Biodistribution studies were conducted in C57BL/6 mice induced with lung tumors (Gangjun et al., 2010 (link)). A549 cell lines (5 × 106 cells) were injected into the mice’s flank region, and tumor development was monitored for 30 days. The animals were then divided equally into three groups: Group I received NF, Group II received NFM, and Group III received Marketed MTX powder for injection. Once tumor growth in the lungs was confirmed, a single dose of PI equivalent to 4 mg/kg was reconstituted in water for injection and administered via the tail vein. After 24 h, the mice were sacrificed, and their lungs, spleens, kidneys, and hearts were excised. The excised organs were homogenized, and the amount of MTX present in each organ was analyzed (Ait Bachir et al., 2018 (link)). MTX in specific organs was extracted by vortexing in acetonitrile, followed by partitioning in a chloroform layer. The quantification of MTX was performed using HPLC with a Disodium phosphate buffer: Acetonitrile ratio of 89:11 on an Agilent 1260 Infinity Capillary LC system with a Phenomenex RP18 column (Subramaniyan et al., 2022 (link)).
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