Laboratory activities to address our first goal include radiolabeling targeting molecules with a positron emitting radionuclide (F-18, Br-76, Ga-68, Cu-64, Zr-89) and evaluating the selectivity of uptake in xenograft tumor models that express varying levels of the desired target using small animal PET imaging (INVEON PET/CT). We have developed a variety of antibody and peptide based targeting molecules that are being evaluated for selectivity to angiogenesis markers, chemokine receptors, growth factor receptors, gastrin releasing hormone receptor, glucagon-like peptide type 1, inflammation, hypoxia, and apoptosis markers. Peptides are modified by conjugation with a metal chelator appropriate for one of the radiometals;incorporation of a cysteine residue to provide a unique sulfhydryl for radiolabeling with 18F-FBEM;and/or reacting a lysine residue with 18F-SFB. These same peptide reactive groups can be radiolabeled with Br-76-containing prosthetic groups. New methods of radiolabeling are also an area of research effort. The wide range of radionuclide half-lives provides the ability to select the appropriate radionuclide for the pharmacokinetics of our radioligand. The automation of frequently practiced radiochemical syntheses is important for safe and reliable preparation of tracers for the many studies required for radiotracer validation. Small animal imaging and dissection studies are used to evaluate the targeting selectivity and pharmacokinetics of biodistribution for our radiolabeled compounds. Targeted optical imaging can be achieved by coupling near IR or visible fluorophores to targeting peptides or small drug-like molecules. We have focused on attaching fluorophores to peptide ligands for angiogenesis, lymphangiogenesis and gastrin releasing hormone tumor markers. This optical technique has wide applications in the study of biological mechanisms in small animals and has application in the clinic for targets accessible by endoscopy. The Laboratory has its own CRI Maestro imaging device for conducting these studies. Both of these imaging modalities require the development of novel targeting agents. We utilize phage display peptide libraries, single chain antibody libraries, and systematic evolution of ligands to develop new probes. For labeling purposes, we are utilizing and developing procedures for site-specific modification of proteins and peptides. This allows the introduction of fluorophores, radionuclide prosthetic groups, or metal chelating agents at sites on the peptide that will not diminish the targeting selectivity of our newly designed probes. In addition, we have exploited our HPLC-MS capabilities to study metabolite profiles and metabolic rates of our novel ligands. All ligands used for the various imaging modalities (radioactive and non-radioactive) can be studied by HPLC-MS. Biodistribution studies can be conducted on many peptides and small drug-like molecules using the sensitivity of mass spectrometry rather than radioactivity or fluorescence. We recently utilized HPLC-MS to evaluate ligand internalization. Through quantitation of a peptide ligand in the various cellular pools, we could determine the amount of internalization of the ligand. Thus, the sensitivity and specificity of HPLC-MS can evaluate many important properties that are necessary in order to fully characterize a novel ligand. In terms of nanomedicine, we are generating novel NanoProbes that exhibit high sensitivity and ultra-low background noise in both cells and in vivo applications. By integrating molecular imaging, nanobioconjugation chemistry and molecular/clinical biology, we are developing imaging probes that can be utilized for imaging disease-related biological processes including but not limited to protease expressions such as matrix metalloproteinases, cathepsins, caspases, apoptosis, and angiogenesis. Furthermore, our probing system can be applied for cell tracking, early diagnosis and monitoring of therapeutic efficacy. We have set nanobioconjugation chemistry particularly for peptides/proteins, biopolymers, and inorganic nanoparticles. These Nanoprobes will take full advantage of different imaging modalities such as optical imaging, MRI, PET, CT and photoacoustic imaging and could provide unique information that impact preclinical and eventually clinical diagnostics. We are developing NanoCarriers that can be utilized for targeted delivery of therapeutics. Various non-toxic and targeted nano-hybrid biomolecules that carry and stabilize therapeutic agents are under development that combine antifouling biopolymers, targeting ligands, self-assembled polymeric nanoparticles, iron oxide/gold nanoparticles and carbon nanotubes. Small chemicals, therapeutic peptides/proteins and siRNA/miRNA can be engineered and formulated by sophisticated nanobioconjugation and encapsulation methods to maximize therapeutic efficacy. To date, various conventional anticancer drugs and newly identified small drug-like molecules in the pipeline are successfully reformulated and their therapeutic efficacies are under evaluation in specific disease animal models. Furthermore, with the help of molecular imaging techniques, carrier systems and their outcomes can be non-invasively tracked and monitored in vivo. We have interest in developing ultrasensitive, simple and cost-effective NanoDiagnostics for screening and early detection of disease-specific biomarkers and drugs using combined inorganic and polymeric nano-platform technology, fluorescence amplification strategy and electrochemistry. Our system is expected to boost the sensitivity of conventional ELISA technology and facilitate the detection of disease-specific antigens in various biological samples in an efficient fashion. This technology can be applied to real-time, cell-based, high-throughput drug screening systems. Currently, various unique systems are under development for antigen detection and for screening drugs modulating apoptosis and kinase pathways. In addition, we are also interested in developing point-of-care (POC) devices that can quickly determine the level of biomarkers for immediate care and measure the progression of specific disease. Nanoparticles can be prepared that have many unique properties. We have interest in developing nano-platforms for therapeutic purposes as well as molecular imaging. Among properties that can be applied for molecular imaging are fluorescence, fluorescence quenching, and magnetic properties that allow tracking by MRI. We are developing nanoplatforms that can be utilized for early detection of disease bio-markers, monitoring for therapeutic efficacy, and for targeted delivery of therapeutics. We believe nanoparticles can be employed to develop simple inexpensive biomarker assay kits that should exceed the sensitivity of ELISA.
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