2.5 Drug assay and release of DOX-loaded beta-CD nanocage
A series of standard DOX stock solutions were prepared, and a standard curve was obtained by measuring the absorbance using a plate reader with a 485 nm excitation filter and a 538 nm emission filter (Thermo Scientific Fluoroskan Ascent FL). Based on the standard curve equation y = 0.04464*x + 0.02131, the drug loading capacity of the beta-CD nanocages was determined to be 37.5% using the formula: the beta-CD nancage drug loading capacity=the loaded DOX content/( the loaded DOX content+beta-CD nanocage content)*100%.
pH 4.5 PBS solution was used to mimic the acidic tumor microenvironment, while a 5 mM GSH solution simulated the reductive tumor microenvironment. pH 7.4 PBS solution was used to represent the normal physiological environment. DOX-loaded beta-CD nanocages (20 mg) were dissolved in each of these three types of solutions (1 ml) and poured into separate Amicon centrifuge tubes. These tubes were then immersed in larger tubes containing the respective release medium (5 ml) and placed in an incubator shaker at 100 rpm at 37 °C. Aliquots were taken from the release medium at predetermined time intervals, and an equal volume of fresh buffer was added each time. The samples were measured using a plate reader with a 96-well plate.