Polyethylene Glycol Camouflaged Earthworm Hemoglobin

Jani, Vivek; Jelvani, Alborz; Moges, Selamawit; Nacharaju, Parimala; Roche, Camille J.; Dantsker, David; Palmer, Andre F.; Friedman, Joel M.; Cabrales, Pedro

doi:10.1371/journal.pone.0170041

Cited by 19 publications

(17 citation statements)

References 43 publications

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“…All of these properties suggest that gLtEc may be an attractive blood substitute for deployment in battlefield scenarios. Lastly, it is also worth mentioning that a PEGylated LtEc developed by Roche et al has a dramatically increased circulation half‐life (66.8 ± 1.8 h) compared to native LtEc (18 ± 0.8 h) . Taken together, both LtEc derivatives demonstrate that LtEc can be synthetically modified to make it an even more attractive blood substitute.…”

Section: Resultsmentioning

confidence: 99%

Glutaraldehyde cross‐linking increases the stability of Lumbricus terrestris erythrocruorin

Rajesh

Zimmerman

Spivack

et al. 2017

Biotechnology Progress

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Since donated red blood cells must be constantly refrigerated, they are not available in remote areas and battlefields. We have previously shown that the hemoglobin of the earthworm Lumbricus terrestris (LtEc) is an effective and safe substitute for donated blood that is stable enough to be stored for long periods at the relatively high temperatures that may be encountered in remote areas. The goal of this study was to further increase the thermal stability of LtEc by covalently cross-linking LtEc with glutaraldehyde (gLtEc). Our results show that the melting temperatures of the gLtEc samples steadily increase as the molar ratio of glutaraldehyde to heme increases (from T = 57°C for native LtEc up to T = 68°C at a ratio of 128:1). In addition, while native LtEc is susceptible to subunit dissociation at alkaline pH (8-10), cross-linking with glutaraldehyde completely prevents dissociation of gLtEc at pH 10. Increasing the molar ratio of glutaraldehyde:heme also significantly increased the oxygen affinity of gLtEc, but this effect was decreased by cross-linking gLtEc in the deoxygenated T state. Finally, while gLtEc samples cross-linked at low G:H ratios (e.g., 2:1) exhibited slight increases in oxidation rate in Tris buffer, no significant difference in oxidation rate was observed between native LtEc and the gLtEc samples in Ringer's Solution, which contains antioxidants. Overall, cross-linking LtEc with glutaraldehyde significantly increases its thermal and structural stability without any loss of function, making gLtEc an attractive blood substitute for deployment in remote areas and battlefields. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:521-528, 2018.

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Section: Resultsmentioning

confidence: 99%

Glutaraldehyde cross‐linking increases the stability of Lumbricus terrestris erythrocruorin

Rajesh

Zimmerman

Spivack

et al. 2017

Biotechnology Progress

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“…Still, physiological in vivo "proof of concept" was missing. In the context of AOC development, normovolemic hemodilution of the rat is a well-established model to test functionality of a novel AOC [16][17][18][19] . Consequently, we used the model of massive hemodilution in the rat to determine the physiological functionality of our capsules.…”

Section: Discussionmentioning

confidence: 99%

“…Herein, we used a rat model of massive hemodilution, where we exchanged about 95% of the blood volume with either capsules in a plasma-like solution (treatment) or the plasma-like solution without capsules (control) and posthumously surveyed hypoxia sensitive organs such as the small intestine and kidney. In the context of AOC development, normovolemic hemodilution of the rat is a well-established model to test their functionality [16][17][18][19] . Our study is aimed at serving as a "proof of concept" that capsules are a life-saving erythrocyte substitute, thereby avoiding the onset of tissue hypoxia even at critically low hematocrit.…”

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confidence: 99%

Albumin-derived perfluorocarbon-based artificial oxygen carriers can avoid hypoxic tissue damage in massive hemodilution

Wrobeln

Jägers

Quinting

et al. 2020

Sci Rep

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Artificial blood for clinical use is not yet available therefore, we previously developed artificial oxygen carriers (capsules) and showed their functionality in vitro and biocompatibility in vivo. Herein, we assessed the functionality of the capsules in vivo in a normovolemic hemodilution rat-model. We stepwise exchanged the blood of male Wistar-rats with medium either in the presence of capsules (treatment) or in their absence (control). We investigated tissue hypoxia thoroughly through online biomonitoring, determination of enzyme activity and pancreatic hormones in plasma, histochemical and immunohistochemical staining of small intestine, heart, liver and spleen as well as in situ hybridization of kidneys. After hemodilution, treated animals show higher arterial blood pressure and have a stable body temperature. Additionally, they show a more stable pH, a higher oxygen partial pressure (pO 2), and a lower carbon dioxide partial pressure (pCO 2). Interestingly, blood-glucose-levels drop severely in treated animals, presumably due to glucose consumption. Creatine kinase values in these animals are increased and isoenzyme analysis indicates the spleen as origin. Moreover, the small intestine of treated animals show reduced hypoxic injury compared to controls and the kidneys have reduced expression of the hypoxia-inducible erythropoietin mRNA. In conclusion, our capsules can prevent hypoxic tissue damage. The results provide a proof of concept for capsules as adequate erythrocyte substitute. The need to develop synthetic products for blood replacement has become more and more important during the last decades. On the one hand, the demographic change leads to increasing requirements for blood, and on the other hand, simultaneously the willingness for blood donation declines 1. Blood with its various characteristics fulfils many functions, recent developments of synthetic blood substitutes focus on its most important function: the transport of physiological gases, in particular of oxygen. Additional key requirements for artificial blood substitutes are optimal size, sterility, biocompatibility, and the ability to be stored without loss of functionality. Moreover, artificial oxygen carriers (AOCs) should have a long intravascular circulation time and universal usage independent of the blood group characteristics 2,3. There are two main approaches to realize the transport of essential gases. The first is the use of the physiological oxygen carrier hemoglobin (hemoglobin-based artificial oxygen carriers, HBOCs); a concept realized e.g. in the prominent product hemopure, that gained pharmaceutical approval in South Africa and Mexico. Further HBOCs (e.g. sanguinate) are currently tested in clinical trials 4. So far, most of the products showed severe side effects so that the manufacturing had to be stopped 5. The second is the work with synthetic materials like perfluorocarbons [perfluorocarbon-based artificial oxygen carriers (PFOCs)]. Depending on the actual partial pressure, PFOCs are able to solve gases physically 6...

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“…Preclinical studies in mice, rats, and hamsters have already shown that both LtEc and AmEc effectively deliver O 2 in vivo without the significant increase in mean arterial pressure observed with some other HBOCs . The circulation half‐life of both LtEc and AmEc are limited to 12 hours, but PEGylation of LtEc has been shown to increase its half‐life up to 70 hours . Transfusions of LtEc and AmEc also do not elicit any changes in animal behavior or health for several months after the initial injection .…”

Section: Discussionmentioning

confidence: 99%

Direct comparison of oligochaete erythrocruorins as potential blood substitutes

Zimmerman

DiIusto

Dienes

et al. 2017

Bioengineering & Transla Med

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While many blood substitutes are based on mammalian hemoglobins (e.g., human hemoglobin, HbA), the naturally extracellular hemoglobins of invertebrates (a.k.a. erythrocruorins, Ecs) are intriguing alternative oxygen carriers. Specifically, the erythrocruorin of Lumbricus terrestris has been shown to effectively deliver oxygen in mice and rats without the negative side effects observed with HbA. In this study, the properties of six oligochaete Ecs (Lumbricus terrestris, Eisenia hortensis, Eisenia fetida, Eisenia veneta, Eudrilus eugeniae, and Amynthas gracilis) were compared in vitro to identify the most promising blood substitute candidate(s). Several metrics were used to compare the Ecs, including their oxidation rates, dissociation at physiological pH, thermal stability, and oxygen transport characteristics. Overall, the Ecs of Lumbricus terrestris (LtEc) and Eisenia fetida (EfEc) were identified as promising candidates, since they demonstrated high thermal and oligomeric stability, while also exhibiting relatively low oxidation rates. Interestingly, the O2 affinity of LtEc (P 50 = 26.25 mmHg at 37 °C) was also observed to be uniquely lower than EfEc and all of the other Ecs (P 50 = 9.29–13.62 mmHg). Subsequent alignment of the primary sequences of LtEc and EfEc revealed several significant amino acid substitutions within the D subunit interfaces that may be responsible for this significant change in O2 affinity. Nonetheless, these results show that LtEc and EfEc are promising potential blood substitutes that are resistant to oxidation and denaturation, but additional experiments will need to be conducted to determine their safety, efficacy, and the effects of their disparate oxygen affinities in vivo.

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Polyethylene Glycol Camouflaged Earthworm Hemoglobin

Cited by 19 publications

References 43 publications

Glutaraldehyde cross‐linking increases the stability of Lumbricus terrestris erythrocruorin

Glutaraldehyde cross‐linking increases the stability of Lumbricus terrestris erythrocruorin

Albumin-derived perfluorocarbon-based artificial oxygen carriers can avoid hypoxic tissue damage in massive hemodilution

Direct comparison of oligochaete erythrocruorins as potential blood substitutes

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