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Transfection method

A bubble and cell technology, applied in the field of transfection, to achieve the effects of low-cost production, cost-effective production, and improved introduction efficiency

Pending Publication Date: 2022-04-01
TAKEDA PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the introduction of target substances (such as nucleic acid, protein, etc.) into cells by combining nanobubble water with ultrasound

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0185] Reference Example 1: Preparation of Ultrafine Bubble Water

[0186] Polysorbate 80 (2 g) (0.1% polysorbate 80) was dissolved in water for injection (2 L) or diluted Mclivaine buffer (pH 3.0, 2 L), and an ultrafine Bubble generator (nanoGALF TM FZ1N-02) to prepare an aqueous solution of ultrafine bubbles. Positively charged ultrafine bubbles can be generated when an acidic buffer (diluted Mclivaine buffer: pH 3.0) is used, and negatively charged ultrafine bubbles can be generated when water for injection is used.

[0187] Gases used for preparation: air (Example 3), C 3 f 8 (Example 1, 2, 4)

[0188] Bubble water flow: about 4.0L / min

[0189] Dissolving pressure: 300KPa±5%

[0190] The prepared ultra-fine bubble water was subjected to autoclaving for 30 min at 121° C.-124° C. using an autoclave as appropriate. After the sterilization, the ultrafine bubble average diameter, the ultrafine bubble density, and the d90 / d10 ratio were measured by a tracking method usin...

Embodiment 2

[0201] Embodiment 2 is introduced with the comparison of microbubble

[0202] C in DMEM medium containing 10% FBS 2 C 12 cells (3×10 4 cells, 0.5 mL) were seeded in 48 wells. After removing the medium, 0.5 mL of ultrafine bubble / DMEM medium or microbubble / DMEM medium containing 5 μg of siRNA labeled with FAM fluorescence (FAM-siRNA) was added. The ultrafine bubble / DMEM medium and the microbubble / DMEM medium prepared in the following manner were used as the transfection medium: 1 L of the ultrafine bubble aqueous solution prepared in Reference Example 1 or commercially available microbubble water Add to DMEM medium powder, and add 2g / L NaHCO 3 and 10% FBS. As the microbubble water, Sonazoid (average diameter: 3 μm) containing phosphatidylserine as a phospholipid in the constituent components was used.

[0203] After adding each FAM-siRNA-containing medium, use an ultrasonic generator (NEPAGENE) at a frequency of 1 MHz and 500 mW / cm 2 The output intensity of ultrasonic ir...

Embodiment 3

[0206] The introduction of embodiment 3 protein to skeletal muscle cell

[0207] C in DMEM medium containing 10% FBS 2 C 12 cells (6×10 4 cells, 1 mL) were seeded in 48 wells. After removing the medium, 1 mL of IgG-FITC solution diluted 100-fold with ultrafine bubble / DMEM medium was added. The ultrafine bubble / DMEM medium prepared in the following manner was used as a transfection medium: 1 L of the ultrafine bubble aqueous solution prepared in Reference Example 1 was added to the DMEM medium powder, and 2 g / L NaHCO 3 and 10% FBS.

[0208] After adding IgG-FITC-containing medium, at 500mW / cm 2 The output intensity and the frequency of 0.5MHz or 3MHz and ultrasonic irradiation using ultrasonic generator (NEPAGENE) for 10s. After ultrasonic irradiation, the cells were cultured for 2 h, then the transfection medium was removed, and the cells were cultured in DMEM medium (cultivation medium) for 48 h.

[0209] After the cultivation was completed, the fluorescence intensity ...

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Abstract

The present invention provides a novel method for safely and efficiently introducing a target substance such as a nucleic acid or a protein into a cell (excluding an immune cell). Specifically, provided are: a delivery system for introducing a target substance into cells (excluding immune cells), the delivery system being prepared by combining ultra-fine bubble water or an aqueous ultra-fine bubble solution comprising phospholipid-free ultra-fine bubbles having an average diameter of 200 nm or less with an ultrasound generator; and a method for improving the delivery ability of nucleic acids, proteins or low molecular weight compounds to cells (excluding immune cells) by using the ultrafine bubble water or the like and ultrasonic waves.

Description

[technical field] [0001] The present invention relates to a system for delivering a target substance to cells (excluding immune cells), the system comprising a combination of ultrafine bubble water containing ultrafine bubbles or an aqueous solution of ultrafine bubbles and an ultrasonic generator; Method for increasing delivery of nucleic acid, protein or low molecular weight compound to cells (excluding immune cells) by using said ultrafine bubble water or aqueous solution and ultrasound; a method comprising nucleic acid, protein or low molecular weight compound and said ultrafine bubble A combined formulation of water or an aqueous solution for delivering said nucleic acid, protein or low molecular weight compound to cells (excluding immune cells) by use in combination with ultrasound irradiation; And a method in which said cells are sonicated to deliver nucleic acids, proteins or low molecular weight compounds to said cells (excluding immune cells); etc. (Background tech...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/26A61K9/10A61K47/32A61K48/00
CPCA61K9/0009A61K9/0019A61K9/5138A61K9/5123C12N15/87C12N15/111C12N2310/14C12N2320/32C12N2310/3517A61K41/0028A61K48/0033A61K48/0083C12N15/113C12N15/88
Inventor 村田直之柳井薫雄
Owner TAKEDA PHARMA CO LTD
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