0 1 mL of BIN concentrate was added to 5 mL of acetonitrile, and

0.1 mL of BIN concentrate was added to 5 mL of acetonitrile, and the concentrate was ultrasounded for 5 minutes to fully extract the brucine, then acetonitrile was added to make up the volume to 10 mL. After being shaken and centrifuged at 12,000 rpm for 20 minutes, the supernatant was collected to measure the the site UV absorption at 263 nm wavelength, and the absorbance A2 was recorded. The UV absorption of the control solution was measured at a wavelength of 263 nm, and the absorbance (As) was recorded. The brucine content in the concentrate was calculated as (A2/As) in mg/mL. In vitro drug release The dialysis method was employed to determine the drug release rate of BIN in vitro. To calculate the stability of the brucine in the release medium, 20 mg of brucine was diluted to 0.01 mg/mL with 0.

5% polyoxyethylene dehydrated sorbitol monooleate PBS solution. The solution was put into a water bath constant temperature oscillator (37��C �� 0.5��C) in the dark and 1.0 mL was sampled at 0, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, and 60 hours, with 20 ��L of this sample used to measure the brucine content. The content at 0 hours was set as 100% and changes in the brucine content were measured. To calculate the drug release of BIN in vitro, dialysis bags were placed in distilled water for 24 hours. Eight milliliters of BIN suspension was put into the dialysis bags, the ends of the dialysis bags were clipped, and the bags were placed into the release medium with magnetic stirring (200 rpm). Of the solution, 1.0 mL was sampled at 0, 0.

5, 1, 2, 4, 8, 12, 24, 36, 48, and 60 hours, with 20 ��L of this sample used to measure the brucine content. The data were corrected in accordance with the degradation curve in the release medium. Concentrations of the brucine in the sample were measured at different time points. The cumulative drug release percentage (Q) was calculated and the cumulative release curve was plotted. Determination of monoclonal antibody on brucine immuno-particle surface The bicinchoninic acid (BCA) method was used to measure the antibody content of samples. After the BSA standard protein solution (10 ��g/mL�C750 ��g/mL) and BCA working solution were allowed to react, the solution��s absorbance at 562 nm was measured and the concentration-absorbance curve and the curve equation were obtained. Forty microliters of BIN suspension reacted with the BCA working solution and its absorbance at 562 nm was determined.

The concentration of the antibody on the brucine nanoparticles was then obtained using the curve equation. Cell targeting and positioning Human hepatoma cells (SMMC-7721) were diluted with 10% fetal calf serum medium (RPMI-1640) Dacomitinib to a cell concentration of 1 �� 105/mL. One milliliter of human hepatoma cells (SMMC-7721) was cultured in an incubator in 5% CO2 at 37��C for 24 hours. The culture fluid was discarded and the cells were washed twice with 0.01 M PBS.

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