Icap qc quadrupole

To measure the cellular uptake of 2–3 and 5–6, ca. 1 million U2OS cells were treated with 2–3 and 5–6 (0.25 μM) at 37 °C for 24 h. After incubation, the media was removed and the cells were washed with PBS (2 mL×3), and harvested. The number of cells was counted at this stage, using a haemocytometer. This mitigates any cell death induced by 2–3 and 5–6 at the administered concentration and experimental cell loss. The cells were centrifuged to form pellets. The cellular pellets were dissolved in 65 % HNO₃ (250 μL) overnight. The cellular pellets were also used to determine the gallium content in the nuclear fraction. The Thermo Scientific NE‐PER Nuclear and Cytoplasmic Extraction Kit was used to extract and separate the nuclear fraction. The fractions were dissolved in 65 % HNO₃ overnight (250 μL final volume). All samples were diluted 5‐fold with water and analysed using inductively coupled plasma mass spectrometry (ICP‐MS, Thermo Scientific iCAP‐Qc quadrupole). Gallium levels are expressed as Ga (ng) per million cells. Results are presented as the mean of four determinations for each data point.

Cellular uptake studies involving the nanoparticle formulations 1:1 NP¹⁵ or 4:1 NP¹⁵ were conducted under various conditions. HMLER-shEcad cells (ca. 1 million) were treated with 1:1 NP¹⁵ or 4:1 NP¹⁵ (both 0.15 µM) at 4 °C or 37 °C for 4 h or 8 h. Experiments were also conducted in the presence of endocytosis inhibitor chloroquine (100 μM, 2 h pre-treatment). After incubation, the media containing the nanoparticle formulations 1:1 NP¹⁵ or 4:1 NP¹⁵ (with or without chloroquine) were aspirated and the remaining adherent cells were thoroughly washed with 2 mL of PBS, three times. The cells were then collected by trypsinisation and centrifuged to form a pellet. The resultant pellets were digested with 65% HNO₃ (250 μL) overnight at room temperature. The solutions were then diluted with MilliQ water and measured by ICP-MS to determine the copper content (Thermo Scientific iCAP-Qc quadrupole). The copper content in each sample (cellular material) is represented as Cu (ng) per million cells (overall n = 4).