HoAcAc Microspheres: In Vitro and In Vivo Stability Studies Although the results from the in vitro and the in vivo stability study show a minimal to no release of holmium from the HoAcAcMS, the in vivo data cannot be related to the chimie douce reaction found in vitro. The phosphate concentration (116 mM) used for the in vitro experiment is substantially higher than the phosphate concentration in human serum (around 1.5 mM (29)). The high phosphate concentration in vitro was required to keep the pH stable throughout the experiment, which, however, might accelerate the exchange of acetylacetonate by phosphate. The exchange of acetylacetonate by phosphate could not be established in vivo since paraffin interfered with the TOF-SIMS measurements, nonetheless, the presence of intact microspheres and the absence of holmium in urine, faeces and blood show that the HoAcAcMS are stable in vivo. The identification of the chemical structure of the HoAcAcMS after intratumoral administration requires further research. A transient rise in ASAT was observed after the administration of the radioactive microspheres, which can be attributed to the subxiphoid manipulation of liver tissue, and radiation effects of the 166HoAcAcMS. No other elevations from baseline levels or differences in serum enzyme levels were found between the radioactive and non-radioactive group (see Fig. 5). ASAT and ALAT are markers for short term liver damage, whereas gamma GT and Alkaline Phosphatase are a measure for long-term (chronic) liver damage. Albumin levels are a marker for the liver function, since the liver is the main site for albumin production (30). The transient rise in ASAT was previously observed after transcatheter radioembolization with both 90Y microspheres in human liver cancer patients (31) and 166HoPLLAMS in healthy pigs (30). This transient rise was attributed to the manipulation of liver tissue and radiation effects of the microspheres (30,31). No changes in serum enzyme levels or albumin levels were observed in the present study, an indication that the liver function was neither impaired by the tumor, nor intratumoral administration of either HoAcAcMS or 166HoAcAcMS. Histological evaluation of liver sections showed intact microspheres amidst necrotic tissue both for HoAcAcMS and 166HoAcAcMS (Fig. 6), which is in agreement with the results from the in vitro stability study showing intact HoAcAcMS after 6 months incubation in buffer (Fig. 2). Both the non-radioactive HoAcAcMS and the 166HoAcAcMS are surrounded by necrotic tissue. The VX2 tumor is a rapid-growing tumor, with a viable rim and a necrotic core. Therefore, the HoAcAcMS are surrounded by necrotic tissue (Fig. 6a). Radiation damage from the 166HoAcAcMS resulted in the necrotic tissue surrounding the cluster of microspheres (Fig. 6b). Possible release of holmium from the HoAcAcMS was determined by measuring the holmium content in bone. 835 Holmium is a bone seeking element, and it has been shown that within 4 days after intravenous administration of holmium nitrate, approximately 55% of the injected dose accumulates in bone (32). Therefore, if holmium (Ho3+) would be released from the HoAcAcMS in vivo, the holmium levels in bone would be elevated. In accordance with the holmium levels in urine and faeces, the holmium levels in all bone samples were below the detection limit and it can be concluded that no release of holmium from the HoAcAcMS has occurred. CONCLUSION This paper demonstrates the stability of HoAcAcMS, both in vitro and in vivo. The in vitro release was approximately 0.5% after 6 months of incubation in buffer. The microspheres remained spherical, and a HoPO4 complex was formed while the surface morphology was retained. No release of holmium was observed after the intratumoral injection of HoAcAcMS in VX2 tumor-bearing rabbits, and the microspheres remained intact in tumor tissue for 1 month. The results confirm the potential of these microspheres as a novel intratumoral radioablation device. ACKNOWLEDGMENTS & DISCLOSURES Financial support by the Dutch Technology Foundation STW, under grant 06069 is gratefully acknowledged. Dr. G.C. Krijger and ms. M.J.J. Koster-Ammerlaan are acknowledged for performing the neutron irradiations. Ms. H.M. de Bruin, Mr. N.J.M. Attevelt and Mr. H.W.G. Vosmeer are gratefully acknowledged for biotechnical assistance. The authors would like to thank prof. dr. K.P. de Jong for valuable discussions. Dr. W.H. Müller and Mr. C.J.M.M. Schneijdenberg are acknowledged for their assistance with the SEM measurements. 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