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Human pluripotent stem cell exosomes loaded with antitumor drugs and preparation method and use of human pluripotent stem cell exosomes

A technology of human pluripotent stem cells and anti-tumor drugs, applied in non-embryonic pluripotent stem cells, artificially induced pluripotent cells, anti-tumor drugs, etc., can solve the problems that the treatment potential of diseases and injuries has not been fully utilized and needs further confirmation , to achieve the effect of reducing dosage and reducing side effects

Pending Publication Date: 2019-08-23
上海市第六人民医院东院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 exosomes loaded with antitumor drugs and preparation method and use of human pluripotent stem cell exosomes
  • Human pluripotent stem cell exosomes loaded with antitumor drugs and preparation method and use of human pluripotent stem cell exosomes
  • Human pluripotent stem cell exosomes loaded with antitumor drugs and preparation method and use of human pluripotent stem cell exosomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Culture of human embryonic stem cells (ESCs) and extraction and identification of exosomes

[0122] 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), cultivated in an incubator (37° C., 5% CO2, saturated humidity), and collected 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 30% sucrose / heavy water density pad (1.210g / cm3), centrifuge at 100000g at 4°C for 210 minutes, collect the bottom 5ml sucrose / heavy water density pad, add PBS to dilute, and transfer to an ultra-thin filter with a molecular weight cut-off of 100KD ...

Embodiment 2

[0127] Culture of human induced pluripotent stem cells (iPSCs) and extraction and identification of exosomes

[0128] 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), cultivated in an incubator (37° C., 5% CO2, saturated humidity), and collected 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...

Embodiment 3

[0133] Human ESC-derived exosomes (ESC-Exos) encapsulated paclitaxel (PTX) by co-incubation method

[0134] ESC-Exos solution is from Example 1. Quantitative standard curve determination of PTX: Accurately weigh 1mg of PTX powder and dissolve it in 1mL of methanol, then dilute the solution with methanol to configure methanol standard solutions of 50μg / mL, 20μg / mL, and 10μg / mL of PTX, HPLC injection. The test conditions are as follows: flow rate 1mL / min; injection volume 10μL; detection wavelength 227nm; column temperature 30°C, separation column: Zorbax Extend-C18Analytical, 4.6×150mm 5-micron.

[0135] After obtaining the corresponding experimental results, the peak area (PA) of the chromatographic peak was plotted as a function of the PTX concentration (C, μg / mL) (see attached Figure 6 shown), obtain the quantitative standard curve of Res under the following chromatographic separation conditions: PA=26.7388C+2.6119

[0136] Encapsulation of PTX: prepare 1 mg / mL PTX in ph...

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Abstract

The invention relates to human pluripotent stem cell exosomes loaded with antitumor drugs and a preparation method and use of the human pluripotent stem cell exosomes. According to the method, the human pluripotent stem cell exosomes are taken as nanoscale carriers of the antitumor drugs, and by performing RGD peptide artificial modification on the surfaces of the exosomes, the exosomes have stronger tumor targeting, thereby constructing a natural drug delivery system easily passing through a blood brain barrier and having the tumor targeting. The human pluripotent stem cell exosomes loaded with the antitumor drugs have both the tumor resistance, immune adjustment, inflammatory resistance and other functions of pluripotent stem cell exosomes and the specific antitumor function of the antitumor drugs; the dual functions of the pluripotent stem cell exosomes and the antitumor drugs can better exert the antitumor effect and greatly reduce the dosage of the antitumor drugs, thereby greatlyreducing the toxic and side effects of the antitumor drugs; by using the nanoscale properties of the exosomes, the exosomes can pass through the blood brain barrier, thereby having unique advantagesfor the treatment of central nervous system tumors; the human pluripotent stem cell exosomes loaded with the drugs are modified by RGD peptides, thereby greatly enhancing tumor targeting of the exosomes.

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 a human pluripotent stem cell exosome loaded with antineoplastic 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 and immune-regulating functions. In recent years, it has been reported that by adding a certain amount of microvesicles (extra...

Claims

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Application Information

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IPC IPC(8): A61K47/69A61K47/64A61K45/00A61P35/00C12N5/074C12N5/0735
CPCA61K45/00C12N5/0606C12N5/0696C12N2500/90
Inventor 邓志锋汪泱杨云龙朱庆伟
Owner 上海市第六人民医院东院
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