Transferin scFc formulation

Preparation of Tf–lipoplexes
Cationic liposomes (Lip) consisting of dioleoyl trimethyl-
ammonium propane (DOTAP) and dioleoyl phosphatidyl-
ethanolamine (DOPE) (Avanti Polar Lipids, Alabaster, AL) at
a 1:1 molar ratio were prepared either by sonication or ethanol
injection as previously described (L. Xu et al., 1997, 1999a,
2001). Tf–Lip–DNA was prepared according to the in vivo for-
mulation described previously (L. Xu et al., 1999a). Briefly, for
a typical preparation with optimized in vivo formulation, 25 ml
of Tf (5 mg/ml, iron-saturated holo-transferrin; Sigma, St.
Louis, MO) and 50 ml of Lip (2 mM total lipids) plus 75 ml of
water were mixed in a polypropylene tube and incubated for
5–15 min at room temperature with frequent rocking. Ten mi-
crograms of plasmid DNA in 120 ml of 20 mM HEPES buffer,
pH 7.4, was added to the tube, mixed immediately and thor-
oughly, and incubated for 20 min at room temperature with fre-
quent rocking. Thirty microliters of 50% dextrose solution was
then added to the tube. The final DNA:lipid:Tf ratio was
1:10:12.5 (mg/nmol/mg). The HEPES buffer can be replaced by
pure water.