Prolonged functional life span of artificial red cells in blood circulation by repeated methylene blue injections

Abstract

Hemoglobin-vesicles (HbVs) are artificial oxygen carriers encapsulating purified and concentrated hemoglobin solution in phospholipid vesicles (liposomes) and their safety and efficacy as a transfusion alternative have been evaluated. Because of the absence of enzymatic methemoglobin reduction system in HbV, the level of ferric methemoglobin (metHb) increases gradually after intravenous administration. Our previous studies clarified that the glycolytic electron energies, charged as NAD(P)H in red blood cells (RBC), are donated to reduce metHb compartmentalized in HbV via a water-soluble electron mediator such as methylene blue [MB; 3,7-bis(dimethylamino)phenothiazinium chloride], which freely shuttle across both RBC biomembrane and HbV lipid membrane. Herein, we tried to test repeated injections of MB after the massive HbV administration (28 mL/kg) to hemorrhagic shocked Wistar rats (n = 3). MB was injected (3.1 mg/kg) at 7, 24 and 48 h after HbV administration. Every MB injection showed rapid reduction of metHb and gradual reversal increase. As a result, the functional life span of HbV was significantly extended over 60 h. It is expected that further optimization of injection scheduling will decrease the total amount of MB and prolong the functional life span of HbV.

Keywords:

• Erythrocytes
• glycolysis
• electron transfer
• blood substitutes
• liposomes

Disclosure statement

Of the authors, H.S. is an inventor holding some patents related to the production and utilization of Hb-vesicles.

Author contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Additional information

Funding

This research was supported by a Grant-in-Aid for Scientific Research [Kiban B, No. 25282136, and Kiban B 17H02087] from the Japan Society for the Promotion of Sciences (JSPS), Health and Labour Sciences Grants (Health Science Research Including Drug Innovation) from the Ministry of Health, Labour and Welfare, Japan, and Strategic Research Platforms for Private University: a matching fund subsidy from Ministry of Education, Culture, Sports, Science and Technology, Japan.