Preparation of immunoliposomes

Immunoliposomes composed of DSPC and cholesterol (molar ratio 2:1) containing DSPE-PEG(2000) maleimide at 1.5 mol% of DSPC as a coupling lipid were prepared according to the method of dried lipid film hydration in PBS (pH 7.4) as previously described.27 DiI (fluorescent lipophilic dye) was added at 0.2 mol% of DSPC, and drugs were incorporated based on their hydrophilicity. The resulting multi-lamellar dispersions were reduced in size and lamellarity by ultrasonication at 60% amplitude for 5 minutes at 65°C. The activated liposome suspension was immediately mixed with thiolated antibody at room temperature. Thiolated antibodies were prepared by conjugating anti-OTR monoclonal antibodies (25 µg) or non-specific rabbit IgG (25 µg) with SPDP (6.25 mg/mL; SPDP/mAb molar ratio =10:1). PD-10 column equilibrated with distilled water was used to remove excess SPDP and fractions containing pyridyldithiopropionated-Ab (PDP-Ab) conjugates (assessed by absorbance at 280 nm) were lyophilized and stored at 4°C under nitrogen gas. PDP-Ab was reduced with 5 mM TCEP for 5 minutes to produce thiolated-Ab (Ab-SH), and absorbance was checked at 280 nm (protein concentration) and 343 nm (SPDP modification) to ensure stability of the compound. Thiolated antibody was mixed immediately with liposomes for 1 hour at room temperature with stirring in the dark. TLX ultracentrifugation (Optima™) was used to remove unconjugated Ab and non-encapsulated drug (100,000× g; 45 minutes). Liposomes were resuspended in PBS (pH 7.4) and stored at 4°C under nitrogen gas and in the dark, and were used within 2 weeks.