Macrophage drug-loaded MP (microparticle) preparation and preparation method thereof
A macrophage and microparticle technology, applied in the field of drug targeting carriers, can solve the problem that small drug molecules cannot be enriched and target macrophages, so as to alleviate the tumor immunosuppressive microenvironment and achieve a good reverse polarization effect. , Improve the effect of reverse polarization effect
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Embodiment 1
[0051] Example 1 Preparation and characterization of mannose-modified macrophage-derived drug-loaded microparticles
[0052] 1. Experimental materials and reagents
[0053] The mouse macrophage cell line RAW264.7, metformin hydrochloride, DSPE-PEG-Mannose, and the ultraviolet device are owned by the conventional cell ultra-clean bench.
[0054] 2. Experimental steps
[0055] 1) In a sterile petri dish with a diameter of 10 cm, use DMEM high-glucose full medium (containing 25 μg / mL DSPE-PEG-Mannose) to culture RAW264.7 mouse macrophages for 3 days, so that DSPE-PEG-Mannose can interact with macrophages Phage cells perform membrane phospholipid exchange.
[0056] 2) When the cell volume reaches 5×10 7 , discard the medium, wash with phosphate buffered saline (PBS), add 4 mL of serum-free DMEM medium, and irradiate with ultraviolet rays for 60 min, then add 1 mL of metformin to make the final concentration of metformin 4 mg / mL.
[0057] 3) 24 hours after metformin administrat...
Embodiment 2
[0061] Example 2 Using ultraviolet rays to induce macrophage apoptosis to produce microparticles modified with mannose and incubated with small molecule drugs to obtain drug-loaded microparticles
[0062] 1. Experimental materials and reagents
[0063] The mouse macrophage cell line RAW264.7, metformin hydrochloride, and DSPE-PEG-Mannose used were the same as in Example 1, and the ultraviolet device was owned by a conventional cell clean bench.
[0064] 2. Experimental steps
[0065] 1) Use DMEM high-glucose full medium to culture RAW264.7 mouse macrophages in a sterile petri dish with a diameter of 10 cm, so that the cell volume reaches 5×10 7 .
[0066] 2) RAW264.7 mouse macrophages (with cell culture medium) were subjected to ultraviolet irradiation for 60 min.
[0067] 3) After 24 hours of ultraviolet irradiation, the supernatant of the apoptotic RAW264.7 macrophage culture medium was gradually separated, and the macrophage microparticles were obtained as in Example 1. ...
Embodiment 3
[0073] Example 3 Targeting of microparticles to M2 macrophages in vitro
[0074] 1. Experimental materials and reagents
[0075] H22 mouse liver cancer cells, DC2.4 mouse dendritic cells, the mouse macrophage cell line RAW264.7 used is the same as in Example 1, IL-4, LPS, IFN-γ, metformin, DSPE-PEG-Mannose Example 1.
[0076] 2. Experimental steps
[0077] 1) Collect the mannose-modified drug-loaded microparticles, and the collection method is the same as in Example 1.
[0078] 2) Add 20ng / mL IL-4 or 20ng / mL IFN-γ and 100ng / mL LPS to RAW264.7 to stimulate and induce for 24 hours to obtain M1 or M2 type macrophages. 2×10 5 M0, M1, M2 macrophages, DC2.4, and H22 were cultured in cell culture plates, and the medium was removed after 12 hours.
[0079] 3) The microparticles were stained with cell membrane red fluorescent dye PKH26.
[0080] 4) After the cells in the cell culture plate were removed from the medium, serum-free medium containing 50 μg (protein amount) of PKH26-la...
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