*2.5. Pharmacokinetics and Biodistribution of N-AI PAI-2 Liposomes in Mice*

Female BALB/c-Fox1nu/Ausb nude immunocompromized mice (5 weeks old) (Australian BioResources, Moss Vale) were housed in isolator cages at the University of Wollongong animal facility. Mice were given food and water ad libitum and kept on a 12-h light/dark cycle for the duration of the experiment. Mice were allowed to acclimatize for 2 weeks before commencement of the experiment. All experiments were conducted in accordance with the 'NHMRC Australian Code for the Care and Use of Animals for Scientific Purposes', which requires 3R compliance (replacement, reduction, and refinement) at all stages of animal care and use, and the approval of the Animal Ethics Committee of the University of Wollongong (Australia) under protocol AE13/18. MDA-MB-231 cells (ATCC; mycoplasma negative and STR profiled) were resuspended in PBS (no Ca/Mg; pH 7.4; Sigma-Aldrich, MO, USA) and counted using Trypan blue (Sigma-Aldrich, MO, USA) and a hemocytometer. Insulin syringe needles (29-gauge; BD Biosciences, NJ, USA) were used to inject 50 µL of cell suspension (containing 2 <sup>×</sup> <sup>10</sup><sup>6</sup> cells) into the upper left mammary fat pad. Mice were injected one cage at a time and the injection order of cages was randomized. Mice were monitored closely following the injection of cells, and the tumors were observed to form at approximately 3 weeks post-injection.

Tumors were <100 mm<sup>3</sup> upon commencement of liposome treatment. Mice were randomly allocated to treatment (*N*-AI liposome or *N*-AI PAI-2 liposome) and time-point (10 min, 3 h, 6 h, 24 h, 48 h or 96 h) groups (4 mice per cohort). Treatments (100 µL; 4 µCi/mouse) were administered intravenously via a single lateral tail-vein injection. Mice that were deemed significantly (±10%) smaller or larger in weight than their cage mates had their dose volume adjusted proportionally, based on their weight, relative to the average of their cage mates. The <sup>3</sup>H-CHE radioactivity (liposome) in the plasma, kidneys, liver, spleen, lungs, tumor and tail (for injection correction) was quantified using previously published methods [33]; further details are provided in Supplementary Information.
