Dil stain

Doxorubicin HCl (DOX) and QC (purity > 95%) were obtained from Ebewe Pharma (Unterach, Austria) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Soybean phosphatidylcholine and DSPE-PEG2000 (distearoyl phosphoethanolamine-polyethylene glycol) were obtained from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol was supplied by Sigma-Aldrich (St. Louis, MO, USA). PBS tablets, dialysis bags (MW  =  12  kDa), DMSO (dimethyl sulfoxide), MTT (3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and paraformaldehyde solution were procured from Sigma-Aldrich (St. Louis, MO). DAPI (4′,6-diamidino2-phenylindole) and DIL Stain (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate) were supplied by Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals, solvents, and salts were of the analytical grade and used without further purification unless specified.

The sulforaphane solution (2-isopropyl-5-methylbenzoquinone), cholesterol, DSPE-PEG 400 (distearoyl phosphoethanolamine-polyethylene glycol 400), and PVA (polyvinyl alcohol) were purchased from Sigma-Aldrich (St. Louis, MO, USA). DSPE-PEG 2000 (distearoyl phosphoethanolamine-polyethylene glycol 2000)SPC80 (soybean phospholipids with 75% phosphatidylcholine), and Dipalmitoylphosphatidylcholine (DPPC) were obtained from Lipoid GmbH (Ludwigshafen, Germany). The dialysis bag (MW = 12 kDa), PBS (phosphate-buffered saline) tablets, MTT (3–(4, 5-dime-thylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide), DMSO (dimethyl sulfoxide), chloroform, isopropanol, methanol, and the paraformaldehyde solution, were procured from Sigma-Aldrich (St. Louis, MO, USA). DIL stain (1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindocarbocyanine perchlorate) and DAPI (40, 6-diamidino2-phenylindole) were supplied from Thermo Fisher Scientific (Waltham, MA). Other reagents used in this investigation were of analytical grade without further purifications.

Both CXCR4-targeted (T140-MB) and non-targeted (NT-MB) MB were produced in-house. The NT-MB’s shell contained a mixture of DPPC, DPPA and DSPE-PEG₂₀₀₀-NH₂ (81.9:8.6:9.5 mole ratio, respectively) (Avanti Lipids, Alabaster, AL, USA) (S1 Table) and in the T140-MB, 4.8% of the DSPE-PEG₂₀₀₀-NH₂ was replaced by the modified lipid, DSPE-PEG₂₀₀₀-T140 (Fig 1A and S2 Table). These components were dissolved in CHCl₃ and aliquoted into 3 mL vials from which the solvent was dried using a N₂ flow and stored at -20⁰C. Before use, the vials were reconstituted with propylene glycol, phosphate-buffered saline (PBS) and glycerol (16:5:1) whilst stirring. After degassing for a minimum of 15 min, the vial was crimp-sealed and the headspace of the vial was gas-exchanged with 99.0% n-perfluorobutane (FluoroMed L.P., Round Rock, TX, USA). MB were formed via standard mechanical agitation techniques using a Vialmix shaker (Bristol-Myers-Squibb, NY, USA) (2x30 sec shaking). Fluorescent MB were developed by adding 2 μg/mL of Dil stain (1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine perchlorate, DilC₁₈(3), Thermo Fisher Scientific, Carlsbad, CA USA).

A 2-well Lab-Tek Chambered Borosilicate coverglass (Fisher Scientific) was coated with 20 mL of 0.1% (w/v) poly-L-Lysine solution (Sigma-Aldrich) for 30 min and dried. A yeast strain (Nup49p-GFP; Yeast GFP Clone Collection; Thermofisher) in which GFP was tagged to the nucleolar protein Nup49 was used. Stock solutions of Dil Stain (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate; Invitrogen), 250 μg/mL, and DAPI (200 μM) (Invitrogen) were prepared in dimethyl
sulfoxide and phosphate-buffered saline, respectively. Freshly isolated nuclei (20 μL) were mixed with 2 μL of Dil Stain and 2 μL of DAPI followed by incubation for 10 min at RT. Fluorescent stained nuclei were then applied to the coated cover glass and imaged with a Zeiss LSM 780 confocal microscope. Western blotting was performed using nuclei from W303 cells as described.^{12 (link)} Anti-nop1p antibody (Santa Cruz Biotechnologies) was used as a nuclear marker at 1:500 dilution.

MCF7 cells were grown on 10 cm plates until complete confluency. Cells were harvested (using EDTA/PBS; Invitrogen) and aliquoted into individual samples containing 1 × 10⁶ cells for labeling. Samples were stained with either anti-rabbit GRP78 ET-21 (Sigma) antibody, or CD24-APC or CD44-FITC (eBioscience) for 1 hr at 4 °C. Primary antibody (GRP78 ET-21) was used at 5 μg/1million cells. When appropriate, cells were incubated with anti-rabbit 488 secondary antibody (Invitrogen) for 1 hr at 4 °C. Cells were then either analyzed via flow cytometry (BD Fortessa) or sorted (BD FACSAria Cell Sorter) based on the staining of both a secondary-only and IgG (Sigma) control (for GRP78-labeled cells) or single-stained controls (APC/FITC). When cells were sorted for the in vivo experiments, all cells were labeled with a Dil Stain (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate) (Invitrogen) immediately following sorting, just before injection, according to the manufacturer’s recommendations.

On day 42, UiPSC-derived neurons with astrocyte co-culture were washed with DPBS and rapidly fixed with 4% PFA for 1–2 min at room temperature. The UiPSC-derived neurons were removed from the fixative and specifically labeled with a Helios Gene Gun (Bio-Rad, 297BR). Bullets were prepared using Dil Stain (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate; Invitrogen, cat. no. D282). A slice with neurons was shot three times by one bullet. We added 100 μL DPBS to the neurons f to prevent them becoming dry, and we incubated the neurons for 3 h in the dark at room temperature. Slices were mounted with antifade mountant. The excitation and emission wavelengths of Dil (D282) were 549 and 565 nm, respectively. Images were captured by a TCS SP8 STED 3X multiphoton confocal microscope and were analyzed using Fiji software (ImageJ).