Preparation method of composite for treating brain glioma

A glioma and complex technology, which is applied in the directions of non-active ingredients medical preparations, medical preparations containing active ingredients, nano-drugs, etc., can solve the problem of low yield, inability to guarantee the purity of exosomes, and complex modification methods. and other problems to achieve the effect of high drug loading

Active Publication Date: 2019-05-10
SOUTHERN MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This modification method is relatively complicated, the purity of the modified exosomes cannot

Method used

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  • Preparation method of composite for treating brain glioma
  • Preparation method of composite for treating brain glioma
  • Preparation method of composite for treating brain glioma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: Preparation of complex (Exosome-DOX Nano)

[0022] 1. Preparation of complex (Exosome-DOX Nano)

[0023] (1) Extraction and purification of exosomes: fresh grapefruit peeled and squeezed for juice, gradient centrifugation of grapefruit juice: 500g, 10min; Centrifuge for 1 h, take the precipitate, resuspend the precipitate with PBS buffer, and extract exosomes by sucrose density gradient centrifugation (8%, 30%, 45%, 60% sucrose-Tris.HCl solution), and use transmission electron microscopy and dynamic light scattering The appearance characteristics and particle size of exosomes were detected, and the concentration of exosomes was detected by BCA protein quantification method, and the obtained exosomes (Exosomes) were subpackaged and frozen at -80°C.

[0024] see figure 1 , transmission electron microscopy showed that the extracted exosomes were particles with a lipid bilayer structure and a spherical shape, and a dynamic light scattering instrument showed ...

Embodiment 2

[0035] Example 2. Composite (Exosome-DOX Nano) toxicity test on glioma cells

[0036] Resuscitate human glioma cells U87 and U251. In the logarithmic growth phase of the cells, inoculate the U87 and U251 cells in a 96-well plate with a cell number of about 3000 per well. Set 5 concentration gradients for the standard, that is, 0.2 μg / mL, 0.1 μg / mL, 0.05 μg / mL, 0.025 μg / mL, 0.0125 μg / mL, 0 μg / mL to act on the cells, and set 5 duplicate holes for each concentration. Routine culture in the dark for 48 hours after adding the drug, add MTT to continue the culture for 4 hours, detect the absorbance of each well at 490nm with a microplate reader, according to the formula relative cell viability (%)=(OD experimental group mean / OD control group mean)×100 % Calculate the relative survival rate of each group of cells respectively, the results are as follows Figure 6 As shown, the inhibitory ability of doxorubicin and doxorubicin nanomedicine to U87 is close, and the toxicity of the com...

Embodiment 3

[0037] Example 3. Distribution of compound exosomes in nude mice with orthotopic glioma implantation

[0038] When the nude mice grow to about 18 g, the nude mice are anesthetized with pentobarbital sodium, and 150,000 U87 glioma cells overexpressing luciferase are planted in the right striatum of the nude mouse brain. After about 3-4 days, the peritoneal The luciferase substrate was injected, and the growth of intracranial tumors in nude mice was observed 10 minutes later using a small animal in vivo imager. When the intracranial tumor grows to an appropriate size, a certain amount of cy7-labeled Exosome-cy7, DOX Nano-cy7, Exosome-DOX Nano-cy7 (the total amount of cy7 in each group is equal) is injected into the tail vein, and the nude mice are killed at 96 hours, and their hearts are collected. , liver, spleen, lung, kidney and brain, respectively observe the distribution of drugs in each organ and the localization of drugs in brain tumors, the results are as follows Figur...

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Abstract

The invention relates to a preparation method of a composite for treating brain glioma. The method includes the following steps that firstly, heparin serves as a carrier to carry cRGD tumor targetingpeptide, the heparin is connected with adriamycin amycin through a chemical acid-sensitive hydrazone bond, and adriamycin amycin nano-medicine is prepared; then under catalysis of EDC/NHS, carboxyl onthe adriamycin amycin nano-medicine is coupled with amino on the surface of exosome together, and therefore nano-particles of the adriamycin amycin nano-medicine are densely distributed on the surfaces of vesicae of the exosome. The composite for treating brain glioma prepared with the method can be targeted on tumors and pass through blood brain barriers and further has the advantage of being high in medicine loading capacity.

Description

technical field [0001] The present invention relates to a medical preparation characterized by the non-active ingredients used, in particular to a complex of nano-medicines containing an organic heterocyclic compound doxorubicin carried by exosomes derived from plant cells, and the complex is suitable for treating brain gel Glioma. Background technique [0002] Glioma (GBM) is a common malignant primary tumor and the most destructive brain tumor. It mainly occurs in adults over the age of 20, with an incidence rate of about 1 / 100,000. The prognosis of glioma is extremely poor , the median survival time is about 14 months, and the 5-year survival rate is only about 5%. Chemotherapy is a common treatment method for glioma, but due to the existence of the blood-brain barrier, there are very few chemotherapy drugs used in the treatment of glioma. Many chemotherapeutic drugs (such as doxorubicin) have a good killing effect on glioma cells, but they are difficult to pass through...

Claims

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Application Information

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IPC IPC(8): A61K47/69A61K31/704A61K47/64A61P35/00B82Y5/00B82Y30/00
Inventor 王莺牛文博
Owner SOUTHERN MEDICAL UNIVERSITY
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