Targeting Human Thrombus by Liposomes Modified with Anti-Fibrin Protein Binders

Abstract

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.

Thrombus Imaging Using 3D Printed Middle Cerebral Artery Model and Preclinical Imaging Techniques: Application to Thrombus Targeting and Thrombolytic Studies

“…In this study, we formulated D7H2-proteoliposomes in the Z-average size of 250 nm, dual-labelled using lyssamine-rhodamine, and Gd-PE for FLI and MRI, respectively. D7H2 protein was previously reported to bind to clots prepared from human whole blood [31]. Here, we also proved the specific binding to artificial clots prepared from the TISSEEL kit (Figure S3).…”

“…Therefore, dual-labelled D7H2-modified liposomes were employed for both clot targeting (whole blood clots), and also clot pre-labelling and consequent imaging using FLI and MRI (artificial fibrin clots). Modification of liposomes using D7H2 and its physical chemical characterization was described in detail previously [ 31 ].…”

“…Fibrin targeted contrasting MRI imaging has already been used in various models [ 8 ], including a rabbit model [ 34 , 35 ]. In the present study, however, a novel fibrin binding system using protein binders and liposomal carriers [ 31 ] with gadolinium-labelled contrast agent was applied [ 36 ].…”

Myomedin scaffold variants targeted to 10E8 HIV-1 broadly neutralizing antibody mimic gp41 epitope and elicit HIV-1 virus-neutralizing sera in mice

“…In contrast to previously used three-helix bundle ABD-derived combinatorial library [30][31][32], here we demonstrate a new loop-randomized scaffold library designed on the structure of domain 10 of human contractile muscle protein myomesin-1. After the screening of randomized library designated as Myomedin library, the best 10E8 binders were identified, produced in prokaryotic expression system, and used as immunogens in experimental mice to stimulate the production of specific antibodies.…”

An Albumin-Binding Domain Peptide Confers Enhanced Immunoprotection Against Viral Myocarditis by CVB3 VP1 Vaccine

“…Herein, we were concerned with the albumin-binding domain (ABD) derived from the streptococcal protein G. ABD is a 46-residue peptides, which has a very high binding affinity with human, monkey and mouse albumins ( 20 ). Previous studies showed that ABD could efficiently direct the coupled substances to dLNs, and could increase their retention times and stabilities in vivo ( 21 ).…”