Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bβ chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.

Department of Biochemistry Institute of Hematology and Blood Transfusion U nemocnice 2094 1 128 20 Praha 2 Czech Republic

Department of Immunology Faculty of Medicine and Dentistry Palacky University Olomouc Hněvotínská 3 775 15 Olomouc Czech Republic

Department of Pharmacology and Immunotherapy Veterinary Research Institute v v i Hudcova 70 621 00 Brno Czech Republic

Laboratory of Ligand Engineering Institute of Biotechnology Czech Academy of Sciences v v i BIOCEV Research Center Průmyslová 595 252 50 Vestec Czech Republic

Malvern Instruments Ltd Enigma Business Park Grove Lane Malvern WR14 1XZ UK

The International Clinical Research Center ICRC and Neurology Department of St Anne's University Hospital in Brno Pekařská 53 656 91 Brno Czech Republic

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