4.1. Peptides Labelling

The peptides used in this work are commercially available and were purchased already coupled with DOTA chelator from Phoenix Europe GmbH (Manheim, Germany; DOTA-NT and DOTA-NMB), AnaSpec Inc. (Fremont, CA, USA; DOTA-PEG(4)-BBN(7-14)) and PolyPeptide (Strasbourg, France; DOTA-NMN), respectively. ⁶⁸Ga used for labelling was obtained from a GMP-grade ⁶⁸Ge/⁶⁸Ga generator (ITG Isotope Technologies Garching GmbH, Munich, Germany) eluted with 0.05 M HCl prepared with suprapure HCl (Merck KGaA, Darmstadt, Germany). Labelling pH was adjusted with ammonium acetate (Carl Roth GmbH, Karlsruhe, Germany), used as 1 M solution. Separation and purification of labelled peptides were performed on Strata™-X solid phase extraction cartridges (Phenomenex Inc., Torrance, CA, USA). Ethanol (Chimreactiv, Bucharest, Romania) was used to recover the compound retained on cartridge. After evaporation of ethanol, the labelled compounds were dissolved in 1 mL saline solution (0.9% NaCl, B. Braun Melsungen AG, Melsungen, Germany), then finally filtered through a 0.22 μm pore size filter (Merck KGaA). Ultrapure water used for solutions preparation and for rinsing was freshly prepared in-house on a Millipore Milli-Q Direct 8 (Merck KGaA). The ⁶⁸Ge/⁶⁸Ga generator was coupled to a programmable synthesis module Galigand GAL-102 (Veenstra Instruments, Joure, The Netherlands). The whole preparation process was implemented and remotely controlled on Galigand GAL-102 labelling module. The peptides were previously dissolved in ultrapure water. Aliquots of 20 nmol of peptide per 50 μL solution were used for each synthesis, except DOTA-PEG(4)-BBN(7-14) for which 40 nmol per 50 μL solution were used. Prior of being loaded into reaction vial, the peptide must be protected by adding a certain amount of buffer (ammonium acetate 1 M). The amount of buffer necessary to adjust pH in the range of 3.8–4.3, suitable for the labelling reaction, was previously determined [28]. In order to be used for biomolecules labelling, ⁶⁸Ga was eluted from the generator using 0.05 M HCl. At this pH value, the gallium is introduced into the process as gallium (III) chloride. Exactly 2 mL of gallium eluate, containing the highest amount of eluted activity, was added over the buffered peptide already loaded into the reaction vial. To shorten the overall preparation time, the labelling temperature was increased to 95 °C; at this value, the duration required for the labelling step is 5 min. The reaction mixture is then passed on the separation cartridge which retains the peptide, while the impurities are evacuated to waste. After rinsing on cartridge by passing 1 mL water with 5% ethanol content, the peptide was recovered with 1 mL of ethanol and sent to the evaporation vial, where the ethanol was evaporated to dryness under vacuum and heating (95 °C). The peptide was dissolved in 1 mL saline solution and transferred to the final product vial by passing through a 0.22 μm pore size filter for sterilization purpose.