Introduction:
Direct clot targeting represents attractive concept for clot imaging as well as targeted delivery of drugs, e.g. thrombolytics. Small protein binders attached to nanoliposomes may target thrombi and deliver drugs although selective affinity to fibrin and not fibrinogen is the main challenge.
Methods:
For identification and preparation of fibrin-specific artificial protein binders derived from scaffolds of albumin-binding domain (ABD) of streptococcal protein G, a highly complex ABD-derived combinatorial library in combination with ribosome display selection was used. In vitro models were used to document delivery of nanoliposomes to human thrombi.
Results:
A recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptides of the Bβ chain fused to TolA protein carrying polyhistidylated tag and Avitag was constructed. Ribosome display was followed by large-scale ELISA screening of protein binders. Only four protein variants had selective affinity to human fibrin - see figure 1A. The most selective, variant D7, was modified by C-terminal FLAG/His
6
or His
6
/His
6
tag in order to be attached onto the surface of nanoliposomes. The electron microscopy then confirmed the structure of nanoliposome-binder particles. Isothermal titration calorimetry provided dissociation constant for liposome-binder metallochelating bond in the range 10
-7
to 10
-9
for mono- and double-HisTag forms. In vitro, in silicone replica of small diameter artery, the confocal and scanning electron microscopy confirmed a successful binding of D7-attached- to-nanoliposomes to fibrin fibres, see figure 1B.
Conclusions:
We developed binders relatively selective to fibrin, attached them to nanoliposomes, and documented targeting of fibrin in vitro. As the next step, selectivity needs to be now documented in animal studies.
The use of protein binders and sorghum crisps as potential ingredients in a cereal bar for dogs