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Transfection agent

A technology of transfection agent and surfactant, applied in the field of transfection agent, can solve problems such as unclear use of transfection agent

Inactive Publication Date: 2011-07-20
3 D MATRIX +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its use as a transfection agent is unclear

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Comparison of Cytotoxicity between Peptide Surfactant and Cationic Liposome Gene Transfer Agent

[0036] Human cultured cells were used to evaluate the cytotoxicity of peptide surfactants and two kinds of cationic liposome gene transfer agents widely used at present.

[0037]

[0038] · Added substances

[0039] 1. Peptide surfactant (sequence: Ac-AAAAAAK-NH 2 , manufactured by Celtek)

[0040] 2. Lipofectamine 2000 (manufactured by Invitrogen Corporation)

[0041] 3. siFECTOR (manufactured by B-Bridge International, Inc.)

[0042] ·cell

[0043] Human malignant glioma cultured cells (U87MG, purchased from ATC C)

[0044] MTT assay reagent (quantification of cell proliferation number)

[0045] CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit (manufactured by Promega KK)

[0046]

[0047] According to the protocol recommended by ATCC, cells were seeded into 96-well plates (cell density: 2×10 4 / well) and incubated overnight. The culture medium w...

Embodiment 2

[0050] Confirmation of the introduction of siRNA into cells using peptide surfactants

[0051] Fluorescence-labeled siRNA was observed under a microscope to confirm that siRNA had been introduced into cells by the peptide surfactant.

[0052]

[0053] ·Peptide surfactant

[0054] Sequence: Ac-AAAAAAK-NH 2 , manufactured by Celtek

[0055] siRNA

[0056] siRNA / MMP-2, manufactured by Santa Cruz Corporation

[0057] ·siRNA fluorescent labeling reagent

[0058] Label IT Cy-3 Labeling Kit, manufactured by Mirus

[0059] ·cell

[0060] Three kinds of human malignant glioma cultured cells (U87MG, T98G, U251MG, respectively purchased from ATC C)

[0061]

[0062] According to the protocol of Mirus Company, siRNA / MMP-2 was labeled with Cy3, a fluorescent siRNA labeling reagent. According to the protocol of ATCC, 5000 cells were inoculated on the culture chamber slide glass (Chambered slide glass) and left overnight. The medium was removed, and siRNA was added to 1 ml of th...

Embodiment 3

[0066] Time-dependent analysis of the amount of siRNA introduced into cells using a peptide surfactant

[0067] By observing fluorescently-labeled siRNA with a microscope, changes over time in the amount of siRNA introduced into cells using a peptide surfactant were studied.

[0068]

[0069] ·Peptide surfactant

[0070] Sequence: Ac-AAAAAAK-NH 2 , manufactured by Celtek

[0071] · Peptide surfactant concentration

[0072] 0, 0.5, 1.0, 5.0, 10.0, 20.0, 25.0μg / mL

[0073] siRNA

[0074] siRNA / MMP-2, manufactured by Santa Cruz Corporation

[0075] ·siRNA fluorescent labeling reagent

[0076] LabelIT Cy-3 Labeling Kit, manufactured by Mirus

[0077] ·cell

[0078] Three kinds of human malignant glioma cultured cells (U87MG, T98G, U251MG, purchased from ATCC)

[0079]

[0080] According to the standard protocol of Mirus Company, siRNA / MMP-2 was labeled with siRNA fluorescent labeling reagent Cy3. The labeled siRNA is mixed with peptide surfactant. For 1 × 10 5 Each...

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Abstract

Disclosed is a transfection agent which has lower toxicity and can be used for clinical purposes. The transfection agent comprises a peptide surfactant.

Description

technical field [0001] The present invention relates to transfection agents containing peptide surfactants. Background technique [0002] Since RNAi can suppress the expression of a specific gene, it is expected to be applied to the treatment of diseases. Conventionally, cationic phospholipids are the main transfection agents used for transfecting siRNA into cells, but their toxicity has been pointed out. [0003] However, considering the clinical application of nucleic acid drugs such as siRNA, the toxicity of transfection agents is expected to be infinitely low, and the development of transfection agents with lower toxicity is expected. [0004] Peptide surfactants are known to have an action of stabilizing membrane proteins such as the G protein-binding receptor bovine rhodopsin, and self-organizing properties (Non-Patent Documents 1 and 2). However, its use as a transfection agent is unclear. [0005] Non-Patent Document 1: X Zhao et al., PNAS, Vol.103, No.47, 17707-1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00A61K47/42A61K48/00A61P35/00A61P43/00C07K7/06
CPCC07K7/06A61K47/42B82Y5/00C12N15/87A61P35/00A61P43/00A61K9/20A61K9/48
Inventor 吉田大藏武井次郎
Owner 3 D MATRIX
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