In vitro Cytotoxicity

The in vitro cytotoxicity of samples was determined by MTT assay. The principle of this assay is based on the fact that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan, which is deposited in cells, while dead cells cannot perform this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and its light absorption value is then measured using an enzyme-linked immunosorbent assay at a wavelength of 540 nm, with the results indirectly reflecting the number of living cells.

The human hepatoma cell line, HepG2, was used for in vitro cytotoxicity experiments. The cell line was grown in Dulbecco’s Modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum, 100 μg/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated in a saturated humidity incubator (37°C, 5% CO₂), washed with phosphate-buffered saline (PBS), and digested using 0.25% trypsin to form a single cell suspension. Cells in logarithmic growth phase were tested.

With reference to the relevant standards (GB/T 16886.5–2017 and ISO 10993–5:2009), 1 g of the blank preparation, P1, P2, P3, and PYT were injected into 5 mL of DMEM, and leached at 37°C for 48 h. Then, samples were filtered through 0.22 μm sterile membranes, and the filtrates used as stock solutions. Next, the stock solutions were diluted in different proportions (1:0; 1:1; 1:2; 1:4; 1:8; 1:16; 1:32; 1:64; 1:128, v/v) of DMEM culture medium, as test solutions.

Briefly, 100 μL of DMEM was added to the peripheral wells of a 96-well tissue culture microtiter plate, and 100 μL of HepG2 cell suspension (1 × 10⁴ cells/mL) added to the remaining wells. After incubating for 24 h, the medium was removed and 100 μL of the different concentrations of stock solutions diluted with fresh medium added to each well, with 100% DMEM as a negative control (n = 3). After incubation for 72 h, 20 μL MTT solution (5 mg/m L) was added to each well and the cells incubated at 37°C for an additional 4 h. Subsequently, the MTT solution was removed, replaced with 100 μL DMSO, and the mixture shaken at a low speed for 10 min on a shaker to fully dissolve the crystalline formazan. The optical density (OD) at 540 nm of each well was measured using a microplate reader. To express the in vitro hemolytic properties of our samples, cell viability was calculated using the following formula:

Cell viability (%) = OD_570S/OD_570N × 100

where OD_570S represents the average optical density of each tested sample group, and the OD_570N represents the average optical density of the negative control group. According to the ISO standard (GB/T 16886.5–2017/ISO 10993–5:2009), cell viability was then classified according to percentage of viable cells, to reflect the degree of toxicity of the material as follows: 0, 100%; 1, 80–100%; 2, 50–80%; 3, 30–50%; 4, 0–30%.

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.