Method for preparing antitumor drug carrier by using rolling circle amplification technology
A rolling circle amplification technology, anti-tumor drug technology, applied in pharmaceutical formulations, medical preparations with inactive ingredients, etc., can solve problems such as affecting the efficiency of carriers entering cells and the biosafety of metal nanoparticles
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Embodiment 1
[0028] Add 20 μL (100μM) SH-DNA to 1mL 15nm gold colloid solution, shake at room temperature overnight, add 100mM PB buffer (pH7.4), the final concentration is 10mM, add aging solution (10 mM PB, 2M NaCl, pH 7.4) The final concentration of salt in the solution is 0.15M. After overnight at room temperature, the mixture solution was centrifuged at 12000 rpm / min for 15 min at 4°C and washed three times. The precipitate was resuspended in 200 μL, pH7.4 PB (10 mM, 0.15 M NaCl) solution and stored at 4°C.
[0029] Add 5 μL of 100 μM pre-circular DNA and 10 μL of 100 μM complementary sequence into a 0.5 mL centrifuge tube and mix evenly, at 90°C for 1 min, when the solution in the tube is naturally cooled to room temperature, add 3 μL of LT4 ligase and 2 μL of LT4 ligase buffer, at 25°C The ligation was carried out for more than 16 hours under ambient conditions. After the end, the enzyme was denatured at 65°C for 10 minutes, then 3 μL T4 polymerase and 6 μL T4 polymerase buffer were...
Embodiment 2
[0032] Add 20 μL (100μM) SH-DNA to 1mL 30nm gold colloid solution, shake overnight at room temperature, add 100mM PBbuffer (pH7.4), the final concentration is 10mM, add aging solution (10 mM PB, 2M NaCl, pH 7.4) after overnight at room temperature ) The final concentration of salt in the solution is 0.15M. After overnight at room temperature, the mixture solution was centrifuged at 10,000 rpm / min for 15 min at 4°C and washed three times. The precipitate was resuspended in 200 μL, pH 7.4 PB (10 mM, 0.15 M NaCl) solution, and stored at 4°C.
[0033]Add 5 μL of 100 μM pre-circular DNA and 10 μL of 100 μM complementary sequence into a 0.5 mL centrifuge tube and mix evenly, at 90°C for 1 min, when the solution in the tube is naturally cooled to room temperature, add 3 μL of LT4 ligase and 2 μL of LT4 ligase buffer, and store at 25°C The ligation was carried out for more than 16 hours. After the end, the enzyme was denatured at 65°C for 10 minutes, then 3 μL T4 polymerase and 6 μL T...
Embodiment 3
[0036] 1 mg of single-walled carbon nanotubes was dissolved in 1 mL of PBS solution at pH 7, 0.4 mL of EDC (400 mM) and NHS (100 mM) solutions were added after ultrasonication for 10 min, and shaken at room temperature for 15 min. The above solution was centrifuged at 15,000 rpm / min at 4°C for 5 min, and repeated twice to remove excess EDC and NHS. Then 20 μL of 1 mg / mL streptavidin solution was added, and the assembly was shaken overnight at room temperature. Then centrifuge at 15,000 rpm / min at 4°C for 10 min, repeat three times to remove unlinked streptavidin, then add 20 μL of biotin-amplification primer DNA (100 μM), shake at room temperature for 1 hour and then centrifuge (4°C, 15,000 rpm / min, 10min) three times. The precipitate was resuspended in 200 μL, pH7.4 PB (10 mM, 0.15 M NaCl) solution, and stored at 4 °C.
[0037] Add 5 μL of 100 μM pre-circular DNA and 10 μL of 100 μM complementary sequence into a 0.5 mL centrifuge tube and mix evenly, at 90°C for 1 min, when...
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