Biological Evaluation of Liposome-Encapsulated Hemoglobin Surface-Modified With a Novel PEGylated Nonphospholipid Amphiphile

Abstract

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.

The Brain Metabolic Activity after Resuscitation with Liposome-Encapsulated Hemoglobin in a Rat Model of Hypovolemic Shock

“…These processing and characterization methods associated with LEH manufacturing are described in detail elsewhere. 12,13 Rat Model of Hypovolemic Shock…”

Artificial Oxygen Carrier to Regulate Hypoxic Signal Transduction

“…However, recent recognition of the deterioration of stored human red blood cells (RBCs) (2) has aggravated the already keen demand/supply imbalance of donated blood for transfusion, fueling the continuing endeavor to come up with new concepts and technologies to overcome problems (1) or fulfill new requirements proclaimed by the National Institutes of Health (NIH) (3). In this issue, it is encouraging to report new utilities and recently developed AOCs from recycled human RBCs (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), unlimited hemoglobin (Hb) from bovine RBCs (18)(19)(20)(21), and other sources (22)(23)(24). Some of them are close to or are currently under clinical trial (20).…”

Hemorrhagic shock-induced cerebral bioenergetic imbalance is corrected by pharmacologic treatment with EF24 in a rat model

“…The LEH was prepared by encapsulating hemoglobin inside the liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, hexadecylcarbamoylmethylhexadecanoate (HDAS) and HDAS-poly(ethylene glycol)-2000. The methods of preparation and characteristics of LEH are described in the supplemental material; they are also a part of previously published work (Nag et al 2013, Agashe et al 2010, Yadav et al 2014b). …”