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Nucleic acid-encapsulating polymer micelle complex and method for producing same

Foreign code F170009172
File No. K10503WO
Posted date Sep 12, 2017
Country China
Application number 201480043775
Gazette No. 105451719
Gazette No. 105451719
Date of filing Aug 5, 2014
Gazette Date Mar 30, 2016
Gazette Date Jun 11, 2019
International application number JP2014070567
International publication number WO2015020026
Date of international filing Aug 5, 2014
Date of international publication Feb 12, 2015
Priority data
  • P2013-163106 (Aug 6, 2013) JP
  • 2014JP70567 (Aug 5, 2014) WO
Title Nucleic acid-encapsulating polymer micelle complex and method for producing same
Abstract This nucleic acid-encapsulating polymer micelle complex is characterized in being formed of: a block copolymer containing an uncharged hydrophilic polymer chain block and a cationic polymer chain block; and two single-stranded DNA molecules comprising mutually complementary base sequences of 1000 or more bases in length, double-stranded DNA of 1000 or more base pairs in length in which at least a part of the double helix structure has dissociated and taken on a single-stranded structure, or one single-stranded DNA molecule of 1000 or more bases in length.
Outline of related art and contending technology BACKGROUND ART
As the next generation therapeutics, through control of gene expression of a gene therapy to treat diseases of the great has been highly expected. The greatest problem in gene therapy by introducing a gene into target cells or tissues is not sufficient transfer efficiency at the time of this point. In particular, in order to achieve based on systematic administration of gene therapy, necessary for the gene in blood collected in the target tissue to circulate in a stable manner, and the target cell for efficient gene expression after entry. Therefore, in order to solve these problems, is to develop a popular to a target cell transfection efficiency or the like, in the target cells for gene expression in a more preferred gene transporter efficiency (gene carrier).
For example, there is known a secondary structure to obtain precisely controlled amounts of polymer molecules are spontaneous creation textured, it is possible to form a microcapsule, higher-order structure body such as vesicles, drug delivery systems in the prior art, has been studied in various fields such as those materials science such utilization of a self-organized polymer of the structure body. For example Patent Document 1 discloses an electrostatic coupling type polymer microcapsule agent carrier, which comprises a non-chargeability segment (non-chargeability polymer embedded chain segment) and the chargeability segment (chargeability polymer embedded chain segment) of a block copolymer, and the inner core portion may have an opposite charge to that on the charge segments of a medicament. On the charge through use of a cationic segment as a segment, the inner core portion so that the DNA.
In addition, various implementations have also been reported with a higher molecular microcapsules with a stabilized approach. For example, in Patent Document 2 discloses an electrostatic coupling type polymer microcapsule agent carrier, wherein the block copolymer cross-linked with each other through via a crosslinking agent to stabilize. Further, in Patent Document 3 discloses an embedded chain segment and a hydrophilic polymer containing a non-chargeability a portion of the side chain of the cationic poly amino acids embedded chain hydrophobic group has been introduced a block copolymer. Through introducing hydrophobic group in a side chain of the block copolymer, such that the surface energy increases, thus enhancing the cohesion of the micro capsule, small core variable, the result is a polymer stabilization of the microcapsules.
Prior Art Document
Patent Document
Patent Document 1: JP Patent No.8-188541 A
Patent Document 2: International Publication Pamphlet No. 2004/105799
Patent Document 3: International Publication Pamphlet No. 2009/113645
Scope of claims [claim1]
1. One encapsulated nucleic acid of the polymer microcapsule complex, characterized in, hydrophilic polymer that contains embedded chain segment and non-chargeability cationic polymer embedded chain segment of the block copolymer, of not less than 1000 bases in length comprising the base sequence of DNA single strand 2 complementary to one another, at least a portion of the double helix structure 1000 base pairs in length of the structure separated from each other a double-stranded DNA or more, or 1000 bases in length of the single-stranded DNA formed above 1,
Embedded chain segments are derived from a hydrophilic polymer selected from non-chargeability polyalkylene glycol, polyoxazoline, polyacrylamide, polymethacrylamide, polyacrylate and polymethacrylate a block of polymer,
Embedded chain segments are derived from a cationic polymer having a cationic group-containing poly amino acids selected from the group in the side chain or a derivative thereof, poly ethyleneimine (PEI) a block of polymer and an acrylic resin, a cationic polymer having a number of recurring units of the embedded chain segment 10 amino-200 amino,
Poly amino acid is selected from the group consisting of polylysine, polyornithine, polyarginine, poly histidine polymer poly homoArgninine well; amino acid derivatives are selected from the group of the aspartic or glutamic acid with the carboxyl group is bonded to the carboxyl group in addition to 1 except for the parts also have selected from amino, imino, quaternary amino groups for a cationic group of a compound that binds the compound 1.

[claim2]
2. The polymer microcapsule encapsulated nucleic acid complex according to claim 1, which is embedded chain segment and a hydrophilic polymer containing a non-chargeability cationic polymer embedded chain segment of the block copolymer, of not less than 1000 bases in length comprising the base sequence of DNA single strand 2 complementary to one another, or at least a portion of the double helix structure 1000 base pairs in length of the structure separated from each other double-stranded DNA formed of the above.

[claim3]
3. The polymer microcapsule encapsulated nucleic acid complex according to claim 1 or 2, wherein, single-stranded DNA to 2000 bases in length or more, double-stranded DNA to 2000 base pairs in length or more.

[claim4]
4. Nucleic acid-encapsulating microcapsule polymer composite according to claim 1 or 2, wherein the use of dynamic light scattering method in an aqueous medium to give an average particle size 100 nm or less.

[claim5]
5. The polymer microcapsule encapsulated nucleic acid complex according to claim 1 or 2, wherein, by electrostatic interactions with the DNA binding of the DNA and a cationic polymer forming the core portion embedded chain segment, the hydrophilic polymer embedded chain segments forming the shell part non-chargeability.

[claim6]
6. Nucleic acid-encapsulating microcapsule polymer composite according to claim 5, wherein, average particle diameter of 50 nm or less of the core portion.

[claim7]
7. The polymer microcapsule encapsulated nucleic acid complex according to claim 1 or 2, which is spherical.

[claim8]
8. Nucleic acid-encapsulating microcapsule polymer composite according to claim 1 or 2, wherein, single-stranded DNA or double-stranded DNA is a linear shape.

[claim9]
9. The polymer microcapsule encapsulated nucleic acid complex according to claim 1 or 2, wherein, at least a portion of the block copolymer cross-linked with each other.

[claim10]
10. The polymer microcapsule encapsulated nucleic acid complex according to claim 1 or 2, wherein, in a cationic polymer chain or side chain of the embedded chain segments are covalently bound to the hydrophobic group.

[claim11]
11. The polymer microcapsule encapsulated nucleic acid complex according to claim 1 or 2, wherein, in a cationic polymer having amine structure or propyl amine structure embedded chain segments of the side chain.

[claim12]
12. Nucleic acid-encapsulating microcapsule polymer according to one method of producing a composite, characterized in, which is one selected from the nucleic acid molecule containing the DNA of the manufacturing method of a composite microcapsules high,
This method comprises the following step: contains the non-hydrophilic polymer embedded chain segment and a cationic polymer on the charge block copolymers of embedded chain, at least a portion of the structure with the duplex dissociation of the double-stranded DNA of more than 1000 base pairs of the state in an aqueous medium are mixed,
Embedded chain segments are derived from a hydrophilic polymer selected from non-chargeability polyalkylene glycol, polyoxazoline, polyacrylamide, polymethacrylamide, polyacrylate and polymethacrylate a block of polymer,
Embedded chain segments are derived from a cationic polymer having a cationic group-containing poly amino acids selected from the group in the side chain or a derivative thereof, poly ethyleneimine (PEI) a block of polymer and an acrylic resin, a cationic polymer having a number of recurring units of the embedded chain segment 10 amino-200 amino,
At least a portion of the structure of the dissociation of the duplex method is to use a heat treatment at 80 °C method of degeneration,
Poly amino acid is selected from the group consisting of polylysine, polyornithine, polyarginine, poly histidine polymer poly homoArgninine well; amino acid derivatives are selected from the group of the aspartic or glutamic acid with the carboxyl group is bonded to the carboxyl group in addition to 1 except for the parts also have selected from amino, imino, quaternary amino groups for a cationic group of a compound that binds the compound 1.

[claim13]
13. Nucleic acid-encapsulating microcapsule polymer of the method for manufacturing a composite according to claim 12, wherein, double-stranded DNA to 2000 base pairs in length or more.

[claim14]
14. Nucleic acid-encapsulating microcapsule polymer of the method for manufacturing a composite according to claim 12 or 13, wherein, double-stranded DNA is a linear shape.

[claim15]
15. Nucleic acid-encapsulating microcapsule method of manufacturing a polymer composite according to claim 12 or 13, wherein, double-stranded DNA double-stranded DNA at 80 °C or more of the denatured.
  • Applicant
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • KATAOKA KAZUNORI
  • OSADA KENSUKE
  • TOCKARY THEOFILUS AGRIOS
IPC(International Patent Classification)
Reference ( R and D project ) PRESTO Molecular technology and creation of new functions AREA
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