Charge conversional ternary polyplex

Inactive Publication Date: 2011-03-10
THE UNIV OF TOKYO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020]The present invention can provide a charge conversional ternary polyplex that is charged negatively under a neutral condition but its charge is converted to give a cation under an acidic condition.
[0021]In the field of gene vector studies, transfection into a toxicity-sensitive primary cell such as HUVEC is extremely difficult. A polyplex of the present invention is capable of delivering a nucleic acid suc

Problems solved by technology

Nonetheless, even in the case of a nonviral vector, the main concern is the conflict between the delivery efficiency and the safety issues (in particular, chemical toxicity).
While most vectors having high transfection efficiency show high toxicity, vectors with low toxicity are often associated with low transfection efficiency.
However, due to the toxicity of these polycations, polyplexes are used only for limited applications[2].
On the contrary, the positively-charge

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Example

EXAMPLE 1

Materials and Methods

[0136]1. Materials

[0137]N,N-dimethylformamide (DMF) (Wako Pure Chemical Industries, Ltd, Japan), dichloromethane (DCM) (Wako, Japan), n-butylamine, ethylenediamine (1,2-diaminoethane), and diethylenetriamine (bis(2-aminoethyl)amine) (Tokyo Chemical Industry Co. Ltd, Japan) were purchased and redistilled before use. Acetic acid and hydrochloric acid were purchased and used without further purification (Wako, Japan). 1-methyl-2-pyrrolidinone (NMP), cis-aconitic anhydride, succinic anhydride and bovine serum albumin were purchased from Sigma (St. Louis, Mo.). β-benzyl-L-aspartate-N-carboxy-anhydride (BLA-NCA) was obtained from Nippon Oil and Fats Co., Ltd. (Tokyo, Japan).

[0138]2. Synthesis

[0139]2-1. Synthesis of PBLA (poly([3-benzyl-L-aspartate)) (2)

[0140]PBLA was prepared by ring-opening polymerization of BLA-NCA initiated by the terminal amino group of n-butylaminc. n-butylamine (0.0417 mmol) was dissolved in 5 mL of DMF / DCM (1:10). BLA-NCA (4.60 mmol) s...

Example

EXAMPLE 2

Experimental Method

[0176](1) Synthesis of Poly(L-lysine) (PLL) Homopolymer

[0177]An eggplant shaped flask provided with a three-way stopcock was vacuum-dried, added with Lys(TFA)-NCA (1.01 g, 3.77 mmol) under an argon (Ar) atmosphere, and then added with DMSO (8 mL) for lysis. As an initiator, n-butylamine (5.4 mg, 73.8 μmol) was added to the monomer solution, and agitated for about 48 hours in a thermostatic bath at 35° C. After the reaction, the polymerization solution was slowly dripped in an excessive amount of thoroughly agitated diethyl ether to precipitate the polymer. Once most polymer has precipitated after about an hour of agitation, supernatant solution was removed by decantation. The precipitated polymer was dried under reduced pressure at room temperature. According to 1H NMR analysis, the chain length (degree of polymerization (DP) of lysine) was 52.

[0178]50 mg of P[lys(TFA)] (DP: 52) was dissolved in 5 ml of MeOH, added with 0.5 ml of 1N NaOH and agitated at 3...

Example

EXAMPLE 3

Experimental Method

[0212](1) Synthesis of N-Succinimidyl Octadecanoate

[0213]N-succinimidyl octadecanoate was synthesized according to a known method [N. M. Howarth, W. E. Lindsell, E. Murray, P. N. Preson, Tetrahedron 61 (2005) 8875-8887]. Stearic acid (1.87 g, 6.56 mmol) and N-hydroxysuccinimide (0.76 g, 6.56 mmol) was dissolved in 80 mL of dichloromethane (DCM), and allowed to react with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (WSC) (1.25 g, 6.56 mmol) for 48 hours. Thereafter, the resultant was washed with water, extracted twice with DCM and dried with MgSO2 to obtain white powder (amount 1.4 g, yield 56%). The conversion ratio of the carboxyl group of stearic acid was 96% as calculated by 1H-NMR.

[0214](2) Synthesis of Stearoyl Group-Introduced Poly(L-Lysine)

[0215]poly(L-Lysine) (molecular weight 20,000) and DIPEA were dissolved in methanol, and allowed to react with N-succinimidyl octadecanoate dissolved in a small amount of dichloromethane at 4° C....

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Abstract

Disclosed is a polymer composite (polyplex) that contains nucleic acid, a cationic polymer, and an anionic polymer. The anionic polymer covers the surface of the composite comprising the cationic polymer and nucleic acid, has a negative charge at neutral pH, and can change so as to have a positive charge at mildly acidic pH.

Description

TECHNICAL FIELD[0001]The present invention relates to a polymer complex (polyplex) containing a nucleic acid for use as a nonviral synthetic vector capable of delivering the nucleic acid to a target cell. More particularly, the present invention relates to a charge conversional ternary polyplex comprising a nucleic acid, a cationic polymer and an anionic polymer, wherein the anionic polymer covers the surface of a complex comprising the cationic polymer and the nucleic acid.BACKGROUND ART[0002]DNA or RNA delivery to a target cell mediated by a nonviral synthetic vector (lipoplex and polyplex) has been widely recognized as a promising alternative method for delivery that uses a viral vector which has been confronting safety issues specific to biological properties[1]. Nonetheless, even in the case of a nonviral vector, the main concern is the conflict between the delivery efficiency and the safety issues (in particular, chemical toxicity). While most vectors having high transfection ...

Claims

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Application Information

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IPC IPC(8): C07K14/00
CPCA61K31/7084A61K31/785A61K48/0041C08G69/10C12N2320/32C08G69/48C12N15/111C12N15/88C12N2310/14C08G69/40A61P35/00A61P43/00
Inventor KATAOKA, KAZUNORILEE, YANNISHIYAMA, NOBUHIROMIYATA, KANJIROOBA, MAKOTOHIKI, SHIGEHIROSANJO, MAIKIM, HYUNJIN
Owner THE UNIV OF TOKYO
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