Non-invasive imaging and cellular tracking of pulmonary emboli by near-infrared fluorescence and positron-emission tomography.

Michael J Page,Fiore Cattaruzza,Charles S Craik,Shaun R Coughlin,André L Lourenço,Helena C Castro,Tovo David,Henry F Vanbrocklin,Aaron M Lebeau

doi:10.1038/ncomms9448

Abstract

Functional imaging of proteolytic activity is an emerging strategy to quantify disease and response to therapy at the molecular level. We present a new peptide-based imaging probe technology that advances these goals by exploiting enzymatic activity to deposit probes labelled with near-infrared (NIR) fluorophores or radioisotopes in cell membranes of disease-associated proteolysis. This strategy allows for non-invasive detection of protease activity in vivo and ex vivo by tracking deposited probes in tissues. We demonstrate non-invasive detection of thrombin generation in a murine model of pulmonary embolism using our protease-activated peptide probes in microscopic clots within the lungs with NIR fluorescence optical imaging and positron-emission tomography. Thrombin activity is imaged deep in tissue and tracked predominantly to platelets within the lumen of blood vessels. The modular design of our probes allows for facile investigation of other proteases, and their contributions to disease by tailoring the protease activation and cell-binding elements.

Highlights

Functional imaging of proteolytic activity is an emerging strategy to quantify disease and response to therapy at the molecular level
Temporin L (TempL) was chosen from this group on the basis of its relatively short sequence, large number of homologues, modest cationic disposition, conformational change into an a-helix on membrane binding and reasonable haemocompatibility to human blood cells (Fig. 1b,g)[19,20]. These factors enable facile synthesis, tunable potency, limited non-specific interactions, robust on and off state definition and the ability to insert into eukaryotic cell membranes
The protease-activated receptor-1 (PAR1) activation site, a natural substrate of thrombin, was chosen to incorporate into the initial design owing to its known high sensitivity to the protease

Summary

Introduction

Functional imaging of proteolytic activity is an emerging strategy to quantify disease and response to therapy at the molecular level. We present a new peptide-based imaging probe technology that advances these goals by exploiting enzymatic activity to deposit probes labelled with near-infrared (NIR) fluorophores or radioisotopes in cell membranes of disease-associated proteolysis This strategy allows for non-invasive detection of protease activity in vivo and ex vivo by tracking deposited probes in tissues. As the concentration for detection is below the high copy number required to permeabilize membranes, the compounds have a good safety profile and do not affect hepatic or renal functions (Supplementary Fig. 4; Supplementary Table 2) We term these protease-responsive constructs restricted interaction peptides (RIPs) and found them capable of the non-invasive imaging of thrombin activity in vivo and at monitoring this activity at the cellular level

Methods

Results

Conclusion