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 (∼15 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, PEG-neutral, anionic, and PEG-anionic LEHs were 14, 40.3, 13.1, and 35.7% of injected dose, respectively; calculated T1/2 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 t1/2 of LEH but also enhances hemoglobin content inside the vesicles for better oxygen-carrying capacity.
Footnotes
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This study was funded by the Office of Naval Research, Washington, DC (award no. N00140010793).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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DOI: 10.1124/jpet.103.060228.
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ABBREVIATIONS: LEH, liposome-encapsulated hemoglobin; DMPG, dimyristoylphosphatidylglycerol; DSPC, distearoylphosphatidylcholine; RES, reticuloendothelial system; PEG, poly(ethylene glycol); PEG-PE, poly(ethylene glycol)-phosphatidylethanolamine; DSPE, distearoylphosphatidylethanolamine; Chol, cholesterol; HMPAO, hexamethylpropyleneamine oxime; O2-Hb, oxyhemoglobin; CO, carbon monoxide; CO-Hb, carbonyl hemoglobin; PBS, phosphate-buffered saline; 99mTc, technetium-99m.
- Received September 18, 2003.
- Accepted December 18, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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