Preparation method of nano-bacterium hybrid system
A bacterial and hybrid technology, applied in the fields of nanotechnology, nanotechnology, nanomedicine, etc., can solve the problems of poor tumor treatment effect, affect the treatment effect, damage the activity and function of bacteria, and achieve significant tumor treatment effect and tumor inhibition. effect of growth
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Embodiment 1
[0025] Take 300 μL of photochemically obtained green fluorescent silicon nanoparticles (SiNPs) (with amino groups on the surface) solution at a concentration of 25 mg / mL, mix 200 μL of a glucose polymer (GP) solution with a concentration of 10 mg / mL into it, and place the mixed solution in After shaking and reacting on a shaker at 70°C for 4-6 hours, add 1 mL of 10 μg / mL sodium borohydride solution, and shake and react overnight at room temperature. After the reaction, in order to remove excess unreacted GP molecules, centrifuge at 7500rpm for 15 minutes each time with a 3K ultrafiltration tube until the solution in the lower layer of centrifugation contains almost no GP molecules. Take the solution of GP-SiNPs in the upper layer; take 100 μL of Add 50 μL of indocyanine green (ICG) solution with a concentration of 200 μg / mL to the solution of GP-SiNPs obtained from the reaction, and place the mixed solution on a shaker at room temperature for 12-24 hours; after the reaction is ...
Embodiment 2
[0031] 1. Transform the plasmid (pRSETB-mCherry) carrying the red fluorescent protein (mCherry) gene into Escherichia coli according to the conventional plasmid transformation method to construct mCherry@EC engineering bacteria with red fluorescence. The mCherry@EC bacteria solution (1.0×10 8 CFU / mL, 200μL) was injected into the mice through the tail vein, and the mice were sacrificed on the 1st, 3rd, 5th, 7th and 15th day, and their hearts, livers, spleens, lungs, kidneys and tumors were taken out and placed in small animal imaging mCherry fluorescence imaging in the instrument to study the distribution of bacteria in organs and tumors.
[0032] Depend on image 3 a) It can be seen from the image that the fluorescent signal of mCherry@EC is always mainly concentrated in the liver in the mice of the control group. As the number of days increases, the fluorescent signal in the liver gradually disappears, and it is basically undetectable on the 15th day to the fluorescent sign...
Embodiment 3
[0036] The in vitro anticancer experiments of TNFα@EC@GP-ICG-SiNPs were mainly completed using transwell (polycarbonate membrane with a pore size of 400nm) and 808-nm laser. First, murine breast cancer 4T1 cells were seeded in a 24-well plate with transwell and cultured for 24 hours. TNFα@EC@GP-ICG-SiNPs cells were resuspended in 300 μL of RPMI-1640 medium containing 10 wt% fetal bovine serum, and the bacterial solution was directly added to the upper transwell chamber of a 24-well plate with 4T1 cells. The 24-well plate was placed under an 808-nm laser, and the temperature was stabilized at 42°C by adjusting the laser power, irradiated for 30 minutes, and then placed in an incubator for stable culture for 24 hours. The 4T1 cells in the lower layer of the transwell were collected, and the cell survival rate was determined by the MTT method. Cells were resuspended with propidium iodide (for dead cells, λex=543nm, λem=560-620nm) and calcein (for live cells, λex=488nm, λem=500-5...
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