Optical Imaging of Cancer-Related Proteases Using Near-Infrared Fluorescence Matrix Metalloproteinase-Sensitive and Cathepsin B-Sensitive Probes

Cathepsin B and matrix metalloproteinase (MMP) play key roles in tumor progression by controlled degradation of extracellular matrix. Consequently, these proteases have been attracted in cancer research, and many imaging probes utilizing these proteases have been developed. Our groups developed cathepsin B and MMP imaging nanoprobes based on polymer nanoparticle platform. Both cathepsin B and MMP imaging probes used near-infrared fluorescence (NIRF) dye and dark-quencher to for high sensitivity, and protease-sensitive peptide sequence in each probe authorized high specificity of the probes. We compared the bioactivities of cathepsin B and MMP sensitive probes in cancer-related environments to investigate the biological property of the probes. As a result, cathepsin B probe showed fluorescence recovery after the probe entered the cytoplasm. This property could be useful to evaluate the cytoplasmic targeted delivery by using probe-conjugated nanoparticles in vivo. On the other hand, MMP probe was superior in specificity in vivo and tissue study. This comparative study will provide precise information about peptide-based optical probes, and allow their proper application to cancer diagnosis

Cell Labeling and Tracking Method without Distorted Signals by Phagocytosis of Macrophages

Cell labeling and tracking are important processes in understanding biologic mechanisms and the therapeutic effect of inoculated cells in vivo. Numerous attempts have been made to label and track inoculated cells in vivo; however, these methods have limitations as a result of their biological effects, including secondary phagocytosis of macrophages and genetic modification. Here, we investigated a new cell labeling and tracking strategy based on metabolic glycoengineering and bioorthogonal click chemistry. We first treated cells with tetra-acetylated N-azidoacetyl-D-mannosamine to generate unnatural sialic acids with azide groups on the surface of the target cells. The azide-labeled cells were then transplanted to mouse liver, and dibenzyl cyclooctyne-conjugated Cy5 (DBCO-Cy5) was intravenously injected into mice to chemically bind with the azide groups on the surface of the target cells in vivo for target cell visualization. Unnatural sialic acids with azide groups could be artificially induced on the surface of target cells by glycoengineering. We then tracked the azide groups on the surface of the cells by DBCO-Cy5 in vivo using bioorthogonal click chemistry. Importantly, labeling efficacy was enhanced and false signals by phagocytosis of macrophages were reduced. This strategy will be highly useful for cell labeling and tracking

The micromechanical properties of bone tissue.

Bone is a living material which has the ability to change its structure to adapt to altered physiological and mechanical environments. Much research has been devoted to characterizing the mechanical behavior and the structure-function relationship. However, the mechanical behavior of bone tissue on a microstructural level is virtually uncharacterized. The purpose of this dissertation was to determine the micro mechanical properties of bone tissue, and to evaluate the effects of mineral density and microstructure on the properties. First, the moduli of human subchondral, trabecular, and cortical bone tissue were determined using three-point bending tests on regularly-shaped beam specimens. The significant moduli differences found between the bone tissues (1.15 GPa for subchondral, 4.59 GPa for trabecular, and 5.44 GPa for cortical bone tissue) may be a result of the differences in microstructure rather than in mineral density. Furthermore, the size-dependency of the cortical bone modulus was found. Second, the fatigue properties of human trabecular and similar-sized cortical bone specimens were determined using cyclic four-point bending tests. Cortical tissue had higher fatigue strength and higher moduli than trabecular tissue, despite its lower mineral density. Different fracture and microdamage patterns observed between the two tissues likely reflect a significant difference in microstructure. Based on the observations, a possible role for cement lines in trabecular tissue was suggested. The results also showed that a higher loading rate resulted in lower fatigue strength of trabecular tissue. Third, the micro mechanical properties of canine trabecular bone tissue were determined and compared to human tissue values in order to evaluate the appropriateness of canine bone as a model for human bone. Canine trabecular tissue exhibited lower mineral content, lower modulus, and lower fatigue strength as compared with human tissue. This can be explained mainly by the differences in microstructure and perhaps ultrastructure, while the mineral density partly contribute to the differences. Different characteristics in fracture surfaces and microdamage patterns support the microstructural or ultrastructural differences between the two species.Ph.D.BioengineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105356/1/9123996.pdfDescription of 9123996.pdf : Restricted to UM users only

Biomimetic aggrecan reduces cartilage extracellular matrix from degradation and lowers catabolic activity in ex vivo and in vivo models.

Aggrecan, a major macromolecule in cartilage, protects the extracellular matrix (ECM) from degradation during the progression of osteoarthritis (OA). However, aggrecan itself is also susceptible to proteolytic cleavage. Here, the use of a biomimetic proteoglycan (mAGC) is presented, which functionally mimics aggrecan but lacks the known cleavage sites, protecting the molecule from proteolytic degradation. The objective of this study is to test the efficacy of this molecule in ex vivo (human OA synovial fluid) and in vivo (Sprague-Dawley rats) osteoarthritic models. These results indicate that mAGC's may protect articular cartilage against the loss of key ECM components, and lower catabolic protein and gene expression in both models. This suppression of matrix degradation has the potential to provide a healthy environment for tissue repair

Biomimetic aggrecan reduces cartilage extracellular matrix from degradation and lowers catabolic activity in ex vivo and in vivo models.

Aggrecan, a major macromolecule in cartilage, protects the extracellular matrix (ECM) from degradation during the progression of osteoarthritis (OA). However, aggrecan itself is also susceptible to proteolytic cleavage. Here, the use of a biomimetic proteoglycan (mAGC) is presented, which functionally mimics aggrecan but lacks the known cleavage sites, protecting the molecule from proteolytic degradation. The objective of this study is to test the efficacy of this molecule in ex vivo (human OA synovial fluid) and in vivo (Sprague-Dawley rats) osteoarthritic models. These results indicate that mAGC's may protect articular cartilage against the loss of key ECM components, and lower catabolic protein and gene expression in both models. This suppression of matrix degradation has the potential to provide a healthy environment for tissue repair

Conjugated polymer nanoparticles for biomedical in vivo imaging

Conjugated polymer nanoparticles, produced by in situ colloidal Knoevenagel polymerization, show advantageous properties (bright emission, colloidal/chemical stability and mesoscopic size range) that allow the successful in vivo application to real-time sentinel lymph node mapping in a mouse model

Optical Imaging of Cancer-Related Proteases Using Near-Infrared Fluorescence Matrix Metalloproteinase-Sensitive and Cathepsin B-Sensitive Probes

Cathepsin B and matrix metalloproteinase (MMP) play key roles in tumor progression by controlled degradation of extracellular matrix. Consequently, these proteases have been attracted in cancer research, and many imaging probes utilizing these proteases have been developed. Our groups developed cathepsin B and MMP imaging nanoprobes based on polymer nanoparticle platform. Both cathepsin B and MMP imaging probes used near-infrared fluorescence (NIRF) dye and dark-quencher to for high sensitivity, and protease-sensitive peptide sequence in each probe authorized high specificity of the probes. We compared the bioactivities of cathepsin B and MMP sensitive probes in cancer-related environments to investigate the biological property of the probes. As a result, cathepsin B probe showed fluorescence recovery after the probe entered the cytoplasm. This property could be useful to evaluate the cytoplasmic targeted delivery by using probe-conjugated nanoparticles in vivo. On the other hand, MMP probe was superior in specificity in vivo and tissue study. This comparative study will provide precise information about peptide-based optical probes, and allow their proper application to cancer diagnosis.</p

Detection of Active Matrix Metalloproteinase‑3 in Serum and Fibroblast-Like Synoviocytes of Collagen-Induced Arthritis Mice

The activity of rheumatoid arthritis (RA) correlates with the expression of proteases. Among several proteases, matrix metalloproteinase-3 (MMP-3) is one of the biological markers used to diagnose RA. The active form of MMP-3 is a key enzyme involved in RA-associated destruction of cartilage and bone. Thus, detection of active MMP-3 in serum or <i>in vivo</i> is very important for early diagnosis of RA. In this study, a soluble MMP-3 probe was prepared to monitor RA progression by detecting expression of active MMP-3 in collagen-induced arthritis (CIA) mice <i>in vivo</i> in both serum and fibroblast-like synoviocytes (FLSs). The MMP-3 probe exhibited strong sensitivity to MMP-3 and moderate sensitivity to MMP-7 at nanomolecular concentrations, but was not sensitive to other MMPs such as MMP-2, MMP-9, and MMP-13. In an optical imaging study, the MMP-3 probe produced early and strong NIR fluorescence signals prior to observation of erythema and swelling in CIA mice. The MMP-3 probe was able to rapidly and selectively detect and monitor active MMP-3 in diluted serum from CIA mice. Furthermore, histological data demonstrated that activated FLSs in arthritic knee joints expressed active MMP-3. Together, our results demonstrated that the MMP-3 probe may be useful for detecting active MMP-3 for diagnosis of RA. More importantly, the MMP-3 probe was able to detect active MMP-3 in diluted serum with high sensitivity. Therefore, the MMP-3 probe developed in this study may be a very promising probe, useful as a biomarker for early detection and diagnosis of RA