A tumor microenvironment-responsive gene nanomicelle and its preparation method and application
A tumor microenvironment and nanomicelle technology is applied in the field of tumor targeted drug delivery, tumor microenvironment responsive gene nanomicelles and their preparation, which can solve the specific targeting and low loading efficiency of non-viral gene carriers. question
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Embodiment 1P
[0060] Embodiment 1PEI-PBA and PEI-MAH-C225 carrier preparation
[0061] (1) Preparation of polyethyleneimine-phenylboronic acid (PEI-PBA):
[0062]
[0063] The synthetic route of PEI-PBA
[0064] According to the above polymer synthesis route, the specific description is as follows:
[0065] Add 4-(bromomethyl)phenylboronic acid and PEI into anhydrous DMSO, and stir the reaction at 80°C for 24 hours. The ratios of 4-(bromomethyl)phenylboronic acid to PEI were 4:1, 6:1 and 8:1, respectively. Finally, the product was dialyzed and freeze-dried to complete the preparation of PEI-PBA. H NMR spectrum such as Figure 4 , indicating that PEI-PBA was successfully synthesized.
[0066] (2) Preparation of polyethyleneimine-maleic anhydride-cetuximab (PEI-MAH-C225):
[0067]
[0068] The synthetic route of PEI-MAH
[0069] According to the above polymer synthesis route, the specific description is as follows:
[0070] (1) Preparation of PEI-C225: Add C225 to 0.1M MES solutio...
Embodiment 2
[0072] Example 2 Loading microRNA-146a (miR-146a) and cetuximab
[0073] Preparation of PEI-PBA-miR146a / PEI-MAH-C225 Nanomicelles
[0074] Put 5mg PEI-PBA and 2.5mg miR-146a into the aqueous solution to form PEI-PBA-miR-146a nanomicelles loaded with genes, then put 10mg PEI-MAH-C225 into the above nanomicelle solution and stir to form PEI-PBA - miR-146a / PEI-MAH-C225 nanomicelles. The results of nanomicelle by atomic force microscope and transmission electron microscope are as follows: Figure 6 , 7 As shown, the prepared nanoparticles are circular and have good dispersibility, and the particle size is mainly concentrated between 100-200nm, especially the particle size of 150nm.
Embodiment 3
[0075] Example 3 Cytotoxicity of PEI-PBA / PEI-MAH-C225 Nanomicelles
[0076] DU145 prostate cancer cells were plated in a 96-well plate, and the density of cells per well was 1×10 5 a, at 37°C, CO 2 Culture overnight in a cell culture incubator with a volume fraction of 5%. Nanomicelles of different concentrations were added to the 96-well plate (the cell group with only the culture medium was the negative control group), and 5 replicate wells were set for each experimental condition. After 24 hours of culture, 20 μL of cck8 (5 mg / mL) solution was added, and the culture was continued for 2 hours. After that, the culture solution was aspirated, tested at 450 nm in a microplate reader, and the cell survival rate was calculated.
[0077] The experimental results are shown in 8. Compared with the control group, the OD value of the cells incubated with nanoparticles within 24 hours did not change significantly, indicating that the nanoparticles have good biocompatibility.
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