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
|
Nr. |
Surname and name |
Position |
|
1. |
Alexandru Lupan
(alexandru.lupan@ubbcluj.ro) |
Project director |
|
2. |
Amr A. A. Attia |
Researcher |
|
3. |
Adrian M. V. Brânzanic |
Researcher |
|
4. |
Vlad Alexandru Toma |
Researcher |
|
5. |
Florina Scurtu |
Researcher |
|
6. |
Maria Ștefana Lehene |
Ph.D. student |
Project budget
|
Year |
Budget (lei) |
|
2020 |
47000 |
|
2021 |
305500 |
|
2022 |
247500 |
|
Total |
600000 |
Results
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).
Publications with
acknowledgement on this project:
1. Carrascoza, F.; Branzanic, A.M.; Silaghi-Dumitrescu, R. The dynamics of hemerythrin and hemerythrin
derivatives. Stud. Univ. Babes-Bolyai Chem. 2021, in
press.
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.