834 peaks of holmium acetylacetonate around 1,518 cm−1 are present in the samples before and after 1 day of incubation in buffer. The intensity of this peak reduces in time and after 1 month it could not be observed anymore. Interestingly, after 4 days a broad peak around 1,100 cm−1 appeared, which can be attributed to stretching bands of phosphate groups (22). TOF-SIMS analysis of HoAcAcMS before and after incubation in buffer showed spherical particles in total ion count mode (Fig. 3). The resulting mass spectrum showed differences between HoAcAcMS incubated in phosphate buffer and HoAcAcMS before incubation in buffer. The negative ions observed in the HoAcAcMS incubated in buffer were oxygen, chlorine, and phosphorous compounds (m/z 31: P-; m/z 63: PO2-; m/z 79 PO3-), which were absent in the spectrum of the non incubated microspheres (Fig. 3). All negative ions were distributed evenly through the microspheres, indicating a complete exchange of acetylacetonate by phosphate in the microspheres. Holmium (m/z 165), and holmium-oxygen (m/z 181) was present in both the incubated and non incubated samples. The incubated samples also showed a large peak of sodium (m/z 23) from the buffer salts, which was only present at the particle surface. The HoAcAcMS before incubation in phosphate buffer show additional peaks in the positive secondary ion mode at m/z 74 and m/z 147, which can be attributed to trimethylsilylgroups from polydimethylsiloxane (23) that is present on the surface of the carbon sticker. The results of IR analysis and TOF-SIMS confirm elemental analysis data that phosphate has replaced acetyla- cetonate after suspension in buffer. This in turn explains the limited release of holmium from the microspheres, since rare earth metal phosphates are practically insoluble in aqueous media; the solubility product of HoPO4 in water is lower than 10−25 M (24). The exchange of acetylacetonate by phosphate, without changing the surface morphology, is a so called chimie douce reaction (25). Chimie douce reactions are topotactic meaning that the products retain the precursor geometry, and usually take place under mild conditions. Beta diketones, like acetylacetonate, are prone to these types of reactions (26,27). The in vivo stability study was carried out to determine the in vivo implications of the results obtained in the in vitro experiment. Radioactive and cold HoAcAcMS were administered intratumorally to VX2 carcinoma-bearing rabbits. All animals recovered from anaesthesia, and the animals received 40±10 MBq 166HoAcAcMS, corresponding to 4±1 mg microspheres intratumorally. Approximately 80% of the intended dose was administered, which was attributed to the premature settling of the microspheres in the syringes. Nuclear imaging and CT imaging showed a selective administration of microspheres in the tumor (Fig. 4). One animal showed uptake of activity in the Bult et al. lungs (approximately 5% of the injected dose) apart from uptake in the tumor, due to the inadvertent delivery of 166HoAcAcMS in a blood vessel surrounding the tumor artery. This animal however, completed the experiment in good health. The inadvertent delivery of activity can be avoided in humans by administering the microspheres under ultrasound guidance, making it easier to distinguish tumor tissue from blood vessels and liver tissue (6). The excretion of holmium after intratumoral adminis- tration of HoAcAcMS to the blood, urine and the faeces was below the detection limit in all samples (0.1 ppm for Ho). Suzuki and co-workers found that release of free holmium (Ho3+) to the urine and faeces occurred rapidly after local intrahepatic administration of the in-situ gel forming complex holmium-166-chitosan (28). Therefore it can be concluded that Ho3+ was not released from the HoAcAcMS, and that the microspheres retained their integrity as confirmed by histology. Fig. 6 Light micrographs at 400x magnification of a H&E stained section of tumor tissue showing microspheres amidst necrotic tissue. (a) non-radioactive HoAcAcMS. (b) 166HoAcAcMS. The microspheres in red are clustered amidst necrotic tissue. The bar represents 20 μm.