No. A9647), 4′,6-diamidino-2-phenylindole (DAPI) (Cat. No. D9542), 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) (Cat. No. D4292), N-N-dimethylformamide (Cat. No. D4551), nonimmune IgG from human serum (Cat. No. I4506), 99.9% hydroxylamine (Cat. No. 55459), 4% paraformaldehyde (Cat. No. P6148), 4B sepharose (Cat. No. 43200), Tween 20 (Cat. No. P9416). Antibodies Inhibitors,research,lifescience,medical and vendors: Alexa Fluor 488 goat-anti-human (H+L) (Molecular Probes, Invitrogen, Cat. No. A-11013), Alexa Fluor 555 goat-anti-human

(H+L) (Molecular Probes, Invitrogen, Cat. No. A-21433), Alexa Fluor 555 donkey-anti-goat (Molecular Probes, Invitrogen, Cat. No. A-21432), Alexa Fluor 488 goat-anti-rabbit (Molecular Probes, Invitrogen, Cat. No. A-31565), Alexa Fluor 555 goat-anti-rabbit (Molecular Probes, Invitrogen, Cat. No. A-21427), monoclonal Inhibitors,research,lifescience,medical anti-human epidermal growth factor receptor antibody (Merckserono, Erbitux), polyclonal rabbit anti-laminin antibody (DAKO, Cat. No. Z0097), and polyclonal goat-anti-mouse albumin (Nordic Biosite, Cat. No. A90-134A). 2.2. Cell Lines The cell lines used in the study were U87mg (American Type Culture Collection [ATCC], Cat. No. HTB-14) and U251mg (Health Protection Agency Culture Collection [HPA Culture Inhibitors,research,lifescience,medical Collection], Cat. No. 09063001). The cell lines U87mg and U251mg were cultured in DMEM supplemented with 10% (FCS) and 1% penicillin/streptomycin.

Cell cultures were kept in a humidified atmosphere containing 5% CO2 buffered with ambient air at 37°C. The cell medium was changed twice a week. 2.3. Liposome Preparation Liposomes were prepared from soy phosphatidylcholine Inhibitors,research,lifescience,medical (soyPC), cholesterol, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (DSPE-PEG2000-Mal), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene

Inhibitors,research,lifescience,medical glycol)-2000] (mPEG2000-PE), and the fluorescent probe DiO in a molar ratio of 65:30:2:3:0.5. The lipids used for this procedure were all dissolved in PI-103 nmr chloroform and transferred to a round-bottom flask. A thin lipid film was formed by evaporating the chloroform with a stream of gaseous nitrogen for 30 minutes at room temperature. The resulting lipid film was hydrated in CYTH4 HEPES Buffer (10mM HEPES, 136mM NaCl, and 1mM EDTA). To ensure that the lipid film was completely dissolved, the flask was immediately vortexed, and to allow complete hydration the flask was incubated at room temperature on a shaker for one hour. The homogenous liposomes were prepared by a manual extrusion technique by passing through polycarbonate membranes 20 times for each filter with pore sizes of 0.2μm, 0.1μm, and finally 0.05μm. 2.4. Formation of Immunoliposomes The anti-human-EGFR antibody (Erbitux) was used to form immunoliposomes (α-hEGFR-IL’s). Control liposomes were either prepared by conjugation with nonimmune IgG from human serum (hHIgG-IL’s) or left unconjugated (naked liposomes).