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Human pluripotent stem cell exosome loaded with photosensitive drugs and preparation and use thereof

A technology of human pluripotent stem cells and photosensitizing drugs, which is applied in the field of human pluripotent stem cell exosomes and their preparation, can solve the problems that the potential of disease and injury treatment is not fully exerted and needs to be further confirmed.

Active Publication Date: 2019-08-16
上海睿泰生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since milk exosomes contain a large number of non-human genes and proteins, their safety through intravenous systemic injection still needs to be further confirmed, and the focus of this study is mainly on the drug carrier function of exosomes. The therapeutic potential of the substances carried by exosomes to diseases and injuries has not been fully utilized

Method used

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  • Human pluripotent stem cell exosome loaded with photosensitive drugs and preparation and use thereof
  • Human pluripotent stem cell exosome loaded with photosensitive drugs and preparation and use thereof
  • Human pluripotent stem cell exosome loaded with photosensitive drugs and preparation and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Culture of human embryonic stem cells (ESCs) and extraction and identification of exosomes

[0057] A layer of embryonic stem cell Matrigel (ESC-Qualified BD Matrigel, BD Sparks, MD, USA), ESCs were moved into the dish, and mTeSR1 serum-free medium (StemCell Vancouver, BC, Canada), in an incubator (37°C, 5% CO 2 , saturated humidity) culture, and collect the culture medium changed every day. Filter the medium through a 0.22 micron pore size filter membrane and centrifuge at 10,000g at 4°C for 30 minutes to remove cell debris; use a 100KD molecular weight ultrafiltration tube and centrifuge (3500g, 15min) to intercept exosomes in the concentrated supernatant to obtain exosomes concentrate; transfer the concentrate to a 30% sucrose / heavy water density pad (1.210 g / cm 3 ), centrifuge at 100,000g at 4°C for 210 minutes, collect the 5ml sucrose / heavy water density pad at the bottom, add PBS to dilute, transfer to an ultrafiltration centrifuge tube with a molecular weigh...

Embodiment 2

[0062] Culture of human induced pluripotent stem cells (iPSCs) and extraction and identification of exosomes

[0063] A layer of embryonic stem cell Matrigel (ESC-Qualified BD Matrigel, BD Sparks, MD, USA), iPSCs were moved into the dish, and mTeSR1 serum-free medium (StemCell Vancouver, BC, Canada), in an incubator (37°C, 5% CO 2 , saturated humidity) culture, and collect the culture medium changed every day. Filter the medium through a 0.22 micron pore size filter membrane and centrifuge at 10,000g at 4°C for 30 minutes to remove cell debris; use a 100KD molecular weight ultrafiltration tube and centrifuge (3500g, 15min) to intercept exosomes in the concentrated supernatant to obtain exosomes concentrate; transfer the concentrate to a 30% sucrose / heavy water density pad (1.210 g / cm 3 ), centrifuge at 100,000g at 4°C for 210 minutes, collect the 5ml sucrose / heavy water density pad at the bottom, add PBS to dilute, transfer to an ultrafiltration centrifuge tube with a mol...

Embodiment 3

[0068] Human ESC-derived exosomes (ESC-Exos) loaded with 8-methoxypsoralen (8-MOP) by freeze-thaw method

[0069] ESC-Exos solution is from Example 1.

[0070] Determination of the quantitative standard curve of 8-MOP: Accurately weigh 1 mg / mL 8-MOP powder and dissolve it in 1 mL of acetonitrile. Utilize acetonitrile to dilute the above solution, configure 8-MOP standard solution of 100, 70, 50, 20, 10, 5 μg / mL, and then perform HPLC quantitative standard curve determination, the chromatographic conditions are as follows:

[0071] Chromatographic column: Zorbax Extend C-18, 150*4.6μm, 5–micro

[0072] Mobile phase: acetonitrile: water = 55:45

[0073] Flow rate: 1mL / min

[0074] Column temperature: room temperature

[0075] Detection wavelength: 215nm

[0076] After obtaining the corresponding experimental results, the peak area (PA) of the chromatographic peak is plotted as a function of the 8-MOP concentration (C, μg / mL) (as attached image 3 ), obtain the quantitative...

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Abstract

The invention relates to a human pluripotent stem cell exosome loaded with photosensitive drugs and preparation and a use thereof. The photosensitive drug is 8-methoxypsoralen, 5-methoxypsoralen, trimethoprim or a traditional Chinese medicine photosensitizer. Compared with the prior art, the exosome derived from embryonic stem cells and induced human pluripotent stem cells is used as a photosensitive drug carrier including 8-methoxypsoralen, constructs a powerful nano-drug delivery system for promoting skin pigment secretion and improving skin functions, and can be used for treating leucoderma, parapsoriasis guttata, psoriasis and other refractory skin diseases.

Description

technical field [0001] The invention belongs to the technical fields of cell biology, molecular biology and drug research and development, and in particular relates to human pluripotent stem cell exosomes loaded with photosensitizing drugs and its preparation method and application. Background technique [0002] Exosomes are a type of extracellular vesicles secreted by cells with a diameter of about 30-150nm, which carry a variety of biologically active substances from donor cells, including DNA, RNA, protein, etc., into effector cells, effectively regulating the body's cell signal transduction guide. Studies have shown that exosomes secreted by stem cells can physiologically repair or remove damaged, diseased and aging cells in the body by delivering biologically active substances derived from stem cells to effector cells, and play anti-inflammatory, immune-regulating, and promote effector cell proliferation, migration and differentiation , Promote angiogenesis and other f...

Claims

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Application Information

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IPC IPC(8): A61K35/545A61K31/37A61K41/00A61K47/46A61P17/00A61P17/06A61P35/00A61P37/08
CPCA61K9/0014A61K31/37A61K35/545A61K41/0066A61K47/46A61K2300/00
Inventor 汪泱吴复跃何振东
Owner 上海睿泰生物科技股份有限公司
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