A Promising Biocompatible Platform: Lipid-Based and Bio-Inspired Smart Drug Delivery Systems for Cancer Therapy.

Designing new drug delivery systems (DDSs) for safer cancer therapy during pre-clinical and clinical applications still constitutes a considerable challenge, despite advances made in related fields. Lipid-based drug delivery systems (LBDDSs) have emerged as biocompatible candidates that overcome many biological obstacles. In particular, a combination of the merits of lipid carriers and functional polymers has maximized drug delivery efficiency. Functionalization of LBDDSs enables the accumulation of anti-cancer drugs at target destinations, which means they are more effective at controlled drug release in tumor microenvironments (TMEs). This review highlights the various types of ligands used to achieve tumor-specific delivery and discusses the strategies used to achieve the effective release of drugs in TMEs and not into healthy tissues. Moreover, innovative recent designs of LBDDSs are also described. These smart systems offer great potential for more advanced cancer therapies that address the challenges posed in this research area.

Spherical polymeric nanoconstructs for combined chemotherapeutic and anti-inflammatory therapies.

Nanoparticles can simultaneously deliver multiple agents to cancerous lesions enabling de facto combination therapies. Here, spherical polymeric nanoconstructs (SPNs) are loaded with anti-cancer - docetaxel (DTXL) - and anti-inflammatory - diclofenac (DICL) - molecules. In vitro, combination SPNs kill U87-MG cells twice as efficiently as DTXL SPNs, achieving a IC50 of 90.5nM at 72h. Isobologram analysis confirms a significant synergy (CI=0.56) between DTXL and DICL. In mice bearing non-orthotopic glioblastoma multiforme tumors, combination SPNs demonstrate higher inhibition in disease progression. At 70days post treatment, the survival rate of mice treated with combination SPNs is of about 70%, against a 40% for DTXL SPNs and 0% for free DTXL. Combination SPNs dramatically inhibit COX-2 expression, modulating the local inflammatory status, and increase Caspase-3 expression, which is directly related to cell death. These results suggest that the combination of anti-cancer and anti-inflammatory molecules constitutes a potent strategy for inhibiting tumor growth.

TNF-α gene silencing using polymerized siRNA/thiolated glycol chitosan nanoparticles for rheumatoid arthritis.

Among various proinflammatory cytokines involved in the pathogenesis of rheumatoid arthritis (RA), tumor necrosis factor (TNF)-α plays a pivotal role in the release of other cytokines and induction of chronic inflammation. Even though siRNA has the therapeutic potential, they have a challenge to be delivered into the target cells because of their poor stability in physiological fluids. Herein, we design a nanocomplex of polymerized siRNA (poly-siRNA) targeting TNF-α with thiolated glycol chitosan (tGC) polymers for the treatment of RA. Poly-siRNA is prepared through self-polymerization of thiol groups at the 5' end of sense and antisense strand of siRNA and encapsulated into tGC polymers, resulting in poly-siRNA-tGC nanoparticles (psi-tGC-NPs) with an average diameter of 370 nm. In the macrophage culture system, psi-tGC-NPs exhibit rapid cellular uptake and excellent in vitro TNF-α gene silencing efficacy. Importantly, psi-tGC-NPs show the high accumulation at the arthritic joint sites in collagen-induced arthritis (CIA) mice. Treatment monitoring data obtained by the matrix metalloproteinase 3-specific nanoprobe and microcomputed tomography show that intravenous injection of psi-tGC-NPs significantly inhibits inflammation and bone erosion in CIA mice, comparable to methotrexate (5 mg/kg). Therefore, the availability of psi-tGC-NP therapy that target specific cytokines may herald new era in the treatment of RA.

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.

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.

The effects of a minimally invasive laser needle system on complete Freund's adjuvant-induced arthritis.

The present study aimed to investigate the effects of a minimally invasive laser needle system (MILNS) on the acute progression of arthritis. Previous studies showed controversial clinical results regarding the effects of low-level laser therapy on arthritis, with the outcomes depending upon stimulation parameters such as laser wavelength and dosage. Based on the positive effects of MILNS on osteoporotic mice, we hypothesized that MILNS could potentially suppress the progression of arthritis owing to its biostimulation effects. Eight C57BL/6 mice with complete Freund's adjuvant (CFA)-induced arthritis were used as acute progression arthritis models and divided into the laser and control groups (n = 4 each). In the laser group, after minimally invasive laser stimulation, laser speckle contrast images (LSCIs) were obtained every 6 h for a total of 108 h. The LSCIs in the control group were obtained without laser stimulation. The effects of MILNS on the acute progression of arthritis were indirectly evaluated by calculating the paw area and the average laser speckle index (LSI) at the arthritis-induced area. Moreover, the macrophage population was estimated in the arthritis-induced area. Compared to the control group, the laser group showed (1) lower relative variations of the paw area, (2) lower average LSI in the arthritis-induced area, and (3) lower macrophage population in the arthritis-induced area. These results indicate that MILNS may suppress the acute progression of CFA-induced arthritis in mice and may thus be used as a potential treatment modality of arthritis in clinics.

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.

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.

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.

"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.

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.

Anti-EGF Receptor Aptamer-Guided Co-Delivery of Anti-Cancer siRNAs and Quantum Dots for Theranostics of Triple-Negative Breast Cancer.

Many aptamers have been evaluated for their ability as drug delivery vehicles to target ligands, and a variety of small interfering RNAs (siRNAs) have been tested for their anti-cancer properties. However, since these two types of molecules have similar physicochemical properties, it has so far been difficult to formulate siRNA-encapsulating carriers guided by aptamers. Here, we propose aptamer-coupled lipid nanocarriers encapsulating quantum dots (QDs) and siRNAs for theragnosis of triple-negative breast cancer (TNBC). Methods: Hydrophobic QDs were effectively incorporated into lipid bilayers, and then therapeutic siRNAs were complexed with QD-lipid nanocarriers (QLs). Finally, anti-EGFR aptamer-lipid conjugates were inserted into the QLs for TNBC targeting (aptamo-QLs). TNBC-targeting aptamo-QLs were directly compared to anti-EGFR antibody-coupled immuno-QLs. The in vitro delivery of therapeutic siRNAs and QDs to target cells was assessed by flow cytometry and confocal microscopy. The in vivo targeting of siRNAs to tumors and their therapeutic efficacy were evaluated in mice carrying MDA-MB-231 tumors. Results: Both types of EGFR-targeting QLs showed enhanced delivery to target cancer cells, resulting in more effective gene silencing and enhanced tumor imaging compared to non-targeting control QLs. Moreover, combinatorial therapy with Bcl-2 and PKC-ι siRNAs loaded into the anti-EGFR QLs was remarkably effective in inhibiting tumor growth and metastasis. Conclusion: In general, the aptamo-QLs showed competitive in vivo delivery and therapeutic efficacy compared to immuno-QLs under the same experimental conditions. Our results show that the anti-EGFR aptamer-guided lipid carriers may be a potential theranostic delivery vehicle for RNA interference and fluorescence imaging of TNBCs.

Visualization of MMP-2 Activity Using Dual-Probe Nanoparticles to Detect Potential Metastatic Cancer Cells.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes capable of degrading extracellular matrix components. Previous studies have shown that the upregulation of MMP-2 is closely related to metastatic cancers. While Western blotting, zymography, and Enzyme-Linked Immunosorbent Assays (ELISA) can be used to measure the amount of MMP-2 activity, it is not possible to visualize the dynamic MMP-2 activities of cancer cells using these techniques. In this study, MMP-2-activated poly(lactic-co-glycolic acid) with polyethylenimine (MMP-2-PLGA-PEI) nanoparticles were developed to visualize time-dependent MMP-2 activities. The MMP-2-PLGA-PEI nanoparticles contain MMP-2-activated probes that were detectable via fluorescence microscopy only in the presence of MMP-2 activity, while the Rhodamine-based probes in the nanoparticles were used to continuously visualize the location of the nanoparticles. This approach allowed us to visualize MMP-2 activities in cancer cells and their microenvironment. Our results showed that the MMP-2-PLGA-PEI nanoparticles were able to distinguish between MMP-2-positive (HaCat) and MMP-2-negative (MCF-7) cells. While the MMP-2-PLGA-PEI nanoparticles gave fluorescent signals recovered by active recombinant MMP-2, there was no signal recovery in the presence of an MMP-2 inhibitor. In conclusion, MMP-2-PLGA-PEI nanoparticles are an effective tool to visualize dynamic MMP-2 activities of potential metastatic cancer cells.

Lanthanide-Doped Nanoparticles for Diagnostic Sensing.

Lanthanide-doped nanoparticles exhibit unique optical properties, such as a long luminescence lifetime (up to several milliseconds), sharp emission peaks, and upconversion luminescence over the range of wavelengths from near-infrared to visible. Exploiting these optical properties, lanthanide-doped nanoparticles have been widely utilized for cellular and small animal imaging with the absence of background autofluorescence. In addition, these nanoparticles have advantages of high signal-to-noise ratio for highly sensitive and selective diagnostic detection. In this review, we summarize and discuss recent progress in the development of highly sensitive diagnostic methods using lanthanide-doped nanoparticles. Combined with a smartphone, portable luminescence detecting platforms could be widely applied in point-of-care tests.

Ameliorating Amyloid-ß Fibrils Triggered Inflammation via Curcumin-Loaded Polymeric Nanoconstructs.

Inflammation is a common hallmark in several diseases, including atherosclerosis, cancer, obesity, and neurodegeneration. In Alzheimer's disease (AD), growing evidence directly correlates neuronal damage with inflammation of myeloid brain cells, such as microglia. Here, polymeric nanoparticles were engineered and characterized for the delivery of anti-inflammatory molecules to macrophages stimulated via direct incubation with amyloid-ß fibers. 200 nm spherical polymeric nanoconstructs (SPNs) and 1,000 nm discoidal polymeric nanoconstructs (DPNs) were synthesized using poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), and lipid chains as building blocks. First, the internalization propensity in macrophages of both nanoparticles was assessed via cytofluorimetric and confocal microscopy analyses, demonstrating that SPNs are by far more rapidly taken up as compared to DPNs (99.6 ± 0.11 vs 14.4 ± 0.06%, within 24 h). Then, Curcumin-loaded SPNs (Curc-SPNs) were realized by encapsulating Curcumin, a natural anti-inflammatory molecule, within the PLGA core of SPNs. Finally, Curc-SPNs were shown to diminish up to 6.5-fold the production of pro-inflammatory cytokines-IL-1ß; IL-6, and TNF-α-in macrophages stimulated via amyloid-ß fibers. Although more sophisticated in vitro models and systematic analyses on the blood-brain barrier permeability are critically needed, these findings hold potential in the development of nanoparticles for modulating inflammation in AD.

Stat3 inhibitor abrogates the expression of PD-1 ligands on lymphoma cell lines.

Recent studies have indicated the significance of immune checkpoint molecules including programmed death-1 (PD-1), cytotoxic T-lymphocyte associated protein 4, and T-cell immunoglobulin and mucin domain-containing molecule-3 for anti-tumor immune responses. We previously investigated PD-1 ligand 1/2 (PD-L1/2) expression in lymphoma cell lines, and found that PD-L1/2 is expressed on the adult T-cell leukemia/lymphoma (ATL-T) and B-cell lymphoma (SLVL) cell lines. In the present study, we investigated whether the Stat3 inhibitor WP1066 abrogated PD-L1/2 expression in lymphoma cell lines. Incubation with WP1066 inhibited lymphoma cell growth and induced cell apoptosis. PD-L1/2 expression in the ATL-T, SLVL, and human brain malignant lymphoma (HKBML) cell lines was significantly abrogated by WP1066 treatment. These data indicated that a Stat3 inhibitor abrogated PD-L1/2 expression in lymphoma cells. Such an inhibitor is therefore considered to be useful for additional immunotherapy in patients with advanced lymphoma.

Dexamethasone-loaded Polymeric Nanoconstructs for Monitoring and Treating Inflammatory Bowel Disease.

Corticosteroids, such as dexamethasone (DEX), are the mainstays for the treatment of moderate to severe inflammatory bowel disease (IBD). However, their relatively poor bioavailability and lack of specificity is often the origin of short and long-term adverse effects. Here, spherical polymeric nanoconstructs (SPNs) encapsulating dexamethasone are proposed for the systemic treatment of IBD. In a mouse model of colitis, the accumulation of SPNs within the inflamed intestine is firstly assessed using near infra-red fluorescent (NIRF) imaging at different stages of the disease - 5, 7 and 10 days of Dextran Sulfate Sodium (DSS) administration. Then, the efficacy of DEX-SPNs is tested in vitro over macrophages and in vivo by monitoring the animal weight, food and water intake; expression of inflammatory cytokines (TNF-α, IL-1ß, IL-6); intestinal density of macrophages; rectal bleeding and histological scoring. 150 nm DEX-SPNs are shown to deposit within the hyper-permeable inflamed intestine in a disease severity-dependent fashion. DEX-SPNs exposed to LPS-stimulated RAW 264.7 cells reduce the expression of inflammatory cytokines as rapidly as free DEX. In DSS-administered mice, DEX-SPNs treatments improve weight loss, reduce the macrophage infiltration, expression of inflammatory cytokines, rectal bleeding and histological scoring, as compared to free DEX. Moreover, DEX-SPNs exert a strong systemic anti-inflammatory effect and facilitate animal recovery. This work confirms the benefits of using sufficiently small nanoconstructs for targeting inflamed, hyper-permeable tissues and efficiently delivering high doses of corticosteroids for the treatment of intestinal and systemic inflammation.

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.

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.