Biodistribution PET/CT Study of Hemoglobin-DFO-89Zr Complex in Healthy and Lung Tumor-Bearing Mice.

Łukasz Kiraga,Daniel Turnham,Tomasz P Rygiel,Adam M Dabkowski,Alessandra Bonamore,Alberto Boffi,Carmelinda Savino,Gabriele Cerutti,Gillian Seaton,Zuzanna Sas,Magdalena Król,Richard Clarkson,Paulina Kucharzewska,Bartłomiej Taciak,Agata Braniewska,Linda Celeste Montemiglio,Damian Strzemecki,Stephen J Paisey

doi:10.3390/ijms21144991

Abstract

Proteins, as a major component of organisms, are considered the preferred biomaterials for drug delivery vehicles. Hemoglobin (Hb) has been recently rediscovered as a potential drug carrier, but its use for biomedical applications still lacks extensive investigation. To further explore the possibility of utilizing Hb as a potential tumor targeting drug carrier, we examined and compared the biodistribution of Hb in healthy and lung tumor-bearing mice, using for the first time 89Zr labelled Hb in a positron emission tomography (PET) measurement. Hb displays a very high conjugation yield in its fast and selective reaction with the maleimide-deferoxamine (DFO) bifunctional chelator. The high-resolution X-ray structure of the Hb-DFO complex demonstrated that cysteine β93 is the sole attachment moiety to the αβ-protomer of Hb. The Hb-DFO complex shows quantitative uptake of 89Zr in solution as determined by radiochromatography. Injection of 0.03 mg of Hb-DFO-89Zr complex in healthy mice indicates very high radioactivity in liver, followed by spleen and lungs, whereas a threefold increased dosage results in intensification of PET signal in kidneys and decreased signal in liver and spleen. No difference in biodistribution pattern is observed between naïve and tumor-bearing mice. Interestingly, the liver Hb uptake did not decrease upon clodronate-mediated macrophage depletion, indicating that other immune cells contribute to Hb clearance. This finding is of particular interest for rapidly developing clinical immunology and projects aiming to target, label or specifically deliver agents to immune cells.

Highlights

Hemoglobin has been recently rediscovered as a drug and oxygen carrier for biomedical applications due to its unique pharmacokinetics properties [1,2,3,4]
In order to track the complex, we used the positron emission tomography–computed tomography (PET/CT) imaging system. In this manuscript we demonstrate the feasibility of the method proving the Hb-DFO conjugation by X-ray crystallography and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), the quantitative 89Zr uptake by Hb-DFO using radiochromatographic methods and analyzing the biodistribution of the Hb-DFO-89Zr complex over time in naïve and lung tumor-bearing mice
The observed molecular weights obtained from the average mass of the doubly charged ions of the hemoglobin α and β chains are in agreement with their calculated values, respectively, 15,126.4 Da for the α chain and 15,867.2 Da for the β chain in their heme free form

Summary

Introduction

Hemoglobin has been recently rediscovered as a drug and oxygen carrier for biomedical applications due to its unique pharmacokinetics properties [1,2,3,4]. Free Hb released from erythrocytes in hemolytic processes is avidly scavenged by haptoglobin (Hp), capable of forming a tight Hp-Hb complex with almost femtomolar affinity in humans, one of the strongest protein–protein interactions observed so far [5]. The Hp-Hb complex is actively scavenged by macrophages through CD163 [7]. This scavenger receptor, selectively expressed by the monocytic cell lineage, especially in liver and spleen, is thought to be essential for Hb clearance in order to prevent the toxic effect of the heme molecule and to avoid kidney filtration with subsequent protein precipitation and tissue damage. Hb clearance mediated by liver is dose-dependent [2]

Methods

Results

Conclusion