828 latter category, the most widely used technique is radio- embolization of liver malignancies using radioactive micro- spheres (8,9). This technique involves the administration of yttrium-90 (90Y) microspheres using a catheter placed in the hepatic artery. Another approach is the direct intratumoral injection of radiolabeled particles or substances. Intra- tumorally administered 90Y glass microspheres have been successfully tested in humans (6). The results were promising: 90.6% of the tumors treated showed a size reduction, and apparent complete tumor necrosis was observed in 8 patients. Although these results were very promising, the extrahepatic delivery of microspheres, especially to the lungs, and the lack of accurate dose estimations were posed as problems to be overcome. Also, intratumoral administration of holmium-166 (166Ho) chelated to chitosan, an in situ gel forming device, has been tested in hepatocellular carcinoma patients with small (≤3 cm) solitary lesions (10). Complete tumor necrosis was observed in 31 of 40 patients, yet the release of holmium from the injection site led to haematologic side effects in 11 of 40 patients. In itself, 166Ho is an ideal isotope since, in contrast to 90Y, it combines the emission of therapeutic beta particles with gamma rays. The gamma rays can be accurately visualized using gamma scintigraphy, which can be used for accurate dosimetric calculations. Moreover, holmium is a paramagnetic and electron dense element, allowing for magnetic resonance imaging and x-ray computed tomography (CT), respec- tively (11). Chitosan as a delivery device for 166Ho however is not ideal due to leakage of 166Ho from the complex, causing haematologic side effects. To further optimize the intratumoral application of 166Ho, holmium microspheres with an ultra high holmium load were proposed as a radioablation device for solid malignancies (12). These microspheres were suitable to deliver sufficient radiation after intratumoral administration, and their efficacy was tested in a renal cell carcinoma tumor- bearing mouse model (13). The microsphere treated tumors did not show a significant growth (141±99 mm3 at time of treatment versus 104±95 mm3 after 2 weeks), whereas the tumor size in the saline treated control group increased dramatically (from 122 ± 33 mm3 to 4,150± 300 mm3 2 weeks post-injection). In addition to the laboratory animal study, a pilot experiment was performed to assess the feasibility and tolerability of intratumoral administration of 166HoAcAcMS in feline liver cancer patients (14). The treatment was well tolerated, and the clinical condition of the cats improved. It was concluded that intratumoral administration of 166HoAcAcMS is efficacious, and not associated with adverse effects. The purpose of the present study was to investigate the long-term in vitro stability of these radioactive holmium acetylacetonate microspheres (166HoAcAcMS). The holmium Bult et al. release from the HoAcAcMS was measured and the surface morphology and the chemical composition of the HoAcAcMS were investigated upon incubation of both non-neutron activated and neutron activated radioactive HoAcAcMS in phosphate buffer at 37°C for up to 26 weeks. The in vivo stability was assessed in VX-2 tumor-bearing rabbits, after intratumoral administration of both non-activated HoAcAcMS and neutron-activated HoAcAcMS. The rabbits were followed for 1 month, during which blood, urine and faeces were collected to determine the excretion of holmium. Histology was performed on tumor tissue, and the holmium content in the femur was determined. MATERIALS AND METHODS Materials All chemicals were commercially available and used as received. Acetylacetone (2,4-pentanedione (AcAc; >99%)), ammonium hydroxide (NH4OH; 29.3% in water), chloroform (CHCl3; HPLC-grade), holmium phosphate (anhydrous (HoPO4; >99.99+%), poly(vinyl alcohol) (PVA; average MW 30,000–70,000, 88% hydrolyzed) and poloxamer 188 (Pluronic® F68, average MW 8350) were purchased from Sigma Aldrich (Steinheim, Germany). Di-sodium hydrogen phosphate dihydrate (Na2HPO4.2H2O; 99%), ethanol (absolute), holmium CertiPUR ICP Standard (1.00 g L−1), sodium azide (NaN3; 99%) and sodium dihydrogen phosphate dihydrate (NaH2PO4.2H2O; 99%) were purchased from Merck (Darmstadt, Germany). Holmium chloride hexahydrate (HoCl3.6H2O) was obtained from Metall rare earth (Shenzhen, China). Dexmedetomedine (Dexdomitor) and carprofen (Rimadyl) were obtained from Pfizer Animal Health B.V. (Capelle a/d IJssel, The Netherlands) and Isoflurane (IsoFlo) was obtained from Abbott Animal Health (Hoofddorp, The Netherlands). Preparation and Neutron Irradiation of HoAcAcMS HoAcAcMS with a size of 15 μm were prepared by a solvent evaporation process previously described (12). The HoAcAcMS (300 or 600 mg) were weighed in high density polyethylene vials (Type A, Posthumus plastics, Beverwijk, the Netherlands). Neutron irradiations were performed in the pneumatic rabbit system operational at the research reactor facility of the Reactor Institute Delft (Delft University of Technology, The Netherlands) (15). Samples were irradiated for either 3 h or 6 h with a thermal neutron flux of 5 × 1012 n cm−2 s−1, and the radioactive 166Ho was left to decay for 1 month in closed vials.