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Substance-containing vesicle, and production method therefor

Foreign code F180009415
File No. AF12-14US3
Posted date Apr 20, 2018
Country United States of America
Application number 201715684086
Gazette No. 20180008550
Gazette No. 10322092
Date of filing Aug 23, 2017
Gazette Date Jan 11, 2018
Gazette Date Jun 18, 2019
International application number JP2014055186
International publication number WO2014133172
Date of international filing Feb 28, 2014
Date of international publication Sep 4, 2014
Priority data
  • P2013-041186 (Mar 1, 2013) JP
  • P2013-176068 (Aug 27, 2013) JP
  • 2014JP55186 (Feb 28, 2014) WO
  • 201514839854 (Aug 28, 2015) US
Title Substance-containing vesicle, and production method therefor
Abstract Provided is a monodisperse agglomerate of a substance-containing vesicle filled with a substance at a concentration higher than conventionally possible. A mixed solution, in which a target substance is included in an aqueous medium, is mixed with a monodisperse agglomerate of a crosslinked vesicle comprising a prescribed polymer which includes a first polymer, i.e. a block copolymer having uncharged hydrophilic segments and first charged segments, and a second polymer having second charged segments carrying a charge opposite to that of the first charged segments, and in which the first polymer and/or the second polymer are/is crosslinked. As a result, the crosslinked vesicle is made to contain the target substance.
Outline of related art and contending technology BACKGROUND ART
It is known that a vesicle can be formed via self-assembly of polymer molecules of which the primary structure has been have been controlled precisely. Such a vesicle is applicable to various molecular designs, and can serve a new function beyond the properties of the original polymers. Accordingly, the vesicle is being considered for use as a carrier for a drug delivery system (DDS) or as a biomaterial or functional material.
Patent Document 1 (JP-H08-188541A, of which the inventors overlap with the present inventors) discloses a drug carrier in the form of an electrostatically-united polymeric micelle formed via self-assembly of molecules of a block copolymer having an uncharged segment and a charged segment.
Non-Patent Document 1 (Schlaad H. et al., Macromolecules, 2003, 36 (5), 1417-1420) discloses a vesicle referred to as “polymersome”, which is formed via self-assembly of molecules of a first block copolymer having poly(1,2-butadiene) block and poly(cesium methacrylate) block and a second block copolymer having polystyrene block and poly(l-methyl-4-vinylpyridinium iodide) block.
Patent Document 2 (WO2006/118260A, of which the inventors overlap with the present inventors) discloses a vesicle formed via self-assembly of a first block copolymer having an uncharged hydrophilic segment and a cationic segment (e.g., PEG-polycation) with a second block copolymer having an uncharged hydrophilic segment and an anionic segment (e.g., PEG-polyanion).
Non-Patent Document 2 (Anraku Y. et al., J. Am. Chem. Soc., 2010, 132 (5), 1631-1636, of which the inventors overlap with the present inventors) discloses a vesicle formed via self-assembly of a block copolymer having an uncharged hydrophilic segment and a charged segment (e.g., PEG-polycation) and a copolymer charged oppositely to the charged segment of the block copolymer (e.g., polyanion).
It is contemplated that such vesicles formed via self-assembly of polymers as mentioned above can encapsulate and carry various substances within their cavities for desired applications (for overview, see, e.g., Non-Patent Document 3: H. Nyin et al. Soft Matter, 2006, 2, 940-949; and Non-Patent Document 4: “Liposome: New Developments in Applications”, supervised by Kazunari AKIYOSHI et al., NTS Inc., 2005).
A typical process of producing a vesicle encapsulating a substance within its cavity (hereinafter also referred to as “substance-encapsulating vesicle”) includes mixing a substance to be encapsulated (hereinafter also referred to as “encapsulation-target substance”) with membrane component polymers or a preformed polymer membrane to cause formation of a polymer vesicle via self-assembly simultaneously with enclosure of the substance into the vesicle cavity (hereinafter also referred to as “simultaneous mixing method”). Examples include: emulsion method (see, e.g., Non-Patent Document 5: F. Szoka, Jr et al., Proc. Natl. Acad. Sci. USA, 1978 75 (9) 4194-4198); and instillation method using organic solution of lipids (see, e.g., Non-Patent Document 6: Batzri, S. et al., Biochim. Biophys Acta 1973, 298, 1015-1019).
However, the simultaneous mixing method has a drawback that presence of the encapsulation-target substance may affect vesicle formation process via self-assembly, thereby preventing formation of a vesicle or, even if not, enclosure of the substance into the vesicle cavity. Another problem involved in this method is that it often requires use of organic solvent which is detrimental to membrane formation, rendering the process complicated and causing damage to the encapsulation-target substance due to the organic solvent. This method has still another drawback that it is difficult to form vesicles having uniform particle size and structure unless carrying out an additional step, which is likely to render the process complicated. Thus, this method lacks versatility, and is not practical as a means for producing various kinds of substance-encapsulating vesicles.
On the other hand, as a general method of producing a particle encapsulating a substance, there is a method in which an encapsulation-target substance is introduced into the cavity of an existing vacant particle such that the substance is enclosed and carried by the particle (hereinafter also referred to as “post-carrying method”) (see, e.g., Non-Patent Document 7: W. Tong et al. J. Phys. Chem. B, 2005, 109, 13159-13165). This method could be an option for producing substance-encapsulating vesicles.
However, application of the post-carrying method to vesicles would require any additional means to introduce an encapsulation-target substance beyond the membrane of a vacant vesicle into the vesicle cavity. A conceivable method includes: making the vacant vesicle swell to relax the membrane; penetrating the encapsulation-target substance into the cavity through cleavage which has occurred on the relaxed membrane; and contracting the membrane to prevent release of the encapsulation-target substance. Another conceivable method includes: opening pores on the membrane of the vacant vesicle; introducing the encapsulation-target substance into the cavity through the pores; and closing the pores to prevent release of the encapsulation-target substance. However, these methods are cumbersome and complicated, too disadvantageous to be put into practical use. In addition, the particle size and the structure of the existing vacant vesicle would probably be disturbed during the process of enclosure and carriage of the encapsulation-target substance. Accordingly, these methods have been considered as being far from practical.
Another published method for lipid bilayer membrane vesicles such as liposomes includes integrating a channel protein into the lipid bilayer membrane (see, e.g., 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). However, this method is not practical either, since the process is cumbersome and complicated and lacks versatility.
Under such circumstance, the present inventors filed a patent application (Patent Document 3: WO2011/145745A) based on a surprising finding that a vacant vesicle formed of a membrane containing a first polymer, which is a block copolymer having an uncharged hydrophilic segment and a first charged segment, and a second polymer, which has a second charged segment oppositely charged to the first charged segment, wherein the membrane defines a cavity, can be used for producing a substance-encapsulating vesicle efficiently with ease by a method including mixing a vacant vesicle in aqueous medium in the presence of a target substance to be encapsulated (which method corresponds to a “post-carrying method” mentioned above), whereby the target substance is encapsulated into the vesicle cavity (inner aqueous phase) via self-assembly of the first and second polymers.
Scope of claims [claim1]
1. A method of producing a target substance-encapsulating vesicle, comprising the steps of:
(a) obtaining an enzyme-encapsulating vesicle comprising a membrane containing a first polymer, which is a block copolymer having a polyethylene glycol segment and a first charged segment, and a second polymer, which has a second charged segment having a charge opposite to the charge of the first charged segment, and an enzyme encapsulated in the vesicle, wherein the first charged segment and the second charged segment are selected from (i) the anionic segment consisting of polyaspartic acid, polyglutamic acid, polycarboxylic acids, and nucleic acids, or (ii) the cationic segment consisting of polyglutamide, polylysine, polyarginine, and polyhistidine, and wherein the enzyme is capable to convert a precursor to a target substance; and
(b) mixing the enzyme-encapsulating vesicle with a precursor of a target substance in an aqueous medium under conditions which provide a lower water solubility for the target substance than for the precursor such that when the enzyme converts the precursor into the target substance, the target substance precipitates and is encapsulated in the enzyme-encapsulating vesicle, thereby forming the target substance-encapsulating vesicle.

[claim2]
2. The method according to claim 1, wherein step (b) is carried out by mixing the enzyme-encapsulating vesicle with an aqueous solution of the precursor.

[claim3]
3. The method according to claim 2, further comprising crosslinking the first and/or the second polymer(s) of the enzyme-encapsulating vesicle before step (b).

[claim4]
4. A substance-encapsulating vesicle produced by the method according to claim 1.

[claim5]
5. A substance-encapsulating vesicle comprising:
a vesicle comprising a membrane containing a first polymer, which is a block copolymer having a polyethylene glycol segment and a first charged segment, and a second polymer, which has a second charged segment having a charge opposite to the charge of the first charged segment, wherein the first charged segment and the second charged segment are selected from (i) the anionic segment consisting of polyaspartic acid, polyglutamic acid, polycarboxylic acids, and nucleic acids, or (ii) the cationic segment consisting of polyglutamide, polylysine, polyarginine, and polyhistidine, and wherein the enzyme is capable to convert a precursor to a target substance;
a substance encapsulated in the vesicle at a concentration exceeding the solubility of the substance to the inner aqueous phase.

[claim6]
6. The substance-encapsulating vesicle according to claim 5, wherein the first and/or the second polymer(s) is(are) crosslinked.

[claim7]
7. A drug delivery system comprising the vesicle according to claim 4.

[claim8]
8. A drug delivery system comprising the vesicle according to claim 5.

[claim9]
9. A method of producing a target substance-encapsulating vesicle, comprising the steps of:
(a) obtaining an enzyme-encapsulating vesicle comprising a membrane containing a first polymer and a second polymer, and an enzyme encapsulated in the vesicle, wherein the enzyme is capable to convert a precursor to a target substance; and
(b) mixing the enzyme-encapsulating vesicle with a precursor of a target substance in an aqueous medium under conditions which provide a lower water solubility for the target substance than for the precursor such that when the enzyme converts the precursor into the target substance, the target substance precipitates and is encapsulated in the enzyme-encapsulating vesicle, thereby forming the target substance-encapsulating vesicle,
(i) wherein the first polymer is a PEG-anionic segment block copolymer represented by formula (I) or (II), and the second polymer is a PEG-cationic segment block copolymer represented by formula (III) or (IV) or a cationic polymer represented by formula (VII) or (VIII), or
(ii) wherein the first polymer is a PEG-cationic segment block copolymer represented by formula (III) or (IV), and the second polymer is a PEG-anionic segment block copolymer represented by formula (I) or (II) or an anionic polymer represented by formula (V) or (VI),
wherein
R1a and R1b each independently represent hydrogen or C1-12 alkyl group, optionally substituted with at least one substituent selected from the group consisting of halogen, aryl, hydroxyl, amino, carboxyl, cyano, formyl, dimethyl acetalated formyl, diethyl acetalated formyl, C1-6 alkoxycarbonyl, C2-7 acylamide, siloxy, tri(C1-6 alkyl)siloxy, and silylamino,
L1 is ―(CH2)b―NH―, and L2 is ―(CH2)c―CO―, where b and c are independently an integer of 1 to 5,
R2a, R2b, R2c and R2d each independently represent a methylene or ethylene group,
R3 represents a hydrogen atom, acetyl, acryloyl, or methacryloyl group,
R4 represents hydroxyl, oxybenzyl, or ―NH―R9 where R9 represents a straight-chain or branched C1-20 alkyl group,
R5a, R5b, R5c and R5d each independently represent hydroxyl, oxybenzyl, or ―NH―(CH2)a―X, where X is (NH(CH2)2)e―NH2 with the proviso that e is an integer of 0 to 5,
R6a and R6b each independently represent a hydrogen atom, ―C(═NH)NH2 or a protecting group selected from the group consisting of a benzyloxycarbonyl group, Boc group, acetyl group and trifluoroacetyl group,
m and n are independently an integer of 5 to 2,000,
t is an integer of 2 to 6,
y and z are independently an integer of 0 to 5,000.

[claim10]
10. The method according to claim 9, wherein step (b) is carried out by mixing the enzyme-encapsulating vesicle with an aqueous solution of the precursor.

[claim11]
11. The method according to claim 10, further comprising crosslinking the first and/or the second polymer(s) of the enzyme-encapsulating vesicle before step (b).

[claim12]
12. A substance-encapsulating vesicle produced by the method according to claim 9.

[claim13]
13. A substance-encapsulating vesicle comprising:
a vesicle comprising a membrane containing a first polymer and a second polymer,
a substance encapsulated in the vesicle at a concentration exceeding the solubility of the substance to the inner aqueous phase,
wherein the first polymer is a PEG-anionic segment block copolymer represented by formula (I) or (II), and the second polymer is a PEG-cationic segment block copolymer represented by either formula (III) or (IV) or a cationic copolymer formula (VII) or (VIII), or
wherein the first polymer is a PEG-cationic segment block copolymer represented by formula (III) or (IV) and the second polymer is a PEG-anionic segment block copolymer represented by either formula (I) or (II) or an anionic polymer represented by formula (V) and/or (VI),
wherein
R1a and R1b each independently represent hydrogen or C1-12 alkyl group, optionally substituted with at least one substituent selected from the group consisting of halogen, aryl, hydroxyl, amino, carboxyl, cyano, formyl, dimethyl acetalated formyl, diethyl acetalated formyl, C1-6 alkoxycarbonyl, C2-7 acylamide, siloxy, tri(C1-6 alkyl)siloxy, and silylamino,
L1 is ―(CH2)b―NH―, and L2 is ―(CH2)c―CO―, where b and c are independently an integer of 1 to 5,
R2a, R2b, R2c and R2d each independently represent a methylene or ethylene group,
R3 represents a hydrogen atom, acetyl, acryloyl, or methacryloyl group,
R4 represents hydroxyl, oxybenzyl, or ―NH―R9 where R9 represents a straight-chain or branched C1-20 alkyl group,
R5a, R5b, R5c and R5d each independently represent hydroxyl, oxybenzyl, or ―NH―(CH2)a―X, where X is (NH(CH2)2)e―NH2 with the proviso that e is an integer of 0 to 5,
R6a and R6b each independently represent a hydrogen atom, ―C(═NH)NH2 or a protecting group selected from the group consisting of a benzyloxycarbonyl group, Boc group, acetyl group and trifluoroacetyl group,
m and n are independently an integer of 5 to 2,000,
t is an integer of 2 to 6,
y and z are independently an integer of 0 to 5,000.

[claim14]
14. The substance-encapsulating vesicle according to claim 13, wherein the first and/or the second polymer(s) is(are) crosslinked.

[claim15]
15. A drug delivery system comprising the vesicle according to claim 12.

[claim16]
16. A drug delivery system comprising the vesicle according to claim 13.
  • Inventor, and Inventor/Applicant
  • Kataoka Kazunori
  • Kishimura Akihiro
  • Anraku Yasutaka
  • Goto Akinori
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
IPC(International Patent Classification)
Reference ( R and D project ) CREST Establishment of Innovative Manufacturing Technology Based on Nanoscience AREA
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