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Gene-loaded exosome as well as preparation method and application thereof

A technology of exosomes and genes, applied in the field of biomedicine, can solve problems such as unsatisfactory effects and limited prevention and treatment methods for heterotopic ossification

Pending Publication Date: 2022-05-10
SHANGHAI SIXTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the embodiments of the present invention is to provide a gene-carrying exosome, which aims to solve the problem that the existing prevention and treatment methods for heterotopic ossification are limited and the effect is not ideal

Method used

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  • Gene-loaded exosome as well as preparation method and application thereof
  • Gene-loaded exosome as well as preparation method and application thereof
  • Gene-loaded exosome as well as preparation method and application thereof

Examples

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Effect test

preparation example Construction

[0033] The embodiment of the present invention also provides a method for preparing the above-mentioned gene-carrying exosomes, comprising the following steps:

[0034] Step S1: reacting phospholipid polyethylene glycol succinimide ester with CD34 antibody to synthesize an exosome treatment solution.

[0035] Step S2: Dispersing secreted exosomes in phosphate buffered saline solution, adding the exosome treatment solution and incubating at room temperature to obtain exosome carriers.

[0036] In the embodiment of the present invention, the exosome membrane is mainly composed of lipid membrane, so fat-soluble compounds are easy to integrate into the exosome membrane. The present invention synthesizes DSPE-PEG-AbCD34 by conjugating CD34 antibody to phospholipid polyethylene glycol succinimide ester (DSPE-PEG-NHS), and then treats exosomes with DSPE-PEG-AbCD34 to convert lipophilic DSPE Fused to the exosome membrane, PEG-AbCD34 is thus carried to the surface of the exosome to ob...

Embodiment 1

[0059] Example 1 Preparation and Characterization of Exosome-DSPE-PEG2000-AbCD34

[0060] In this example, exosomes were isolated from the culture medium of mouse aortic endothelial cells by mixing 5 mL of phospholipid polyethylene glycol succinimide ester (DSPE-PEG2000-NHS) (0.1 M) with 10 uL of excess CD34 Antibody (AbCD34) (1mg / mL) was reacted at room temperature for 24h to synthesize exosome treatment solution (DSPE-PEG2000-AbCD34). Then the prepared exosomes were dispersed in 5 mL of PBS, and the protein content was adjusted to 50 mg / mL, and 0.1 mL of DSPE-PEG2000-AbCD34 solution with a concentration of 20 mg / mL was added to 2 ml of exosome dispersion liquid, blown gently Disperse the mixture and incubate at room temperature for 1 h. Then, transfer the dispersion to a special centrifuge tube, and ultracentrifuge at 120,000 rpm at 4°C for 70 min. Aspirate the upper layer solution to remove unbound DSPE-PEG2000-AbCD34, and the resulting precipitate is the exosome carrier ...

Embodiment 2

[0063] Example 2 Detection of biocompatibility, stability, targeting and cell internalization of Exosome-DSPE-PEG2000-AbCD34

[0064] Biocompatibility, stability, targeting, and cellular internalization are important properties of gene carriers. The present invention uses the CCK-8 method to detect the effects of different concentrations of Exosome-DSPE-PEG2000-AbCD34 on the viability of mouse aortic endothelial cells, and the results show that all concentrations of Exosome-DSPE-PEG2000-AbCD34 have no effect on the survival rate of the cells ( Figure 5 ).

[0065] Further, the present invention tested the stability of Exosome-DSPE-PEG2000-AbCD34 at 4°C and 37°C, and the results showed that Exosome-DSPE-PEG2000-AbCD34 could be stored stably at 4°C and 37°C for at least 8 days ( Image 6 ).

[0066] Further, the present invention observed the targeting effect of Exosome-DSPE-PEG-2000AbCD34 on mouse aortic endothelial cells through laser confocal microscopy, and the results s...

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Abstract

The invention is applicable to the technical field of biomedicine, and provides a gene-loaded exosome as well as a preparation method and application thereof, and the gene-loaded exosome is obtained by loading an SMAD7 gene into an exosome carrier; wherein the exosome carrier is formed by conjugating a CD34 antibody and phospholipid polyethylene glycol succinimide ester and then binding the conjugated antibody and phospholipid polyethylene glycol succinimide ester with the surface of a secretory exosome. A CD34 antibody is conjugated with phospholipid polyethylene glycol succinimide ester and then is combined with the surface of a secretory exosome to synthesize an exosome carrier with a high endothelial cell targeting characteristic, and then an SMAD7 gene is loaded into the exosome carrier, so that a novel gene-loaded exosome is prepared; a new preclinical research strategy is provided for targeted therapy of HO, and a certain theoretical basis is provided for further research and elaboration of pathogenesis of HO.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a gene-carrying exosome and its preparation method and application. Background technique [0002] Heterotopic ossification (HO) is an abnormal bone formation process that occurs in muscle or connective tissue, mostly around large joints, such as hip and knee joints, as a complication of central nervous system diseases, common in neurologically paralyzed patients. HO can directly reduce the range of motion of adjacent joints, limit joint motion, and even cause joint stiffness and loss of function. It may also cause neurologic symptoms, pressure damage to peripheral nerves and pressure sores, forming localized pressure ulcers. Compared with normal bone, heterotopic ossified bone has higher metabolic activity, bone deposition rate, suture width, and osteoblast number. The disability rate of this disease is as high as 10% to 36%, and the high-incidence age group is...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/12A61K47/24A61K47/42A61K47/46A61K48/00A61P19/08
CPCC12N5/069C12N15/85C07K14/4703A61K48/005A61K48/0008A61K47/42A61K47/24A61K47/46A61P19/08C12N2510/00C12N2800/107
Inventor 仲飙
Owner SHANGHAI SIXTH PEOPLES HOSPITAL
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