Erythrocytes as bioreactors to decrease excess ammonium concentration in blood

Abstract

Increased blood ammonium concentrations cause neurological complications. Existing drugs are not always sufficiently effective. Alternatively, erythrocytes-bioreactors (EBRs) loaded with enzymes utilizing ammonium, were suggested for ammonium removal from blood. However all they worked only for a short period of time. The reasons for this were not investigated. In this study, EBR mathematical models were developed and analysed based on the reactions of glycolysis and different enzymes utilizing ammonium, which showed that the efficiency and duration of EBRs’ functioning could be limited due to low permeability of the cell membrane for some key substrates and products. A new enzyme system including glutamate dehydrogenase and alanine aminotransferase was proposed and realised experimentally, which was not limited by cell membrane permeability for glutamate and α-ketoglutarate due to creating metabolic pathway where these metabolites were produced and consumed cyclically. New bioreactors removed ammonium in vitro at the rate of 1.5 mmol/h × lRBCs (for human bioreactors) and in vivo in a model of hyperammoniemia in mice at the rate of 2.0 mmol/h × lRBCs (for mouse bioreactors), which correlated with model calculations. Experimental studies proved the proposed mathematical models are correct. Mathematical simulation of erythrocyte-bioreactors opens new opportunities for analysing the efficiency of any enzyme included in erythrocytes.

Erythrocytes as Carriers: From Drug Delivery to Biosensors

“…Moreover, they continued to work even 2 h after the administration, which distinguished them from the bioreactors described previously in the literature [72,74]. The authors of [75] calculated that under physiological conditions transfusion of 200 mL of such EBRs to a patient will lead to a decrease in the plasma ammonium concentration by 6 mM/day, which is 10 times higher than similar values (600 µM/day) for the best drugs to reduce ammonium concentration currently available.…”

“…Using mathematical models of EBRs created in [75], it was shown that the reason for this behavior is the depletion of the substrates inside the cell (l-glutamic acid or α-ketoglutarate), which are consumed during the utilization of ammonium by these enzymes, but are unable to enter the cell from the bloodstream. The authors of [75] proposed a new promising system to create ammonium-removing EBRs, based on the RBCs entrapment of a tandem from two enzymes-glutamate dehydrogenase and alanine aminotransferase. As a result, a new metabolic pathway was created in the erythrocytes, in which α-ketoglutarate and l-glutamic acid were produced and consumed in a cyclic process.…”

“…In this case, the researchers proposed for incorporation into the RBCs a new enzyme system consisting of two enzymes that provided cyclic consumption and production of the necessary metabolites inside the cell. This made the process independent of the transport of these metabolites [75]. Another area of modern developments to improve the delivery of drugs that can affect the metabolism of RBCs is associated with the replacement of RBCs with artificial RBCs or hybrid nanoparticles, the surface of which contains fragments of the RBC membrane, to ensure their long lifetime in the bloodstream [9].…”

Preclinical developments of enzyme-loaded red blood cells

“…However, in both the cited studies, enzyme-loaded RBCs worked only for a short period of time, after which blood ammonia concentration decreased approximately at the same rate in treated and control hyperammonaemic mice. Recently [89], it has been supposed that the poor efficiency and duration in the performances of these bioreactors could be due to the low permeability of RBC membranes to α-ketoglutarate and L-glutamic acid. Following these assumptions, an interesting mathematical model allowing to analyze the efficiency of a new enzyme system to load into RBCs has been developed and tested in hyperammonaemic mice.…”

Theoretical Analysis of the Built-in Metabolic Pathway Effect on the Metabolism of Erythrocyte-Bioreactors That Neutralize Ammonium

“…Erythrocytes containing artificially-embedded metabolic pathways capable of working inside the cell, but absent in a normal erythrocyte, are called erythrocyte-bioreactors (EBRs) [4,5]. Various scientific groups have created EBRs at different times to remove ethanol [6,7], methanol [8,9], ammonium [10][11][12], asparagine [13][14][15], and other compounds from the bloodstream [16][17][18].…”