Modulation of the Cellular Uptake of DNA Origami through Control over Mass and Shape.

Designer nanoparticles with controlled shapes and sizes are increasingly popular vehicles for therapeutic delivery due to their enhanced cell-delivery performance. However, our ability to fashion nanoparticles has offered only limited control over these parameters. Structural DNA nanotechnology has an unparalleled ability to self-assemble three-dimensional nanostructures with near-atomic resolution features, and thus, it offers an attractive platform for the systematic exploration of the parameter space relevant to nanoparticle uptake by living cells. In this study, we examined the cell uptake of a panel of 11 distinct DNA-origami shapes, with the largest dimension ranging from 50-400 nm, in 3 different cell lines. We found that larger particles with a greater compactness were preferentially internalized compared with elongated, high-aspect-ratio particles. Uptake kinetics were also found to be more cell-type-dependent than shape-dependent, with specialized endocytosing dendritic cells failing to saturate over 12 h of study. The knowledge gained in the current study furthers our understanding of how particle shape affects cellular uptake and heralds the development of DNA nanotechnologies toward the improvement of current state-of-the-art cell-delivery vehicles.

Quantitative Imaging of Cerebral Thromboemboli In Vivo: The Effects of Tissue-Type Plasminogen Activator.

BACKGROUND AND PURPOSE: Quantitative imaging for the noninvasive assessment of thrombolysis is needed to advance basic and clinical thrombosis-related research and tailor tissue-type plasminogen activator (tPA) treatment for stroke patients. We quantified the evolution of cerebral thromboemboli using fibrin-targeted glycol chitosan-coated gold nanoparticles and microcomputed tomography, with/without tPA therapy. METHODS: We injected thrombi into the distal internal carotid artery in mice (n=50). Fifty-five minutes later, we injected fibrin-targeted glycol chitosan-coated gold nanoparticles, and 5 minutes after that, we treated animals with tPA or not (25 mg/kg). We acquired serial microcomputed tomography images for 24 hours posttreatment. RESULTS: Thrombus burden at baseline was 784×103±59×103 µm2 for the tPA group (n=42) and 655×103±103×103 µm2 for the saline group (n=8; P=0.37). Thrombus shrinkage began at 0.5 to 1 hour after tPA therapy, with a maximum initial rate of change at 4603±957 µm2/min. The rate of change lowered to ≈61% level of the initial in hours 1 to 2, followed by ≈29% and ≈1% in hours 2 to 3 and 3 to 24, respectively. Thus, 85% of total thrombolysis over 24 hours (≈500 µm2, equivalent to 64% of the baseline thrombus burden) occurred within the first 3 hours of treatment. Thrombus burden at 24 hours could be predicted at around 1.5 to 2 hours. Saline treatment was not associated with significant changes in the thrombus burden. Infarct size was smaller in the tPA group versus saline group (18.1±2.3 versus 45.8±3.3 mm2; P<0.01). Infarct size correlated to final thrombus burden (r=0.71; P<0.01). Time to thrombolysis, completeness of thrombolysis, and tPA therapy were independent predictors of infarct size. CONCLUSIONS: Thromboembolic burden and the efficacy of tPA therapy can be assessed serially, noninvasively, and quantitatively using high-resolution microcomputed tomography and a fibrin-binding nanoparticle imaging agent.

Bioorthogonal Copper Free Click Chemistry for Labeling and Tracking of Chondrocytes In Vivo.

Establishment of an appropriate cell labeling and tracking method is essential for the development of cell-based therapeutic strategies. Here, we are introducing a new method for cell labeling and tracking by combining metabolic gylcoengineering and bioorthogonal copper-free Click chemistry. First, chondrocytes were treated with tetraacetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz) to generate unnatural azide groups (-N3) on the surface of the cells. Subsequently, the unnatural azide groups on the cell surface were specifically conjugated with near-infrared fluorescent (NIRF) dye-tagged dibenzyl cyclooctyne (DBCO-650) through bioorthogonal copper-free Click chemistry. Importantly, DBCO-650-labeled chondrocytes presented strong NIRF signals with relatively low cytotoxicity and the amounts of azide groups and DBCO-650 could be easily controlled by feeding different amounts of Ac4ManNAz and DBCO-650 to the cell culture system. For the in vivo cell tracking, DBCO-650-labeled chondrocytes (1 × 10(6) cells) seeded on the 3D scaffold were subcutaneously implanted into mice and the transplanted DBCO-650-labeled chondrocytes could be effectively tracked in the prolonged time period of 4 weeks using NIRF imaging technology. Furthermore, this new cell labeling and tracking technology had minimal effect on cartilage formation in vivo.

In vivo monitoring of angiogenesis in a mouse hindlimb ischemia model using fluorescent peptide-based probes.

Vascular endothelial growth factor receptor (VEGFR) and matrix metalloproteinase (MMP) are up-regulated in ischemic tissue and play pivotal roles in promoting angiogenesis. The purpose of the present study was to evaluate two fluorophore-conjugated peptide probes specific to VEGFR and MMP for dual-targeted in vivo monitoring of angiogenesis in a murine model of hindlimb ischemia. To this end, VEGFR-Probe and MMP-Probe were developed by conjugating distinct near-infrared fluorophores to VEGFR-binding and MMP substrate peptides, respectively. VEGFR-Probe exhibited specific binding to VEGFR on HUVECs, and self-quenched MMP-Probe produced strong fluorescence intensity in the presence of MMPs in vitro. Subsequently, VEGFR-Probe and MMP-Probe were successfully utilized for time course in vivo visualization of VEGFR or MMP, respectively. Simultaneous visualization provided information regarding the spatial distribution of these proteins, including areas of co-localization. This dual-targeted in vivo imaging approach will be useful for understanding the detailed mechanism of angiogenesis and for evaluating therapeutic angiogenesis.

Cathepsin†B-Specific Metabolic Precursor for In†Vivo Tumor-Specific Fluorescence Imaging.

Recently, metabolic glycoengineering with bioorthogonal click reactions has focused on improving the tumor targeting efficiency of nanoparticles as delivery vehicles for anticancer drugs or imaging agents. It is the key technique for developing tumor-specific metabolic precursors that can generate unnatural glycans on the tumor-cell surface. A cathepsin B-specific cleavable substrate (KGRR) conjugated with triacetylated N-azidoacetyl-d-mannosamine (RR-S-Ac3 ManNAz) was developed to enable tumor cells to generate unnatural glycans that contain azide groups. The generation of azide groups on the tumor cell surface was exogenously and specifically controlled by the amount of RR-S-Ac3 ManNAz that was fed to target tumor cells. Moreover, unnatural glycans on the tumor cell surface were conjugated with near infrared fluorescence (NIRF) dye-labeled molecules by a bioorthogonal click reaction in cell cultures and in tumor-bearing mice. Therefore, our RR-S-Ac3 ManNAz is promising for research in tumor-specific imaging or drug delivery.

Tumor-homing glycol chitosan-based optical/PET dual imaging nanoprobe for cancer diagnosis.

Imaging techniques including computed tomography, magnetic resonance imaging, and positron emission tomography (PET) offer many potential benefits to diagnosis and treatment of cancers. Each method has its own strong and weak points. Therefore, multimodal imaging techniques have been highlighted as an alternative method for overcoming the limitations of each respective imaging method. In this study, we fabricated PET/optical activatable imaging probe based on glycol chitosan nanoparticles (CNPs) for multimodal imaging. To prepare the dual PET/optical probes based on CNPs, both (64)Cu radiolabeled DOTA complex and activatable matrix metalloproteinase (MMP)-sensitive peptide were chemically conjugated onto azide-functionalized CNPs via bio-orthogonal click chemistry, which was a reaction between azide group and dibenzyl cyclooctyne. The PET/optical activatable imaging probes were visualized by PET and optical imaging system. Biodistribution of probes and activity of MMP were successfully measured in tumor-bearing mice.

Advancing cancer immunotherapy through siRNA-based gene silencing for immune checkpoint blockade.

Cancer immunotherapy represents a revolutionary strategy, leveraging the patient's immune system to inhibit tumor growth and alleviate the immunosuppressive effects of the tumor microenvironment (TME). The recent emergence of immune checkpoint blockade (ICB) therapies, particularly following the first approval of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors like ipilimumab, has led to significant growth in cancer immunotherapy. The extensive explorations on diverse immune checkpoint antibodies have broadened the therapeutic scope for various malignancies. However, the clinical response to these antibody-based ICB therapies remains limited, with less than 15% responsiveness and notable adverse effects in some patients. This review introduces the emerging strategies to overcome current limitations of antibody-based ICB therapies, mainly focusing on the development of small interfering ribonucleic acid (siRNA)-based ICB therapies and innovative delivery systems. We firstly highlight the diverse target immune checkpoint genes for siRNA-based ICB therapies, incorporating silencing of multiple genes to boost anti-tumor immune responses. Subsequently, we discuss improvements in siRNA delivery systems, enhanced by various nanocarriers, aimed at overcoming siRNA's clinical challenges such as vulnerability to enzymatic degradation, inadequate pharmacokinetics, and possible unintended target interactions. Additionally, the review presents various combination therapies that integrate chemotherapy, phototherapy, stimulatory checkpoints, ICB antibodies, and cancer vaccines. The important point is that when used in combination with siRNA-based ICB therapy, the synergistic effect of traditional therapies is strengthened, improving host immune surveillance and therapeutic outcomes. Conclusively, we discuss the insights into innovative and effective cancer immunotherapeutic strategies based on RNA interference (RNAi) technology utilizing siRNA and nanocarriers as a novel approach in ICB cancer immunotherapy.

Applications of Cu2+-Loaded Silica Nanoparticles to Photothermal Therapy and Tumor-Specific Fluorescence Imaging.

Copper-based nanomaterials have been employed as therapeutic agents for cancer therapy and diagnosis. Nevertheless, persistent challenges, such as cellular toxicity, non-uniform sizes, and low photothermal efficiency, often constrain their applications. In this study, we present Cu2+-loaded silica nanoparticles fabricated through the chelation of Cu2+ ions by silanol groups. The integration of Cu2+ ions into uniformly sized silica nanoparticles imparts a photothermal therapy effect. Additionally, the amine functionalization of the silica coating facilitates the chemical conjugation of tumor-specific fluorescence probes. These probes are strategically designed to remain in an 'off' state through the Förster resonance energy transfer mechanism until exposed to cysteine enzymes in cancer cells, inducing the recovery of their fluorescence. Consequently, our Cu2+-loaded silica nanoparticles demonstrate an efficient photothermal therapy effect and selectively enable cancer imaging.

DNA origami vaccine (DoriVac) nanoparticles improve both humoral and cellular immune responses to infectious diseases.

Current SARS-CoV-2 vaccines have demonstrated robust induction of neutralizing antibodies and CD4+ T cell activation, however CD8+ responses are variable, and the duration of immunity and protection against variants are limited. Here we repurposed our DNA origami vaccine platform, DoriVac, for targeting infectious viruses, namely SARS-CoV-2, HIV, and Ebola. The DNA origami nanoparticle, conjugated with infectious-disease-specific HR2 peptides, which act as highly conserved antigens, and CpG adjuvant at precise nanoscale spacing, induced neutralizing antibodies, Th1 CD4+ T cells, and CD8+ T cells in naïve mice, with significant improvement over a bolus control. Pre-clinical studies using lymph-node-on-a-chip systems validated that DoriVac, when conjugated with antigenic peptides or proteins, induced promising cellular immune responses in human cells. These results suggest that DoriVac holds potential as a versatile, modular vaccine platform, capable of inducing both humoral and cellular immunities. The programmability of this platform underscores its potential utility in addressing future pandemics.

Fine tuning of CpG spatial distribution with DNA origami for improved cancer vaccination.

Multivalent presentation of ligands often enhances receptor activation and downstream signalling. DNA origami offers a precise nanoscale spacing of ligands, a potentially useful feature for therapeutic nanoparticles. Here we use a square-block DNA origami platform to explore the importance of the spacing of CpG oligonucleotides. CpG engages Toll-like receptors and therefore acts to activate dendritic cells. Through in vitro cell culture studies and in vivo tumour treatment models, we demonstrate that square blocks induce Th1 immune polarization when CpG is spaced at 3.5 nm. We observe that this DNA origami vaccine enhances DC activation, antigen cross-presentation, CD8 T-cell activation, Th1-polarized CD4 activation and natural-killer-cell activation. The vaccine also effectively synergizes with anti-PD-L1 for improved cancer immunotherapy in melanoma and lymphoma models and induces long-term T-cell memory. Our results suggest that DNA origami may serve as a platform for controlling adjuvant spacing and co-delivering antigens in vaccines.

Rapid and sensitive LC-MS/MS method for determination of megestrol acetate in human plasma: application to a human pharmacokinetic study.

A rapid, simple and fully validated LC-MS/MS method was developed and validated for the determination of megestrol acetate in human plasma using tolbutamide as an internal standard (IS) after one-step liquid-liquid extraction with methyl-tert-butyl-ether. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions m/z 385.5 → 267.1 for megestrol acetate and m/z 271.4 → 155.1 for IS. Chromatographic separation was performed on a YMC Hydrosphere C18 column with an isocratic mobile phase, which consisted of 10 mm ammonium formate buffer (adjusted to pH 5.0 with formic acid)-methanol (60:40, v/v) at a flow rate of 0.4 mL/min. The achieved lower limit of quantitation (LLOQ) was 1 ng/mL (signal-to-noise ratio > 10) and the standard calibration curve for megestrol acetate was linear (r > 0.99) over the studied concentration range (1-2000 ng/mL). The proposed method was fully validated by determining its specificity, linearity, LLOQ, intra- and inter-day precision and accuracy, recovery, matrix effect and stability. The validated LC-MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of megestrol acetate after oral administration of a single dose 800 mg of megestrol acetate (Megace™) to five healthy Korean male volunteers under fed conditions.

Multifunctional nanoparticles for multimodal imaging and theragnosis.

Nanomedicine is the biomedical application of nanoscale materials for diagnosis and therapy of disease. Recent advances in nanotechnology and biotechnology have contributed to the development of multifunctional nanoparticles as representative nanomedicine. They were initially developed to enable the target-specific delivery of imaging or therapeutic agents for biomedical applications. Due to their unique features including multifunctionality, large surface area, structural diversity, and long circulation time in blood compared to small molecules, nanoparticles have emerged as attractive preferences for optimized therapy through personalized medicine. Multimodal imaging and theragnosis are the cutting-edge technologies where the advantages of nanoparticles are maximized. Because each imaging modality has its pros and cons, the integration of several imaging agents with different properties into multifunctional nanoparticles allows precise and fast diagnosis of disease through synergetic multimodal imaging. Moreover, nanoparticles are not only used for molecular imaging but also applied to deliver therapeutic agents to the disease site in order to accomplish the simultaneous imaging and therapy called theragnosis. This tutorial review will highlight the recent advances in the development of multifunctional nanoparticles and their biomedical applications to multimodal imaging and theragnosis as nanomedicine.

Controlled spatial characteristics of ligands on nanoparticles: Determinant of cellular functions.

Interactions of various ligands and receptors have been extensively investigated because they regulate a series of signal transduction leading to various functional cellular outcomes. The receptors on cell membrane recognize their specific ligands, resulting in specific binding between ligands and receptors. Accumulating evidence reveals that the receptors recognize the difference on the spatial characteristics of ligands as well as the types of ligands. Thus, control on spatial characteristics of multiple ligands presented on therapeutic nanoparticles is believed to impact the cellular functions. Specifically, the localized and multivalent distribution of ligands on nanoparticles can induce receptor oligomerization and receptor clustering, controlling intensity or direction of signal transduction cascades. Here, we will introduce recent studies on the use of material-based nanotechnology to control spatial characteristics of ligands and their effect on cellular functions. These therapeutic nanoparticles with controlled spatial characteristics of ligands may be a promising strategy for maximized therapeutic outcome.

Predicting response to anti-EGFR antibody, cetuximab, therapy by monitoring receptor internalization and degradation.

Anti-epidermal growth factor receptor (EGFR) antibody, cetuximab, therapy has significantly improved the clinical outcomes of patients with colorectal cancer, but the response to cetuximab can vary widely among individuals. We thus need strategies for predicting the response to this therapy. However, the current methods are unsatisfactory in their predictive power. Cetuximab can promote the internalization and degradation of EGFR, and its therapeutic efficacy is significantly correlated with the degree of EGFR degradation. Here, we present a new approach to predict the response to anti-EGFR therapy, cetuximab by evaluating the degree of EGFR internalization and degradation of colorectal cancer cells in vitro and in vivo. Our newly developed fluorogenic cetuximab-conjugated probe (Cetux-probe) was confirmed to undergo EGFR binding, internalization, and lysosomal degradation to yield fluorescence activation; it thus shares the action mechanism by which cetuximab exerts its anti-tumor effects. Cetux-probe-activated fluorescence could be used to gauge EGFR degradation and showed a strong linear correlation with the cytotoxicity of cetuximab in colorectal cancer cells and tumor-bearing mice. The predictive ability of Cetux-probe-activated fluorescence was much higher than those of EGFR expression or KRAS mutation status. The Cetux-probes may become useful tools for predicting the response to cetuximab therapy by assessing EGFR degradation.

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.

Therapeutic effect of NLRP3 inhibition on hearing loss induced by systemic inflammation in a CAPS-associated mouse model.

BACKGROUND: Cryopyrin-associated periodic syndrome (CAPS) is an inherited autoinflammatory disease caused by a gain-of-function mutation in NLRP3. Although CAPS patients frequently suffer from sensorineural hearing loss, it remains unclear whether CAPS-associated mutation in NLRP3 is associated with the progression of hearing loss. METHODS: We generated a mice with conditional expression of CAPS-associated NLRP3 mutant (D301N) in cochlea-resident CX3CR1 macrophages and examined the susceptibility of CAPS mice to inflammation-mediated hearing loss in a local and systemic inflammation context. FINDINGS: Upon lipopolysaccharide (LPS) injection into middle ear cavity, NLRP3 mutant mice exhibited severe cochlear inflammation, inflammasome activation and hearing loss. However, this middle ear injection model induced a considerable hearing loss in control mice and inevitably caused an inflammation-independent hearing loss possibly due to ear tissue damages by injection procedure. Subsequently, we optimized a systemic LPS injection model, which induced a significant hearing loss in NLRP3 mutant mice but not in control mice. Peripheral inflammation induced by a repetitive low dose of LPS injection caused a blood-labyrinth barrier disruption, macrophage infiltration into cochlea and cochlear inflammasome activation in an NLRP3-dependent manner. Interestingly, both cochlea-infiltrating and -resident macrophages contribute to peripheral inflammation-mediated hearing loss of CAPS mice. Furthermore, NLRP3-specific inhibitor, MCC950, as well as an interleukin-1 receptor antagonist significantly alleviated systemic LPS-induced hearing loss and inflammatory phenotypes in NLRP3 mutant mice. INTERPRETATION: Our findings reveal that CAPS-associated NLRP3 mutation is critical for peripheral inflammation-induced hearing loss in our CAPS mice model, and an NLRP3-specific inhibitor can be used to treat inflammation-mediated sensorineural hearing loss. FUNDING: National Research Foundation of Korea Grant funded by the Korean Government and the Team Science Award of Yonsei University College of Medicine.

Direct thrombus imaging as a means to control the variability of mouse embolic infarct models: the role of optical molecular imaging.

BACKGROUND AND PURPOSE: High experimental variability in mouse embolic stroke models could mask the effects of experimental treatments. We hypothesized that imaging thrombus directly would allow this variability to be controlled. METHODS: We optically labeled thrombi with a near-infrared fluorescent (NIRF) probe C15 that is covalently linked to fibrin by factor-XIIIa. Labeled thrombus was injected into the left distal internal carotid artery (ICA) of C57/BL6 mice (n=47), near its bifurcation, and laser-Doppler cerebral-blood-flow (CBF) was assessed for 30 minutes. NIRF thrombus imaging was done ex vivo at 24 hours. RESULTS: CBF variably decreased to 43.9±17.3% at 5 minutes (rCBF; 11.2∼80.4%). NIRF thrombus imaging at 24 hours showed variability in distribution (ICA bifurcation, adjacent and/or remote areas) and burden (2279±1270 pixels; 0∼5940 pixels). Final infarct size was also variable (21.0±10.3%; 4.7∼60.3% of the bihemispheric volume). Despite this heterogeneity, a strong thrombus-infarct correlation was maintained. The left hemispheric target infarct size (% of the hemisphere) correlated with thrombus burden, as a stronger predictor of infarct volume (P<0.001, r=0.50) than rCBF (P=0.02, r=-0.34). The infarct size was best predicted by a combination of thrombus imaging and CBF: left-hemispheric big-thrombi (>1865 pixels)/low-rCBF (≤42%) had an infarct volume of 56.9±10.4% (n=12), big-thrombi/high-rCBF had 45.9±23.5% (n=11), small-thrombi/low-rCBF 35.7±17.3% (n=11) and small-thrombi/ high-rCBF 27.3±16.4% (n=12). CONCLUSIONS: This is the first study to demonstrate that the highly heterogeneous nature of the mouse embolic stroke model can be characterized and managed by using near-infrared fluorescent thrombus imaging combined with CBF monitoring to stratify animals into useful subgroups.

Real time, high resolution video imaging of apoptosis in single cells with a polymeric nanoprobe.

We report a new apoptosis nanoprobe (Apo-NP) designed on the basis of a polymer nanoparticle platform. This simple one-step technique is capable of boosting fluorescence signals upon apoptosis in living cells, enabling real-time imaging of apoptosis in single cells and in vivo. The Apo-NP efficiently delivers chemically labeled, dual-quenched caspase-3-sensitive fluorogenic peptides into cells, allowing caspase-3-dependent strong fluorescence amplification to be imaged in apoptotic cells in real-time and at high resolution. The design platform of the Apo-NP is flexible and can be fine-tuned for a wide array of applications such as identification of caspase-related apoptosis in pathologies and for monitoring therapeutic efficacy of apoptotic drugs in cancer treatment.

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.

Application of near-infrared fluorescence imaging using a polymeric nanoparticle-based probe for the diagnosis and therapeutic monitoring of colon cancer.

BACKGROUND: Early and accurate detection of adenomatous colonic polyps is a major concern in the prevention of colon cancer. Near-infrared fluorescence (NIRF) imaging with optical probes targeting specific peptides enables the noninvasive visualization and characterization of lesions. Matrix metalloproteinases (MMPs) are known to play an important role in tumorigenesis and tumor progression. AIM: To investigate the effectiveness of NIRF imaging, with a novel MMP-activatable probe based on a polymeric nanoparticle platform, in the colon cancer models. METHODS: We used an azoxymethane (AOM)-induced mouse colon cancer model resembling human sporadic colon cancer and an MMP-positive xenograft tumor model. MMP expression was evaluated by Western blotting, real-time PCR, and immunohistochemical staining. NIRF imaging was performed with a novel MMP-activatable probe, an MMP-inactivatable probe, and saline. In addition, we observed the change of NIRF signal intensity after intratumoral administration of an MMP-inhibitor. RESULTS: Multiple tumors with various sizes developed in AOM-treated mouse colons, progressing from adenomas to adenocarcinomas, with MMP expression progressively increasing in the normal-adenoma-adenocarcinoma sequence. In mice injected with the MMP-activatable probe, the NIRF signal also increased in this sequence and was highly correlated with MMP expression (p < 0.001). Tumor-background-ratios (TBR) of adenocarcinoma to adjacent normal mucosa by a novel probe were significantly higher than that of adenoma (p < 0.001). In both the AOM and xenograft models, NIRF signals of tumors decreased after treatment with an MMP-inhibitor. CONCLUSIONS: NIRF imaging using a polymeric nanoparticle-based probe may be useful for detecting early stage disease and for assessing treatment response.