Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Summary

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., ⁶⁸Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Abstract

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using ⁶⁸Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

Introduction

One topic that is of utmost relevance is research in the breast cancer field. Breast cancer remains a frequently occurring cancer, accounting for roughly 1/3^rd of all cancers in women. The treatment is tailored to the biological and histological characteristics of the tumor and to the stage of the disease. The chance of survival is generally good unless the tumor has already metastasized, in which case the 5-year survival is only about 30%¹. Other gynecologic cancers suffer from a similar fate, with, for example, ovarian cancer showing > 95% 5-year survival for stage 1 tumors but only 15% for metastasized stage 4 tumors

Protocol

The research protocol adheres to the highest standards of animal welfare and is in strict accordance with the Animal Care Guidelines of University Hospital RWTH Aachen. We are committed to ensuring the ethical and humane treatment of all animals involved in the studies, and the procedures are reviewed and approved by the local animal ethics committee. All animal experiments were approved by a German competent authority (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, LANUV) for compliance with.......

Discussion

Radiosynthesis
The radiosynthesis described here is typical for a new ⁶⁸Ga-labeled compound - short synthesis time, with emphasis on suitable pH and avoiding metals whenever possible. For this, it is important to strictly follow the order in which the components are added. In any case, the pH value of the ⁶⁸Ga solution must first be adjusted to pH 4 with 3 M NH₄OAc; otherwise, the nanobody may degrade if the pH is too acidic. The general concepts of ⁶⁸Ga-.......

Materials

Name	Company	Catalog Number	Comments
Equipment
Activity meter ISOMED 2010	Nuviatech Healthcare	-
Centrifuge MIKRO 185	Andreas Hettich GmbH & Co. KG	1203
Endotoxin testing Endosafe nexgen-PTS	Charles River	-
Heating block NANOCOLOR VARIO C2	Macherey-Nagel	919350
HPLC system, including radio detector	Knauer & Raytest	-
Image analysis software Pmod 4.4	PMOD Technologies LLC	-
Small animal PET/CT system β-CUBE and X-CUBE	Molecubes NV	-
TLC MiniGITA*	Elysia-Raytest	-
Materials
0.3 mm diameter PE10 tube	fisher scientific	22-204008
30G needle	B|Braun	4656300
Centrifugal filter; 10 kDa MWCO, 0.5 mL	Millipore	UFC501008
Chromatography paper strip iTLC-SG	Agilent Technologies	SGI0001
Endotoxin Cartridge, 0.05 EU/ml sensitivity	Charles River	PTS-2005
HPLC Column Biosep SEC-s2000	Phenomenex	-
Microcentrifuge tube (1.5 mL)	Eppendorf	0030125150
pH strip 0.0 - 6.0	Merck KGaA	109531
pH strip 0-14	Merck KGaA	109535
PS-H+ SPE cartridge	Macherey Nagel GmbH & Co. KG	731861
Sterile vial 10 mL	ALK Life Science Solutions	SEV100
Reagents
68Ge/68Ga-Generator	NRF-iThembaLABS	-
Ammoniumacetate	Merck KGaA	101116
Citric acid	Merck KGaA	100241
Hydrochloric acid	Merck KGaA	320331
NaCl	Merck KGaA	S9888
Nanobody NM-02	Radiopharm Theranostics	-
P-SCN-Bz-DOTA-GA	CheMatech	C115
Trifluoracetic acid	Merck KGaA	T6508
Ultrapure water	Merck KGaA	101262