Lyophilization of TC-99m-Hynic Labeled Peg-Liposomes

Abstract

The development of long circulating liposomes represented a major step forward towards the use of radiolabeled liposomes in nuclear medicine. The long circulation property markedly improves their uptake and consequently visualization of sites of infection and inflammation. Previously, we have developed a rapid and convenient method to label polyethylene glycol (PEG)-lipo-somes with technetium-99m (Tc-99m). PEG-liposomes containing the technetium-chelator hydrazino nicotinamide (HYNIC) could be labeled with Tc-99m with high efficiency. We showed that these Tc-99m-HYNIC labeled PEG-liposomes have excellent in vivo imaging characteristics in several pre-clinical and clinical studies. However, an important limitation associated with the use of HYNIC-PEG-liposome formulation as radiopharmaceutical is that their labeling efficiency decreases markedly within 3 months. In this paper we present a lyophilization method for HYNIC-PEG-liposomes using sucrose as a lyopro-tectant. The long-term stability of these liposomes in terms of the particle size and labeling efficiency upon reconstitution were determined. Additionally, the in vivo behavior of reconstituted radiolabeled liposomes in a rat model of focal infection was studied at two time-points after preparation.

Increasing the duration of the dehydration step significantly reduced the mean particle size upon reconstitution. Increasing the storage temperature from -20^°C to +4^°C also improved the particle size distribution upon reconstitution. The labeling efficiency for both freeze-dried preparations remained high during the 1 year-storage period and was always higher than 86%, but decreased for the control liposomes. Eight months after preparation, these liposomes had a labeling efficiency as low as 6%, whereas both freeze-dried preparations could still be labeled with an efficiency of 90%. The in vivo studies showed that there was no major difference in the biodistribution of the radiolabeled liposomes between 3 and 30 weeks post-preparation in rats with an Staphylococcus aureus abscess, indicating an acceptable long-term shelf-life of both freeze-dried liposome preparations. Abscesses were visualized from 2 hours post injection onwards.

In conclusion, a freeze-drying method which improved the long term shelf-life of HYNIC-PEG-liposomes is presented. The in vivo behavior of Tc-99m-PEG-liposomes, reconstituted 30 weeks after preparation, was similar to the biodistribution obtained with the non-freeze-dried preparation. The splenic uptake of these liposomes was slightly increased.