Functionalization and radiolabeling of M13 bacteriophages for PET imaging

Objectives and Introduction
Parkinson’s disease is caused by degeneration of nigro-striatal dopaminergic neurons and denervation of the target neurons in the neostriatum. The resulting disruption of dopaminergic modulation produces an imbalance between antagonistic pathways in the basal ganglia leading to rigidity, tremor, and bradykinesia [1]. One of several treatment options is the so called deep-brain-stimulation (DBS), whereby an electrode is implanted to re-equilibrate the nervous pathways and rescue the pathological imbalance. Though highly effective, DBS is linked to a very complex surgical procedure and can lead to adverse neurological effects [2,3].
The goal of this project is to enable a selective stimulation of striatal dopaminoceptive neurons from outside the brain through polymeric photovoltaic nanoparticles which are transported to the neostriatum using an engineered M13 bacteriophage as nanocarrier. These phages were chosen as a biovector since their filamentous envelop, formed by the major coat protein P8, offers a large surface area which can be modified easily. To monitor its biodistribution in the organism, the engineered bacteriophage is being with [64Cu]CuCl2 enabling PET imaging.

Methods:

Methods for analysis of the phages by TLC, HPLC and MALDI-TOF MS have been developed. To allow labeling with 64Cu, the bacteriophages were functionalized with 1,4,7‑triazacyclononane,1‑glutaric acid‑4,7-acetic acid (NODA-GA) and the conjugation reaction was analyzed by MALDI-TOF MS. The M13-NODA-GA conjugates were purified using HPLC-SEC. After labeling of the M13-NODA-GA conjugates with inhouse produced [64Cu]CuCl2 and purification by spin filtration, the radiolabeling efficiency was analyzed by radio-TLC and radio-HPLC-SEC. PET imaging was carried out in mice and scans were taken every 15 min up to 1 h after intravenous injection. The mice were sacrificed 70 min post-injection and the radioactivity accumulated in different organs was measured.

Results:

Conjugation of the NODA-GA chelator to the major capsid protein P8 of the phages was confirmed by MALDI-TOF MS. Subsequent radiolabeling of the bioconjugates was achieved with a specific activity of 17 MBq/pmol and a radiochemical purity of 98.5% was obtained as determined by radio-TLC as well as radio-HPLC-SEC. A rapid accumulation of the radiolabeled M13-NODA-GA conjugates in the murine liver was observed by PET imaging 15 min post-injection. According to ex vivo analysis, approximately 80% of the injected dose was accumulated in the liver, and smaller amounts were detected in spleen (~ 5%) and in the gastrointestinal tract (<1%).

Conclusions:

The present study shows for the first time the successful chemical modification and 64Cu-labeling of NODA-GA-functionalized M13 bacteriophages as well as their biodistribution. Furthermore, a set of analytical methods is presented allowing the assessment of bacteriophage purity, integrity as well as stability in future studies.

Acknowledgements:

The research work was financially supported by the EU Joint Programme – Neurodegenerative Disease Research (NeuroPhage, Project ID: JPND2020-568-126). The financial support (Project ID: 01ED2108) by the German Federal Ministry of Education and Research (BMBF) is gratefully acknowledged.

References:

[1] Balestrino R and Schapira A H V, Eur. J. Neurol. 2020; 27: 27–42.
[2] Stoker T B et al., Front. Neurosci. 2018; 12:693.
[3] Krack P et al., Mov. Disord., 2019; 34: 12