Li C, Wang W, Wu Q, Ke S, Houston J, Sevick-Muraca E, Dong L, Chow D, Charnsangavej C, Gelovani JG. allow us to understand the biodistribution and pharmacokinetics of antibody-conjugated drugs and radionuclides targeted to tumors. In this manner, tumor growth can be monitored and effectiveness of therapy can be evaluated. In this study, anti-P-selectin scFv antibodies have been labeled with fluorescent optical probes and radionuclides for near-infrared optical imaging and gamma video camera imaging of P-selectin expression in radiation treated lung tumor xenografts. Tumor targeting efficacy and kinetics profiles were observed and evaluated using complementary optical and nuclear imaging modalities. Nuclear imaging modalities such as PET, SPECT and gamma video camera imaging typically exhibit high sensitivity in deep tissues, and increased spatial resolution but poor temporal resolution as compared to optical imaging.18,20 Optical imaging modalities have the advantage of higher temporal resolution, safer use since they do not use ionizing radiation or radioactive materials, but they provide lower spatial resolution and sensitivity in deep tissues.4,5,16,35,38 A major limitation of optical imaging is the high absorption and scattering that occurs in biological tissues and the limited penetration of the light through the body. Near-infrared fluorescence (NIR) imaging is usually a particular kind of optical imaging that exploits the near-infrared range in the spectra to bypass the typical absorption and autofluorescence problems seen in optical imaging of biological tissues.4,35 Typically, tissues exhibit a high photon absorbance in both the visible wavelength range (350?650 nm) and the infrared range (above 900 nm).4 However, in the NIR range of 650?900 nm the absorbance of water and tissues in the body is at a minimum and thus allows photons to penetrate tissue more efficiently and minimizes scattering.4,5,16,35,38 Functional imaging of molecularly based events such as tumor-specific binding can be performed using both nuclear and optical imaging modalities in order to provide complementary information.6,24,31 In this study, fluorescent probes in the NIR range were conjugated to anti-P-selectin scFvs for NIR optical imaging, and complementary data was gathered using 111In labeled anti-P-selectin scFvs with gamma camera imaging. METHODS Drug delivery methods generally rely on the use of targeting agents to deliver the conjugate to the appropriate site, and therapeutic agents to produce the desired therapeutic Chuk effect at that site. In order to create optimal drug delivery vehicles, biodistribution and kinetics of the targeting agent and its receptor must first be analyzed and validated. Optical imaging and nuclear imaging are commonly utilized for these purposes and provide complementary information regarding targeting kinetics and biodistribution. This section focuses on the methods utilized for preliminary imaging studies in order to explore the potential of P-selectin targeting for radiation-guided drug delivery. Antibody Selection All antibodies were screened from phage display libraries and developed in the Molecular Acknowledgement Core Laboratory at Vanderbilt University or college. The antibodies with highest affinity for P-selectin were then selected for and imaging studies offered here. The altered antibody used in the nuclear imaging studies was developed in collaboration with Dr. Martin Brechbiel in the radiochemistry laboratory at the National Institutes of Health. In vitro Model Immunofluorescence Assay Main culture Ki 20227 human umbilical vein endothelial cells (HUVECs) were cultured till 80% confluency in Lab-Tek II chamber slide wells (Nunc International, Naperville, IL) in endothelial cell medium and incubated 37 C in a humidified 5% CO2 atmosphere. Cells were then incubated with human TNF(500 mM) or treated with 3 Gy radiation for 30 min. After treatment, cells were fixed with 4% paraformaldehyde for 10 min at room temperature, and washed 3 times with antibody buffer (4 g Ki 20227 bovine serum albumin, 0.1 g sodium azide, 0.75 g glycine, and 100 stability Ki 20227 of the radionuclide.36 To enable conjugation of scFv 10A to CHX-A DTPA, a cysteine residue was engineered Ki 20227 into the amino acid sequence of the scFv. The synthesis of 111In-CHX-A DTPA-scFv10Acys conjugate was achieved through conjugation of scFv 10Acys-CHX-A DTPA with 111In using thiol chemistry of the cysteine residue. The reaction was performed by adding 5 imaging studies. Statistical Analysis One-way analysis of variance (ANOVA) was utilized for statistical evaluation. Means were compared by using Student’s NIR fluorescence imaging was performed with a Xenogen IVIS 200 small animal Ki 20227 imaging system (Xenogen Inc., Alameda CA, USA) with a Cy7 filter set (excitation at 680 nm and emission at 775 nm). Nude.