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

DNA Amplification in Neutral Liposomes for Safe and Efficient Gene Delivery

In general, traditional gene carriers contain strong cationic charges to efficiently load anionic genes, but this cationic character also leads to destabilization of plasma membranes and causes severe cytotoxicity. Here, we developed a PCR-based nanofactory as a safe gene delivery system. A few template plasmid DNA can be amplified by PCR inside liposomes about 200 nm in diameter, and the quantity of loaded genes highly increased by more than 8.8-fold. The liposome membrane was composed of neutral lipids free from cationic charges. Consequently, this system is nontoxic, unlike other traditional cationic gene carriers. Intense red fluorescent protein (RFP) expression in CHO-K1 cells showed that the amplified genes could be successfully transfected to cells. Animal experiments with the luciferase gene also showed <i>in vivo</i> gene expression by our system without toxicity. We think that this PCR-based nanofactory system can overcome the toxicity problem that is the critical limitation of current gene delivery to clinical application

Prediction of Antiarthritic Drug Efficacies by Monitoring Active Matrix Metalloproteinase‑3 (MMP-3) Levels in Collagen-Induced Arthritic Mice Using the MMP‑3 Probe

Active matrix metalloproteinase-3 (MMP-3) is a prognostic marker of rheumatoid arthritis (RA). We recently developed an MMP-3 probe that can specifically detect the active form of MMP-3. The aim of this study was to investigate whether detection and monitoring of active MMP-3 could be useful to predict therapeutic drug responses in a collagen-induced arthritis (CIA) model. During the period of treatment with drugs such as methotrexate (MTX) or infliximab (IFX), MMP-3 mRNA and protein levels were correlated with fluorescence signals in arthritic joint tissues and in the serum of CIA mice. Also, bone volume density and erosion in the knee joints and the paws of CIA mice were measured with microcomputed tomography (micro-CT), X-ray, and histology to confirm drug responses. In joint tissues and serum of CIA mice, strong fluorescence signals induced by the action of active MMP-3 were significantly decreased when drugs were applied. The decrease in RA scores in drug-treated CIA mice led to fluorescence reductions, mainly as a result of down-regulation of MMP-3 mRNA or protein. The micro-CT, X-ray, and histology results clearly showed marked decreases in bone and cartilage destruction, which were consistent with the reduction of fluorescence by down-regulation of active MMP-3 in drug-treated CIA mice. We suggest that the MMP-3 diagnostic kit could be used to detect and monitor the active form of MMP-3 in CIA mice serum during a treatment course and thereby used to predict the drug response or resistance to RA therapies at an earlier stage. We hope that monitoring of active MMP-3 levels in arthritis patients using the MMP-3 diagnostic kit will be a promising tool for drug discovery, drug development, and monitoring of drug responses in RA therapy

Precise Targeting of Liver Tumor Using Glycol Chitosan Nanoparticles: Mechanisms, Key Factors, and Their Implications

Herein, we elucidated the mechanisms and key factors for the tumor-targeting ability of nanoparticles that presented high targeting efficiency for liver tumor. We used several different nanoparticles with sizes of 200–300 nm, including liposome nanoparticles (LNPs), polystyrene nanoparticles (PNPs) and glycol chitosan-5β-cholanic acid nanoparticles (CNPs). Their sizes are suitable for the enhanced permeation and retention (EPR) effect in literature. Different <i>in vitro</i> characteristics, such as the particle structure, stability, and bioinertness, were carefully analyzed with and without serum proteins. Also, pH-dependent tumor cell uptakes of nanoparticles were studied using fluorescence microscopy. Importantly, CNPs had sufficient stability and bioinertness to maintain their nanoparticle structure in the bloodstream, and they also presented prolonged circulation time in the body (blood circulation half-life <i>T</i>_1/2 = about 12.2 h), compared to the control nanoparticles. Finally, employing liver tumor bearing mice, we also observed that CNPs had excellent liver tumor targeting ability <i>in vivo</i>, while LNPs and PNPs demonstrated lower tumor-targeting efficiency due to the nonspecific accumulation in normal liver tissue. Liver tumor models were produced by laparotomy and direct injection of HT29 tumor cells into the left lobe of the liver of athymic nude mice. This study provides valuable information concerning the key factors for the tumor-targeting ability of nanoparticles such as stability, bioinertness, and rapid cellular uptake at targeted tumor tissues

Chemical Tumor-Targeting of Nanoparticles Based on Metabolic Glycoengineering and Click Chemistry

Tumor-targeting strategies for nanoparticles have been predominantly based on optimization of physical properties or conjugation with biological ligands. However, their tumor-targeting abilities remain limited and insufficient. Furthermore, traditional biological binding molecules have intrinsic limitations originating from the limited amount of cellular receptors and the heterogeneity of tumor cells. Our two-step <i>in vivo</i> tumor-targeting strategy for nanoparticles is based on metabolic glycoengineering and click chemistry. First, an intravenous injection of precursor-loaded glycol chitosan nanoparticles generates azide groups on tumor tissue specifically by the enhanced permeation and retention (EPR) effect followed by metabolic glycoengineering. These ‘receptor-like’ chemical groups then enhance the tumor-targeting ability of drug-containing nanoparticles by copper-free click chemistry <i>in vivo</i> during a second intravenous injection. The advantage of this protocol over traditional binding molecules is that there are significantly more binding molecules on the surface of most tumor cells regardless of cell type. The subsequent enhanced tumor-targeting ability can significantly enhance the cancer therapeutic efficacy in animal studies

Facile Method To Radiolabel Glycol Chitosan Nanoparticles with ⁶⁴Cu via Copper-Free Click Chemistry for MicroPET Imaging

An efficient and straightforward method for radiolabeling nanoparticles is urgently needed to understand the <i>in vivo</i> biodistribution of nanoparticles. Herein, we investigated a facile and highly efficient strategy to prepare radiolabeled glycol chitosan nanoparticles with ⁶⁴Cu via a strain-promoted azide–alkyne cycloaddition strategy, which is often referred to as click chemistry. First, the azide (N₃) group, which allows for the preparation of radiolabeled nanoparticles by copper-free click chemistry, was incorporated to glycol chitosan nanoparticles (CNPs). Second, the strained cyclooctyne derivative, dibenzyl cyclooctyne (DBCO) conjugated with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator, was synthesized for preparing the preradiolabeled alkyne complex with ⁶⁴Cu radionuclide. Following incubation with the ⁶⁴Cu-radiolabeled DBCO complex (DBCO-PEG₄-Lys-DOTA-⁶⁴Cu with high specific activity, 18.5 GBq/μmol), the azide-functionalized CNPs were radiolabeled successfully with ⁶⁴Cu, with a high radiolabeling efficiency and a high radiolabeling yield (>98%). Importantly, the radiolabeling of CNPs by copper-free click chemistry was accomplished within 30 min, with great efficiency in aqueous conditions. In addition, we found that the ⁶⁴Cu-radiolabeled CNPs (⁶⁴Cu-CNPs) did not show any significant effect on the physicochemical properties, such as size, zeta potential, or spherical morphology. After ⁶⁴Cu-CNPs were intravenously administered to tumor-bearing mice, the real-time, <i>in vivo</i> biodistribution and tumor-targeting ability of ⁶⁴Cu-CNPs were quantitatively evaluated by microPET images of tumor-bearing mice. These results demonstrate the benefit of copper-free click chemistry as a facile, preradiolabeling approach to conveniently radiolabel nanoparticles for evaluating the real-time <i>in vivo</i> biodistribution of nanoparticles