In vitro Anticancer Activity

To test the in vitro anticancer effects of SN and/or 5-FU solutions, according to the literature^12–20 and preliminary experimental results, the stock solution concentration of 5-FU was 640 μg/mL, while that of SN was 1280 μg/mL, and the stock combination solution containing was 640 μg/mL 5-FU + 1280 μg/mL +SN. Four groups were included in the experiment as follows: negative control group (only DMEM culture medium added), 5-FU monotherapy group, SN monotherapy group, and the combination group. All solutions were formulated and diluted with DMEM medium at ratios of 1:0, 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128 (v/v), then filtered through a 0.22 µm microporous membrane before use in experiments. Cell proliferation was determined in vitro by MTT assay, as described above, except that cell viability was detected at 24, 48, and 72 h. Based on the results of these experiments, drug loading of SN in precursor injections was set to 0.5%, that of 5-FU to 0.25%, and the combined drug loading to 0.5% + 0.25% (SN + 5-FU). Drug-loaded precursor solutions were extracted as described above and diluted with the culture solution to obtain a gradient of sample concentrations.

To analyze the drug interaction between SN and 5-FU, the co-administration efficacy analysis software, CalcuSyn 2.1, was used to analyze the inhibition rates (IRs) according to Chou and Talalay,^28,29 and combination index (CI) values, a dose-effect curve, and a median-effect plot were obtained. The median-effect plot is a graph of x = log (D) vs y = log (fa/fu), based on the median effect equation (a general equation for the dose-effect relationship):

fa/fu = (D/Dm)m

where fa is the fraction affected by the dose, fu is the fraction unaffected (fu = 1 – fa), D is the drug concentration or dose, Dm is the median-effect dose or concentration (usually expressed as ED50 or IC50), m is an exponent signifying the sigmoidicity (shape) of the dose-effect curve; m = 1, >1, and <1 indicate hyperbolic, sigmoidal, and negative sigmoidal shapes, respectively. CI is a quantitative measure of the degree of drug interaction, in terms of additive effect (CI = 1), synergism (CI < 1), or antagonism (CI > 1), for a given endpoint of the effect measurement.