Bransonic 12

We characterized commercially pure titanium particles (Johnson Matthey Chemicals, Ward Hill, MA, USA) in our previous studies, showing a size range considered clinically relevant (size range of 1–15 µm; 75% were < 4 µm in diameter)^{18 (link),22 (link),23 (link)}. The titanium particles were sterilized by incubation in isopropanol at RT and dried for 48 h under UV light in a laminar flow hood^{18 (link),22 (link)–24 (link)}. The suspensions of titanium particles employed in this study had endotoxin levels of < 0.015 endotoxin units (EU)/mL, as demonstrated using the E-TOXATE assay for detection and semi-quantification of endotoxins (Sigma, Madrid, Spain). We incubated 0.1 mL of the particle suspensions (resuspended in endotoxin-free water at 5 mg/mL) with E-TOXATE reagent at 37 °C for 1 h. Endotoxin-free water and endotoxin standards derived from Escherichia coli were diluted at 0.015–4 EU/mL and incubated in parallel with E-TOXATE reagent. Gel formation was absent in samples that contained endotoxin levels below the detection limit of the assay.
As described in our previous studies^{18 (link),22 (link),23 (link)}, the particles were resuspended at 20 mg/mL in growth medium, sonicated for 10 min at maximum power in a bath sonicator (Bransonic 12, Branson Ultrasonidos SAE, Barcelona, Spain) and thoroughly vortexed. Immediately after, the particle suspension was added to the cells.

HOla was prepared using two different procedures: i) the disassembly/reassembly method^{22 (link)} and ii) the drug complexation with Cu(II)^{24 (link)}. i) HFn nanoparticles (2 mg) were disassembled in 1.5 mL of 0.15 M NaCl by raising the pH to 12. Then, Olaparib powder (0.5 mg; Alsachim, C3734) previously solubilized by 1 h of sonication (Bransonic 12, Branson) in NaOH 0.1 M (0.5 mL) was added to the solution of disassembled HFn and incubated for 10 minutes at room temperature (RT). Then, the pH value was lowered to 7.5 using 0.1 M HCl. The resulting solution was stirred at RT for 2 h in order to favor the nanoparticle refolding. The solution was concentrated through a 100 kDa Amicon filter (Millipore) and loaded to Zeba™ Spin Desalting Column (Thermo Fischer Scientific) to remove free Ola. ii) Olaparib powder, solubilized as previously described, was incubated for 10 min at RT with 10 mM CuSO₄ obtaining a Cu(II)-Ola complex. The complexed drug was added to HFn (2 mg) and incubated 2 h at RT. HOla was separated from free Ola by a gel filtration to Sephadex G-25 column. The amount of HFn in HOla sample was determined assessing the protein content by Bradford assay, while the amount of encapsulated Ola was determined by quantitative MS analysis, as described below.

The freeze-dried media and cell extract samples were dissolved in equal volume, 200 µL and 400 µL, respectively, of double distilled deionized water. To ensure complete solubilization, samples were subjected to vortexing for 5 min using the Vortex V-1 Plus (Scientifix) and sonication for 10 min (Bransonic 12, Branson) thrice, each followed by centrifugation at 18,000× g for 30 min using a Velocity 15µR Microcentrifuge (Dynamica). Supernatants were transferred to mass spec vials for analysis.