Alt and ast activity assay kits

ALT and AST activity assay kits (Jiancheng, Nanjing, China) were used to detect the activity changes of ALT and AST in mouse serum. After adding mouse serum and reagents to the 96-well plate, placed at room temperature for 15 min, and measured the OD value at a wavelength of 510 nm with a microplate reader.

A HER2-positive NCI-N87 xenograft mouse model was adopted to evaluate the in vivo antitumor activity of these conjugates. The model was established by subcutaneous inoculation of 5.0 × 10⁶ NCI-N87 cells per mouse in the right flanks of female BALB/c nude mice. When the tumor volume reached approximately 210 mm³, the mice were randomized into four groups (n = 5) and injected with various dosages of the conjugates once every three days for a total of four times, and PBS injection was used as the control. The tumor size and weight were monitored every three days until day 31. The tumor dimensions were measured with a digital caliper, and their volumes were calculated using the formula length × (width)²/2.
On day 31 post first injection of the conjugates, blood samples (200 µL) were collected from the mice receiving treatment with a 1.5 mg/kg dose injection and the serum was separated by centrifugation at 3000 rpm for 10 min. The levels of ALT and AST in serum were quantified by ALT and AST activity assay kits (Nanjing Jiancheng Bioengineering Institute), respectively. The mice were then euthanized, and their tissues and organs were collected and fixed with 4% paraformaldehyde. The samples were embedded in paraffin, stained with hematoxylin and eosin (H&E), and visualized by a Nikon eclipse E100 microscope for histopathological analysis.