Near-infrared Fluorescence Enhancement Research Articles

(Invited) Quantitative in Vitro/In Vivo Fluorescence Bioimaging with Graphene Quantum Dots

Graphene Quantum Dots (GQDs) are novel carbon nanomaterials consisting of few layers of partially functionalized graphitic carbon. Their nanometer-scale size together with functional groups render GQDs more water soluble and biocompatible than many other nanocarbon constructs. Like many nanocarbons, GQDs possess structure-dependent optical properties, exhibiting confinement-defined fluorescence in the visible and defect-originated emission in the near-infrared (NIR). These properties give GQDs a certain advantage for applications in therapeutic tracking, imaging, analyte detection and targeted drug/gene delivery. In addition to tracing the location of therapeutic or analyte, GOD fluorescence allows for several modes of quantitative assessment within these applications. In vitro it is utilized to evaluate relative amounts of therapeutics or analytes present within the cells at a certain time period. In vivo NIR emission of the GQDs-based sensors implanted under the skin can help detect trace quantities of analyte in the body, while their NIR fluorescence in animal organs can quantify relative therapeutic uptake over time.The present work introduces novel studies and analytical tools exploring and enhancing such imaging modalities. Although GQD in vitro fluorescence microscopy imaging is often used to quantitate their (and payload) uptake and excretion, this method suffers from multiple discrepancies including the analysis of dead cells, cellular debris and non-internalized nanomaterials, and can be also time and resource-consuming. Our work aiming to circumvent these issues utilizes Artificial Intelligence (AI) approach to provide more accurate quantification of the uptake/excretion of the GQDs and their payload. In this application the AI algorithms developed in house do not only optimize the imaging, but also provide automated image analysis streamlining the assessment of a variety of GQD biomedical applications including therapeutic delivery and targeting. In vivo quantitative animal imaging with several different types of GQDs, from pristine to doped with rare earth metals for NIR fluorescence enhancement, is performed to evaluate GQD organ accumulation over time. Defect or dopant-originated NIR GQD fluorescence in the range of 900 – 1060 nm due to its high penetration depth can be observed from the organs of sedated live mice and utilized for GQD uptake tracking. NIR imaging of the excised organs allows for quantitative biodistribution assessment with further microscopic confirmation of the GQD content in organ tissue slices. Quantitative optical imaging and analysis presented in this work for GQDs can aid the further advancement of these novel nanomaterials in therapeutic delivery and analyte detection applications in vitro and in vivo.

A novel aminopeptidase N triggered near-infrared fluorescence probe for imaging enzyme activity in cells and mice

Aminopeptidase N (APN) is an important proteinase, and the abnormal level of APN is closely related to many diseases. Thus, the specific detection and tracking of APN activity is of great significance in the fields of clinical diagnosis and drug development. Herein, we designed and synthesized an APN-activated near-infrared (NIR) fluorescent probe ZY-APN and successfully applied it for in vitro and in vivo enzyme detection. Linking L-alanine recognition moiety to the xanthene dye through amide bond endowed ZY-APN with specific response towards APN without being influenced by other bio-species. After reaction with APN, the probe exhibited a notable NIR fluorescence enhancement at 730 nm. With this probe, we could distinguish between normal cells and tumor cells due to the differences of APN activity. Besides, we have realized the visualization of high expression of APN in tumor-bearing mice. Hence, probe ZY-APN may display potential application in the diagnosis and treatment of APN-related diseases, especially cancer.

A near-infrared emitting “off-on” fluorescent probe for bioimaging of Pd(Ⅱ) ions in living cells and mice

BackgroundThe surging consumption of palladium in modern industry has given rise to its accumulation in the ecosystem, posing conspicuous toxicity to aquatic organisms and human health. The investigation of palladium in biological systems is highly demanded for the in-depth understanding of its dynamics and behaviors. Fluorescence imaging serves as a powerful approach to assess palladium species in biological systems, and currently most of the sensing probes are applicable to living cells. Effective tracking of palladium species in living organisms is challenging, which requires sufficient hydrophilicity and imaging depth of the probes. ResultsBased on an intramolecular charge transfer (ICT) mechanism, a distyryl boron dipyrromethene (BODIPY) derivative (DISBDP-Pd) has been prepared for the near-infrared (NIR) fluorescence imaging of Pd2+ ions. Two additional methoxy triethylene glycol (TEG) chains could serve as flexible and hydrophilic moieties to enhance the aqueous solubility and cell permeability of the extended conjugate. Solution studies revealed that DISBDP-Pd exhibited a NIR fluorescence enhancement signal exclusively to Pd2+ ions (detection limit as low as 0.85 ppb) with negligible interference from Pd0 species and other closely related metal ions. Computational calculations have been performed to rationalize the binding mode and the mechanism of action. Fluorescence imaging assays have been conducted on A549 human non-small cell lung carcinoma cells and mouse models. Exhibiting negligible cytotoxicity, DISBDP-Pd demonstrated concentration-related fluorescence enhancement signals in response to Pd2+ ions in living cells and mice. SignificanceDISBDP-Pd exhibits advantages over many small molecule palladium probes in terms of satisfactory aqueous solubility, high sensitivity and selectivity, and biocompatible NIR emission property, which are particularly favorable for the sensing application in biological environments. The design strategy of this probe can potentially be adopted for the functionalization of other BODIPY probes implemented for NIR fluorescence bioimaging.

A near-infrared fluorescence enhancement strategy of amorphous silicon nanoparticles for night vision imaging and visualizing latent fingerprints

A broadband near-infrared fluorescence enhancement strategy and night vision imaging and potential fingerprint recognition of amorphous silicon nanoparticles.

Near-Infrared Fluorescence Enhancement by Deuteration of Phthalocyanine.

Organic compounds with near-IR (NIR) fluorescence have many potential applications in materials and life sciences, but the much weaker intensity of fluorescence in the NIR region than in the UV-visible region is a major obstacle. Herein we show that deuteration of phthalocyanines, a representative class of organic NIR dyes, increases both the fluorescence quantum yield and the fluorescence lifetime compared with non-deuterated phthalocyanines.

Novel Near‐Infrared Fluorescent Nanoprobe Synthesized by the RAFT‐Mediated PISA Strategy for Hypoxia‐Triggered Tumor Imaging and Azoreductase‐Responsive Drug Release

AbstractDrug carriers and visual tracking play an important role in accurate and efficient drug delivery systems for targeted drug therapy. Polymerization‐induced self‐assembly (PISA), a powerful strategy for the fabrication of stimuli‐responsive polymeric nanoparticles has been a focus over the past decade due to its promising applications in biomedical fields such as targeted drug delivery and fluorescent nanoprobes used for tumor imaging. Herein, hypoxia‐responsive near‐infrared (NIR) fluorescent nanoprobes of amphiphilic block copolymers (PPEGMA14‐ADP‐Azo‐PBzMAx) are reported, the first example synthesized by RAFT‐mediated PISA strategy in situ to realize synchronous drug release and cell imaging. First, a series of nanoparticles with different morphologies and sizes, including spheres and vesicles, are synthesized by the PISA strategy using PPEGMA14‐ADP‐Azo‐CDPA as macro‐RAFT agents link with near‐infrared Aza‐BODIPY fluorogen and azobenzene groups. Hypoxia‐triggered dissociation of the nanoprobes and NIR fluorescence emission is then observed because of the cleavage of azo bonds. Meanwhile, the in situ DOX‐loaded nanoprobes by the PISA demonstrate synchronous drug release and NIR fluorescence enhancement against mouse colon cancer cells (CT26) under hypoxia (1% O2) compared with under normoxia (16% O2).

Plasmon-induced near-infrared fluorescence enhancement of single-walled carbon nanotubes

Single-walled carbon nanotubes (SWCNTs) emit near-infrared (NIR) fluorescence that is ideal for optical sensing. However, the low quantum yields diminish the sensor’s signal-to-noise ratio and limits the penetration depths for in vivo measurements. In this study, we perform a systematic investigation of the plasmonic effects of Ag and Au nanoparticles of various geometries to tune and even enhance the fluorescence intensity of single-stranded DNA-wrapped SWCNTs (ssDNA-SWCNTs). We observe a chirality-dependent NIR fluorescence enhancement that varies with both nanoparticle shape and material, with Au nanorods increasing (7, 5) and (7, 6) chirality emissions by 80% and 60% and Ag nanotriangles increasing (7, 5) and (6, 5) emissions by 200% and 240%, respectively. The chirality-dependent enhancement was modeled using finite element modeling (FEM), which confirms contributions not only from a plasmon-induced localized increase in electron density but also from the radiative recombination of dark exciton states from the resulting electromagnetic field. Finally, we demonstrate the application of these nanoparticles in enhancing the single-molecule fluorescence of individual SWCNTs imaged in a custom-built confocal setup. The plasmonically coupled sensors show four orders of magnitude greater sensitivity towards ferricyanide, a model analyte, compared to the non-coupled sensors. Plasmonic nanoparticles thus provide a tunable means of modulating SWCNT fluorescence to study fundamental transitions of otherwise forbidden states and to improve the optical sensing performance.

Biothiol-triggered H2S release from a near-infrared fluorescent H2S donor promotes cutaneous wound healing

Hydrogen sulfide (H2S) plays crucial roles in antioxidation, anti-inflammation, and cytoprotection. Despite substantial progress in the design and synthesis of activatable H2S donors, methods for high-precision detection and imaging of released H2S in living systems have been lacking. In this study, a biothiol-activated near-infrared (NIR) fluorescent H2S donor, PRO-ST, was developed for real-time visualization of H2S release. PRO-ST consists of a dicyanoisophorone-based NIR fluorescence moiety (TCOO), a biothiol-trigger moiety (4-isothiocyanate benzyl alcohol), and a sulfur-source group (thiophosgene). PRO-ST exhibits high NIR fluorescence enhancement (45-fold), outstanding H2S release efficiency (73%), controllable H2S release (60 min), and excellent cell compatibility. These distinctive features enable PRO-ST to be applied in visualizing H2S release in cells, zebrafish, and mice. Moreover, PRO-ST exhibits excellent performance in visualizing real-time anti-inflammation and wound healing enhancement in biological systems, as confirmed by in situ visualization of H2S release. Thus, PRO-ST provides a versatile and effective method to detect and visualize H2S release, elucidate the mechanisms underlying wound healing, or optimize interventional therapy.

A nitroreductase-responsive near-infrared phototheranostic probe for in vivo imaging of tiny tumor and photodynamic therapy

The hypoxia-activated and nitroreductase-responsive phototheranostic probe has been developed by incorporating a nitro group into a hemicyanine fluorophore. The probe displays extremely sensitive and selective near-infrared fluorescence enhancement to nitroreductase with the detection limit of 2.10 ng/mL. The detection mechanism relies on the nitroreductase-catalyzed reduction of the nitro group to an amino group, along with the generation of the fluorophore. The availability of the probe in fluorescence imaging and photodynamic therapy was demonstrated at cellular level and in vivo. The probe can image endogenous nitroreductase and the hypoxia status of living cells. The probe also exhibits significant phototoxicity to hypoxia tumor cells under the 660 nm laser irradiation. More importantly, the probe has been successfully utilized in imaging tiny tumor (about 6 mm3) and tumor photodynamic therapy in vivo. The proposed probe integrates accurate near-infrared fluorescence imaging and photodynamic therapy into the same molecule, which probably become a promising agent in the early diagnosis and therapy of tumors.

A near-infrared turn-on fluorescent probe for the rapid detection of selenocysteine and its application of imaging in living cells and mice

Selenocysteine (Sec) plays critical roles in many physiological and pathological processes of human beings, thus it is of great significance to develop accurate methods for detecting and visualizing Sec. Herein, a new anthracene derivative (YZ-M5) as a near-infrared (NIR) fluorescent probe for sensing Sec was designed. Based on the target-induced modulation of the intramolecular charge transfer (ICT) process of the probe, YZ-M5 can display a rapid strong NIR fluorescence enhancement at 660 nm for Sec within 5 min. The quantitative detection range for Sec is 0–40 μM with a low detection limit of 18.5 nM. YZ-M5 also showed high selectivity and sensitivity, as well as the capability for identification of Sec in living cells and mice.

Mitochondria-targeted NIR fluorescent probe for sensing Hg2+/HSO3− and its intracellular applications

Mercury and sulfur dioxide (SO2) are common pollutants in the ecological environment, which are important factors causing many diseases of organisms. The lack of appropriate analytical tools has limited the further understanding of the relationship between ionic mercury (Hg2+) and SO2. Herein, a bifunctional fluorescent probe LJ was designed and explored to simultaneously detect Hg2+ and SO2 via desulfurization reaction and Michael addition reaction, respectively. Probe LJ showed distinct fluorescence responses which a large near-infrared fluorescence enhancement towards Hg2+ at λem = 713 nm and a blue shift at λem = 450 nm towards SO2 without any spectral cross interferences. To the best of our knowledge, this is the first fluorescent probe with dual fluorescent emission channels to detect Hg2+ and SO2 with the detection limit of 187 nM and 354 nM, respectively. Moreover, cell fluorescent imaging experiments indicated that the probe was mitochondria targetable and provided evidence that SO2 could be used as an antidote to attenuate the toxicity of Hg2+ in living cells.

An Activatable Near-Infrared Fluorescence Hydrogen Sulfide (H2S) Donor for Imaging H2S Release and Inhibiting Inflammation in Cells.

Hydrogen sulfide (H2S) is a vital endogenous signal molecule that exerts critical physiological functions such as biological regulation and cytoprotection. Despite significant progress in developing H2S donors, site-specific delivery and controllable release of H2S in biological systems remain a key challenge. Herein, we develop new Cys-triggered fluorescent H2S donor Pro-S that is composed of a dicyanoisophorone-based near-infrared (NIR) fluorescent dye and a thiocarbamate moiety. The H2S donor releases H2S under the attack of Cys, accompanied by the release of a fluorescent reporter, which enables the real-time capturing of H2S by fluorescence spectroscopy or microscopy. Pro-S exhibits strong NIR fluorescence enhancement (70-fold), excellent controllable H2S release (30 min), high H2S release efficiency (62%), and well live-cell compatibility, allowing for visualization of H2S release in cells and zebrafish. Moreover, Pro-S presents a good effect of anti-inflammation in RAW 264.7 cells. This work provides a new idea for the design of H2S donors, which may be beneficial to the comprehension of the potential mechanism of inflammation and optimization of treatment strategies.

Nucleolin-Targeted Ratiometric Fluorescent Carbon Dots with a Remarkably Large Emission Wavelength Shift for Precise Imaging of Cathepsin B in Living Cancer Cells.

As one of the most promising biomarkers for numerous malignant tumors, accurate and reliable reporting of Cathepsin B (CTSB) activity is of great significance to achieve efficient diagnosis of cancers at an early stage and predicting metastasis. Here, we report a vigorous ratiometric fluorescent method integrating a cancer-targeting recognition moiety with a remarkably large emission wavelength shift into a single matrix to report CTSB activity sensitively and specifically. As a proof of concept, we synthesized amine-rich carbon quantum dots (CQDs) with a blue fluorescence, which offered an efficient scaffolding to covalently assemble the nucleolin-targeting recognition nucleic acid aptamer AS1411 and a CTSB-cleavable peptide substrate Gly-Arg-Arg-Gly-Lys-Gly-Gly-Cys-COOH that tethered with a near-infrared (NIR) fluorophore chlorin e6 (Ce6-GRRGKGGC, Ce6-Pep), enabling a cancer-targeting and CTSB stimulus-responsive ratiometric nanoprobe AS1411-Ce6-CQDs. Owing to the efficient fluorescence resonance energy transfer (FRET) process from the CQDs to Ce6 inside the assembly of nanoprobe, the blue fluorescence of CQDs at ∼450 nm was remarkably quenched, along with an obvious NIR fluorescence enhancement of Ce6 at ∼650 nm. After selective entry into cancer cells via nucleolin-mediated endocytosis, the overexpressed CTSB in lysosome could cleave Ce6-Pep and trigger the Ce6 moiety dissociation from AS1411-Ce6-CQDs, thus leading to the termination of FRET process, achieving the efficient ratiometric fluorescence response toward endogenous CTSB with a remarkably large emission wavelength shift of ∼200 nm from NIR to blue emission region. Notably, the nanoprobe AS1411-Ce6-CQDs exhibited an excellent specificity for ratiometric fluorescent sensing of CTSB activity with an ultralow detection limit of 0.096 ng/mL, demonstrating its promising use for early precise cancer diagnosis in the near future.

Engineering of a Dual-Recognition Ratiometric Fluorescent Nanosensor with a Remarkably Large Stokes Shift for Accurate Tracking of Pathogenic Bacteria at the Single-Cell Level.

Rapid, accurate, reliable, and risk-free tracking of pathogenic microorganisms at the single-cell level is critical to achieve efficient source control and prevent outbreaks of microbial infectious diseases. For the first time, we report a promising approach for integrating the concepts of a remarkably large Stokes shift and dual-recognition into a single matrix to develop a pathogenic microorganism stimuli-responsive ratiometric fluorescent nanoprobe with speed, cost efficiency, stability, ultrahigh specificity, and sensitivity. As a proof-of-concept, we selected the Gram-positive bacterium Staphylococcus aureus (S. aureus) as the target analyte model, which easily bound to its recognition aptamer and the broad-spectrum glycopeptide antibiotic vancomycin (Van). To improve the specificity and short sample-to-answer time, we employed classic noncovalent π-π stacking interactions as a driving force to trigger the binding of Van and aptamer dual-functionalized near-infrared (NIR) fluorescent Apt-Van-QDs to the surface of an unreported blue fluorescent π-rich electronic carbon nanoparticles (CNPs), achieving S. aureus stimuli-responsive ratiometric nanoprobe Apt-Van-QDs@CNPs. In the assembly of Apt-Van-QDs@CNPs, the blue CNPs (energy donor) and NIR Apt-Van-QDs (energy acceptor) became close to allow the fluorescence resonance energy transfer (FRET) process, leading to a remarkable blue fluorescence quenching for the CNPs at ∼465 nm and a clear NIR fluorescence enhancement for Apt-Van-QDs at ∼725 nm. In the presence of S. aureus, the FRET process from CNPs to Apt-Van-QDs was disrupted, causing the nanoprobe Apt-Van-QDs@CNPs to display a ratiometric fluorescent response to S. aureus, which exhibited a large Stokes shift of ∼260 nm and rapid sample-to-answer detection time (∼30.0 min). As expected, the nanoprobe Apt-Van-QDs@CNPs showed an ultrahigh specificity for ratiometric fluorescence detection of S. aureus with a good detection limit of 1.0 CFU/mL, allowing the assay at single-cell level. Moreover, we also carried out the precise analysis of S. aureus in actual samples with acceptable results. We believe that this work offers new insight into the rational design of efficient ratiometric nanoprobes for rapid on-site accurate screening of pathogenic microorganisms at the single-cell level in the early stages, especially during the worldwide spread of COVID-19 today.

Dual-functional multi-application probe: Rapid detection of H2S and colorimetric recognition of HSO3− in food and cell

Since hydrogen sulfide (H2S) and bisulfite (HSO3−) are widely used in the biological environment and industrial production, many fluorescent probes have been developed to detect them individually. However, designing and synthesizing a sensor that recognizes both H2S and HSO3− remains a challenge. In this study, we developed a new dual-function probe TCF-DNs for the dual channel detection of H2S and HSO3− in EtOH/H2O (2/8, v/v, HEPES 10 mM, pH = 7.4). The color of TCF-DNs changed from lilac to light-blue on treatment with H2S, and a visible color change from lilac to colorless occurred on treatment with HSO3− as well. The easy-to-synthesize TCF-DNs can recognize H2S through rapid near-infrared fluorescence enhancement with a detection limit of 13.5 nM, however, the probe exhibits fluorescence quenching toward HSO3−. The sensing mechanism of TCF-DNs is based on the removal of 2,4-dinitrobenzenesulfonate unit by HS− and the occurrence of a nucleophilic addition for HSO3−. In addition, the application study indicates that probe TCF-DNs can detect HSO3− in granulated sugar, and sensitively detect H2S in red wine, real water, living cells. Meanwhile, TCF-DNs can be used to prepare a cheap test paper strip for the rapid and qualitative detection of H2S by the naked eye and to serve as an indicator to detect H2S gas.

Rational design of a near-infrared fluorescence probe for highly selective sensing butyrylcholinesterase (BChE) and its bioimaging applications in living cell

In the current work, a near-infrared (NIR) fluorescent probe (CyClCP) was developed for fast (35 min), highly sensitive (LOD of 3.75 U/L) and selective response to BChE in vitro and in vivo. Upon the addition of BChE, CyClCP could be efficiently activated with remarkable NIR (λem = 708 nm) fluorescence enhancement and obvious absorbance red shift (581 nm–687 nm). Specifically, according to the subtle differences structural features and substrate preference between BChE and its sister enzyme AChE, CyClCP was constructed by introducing chlorine atom at the ortho-position of the phenolic hydroxyl in the previous reported probe (CyCP). Fortunately, CyClCP exhibited better selectivity towards BChE over AChE compared with CyCP. This molecular design strategy was further rationalized by docking molecular of fluorescence probes (CyClCP and CyCP) and enzymes (BChE and AChE). Finally, CyClCP was membrane permeable and successfully applied to image endogenous BChE level in HepG2 and LO2 cells. Therefore, CyClCP could serve as a promising tool for BChE-related physiological function studies in complex biological systems.

(Invited) Plasmon Induced Near-infrared Fluorescence Enhancement of Single-walled Carbon Nanotubes

The near-infrared emissions of single-walled carbon nanotubes (SWCNTs) falls within the optical transparency window of biological tissue, allowing deep-tissue, in vivo and in vitro imaging. However, the relatively low photoluminescence quantum yield of SWCNTs, which typically lies in the range of 0.1 - 1.5%, limits the penetration depth and sensitivity of these fluorophores when used as imaging agents and optical sensors. In the present work, we enhance the fluorescence emission of SWCNTs up to 10-fold by employing different localized surface plasmon resonance (LSPR) nanostructures. We observe a chirality-dependent enhancement that varies with the nanostructure geometry and material. The proposed mechanism is based on changes in the charge density following nanoparticle conjugation with the SWCNT. In agreement with this mechanism, the enhancement and quenching effects show a strong correlation with the distance between the SWCNTs and the nanostructures. Finally, we demonstrate the applicability of this technology in a single-molecule imaging SWCNT application.

Tunable Three-Dimensional Plasmonic Arrays for Large Near-Infrared Fluorescence Enhancement.

Metal-enhanced fluorescence (MEF), resulting from the near-field interaction of fluorophores with metallic nanostructures, has emerged as a powerful tool for dramatically improving the performance of fluorescence-based biomedical applications. Allowing for lower autofluorescence and minimal photoinduced damage, the development of multifunctional and multiplexed MEF platforms in the near-infrared (NIR) windows is particularly desirable. Here, a low-cost fabrication method based on nanosphere lithography is applied to produce tunable three-dimensional (3D) gold (Au) nanohole-disc arrays (Au-NHDAs). The arrays consist of nanoscale glass pillars atop nanoholes in a Au thin film: the top surfaces of the pillars are Au-covered (effectively nanodiscs), and small Au nanoparticles (nanodots) are located on the sidewalls of the pillars. This 3D hole-disc (and possibly nanodot) construct is critical to the properties of the device. The versatility of our approach is illustrated through the production of uniform and highly reproducible Au-NHDAs with controlled structural properties and tunable optical features in the NIR windows. Au-NHDAs allow for a very large NIR fluorescence enhancement (more than 400 times), which is attributed to the 3D plasmonic structure of the arrays that allows strong surface plasmon polariton and localized surface plasmon resonance coupling through glass nanogaps. By considering arrays with the same resonance peak and the same nanodisc separation distance, we show that the enhancement factor varies with nanodisc diameter. Using computational electromagnetic modeling, the electric field enhancement at 790 nm was calculated to provide insights into excitation enhancement, which occurs due to an increase in the intensity of the electric field. Fluorescence lifetime measurements indicate that the total fluorescence enhancement may depend on controlling excitation enhancement and therefore the array morphology. Our findings provide important insights into the mechanism of MEF from 3D plasmonic arrays and establish a low-cost versatile approach that could pave the way for novel NIR-MEF bioapplications.

Near-infrared fluorescence probes to detect reactive oxygen species for keloid diagnosis.

Development of molecular probes for the detection of reactive oxygen and nitrogen species (RONS) is important for the pathology and diagnosis of diseases. Although an abnormally high RONS level has been identified in keloids - a benign dermal tumour developed after lesion, the ability of employing RONS probes for keloid detection has not yet been exploited. Herein, we report two near-infrared (NIR) fluorescent probes (CyTF and CyBA) that can specifically distinguish keloid fibroblasts from normal dermal fibroblasts. Both CyTF and CyBA show a 15-fold NIR fluorescence enhancement at 717 nm upon reaction with RONS. However, because CyTF has higher specificity towards ONOO- than CyBA, CyTF can detect stimulated fibroblasts in a more sensitive way, showing 3.76 and 2.26-fold fluorescence increments in TGF-β1 stimulated dermal fibroblasts and keloid fibroblasts, respectively. Furthermore, CyTF permits specific detection of implanted keloid fibroblasts in a xenograft live mouse model. Our work thus developed a new optical imaging approach that has the potential for early diagnosis and drug screening of keloids.

Development of a new bioactivatable fluorescent probe for quantification of apolipoprotein A-I proteolytic degradation in vitro and in vivo

Background and aimsThe potential benefits of high-density lipoproteins (HDL) against atherosclerosis are attributed to its major protein component, apolipoprotein A-I (apoA-I). Most of the apoA-I in the vascular wall appears to be in its lipid-poor form. The latter, however, is subjected to degradation by proteases localized in atherosclerotic plaques, which, in turn, has been shown to negatively impact its atheroprotective functions. Here, we report the development and in vivo use of a bioactivatable near-infrared full-length apoA-I-Cy5.5 fluorescent probe for the assessment of apoA-I-degrading proteolytic activities. MethodsFluorescence quenching was obtained by saturation of Cy5.5 fluorophore molecules on apoA-I protein. ApoA-I cleavage led to near-infrared fluorescence enhancement. In vitro proteolysis of the apoA-I probe by a variety of proteases including serine, cysteine, and metalloproteases resulted in an up to 11-fold increase in fluorescence (n = 5, p ≤ 0.05). ResultsWe detected activation of the probe in atherosclerotic mice aorta sections using in situ zymography and showed that broad-spectrum protease inhibitors protected the probe from degradation, resulting in decreased fluorescence (−54%, n = 6 per group, p ≤ 0.0001). In vivo, the injected probe showed stronger fluorescence emission in the aorta of human apoB transgenic Ldlr−/− atherosclerotic mice (ATX) as compared to wild-type mice. In vivo observations were confirmed by ex vivo aorta imaging quantification where a 10-fold increase in fluorescent signal in ATX mice (p ≤ 0.05 vs. control mice) was observed. ConclusionsThe use of this probe in different applications may help to assess new molecular mechanisms of atherosclerosis and may improve current HDL-based therapies by enhancing apoA-I functionality.