The GS-nitroxide JP4-039 has been described in detail previously (17 (link)-21 (link)). JP4-039 (21 (link)) was formulated at final drug concentrations of 8 mg/ml in cationic mutilamellar liposomes termed F-15. F-15 is a unique form of multilamellar liposome (N,N-dioleylamine amido-L-glutamate), which was utilized as a solvent for JP4-039 in order to facilitate adherence of the drug to the esophageal mucosa. The drug is entrapped between lipid bilayers and allows slow release over time from the liposome particles. F-15 was cationically charged to facilitate surface coating and retention for esophageal mucosa. Its composition was: soy PC: Tween-80: N,N-dioleylamine amido-L-glutamate (4:1:1w/w) with a final drug concentration of 8 mg/ml in PBS. It has low toxicity to cultured mammalian cells (>0.5 mg/ml).
Soy phosphatidyl choline, Lissamine rhodamine-phycoerythrin were obtained from Avanti Polar Lipids (Alabaster, AL, USA); Tween-80, tert-boc-L-glutamic acid, oleylamine, dicyclohexylcarbodiimide, N-hydroxysuccinimide, trifluoroacidic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's phospate-buffered saline (d-PBS) was obtained from Lonza (Walkersville, MD, USA). A cationic lipid, L-glutamic acid-1,5,-dioleyl amide [NH2-L-Glu(NHC18H36)2] was synthesized using a modified route as previously described (37 (link)), by coupling tert-boc-L-glutamic acid and oleylamine with dicyclohexylcarbodiimide and N-hydroxysuccinimide as the coupling agents, followed by use of trifluoroacidic acid as the deprotecting agent.
The lipid mixture (6 mg) and drug to be encapsulated (1 mg) were dissolved in 100 μl tert-butanol, frozen on dry ice, and lyophilized overnight into a cake. The next day, a 62.5 μl d-PBS was added to the lipid cake which was allowed to hydrate for 24 h at room temperature. Cationic liposomes were prepared from the hydrated lipid suspension by manual homogenization using a pair of custom-made tight-fit tube and pestle until a homogeneous consistency were reached. Finally, the liposome suspension was removed from the tube and another 62.5 μl d-PBS was used to rinse the tube and pestle and the wash solution was combined with the liposome suspension. Thus, 1 mg JP4-039 was formulated in 225 μl volumes. The final particle sizes were measured by a laser dynamic scattering method (NP-4 Particle Sizer, Beckman Coutler, Inc., Brea, CA, USA) and found to be in the range of 200-300 nm with a mean of ~255 nm in diameter. Each mouse received an intraesophageal injection of 110 μls of F15 formulation containing 400 μg JP4-039. To determine whether Tween-80 was required for effective uptake, an identical formulation (F14) without Tween-80 was tested.
Soy phosphatidyl choline, Lissamine rhodamine-phycoerythrin were obtained from Avanti Polar Lipids (Alabaster, AL, USA); Tween-80, tert-boc-L-glutamic acid, oleylamine, dicyclohexylcarbodiimide, N-hydroxysuccinimide, trifluoroacidic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's phospate-buffered saline (d-PBS) was obtained from Lonza (Walkersville, MD, USA). A cationic lipid, L-glutamic acid-1,5,-dioleyl amide [NH2-L-Glu(NHC18H36)2] was synthesized using a modified route as previously described (37 (link)), by coupling tert-boc-L-glutamic acid and oleylamine with dicyclohexylcarbodiimide and N-hydroxysuccinimide as the coupling agents, followed by use of trifluoroacidic acid as the deprotecting agent.
The lipid mixture (6 mg) and drug to be encapsulated (1 mg) were dissolved in 100 μl tert-butanol, frozen on dry ice, and lyophilized overnight into a cake. The next day, a 62.5 μl d-PBS was added to the lipid cake which was allowed to hydrate for 24 h at room temperature. Cationic liposomes were prepared from the hydrated lipid suspension by manual homogenization using a pair of custom-made tight-fit tube and pestle until a homogeneous consistency were reached. Finally, the liposome suspension was removed from the tube and another 62.5 μl d-PBS was used to rinse the tube and pestle and the wash solution was combined with the liposome suspension. Thus, 1 mg JP4-039 was formulated in 225 μl volumes. The final particle sizes were measured by a laser dynamic scattering method (NP-4 Particle Sizer, Beckman Coutler, Inc., Brea, CA, USA) and found to be in the range of 200-300 nm with a mean of ~255 nm in diameter. Each mouse received an intraesophageal injection of 110 μls of F15 formulation containing 400 μg JP4-039. To determine whether Tween-80 was required for effective uptake, an identical formulation (F14) without Tween-80 was tested.