Nano-drug carrier loading anticancer drugs, and preparation method and application of nano-drug carrier
A nano-drug carrier, anti-tumor drug technology, applied in the field of immunology and biomedicine, nanotechnology
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Embodiment 1
[0032] Example 1, Preparation and Characterization of Pyrococcus Furious Ferritin Nano-Drug Carrier Fn-Dox
[0033] Pyrococcus furiosus ferritin pfFn (Fn) is highly similar to ferritin from other species (human, mouse) in structure, and can self-assemble into a 24-mer spherical protein shell structure in a natural state, with an inner diameter of 8nm and an outer diameter of 12nm. As mentioned above, the structural characteristics of ferritin make it an obvious advantage as a drug carrier. According to the current research, the loading capacity of a single ferritin molecule to the first-line chemotherapy drug doxorubicin is about 20-40 molecules1-6, and the ferritin molecule modified by PAS polypeptide can load about 90 molecules of doxorubicin (Elisabetta Falvo. et al., Biomac, 2015). Our work found that using a relatively mild method of urea denaturation and renaturation to prepare ferritin doxorubicin nanoparticles, the doxorubicin loading capacity of Pyrococcus furiosus f...
Embodiment 2
[0044] Example 2. Preparation and characterization of liver cancer-targeted ferritin nano-drug carrier HccFn-Dox
[0045] By means of gene fusion, the SP94 liver cancer targeting peptide (SEQ ID NO: 2) is fused and expressed at the N-terminal of Pyrococcus furiosus ferritin (SEQ ID NO: 1) through the adapter protein (SEQ ID NO: 3), and we fused this The protein was named HccFn (SEQ ID NO: 4).
[0046] With the gradient urea dialysis method as described in Example 1, doxorubicin is loaded into the HccFn lumen ( figure 2 A). Superdex 200pg molecular sieves detect the ultraviolet absorption of A280nm and A485nm, and it can be seen that doxorubicin is successfully loaded into the inner cavity of HccFn ( figure 2 B). TEM ( figure 2 C) and dynamic light scattering ( figure 2 D) The analysis results also show that ferritin (Fn), HccFn and HccFn-Dox of Pyrococcus furiosus all maintain good uniformity and spherical shell structure. The results of native gel electrophoresis ( ...
Embodiment 3
[0048] Example 3. Liver cancer-targeting characterization of liver cancer-targeting ferritin nano-drug carrier HccFn-Dox in vivo and in vitro
[0049] 1. Cell targeting of HccFn-Dox
[0050] First, we use FITC to fluorescently label Fn and HccFn protein shells. The FITC fluorescent labeling method is as follows: Dissolve 1mg of Fn or HccFn protein shells in 0.1M carbonate buffer solution (pH9.0) (final protein concentration 1mg / ml), add 50 microliters of FITC solution (1mg / ml, dissolved in DMSO), and mix overnight at 4°C in the dark. Centrifuge at 12,000 g for 10 min to take the supernatant, and replace the PBS buffer with an ultrafiltration concentrator until the filtrate is colorless. FITC concentration was determined by UV absorbance (Nanodrop 2000).
[0051] Mix 1 μg / ml FITC-Fn and FITC-HccFn with 100 μL hepatoma cell HepG2 (2.5×10 6 cells / mL) were co-incubated for 30 minutes, observed by laser confocal scanning microscope, HccFn specifically bound to the surface of He...
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