Composite exosome loaded with cRGD and small-molecule antitumor drug as well as preparation method and application of composite exosome

An anti-tumor drug and small molecule technology, which is applied in the direction of anti-tumor drugs, drug combinations, medical preparations of non-active ingredients, etc., can solve the problems of narrow therapeutic window, poor solubility of triptolide, strong liver and kidney toxicity, etc. Achieve the effect of reducing toxic and side effects, excellent physical and chemical properties, and good targeting

Pending Publication Date: 2022-06-07
FUDAN UNIV SHANGHAI CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantages of triptolide, such as poor solubility, narrow therapeutic window, and strong liver and kidney toxicity, severely limit its clinical application.

Method used

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  • Composite exosome loaded with cRGD and small-molecule antitumor drug as well as preparation method and application of composite exosome
  • Composite exosome loaded with cRGD and small-molecule antitumor drug as well as preparation method and application of composite exosome
  • Composite exosome loaded with cRGD and small-molecule antitumor drug as well as preparation method and application of composite exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Preparation and characterization of EXO

[0056]1. Exosome extraction: Take the cells in the logarithmic growth stage, when the cell fusion reaches 70%-80%, discard the culture medium, PBS wash 2-3 times, and replace it with serum-free medium for starvation culture. After 12 h of culture, collect the cell supernatant and isolate the exosomes by gradient ultracentrifugation: at 4 °C, centrifuge the cell supernatant 300 g for 10 min to remove the cells; Centrifuge at 2000 g for 10 min to remove dead cells; Centrifuge at 10,000 g for 30 min to remove cell debris; 120000 g ultra-high-speed centrifugation for 70 min, collection of pellet, PBS resuspending; Perform a 120,000 g centrifugation again for 70 min, collect the pellet, and after resuspending with PBS, store at -80 °C for follow-up experiments.

[0057] 2. Exosome characterization: transmission electron microscopy experiment: resuspend EXO with 100 μL 2% paraformaldehyde, take 5 μL EXO suspension and drop it o...

Embodiment 2

[0061] Example 2: Preparation of cRGD-EXO

[0062] 1. Preparation: DSPE-PEG2000-c (RGDfk) (hereinafter referred to as cRGDfk) was treated at 60 °C in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES) for 15 min to form a micelle.

[0063] The micelle is sonicated at 10 μm amplitude for 2×5s to reduce the size of the micelle and thus facilitate its separation from EXO. Mix the EXO suspension with the above suspension at 40 °C for 2 h. Immediately cool to 4 °C, centrifuge at 120,000 × g for 70 min to purify exosomes to give cRGD-EXO.

[0064]2. ExO and cRGDfk mass ratio optimization: Taking the average fluorescence intensity of cell uptake as the index, the mass ratio of EXO and cRGDfk is set to 5:1, 1:1, 1:5, 1:10, 1:15 (μg protein: μg), and the flow cytometry is used for one-way optimization. The specific procedure of the flow cytometer is as follows: A375 cells are × 50 5 Cells / mL density inoculated in 6-well plates at 37 °C, 5% CO 2 Incubate in an incubator for 2...

Embodiment 3

[0067] Example 3: preparation and characterization of cRGD-EXO / TPL

[0068]1. Preparation and drug loading optimization of cRGD-EXO / TPL: at room temperature, mix cRGD-EXO with TPL evenly, incubate the shaker for 90 min, and the speed is 200 rpm. In order to select the optimal ratio of cRGD-EXO and TPL, the mass ratios of cRGD-EXO and TPL were set to 10:1, 5:1, 1:1, 1:5, 1:10 (μg protein: μg) based on the drug encapsulation rate and drug loading, and the one-way investigation was carried out. Finally, the purified cRGD-EXO containing TPL is purified by ultracentrifugation to obtain a purified cRGD-EXO / TPL. TPL content in cRGD-EXO / TPL was detected by high performance liquid chromatography.

[0069] 2. cRGD-EXO / TPL characterization: Transmission electron microscopy experiment: cRGD-EXO / TPL are fixed, dried, cleaned, negatively stained, and naturally dried, respectively, detected on TEM, set the acceleration voltage to 80kV, observed and photographed.

[0070] Particle size analysis...

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Abstract

The invention relates to the technical field of pharmaceutical preparations, in particular to a composite exosome loaded with polypeptide and a small-molecule antitumor drug as well as a preparation method and application of the composite exosome. According to the composite exosome drug delivery system, exosome (Exosome, EXO) derived from human umbilical cord mesenchymal stem cells is used as a carrier, annular polypeptide cRGD is embedded on the membrane surface of the carrier so as to be delivered to melanoma cells with high expression of alpha v beta 3 integrin receptors in a targeted mode, small-molecule anti-tumor drugs are entrapped in the exosome membrane, and the drug delivery efficiency is improved. And constructing a composite exosome drug delivery system cRGD-EXO/small molecule antitumor drug. In-vivo and in-vitro experiments show that the drug delivery system has good tumor targeting property, and shows a remarkable tumor inhibition effect while reducing the toxic and side effects of the drug. The invention provides a small molecule drug delivery system which is good in targeting property, high in biological safety and exact in tumor inhibition effect for tumor treatment with high expression of an alpha v beta 3 integrin receptor.

Description

Technical field [0001] The present invention relates to the field of pharmaceutical formulation technology, in particular, is a composite exosome loaded with cRGD and small molecule antitumor drugs and preparation methods and applications thereof. Background [0002] Integrins are a class of glycoprotein receptors located on most cell membranes that mediate adhesion between cells and between cells and the extracellular matrix, and signaling between cells and the extracellular matrix. Alphavβ3 integrin is the most important member of the 24 integrins, which play an important role in tumor metastasis, invasion and vascular formation. αvβ3 integrin receptors are specifically expressed on activated endothelial cells and certain tumor cells (e.g., human malignant melanoma, prostate cancer, glioblastoma, and breast cancer), but are not specific or rarely expressed on quiescent endothelial cells. Therefore, this feature can be used to target the tumor site or tumor blood vessels. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/69A61K47/62A61K31/585A61K45/00A61P35/00C12N5/10
CPCA61K47/6901A61K47/62A61K31/585A61K45/00A61P35/00C12N5/0662C12N5/0645C12N5/0646C12N5/0636C12N5/0693C07K7/64
Inventor 刘继勇顾永卫杜月武鑫姜良弟李丹唐晓萌李爱雪赵语南
Owner FUDAN UNIV SHANGHAI CANCER CENT
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