New directions of activity-based sensing for in vivo NIR imaging

In vivo imaging is a powerful approach to study biological processes. Beyond cellular methods, in vivo studies allow for biological stimuli (small molecules or proteins) to be studied in their native environment. This has the potential to aid in the discovery of new biology and guide the development of diagnostics and therapies for diseases. To ensure selectivity and an observable readout, the probe development field is shifting towards activity-based sensing (ABS) approaches and near-infrared (NIR) imaging modalities. This perspective will highlight recent in vivo ABS applications that utilize NIR imaging platforms.

In vivo imaging is a powerful approach to study biological processes.     

In vivo monitoring of tissue regeneration using a ratiometric lysosomal AIE probe

Tissue regeneration is a crucial self-renewal capability involving many complex biological processes. Although transgenic techniques and fluorescence immunohistochemical staining have promoted our understanding of tissue regeneration, simultaneous quantification and visualization of tissue regeneration processes is not easy to achieve. Herein, we developed a simple and quantitative method for the real-time and non-invasive observation of the process of tissue regeneration. The synthesized ratiometric aggregation-induced-emission (AIE) probe exhibits high selectivity and reversibility for pH responses, good ability to map lysosomal pH both in vitro and in vivo, good biocompatibility and excellent photostability. The caudal fin regeneration of a fish model (medaka larvae) was monitored by tracking the lysosomal pH change. It was found that the mean lysosomal pH is reduced during 24–48 hpa to promote the autophagic activity for cell debris degradation. Our research can quantify the changes in mean lysosomal pH and also exhibit its distribution during the caudal fin regeneration. We believe that the AIE-active lysosomal pH probe can also be potentially used for long-term tracking of various lysosome-involved biological processes, such as tracking the stress responses of tissue, tracking the inflammatory responses, and so on.

An AIE-active ratiometric probe for the first time achieved the long-term quantification of lysosomal pH during the medaka larva''s caudal fin regeneration.     

In situ visualization of peroxisomal viscosity in the liver of mice with non-alcoholic fatty liver disease by near-infrared fluorescence and photoacoustic imaging

Non-alcoholic fatty liver disease (NAFLD) can gradually develop into hepatic failure, and early diagnosis is crucial to improve treatment efficiency. The occurrence of NAFLD is closely related to lipid metabolism. Peroxisomes act as the first and main site for lipid metabolism in the hepatocytes, so abnormal lipid metabolism might directly affect peroxisomal viscosity. Herein, we developed a new near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging probe (PV-1) for the real-time visualization of peroxisomal viscosity in vivo. This PV-1 encompasses the malononitrile group as the rotor, which emits strong NIRF (at 705 nm) and PA (at 680 nm) signals when rotation is hindered as viscosity increases. Through dual-mode imaging, we discovered distinctly higher viscosity in the liver of NAFLD mice for the first time. We further found the remarkable amelioration of NAFLD upon treatment with N-acetylcysteine (NAC). Therefore, we anticipate that the PV-1 imaging method is promising for the early diagnosis and prognostic evaluation of NAFLD.

We report a novel near-infrared fluorescence/photoacoustic imaging method for peroxisomal viscosity, enabling an accurate diagnosis and drug evaluation of non-alcoholic fatty liver disease.     

A FRET-based fluorescent Zn2+ sensor: 3D ratiometric imaging,flow cytometric tracking and cisplatin-induced Zn2+ fluctuation monitoring

Monitoring labile Zn²⁺ homeostasis is of great importance for the study of physiological functions of Zn²⁺ in biological systems. Here we report a novel ratiometric fluorescent Zn²⁺ sensor, CPBT, which was constructed based on chelation-induced alteration of FRET efficiency. CPBT was readily cell membrane permeable and showed a slight preferential localization in the endoplasmic reticulum. With this sensor, 3D ratiometric Zn²⁺ imaging was first realized in the head of zebra fish larvae via Z-stack mode. CPBT could track labile Zn²⁺ in a large number of cells through ratiometric flow cytometric assay. More interestingly, both ratiometric fluorescence imaging and flow cytometric assay demonstrated that the labile Zn²⁺ level in MCF-7 cells (cisplatin-sensitive) decreased while that in SKOV3 cells (cisplatin-insensitive) increased after cisplatin treatment, indicating that Zn²⁺ may play an important role in cisplatin induced signaling pathways in these cancer cells.

A Zn²⁺ sensor exhibiting 3D ratiometric imaging and flow cytometric ability was constructed based on the FRET mechanism, and cisplatin-induced endogenous labile Zn²⁺ fluctuations were monitored in real time.     

A facile combinatorial approach to construct a ratiometric fluorescent sensor: application for the real-time sensing of cellular pH changes

Realtime monitoring of the cellular environment, such as the intracellular pH, in a defined cellular space provides a comprehensive understanding of the dynamics processes in a living cell. Considering the limitation of spatial resolution in conventional microscopy measurements, multiple types of fluorophores assembled within that space would behave as a single fluorescent probe molecule. Such a character of microscopic measurements enables a much more flexible combinatorial design strategy in developing fluorescent probes for given targets. Nanomaterials with sizes smaller than the microscopy spatial resolution provide a scaffold to assemble several types of fluorophores with a variety of optical characteristics, therefore providing a convenient strategy for designing fluorescent pH sensors. In this study, fluorescein (CF) and tetramethylrhodamine (CR) were assembled on a DNA nanostructure with controlling the number of each type of fluorophore. By taking advantage of the different responses of CF and CR emissions to the pH environment, an appropriate assembly of both CF and CR on DNA origami enabled a controlled intensity of fluorescence emission and ratiometric pH monitoring within the space defined by DNA origami. The CF and CR-assembled DNA origami was successfully applied for monitoring the intracellular pH changes.

A combinatorial assembly of two types of intensity-based fluorophores on a DNA nanostructure provided a ratiometric pH probe with high emission intensity for monitoring intracellular pH changes.     

A ratiometric photoelectrochemical microsensor based on a small-molecule organic semiconductor for reliable in vivo analysis

Photoelectrochemical (PEC) sensing has been developing quickly in recent years, while its in vivo application is still in the infancy. The complexity of biological environments poses a high challenge to the specificity and reliability of PEC sensing. We herein proposed the concept of small-molecule organic semiconductor (SMOS)-based ratiometric PEC sensing making use of the structural flexibility as well as readily tunable energy band of SMOS. Xanthene skeleton-based CyOH was prepared as a photoactive molecule, and its absorption band and corresponding PEC output can be modulated by an intramolecular charge transfer process. As such, the target mediated shift of absorption offered the opportunity to construct a ratiometric PEC sensor. A proof-of-concept probe CyOThiols was synthesized and assembled on a Ti wire electrode (TiWE) to prepare a highly selective microsensor for thiols. Under two monochromatic laser excitation (808 nm and 750 nm), CyOThiols/TiWE offered a ratiometric signal (j₈₀₈/j₇₅₀), which exhibited pronounced capacity to offset the disturbance of environmental factors, guaranteeing its reliability for application in vivo. The ratiometric PEC sensor achieved the observation of bio-thiol release induced by cytotoxic edema and fluctuations of thiols in drug-induced epilepsy in living rat brains.

The first small-molecule organic semiconductor-based ratiometric photoelectrochemical sensor was proposed, which exhibited pronounced selectivity and capacity to offset environmental disturbance, guaranteeing its reliability for in vivo analysis.     

Tuning molecular aggregation to achieve highly bright AIE dots for NIR-II fluorescence imaging and NIR-I photoacoustic imaging

Currently, bright aggregation-induced emission luminogens (AIEgens) with high photoluminescence quantum yields (PLQYs) in the NIR-II region are still limited, and thus an efficient strategy to enhance NIR-II fluorescence performance through tuning molecular aggregation is proposed here. The synthesized donor–acceptor tailored AIEgen (DTPA-TBZ) not only exhibits an excellent absorptivity in the NIR-I region, but also good fluorescence signals in the NIR-II region with an emission extending to 1200 nm. Benefiting from such improved intramolecular restriction and aggregation, a significant absolute PLQY value of 8.98% was obtained in solid DTPA-TBZ. Encouragingly, the resulting AIE dots also exhibit a high relative PLQY of up to 11.1% with IR 26 as the reference (PLQY = 0.5%). Finally, the AIE dots were applied in high performance NIR-II fluorescence imaging and NIR-I photoacoustic (PA) imaging: visualization of abdominal vessels, hind limb vasculature, and cerebral vessels with high signal to background ratios was performed via NIR-II imaging; Moreover, PA imaging has also been performed to clearly observe tumors in vivo. These results demonstrate that by finely tuning molecular aggregation in DTPA-TBZ, a good NIR-I absorptivity and a highly emissive fluorescence in the NIR-II region can be achieved simultaneously, finally resulting in a promising dual-modal imaging platform for real-world applications to achieve precise cancer diagnostics.

A highly efficient dual-modal imaging platform by using bright AIE dots was constructed to achieve precise cancer diagnostics.     

High-efficiency dynamic sensing of biothiols in cancer cells with a fluorescent β-cyclodextrin supramolecular assembly

A unique fluorescent supramolecular assembly was constructed using coumarin-modified β-cyclodextrin as a reversible ratiometric probe and an adamantane-modified cyclic arginine–glycine–aspartate peptide as a cancer-targeting agent via host–guest inclusion complexation. Importantly, the coumarin-modified β-cyclodextrin not only showed higher sensitivity than the parent coumarin derivatives owing to the presence of numerous hydroxyl groups on the cyclodextrin but also provided a hydrophobic cavity for encapsulation of a cancer-targeting agent. The assembly showed a reversible and fast response to biothiols with a micromolar dissociation constant, as well as outstanding cancer cell permeability, which can be used for high-efficiency real-time monitoring of biothiols in cancer cells. This supramolecular assembly strategy endows the fluorescent probe with superior performance for dynamic sensing of biothiols.

A unique fluorescent supramolecular assembly was constructed from coumarin-modified β-cyclodextrin and an adamantane-modified cyclic arginine–glycine–aspartate peptide for high-efficiency real-time monitoring of biothiols in cancer cells.     

A fluorescent molecular imaging probe with selectivity for soluble tau aggregated protein

Soluble forms of aggregated tau misfolded protein, generally termed oligomers, are considered to be the most toxic species of the different assembly states that are the pathological components of neurodegenerative disorders. Therefore, a critical biomedical need exists for imaging probes that can identify and quantify them. We have designed and synthesized a novel fluorescent probe, pTP-TFE for which binding and selectivity profiles towards aggregated tau and Aβ proteins were assessed. Our results have shown pTP-TFE to be selective for early forms of soluble tau aggregates, with high affinity of dissociation constants (K_d) = 66 nM, and tenfold selectivity over mature tau fibrils. Furthermore, we found that pTP-TFE is selective for tau over Aβ aggregates and had good cell permeability. This selectivity of pTP-TFE towards early forms of aggregated tau protein ex vivo was also supported with studies on human brain tissue containing tau and Aβ pathology. To the best of our knowledge, this is the first fluorescent molecule to be reported to have this form of selectivity profile, which suggests that pTP-TFE is a unique probe candidate for imaging-based detection of early stages of Alzheimer''s disease and other tauopathies.

pTP-TFE imaging probe can distinguish soluble tau aggregated proteins from other aggregated proteins enabling earlier detection of neurodegenerative diseases.     

Protein encapsulation: a new approach for improving the capability of small-molecule fluorogenic probes

Herein, we report a protein-based hybridization strategy that exploits the host-guest chemistry of HSA (human serum albumin) to solubilize the otherwise cell impermeable ONOO⁻ fluorescent probe Pinkment-OAc. Formation of a HSA/Pinkment-OAc supramolecular hybrid was confirmed by SAXS and solution-state analyses. This HSA/Pinkment-OAc hybrid provided an enhanced fluorescence response towards ONOO⁻versusPinkment-OAc alone, as determined by in vitro experiments. The HSA/Pinkment-OAc hybrid was also evaluated in RAW 264.7 macrophages and HeLa cancer cell lines, which displayed an enhanced cell permeability enabling the detection of SIN-1 and LPS generated ONOO⁻ and the in vivo imaging of acute inflammation in LPS-treated mice. A remarkable 5.6 fold (RAW 264.7), 8.7-fold (HeLa) and 2.7-fold increased response was seen relative to Pinkment-OAc alone at the cellular level and in vivo, respectively. We anticipate that HSA/fluorescent probe hybrids will soon become ubiquitous and routinely applied to overcome solubility issues associated with hydrophobic fluorescent imaging agents designed to detect disease related biomarkers.

Herein, we report a protein-based hybridization strategy that exploits the host–guest chemistry of HSA (human serum albumin) to solubilize the otherwise cell impermeable ONOO⁻ fluorescent probe Pinkment-OAc.     

A pH ratiometrically responsive surface enhanced resonance Raman scattering probe for tumor acidic margin delineation and image-guided surgery

Surgery remains the mainstay for most solid tumor treatments. However, surgeons face challenges in intra-operatively identifying invasive tumor margins due to their infiltrative nature. Incomplete excision usually leads to early recurrence, while aggressive resection may injure adjacent functional tissues. Herein, we report a pH responsive ratiometric surface-enhanced Raman scattering (SERRS) probe that determined physiological pHs with a high sensitivity and tissue penetration depth via an innovative mechanism named spatial orientation induced intramolecular energy transfer (SOIET). Due to the positive correlation between tumor acidity and malignancy, an acidic margin-guided surgery strategy was implemented in live animal models by intra-operatively assessing tissue pH/malignancy of the suspicious tissues in tumor cutting edges. This surgery remarkably extended the survival of animal models and minimized their post-surgical complications, showing promise in precisely identifying invasive tumor boundaries and achieving a balance between maximum tumor debulking and minimal functional impairment.

A novel pH ratiometrically responsive surface-enhanced resonance Raman scattering (SERRS) probe was developed for tumor acidic margin delineation and image-guided surgery.     

Regulation of redox balance using a biocompatible nanoplatform enhances phototherapy efficacy and suppresses tumor metastasis

Many cancer treatments including photodynamic therapy (PDT) utilize reactive oxygen species (ROS) to kill tumor cells. However, elevated antioxidant defense systems in cancer cells result in resistance to the therapy involving ROS. Here we describe a highly effective phototherapy through regulation of redox homeostasis with a biocompatible and versatile nanotherapeutic to inhibit tumor growth and metastasis. We systematically explore and exploit methylene blue adsorbed polydopamine nanoparticles as a targeted and precise nanocarrier, oxidative stress amplifier, photodynamic/photothermal agent, and multimodal probe for fluorescence, photothermal and photoacoustic imaging to enhance anti-tumor efficacy. Remarkably, following the glutathione-stimulated photosensitizer release to generate exogenous ROS, polydopamine eliminates the endogenous ROS scavenging system through depleting the primary antioxidant, thus amplifying the phototherapy and effectively suppressing tumor growth in vitro and in vivo. Furthermore, this approach enables a robust inhibition against breast cancer metastasis, as oxidative stress is a vital impediment to distant metastasis in tumor cells. Innovative, safe and effective nanotherapeutics via regulation of redox balance may provide a clinically relevant approach for cancer treatment.

Amplified oxidative stress achieved by modulating redox homeostasis with PDA–MB for highly effective synergistic phototherapy to inhibit primary tumors and metastases.     

In vivo therapeutic response monitoring by a self-reporting upconverting covalent organic framework nanoplatform

The real-time and in situ monitoring of reactive oxygen species (ROS) generation is critical for minimizing the nonspecific damage derived from the high doses of ROS required during the photodynamic therapy (PDT) process. However, phototherapeutic agents that can generate ROS-related imaging signals during PDT are rare, hampering the facile prediction of the future therapeutic outcome. Herein, we develop an upconverting covalent organic framework (COF) nanoplatform via a core-mediated strategy and further functionalized it with a singlet oxygen reporter for the efficient near-infrared activated and in situ self-reporting of PDT. In this work, the COF photodynamic efficacy is greatly improved (12.5 times that of irregular COFs) via tailoring the size. Furthermore, this nanoplatform is able to not only produce singlet oxygen for PDT, but it can also emit singlet oxygen-correlated luminescence, allowing the real-time and in situ monitoring of the therapeutic process for cancer cells or solid tumors in vivo via near-infrared luminescence imaging. Thus, our core-mediated synthetic and size-tailored strategy endows the upconverting COF nanoplatform with promising abilities for high-efficacy, deep-tissue, precise photodynamic treatment.

An upconverting covalent organic framework nanoplatform is designed for the first time for the near-infrared activated in situ self-reporting of photodynamic therapy in vivo.     

Engineering multifunctional metal/protein hybrid nanomaterials as tools for therapeutic intervention and high-sensitivity detection

Protein-based hybrid nanomaterials have recently emerged as promising platforms to fabricate tailored multifunctional biologics for biotechnological and biomedical applications. This work shows a simple, modular, and versatile strategy to design custom protein hybrid nanomaterials. This approach combines for the first time the engineering of a therapeutic protein module with the engineering of a nanomaterial-stabilizing module within the same molecule, resulting in a multifunctional hybrid nanocomposite unachievable through conventional material synthesis methodologies. As the first proof of concept, a multifunctional system was designed ad hoc for the therapeutic intervention and monitoring of myocardial fibrosis. This hybrid nanomaterial combines a designed Hsp90 inhibitory domain and a metal nanocluster stabilizing module resulting in a biologic drug labelled with a metal nanocluster. The engineered nanomaterial actively reduced myocardial fibrosis and heart hypertrophy in an animal model of cardiac remodeling. In addition to the therapeutic effect, the metal nanocluster allowed for in vitro, ex vivo, and in vivo detection and imaging of the fibrotic disease under study. This study evidences the potential of combining protein engineering and protein-directed nanomaterial engineering approaches to design custom nanomaterials as theranostic tools, opening up unexplored routes to date for the next generation of advanced nanomaterials in medicine.

Engineering protein-based hybrids by combining protein engineering and nanotechnology: a protein-nanocluster hybrid for theranostic use in myocardial fibrosis shows the potential to create tailored multifunctional biologics for biomedicine.     

Development of photo- and chemo-stable near-infrared-emitting dyes: linear-shape benzo-rosol and its derivatives as unique ratiometric bioimaging platforms

Microscopic imaging aided with fluorescent probes has revolutionized our understanding of biological systems. Organic fluorophores and probes thus continue to evolve for bioimaging applications. Fluorophores such as cyanines and hemicyanines emit in the near-infrared (NIR) region and thus allow deeper imaging with minimal autofluorescence; however, they show limited photo- and chemo-stability, demanding new robust NIR fluorophores. Such photo- and chemo-stable NIR fluorophores, linear-shape π-extended rosol and rosamine analogues, are disclosed here which provide bright fluorescence images in cells as well as in tissues by confocal laser-scanning microscopy. Furthermore, they offer unique ratiometric imaging platforms for activatable probes with dual excitation and dual emission capability, as demonstrated with a 2,4-dinitrophenyl ether derivative of benzo-rosol.

NIR-emitting benzo-rosol and -rosamine dyes offer novel ratiometric imaging platforms with high pohoto- and chemo-stability.     

Self-assembled amphiphilic fluorescent probe: detecting pH-fluctuations within cancer cells and tumour tissues

Abnormal anaerobic metabolism leads to a lowering of the pH of many tumours, both within specific intracellular organelles and in the surrounding extracellular regions. Information relating to pH-fluctuations in cells and tissues could aid in the identification of neoplastic lesions and in understanding the determinants of carcinogenesis. Here we report an amphiphilic fluorescent pH probe (CS-1) that, as a result of its temporal motion, provides pH-related information in cancer cell membranes and selected intracellular organelles without the need for specific tumour targeting. Time-dependent cell imaging studies reveal that CS-1 localizes within the cancer cell-membrane about 20 min post-incubation. This is followed by migration to the lysosomes at 30 min before being taken up in the mitochondria after about 60 min. Probe CS-1 can selectively label cancer cells and 3D cancer spheroids and be readily observed using the green fluorescence channel (λ_em = 532 nm). In contrast, CS-1 only labels normal cells marginally, with relatively low Pearson''s correlation coefficients being found when co-incubated with standard intracellular organelle probes. Both in vivo and ex vivo experiments provide support for the suggestion that CS-1 acts as a fluorescent label for the periphery of tumours, an effect ascribed to proton-induced aggregation. A much lower response is seen for muscle and liver. Based on the present results, we propose that sensors such as CS-1 may have a role to play in the clinical and pathological detection of tumour tissues or serve as guiding aids for surgery.

A self-assembled amphiphilic fluorescent probe allows pH-fluctuations within cancer cells and tumour tissues to be readily detected.     

NIR-II cell endocytosis-activated fluorescent probes for in vivo high-contrast bioimaging diagnostics

Fluorescence probes have great potential to empower bioimaging, precision clinical diagnostics and surgery. However, current probes are limited to in vivo high-contrast diagnostics, due to the substantial background interference from tissue scattering and nonspecific activation in blood and normal tissues. Here, we developed a kind of cell endocytosis-activated fluorescence (CEAF) probe, which consists of a hydrophilic polymer unit and an acid pH-sensitive small-molecule fluorescent moiety that operates in the “tissue-transparent” second near-infrared (NIR-II) window. The CEAF probe stably presents in the form of quenched nanoaggregates in water and blood, and can be selectively activated and retained in lysosomes through cell endocytosis, driven by a synergetic mechanism of disaggregation and protonation. In vivo imaging of tumor and inflammation with a passive-targeting and affinity-tagged CEAF probe, respectively, yields highly specific signals with target-to-background ratios over 15 and prolonged observation time up to 35 hours, enabling positive implications for surgical, diagnostic and fundamental biomedical studies.

A Cell Endocytosis-Activated Fluorescent (CEAF) probe triggered by disaggregation and protonation is designed for high contrast in vivo bioimaging and diagnostics in the second near-infrared window (1000–1700 nm).     

Late-stage functionalisation of alkyne-modified phospha-xanthene dyes: lysosomal imaging using an off–on–off type of pH probe

Near-infrared (NIR) fluorescent molecules are of great importance for the visualisation of biological processes. Among the most promising dye scaffolds for this purpose are P O-substituted phospha-xanthene (POX) dyes, which show NIR emission with high photostability. Their practical utility for in vitro and in vivo imaging has recently been demonstrated. Although classical modification methods have been used to produce POX-based fluorescent probes, it is still a challenge to introduce additional functional groups to control the localisation of the probe in cells. Herein, we report on the development of POXs that bear a 4-ethynylphenyl group on the phosphorus atom. These dyes can subsequently be functionalised with azide-tagged biomolecules via a late-stage Cu-catalysed azide/alkyne cycloaddition (CuAAC) reaction, thus achieving target-selective labelling. To demonstrate the practical utility of the functionalised POXs, we designed a sophisticated NIR probe that exhibits a bell-shaped off–on–off pH-response and is able to assess the degree of endosomal maturation.

A series of NIR-emissive phospha-xanthene dyes bearing an ethynyl group are reported. The late-stage functionalisation of the NIR dyes enables creation of multi-functionalised fluorescent probes that can be designed to target organelles of interest.     

Enzyme-activatable fluorescent probes for β-galactosidase: from design to biological applications

β-Galactosidase (β-gal), a typical hydrolytic enzyme, is a vital biomarker for cell senescence and primary ovarian cancers. Developing precise and rapid methods to monitor β-gal activity is crucial for early cancer diagnoses and biological research. Over the past decade, activatable optical probes have become a powerful tool for real-time tracking and in vivo visualization with high sensitivity and specificity. In this review, we summarize the latest advances in the design of β-gal-activatable probes via spectral characteristics and responsiveness regulation for biological applications, and particularly focus on the molecular design strategy from turn-on mode to ratiometric mode, from aggregation-caused quenching (ACQ) probes to aggregation-induced emission (AIE)-active probes, from near-infrared-I (NIR-I) imaging to NIR-II imaging, and from one-mode to dual-mode of chemo-fluoro-luminescence sensing β-gal activity.

This review highlights the molecular design strategy of β-galactosidase-activatable probes from turn-on mode to ratiometric mode, from ACQ to AIE-active probes, from NIR-I to NIR-II imaging and dual-mode of chemo-fluoro-luminescence imaging.