A biomimetic approach to remove free hemoglobin from plasma inspired by bacterial iron capturing systems - Finanziamento dell’Unione Europea – NextGenerationEU – missione 4, componente 2, investimento 1.1.

Intravascular hemolysis consists in the abnormal lysis of red blood cells that causes the release of hemoglobin (Hb) in the bloodstream. Several causative agents can induce hemolysis, ranging from infectious diseases to genetic and autoimmune disorders. Extracorporeal blood treatments are often associated with the risk of hemolysis. Free plasma hemoglobin (fp-Hb) can cause hemoglobinuria, renal failure, vasoconstriction, and thrombosis; the release of heme and iron from fp-Hb triggers oxidative damage. No efficient fp-Hb capturing systems are available to date. The aim of the project is to develop a device able to remove fp-Hb from plasma, based on a biomimetic approach inspired by a bacterial iron-capturing protein named IsdB, a Staphylococcus aureus hemophore that specifically binds Hb. The cryo-EM structure of the IsdB:Hb complex will be exploited as a molecular template for the development of peptides and peptidomimetics able to sequester fp-Hb. The main objectives of the project are: 1) identify Hb-binding element(s) starting from IsdB protein scaffold, 2) graft Hb-binding elements on a solid support, and 3) test the in-house built device for fp-Hb cleanup from Hb solutions or plasma samples. Peptides and peptidomimetics will be used to mimic the IsdB scaffold for Hb binding, since they are more suitable than full-length IsdB in terms of yield of production, stability, and possibility of derivatization. A series of chemical modifications of peptides will be investigated aiming at increasing both proteolytic and conformational stability. Based on the structural information on the IsdB:Hb complex, peptide/peptidomimetic best candidates will be selected through bioinformatics and molecular dynamic-based tools. Possible mutations will also be considered in order to optimize peptide-Hb interactions. The synthesis of the identified compounds will be performed by microwave-assisted solid-phase peptide synthesis. The secondary structure of the peptides will be assessed by circular dichroism and the binding to Hb will be investigated by different complementary techniques: fluorescence spectroscopy, STD-NMR, ITC, and MST. An iterative process will lead to optimization of the peptide structures in terms of stability and efficacy in the binding of Hb. Peptides and peptidomimetics selected as best binders of Hb in solution will then be grafted on a biocompatible support composed of mesoporous silica nanoparticles (MSNs) with dimensions below 100 nm, suitable to capture a high amount of fp-Hb. The effect of grafting on the properties of the peptides/peptidomimetics will be evaluated by experimental and computational methods. Functionalized MSNs will be packed in a plastic housing simulating a hemoperfusion module and tested for their capacity to bind Hb in buffer solutions and in hemolyzed plasma samples. The device will represent a “proof-of-concept” of the possibility to produce a Hb binding module for extracorporeal blood treatment.