Biological evaluation of liposome-encapsulated hemoglobin (LEH) surface-modified with a novel PEGylated non-phospholipid amphiphile

摘要

Traumatic injury is often associated with hemorrhagic shock. Liposome-encapsulated hemoglobin (LEH) is being developed as an artificial oxygen carrier to address post-hemorrhage oxygen and volume deficit. Here, we report a new composition of LEH based on the use of a PEG-non-phospholipid, hexadecylcarbamoylmethylhexadecanoate-PEG2K (HDAS-PEG2K) for modifying the surface of LEH particles. LEH was manufactured by high-pressure homogenization method using dipalmitoylphosphatidylcholine (~38 mol%), cholesterol (~38 mol%), HDAS (~20 mol%), and highly-purified stroma-free human hemoglobin. HDAS-PEG2K was post-inserted into the resultant LEH to generate HDAS-PEG2K-LEH. We investigated HDAS-PEG2K-LEH in mice models for the potential immune response. At the same time the preparation was tested in a rat model to study the effect of repeated HDAS-PEG2K-LEH injection over 4 weeks. We found that HDAS-PEG2K modification substantially reduced the circulating levels of anaphylotoxins C3a and C5a, as well as plasma levels of thromboxane B2 in mice. Repeated injections of HDAS-PEG2K-LEH in rats did not appear to alter its clearance profile after 4 weeks of treatment. No antibody response against human hemoglobin and PEG was detected in rat plasma. Histological observations of lung, liver, spleen, and kidney were inconspicuous between saline-treated rats and HDAS-PEG2K-LEH-treated rats. Immunohistochemical staining for rat heme oxygenase-1 (HO-1) did not show induced expression of HO-1 in these organs. These results suggest that the new surface modification of LEH is immune neutral and does not adversely affect histology even after repeated administration.