1 2 distearoyl sn glycero 3 phosphoethanolamine

For in vivo delivery, siRNA was mixed with 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) from Avanti Polar Lipids Inc in the presence of excess tertiary butanol at a ratio of 1:10 siRNA:DOPC (weight: weight) as described previously^{15 (link)}. PEG-modifed nanoliposomes were prepared using DOPC and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) with various PEG linker lengths, including PEG 1000, PEG 2000 and PEG 5000 (Avanti Polar Lipids Inc). DOPC was mixed with DSPE-PEG (10:1) and later mixed with Si14 or luciferase targeting siRNA at 10:1 lipid to siRNA ratio in excess tertiary butanol and lyophilized overnight. The mean size of the liposomes was about 78 nm by Zetasizer Nano (Malvern). Prior to in vivo administration, the lyophilized mixture was reconstituted with saline at a concentration of 1.5 mg/ml to achieve the desired dose in 100 μl per i.v. injection via tail vein. Folate bound DSPE-PEG- was purchased from Avanti Polar Lipids Inc. Preparation of folate or peptide coated-PEG-modified liposomal–siRNA nanovectors were done using 10:1 ratio of DOPC:DSPE-PEG-linker containing maleimide functional group (Avanti polar lipids Inc) that was used for conjugation with RGD (Arg-Gly-Asp-D-Phe-Cys), or bFGFR1 binding peptide (Lys-Arg-Thr-Gly-Gln-Tyr-Lys-Leu-Cys^{16 (link)}; Peptide International Inc)

Fmoc-protected amino acids, resins, and coupling reagents for solid-phase synthesis were purchased from EMD Millipore. Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) was purchased from Sigma-Aldrich. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-mPEG1000 (mPEG1000-DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000-azide (N₃-PEG2000-DSPE), and cholesterol (Chol) were purchased from Avanti Polar Lipids. Cyanine 5.5-NHS ester was purchased from Lumiprobe. Cellulose acetate (CA) syringe filters were purchased from Macherey-Nagel Inc. Sephadex^® G-50 and LH-20 were purchased from GE Healthcare. LIVE/DEAD^® fixable green dead cell stain was purchased from ThermoFisher. AlexaFluor^® 680-Wheat Germ Agglutinin conjugate (WGA) and LysoTracker^® Blue DND-22 were purchased from Life Technologies. Cell culture reagents such as DMEM, FBS, trypsin, and PBS were purchased from Atlanta Biologicals. Ultrapure water (18 MΩ) was used for preparation of all buffers and in all experiments. All solvents were of analytical grade, purchased from commercial sources and used without further purification, except DMF which was dried over CaH₂ under N₂, filtered, and distilled under reduced pressure.