HoAcAc Microspheres: In Vitro and In Vivo Stability Studies In Vitro Degradation of the Microspheres The release of Ho3+-ions from HoAcAcMS was determined as described by Zielhuis et al. (16). Neutron irradiated microsphere samples (50 mg) were suspended in 100 μl of 2% Pluronic® F68 aqueous solution and incubated in test tubes containing 5 mL isotonic phosphate buffer (116 mM, 39 mmol of NaH2PO4·2H2O and 77 mmol of Na2HPO4·2H2O in 1 L, pH 7.4) with 0.05% NaN3 to prevent bacterial growth. All samples were prepared in duplicate. The tubes were placed in a water bath at 37°C and continuously shaken and at predetermined time points (1 day, 4 days, 1, 2, 4, 8, 12 and 24 weeks) the test tubes were centrifuged, and the supernatant was collected. The microspheres were washed three times with water for injection and dried at 50°C. The microspheres and the supernatant were used for further analysis. Particle Surface Morphology Surface morphology of the HoAcAcMS after incubation was studied using a Phenom (Phenom World BV, Eindhoven, The Netherlands) scanning electron microscope. The samples were placed on aluminium stubs and sputter coated with a 6 nm Pt layer, and images were acquired at a voltage of 5 kV. Elemental Analysis The holmium, carbon, oxygen, hydrogen and phosphorous content was determined before and after incubation of HoAcAcMS in phosphate buffer by H. Kolbe (Mulheim an der Ruhr, Germany). Infrared Spectroscopy Fourier Transform Infrared spectroscopy was used to ascertain the presence of acetylacetonate in the microspheres. Infrared spectra of dried microspheres were acquired on a BIO-RAD FTS6000 FT-IR (BIO-RAD, Cambridge, MA, USA). The spectra were recorded from 400 to 4,000 cm−1 using KBr pellets, by accumulating 256 scans per spectrum. Secondary Ion Mass Spectrometry Time of flight secondary ion mass spectrometry (TOF- SIMS) analysis was performed to obtain mass spectral information about the molecular composition and distribution on the surface of the microspheres before and after incubation in buffer (17). Samples were fixed on a conductive carbon sticker on a stainless steel sample holder, and measured using a TRIFT II mass spectrometer 829 (Physical Electronics, Eden Prairie, MN) equipped with a Au primary ion gun. 22 keV Au+ primary ions were used for analysis. SIMS spectra were measured both in the positive and the negative secondary ion mode with a secondary ion energy of 7 keV. In Vivo Degradation Study Animal Model All experimental protocols and procedures were approved by the local experimental animal welfare committee and conform to national and European regulations for animal experimentation. Eight adult female New Zealand White rabbits of approximately 3.5 kg were purchased from Harlan (Horst, The Netherlands). The animals were housed individually to allow for urine and faeces collection and were fed approximately 100 g complete diet pellets for rabbits daily. Water was supplied ad libitum. Tumor Cells The VX2 cell line was a kind gift from dr. R.J.J. van Es (department of Oral and Maxillofacial surgery, University Medical Centre Utrecht, Utrecht, The Netherlands) (18). The VX2 cells were propagated by subcutaneous passage in the rabbit hind limb. After dissecting the tumor, small portions of viable tumor (2 mm in diameter) were selected for intrahepatic implantation as described by Nijsen et al. (19). Analgesia, Sedation and Euthanasia Analgesia during and 2 days post laparotomy was achieved with carprofen s.c. 4 mg kg−1 bodyweight. General anaesthesia was induced by ketamine i.m. at 30 mg kg−1 bodyweight and xylazine i.m. at 4 mg kg−1 bodyweight. Anaesthesia was maintained by inhalation of isoflurane Fig. 1 Release of holmium from HoAcAcMS (diamonds) and 166HoAcAcMS irradiated for either 3 h (triangles) or 6 h (squares) during incubation in phosphate buffer for 6 months. The data are presented as the mean of two measurements.