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

外国特許コード F170009172
整理番号 K10503WO
掲載日 2017年9月12日
出願国 中華人民共和国
出願番号 201480043775
公報番号 105451719
公報番号 105451719
出願日 平成26年8月5日(2014.8.5)
公報発行日 平成28年3月30日(2016.3.30)
公報発行日 令和元年6月11日(2019.6.11)
国際出願番号 JP2014070567
国際公開番号 WO2015020026
国際出願日 平成26年8月5日(2014.8.5)
国際公開日 平成27年2月12日(2015.2.12)
優先権データ
  • 特願2013-163106 (2013.8.6) JP
  • 2014JP70567 (2014.8.5) WO
発明の名称 (英語) Nucleic acid-encapsulating polymer micelle complex and method for producing same
発明の概要(英語) 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.
従来技術、競合技術の概要(英語) 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
特許請求の範囲(英語) [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.
  • 出願人(英語)
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • 発明者(英語)
  • KATAOKA KAZUNORI
  • OSADA KENSUKE
  • TOCKARY THEOFILUS AGRIOS
国際特許分類(IPC)
参考情報 (研究プロジェクト等) PRESTO Molecular technology and creation of new functions AREA
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