Delsa nano

Particle size and polydispersity index (PDI) were determined by dynamic light scattering (Beckman Coulter Delsa^™Nano) at 25°C. TyroSphere liquid dispersions were diluted 10 times for size measurement and were examined in triplicate by normalized intensity distribution for cumulants, size distribution, and polydispersity. An average of 50 measurements were recorded per replicate (Kilfoyle et al., 2012 (link)).

Superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized using coprecipitation method.^{45 (link)} Prior to the synthesis, 0.5 M NaOH solution in deionized water (DIW) was prepared in a three-neck 500 mL round-bottom flask (RBF) and degassed by bubbling N₂ under stirring at room temperature (RT) for 30 min, followed by degas under vacuum under stirring at RT for another 30 min, and then it was heated to 40 °C. Then ferric chloride FeCl₃·H₂O (6.56 g, 0.024 mol) and ferrous chloride FeCl₂·4H₂O (2.48 g, 0.012 mol) were dissolved in 25 mL of degassed 0.4 M HCl solution in DIW and then added to the RBF through a septum. The RBF was heated at 80 °C under strong stirring for 1 h. SPIONs were precipitated using a strong neodymium N52 magnet, and then the reaction mixture was decanted. The SPIONs were washed five times by dispersing them back in EtOH (300 mL) with probe sonication for 10 min, followed by magnet precipitation and decantation of the liquid. A dry powder of SPIONs was obtained by drying under vacuum on a rotary evaporator. The product was characterized by Fourier transform infrared (FTIR) spectroscopy. Hydrodynamic size and zeta potential were characterized by dynamic light scattering using Beckman Coulter Delsa Nano.

Liposomes were prepared by the thin film hydration method ^{20 (link)–22 (link)}. A lipid mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids, Alabaster, AL, USA) and cholesterol (Sigma, St. Louis, MO, USA) 2:1 molar ratio was dissolved in a 9:1 chloroform : methanol solution. The lipid mixture underwent vacuum evaporation to form a dry lipid film. T-butanol was used to dissolve the lipid thin film and freeze-dried to form a lipid cake. The lipid cake was hydrated with phosphate buffered saline (PBS), DMED solution (1 mg/mL PBS; Sigma, St. Louis, MO, USA), DexP solution (50 mg/mL PBS; Sigma, St. Louis, MO, USA), or 250 mM (NH₄)₂SO₄. The liposomes then underwent ten freeze-thaw cycles followed by dialysis against PBS for 48 hours. Bup (Sigma, St. Louis, MO, USA) was loaded into liposomes using a previously reported remote loading technique ^{23 (link)}. Briefly, after dialysis in PBS, the liposomes that were hydrated with 250 mM (NH₄)₂SO₄ were loaded with Bup upon incubation with a 50 mg/mL Bup hydrochloride solution at 50°C for 1 h. The liposomes were then dialyzed against PBS for 48 hours.
Liposome size was determined by dynamic light scattering (Delsa Nano, Beckman Coulter, Brea, CA, USA). Drug loading was determined by HPLC after disrupting the liposomes with 100 mM octyl β-D-glucopyranoside (Sigma, St. Louis, MO, USA).