Targeted composite exosome and preparation method thereof
An exosome and targeting technology, applied in the field of medicine, can solve problems such as limitation, reduction of drug efflux, lack of active targeting ability of tumor cells or inflammatory cells, and achieve high active targeting ability, long residence time, Aggregation and Retention Time Reduction Effects
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[0033] The present invention provides a method for preparing targeted complex exosomes, comprising the following steps:
[0034] 1) Targeting modification of exosomes with targeting polypeptides to obtain membrane-modified exosomes with targeting polypeptides;
[0035] 2) Mix the membrane-modified exosomes with targeting polypeptides, drugs, nanometer iron ferric oxide aqueous solution, and electroporation buffer in step 1), and perform electroporation treatment at 2-6°C to obtain the Targeted composite exosomes were incubated at 35-40°C for 25-35 minutes to obtain targeted composite exosomes.
[0036] The present invention uses the targeting polypeptide to carry out targeted modification on exosomes to obtain the exosomes whose membrane is modified with the targeting polypeptide. In the present invention, the method for targeted modification preferably includes the following steps:
[0037] a. After mixing PBS, NHS and 4-pentynoic acid, adjust the pH to 7.2-7.5, and shake a...
Embodiment 1
[0060] 1. Preparation of membrane-modified exosomes with targeting polypeptides
[0061] Add 35 mg of NHS and 29 mg of 4-pentynoic acid to 1 mL of PBS, adjust the pH to 7.4 with sodium bicarbonate, place on ice and shake at a frequency of 80 times / min for 1 h to obtain the first reaction system.
[0062] Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to the first reaction system, place it on ice and vibrate at a frequency of 80 times / min for 1 h to obtain the second Two reaction system (the final concentration of EDC is 0.3 mmol). Take 4 μL of the second reaction system and add it to 150 μL of PBS containing 160 μg of exosomes (protein), shake at room temperature (20°C) at a frequency of 80 times / min for 24 hours, and then use a 100KDa membrane to remove impurities twice to obtain a membrane Exosomes linked with acetylene bonds. Mix the obtained exosomes with acetylenic bonds in the membrane with 300 μL PBS solution, add 14 μL of anhydrous copper sulfa...
Embodiment 2
[0071] The targeting properties of the synthesized targeted compound exosomes were verified from the level of cells and tumor-bearing mice respectively, and the cell level was observed by confocal microscope. The specific method is as follows:
[0072] 1) Prepare RGE-Exo-SPION / Cur 1000, free-Exos each 1000ug (total protein) / 1mL, 100nM RGE peptide solution 10mL, wherein RGE-Exo-SPION / Cur and free-Exos are respectively CM according to the steps 4.2.4.2 - DiI stained membrane to obtain CM-DiI labeled Exos;
[0073] 2) Select the target cells (U251) and non-target cells (Bel-7404) in the logarithmic growth phase and inoculate them in confocal culture dishes respectively to maintain a good cell state and a suitable cell density (about 1×105 / well), RGE peptide solution 1nM (ensure sufficient amount) was incubated with some U251 cells at room temperature for 4 hours, and the unbound peptide was absorbed to obtain RGE-blocked U251 cells;
[0074] 3) Co-incubate with cells according t...
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