PN-III-P2-2.1-PED-2019-2293 financed by UEFISCDI Romania under contract 368PED/2.11.2020



Semi-artificial oxygen carriers


The aim of the project is to demonstrate a viable product that can be used in emergency medicine and in surgery for transfusions, based on hemerythrin (Hr) as an oxygen-carrying protein.  Recombinant-Hr-based copolymers have so far been characterized spectroscopically and biochemically as well as from point of view of immune response, of the biochemical and hematological parameters, oxidative stress, and shown to perform at least as well as canonical, hemoglobin-based, counterparts.  Having previously formulated the concept of using Hr, chemically and genetically-derivatized, for the purpose of use as blood substitute, we propose here to experimentally demonstrate at laboratory level the feasibility of these products by employing a complex multidisciplinary set of methods: analysis of immunological parameters (amounts of immunoglobulins, complement C3, C reactive protein), hematological parameters (concentration of leukocytes, lymphocytes, granulocytes, thrombocyte, red blood cells, hemoglobin), biochemical parameters (potassium, sodium, calcium, content of the blood respiratory gases, specific parameters of renal function, and also of some cellular enzymes), coagulation, oxidative stress (catalase, malondialdehyde), alongside electron paramagnetic spectroscopy – steered molecular dynamics on hemerythrin, on its three chemically derivatized versions (glutaraldehyde-polymerized, glutaraldehyde copolymer with human serum albumin, polyethyleleneglycol-derivatized ) and two of its already generated and characterized mutants designed for selective derivatization with polyethyleneglycol. Biochemical and physiological parameters from standard animal experiments will be matched against molecular dynamics data on Hr and its derivatives and on their spin-labelled versions whose fate, location and stability will be monitored by electron paramagnetic (EPR) spectroscopy.



The list of people involved in this project



Surname and name



Alexandru Lupan (

Project director


Amr A. A. Attia



Adrian M. V. Brânzanic



Vlad Alexandru Toma



Florina Scurtu



Maria Ștefana Lehene

Ph.D. student




Project budget



Budget (lei)














Stage 1 of the grant (ending at 31/12/2020) had as objectives the production of competent cells that express hemerythrin, production of hemerythrin stocks for preliminary biochemical analysis, and molecular mechanics calculations run on hemerythrin and derivatives. More specifically, the activity involved was to preapare competent cells, culture them, and to confirm by purification that Hr has been produced. Also, the identification / preparation of protocol and calculations of molecular mechanics and short-term dynamics for hemerythrin and its derivatives. These objectives were reached 100%, cells overexpressing Hr were produced, initial Hr stocks were prepared and molecular dynamics protocols were selected (three lines of study – YASARA, AMBER, Materials Studio).


Stage 2 of the grant (ending at 31/12/2021) had as objectives the preparation and biochemical analysis of stocks of 4 SARTOC. Also, molecular dynamics performed and analyzed on 4 variants of SARTOC. Also, preliminary tests on 4 SARTOC models in animal models and last but not least, preparation of manuscripts. In brief, nine SARTOC prepared/analyzed were control Hr, K92H Hr, K92C Hr, pegylated native Hr, pegylated K92C Hr, glutaraldehyde-polymerized Hr, Hr-albumin copolymer, as well as two control SARTOC based on hemoglobin (glutaraldehyde[polymerized ovine and bone hemoglobin, respectively). Four SARTOC were tested/analyzed in vivo – glutaraldehyde-polymerized Hr, Hr-albumin copolymer, pegylated Hr and, as a control, ovine hemoglobin polymerized with glutaraldehyde. Molecular dynamics simulations were performed on five SARTOC candidates – native monomeric Hr, native octameric Hr, native octameric Hr derivatized at Cys51, and a control set of tests on hemoglobin. Five manuscripts were prepared, of which three centered exclusively on Hr SARTOC and two report control or preliminary testing of methodology to be employed for SARTOC analysis: (i) on hemerythrin mutant preparation and characterization (to be submitted during Stage 3), (ii) on the performance of Hr-albumin copolymers in vivo as compared to simple Hr polymers (submitted, revision requested by Reviewers and performed), (iii) on Hr stability with molecular dynamics with native vs derivatized forms (published as [1]), (iv) on free radical reactivity of alternate SARTOC material, hemoglobin (published as [2], employing molecular dynamics in the YASARA package, which was then also successfully applied on Hr in paper [1] mentioned above), and (v) one centered on biopolymer interaction with solid surfaces (published as [3], a preliminary test/analysis along the Materials Studio route for Hr molecular dynamics).


Stage 3 of the grant (ending at 31/12/2020) had as objectives experiments performed on animal models for 4 SARTOC, dynamics analyses for SARTOC optimization, publication of 3 articles, and, most importantly, identifying the best SARTOC to initiate practical/commercial applications and clinical trials. These objectives were reached 100% as detailed below. Thus, experiments were performed/continued/finalized on animal models with 4 SARTOC candidates (Hr derivatives, Hb derivatives, as well as control dextran plasma expander). Dynamics analyses based on molecular mechanics, DFT and experimental spectroscopy (EPR, NMR, fluorescence, UV-vis, resonance Raman) were performed on 7 SARTOC materials. Three papers were published, three more manuscripts were submitted, and a number of three more manuscripts are in work (an overall total of 9 papers anticipated that will be based on funding from this grant. Molecular dynamics and animal model studies were completed, and a conclusion has been reached as to the best SARTOC candidate for subsequent clinical trials – namely, GPOH (see below), followed by (and outperforming) the Hr-albumin copolymer.




1. Carrascoza, F.; Branzanic, A.M.; Silaghi-Dumitrescu, R. The dynamics of hemerythrin and hemerythrin derivatives. Stud. Univ. Babes-Bolyai Chem. 2021, LXVI, 397-404.

2. Carrascoza, F.; Silaghi-Dumitrescu, R. The dynamics of hemoglobin-haptoglobin complexes. Relevance for oxidative stress. J. Mol. Struct. 2022, 1250, 131703, doi:10.1016/j.molstruc.2021.131703.

3. Irsai, I.; Brânzanic, A.M.V.; Silaghi-dumitrescu, R. Polylactic acid interactions with bioceramic surfaces. Stud. Univ. Babes-Bolyai Chem. 2021, LXVI, 107–121, doi:10.24193/subbchem.2021.3.06.

4. M.-K. Arkosi, A.C. Mot, I. Lupan, R. Silaghi-Dumitrescu, Selective polyethylene glycol attachment to hemerythrin for potential use in blood substitutes, Proteinn J., submitted, doi:10.21203/

5. I. Irsai, P.Szilard, R. Silaghi-Dumitrescu, Glutaraldehyde-polymerized hemerythrin: assessment of performance as oxygen carrier in hemorrhage models, Bioinorg. Chem. Appl., submitted.