Erythrocyte membrane bionic nano-drug and preparation method thereof
A technology of erythrocyte membrane and biomimetic nanotechnology, which is applied in antibacterial drugs, drug combinations, pharmaceutical formulations, etc., can solve the problems of limited target sites of macrophage membrane vesicles, limitation of the size and quantity of loaded drugs, and inability to obtain a large number of cell membranes. , to achieve the effects of easy clinical transformation, avoiding midway leakage, and avoiding rapid metabolism
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Embodiment 1
[0044] Example 1 Preparation and Characterization of Triptolide Nanomedicine Encapsulated by Mannose Modified Erythrocyte Membrane
[0045] A preparation method of erythrocyte membrane biomimetic nano-medicine, comprising the following steps:
[0046] (1) collecting blood cells to obtain red blood cell membranes;
[0047] (2) Obtaining sugar-modified red blood cell membranes;
[0048] (3) Coating the sugar-modified erythrocyte membrane onto the hydrophobic small molecule drug nanoparticles to form a core-shell structure.
[0049] The specific operation steps are as follows:
[0050] After the animals were anesthetized with 5% chloral hydrate, the blood was taken from the orbit and placed in an anticoagulant tube. Let stand at 2-8°C for less than 1 hour, remove serum and collect blood cells by centrifugation, add 5 times the volume of 0.25× phosphate buffer, place in a refrigerator at 4°C for hypotonic hemolysis for 1-2 hours, collect the precipitate by centrifugation, and t...
Embodiment 2
[0053] Example 2 Detection of the uptake rate of nanomedicine by macrophages and T cells
[0054] Take 5×10 5 The cell density of cells / well was plated in a 6-well culture plate, and after 12-16 hours of culture When the cell coverage rate reaches 80%, the cytokine IL-4 at a concentration of 100 ng / mL and the cytokine TNF-a at a concentration of 10 ng / mL are added to co-culture for 4-6 hours to activate the cells. After that, within 6 hours, 50 μL of triptolide nanoparticles (TPNs) labeled with cyanine dye 5.5 (Cy5.5) or mannose-modified erythrocyte membrane triptolide nanoparticles (manRTPNs) were injected every one hour. ) into the prepared cells for co-cultivation in the dark. After digestion with trypsin, the cells were washed 3 times with ice-cold PBS, and finally the collected cells were detected by flow cytometry with FACS Canto II, and the data were analyzed using Flow Jo software. The mean fluorescence intensity (MFI) reflects the amount of nanoparticles ingested. ...
Embodiment 3
[0055] Example 3 Expression of activated macrophages and T cell surface molecules and localization of nanomedicines
[0056] 5 x 10 per well 5 The macrophage cell line RAW264.7 and the T cell line CTLL-2 were inoculated in an 8-well plate for 12 hours and cultured for 6 hours by adding IL-4 at a concentration of 100 ng / mL and TNF-α at a concentration of 10 ng / mL. . Afterwards, they were incubated with different nanomaterials triptolide nanoparticles (TPNs) and mannose-modified erythrocyte membrane triptolide nanomedicines (manRTPNs) for 3 hours. Then, the cells were fixed with 4% paraformaldehyde for 10 min and treated with 0.1% Triton TM Permeabilize with X-100 for 10 minutes, block with 1% BSA for 1 hour and label and incubate with 2 μg / mL anti-mannose receptor / AF488-conjugated antibody and rabbit anti-CD2 / AF488-conjugated antibody for 3 hours at room temperature. After washing three times with phosphate buffer, add 4',6-diamidino-2-phenylindole (DAPI) dye and treat for 1...
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