Fabrication of Lipid-Magnetic Nanovesicles

25 mg of 1-stearoyl-rac-glycerol (Sigma-Aldrich), 2.5 mg of oleic acid (Sigma-Aldrich), 2.5 mg of 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (Sigma-Aldrich), 4 mg of mPEG-DSPE_5k (Sigma-Aldrich), and 2.5 mg of temozolomide (Sigma-Aldrich) (when TMZ-LMNVs are fabricated), are mixed with 84.5 μl of an ethanol solution of superparamagnetic iron oxide nanoparticles (15 wt%; US Research Nanomaterials Inc.), inside a 6 ml glass vial. Subsequently, the vial is placed inside an ultrasonic bath (Elmasonic S 35w) set at 70 °C in order to melt the lipids and to allow ethanol to evaporate. After ethanol evaporates, 3 ml of a pre-warmed (70 °C) Tween® 80 (Sigma-Aldrich) solution (1.0 wt%) were added to the lipid mixture and immediately sonicated using an ultrasonic homogenizer (Fisherbrand™ Q125 Sonicator) for 15 min (amplitude 30%, 120 W). After the ultrasonic homogenization, the hot mixture is transferred to a high pressure homogenizer (HPH, EmulsiFlex-B15 from Avestin), where the sample is further homogenized by passing it 5 times through the homogenizer at a pressure of 100 000 psi. After the homogenization, the LMNVs are placed for 30 min at 4 °C to allow the lipid-based structures to stabilize. The LMNVs are purified by centrifugation and washing with ultrapure (Mili-Q) water (3 times for 30 min at 4 °C).