Exogenous mitochondrial vector and compound and preparation method and application of exogenous mitochondrial vector and compound
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A mitochondrial carrier, mitochondrial technology, applied in the field of biomedicine
Pending Publication Date: 2021-06-22
CHINA PHARM UNIV
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At present, the treatment of mitochondrial defect-related diseases mainly focuses on symptomatic treatment (for example, the use of anticonvulsant drugs to treat epilepsy caused by ME
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Embodiment 1
[0034] The mitochondrial donors in this example are selected from various tumor cell lines such as HeLa, MCF-7, HepG-2, L0 2 , LX 2 , H293T and other normal cell lines, and one or more of autologous tissues and organs. Extract exogenous mitochondria Mito using the Mitochondria Extraction Kit according to the instructions. The isolated exogenous mitochondria were lysed, and the mitochondrial protein content was detected using the BCA protein kit, and finally the linear relationship between the mitochondrial protein content and the number of cells was determined.
Embodiment 2
[0036] The preparation method of above-mentioned coating-mito and coating-mito / DNA specifically comprises the following steps:
[0037] 1) Isolated exogenous mitochondria with complete biological functions are extracted from animal tissues or cells cultured in vitro to become mito.
[0038] 2) Prepare a material solution with good biocompatibility, and the final concentration range is 0.1-10 mg / ml.
[0039] 3) Resuspend the isolated exogenous mitochondria in 50-200 μL of a material solution with good biocompatibility, wherein the volume of the material solution with good biocompatibility is determined according to the amount of extracted exogenous mitochondria.
[0040] 4) Incubate the isolated exogenous mitochondria and the material solution with good biocompatibility at room temperature for 30 min.
[0041] 5) Centrifuge at 14,000 rpm for 10 min to obtain a precipitate, and wash the precipitate with phosphate-buffered saline (PBS) several times to obtain the isolated exogen...
Embodiment 3
[0044] Calculate the number of mitochondrial protein from the coating-mito obtained in Example 2 according to the linear graph of mitochondrial protein and cell number in Example 1, and dilute it to 2 μg / μL, 4 μg / μL, 8 μg / μL and 16 μg / μL, vortexed with 2 μL (0.2 μg / μL) gene solution for 10 s, and incubated at room temperature for 30 min to obtain the coating-mito-DNA complex. Finally, the binding ability of the material and the gene was detected by gel electrophoresis experiment. Experimental results such as figure 2 As shown, it shows that coating-mito binds to DNA at a mass ratio of 40:1.
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Abstract
The invention discloses an exogenous mitochondrial vector and a compound as well as a preparation method and application of the exogenous mitochondrial vector and the compound; in-vitro exogenous mitochondria is modified by one or more materials with good biocompatibility, a functional gene and/or drug is encapsulated by the modified in-vitro exogenous mitochondria, finally, the two components are jointly delivered to diseased cells to treat mitochondrial defect related diseases. A delivery system is composed of the in-vitro exogenous mitochondria with intact functions, a material with good biocompatibility and the functional gene/drug. Mitochondria mito extracted from autologous or allogenic tissues and cells is subjected to special treatment with the materials with good biocompatibility to form coating-mito, then the coating-mito is mixed and incubated with the functional gene and/or drug according to a certain proportion, and the exogenous mitochondria drug compound is formed through non-covalent acting force. The exogenous mitochondria drug compound can be efficiently taken by damaged cells, and can release normal mitochondria and the related functional gene and/or drug in cytoplasm.
Description
technical field [0001] The invention relates to a method for transplanting exogenous mitochondria and using the mitochondria as a functional gene and / or drug delivery carrier, which can achieve safe and effective treatment of a series of mitochondrial defect-related diseases, and belongs to the technical field of biomedicine. Background technique [0002] Mitochondria is an organelle surrounded by two membranes that exists in most cells. It is an important place for oxidative metabolism in eukaryotes, and it is also a place for final oxidation of sugars, fats and amino acids to release energy. It is called "energy factory". ". The nuclear genome and the mitochondrial genome jointly maintain the normal functions of human cells. Among them, mtDNA is a circular double-stranded DNA molecule of about 16.6kb, which is responsible for encoding 13 mitochondria-related proteins. Mitochondrial defect-associated diseases are caused by mutations in mitochondrial or nuclear genes that e...
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