Bionic binary cooperative nano-carrier as well as preparation method and application thereof
A nano-carrier and nano-particle technology, applied in the field of bionic binary synergistic nano-carriers and its preparation, can solve problems such as normal tissue damage, achieve precise treatment, avoid damage, and improve the effect of enrichment
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Embodiment 1I
[0039] Example 1 Preparation of ICG Membrane Breaking Biomimetic Binary Synergistic Nanocarrier
[0040] 1. Preparation of apoferritin nanocages (HFn) and ferritin nanoparticles (M-HFn)
[0041] (1) Preparation of apoferritin nanocages (HFn) by genetic engineering
[0042] The positive strain expressing apoferritin nanocages was constructed by Hubei Bios Biotechnology Co., Ltd., which provides services such as plasmid construction, and the cells are also derived from this company.
[0043] RNA was extracted from human skeletal muscle cells, reverse-transcribed into cDNA as a template; human FTH1 gene was amplified, and the PCR primers were as follows (the black underline is the restriction site):
[0044] F:5'-A GTC GCC CAT ATG ACG ACC GCG TCC-3'(Nde I)
[0045] R:5'-GCC GGA TCC TTA GCT TTC ATT ATCA C-3'(Bam HI)
[0046] After the PCR product was purified and recovered, it was ligated with the pET-30a(+) carrier by double enzyme digestion, and the product was transfor...
Embodiment 2
[0058] Example 2 Preparation of DIR Membrane Breaking Biomimetic Binary Synergistic Nanocarriers
[0059] 1. Preparation of apoferritin nanocages (HFn) and ferritin nanoparticles (M-HFn)
[0060] (1) Preparation of apoferritin nanocages by genetic engineering
[0061] RNA was extracted from human skeletal muscle cells, reverse-transcribed into cDNA as a template; human FTH1 gene was amplified, and the PCR primers were as follows (the black underline is the restriction site):
[0062] F:5'-A GTC GCC CAT ATG ACG ACC GCG TCC-3'(Nde I)
[0063] R:5'-GCC GGA TCC TTA GCT TTC ATT ATCA C-3'(Bam HI)
[0064] After the PCR product was purified and recovered, it was ligated with the pET-30a(+) carrier by double enzyme digestion, and the product was transformed into XL2-Blue competent cells, screened on a Kan+ resistance plate, and colonies were picked and identified before being sent for sequencing. The plasmids with correct sequencing were transferred into competent BL21(DE3)E....
Embodiment 3
[0075] Embodiment 3 animal experiments
[0076] 1. Construction of BALB / c mouse subcutaneous tumor model
[0077] Prepare a single-cell suspension from tumor cells in logarithmic growth phase, wash 3 times with serum-free medium, and adjust the cell concentration to 1*10 7 / mL, animal experiments were carried out in a clean bench. Inject the cell suspension into the subcutaneous inner thigh of nude mice with a TB empty needle (choose the place with rich blood vessels), inoculate each mouse with 0.1mL, containing 1*10 cells 6 , the miliary size of subcutaneously transplanted tumors (about 10 days, about 60-80mm in size) 3 ) to start the intervention treatment, and start to measure the long diameter and short diameter of the tumor with a vernier caliper.
[0078] 2. Experimental steps
[0079] The tumor-bearing mice at the age of 4 to 6 weeks were randomly divided into 4 groups, 6 in each group, the first group was the PBS group, and the second group was the non-targeted env...
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