Novel Hemoglobin from Synechocystis sp. PCC 6803: Shedding Light on the Structure-Function Relationship and Its Biotechnological Applications

Cyanobacteria are oxygenic photosynthetic prokaryotes, practically present in every plausible environment on the earth. In 1996, the first cyanobacterial genome was sequenced from Synechocystis sp. PCC 6803 and the cyanobacterial genome database has been continuously growing with genomes from more than 300 cyanobacterial and other related species, so far. Synechocystis sp. PCC 6803 is one of the best-characterized cyanobacteria and has developed into a model cyanobacterium that scientists are using throughout the world. At the same time, the field of hemoglobin was undergoing a breakthrough with the identification of new globins in all three kingdoms of life including cyanobacteria. Since then, the newly identified globins in the cyanobacteria are raising intriguing questions about their structure and physiological functions, which are quite different from vertebrate’s hemoglobin and myoglobin. These hemoglobins have displayed unprecedented stability, unique heme coordination, novel conformational changes, and other properties that are not often observed in the globin superfamily. This chapter provides an overview of the unique globin from Synechocystis sp. PCC 6803, its interacting protein partners, proposed functions, and its biotechnological implications including potential in the field of artificial oxygen carriers.

Bacterial safety study of the production process of hemoglobin-based oxygen carriers

Hemoglobin microparticles (HbMP) produced with a three-step procedure including co-precipitation of hemoglobin with manganese carbonate, protein crosslinking and dissolution of the carbonate template were shown to be suitable for application as artificial oxygen carriers. First preclinical safety investigations delivered promising results. Bacterial safety plays a decisive role during the production of the HbMP. Therefore, bioburden and endotoxin content of the starting materials (especially hemoglobin) and the final particle suspension are intensively tested. However, some bacteria may not have been detected by the standard tests due to low concentration. The aim of this study was to investigate how these bacteria would behave in the fabrication process. Biocidal effects are known for glutaraldehyde and for ethylenediaminetetraacetic acid, chemicals that are used in the fabrication process of HbMP. It could be shown that both chemicals prevent bacterial growth at the concentrations used during the HbMP fabrication. In addition, the particle production was carried out with hemoglobin solutions spiked with Escherichia coli or Staphylococcus epidermidis. No living bacteria could be detected in the final particle suspensions. Therefore, we conclude that the HbMP fabrication procedure is safe in respect of a bacterial contamination.

Development of a Lyophilization Process for Long-Term Storage of Albumin-Based Perfluorodecalin-Filled Artificial Oxygen Carriers

Every day, thousands of patients receive erythrocyte concentrates (ECs). They are indispensable for modern medicine, despite their limited resource. Artificial oxygen carriers (AOCs) represent a promising approach to reduce the need for ECs. One form of AOCs is perfluorodecalin-filled albumin-based nanocapsules. However, these AOCs are not storable and need to be applied directly after production. In this condition, they are not suitable as a medicinal product for practical use yet. Lyophilization (freeze drying) could provide the possibility of durable and applicable nanocapsules. In the present study, a suitable lyophilization process for perfluorodecalin-filled nanocapsules was developed. The nanocapsules were physicochemically characterized regarding capsule size, polydispersity, and oxygen capacity. Even though the perfluorodecalin-filled albumin-based nanocapsules showed a loss in oxygen capacity directly after lyophilization, they still provided a remarkable residual capacity. This capacity did not decline further for over two months of storage. Furthermore, the nanocapsule size remained unaltered for over one year. Therefore, the AOCs were still applicable and functional after long-term storage due to the successful lyophilization.

Translational research of hemoglobin vesicles as a transfusion alternative

: Clinical situations arise in which blood for transfusion becomes scarce or unavailable. Considerable demand for a transfusion alternative persists because of various difficulties posed by blood donation and transfusion systems. Hemoglobin-vesicles (HbV) are artificial oxygen carriers being developed for use as a transfusion alternative. Just as biomembranes of red blood cells (RBCs) do, phospholipid vesicles (liposomes) for Hb encapsulation can protect the human body from toxic effects of molecular Hb. The main HbV component, Hb, is obtained from discarded human donated blood. Therefore, HbV can be categorized as a biologic agent targeting oxygen for peripheral tissues. The purification procedure strictly eliminates the possibility of viral contamination. It also removes all concomitant unstable enzymes present in RBC for utmost safety from infection. The deoxygenated HbVs, which are storable for over years at ambient temperature, can function as an alternative to blood transfusion for resuscitation from hemorrhagic shock and O2 therapeutics. Moreover, a recent study clarified beneficial effects for anti-oxidation and anti-inflammation by carbon monoxide (CO)-bound HbVs. Autoxidation of HbV (HbO2 → metHb + O2-.) is unavoidable after intravenous administration. Co-injection of methylene blue can extract the intraerythrocytic glycolytic electron energy effectively and reduce metHb. Other phenothiazine dyes can also function as electron mediators to improve the functional life span of HbV. This review paper summarizes recent progress of the research and development of HbV, aimed at clinical applications.

Quality by design approach identifies critical parameters driving oxygen delivery performance in vitro for perfluorocarbon based artificial oxygen carriers

Perfluorocarbons (PFCs) exhibiting high solubility for oxygen are attractive materials as artificial oxygen carriers (AOC), an alternative to whole blood or Haemoglobin-based oxygen carriers (HBOCs). PFC-based AOCs, however, met clinical translation roadblocks due to product quality control challenges. To overcome these issues, we present an adaptation of Quality by Design (QbD) practices to optimization of PFC nanoemulsions (PFC-NEs) as AOCs. QbD elements including quality risk management, design of experiments (DoE), and multivariate data analysis facilitated the identification of composition and process parameters that strongly impacted PFC colloidal stability and oxygen transport function. Resulting quantitative relationships indicated a composition-driven tradeoff between stability and oxygen transport. It was found that PFC content was most predictive of in vitro oxygen release, but the PFC type (perfluoro-15-crown-5-ether, PCE or perfluorooctyl bromide, PFOB) had no effect on oxygen release. Furthermore, we found, under constant processing conditions, all PFC-NEs, comprised of varied PFC and hydrocarbon content, exhibited narrow droplet size range (100–150 nm) and narrow size distribution. Representative PFOB-NE maintained colloidal attributes upon manufacturing on larger scale (100 mL). QbD approach offers unique insights into PFC AOC performance, which will overcome current product development challenges and accelerate clinical translation.