One-Year Observation of Wistar Rats after Intravenous Infusion of Hemoglobin-Vesicles (Artificial Oxygen Carriers)

Abstract

Hemoglobin-vesicles (HbV) or liposome-encapsulated Hb are artificial oxygen carriers. Our previous studies of the bolus infusion of HbV into Wistar rats showed that HbV was captured by the reticuloendothelial system from the blood stream and degraded completely with no deteriorative effect for 2 weeks. However, one authority on artificial organs research suggested conducting a one-year observation because he experienced, with one lipid-emulsified perfluorocarbon (PFC), that rats died within one year from a pulmonary abnormality after receiving the PFC emulsion due to the unstable dispersion state (personal communication). We thought this would never happen for HbV because the dispersion state of HbV is stable with PEG-modification. To confirm this, we made one-year observations after HbV infusion as suggested. Five male Wistar rats intravenously received 20 ml/kg HbV suspended in saline ([Hb] = 10 g/dL). They were housed in separated cages and provided with food and water ad libitum. All rats survived one year, and were apparently healthy. Their body weights (821+/-75 g) reflected obesity from their confinement in small cages. No histopathological abnormality was found in the lung. Plasma biochemical analyses showed overall normal organ functions. In our previous report, plasma lipid levels increased transiently at 1 or 2 days; then they reverted to the control level at 7 days. One year later, the rats showed much higher plasma lipid levels, a symptom of hyperlipidemia that is attributable to obesity and aging. It seemed the transient increases at the early days had no impact compared with the levels of hyperlipemia of the old rats.

Simple Method for Preparing Poly(ethylene glycol)-Surface-Conjugated Liposome-Encapsulated Hemoglobins: Physicochemical Properties, Long-Term Storage Stability, and Their Reactions with O2, CO, and NO

“…The most common lipid used to partially form the membrane of PEG-LEHs consists of dipalmitoylphosphatidylcholine (DPPC). Unfortunately, DPPC has a phase transition temperature of 41°C, 8–10, 14–18, 25, 28, 29, 34–39 which is close to the core body temperature (37°C). Therefore, transfusion of DPPC containing PEG-LEHs will result in increased PEG-LEH membrane fluidity, which will eventually lead to liposomal membrane destabilization and subsequent release of free Hb into the blood stream.…”

“…8–10, 14–18, 25, 28, 29, 34–39 Scale up of this process is challenging, as it requires large ultracentrifuges capable of attaining high speeds (100,000–200,000 g ). In this work, TFF using HF cartridges of a defined molecular weight cut off (500 kDa) were used to completely remove all unencapsulated Hb and concentrate the final PEG-LEH dispersion.…”

Biological Responsiveness and Metabolic Performance of Liposome-Encapsulated Hemoglobin (Hemoglobin-Vesicles) in Apolipoprotein E-Deficient Mice after Massive Intravenous Injection

“…The injected volume of HbV (2000 mg Hb/kg, 20 mL/kg) is the same as our previous safety and toxicology studies of HbV using rodent 7,8,14) that corresponded to nearly 28% of mouse blood volume (72 mL/ kg). 15) At days 1, 3, 7 and 14 after the HbV injection, five mice were randomly selected from each group for collection of blood and organs.…”

Long-Term Stored Hemoglobin-Vesicles, a Cellular Type of Hemoglobin-Based Oxygen Carrier, Has Resuscitative Effects Comparable to That for Fresh Red Blood Cells in a Rat Model with Massive Hemorrhage without Post-Transfusion Lung Injury

“…The transport capacity of O 2 by HbV is equivalent to that for RBC, and HbV shows resuscitative effects that are comparable to RBC in hemorrhagic shock rat models [5–7]. In addition, HbV has been shown to be safe and biocompatible based on in vitro and in vivo experiments as follows: the absence of viral contamination [8], high biocompatibility in systemic immune responses [9], no innate toxicity [10–12], readily metabolized and excreted under both healthy and pathological conditions [13–15]. Furthermore, the structure and physicochemical characteristics of HbV in the liquid state remain about the same during storage for periods of over 1 year at 4, 23, and 40°C, indicating that a HbV suspension can be stored at room temperature for at least 1 year [16].…”