Q125 sonicator

The preparation of the magnetic MNVs was performed by following a procedure optimized from a previous work.^[20 (link)
^] Briefly, 17.5 mg of 1‐stearoyl‐rac‐glycerol (GMS, Sigma‐Aldrich) and 2 mg of DPPC (Sigma‐Aldrich) were mixed with 500 µL of a 10 mg mL⁻¹ solution of IONPs in chloroform and heated at 70 °C. 3 mL of an aqueous solution (1.0 wt%) of Tween 80 (Sigma‐Aldrich), previously heated at 70 °C, were added to the melted lipid/IONPs mixture; the dispersion was then vortexed and sonicated for 10 min (amplitude 90%) with an ultrasonic tip (Fisherbrand Q125 Sonicator). MNVs were cooled down at 4 °C for 30 min and purified by centrifugation (16 000 g, 90 min, at 4 °C for three times), and finally redispersed in Milli‐Q water (Millipore). Thereafter, MNVs were coated with CM extracted from patient cells to obtain CDMNVs. Around 5 mg of MNVs were mixed with five pellets (deriving from about 2.4 × 10⁶ cells) of CM extracts in 3 mL of sterile Milli‐Q water. The dispersion was sonicated with an ultrasonic tip for a total of 20 min at 20% amplitude (in “pulse” mode: 59 s ON, 20 s OFF) in an ice bath to avoid thermal denaturation and defolding of membrane proteins. Thereafter, CDMNVs were purified by three steps of centrifugation (16 000 g, 90 min, 4 °C) and finally redispersed in Milli‐Q water.
Nanovectors coated with deproteinated cell membrane extracts (CD*MNVs) were prepared following the same procedure. Drug‐loaded nanovectors (Reg‐CDMNVs) were synthesized by adding and 1 mg of regorafenib (MedChemExpress) to the initial lipid mixture. Nanovectors labeled with fluorescent Vybrant DiO cell‐labeling dye (Invitrogen) were analogously prepared, with the addition of 10 µL of dye in the lipid mixture.
The concentrations of the nanovector dispersions were assessed by weighting the dried pellets of known volumes of each dispersion upon freeze‐drying.

Nutlin-3a loaded P(VDF-TrFE) nanoparticles (Nut-PNPs) were synthesized and surface-functionalized with a peptide that corresponds to a fragment of apolipoprotein E (ApoE; GenScript), as previously described in a work of our group [15 (link)]. Briefly, 2 ​mL of 5 ​mg/mL P(VDF-TrFE) (45:65; Piezotech) and 200 ​μL of 5 ​mg/mL Nutlin-3a (Sigma-Aldrich) in acetone (Sigma-Aldrich) were placed into 4.5 ​mL of a 1 ​mg/mL 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG; Nanocs) dispersion in water under stirring. The obtained mixture underwent three consecutive cycles of sonication (ultrasonic tip; Fisherbrand™ Q125 Sonicator) and purification (Amicon® centrifuge filters, Ultra-4 Centrifugal Filter Unit, 100 ​kDa; Sigma-Aldrich) with the pellet suspended in water (4 ​mL). The polymeric core of the nanoparticles was recrystallized by refluxing (90°C for 60 ​min) and then by progressively cooling down to 25°C (−1°C/min). Subsequently, 1 ​mg of DSPE-PEG/DSPE-PEG-maleimide (1:1) was added for the stabilization of the nanoparticle dispersion, which was sonicated for 10 ​min (ultrasonic tip, 70% amplitude; Fisherbrand™ Q125 Sonicator). Finally, three consecutive centrifugation steps were performed at 15°C (2460 ​g for 15 ​min; Amicon® centrifuge filters, Ultra-4 Centrifugal Filter Unit, 100 ​kDa; Sigma-Aldrich) to remove the excess of lipids. The bioconjugation of the nanoparticles with the ApoE peptide was carried out through the maleimide-thiol click reaction [20 (link)] by incubating the 141–150 residues of the ApoE (200 ​μL of a 2 ​mg/mL water suspension) to 4 ​mL of the 2 ​mg/mL nanoparticle dispersion for 4 ​h at 4°C under shaking. Three centrifugation steps were then carried out as described above to remove the non-bounded peptide. ApoE-PNPs were synthesized following the same protocol used for ApoE-Nut-PNPs, without adding the drug in the polymer/acetone solution. Nut-PNPs and PNPs were obtained skipping the functionalization step. The fluorescent staining of the nanoparticles was obtained by adding 5 ​μL DiO fluorescent dye (Vybrant™; Invitrogen) to the 2 ​mL polymer/acetone initial solution.
Morphologic analysis of PNPs, Nut-PNPs, ApoE-PNPs, and ApoE-Nut-PNPs was performed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Concerning TEM, a drop of 70 ​μg/mL nanoparticle dispersions was cast on an ultrathin amorphous carbon film-coated Cu grid composed of 150 mesh and, subsequently, imaging was performed with a JEOL 1011 operating at 100 ​kV. For SEM imaging, a drop of nanoparticle dispersions was deposited on a silicon wafer. Then, samples were imaged with a Helios NanoLab 600i Dual Beam™ FIB/SEM FEI after gold-sputtering (30 ​mA for 1 ​min) with a Quorum Tech Q150RES Gold Sputter Coater. The nanoparticle size was measured from the TEM images by using ImageJ software, and data were reported as average diameter ​± ​standard deviation.
The hydrodynamic size of PNPs, Nut-PNPs, ApoE-PNPs, and ApoE-Nut-PNPs (500 ​μg/mL) were investigated in water at 37°C by using a Nano Z-Sizer 90 (Malvern Instrument); the ζ-potential measurements were performed in the same conditions. The hydrodynamic size and ζ-potential measurements are shown as the mean ​± ​standard deviation of three different measurements with 10 runs for each of them. CONTIN analysis was used to obtain the intensity distribution, and the value of the hydrodynamic diameter and the polydispersity index (PdI) was assessed by cumulant analysis. Furthermore, stability of PNPs, Nut-PNPs, ApoE-PNPs, and ApoE-Nut-PNPs was assessed at a concentration of 500 ​μg/mL in plasma obtained from mice blood (see Section 2.4 for details), at 37°C for 14 days, periodically performing dynamic light scattering measurements.