Advanced micro osmometer

Two groups of blank SUVs
having membrane lipid composition identical to that of Doxil and Lipodox,
but differing in their intraliposomal concentrations of ammonium or
sulfate ions, were prepared as previously described.^{5 (link),10 (link)} In summary, in the first group, ammonium sulfate solutions of different
concentrations (100, 150, 200, 250, and 300 mM, ammonium-to-sulfate
mole ratio = 2:1) were used. In the second group, an ammonium sulfate
solution (100 mM) mixed with sodium sulfate or ammonium chloride (both
at 150 mM) was used. Namely, in the second group, the ratio of ammonium
to sulfate varied. Osmolality of the above solutions was measured
using the freezing point depression method (Advanced Micro-Osmometer,
model 3320, Advanced Instruments, MA). The transmembrane ion gradients
were created by diafiltration using Labscale TFF system (Millipore,
MA) against 10% sucrose. The desired PLDs prepared by adding doxorubicin
hydrochloride solution to these SUVs to a final doxorubicin concentration
of 2 to ∼16 mg/mL total lipids followed by incubation at 60
°C for 30 min. Histidine buffer (pH 6.5, to a final 10 mM concentration)
was added after cooling. The PLD dispersion’s osmolality is
350–360 mOsm/kg.^{8 (link)} The blank liposomes
and PLDs were stored at 2–8 °C until use.