Aeju Lee

Polymeric Nanoparticle-Based Activatable Near-Infrared Nanosensor for Protease Determination In Vivo

We report here a new protease activatable strategy based on a polymer nanoparticle platform. This nanosensor delivers chemically labeled matrix metalloproteinase (MMP)-activatable fluorogenic peptides to the specific MMPs of interest in vivo. Intravenous administration of the nanosensor in an MMP-positive SCC-7 xenograft tumor and a colon cancer mouse model verified the enzyme specificity of the nanosensor in vivo. The design platform of the nanosensor is flexible and can be fine-tuned for a wide array of applications such as the detection of biomarkers, early diagnosis of disease, and monitoring therapeutic efficacy.

Early diagnosis of arthritis in mice with collagen-induced arthritis, using a fluorogenic matrix metalloproteinase 3–specific polymeric probe

OBJECTIVE Early treatment based on an early diagnosis of rheumatoid arthritis (RA) could halt progression of the disease, but early diagnosis is often difficult. Matrix metalloproteinase 3 (MMP-3) is thought to be particularly important in the pathogenesis of RA. The aim of this study was to investigate whether an MMP-3-specific polymeric probe could be used for early diagnosis and for visualizing the progression of arthritis, using a near-infrared fluorescence (NIRF) imaging system. METHODS The MMP-3-specific polymeric probe was developed by conjugating NIRF dye, MMP substrate peptide, and dark quencher to self-assembled chitosan nanoparticles. One hour after intravenous administration of the probe, fluorescent images of mice with collagen-induced arthritis at different stages of disease development were obtained. The correlation between the fluorescence recovered in in vivo imaging when using an MMP-3-specific polymeric probe and up-regulated MMP-3 activity in the joint tissues was evaluated by Western blotting and immunohistochemical staining. Histologic analysis and micro-computed tomography (micro-CT) were also used to assess arthritis progression. RESULTS A significantly higher NIRF signal was recovered from arthritic joints compared with normal joints at 14 days after the first immunization, before any erythema or swelling could be observed with the naked eye or any erosion was detected by histologic analysis or micro-CT. The results of immunohistochemical analysis and Western blotting confirmed that the fluorescence recovered in the in vivo imaging was related to up-regulated MMP-3 activity in the joint tissues. CONCLUSION An MMP-3-specific polymeric probe provided clear early diagnosis of arthritis and visualization of arthritis progression using an NIRF imaging system. This approach could be used for early diagnosis and for monitoring drug and surgical therapies in individual cases.

A novel near-infrared fluorescence chemosensor for copper ion detection using click ligation and energy transfer

A novel near-infrared fluorescence (NIRF) copper sensor allows rapid and ultra-sensitive detection of copper ions with excellent selectivity and specificity due to the specificity of click ligation and effective dark-quenching mechanism.

Cathepsin B-sensitive nanoprobe for in vivo tumor diagnosis

We developed a cathepsin B-sensitive nanoprobe (CB-NP) with a cathepsin B substrate peptide probe and tumor-targeting glycol chitosan nanoparticles. The CB-NP could accumulate in tumor tissues, enter into tumor cells, and generate fluorescent signals in cytosol in response to cathepsin B. The results of non-invasive fluorescence imaging in a tumor-bearing mouse model demonstrated the potential of CB-NP for tumor diagnosis in clinical fields.

Glycol Chitosan-Based Fluorescent Theranostic Nanoagents for Cancer Therapy

Theranostics is an integrated nanosystem that combines therapeutics with diagnostics in attempt to develop new personalized treatments with enhanced therapeutic efficacy and safety. As a promising therapeutic paradigm with cutting-edge technologies, theranostic agents are able to simultaneously deliver therapeutic drugs and diagnostic imaging agents and also monitor the response to therapy. Polymeric nanosystems have been intensively explored for biomedical applications to diagnose and treat various cancers. In recent years, glycol chitosan-based nanoagents have been developed as dual-purpose materials for simultaneous diagnosis and therapy. They have shown great potential in cancer therapies, such as chemotherapeutics and nucleic acid and photodynamic therapies. In this review, we summarize the recent progress and potential applications of glycol chitosan-based fluorescent theranostic nanoagents for cancer treatments and discuss their possible underlying mechanisms.

Measurement of MMP activity in synovial fluid in cases of osteoarthritis and acute inflammatory conditions of the knee joints using a fluorogenic peptide probe-immobilized diagnostic kit

Purpose: A fluorogenic peptide probe-immobilized diagnostic kit was used to analyze MMP activity in the synovial fluids (SFs) from patients with osteoarthritis (OA) and acute inflammatory conditions of the knee joint. Methods: The MMP diagnostic kit containing a polymer-conjugated MMP probe immobilized on a 96-well plate was utilized for high-throughput screening of MMP activity in SFs from OA patients (n = 33) and patients with acute inflammatory conditions of the knee joint (n = 5). Results: Compared to SF from OA patients, SF from patients with acute inflammatory conditions of the knee joint presented stronger NIR fluorescent signals. In gelatin zymography, most samples from patients with acute inflammatory conditions of the knee joint also displayed 92 kDa (pro-form) MMP-9 and faint 84 kDa (active form) MMP-9, while SF from OA patients did not display detectable MMP-9 activity . Conclusion: The presence of a strong fluorescence signal from the MMP diagnostic kit corresponded well with patients with acute inflammatory conditions of the knee joint. The results suggest that our MMP diagnostic kit can be useful in differentiation between early stages of OA and acute inflammatory conditions of the knee joint.

Optimization of matrix metalloproteinase fluorogenic probes for osteoarthritis imaging.

Among the classical collagenases, matrix metalloproteinase-13 (called MMP-13, collagenase-3) is one of the most important components for cartilage destruction of osteoarthritis (OA) developments. Despite many efforts, the detection methods of MMP-13 activity have been met with limited success in vivo, in part, due to the low sensitivity and low selectivity by homology of MMP family. Previously, we demonstrated the use of strongly dark-quenched fluorogenic probe allowed for the visual detection of MMP-13 in vitro and in OA-induced rat models. In this study, we described the optimization of MMP-13 fluorogenic probe for OA detection in vivo. Three candidate probes demonstrated recovered fluorescent intensity proportional with MMP-13 concentrations, respectively; however, Probe 2 exhibited both high signal amplification and selective recognition for MMP-13, not MMP-2 and MMP-9 in vitro. When Probe 2 was applied to OA-induced rat models, clear visualization of MMP-13 activity in OA-induced cartilage was obtained. Optimized MMP-13 fluorogenic probe can be applied to detect and image OA and have potential for evaluating the in vivo efficacy of MMP-13 inhibitors which are being tested for therapeutic treatment of OA.

One-Step Detection of Matrix Metalloproteinase Activity Using a Fluorogenic Peptide Probe-Immobilized Diagnostic Kit

Matrix metalloproteinases (MMPs) have been shown to be abundant in pathological conditions such as cancer, osteoarthritis (OA), and rheumatoid arthritis (RA). The extent of MMPs detected in biological samples provides important clinical information for diagnosis, prognosis, and therapeutic monitoring of various diseases relating with MMPs. Herein, we developed a new high-throughput MMP diagnostic kit (MMP-D-KIT) based on a 96-well plate by immobilizing MMP-13 specific fluorogenic peptide probes (MMP peptide probe), which is a pair consisting of a near-infrared (NIR) fluorophore (Cy5.5) and a quencher (BHQ-3), onto the biocompatible glycol chitosan (GC) polymer anchored 96-well plate. When MMP enzymes were simply added and incubated in a MMP-D-KIT, the fluorescence of each well was recovered and the fluorescence intensity showed distinct difference within minutes through NIR fluorescence imaging system. The fluorescence was recovered not only by MMP-13 activity, but also by other MMPs activity. Furthermore, recovery of NIR fluorescent signals in MMP-D-KIT was proportional to concentrations of immobilized MMP peptide probe-GC conjugates and, importantly, MMP concentration. The MMP-D-KIT is most specific for target MMP, compared with other enzymes including caspase-3 and 20s proteasome. Additionally, the MMP-D-KIT was used to detect MMP activity in biological samples such as synovial fluid from 12 OA patients (grades 1-4 based on the Kellgren-Lawrence grading scale). It was found that the fluorescence intensity measured using MMP-D-KIT decidedly correlates with the progression of OA. The MMP-D-KIT could be applicable in detecting MMP activities in various biological samples and evaluating the effects of MMP inhibitors in a rapid and easy fashion.

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 mAGCs 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.

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 in vivo 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 in vivo 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.