Neutral and Anionic Liposome-Encapsulated Hemoglobin: Effect of Postinserted Poly(ethylene glycol)-distearoylphosphatidylethanolamine on Distribution and Circulation Kinetics

Abstract

To prepare long-circulating liposomes, poly(ethylene glycol) (PEG)-lipid is usually mixed with other lipid components before vesicle formation. PEG-lipids can also be postinserted in the outer layer of liposomes after the preparation. In this study, PEG-distearoylphosphatidylethanolamine was incorporated by postinsertion technique into liposome-encapsulated hemoglobin (LEH) carrying neutral or negative charge. Postinsertion technique improved the encapsulation efficiency of hemoglobin from about 0.0017 to 0.017 (hemoglobin/phospholipid, molar ratio) for a similar lipid composition. Thus, neutral, anionic, PEG-neutral, and PEG-anionic LEHs were made and labeled with technetium-99m to follow their biodistribution. A small dose of LEH (ϳ15 mg of phospholipid) was injected intravenously in rabbits, and its distribution was monitored by blood sampling, gamma camera imaging, and tissue radioactivity counting on necropsy. The 24-h blood levels of neutral, PEGneutral, anionic, and PEG-anionic LEHs were 14, 40.3, 13.1, and 35.7% of injected dose, respectively; calculated T 1/2 values of circulation were 8.9, 19.3, 9.6, and 16.5 h, respectively. PEGylation also influenced accumulation of LEH in the reticuloendothelial system. Liver uptake of neutral LEH dropped from 52.1 to 19.1%, whereas that of anionic LEH came down from 35.3 to 11.5% on PEGylation. In contrast, PEGylation increased the spleen uptake by 8.5-and 2.5-fold for neutral and anionic LEH, respectively. The results demonstrate that PEGylation by postinsertion not only improves the circulation t 1/2 of LEH but also enhances hemoglobin content inside the vesicles for better oxygen-carrying capacity.

Insertion of poly (ethylene glycol)-lipid reduces the liposome-encapsulated hemoglobin-induced thrombocytopenic reaction

“…This technique, called post-insertion, is especially useful in the case of LEH. Post-insertion technique not only doubles the circulation T 1/2 of LEH, it also improves the encapsulation efficiency of hemoglobin [12,32] .…”

“…Cholesterol (Chol) was purchased from Calbiochem (La Jolla, CA) and α-tocopherol was purchased from Aldrich (Waukegan, IL). Glutathione (GSH), octyl-β-glucoside (OBG), and pyridoxal-5' phosphate (PLP) were from Sigma (St. Louis, MO [12,13] .…”

“…The method of LEH manufacturing, including PEGylation by post-insertion of PEG-DSPE has been reported elsewhere [1,12] . Briefly, the lipid phase was homogenized in a microfluidizer (M110-T, Microfluidics Corp., Newton, MA) with hemoglobin containing pyridoxal-5' phosphate as an allosteric modifier.…”

“…PEGylation makes liposomes more compatible in vivo, and helps evade opsonization and RES uptake. The resultant "sterically stabilized liposomes" circulate in blood for a prolonged time period [12] . In this work, we studied the effect of postinsertion of PEG-lipid in the outer phospholipid layer of neutral and negatively charged LEH on the circulation of rabbit platelets in vivo.…”

Hematological Indices for Mice Bearing Ehrlich Tumor When Treated by Liposomes Encapsulated Hemoglobin and Irradiation

“…Hemoglobin (Hb)-based oxygen carriers are classified into acellular chemically modified Hbs and encapsulated Hbs (4,5) . The cellular structure of HbV has characteristics that resemble those of RBCs.…”