セグメントであるポリアスパラギン酸(重合度約75)とからなるアニオン性ブロックコポリマーPEG-P(Asp)を使用した。一部のポリアスパラギン酸のN末端をCy3により蛍光標識した。
上記で得られた空ベシクル含有溶液0.5mLを、PEG-P(Asp)に含まれるカルボキシル基に対して0.5等量のEDC(同仁化学研究所製)を含む溶液(pH7.4の10mMリン酸緩衝液(0mM NaCl)中)に加え、室温で12時間静置し、空ベシクルを架橋して空の架橋ベシクル含有溶液を得た。
被内包物質として、フルオレセインイソチオシアナート-デキストラン(Sigma-Aldrich製,以下「FITC-Dex40k」と表記する場合がある)(重量平均分子量40,000)を使用した。
表2に示す通り、FITC-Dex40k内包ベシクル1個あたりのFITC-Dex40kの個数は、空の架橋ベシクルと混合するFITC-Dex40k溶液の濃度の上昇ととともに増加した。
実施例I-1と同様に、第1及び第2の重合体を各々、ポリマー濃度が2.0mg/mLとなるように10mMリン酸緩衝液(pH7.4)(水性媒体)に溶解させた。この際、第1の重合体の溶液には、第2の重合体の溶液と混合後のFITC-Dex40k最終濃度が5~50mg/mL、荷電ポリマー(第1及び第2の重合体)最終濃度が1.0mg/mLとなるように、所定のFITC-Dex40kを加えた。
実施例I-1と同様に、第1及び第2の重合体を各々、ポリマー濃度が1.0mg/mLとなるように10mMリン酸緩衝液(pH7.4)(水性媒体)に溶解させた。得られた第1の重合体の溶液と第2の重合体の溶液とを、第1の重合体と第2の重合体の電荷比が等しくなる(すなわちC/A比=1.0となる)ようにエッペンドルフチューブに入れて混合し、ボルテックスミキサーを使用して2000rpmで2分間攪拌することにより、第1及び第2の重合体の自己組織化集合体を含有する溶液を得た。DLSの結果から、平均粒径は101.8nm、PDI0.081であった。
実施例I-1と同様に、第1及び第2の重合体を各々、ポリマー濃度が1.0mg/mLとなるように10mMリン酸緩衝液(pH7.4)(水性媒体)に溶解させた。得られた第1の重合体の溶液と第2の重合体の溶液とを、第1の重合体と第2の重合体の電荷比が等しくなる(すなわちC/A比=1.0となる)ようにエッペンドルフチューブに入れて混合し、ボルテックスミキサーを使用して2000rpmで2分間攪拌することにより、第1及び第2の重合体の自己組織化集合体を含有する溶液を得た。
比較例I-1において、ベシクル形成プロセス(特に撹拌混合状況)が溶液粘度の影響を受けることが示唆されたので、FITC-Dex(重量平均分子量4000,10000,40000)の濃度と粘度との関係を調べた。
粘度(Pa・s)の測定結果を表3に示す。
(1)シトクロムc内包架橋ベシクルの調製
比較例I-2と同様にして、空ベシクルへのシトクロムc(Sigma-Aldrich社製,分子量12,327Da)の封入を行った。その具体的な方法は、以下の通りである。
得られたシトクロムc内包架橋ベシクル含有溶液0.5mLに、FITC-Dex4k溶液(重量平均分子量:4000,濃度:50mg/mL,容量:0.5mL)を加え、ボルテックスミキサーを使用して2000rpmで2分間攪拌し、シトクロムc内包架橋ベシクルにFITC-Dex4kを封入し、シトクロムc及びFITC-Dex4k内包架橋ベシクル含有溶液を調製した。
実施例I-1と同様に調製した空ベシクルに、PEG-P(Asp)に含まれるカルボキシル基に対して1.0等量のEDCを加えて空の架橋ベシクルを作製した。この時、平均粒径は101nm、PDIは0.084であった。スルホン化フタロシアニン(AlPcS2a)(Frontier Scientific社製)の分散液を種々の濃度(0、1、2、10、20、50mg/mL)で調製し、空の架橋ベシクルに、ベシクル濃度が1mg/mLになるように混合し、2分間の超音波照射を行った。なお、ベシクル濃度は、蛍光標識Cy3により定量した。
比較例I-2と同様に、第1及び第2の重合体を各々、ポリマー濃度が2.0mg/mLとなるように10mMリン酸緩衝液(pH7.4)(水性媒体)に溶解させた。得られた第1の重合体の溶液と第2の重合体の溶液とを、第1の重合体と第2の重合体の電荷比が等しくなる(すなわちC/A比=1.0となる)ようにエッペンドルフチューブに入れて混合し、ボルテックスミキサーを使用して2000rpmで2分間攪拌することにより、第1及び第2の重合体の自己組織化集合体を含有する溶液を得た。得られた溶液0.5mLに、混合後のAlPcS2a濃度が2.0mg/mL、荷電ポリマー濃度が1.0mg/mLとなるように、所定のAlPcS2a溶液を加え、再びボルテックスミキサーで2000rpm、2分間攪拌した。続いて、PEG-P(Asp)に含まれるカルボキシル基に対して10等量のEDCを含む溶液(pH7.4の10mMリン酸緩衝液(0mM NaCl)中)を加え、室温で12時間静置した。ベシクルに内包されず溶液中に遊離状態で残存するAlPcS2aを実施例I-1と同様に遠心限外濾過により除去した。DLS測定から、平均粒径は104nm、PDIは0.062であった。封入されたAlPcS2aの量を図10(A)に示す比色試験により、実施例I-3の結果と比べると、実施例I-3の方が、同一濃度のAlPcS2aを接触した場合においてもより多くの量が封入されていることが判った。また、混合後のAlPcS2a濃度が2.0mg/mLより大きい場合、粒径とPDIの維持が困難であった。
また、実施例I-3及び比較例I-3のベシクル溶液(何れもAlPcS2a濃度20mg/mL)を、荷電ポリマー濃度0.5mg/mLまで希釈し、分光光度計で吸光スペクトルを測定した。その結果を図10(B)に示す。やはり実施例I-3の方が、比較例I-3と比べて、同一濃度のAlPcS2aを接触した場合においても、より多くの量が封入されていることが判った。
第1の重合体として、非荷電親水性セグメントであるポリエチレングリコール(重量平均分子量約2300)(以降「PEG」と表示する場合がある)と、アニオン性セグメントであるポリアスパラギン酸(重合度約79)とからなるアニオン性ブロックコポリマーPEG-P(Asp)を使用した。第2の重合体として、ポリ(ジアミノペンタン構造含有アスパラギン誘導体)(重合度約71)(以降「P(Asp-AP)」と表示する場合がある)からなるカチオン性ホモポリマーHomo-P(Asp-AP)を使用した。第1及び第2の重合体を各々、ポリマー濃度が1.0mg/mLとなるように10mMリン酸緩衝液(pH7.4;水性媒体)に溶解させた。得られた第1の重合体の溶液35.5μLと第2の重合体の溶液50μLを、電荷比が等しくなる(すなわちC/A比=1.0となる)ようにエッペンドルフチューブに入れて混合し、ボルテックスミキサーを使用して2000rpmで2分間攪拌し、第1及び第2の重合体の自己組織化により形成されたベシクル(空ベシクル)を含有する溶液を得た。ここに、最終濃度が5,10,20,150mMとなるように所定量のNaClを含む10mMリン酸緩衝液(pH7.4)を加え、一晩静置した。
第1の重合体として、非荷電親水性セグメントであるポリエチレングリコール(重量平均分子量約2000)(以降「PEG」と表示する場合がある)と、アニオン性セグメントであるポリアスパラギン酸(重合度約75)とからなるアニオン性ブロックコポリマーPEG-P(Asp)を使用した。アニオン性ポリマーの末端をCy3で標識した(アニオン性ポリマー数の10%を標識)。第2の重合体として、ポリ(ジアミノペンタン構造含有アスパラギン誘導体)(重合度約82)(以降「P(Asp-AP)」と表示する場合がある)からなるカチオン性ホモポリマーHomo-P(Asp-AP)を使用した。それぞれを1mg/mLとなるようにpD=7.4のD2Oに溶解した。
系中に含まれるCy3-PEG-P(Asp)のカルボキシル基に対して10,5,1,0.5,0.1等量分のEDC溶液(D2O,pD=7.4)を添加し、空の架橋ベシクル含有溶液を調製した。
全反射赤外分光法によって得られた1550-1600cm-1のピークを、解析ソフトウェア(IGOR Pro)を使用して分離し、COO-に対応する面積の減少を定量することで架橋率を算出した(表7)。
実施例I-1と同様にして調製した空ベシクルに、実施例I-1と同様にしてフルオレセインイソチオシアナート-デキストラン(重量平均分子量4000)(Sigma-Aldrich製,以下「FITC-Dex4k」と表記する場合がある)を封入し、得られたFITC-Dex4k内包非架橋ベシクルについて、動的光散乱法により粒度分布、平均粒径、多分散指数(PDI)を測定した。その結果、FITC-Dex4kの濃度が10mg/mLのときの平均粒径は107.4nm、PDIは0.067、FITC-Dex4kの濃度が15mg/mLのときの平均粒径は109.2nm、PDIは0.078であり(空ベシクルの平均粒径は101.4nm,PDIは0.070)、空ベシクルに、その単分散性が維持されつつ、FITC-Dex4kが封入されたことが確認された。
実施例I-1と同様にして調製した空ベシクルを超音波処理(2分)し、超音波処理後のベシクルについて、動的光散乱法により粒度分布、平均粒径、多分散指数(PDI)を測定した。その結果、超音波処理を氷浴中で実施した場合、平均粒径は112.8nm、PDIは0.098であり、ベシクル溶液に異変は見られなかったが、超音波処理を氷浴中で実施しなかった場合、平均粒径は3606nm、PDIは0.301であり、ベシクル溶液は白濁した。この結果から、ベシクルに対する超音波処理は、ベシクルが過度に加熱されないように実施すること(例えば、超音波処理を非連続的に実施すること、超音波処理を連続的に実施する場合にはベシクルを冷却すること)が望ましいと考えられる。
・シトシンデアミナーゼ封入架橋ベシクルの調製及び評価:
シトシンデアミナーゼ(cytosine deaminase:CD)(シトシンデアミナーゼ硫酸アンモニウム懸濁物(Cytosine deaminase ammonium sulfate suspension)、CALZYME製)を封入した架橋ベシクルを、以下の手順で作成した。なお、CDは5-フルオロシトシン(以下適宜「5-FC」と略す。)を5-フルオロウラシル(以下適宜「5-FU」と略す。)に変換する酵素である。
CD封入架橋ベシクルによる5-FCから5-FUへの変換効率を以下の手順で評価した。上記手順で調製したCD封入架橋ベシクルの分散液0.15mLを、0.01mg/mLの5-FC溶液0.15mlと混合し、混合液中の5-FU濃度を液体クロマトグラフィー法により継時的に測定した。結果を図13のグラフに示す。架橋ベシクルに封入されたCDにより5-FCが5-FUに変換された。その変換速度(酵素活性)は0.03U/mLであった。
CD封入架橋ベシクルによる酵素活性の安定性を以下の手順で評価した。上記手順で調製したCD封入架橋ベシクルを、10mM PBS(pH7.4、150mM NaCl)中、4℃で保存した。調製から0日、5日、7日及び14日の時点で、上記と同様の条件で5-FC溶液と混合し、40分間後の混合液中の5-FU濃度を上記手法で測定した。結果を図14のグラフに示す。調製後少なくとも7日間は酵素活性が維持された。
マウス大腸がん細胞株C26を移植したBALB/cマウス(6週齢)を5匹ずつ8群(グループ1~8)用意し、各グループに対し、上記手順で調製したCD封入架橋ベシクル(0.5U/mL)、5-FC、5-FU、又はPBS各200μLを、以下の表9に示すタイミングで静脈内投与した。5-FC及び5-FUは、マウス体重に対し10mg/kg(体重)又は80mg/kg(体重)となるように調整した。投与後、各マウスの生存期間中、最長28日間に亘って、腫瘍体積を測定すると共に、各マウスの体重を測定した。腫瘍体積は、ノギスを用いて腫瘍の長径及び短径を測定し、以下の計算式により算出した。
V=(a2×b)/2
V:腫瘍体積、a:長径、b:短径
マウス大腸がん細胞株C26を移植したBALB/cマウス(7週齢)3匹を用意し、Cy5で標識した上記のCD封入架橋ベシクル(0.5U/mL)200μLを静脈内投与した。投与後24時間及び72時間の時点で、Cy5標識に基づき、血中CD量を測定した。
血中CD量の測定結果を図18のグラフに示す。CD封入架橋ベシクルによれば、投与から24時間後及び72時間後の何れにおいても、血中に高い濃度のCD量が残存していた。よってCD封入架橋ベシクルは血中滞留性に優れていることが分かる。
実施例I-1と同様に調製した架橋前の空ベシクル分散液に、CD分散液を加え、ボルテックスミキサーを使用して2000rpmで2分間攪拌混合することにより、荷電ポリマー(第1及び第2の重合体)濃度が約0.9mg/mL、混合後のCD最終濃度が1mg/mLのベシクル分散液を調製した。この分散液について動的光散乱(DLS)法による測定を行い、平均粒径及び多分散指数(PDI)を求めたが、均一な粒子形成は確認できず、白濁した状態であった。
[実施例II-1]MSN内包ベシクル1
・材料:
第1の重合体として、非荷電親水性セグメントであるポリエチレングリコール(PEG;分子量約2000)と、アニオン性セグメントであるポリアスパラギン酸(P(Asp);重合度約75)とからなるアニオン性ブロックコポリマー(PEG-P(Asp);ゼータ電位-30.6mV)を用いた。
上記の第1及び第2の重合体を各々10mMリン酸緩衝液(pH7.4)(水性媒体)に溶解させ、各重合体濃度1.0mg/mLの溶液を調製した。また、上記のMSNを10mMリン酸緩衝液(pH7.4)(水性媒体)に超音波照射により分散させ、MSN濃度1.0mg/mLの分散液を調製した。エッペンドルフチューブ中で、上記の第2の重合体の溶液に、上記のMSNの分散液を、MSN濃度が総ポリマー濃度(第1及び第2の重合体の総濃度)の20w/w%となるように添加し、ボルテックスミキサーで約2000rpm、2分間撹拌し、その後8分間静置した。得られたエッペンドルフチューブ中の分散液に、上記の第1の重合体の溶液を、第2の重合体と電荷比が等しくなる(即ちC/A比=1.0となる)ように入れて混合し、ボルテックスミキサーで約2000rpm、2分間攪拌することにより、第1及び第2の重合体の自己組織化により形成されたMSN内包ベシクル(吸着材内包ベシクル)を含有する溶液を得た。
上記のMSN内包ベシクル含有溶液を、PEG-P(Asp)に含まれるカルボキシル基に対して10等量のEDC(同仁化学研究所製、WSC)を含む溶液に加え、一晩静置してEDC反応による架橋を行った。反応の副生成物を遠心限外濾過(VIVASPIN 20、sartorius stedium biotech社製、分画分子量30万;2000rpm、25℃)により除去して精製することにより、架橋されたMSN内包ベシクルを含有する溶液を得た。
また、MSN内包ベシクルのMSN内包率を調べるため、以下の実験を行った。MSNにフルオレセインイソチオシアネート(Fluorescein isothiocyanate:FITC)を吸着させたFITC標識MSNを用い、上述と同様の手法にて、架橋MSN内包ベシクルを含有する溶液を調製した。得られた架橋MSN内包ベシクル含有溶液について、キャピラリー電気泳動(Beckman coulter Inc.製P/ACE MDQ)による測定を行った。検出器としては蛍光検出器を用い、キャピラリーとしては内径75μmのもの(polymicro Technology製TSP075375)を使用し、泳動液としては200mMグリシン-水酸化ナトリウム緩衝液(pH8.0)を用いた。MSN内包ベシクルと、ベシクルに内包されていないMSNとは、表面電荷の違いに応じて、キャピラリー電気泳動により分離される。得られたクロマトグラムを図21に示す。本結果によればベシクルに内包されているMSNは85%であった。
MSN内包ベシクルの血中滞留性を、以下の手順により評価した。
まず、以下のサンプル(i)~(iv)を調製した。
(ii)Cy5標識MSN内包ベシクルのサンプル:MSNの代わりに、上記(i)で使用したCy5標識MSNを用いた他は、上述と同様の手順により調製されたMSN内包ベシクル含有溶液。
(iii)MSN内包Cy5標識ベシクルのサンプル:PEG-P(Asp)の代わりに、Cy5で標識したPEG-P(Asp)(PEG-P(Asp)に対するCy5の比率10mol%)を用いた他は、上述と同様の手順により調製された、MSN内包ベシクル含有溶液。
上述の実施例II-1の手順において、上記の第1及び第2の重合体の各溶液並びにMSNの分散液の混合順を変更し、先に第1の重合体の溶液をMSNの分散液と混合し、得られた混合液に第2の重合体の溶液を混合した他は、実施例II-1と同様の手順に従うことにより、MSNを内包するベシクル(MSN内包ベシクル:吸着材内包ベシクル)を含有する溶液を得た。
得られたMSN内包ベシクル含有溶液を用い、実施例II-1と同様の手順に従って架橋を行うことにより、架橋されたMSN内包ベシクルを含有する溶液を得た。
上述の実施例II-1の手順において、上記の第1及び第2の重合体の各溶液並びにMSNの分散液の混合順を変更し、先に第1の重合体の溶液と第2の重合体の溶液とを混合し、得られた混合液にMSNの分散液を混合した他は、実施例II-1と同様の手順に従うことにより、MSNを内包するベシクル(MSN内包ベシクル:吸着材内包ベシクル)を含有する溶液を得た。
・アミノ化MSN内包ベシクルの調製:
(3-アミノプロピル)トリメトキシシラン((3-Aminopropyl)trimethoxysilane:APTS)50μLを、蒸留水4mLと共に常温で1時間撹拌して溶解させた。続いて、上述の実施例II-1と同様の手順で得られたMSN内包ベシクル(吸着材内包ベシクル)の10mg/mL分散液1mLを加えて更に24時間撹拌し、内封されているMSNにAPTSによる表面処理を施した。その後、限外濾過(分画分子量30万、20%エタノールで3回、その後10mM PBS(pH7.4)で5回)で精製し、アミノ化MSN内包ベシクルを得た。
表面処理前の未処理MSN内包ベシクル及びアミノ化MSN内包ベシクルについて、動的光散乱(DLS)法による測定を行い、平均粒径及び多分散指数(PDI)を求めた。その結果を以下の表11に示す。また、未処理MSN内包ベシクル及びアミノ化MSN内包ベシクルの透過型電子顕微鏡写真をそれぞれ図28(a)及び(b)に示す。
また、未処理MSN内包ベシクル及びアミノ化MSN内包ベシクルについて、TNBSアッセイによりアミノ基の量を測定した。具体的には、0.15Mホウ酸ナトリウム及び0.01M亜硫酸ナトリウム水溶液からなるバッファー200μLと、2,4,6-トリニトロベンゼンスルホン酸(2,4,6-trinitrobenzene sulfonic acid:TNBS)の0.1%水溶液50μLと、未処理MSN内包ベシクル又はアミノ化MSN内包ベシクルの2mg/mL分散液50μLとを混合し、37℃で一晩放置した後、波長420nmの紫外線(UV)吸光度を測定した。得られた吸光度から、各MSN内包ベシクル中に存在するアミノ基数を求めた。その結果、アミノ化MSN内包ベシクルでは未処理MSN内包ベシクルよりもMSN1mgあたり8.14×1019個多くのアミノ基が検出された。
・メルカプト化及びスルホニル化MSN内包ベシクルの調製:
(3-メルカプトプロピル)トリメトキシシラン((3-Mercaptopropyl)trimethoxysilane:MPTS)50μLを、1%酢酸水溶液4mLと共に常温で1時間撹拌して溶解させた。続いて、上述の実施例II-1と同様の手順で得られたMSN内包ベシクル(吸着材内包ベシクル)の10mg/mL分散液1mLを加えて更に24時間撹拌することにより、内封されているMSNにMPTSによる表面処理を施した。その後、限外濾過(分画分子量30万、1%酢酸水溶液で3回、その後水で5回)で精製し、メルカプト化MSN内包ベシクルを得た。続いて、このメルカプト化MSN内包ベシクルの分散液に30%過酸化水素水溶液1mL及び濃硫酸10μLを加え、12時間撹拌することによりメルカプト基を酸化してスルホニル基へと変換した。5M水酸化ナトリウム水溶液で中和し、水で希釈した後、限外濾過(分画分子量30万、10mM PBS(pH7.4)で5回)で精製し、スルホニル化MSN内包ベシクルを得た。
表面処理前の未処理MSN内包ベシクル、メルカプト化MSN内包ベシクル、及びスルホニル化MSN内包ベシクルについて、動的光散乱(DLS)法による測定を行い、平均粒径及び多分散指数(PDI)を求めた。結果を以下の表12に示す。また、未処理MSN内包ベシクル及びスルホニル化MSN内包ベシクルの透過型電子顕微鏡写真をそれぞれ図29(a)及び(b)に示す。
また、未処理MSN内包ベシクル及びスルホニル化MSN内包ベシクルについて、X線分析顕微鏡(XGT-5200WR、(株)堀場製作所)により硫酸基の有無を確認した。得られたX線分析スペクトルを図30に示す。スルホニル化MSN内包ベシクルでは硫酸基に対応するSのピークが確認されたのに対して、未処理MSN内包ベシクルではSのピークは確認されなかった。
・ローズベンガル吸着アミノ化MSN内包ベシクルの調製:
実施例II-3Aで得られたアミノ化MSN内包ベシクルの分散液にローズベンガル(rose bengal:以下適宜「RB」と略す。)を加えて混合し、アミノ化MSN内包ベシクルにローズベンガルを吸着させた。未吸着ローズベンガルは限外濾過(分画分子量30万)で除去した。得られたRB吸着アミノ化MSN内包ベシクルのRB含有率は3.2w/w%であった。ローズベンガルの化学式を以下に示す。
このRB吸着アミノ化MSN内包ベシクルについて、10mM PBS(pH7.4、150mM NaCl)中37℃でのローズベンガルの放出特性を測定した。得られた結果を図31のグラフに示す。24時間で約95%のローズベンガルが放出された。
・ゲムシタビン吸着スルホニル化MSN内包ベシクルの調製:
実施例II-3Bで得られたスルホニル化MSN内包ベシクルの分散液にゲムシタビン(gemcitabine:以下適宜「GEM」と略す。)を加えて混合し、スルホニル化MSN内包ベシクルにゲムシタビンを吸着させた。未吸着のゲムシタビンは限外濾過(分画分子量30万)で除去した。得られたGEM吸着スルホニル化MSN内包ベシクルのGEM含有率は7.9w/w%(10mM PBS用サンプル)及び8.1w/w%(疑似体液用サンプル)であった。
得られたGEM吸着スルホニル化MSN内包ベシクルについて、10mM PBS(pH7.4、150mM NaCl)又は疑似体液(Simulated Body Fluid:SBF)中での37℃でのゲムシタビンの放出特性を測定した。なお、疑似体液は、T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi and T. Yamamuro, "Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W", J. Biomed. Mater. Res., 24, 721-734 (1990)の記載に従って作成した。得られた結果を図32のグラフに示す。10mM PBS中と疑似体液中とでは大きく異なるゲムシタビン放出特性が得られた。これはイオン強度やイオン種の違いによる影響と考えられる。
また、GEM吸着スルホニル化MSN内包ベシクルのA549細胞(ヒト肺胞基底上皮腺癌細胞)に対する細胞取り込み特性及び殺細胞効果を、次のサンプルを使用し、以下の手順で評価した。
サンプル番号1:GEM吸着スルホニル化MSN内包ベシクル
サンプル番号2:スルホニル化MSN内包ベシクル
サンプル番号3:GEM吸着スルホニル化MSN
サンプル番号4:スルホニル化MSN
サンプル番号5:空ベシクル
サンプル番号6:GEM単独
また、A549細胞の代わりにC26細胞(ラット骨芽細胞様細胞)を用いて、以下に説明する点以外は、上記と同様の手順で細胞取り込み特性及び殺細胞効果を評価した。GEM吸着スルホニル化MSN内包ベシクル(サンプル番号1)、GEM吸着スルホニル化MSN(サンプル番号3)及びゲムシタビン単独(サンプル番号6)の添加量は、ゲムシタビンが培地中0.1μg/mLの濃度となる量とし、スルホニル化MSN内包ベシクル(サンプル番号2)及び空ベシクル(サンプル番号5)の添加量は、MSN内包ベシクル換算量でGEM吸着スルホニル化MSN内包ベシクル(サンプル番号1)と同量となるように調整し、スルホニル化MSN(サンプル番号4)の添加量は、MSN換算量でGEM吸着スルホニル化MSN(サンプル番号3)と同量となるように調整した。
また、殺細胞作用の評価結果として、48時間培養後の結果を図36(a)に、72時間培養後の結果を図36(b)に示す。A549細胞の場合と同様、C26細胞についても、GEM吸着スルホニル化MSN内包ベシクル(サンプル番号1)には、GEM吸着スルホニル化MSN(サンプル番号3)及びゲムシタビン単独(サンプル番号6)と同程度の強い殺細胞効果が認められた。また、スルホニル化MSN(サンプル番号4)には弱い殺細胞効果が認められたが、スルホニル化MSN内包ベシクル(サンプル番号2)及び空ベシクル(サンプル番号5)には殺細胞効果は認められなかった。
ヒト肺がん細胞株A549を移植したBALB/cヌードマウス(7週齢、各サンプル)を5匹ずつ7群用意し、うち6群にはそれぞれ上記サンプル1~6を静脈内投与した(グループ1~6)。各サンプルの投与量は200μLとし、各サンプルの濃度は、GEM吸着スルホニル化MSN内包ベシクル(サンプル番号1)、GEM吸着スルホニル化MSN(サンプル番号3)及びゲムシタビン単独(サンプル番号6)については、マウス体重に対してゲムシタビンが5mg/kg(体重)となるように調整し、スルホニル化MSN内包ベシクル(サンプル番号2)及び空ベシクル(サンプル番号5)の添加量は、MSN内包ベシクル換算量でGEM吸着スルホニル化MSN内包ベシクル(サンプル番号1)と同量となるように調整し、スルホニル化MSN(サンプル番号4)の添加量は、MSN換算量でGEM吸着スルホニル化MSN(サンプル番号3)と同量となるように調整した。また、残る1群にはPBS200μLを投与した(グループ7)。投与後28日間にわたって、腫瘍体積を測定した。腫瘍体積は、ノギスを用いて腫瘍の長径及び短径を測定し、以下の計算式により算出した。
V=(a2×b)/2
V:腫瘍体積、a:長径、b:短径
ヒト肺がん細胞株A549を移植したBALB/cヌードマウス(7週齢、各サンプル)を3匹ずつ4群用意した。うち2群にはMSNをCy5で標識したGEM吸着スルホニル化MSN内包ベシクル(サンプル番号1)を静脈内投与し(グループA1及びA2)、残る2群にはCy5で標識したGEM吸着スルホニル化MSN(サンプル番号3)を静脈内投与した(グループB1及びB2)。各サンプルの投与量は200μLとし、各サンプルの濃度は、マウス体重に対してゲムシタビンが5mg/kg(体重)となるように調整した。投与後24時間(グループA1及びB1)及び72時間(グループA2及びB2)の時点で動物を屠殺し、上記と同様の手法により、Cy5蛍光量に基づいて、GEM吸着スルホニル化MSN内包ベシクル及びGEM吸着スルホニル化MSNの血中濃度及び腫瘍における濃度を測定した。
また、GEM吸着スルホニル化MSN内包ベシクル及びGEM吸着スルホニル化MSNの腫瘍における濃度の測定結果を図39のグラフに示す。GEM吸着スルホニル化MSN内包ベシクル投与群(グループA1及びA2)では、GEM吸着スルホニル化MSN投与群(グループB1及びB2)と比較して、投与後24時間及び72時間の何れの時点でも腫瘍における濃度が遥かに高く、腫瘍集積性に優れていることが分かる。
[実施例III-1]β-ガラクトシダーゼ内包架橋ベシクルによるインディゴ系染料内包ベシクルの製造
・材料:
第1の重合体として、非荷電親水性セグメントであるポリエチレングリコール(PEG;分子量約2000)と、アニオン性セグメントであるポリアスパラギン酸(P(Asp);重合度約75)とからなるアニオン性ブロックコポリマー(PEG-P(Asp);ゼータ電位-30.6mV)を用いた。
被内包物質たる酵素として、β-ガラクトシダーゼを用いた。
第1及び第2の重合体を各々、10mMリン酸緩衝液(pH7.4)(水性媒体)に溶解させ、各ポリマー濃度1.0mg/mLの溶液を調製した。また、β-ガラクトシダーゼ(酵素)を濃度1mg/mLとなるように、10mMリン酸緩衝液(pH7.4、150mM塩化ナトリウム含有)に溶解させ、溶液を調製した。得られた第1の重合体の溶液及び第2の重合体の溶液を、電荷比が等しくなる(即ちC/A比=1.0となる)ようにエッペンドルフチューブに入れて混合し、ボルテックスミキサーで約2000rpm、2分間攪拌することにより、第1及び第2の重合体の自己組織化により形成されたベシクル(空ベシクル)を含有する溶液を得た。
X-gal(5-ブロモ-4-クロロ-3-インドリル-β-D-ガラクトピラノシド:水溶性前駆体)を、濃度5mg/mLとなるように、10mMリン酸緩衝液(pH7.4、150mM塩化ナトリウム含有)/ジメチルホルムアミド混液(4:1)に溶解させ、溶液を調製した(なお、X-galは、β-ガラクトシダーゼの酵素作用によって、インディゴ系染料(5,5’-ジブロモ-4,4’-ジクロロ-インディゴ)に転換される。)。総ポリマー濃度0.5mg/mLの上記の架橋β-ガラクトシダーゼ内包ベシクル含有溶液50μLに、上記のX-gal溶液10μLを添加し、さらに10mMリン酸緩衝液190μL加えて37℃で24時間放置し、β-ガラクトシダーゼ(酵素)によるX-gal(水溶性前駆体)からインディゴ系染料(5,5’-ジブロモ-4,4’-ジクロロ-インディゴ:低水溶性物質)への転換反応を進行させた。その後、0.45μmのPESフィルターで濾過し、更に遠心限外濾過(VIVASPIN 20、sartorius stedium biotech社製、分画分子量30万を使用;2000rpm、25℃)により精製を行い、インディゴ系染料内包ベシクル(実施例III-1の低水溶性物質内包ベシクル)を含有する溶液を得た。
実施例III-1と同様の手順に従って調製された空ベシクル含有溶液に対して、β-ガラクトシダーゼ溶液を加えて混合することなく、そのまま実施例III-1と同様の手順に従ってEDC反応による架橋を行い、架橋された空ベシクル含有溶液を得た。
得られた架橋空ベシクル含有溶液について、DLS測定を行い、粒度分布、平均粒径及びPDIを求めた。粒度分布のグラフを図44に示す。平均粒径98nmで単分散な粒子の形成が認められた。PDIは0.076であった。
また、このインディゴ系染料生成後の空ベシクル含有溶液について、透過型電子顕微鏡(TEM)による形態観察を行った。TEM(日本電子製JEM-1400)により得られたTEM写真を図46に示す。図46によれば、β-ガラクトシダーゼとX-galの反応により低水溶性のインディゴ系染料が生成したものの、ベシクルに内包されているインディゴ系染料は殆ど認められなかった。
Claims (41)
- 非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体と、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体とを含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、
前記架橋膜によって包囲された内水相と
を含んでなり、前記内水相に目的物質が内包された物質内包架橋ベシクルの単分散集合体であって、
前記内水相に含有される前記目的物質の濃度が、
前記第1及び/又は前記第2の重合体が架橋されていない点及び前記目的物質が内包されていない点で前記物質内包架橋ベシクルの単分散集合体と異なる空の非架橋ベシクルの単分散集合体を、前記物質内包架橋ベシクルの内水相と同一濃度の前記目的物質を水性媒体とともに含有する混合液中で混合した場合に、
前記目的物質が内包された物質内包非架橋ベシクルの単分散集合体の形成を阻害する濃度である、前記物質内包架橋ベシクルの単分散集合体。 - 0.2以下の多分散指数を有する、請求項1に記載の物質内包架橋ベシクルの単分散集合体。
- 前記目的物質の重量平均分子量が10000~40000であり、前記内水相に含有された前記目的物質の濃度が5mg/mLを上回る、請求項1又は2に記載の物質内包架橋ベシクルの単分散集合体。
- 前記第1及び/又は前記第2の重合体が、カチオン基間に形成された架橋結合、アニオン基間に形成された架橋結合、及びカチオン基とアニオン基との間に形成された架橋結合からなる群より選択された1種又は2種以上の架橋結合によって架橋されており、前記架橋結合が形成された割合が、前記架橋膜に含まれるカチオン基及び/又はアニオン基の総モル数の35%以上である、請求項3に記載の物質内包架橋ベシクルの単分散集合体。
- 非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体と、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体とを含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、前記架橋膜によって包囲された内水相とを含んでなり、前記内水相に第1の目的物質及び前記第1の目的物質よりも分子量の小さい第2の目的物質が内包された物質内包架橋ベシクルであって、
前記第1の目的物質が、前記第2の目的物質の不存在下で前記内水相に含有されている場合よりも安定化されている、前記物質内包架橋ベシクル。 - 前記第2の目的物質がクラウディング剤である、請求項5に記載の物質内包架橋ベシクル。
- 物質内包ベシクルを製造する方法であって、
非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体と、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体とを含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、
前記架橋膜によって包囲された内水相と
を含んでなり、前記内水相に目的物質が内包されていない空の架橋ベシクルの単分散集合体を、
前記目的物質を水性媒体とともに含有する混合液中で混合し、
前記第1及び前記第2の重合体を含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、
前記架橋膜によって包囲された内水相と
を含んでなり、前記内水相に前記目的物質が内包された物質内包架橋ベシクルの単分散集合体を形成させる工程を含んでなる、前記方法。 - 前記空の架橋ベシクルの単分散集合体、前記物質内包架橋ベシクルの単分散集合体、前記空の非架橋ベシクルの単分散集合体、及び前記物質内包非架橋ベシクルの単分散集合体が、0.2以下の多分散指数を有する、請求項7に記載の方法。
- 前記目的物質の重量平均分子量が10000~40000であり、前記混合液に含有される前記目的物質の濃度が5mg/mLを上回る、請求項7又は8に記載の方法。
- 前記空の架橋ベシクル及び前記物質内包架橋ベシクルにおいて、前記第1及び/又は前記第2の重合体が、カチオン基間に形成された架橋結合、アニオン基間に形成された架橋結合、及びカチオン基とアニオン基との間に形成された架橋結合からなる群より選択された1種又は2種以上の架橋結合によって架橋されており、前記架橋結合が形成された割合が、前記架橋膜に含まれるカチオン基及び/又はアニオン基の総モル数の35%以上である、請求項9に記載の方法。
- 前記物質内包架橋ベシクルの単分散集合体を、前記第1及び/又は前記第2の重合体と反応し得る架橋剤と反応させる工程をさらに含む、請求項7~10のいずれか1項に記載の方法。
- 物質内包ベシクルを製造する方法であって、
非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体と、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体とを含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、
前記架橋膜によって包囲された内水相と
を含んでなり、前記内水相に第1の目的物質が内包された第1の物質内包架橋ベシクルを、
前記第1の目的物質よりも分子量の小さい第2の目的物質を水性媒体とともに含有する混合液中で混合し、
前記第1及び前記第2の重合体を含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、
前記架橋膜によって包囲された内水相と
を含んでなり、前記内水相に前記第1及び前記第2の目的物質が内包された第2の物質内包架橋ベシクルを形成させる工程を含んでなる、前記方法。 - 前記第1の物質内包架橋ベシクルが、その単分散集合体であり、前記第2の物質内包架橋ベシクルが、その単分散集合体である、請求項12に記載の方法。
- 前記混合液に含有される前記第2の目的物質の濃度が、
前記第1及び/又は前記第2の重合体が架橋されていない点で前記第1の物質内包架橋ベシクルの単分散集合体と異なる第1の物質内包非架橋ベシクルの単分散集合体を前記混合液中で混合した場合に、
前記第1及び前記第2の目的物質が内包された物質内包非架橋ベシクルの単分散集合体の形成を阻害する濃度である、請求項13に記載の方法。 - 前記第1及び前記第2の重合体を含んでなり、前記第1及び/又は前記第2の重合体が架橋された架橋膜と、
前記架橋膜によって包囲された内水相と
を含んでなり、前記内水相に前記第1及び前記第2の目的物質のいずれも内包されていない空の架橋ベシクルを、
前記第1の目的物質を水性媒体とともに含有する混合液中で混合し、必要に応じて、前記第1及び/又は前記第2の重合体と反応し得る架橋剤と反応させ、
前記第1の物質内包架橋ベシクルを形成させる工程をさらに含んでなる、請求項12~14のいずれか1項に記載の方法。 - 前記空の架橋ベシクルが、その単分散集合体であり、前記第1の物質内包架橋ベシクルが、その単分散集合体である、請求項15に記載の方法。
- 前記混合液に含有される前記第1の目的物質の濃度が、
前記第1及び/又は前記第2の重合体が架橋されていない点で前記空の架橋ベシクルの単分散集合体と異なる空の非架橋ベシクルの単分散集合体を前記混合液中で混合した場合に、
前記第1の目的物質が内包された物質内包非架橋ベシクルの単分散集合体の形成を阻害する濃度である、請求項16に記載の方法。 - 前記第2の目的物質がクラウディング剤である、請求項12~17のいずれか1項に記載の方法。
- 非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体、及び、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体を含む膜からなるベシクルと、当該ベシクルに内包された吸着材粒子とを含み、前記第1及び第2の重合体の少なくとも一方が前記吸着材粒子に吸着されてなる吸着材内包ベシクル。
- 前記第1及び/又は第2の重合体が架橋されてなる、請求項19に記載の吸着材内包ベシクル。
- 前記吸着材粒子がシリカ粒子である、請求項19又は20に記載の吸着材内包ベシクル。
- 前記吸着材粒子の平均粒径が40nm~10μmである、請求項19~21のいずれか1項に記載の吸着材内包ベシクル。
- 前記吸着材粒子が表面処理されてなる、請求項19~22のいずれか1項に記載の吸着材内包ベシクル。
- 前記吸着材粒子に低分子化合物が吸着されてなる、請求項19~23のいずれか1項に記載の吸着材内包ベシクル。
- 非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体、及び、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体を含む膜からなるベシクルに、吸着材粒子が内包されてなる吸着材内包ベシクルを製造する方法であって、
(a)前記第1及び第2の重合体のうち一方を前記吸着材粒子と混合し、前記吸着材粒子に吸着させる工程、並びに、
(b)前記工程(a)の混合物を前記第1及び第2の重合体のうち他方と更に混合し、前記吸着材粒子の周囲に前記第1及び第2の重合体を含む膜からなるベシクルを形成させ、吸着材内包ベシクルとする工程
を含む方法。 - (c)前記工程(b)のベシクル中の前記第1及び/又は前記第2の重合体を架橋する工程を更に含む、請求項25に記載の方法。
- 前記吸着材粒子がシリカ粒子である、請求項25又は26に記載の方法。
- 前記吸着材粒子の平均粒径が40nm~10μmである、請求項25~27のいずれか1項に記載の方法。
- 前記吸着材粒子を表面処理する工程を更に含む、請求項25~28のいずれか1項に記載の方法。
- 前記吸着材粒子に低分子化合物が吸着されてなる、請求項25~29のいずれか1項に記載の方法。
- 非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体、及び、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体を含む膜からなるベシクルに、目的物質が内包されてなる物質内包ベシクルを製造する方法であって、
(a)前記目的物質よりも水溶性の高い前駆体を前記目的物質に転換し得る酵素が、前記第1及び第2の重合体を含む膜からなるベシクルに内包されてなる酵素内包ベシクルを用意する工程、及び、
(b)前記前駆体よりも前記目的物質に対して低い溶解性を示す条件下で、前記酵素内包ベシクル内に前記前駆体を浸透させ、前記酵素によって前記前駆体を前記目的物質に転換することにより、前記目的物質を析出させて前記酵素内包ベシクルに内包させ、物質内包ベシクルとする工程
を含む方法。 - 前記工程(b)において、前記酵素内包ベシクルを前記前駆体の水溶液と混合することにより、前記酵素内包ベシクル内に前記前駆体を浸透させる請求項31に記載の方法。
- 前記工程(b)の前に、前記酵素内包ベシクルの前記第1及び/又は前記第2の重合体を架橋する工程を更に含む、請求項32に記載の方法。
- 請求項31~33のいずれか1項に記載の方法により製造される物質内包ベシクル。
- 非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体、及び、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体を含む膜からなるベシクルに、前記目的物質よりも水溶性の高い前駆体から転換され得る低水溶性物質が、前記前駆体を前記低水溶性物質に転換し得る酵素と共に内包されてなる、低水溶性物質内包ベシクル。
- 前記低水溶性物質が、内水相に対する前記低水溶性物質の溶解度を超える濃度で内包されてなる、請求項34又は35に記載の低水溶性物質内包ベシクル。
- 前記第1及び/又は前記第2の重合体が架橋されてなる、請求項34~36のいずれか1項に記載の低水溶性物質内包ベシクル。
- 請求項1~4のいずれか1項に記載のベシクルの単分散集合体、及び/又は、請求項5、6、19~22及び34~37のいずれか1項に記載のベシクルを含む薬物送達系。
- 対象に薬物を送達するための方法であって、
(a)非荷電親水性セグメント及び第1の荷電性セグメントを有するブロック共重合体である第1の重合体、及び、前記第1の荷電性セグメントとは反対の電荷に帯電した第2の荷電性セグメントを有する第2の重合体を含む膜からなるベシクルに、前記薬物の前駆体を前記薬物に転換し得る酵素が内包されてなる酵素内包ベシクルを用意する工程、及び、
(b)対象の所定の部位で、前記酵素内包ベシクル内に前記前駆体を浸透させ、前記酵素によって前記前駆体を前記薬物に転換することにより、前記薬物を形成する工程
を含む方法。 - 前記前駆体の水溶性がが前記薬物よりも低いとともに、前記工程(b)において、前記前駆体よりも前記薬物に対して低い溶解性を示す条件下で、前記酵素内包ベシクル内に前記前駆体を浸透させる、請求項39に記載の方法。
- 前記工程(b)において、前記薬物を析出させて前記酵素内包ベシクルに内包させ、薬物内包ベシクルを形成することを更に含む、請求項39又は40に記載の方法。
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US11103461B2 (en) | 2015-12-22 | 2021-08-31 | The Trustees Of Princeton University | Process for encapsulating soluble biologics, therapeutics, and imaging agents |
US11737981B2 (en) | 2017-09-12 | 2023-08-29 | The Trustees Of Princeton University | Cellulosic polymer nanoparticles and methods of forming them |
US11731099B2 (en) | 2018-07-20 | 2023-08-22 | The Trustees Of Princeton University | Method for controlling encapsulation efficiency and burst release of water soluble molecules from nanoparticles and microparticles produced by inverse flash nanoprecipitation |
WO2020116635A1 (ja) | 2018-12-07 | 2020-06-11 | 国立大学法人 東京大学 | 輸送担体を用いた環状化合物の送達 |
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JP6049854B2 (ja) | 2016-12-27 |
US10471019B2 (en) | 2019-11-12 |
US20180008549A1 (en) | 2018-01-11 |
EP2962752A4 (en) | 2016-09-07 |
US20160051484A1 (en) | 2016-02-25 |
US20180008550A1 (en) | 2018-01-11 |
CN105188905A (zh) | 2015-12-23 |
JPWO2014133172A1 (ja) | 2017-02-09 |
US10322092B2 (en) | 2019-06-18 |
CN109663550B (zh) | 2021-08-06 |
CN109395678A (zh) | 2019-03-01 |
CN109663550A (zh) | 2019-04-23 |
CN105188905B (zh) | 2019-01-15 |
EP2962752A1 (en) | 2016-01-06 |
US9782358B2 (en) | 2017-10-10 |
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